WO2011083140A1

WO2011083140A1 - Immunoglobulin single variable domain directed against human cxcr4

Info

Publication number: WO2011083140A1
Application number: PCT/EP2011/050156
Authority: WO
Inventors: Maarten Dewilde; Maria Gonzalez Pajuelo; Peter Vanlandschoot; Karen Cromie; Els Pattyn; Benedikte Serruys; Catelijne Stortelers; Beatrijs Strubbe
Original assignee: Ablynx Nv
Priority date: 2010-01-08
Filing date: 2011-01-07
Publication date: 2011-07-14
Also published as: WO2011083141A2; WO2011083141A3

Abstract

The present invention relates to amino acid sequences that are immunoglobulin single variable domains (as defined herein) directed against CXCR4. In particular the present invention relates to immunoglobulin single variable domains directed CXCR4 with improved potency and constructs comprising the same. Thus, the invention relates to compounds, constructs, proteins or polypeptides that comprise or essentially consist of one or more such amino acid sequences.

Description

IMMUNOGLOBULIN SINGLE VARIABLE DOMAIN DIRECTED AGAINST HUMAN CXCR4

BACKGROUND OF THE INVENTION

: The present invention relates to amino acid sequences that are immunoglobulin single variable domains (as defined herein) directed against CXCR4.

The invention further relates to compounds, constructs, proteins or polypeptides that comprise or essentially consist of one or more such amino acid sequences.

Further aspects, embodiments, features, advantages, uses, applications and advantages from the present invention will become clear from the further description herein.

The international application WO 09/138519 by Ablynx N.V. entitled Amino acid sequences directed against CXCR4 and other GPCRs and compounds comprising the same^''' describes amino acid sequences against G-protein coupled receptors (GPCRs) and in particular human CXCR4, Genbank accession number AF005058.

WO 09/138519 describes a number of amino acid sequences and in particular VHHs and constructs thereof that are directed against human CXCR4 (see for example the amino acid sequences mentioned such as SEQ ID NO: 238 and SEQ ID NO: 239 in Table B-1.1 of WO 09/138519). WO 09/138519 also describes multivalent, multispecific and/or biparatopic constructs (as defined in WO 09/138519) that are directed against human CXCR4. Reference is for example made to the constructs referred to in Example 4 of WO 09/138519 such as SEQ ID NO: 264 in Table B-5 of WO 09/138519).

One particularly preferred example of an amino acid sequence against human CXCR4 from WO 09/138519 is the sequence called 238D2 (see SEQ ID NO: 238 in WO 09/138519 and SEQ ID NO: 445 herein)

One other particularly preferred example of an amino acid sequence against the human

CXCR4 from WO 09/138519 is the sequence called 238D4 (see SEQ ID NO: 239 in WO 09/138519 and SEQ ID NO: 447 herein)

WO 09/138519 further gives some non-limiting examples of multivalent, multispecific and/or biparatopic constructs that comprise 238D2 and/or 238D4 (see for example SEQ ID NO's: 261 to 266 in WO 09/138519 and in particular 238D2-20GS-238D4).

The non-prepublished US application 61/358,495 by applicant filed on June 25, 2010 entitled "Improved immunoglobulin single variable domains and constructs thereof directed against CXCR-4" describes a number of sequence-optimized/improved variants of the amino acid sequences 238D2 and 238D4, as well as multivalent, muitispecific and/or biparatopic constructs that comprise such improved variants as building blocks.

The PCT application PCT/EP2010/064766 by Ablynx N.V. filed on October 4, 2010 and entitled "Immunoglobulin single variable domains directed against human CXCR-4 and other cell-associated proteins and methods to generate them " describes the binding epitope of amino acid sequences 238D2 and 238D4 as well as a number of further immunoglobulin single variable domains capable of binding to the same epitope.

For the role of CXCR-4 and anti -CXCR-4 therapy in various forms of cancer, further reference is for example made to the reviews by Burger and Kipps, Blood, 2006; Dorsam and Gutkind 2007, Nat Rev Cancer, 2007; Kryczek et al, Am J Physiol Cell Physiol, 2007;

Balkwill, Nat Rev Cancer, 2004; and for example to Mueller et al, Nature, 2001 , 50-56

(breast cancer); Nervi et al., Blood, 2009, 1 19, 24, p.6206-62-14 (AML); Redjal et al., Clin.

Cancer Res., 2006, 12(22), 2006, 6765-6771 (gliomas); Rubin et al., PNAS, 100, 23, 20-03,

13513-13518 (brain tumors); Jin et al, Mol. Cancer Ther. 2008; 7: 48-58 (CML) and Zeng et al., Blood, 1 13, 24, 2009, 6215-6224.

SUMMARY OF THE INVENTION

Unless explicitly mentioned otherwise herein, all terms mentioned herein have the meaning given in WO 09/138519 (or in the prior art cited in WO 09/138519), in US

61/358,495 or in PCT/EP2010/064766. Also, where a method or technique is not specifically described herein, it can be performed as described in WO 09/138519 (or in the prior art cited in WO 09/138519), in US 61/358,495 or in PCT/EP2010/064766.

For example, the term "Nanobody®" is as defined in WO 09/138519, and thus in a specific aspect generally denotes a VHH, a humanized VHH or a camelized VH (such as a camelized human VH) or generally a sequence optimized VHH (such as e.g., optimized for chemical stability and/or solubility, maximum overlap with known human framework regions and maximum expression). [Note: Nanobody®, and Nanobodies® are trademarks of Ablynx N. V.] For a further description of V_HH'S and Nanobodies®, reference is made to the review article by Muyldermans in Reviews in Molecular Biotechnology (74(2001), 277-302); as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Abiynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO

06/122825, by Abiynx N.V. and the further published patent applications by Abiynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application WO 06/040153, which list and references are incorporated herein by reference. As described in these references, generally, Nanobodi.es® (in particular VHH sequences and partially humanized. Nanobodies®) can in particular be characterized by the presence of one or more "Hallmark residues⁷' in one or more of the framework sequences, as described further herein.

In particular, the following terms have the same meaning as given on, and/or where applicable can be determined in the manner described in, pages 62-75 of WO 09/138519: "agonist", "antagonist, "inverse agonist", "non-polar, uncharged amino acid residue", "polar uncharged amino acid residue", "polar, charged amino acid residue", "sequence identity", "exactly the same" and "amino acid difference" (when referring to a sequence comparison of two amino acid sequences), "(in) essentially isolated (form) ", "domain", "binding domain", "antigenic determinant "epitope", "against" or "directed against" (an anti gen), "spe /rc/r ' and "half-life". In addition, the terms "modulating" and "to modulate", "interaction site", "specific for", "cross-block", "cross-blocked'' and "cross-blocking" and "essentially independent of the pW are as defined on (and/or can be determined as described on) pages 74-79 of WO 10/130832 of Abiynx N.V. Also, when referring to a construct, compound, protein or polypeptide of the invention, terms like "monovalent" , "bivalent" (or "multivalent"), "bispecific" (or "multispecific"), and "biparatopic" (or "multipar atopic") may have the meaning given in WO 09/138519 and WO 10/130832.

When a term is not specifically defined, it has its usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd.Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987); Lewin, "Genes Π", John Wiley & Sons, New York, N.Y., (1985); Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering", 2nd edition, University of California Press, Berkeley, CA (1981); Roitt et al., "Immunology" (6th. Ed.), Mosby/Elsevier,

Edinburgh (2001); Roitt et al, Roit s Essential Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science

Publishing/Churchill Livingstone, New York (2005), as well as to the general background art cited herein. It should be appreciated that, as used herein, "essentially consisting of and "consisting essentially of, and like terms, can be used, interchangeably and have the same meaning.

Also, herein, the amino acid residues of a Nanobody® are numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and

Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195; or referred to herein. According to this numbering, FR1 of a Nanobody® comprises the amino acid residues at positions 1-30, CDRl of a Nanobody® comprises the amino acid residues at positions 31-35, FR2 of a Nanobody® comprises the amino acids at positions 36-49, CDR2 of a Nanobody® comprises the amino acid residues at positions 50-65, FR3 of a Nanobody® comprises the amino acid residues at positions 66-94, CDR3 of a Nanobody® comprises the amino acid residues at positions 95-102, and FR4 of a Nanobody® comprises the amino acid residues at positions 103-113. [In this respect, it should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDR's may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. Generally, however, it can be said that, according to the numbering of Kabat and irrespective of the number of amino acid residues in the CDR's, position 1 according to the Kabat numbering corresponds to the start of FR1 and vice versa, position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa, position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa, and position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.]. Alternative methods for numbering the amino acid residues of VH domains, which methods can also be applied in an analogous manner to VHH domains from Camelids and to Nanobodies®, are the method described by Chothia et al. (Nature 342, 877-883 (1989)), the so-called "AbM definition" and the so-called "contact definition". However, in the present description, aspects and figures, the numbering according to Kabat as applied to VHH domains by Riechmann and Muyldermans will be followed, unless indicated otherwise.

Generally, the anti-human CXCR4 amino acid sequences and constructs from WO 09/138519, from US 61/358,495, US 61/358,495 and from PCT/EP2010/064766 show excellent biological activity and other desired properties. However, this does not mean that either alternative anti-human CXCR4 amino acid sequences and constructs and/or an anti- human CXCR4 amino acid sequences and constructs that would have (even further) improved properties would not be a valuable addition to the art.

The invention provides such alternative and/or improved anti human CXCR4 amino acid sequences (also referred to herein as "amino acid sequence (s) of the invention" or " Immunoglobulin Single Variable Domains (ISVs) of the invention^'") and constructs comprising the same (also referred to herein as "constructs of the invention" or "polypeptides of the invention").

The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to herein as "nucleic acids of the invention" or "nucleotide sequences of the invention"): to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.

In particular, the invention provides: - amino acid sequences binding CXCR-4, which are an alternative to the sequences from WO 09/138519 and US 61/358,495 in that, for example and without limitation, they can bind to a different epitope, domain or (extracellular) loop of CXCR-4 than the sequences 238D2 or 238D4 and their improved variants; and/or which are an alternative to the sequences from WO 09/138519 and US 61/358,495 in that, for example and without limitation, they belong to a different family (as described herein) than the sequences 238D2 or 238D4 and their improved variants;

and/or - amino acid sequences binding CXCR-4, which are improved compared to the

sequences 238D2 and/or 238D4 and their improved variants in that, for example and without limitation, they show greater potency in a relevant assay (for example but without limitation, in the Jurkat assay described in Example 1, C-4; and/or the cAMP inhibition assay described in Example 1, C-3);

and/or

constructs, compounds and polypeptides comprising one or more of such amino acid sequences, as further described herein.

Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.

More in particular, in a preferred but non-limiting aspect, the invention provides the following amino acid sequences: 4CXCR010E09 (SEQ ID NO: 189, also referred to herein as "10E9"), 4CXCR281E10 (SEQ ID NO: 190, also referred to herein as "281E1.0"),

4CXCR010E12 (SEQ ID NO: 191, also referred to herein as "10E12"), 4CXCR010A10 (SEQ ID NO: 192, also referred to herein as "10A10"), 4CXCR010G10 (SEQ ID NO: 193, also referred to herein as "10G10"), 4CXCR014A02 (SEQ ID NO: 194, also referred to herein as "14A2"), 4CXCR015A01 (SEQ ID NO: 195, also referred to herein as "15ΑΓ), 4CXCR015H03 (SEQ ID NO: 196, also referred to herein as "15H3") and 4CXCR283B06 (SEQ ID NO: 197, also referred to herein as "283B6"), as well as humanized and/or otherwise sequence-optimized variants thereof (as further described herein), of which: - the amino acid sequences 4CXCR010E09 (SEQ ID NO: 189), 4CXCR281E10 (SEQ

ID NO: 190), 4CXCR010E12 (SEQ ID NO: 191), 4CXCR010A10 (SEQ ID NO: 192), 4CXCR010G10 (SEQ ID NO: 193), 4CXCR014A02 (SEQ ID NO: 194), 4CXCR015A01 (SEQ ID NO: 195), 4CXCR015H03 (SEQ ID NO: 196) and 4CXCR283B06 (SEQ ID NO: 197) all show a better potency than 238D2 and 238D4 in the Jurkat assay described. in Example 1, C-4; the amino acid sequences 4CXCR010E09 (SEQ ID NO: 189), 4CXCR281E10 (SEQ ID NO: 190), 4CXCR010E12 (SEQ ID NO: 191 ), 4CXCR010A10 (SEQ ID NO: 192), 4CXCR010G10 (SEQ ID NO: 193), 4CXCR034A02 (SEQ ID NO: 194), 4CXCR015A01 (SEQ ID NO: 195), 4CXCR015H03 (SEQ ID NO: 196) and 4CXCR283B06 (SEQ ID NO: 197) all show a better potency than 238D2 in the cAMP inhibition assay described in

Example 1, C-3;

the amino acid sequences 4CXCR010E09 (SEQ ID NO: 189),4CXCR281E10 (SEQ ID NO: 190), 4CXCR010E12 (SEQ ID NO: 191), 4CXCR010G10 (SEQ ID NO: 193), 4CXCR015A01 (SEQ ID NO: 195), show a better potency than 238D4 in the cAMP inhibition assay described in Example 1 , C-3; with 4CXCR014A02 (SEQ ID NO: 194) showing a potency comparable to 238D4 in the same assay;

the amino acid sequences 4CXCR010E12 (SEQ ID NO: 191), 4CXCR010A10 (SEQ ID NO: 192), 4CXCR010G10 (SEQ ID NO: 193), 4CXCR014A02 (SEQ ID NO: 194), 4CXCR015A01 (SEQ ID NO: 195), 4CXCR015H03 (SEQ ID NO: 196) and 4CXCR283B06 (SEQ ID NO: 197), belong to different families and/or bind to different epitopes, domains or loops on CXCR-4 and/or have different "footprints" (see Example 1, D-4 and Example 1.9 of PCT/EP2010/064766,) than 238D2 and 238D4 (see Table C-2), and which each also belong to different families and/or bind to different epitopes from each other (except for 4CXCR283B06 and 4CXCR014A02, which belong to the same family).

As will become clear from the further description herein, the amino acid sequences provided by the invention are immunoglobulin single variable domains (or ISV's" for short) or amino acid sequences that, under suitable conditions (such as physiological conditions), are capable of forming an immunoglobulin single variable domain, or are suitable antigen- binding fragments of the same (as further described herein).

In particular, the amino acid sequences of the invention comprise or essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which the framework sequences and CDR's are as defined herein. Each amino acid sequence/ISV of the invention is such that it comprises an immunoglobulin fold or may be an amino acid sequence that, under suitable conditions (such as physiological conditions) is capable of forming an immunoglobulin fold {i.e., by folding), and in particular such that it forms (or under such suitable conditions is capable of fonning), an immunoglobulin single variable domain (i.e., an immunoglobulin variable domain that comprises a functional antigen binding site and does not require an interaction with another immunoglobulin variable domain (such as a VH-VL interaction) to form a functional antigen binding site).

For example, any ISV as described herein may be a light chain variable domain sequence (e.g.,, a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence) or a suitable fragment thereof. When the amino acid sequence of the invention is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a VH sequence that is derived from a human antibody) or it may be a so- called VHH-sequence (as defined herein) that is derived from a so-called "heavy chain antibody" (as defined herein).

In particular, any ISV described herein may be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" (or an amino acid sequence that is suitable for use as a d Ab) or a Nanobody® (as defined herein, and including but not limited to a VFIH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term "dAb's", reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544- 6), to Holt et al, Trends Biotechnol., 2003, 21(1 1):484-490; as well as to for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629).

As further described herein, the ISV's of the invention can either be used per se -i.e., in the form of a (monovalent) protein or polypeptide that comprises or essentially consists of such a preferred immunoglobulin single variable domains)- and/or that can be used as building blocks for making compounds or constructs that comprise one or more of ISV's of the invention and one or more further binding domains, binding units and/or other functional groups or functionalities. For example, in a preferred aspect, the ISV's of the invention may be used as building blocks for providing a range of different multivalent (such as bi- or trivalent), multispecific (such as such as bi- or trispecific) or multiparatopic (such as biparatopic) constructs as further described herein, which may also have a tailored or increased half-life or other desirable properties and/or functionalities. For this reason, the amino acid sequences/ISV's of the invention are also generically referred to herein as "building blocks" and specifically referred to herein as "building blocks of the invention".

For the purposes of the present description and claims (with each of the following ISV's/sequences being a specific aspect of the invention):

10E9-tvpe sequences: a "10E9-type sequence", "10E9-type ISV or "10E9- type building block" is defined as an ISV (as described herein) that has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0 10^"9M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM), and that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR010E09 (SEQ ID NO: 189); and/or (ii) competes with 4CXCR010E09 (SEQ ID NO: 189) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR010E09

(SEQ ID NO: 189) to human CXCR-4. In one particular but non-limiting aspect, such a 10E9-type sequence belongs to the same family as 4CXCR010E09 (SEQ ID NO: 189). Preferably, a 10E9-type sequence also has an IC₅o in cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than lOOOnM, such as better than 750nM, such as better than 500nM. Some preferred, but non-limiting examples of 10E9-type sequences are 4CXCR010E09 (SEQ ID NO: 189) and the "10E9-like sequences", ^uI0E9-like ISV or ⁱⁱ10E9-like building blocks^'" described herein.

281E10-type sequences: a "281EW-type sequence''. "28IE10-type ISV or "281E10-type building block" is defined as an ISV (as described herein) that has an IC₅0 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM), and that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR281E10 (SEQ ID NO: 190); and/or (ii) competes with 4CXCR281E10 (SEQ ID NO: 190) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in

Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR281E10 (SEQ ID NO: 190) to human CXCR-4. In one particular but non-limiting aspect, such a 281E10-type sequence belongs to the same family as 4CXC 281E10 (SEQ ID NO: 190). Preferably, a 281E10-type sequence also has an IC₅o in cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than lOOOnM, such as better than 750nM, such as better than 500nM. Some preferred, but non-limiting examples of 281E10-type sequences are 4CXCR281E10 (SEQ ID NO: 190) and the "281E10-iike sequences", "281E10-Iike ISV" of^u281E10-Hke building blocks described herein.

10E12-tvne sequences: a ⁱ~10E12-type sequence", 10E12-type IS I or "10E12-type building block" is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as

4CXCR010E12 (SEQ ID NO: 191); and/or (ii) competes with 4CXCR010E12 (SEQ ID NO : 191) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR010E12 (SEQ ID NO: 191) to human CXCR-4. In one particular but non- limiting aspect, such a 10E12-type sequence belongs to the same family as

4CXCR010E12 (SEQ ID NO: 191). Most preferably, 10E12-type sequence has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xlO^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM). Preferably, a 10E12-type sequence further has an IC₅₀ in cAMP assay of Example 1 , C-3; of that is either (i) better than the IC₅0 value of 238D2 in the same assay and/or (ii) better than lOOOnM, such as better than 750nM, such as better than 500nM. Some preferred, but non-limiting examples of 10E12- type sequences are 4CXCR010E12 (SEQ ID NO: 191) and the ⁱⁱ10E12-like sequences", "I0E12-like ISV" or ^{^}WEH-like building blocks" described herein.

lOAlO-type sequences: a "JOAIO-npe sequence", "1 OA 10 -type ISV or "lOAlO-type building block" is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as

4CXCR010A10 (SEQ ID NO: 192); and/or (ii) competes with 4CXCR010A10 (SEQ ID NO: 192) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR010A10 (SEQ ID NO: 192) to human CXCR-4. In one particular but non- limiting aspect, such a 10A10-type sequence belongs to the same family as

4CXCR010A10 (SEQ ID NO: 192). Most preferably, 10A10-type sequence has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xl.0^"9 M (7.0E-9M or 7.0 nM). in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM). Preferably, a 10A10-type sequence further has an IC50 in cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than !OOOnM, such as better than 750nM, such as better than 500nM. Some preferred, but non-limiting examples of 10A10-type sequences are 4CXCR010A10 (SEQ ID NO: 192) and the "WAlO-like sequences", "lOAJO-like ISV" or "JOAW-like building blocks" described herein.

lOGlO-type sequences: a "lOGlO-rype sequence", ''lOGlO-type ISV or "WGJO-type building block" is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as

4CXCR010G10 (SEQ ID NO: 193); and/or (ii) competes with 4CXCR010G10 (SEQ ID NO: 193) for binding to huma CXCR-4 (in particular-, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR010G10 (SEQ ID NO: 193) to human CXCR-4. In one particular but non- limiting aspect, such a lOGlO-type sequence belongs to the same family as

4CXCR010G10 (SEQ ID NO: 193). Most preferably, lOGlO-type sequence has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^' M (5.0E-9M or 5.0 nM). Preferably, a lOGlO-type sequence further has an IC50 in cAMP assay of Example 1, C-3; of that is either (i) better

herein.

14A2-type sequences: a "l4A2-t)pe sequence", J4A2-type ISV" or "14A2-type building block'^'' is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR014A02 (SEQ ID NO: 194); and/or (ii) competes with 4CXCR014A02 (SEQ ID NO: 194) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR014A02 (SEQ ID NO: 194) to human CXCR-4. In one particular but non-limiting aspect, such a 14A2-type sequence belongs to the same family as 4CXCR014A02 (SEQ ID NO: 194). Most preferably, 14A2-type sequence has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xlG^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM). Preferably, a 14A2-type sequence further has an IC₅₀ in cAMP assay of Example 1, C-3; of that is either (i) better than the IC₅₀ value of 238D2 in the same assay and/or (ii) better than ΙΟΟΟηΜ, such as better than 750nM, such as better than 500nM. Some preferred, but non-limiting examples of 14A2-type sequences, are 4CXCR014A02 (SEQ ID NO: 194) and the "14A2-like sequences", ^uI4A2-like ISV or "14A2-like building blocks" described herein. [Note: because 14A2 and 283B6 belong to the same family and have an essentially similar footprint, it is expected that a substantial part of the 283B6-type sequences will also be 14A2-type sequences and visa-versa].

15Al-type sequences: a "ISA l-type sequence", 'J5A J-type ISV or ISAl-iype building block" is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR015A01 (SEQ ID NO: 195); and/or (ii) competes with 4CXCR015A01 (SEQ ID NO: 195) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3). and/or cross-blocks (as defined herein) the binding of 4CXCR015A01 (SEQ ID NO: 195) to human CXCR-4. In one particular but non-limiting aspect, such a 15Al-type sequence belongs to the same family as 4CXCR015A01 (SEQ ID NO: 195). Most preferably, 15Al-type sequence has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM). Preferably,_a 15Al-type sequence further has an IC₅₀ in cAMP assay of.Example 1, C-3; of that is either (i) better than the IC₅o value of 238D2 in the same assay and/or (ii) better than 1 OOOnM, such as better than 750nM, such as better than 500nM. Some preferred, but non-limiting examples of 15Al-type sequences are 4CXCR015A01 (SEQ ID NO: 195) and the "15A1-Uke sequences", "lSAl-lihe ISV or "15AI~like building blocks" described herein.

15H3-type sequences: a "15H3-type sequence", "15H3-type ISV or "15H3-lype building block" is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as 4CXCR015H03 (SEQ ID NO: 196); and/or (ii) competes with 4CXCR015H03 (SEQ ID NO: 196) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross -blocks (as defined herein) the binding of 4CXCR015H03 (SEQ ID NO: 196) to human. CXCR-4. In one particular but non-limiting aspect, such a 15H3-type sequence belongs to the same family as 4CXCR0151103 (SEQ ID NO: 196). Most preferably, 15H3-type sequence has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM). Preferably, a 15H3-type sequence further has an IC₅₀in . cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than !OOOnM, such as better than 750nM, such as better than 500nM. Some preferred, but non-limiting examples of 15H3-type sequences are 4CXCR015H03 (SEQ ID NO: 196) and the "15H3-Iite sequences", "ISHJ-like ISV or i^l15H3-like building blocks" described herein.

283B6-type sequences: a "283B6-iype sequence", ^Li283B6~type ISV or "283B6-type building block^"1 is defined as an ISV (as described herein) that either (i) binds to the same epitope, domain or extracellular loop on human CXCR-4 as

4CXCR283B06 (SEQ ID NO: 197); and/or (ii) competes with 4CXCR283B06 (SEQ ID NO: 197) for binding to human CXCR-4 (in particular, in one or both of the competition assays described in Example 1, D-3), and/or cross-blocks (as defined herein) the binding of 4CXCR283B06 (SEQ ID NO: 197) to human CXCR-4. In one particular but non- limiting aspect, such a 283B6-type sequence belongs to the same family as

4CXCR283B06 (SEQ ID NO: 197). Most preferably, 283B6-type sequence has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM). Preferably, a 283B6-type sequence further has an IC50 in cAMP assay of Example 1, C-3; of that is either (i) better than the IC50 value of 238D2 in the same assay and/or (ii) better than ΙΟΟΟηΜ, such as better than 750nM, such as better than 500nM. Some preferred, but non-limiting examples of 283B6-type sequences are 4CXCR283B06 (SEQ ID NO: 197) and the "283B6-like sequences^'', ^li283B6-like ISV or ⁱⁱ283B6-like building blocks" described herein. [Note: because 14A2 and 283B6 belong to the same family and have an essentially similar footprint, it is expected that a substantial part of the 283B6-type sequences will also be 14A2-type sequences and visa- ersa].

The framework sequences and CDR's of the sequences 10E9, 281E10, 10E12, 10A10,G10, 14A2, 15A1, 15H3 and 283B6 are given in Table B-l below. Tab!e B-1: frameworks and CDR's of 10E9, 281ΕΪΘ, 10E12, 10A10, lOGlO, 14A2, 15A1, 15H3 and 283B6

Also, for the purposes of the present description and claims (with each of the following s/sequences being a specific aspect of the invention): 10E9-like sequences: a "10E9-like sequence", "J 0E9-like LSI" or J 0E9- like building block" is defined as an ISV (as described herein) that comprises:

a) a CDR1 which comprises or essentially consists of either (i) the amino acid sequence SYAMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SYAMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence TISWRGDRKYYSESVKD or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence TISWRGDRKYYSESVKD; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid, difference(s) (as defined herein) with the amino acid sequence TISWRGDRKYYSESVKD; and/or

c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence DRAPYGSGSPDVKQYEH or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence DRAPYGSGSPDVKQYEH; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence DRAPYGSGSPDVKQYEH; in which the framework sequences present in such an ISV are as further described herein, and in which. CDRI, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0 10^~9 M (5.0E-9M or 5.0 nM).

Preferably, in such a 10E9-like sequence, CDRi and CDR2 are as defined under a) and b), respectively; or CDRI and CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 10E9-like sequence, CDRI, CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such an 10E9-like sequence, CDRI, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC₅o in the Jurkat assay of Example I, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E- 9M or 5.0 nM). For example, in such an 10E9-like sequence: CDR1 may comprise or essentially consist of the amino acid sequence SYAMG (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence TISWRGDRKYYSESVKD (with CDRl and CDR3 being as defined under a) and c)_; respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence DRAPYGSGSPDVKQYEH (with CDRl and CDR2 being as defined under a) and b), respectively). Particularly, when an 10E9-Iike sequence is according to this aspect: CDRl may comprise or essentially consist of the amino acid sequence SYAMG and CDR2 may comprise or essentially consist of the amino acid sequence

TISWRGDRKYYSESVKD (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence SYAMG and CDR3 may comprise or essentially consist of the amino acid sequence DRAPYGSGSPDVKQYEH (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence TISWRGDRKYYSESVKD and CDR3 may comprise or essentially consist of the amino acid sequence DRAPYGSGSPDVKQYEH (with CDRl being as defined under a) above). Again, in such 10E9-like sequences, CDRl, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect, a ⁱⁱJ0E9-Iike sequence", ^iL10E9-like ISV" or "10E9-like building block"' is an ISV that comprises:

d) a CDRl which is either (i) the amino acid sequence SYAMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SYAMG; and/or

e) a CDR2 which is either (i) the amino acid sequence

TISWRGDRKYYSESVKD or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence TISWRGDRKYYSESVKD; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence TISWRGDRKYYSESVKD; and/or

f) a CDR3 which is either (i) the amino acid sequence

DRAPYGSGSPDVKQYEH or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence DRAPYGSGSPDVKQYEH; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence DRAPYGSGSPDVKQYEH; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the 10E9-Hke ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

Preferably, in a 10E9~like sequence according to this specifically preferred aspect, CDRl and CDR2 are as defined under d) and e), respectively; or CDRl and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 10E9-like sequence, CDRl, CDR2 and CDR3 are all as defined under d). e) and f), respectively. Again, in such an 10E9-like sequence. CDRl, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xlO^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

■ For example, in a 10E9-like sequence according to this specifically preferred aspect: CDRl is the amino acid sequence SYAMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

TISWRGDRKYYSESVKD (with CDRl and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence DRAPYGSGSPDVKQYEH (with CDRl and CDR2 being as defined under d) and e), respectively). Particularly, when an 10E9-like sequence is according to this aspect: CDRl is the amino acid sequence SYAMG and CDR2 is the amino acid sequence TISWRGDRKYYSESVKD (with CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence SYAMG and

CDR3 is the amino acid sequence DRAPYGSGSPDVKQYEH (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence

TISWRGDRKYYSESVKD and CDR3 is DRAPYGSGSPDVKQYEH (with CDRl being as defined under d) above). Again, in such 10E9-like sequences, CDRl, CDR2 and CDR3 are preferably such that the 10E9-like ISV has an IC₅₀ in the Jurkat assay of Example 1,

C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM). In a particularly preferred 10E9-like sequence: CDR1 is the amino acid sequence SYAMG, CDR2 is the amino acid sequence TI S WRGDRK Y Y S ES V KD ; and CDR3 is the amino acid sequence DRAPYGSGSPDV QYEH.

In all the 10E9-like sequence described in this paragraph A), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%, such, as at least 95%o sequence identity with the framework sequences of 10E9 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 10E9 while disregarding the CDR^'s in the calculation). Again, the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 10E9-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 10E9-like sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR010E09 (SEQ ID NO: 189). For example, in an I0E9-Iike sequence according to this aspect, the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be SYAMG (CDR1); TISWRGDRKYYSESVKD (CDR2); and

DRAPYGSGSPDVKQYEH (CDR3). Again, preferably, the combination of CDR's and frameworks present in such a 10E9-like ISV are preferably such that the resulting 10E9- like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xI0^"9M (5.0E-9M or 5.0 nM).

In one particular aspect, any 10E9~like sequence may be a humanized sequence, as further described herein. 281 El (Mike sequences: a "281 El 0-like sequence", "281 El 0-like ISV or

"281 El 0-like building block" is defined as an ISV (as described herein) that comprises: a) a CDR1 which comprises or essentially consists of either (i) the amino acid sequence NYAMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence NYAMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence AITRSGVRSGVSAIYGDSVKD or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AITRS G VRS G VS AI YGDS V D ; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence AITRSGVRSGVSAIYGDSVKD; and/or

c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence SAIGSGALRRFEYDY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence SAIGSGALRRFEYDY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SAIGSGALRRFEYDY; in which the framework sequences present in such an IS V are as further described herein, and in which CDRl , CDR2 and CDR3 are preferably such that the 281E10-like ISV has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^" M (5.0E-9M or 5.0 nM).

Preferably, in such a 281 El 0-like sequence, CDRl and CDR2 are as defined under a) and b), respectively; or CDRl and CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 28 IE 3.0-like sequence, CDRl, CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such, an 281 El 0-like sequence, CDRl, CDR2 and CDR3 are preferably such that the 281 El 0-like ISV has an IC50 in the Jurkat assay of Example 1, C- 4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9M (5.0E-9M or 5.0 n ).

For example, in such an 281E10-like sequence: CDRl may comprise or essentially consist of the amino acid sequence NYAMG (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence AITRSGVRSGVSAIYGDSVKD (with CDRl and CDR3 being as defined under a) and c), respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence SAIGSGALRRFEYDY (with CDRl and CDR2 being as defined under a) and b), respectively). Particularly, when an 281E10-like sequence is according to this aspect: CDRl may comprise or essentially consist of the amino acid sequence NYAMG and CDR2 may comprise or essentially consist of the amino acid sequence AITRSG VRS G VS AI YGD S VKD (with CDR3 being as defined under c) above); and/or CDR1 may comprise or essentially consist of the amino acid sequence NYAMG and CDR3 may comprise or essentially consist of the amino acid sequence SAIGSGALRRFEYDY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence

AITRSGVRSGVSAIYGDSVKD and CDR3 may comprise or essentially consist of the amino acid sequence SAIGSGALRRFEYDY (with CDR1 being as defined under a) above). Again, in such 281E10-like sequences, CDRL CDR2 and CDR3 are preferably such that the 281E10-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect, a "281E10-like sequence", "I&IEIQ-Vike ISV or ^u281E10-like building block" is an ISV that comprises:

d) a CDR1 which is either (i) the amino acid sequence NYAMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence NYAMG; and/or

e) a CDR2 which is either (i) the amino acid sequence

AITRSGVRSGVSAIYGDSVKD or (ii) an amino acid sequence that has at least 80%, suc as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AITRSGVRSGVSAIYGDSVKD; or (iii) ell! 3. ΠΙΠΟ acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence AITRSGVRSGVSAIYGDSVKD; and/or

f) a CDR3 which is either (i) the amino acid sequence

SAIGSGALRRFEYDY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence SAIGSGALRRFEYDY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SAIGSGALRRFEYDY; in which the framework sequences present in such an ISV are as further described herein, and in which CDR1, CDR2 and CDR3 are preferably such that the 281E10-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM). Preferably, in a 28 lElO-like sequence according to this specifically preferred aspect, CDRl and CDR2 are as defined under d) and e), respectively; or CDRl and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 281 El 0-like sequence, CDRl, CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an 281E10-like sequence, CDRl, CDR2 and CDR3 are preferably such that the 281E10-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM).

For example, in a 281 El 0-like sequence according to this specifically preferred aspect: CDRl is the amino acid sequence NYAMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

AITRSGVRSGVSAIYGDSVKD (with CDRl and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence SA1GSGALRRFEYDY (with CDRl and CDR2 being as defined under d) and e), respectively). Particularly, when an 281E10-like sequence is according to this aspect: CDRl is the amino acid sequence

NYAMG and CDR2 is the amino acid sequence AITRSGVRSGVSAIYGDSVKD (with CDR3 being as defined under f) above): and/or CDRl is the amino acid sequence NYAMG and CDR3 is the amino acid sequence SAIGSGALRRFEYDY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence

AITRSGVRSGVSAIYGDSVKD and CDR3 is SAIGSGALRRFEYDY (with CDRl being as defined under d) above). Again, in such 281E10-like sequences, CDRl, CDR2 and CDR3 are preferably such that the 281 El 0-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

In a particularly preferred 281E10-like sequence: CDRl is the amino acid sequence NYAMG, CDR2 is the amino acid sequence AITRSGVRSGVSAIYGDSVKD; and CDR3 is the amino acid sequence SAIGSGALRRFEYDY.

In all the 281 ElO-I ke sequence described in this paragraph B), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%o, such as at least 95% sequence identity with the framework sequences of 281 El 0 (which, for example, can be determined by detennining the overall degree of sequence identity of a given sequence with the sequence of 28 IE 10 while disregarding the CDR's in the calculation). Again, the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 281E10-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^'9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 281E10-like sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR281 E10 (SEQ ID NO: 190). For example, in an 28 lE10~like sequence according to this aspect, the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be NYAMG (CDR1); AITRS GVRSGVS AI YGD S VKD (CDR2); and

SAIGSGALRRFEYDY (CDR3). Again, preferably, the combination of CDR's and frameworks present in such a 28 IE] 0-like ISV are preferably such that the resulting 281E10-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^" ⁹ M (7.0E-9M or 7.0 nM), in particular better than S.OxlO^"9 M (5.0E-9M or 5.0 nM). In one particular aspect, any 281 El 0-like sequence may be a humanized sequence, as further described herein.

10E12-Iike sequences; a ^ JOE J 2 -like sequence", 10E12-like ISV" or "lOEll-Uke building block" is defined as an ISV (as described herein) that comprises: a) a CDR1 which comprises or essentially consists of either (i) the amino acid sequence SYDMA or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SYDMA; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence AIRWTGSSTYYADSVKG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AIRWTGSSTYYADSVKG; or (iii) an amino acid sequence that has only 7, 6, 5. 4. 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence AIRWTGSSTYYADSVKG; and/or

c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence RDTGRYRSRTYDY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence RDTGRYRSRTYDY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence RDTGRYRSRTYDY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the 10E12-like ISV has an IC₅₀ in the Jurkat assay of Example 1 , C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 (5.0E-9M or 5.0 nM).

Preferably, in such a 10E12-Ii.ke sequence, CDRl and CDR2 are as defined under a) and b), respectively; or CDRl and CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 10E12-like sequence, CDRl, CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such an 10E12-like sequence, CDRl, CDR2 and CDR3 are preferably such that the 10E12-like ISV has an IC₅o in the Jurkat assay of Example 1, C- 4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or S.O nM).

For example, in such an 10E12-like sequence: CDRl. may comprise or essentially consist of the amino acid sequence S YDMA (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence AIRWTGSSTYYADSVKG (with CDRl and CDR3 being as defined under a) and c)_; respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence RDTGRYRSRTYDY (with CDRl and CDR2 being as defined under a) and b), respectively). Particularly, when an 10E12-like sequence is according to this aspect: CDRl may comprise or essentially consist of the amino acid sequence SYDMA and CDR2 may comprise or essentially consist of the amino acid sequence

AIRWTGSSTYYADSVKG (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence SYDMA and CDR3 may comprise or essentially consist of the amino acid sequence RDTGRYRSRTYDY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence AIRWTGSSTYYADSVKG and CDR3 may comprise or essentially consist of the amino acid sequence RDTGRYRSRTYDY (with CDRl being as defined under a) above). Again, in such 10E12-like sequences, CDRl, CDR2 and CDR3 are preferably such that the 10E12-like ISV has an IC₅o in the Jurkat assay of Example 1 , C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^'9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect, a "WEH-Iike sequence", ⁱ10El2-like ISV or "10E12- like building block" IS Xl ISV that comprises:

d) a CDRl which is either (i) the amino acid sequence SYDMA or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SYDMA; and/or

e) a CDR2 which is either (i) the amino acid sequence

AIRWTGS STY Y AD S V G or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AIRWTGSSTYYADSVKG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence AIRWTGSSTYYADSVKG; and/or

f) a CDR3 which is either (i) the amino acid sequence RDTGRYRSRTYDY or (ii) an amino acid sequence that has at least 80%, such as at least 85%>, for example at least 90% or more than 95% sequence identity with the amino acid sequence RDTGRYRSRTYDY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence

RDTGRYRSRTYDY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the 10E12-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

Preferably, in a 10E12-like sequence according to this specifically preferred aspect CDRl and CDR2 are as defined under d) and e), respectively; or CDRl and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 10E12-like sequence, CDRl, CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an 10E12-like sequence, CDRl, CDR2 and CDR3 are preferably such that the 10E12-like ISV has an iC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xlO^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM). For example, in a 10E12-like sequence according to this specifically preferred aspect: CDR1 is the amino acid sequence SYDMA (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

AIRWTGSSTYYADSVKG (with CDR1 and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence RDTGRYRSRTYDY (with

CDR1 and CDR2 being as defined under d) and e), respectively). Particularly, when an 10E12-like sequence is according to this aspect: CDR1 is the amino acid sequence SYDMA and CDR2 is the amino acid sequence AIRWTGSSTYYADSVKG (with CDR3 being as defined under f) above); and/or CDR1 is the amino acid sequence SYDMA and CDR3 is the amino acid sequence RDTGRYRSRTYDY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence AIRWTGSSTYYADSVKG and CDR3 is RDTGRYRSRTYDY (with CDR1 being as defined under d) above). Again, in such 10E12-like sequences, CDR1, CDR2 and CDR3 are preferably such that the 10E12-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^" M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^~9M (5.0E-9M or 5.0 nM).

In a particularly preferred 10E12-like sequence: CDR1 is the amino acid sequence SYDMA, CDR2 is the amino acid sequence AIRWTGSSTYYADSVKG; and CDR3 is the amino acid sequence

sequence described in this paragraph C), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity with the framework sequences of 10E12 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 10E12 while disregarding the CDR's in the calculation). Again, the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 10E12-like ISV has an IC₅0 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO^"9 M (7.0E-9M or 7.0 nM). in particular better than 5.0xl0^'9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 10E12-Iike sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR010E12 (SEQ ID NO: 191). For example, in an 10E12-like sequence according to this aspect, the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be SYDMA (CDRl); AIRWTGSSTYYADSV G (CDR2); and

RDTG YRS RT YD Y (CDR3). Again, preferably, the combination of CDR's and frameworks present in such a 10E12-like ISV are preferably such that the resulting 10E12-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0 10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In one particular aspect, any I0E12-like sequence may be a humanized sequence, as further described herein. lOAI O-Hke sequences: a '"lOAlO-like sequence", 'ΊΟΑ ίΟ-lik ISV" or "WAlO- ike building block" is defined as an ISV (as described herein) that comprises: a) a CDRl which comprises or essentially consists of either (i) the amino acid sequence DTGTMA or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence DTGTMA; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence AINSGTTNYADSVKG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AINSGTTNYADSVKG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence AINSGTTNYADSVKG; and/or

c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence RVSGWRTRYDY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence RVSGWRTRYDY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence RVSGWRTRYDY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the lOAlO-like ISV has an IC50 in the Jurkat assay of Example I , C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM).

Preferably, in such a lOAIO-Hke sequence, CDRl and CDR2 are as defined under a) and b), respectively; or CDRl and CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 10A10-like sequence, CDRl , CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such an lOAlO-like sequence, CDRl, CDR2 and CDR3 are preferably such that the l OAlO-like ISV has an IC50 in the Jurkat assay of Example 1 , C- 4; of better than 7.0x10^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or S.O nM).

For example, in such an 10A10-like sequence: CDRl may comprise or essentially consist of the amino acid sequence DTGTMA (with CDR2 and CDR3 being as defined under b) and c). respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence AINSGTTN Y ADS V G (with CDRl and CDR3 being as defined under a) and c) , respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence RVSGWRTRYDY (with CDRl and CDR2 being as defined under a) and b), respectively). Particularly, when an 10A10-like sequence is according to this aspect: CDRl may comprise or essentially consist of the amino acid sequence DTGTMA and CDR2 may comprise or essentially consist of the amino acid sequence

AINSGTTNYADSVKG (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence DTGTMA and CDR3 may comprise or essentially consist of the amino acid sequence RVSGWRTRYDY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence AINSGTTNYADSVKG and CDR3 may comprise or essentially consist of the amino acid sequence RVSGWRTRYDY (with CDRl being as defined under a) above). Again, in such 10A10-like sequences, CDRl, CDR2 and CDR3 are preferably such that the 10A10-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect, a "lOAlO-like sequence", "lOAlO-like ISV^" or "10A10- like building block?^'' is an ISV that comprises:

d) a CDRl which is either (i) the amino acid sequence DTGTMA or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence DTGTMA; and/or

e) a CDR2 which is either (i) the amino acid sequence

AINSGTTNYADSVKG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AINSGTTNYADSVKG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence AIN S GTTN Y AD S VKG; and/or

f) a CDR3 which is either (i) the amino acid sequence RVSGWRTRYDY or

(ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence

RVSGWRTRYDY; or (iii) an amino acid sequence that has only 7, 6, 5, 4. 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence

RVSGWRTRYDY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl , CDR2 and CDR3 are preferably such that the 1 OA 10-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0χ10^"9 Μ (7.0E-9M or 7.0 nM), in particular better than 5.0 10^"9 M (5.0E-9M or 5.0 nM).

Preferably, in a 10A10-like sequence according to this specifically preferred aspect, CDRl and CDR2 are as defined under d) and e), respectively; or CDRl and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 1 OA 10-like sequence. CDRL CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an 10A10-like sequence, CDRl, CDR2 and CDR3 are preferably such that the 1 OA 1.0-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xlO^" M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

For example, in a 10A10-like sequence according to this specifically preferred aspect: CD l is the amino acid sequence DTGTMA (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

AINSGTTNYADSVKG (with CDRl and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence RVSGWRTRYDY (with CDRl and CDR2 being as defined under d) and e), respectively). Particularly, when an 10A10- like sequence is according to this aspect: CDRl is the amino acid sequence DTGTMA and CDR2 is the amino acid sequence AINSGTTNYADSVKG (with CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence DTGTMA and CDR3 is the amino acid sequence RVSGWRTRYDY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence AINSGTTNYADSVKG and CDR3 is RVSGWRTRYDY (with CDRl being as defined under d) above). Again, in such 10A10- like sequences, CDR1 , CDR2 and CDR3 are preferably such that the 10A10 ike ISV has an IC₅₀ in the Jurkat assay of Example 1 , C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl 0^"9 M (5.0E-9M or 5.0 nM).

In a particularly preferred l OAlO-like sequence: CDR1 is the amino acid sequence DTGTMA, CDR2 is the amino acid sequence AIN SGTTNY ADS V G; and CDR3 is the amino acid sequence RVSGWRTRYDY.

In all the 10A10-like sequence described in this paragraph D), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%. such as at least 95% sequence identity with the framework sequences of 10A10 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 1 OA 10 while disregarding the CDR's in the calculation). Again, the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 10A10-like ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 10A10-like sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR010A10 (SEQ ID NO: 192). For example, in an 10A10-like sequence according to this aspect, the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be DTGTMA (CDR1 ); AINSGTTN YAD S VKG (CDR2); and

RVSGWRTRYDY (CDR3). Again, preferably, the combination of CDR's and frameworks present in such a l OAl O-like ISV are preferably such that the resulting lOAlO-like ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xl0^"9

M (7.0E-9M or 7.0 nM), in particular better than 5.0 l 0^"9 M (5.0E-9M or 5.0 nM).

In one particular aspect, any 10A10-like sequence may be a humanized sequence, as further described herein. lOGl O-like sequences: a "JOGIO-Iike sequence", "lOGlO-lih ISV or l OGlO-like building block" is defined as an ISV (as described herein) that comprises: a) a CDRl which comprises or essentially consists of either (i) the amino acid sequence SYAMA or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SYAMA; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence AIRWSGGRATKYADSVRG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AIRWSGGRATKYADSVRG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence AIRWSGGRATKYADSVRG; and/or c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence QTYYRSGLASTRDFDS or (ii) an amino acid sequence that has at least 80%), such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence QTYYRSGLASTRDFDS; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence QTYYRSGLASTRDFDS; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the !OGlO-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

Preferably, in such a lOGlO-like sequence, CDRl and CDR2 are as defined under a) and b), respectively; or CDRl and CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a lOGlO-like sequence, CDRl, CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such an l OGlO-like sequence, CDRl, CDR2 and CDR3 are preferably such that the lOGlO-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C- 4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

For example, in such an lOGlO-like sequence: CDRl may comprise or essentially consist of the amino acid sequence SYAMA (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence AIRWSGGRATKYADSVRG (with CDRl and CDR3 being as defined under a) and c), respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence QTYYRSGLASTRDFDS (with CDRl and CDR2 being as defined under a) and b), respectively). Particularly, when an l OGlO-like sequence is according to this aspect: CDRl may comprise or essentially consist of the amino acid sequence SYAMA and CDR2 may comprise or essentially consist of the amino acid sequence

AIRWS GGRATKY AD S VRG (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence SYAMA and CDR3 may comprise or essentially consist of the amino acid sequence

QTYYRSGLASTRDFDS (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence

AIRWSGGRATKYADSVRG and CDR3 may comprise or essentially consist of the amino acid, sequence QTYYRSGLASTRDFDS (with CDRl being as defined under a) above). Again, in such l OGlO-like sequences, CDRl , CDR2 and CDR3 are preferably such that the lOGlO-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect a "lOGlO-like sequence", "lOGlO-like ISV or "lOGJO- like building block" is an ISV that comprises:

d) a CDRl which is either (i) the amino acid sequence SYAMA or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence SYAMA; and/or

e) a CDR2 which is either (i) the amino acid sequence

AIRWSGGRATKYADSVRG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence AIRWSGGRATKYADSVRG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence AIRWSGGRATKYADSVRG; and/or

f) a CDR3 which is either (i) the amino acid sequence

QTYYRSGLASTRDFDS or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence QTYYRSGLASTRDFDS; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3. 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence QTYYRSGLASTRDFDS; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the !OGlO-like ISV has an IC₅0 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than S.OxlO^"9 M (5.0E-9M or 5.0 nM).

Preferably, in a lOGlO-like sequence according to this specifically preferred aspect,

CDRl and CDR2 are as defined under d) and e), respectively; or CD l and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a lOGlO-like sequence, CDRl , CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an lOGlO-like sequence, CDRl, CDR2 and CDR3 are preferably such that the lOGlO-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^~9 M (5.0E-9M or 5.0 nM).

For example, in a lOGlO-like sequence according to this specifically preferred aspect: CDRl is the amino acid sequence SYAMA (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

AIRWSGGRATKYAD S VRG (with CDRl and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence QTYYRSGLASTRDFDS (with CDRl and CDR2 being as defined under d) and e), respectively). Particularly, when an lOGlO-like sequence is according to this aspect: CDRl is the amino acid sequence SYAMA and CDR2 is the amino acid sequence AIRWSGGRATKYADSVRG (with

CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence

SYAMA and CDR3 is the amino acid sequence QTYYRSGLASTRDFDS (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence

AIRWSGGRATKYADSVRG and CDR3 is QTYYRSGLASTRDFDS (with CDRl being as defined under d) above). Again, in such lOGlO-like sequences, CDRl, CDR2 and

CDR3 are preferably such that the lOGlO-like ISV has an IC₅o in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^~9 M (5.0E-9M or 5.0 nM).

In a particularly preferred 10G10-li.ke sequence: CDRl is the amino acid sequence SYAMA, CDR2 is the amino acid sequence AIRWSGGRATKYADSVRG; and CDR3 is the amino acid sequence QTYYRSGLASTRDFDS.

In all the lOGlO-like sequence described in this paragraph E), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity with the framework sequences of 10G10 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 10G10 while disregarding the CDR' s in the calculation). Again, the combination of CDR's and. frameworks present in a given sequence are preferably such that the resulting 10G10-like ISV has an IC50 in the Jurkat assay of Example I , C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl 0^"9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 10G10-like sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR010G10 (SEQ ID NO: 193). For example, in an 10G10-like sequence according to this aspect, the CDR' s may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be SYAMA (CDR1); AIRWSGGRATKYADSVRG (CDR2); and

QTYYRSGLASTRDFDS (CDR3). Again, preferably, the combination of CDR's and frameworks present in such a 10G10-like ISV are preferably such that the resulting l OGJ O-like ISV has an IC₅0 in the Jurkat assay of Example 1 , C~4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM).

In one particular aspect, any lOGlO-Iike sequence may be a humanized sequence, as further described herein.

14A2-Iike sequences: a ⁱⁱ14A2-like sequence", HA 2-1 ike ISV or "14A2- like building block" is defined as an ISV (as described herein) that comprises:

a) a CDR1 which comprises or essentially consists of either (i) the amino acid sequence IRNMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence IRNMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence TISSGGNKDYTDAVKD or (ii) an amino acid sequence that has at least 80%), such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence TISSGGNKDYTDAVKD; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence TISSGGNKDYTDAVKD; and/or c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence EAGTGWATRRGYTY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence EAGTGWATRRGYTY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence EAGTGWATRRGYTY; in which the framework sequences present in such an ISV are as further described herein, and in which CDR1. CDR2 and CDR3 are preferably such that the 14A2~like ISV has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^~9 M (5.0E-9M or 5.0 nM).

Preferably, in such a 14A2-like sequence, CDR1 and CDR2 are as defined under a) and b), respectively; or CDR1 and CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 14A2-like sequence, CDR1, CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such an 14A2-like sequence, CDR1, CDR2 and CDR3 are preferably such that the 14A2-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0 l0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E- 9M or 5.0 nM).

For example, in such an 14A2-like sequence: CDR1 may comprise or essentially consist of the amino acid sequence IRNMG (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence T1S S GGN D YTD A VKD (with CDR1 and CDR3 being as defined under a) and c), respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence EAGTGWATRRGYTY (with CDR1 and CDR2 being as defined under a) and b), respectively). Particularly, when an 14A2-like sequence is according to this aspect: CDR1 may comprise or essentially consist of the amino acid sequence IRNMG and CDR2 may comprise or essentially consist of the amino acid sequence

TI S S GGNKD YTD A VKD (with CDR3 being as defined under c) above); and/or CDR1 may comprise or essentially consist of the amino acid sequence IRNMG and CDR3 may comprise or essentially consist of the amino acid sequence EAGTGWATRRGYTY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence TISSGGNKDYTDAVKD and CDR3 may comprise or essentially consist of the amino acid sequence EAGTGWATRRGYTY (with CDRl being as defined under a) above). Again, in such 14A2-like sequences, CDRl, CDR2 and CDR3 are preferably such that the 14A2-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect, a "14A2-Hke sequence", "14A2-like ISV or ⁱⁱ14A2-like building block" is an ISV that comprises:

d) a CDRl which is either (i) the amino acid sequence IRNMG or (ii) an

amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence IRNMG; and/or

e) a CDR2 which is either (i) the amino acid sequence

TISSGGNKDYTDAV D or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence TISSGGNKDYTDAVKD; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence TISSGGNKDYTDAVKD; and/or

f) a CDR3 which is either (i) the amino acid sequence

EAGTGWATRRGYTY or (ii) an amino acid sequence that has at least 80%. such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence EAGTGWATRRGYTY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence EAGTGWATRRGYTY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the 14A2-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x1.0^'9 M (5.0E-9M or 5.0 nM).

Preferably, in a 14A2-like sequence according to this specifically preferred aspect, CDRl and CDR2 are as defined under d) and e), respectively; or CDRl and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 14A2-like sequence, CDRl, CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an 14A2-like sequence, CDRl, CDR2 and CDR3 are preferably such that the 14A2-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0x10^'9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

For example, in a 14A2-like sequence according to this specifically preferred aspect: CDRl is the amino acid sequence IRNMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

TISSGGNKDYTDAVKD (with CDRl and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence EAGTGWATRRGYTY (with CDRl and CDR2 being as defined under d) and e), respectively). Particularly, when an 14A2-like sequence is according to this aspect: CDRl is the amino acid sequence IRNMG and CDR2 is the amino acid sequence TISSGGNKDYTDAVKD (with CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence IRNMG and CDR3 is the amino acid sequence EAGTGWATRRGYTY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence TISSGGNKDYTDAVKD and CDR3 is EAGTGWATRRGYTY (with CDRl being as defined under d) above). Again, in such 14A2-like sequences, CDRl, CDR2 and CDR3 are preferably such that the 14A2- like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

In a particularly preferred 14A2-like sequence: CDRl is the amino acid sequence IRNMG, CDR2 is the amino acid sequence TISSGGNKDYTDAVKD; and CDR3 is the amino acid sequence EAGTGWATRRGYTY.

In all the 14A2-like sequence described in this paragraph F), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%, such as at least 95 > sequence identity with, the framework sequences of 14A2 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 14A2 while disregarding the CDR's in the calculation). Again, the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 14A2-like ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xlO^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 14A2-Iike sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR014A02 (SEQ ID NO: 194). For example, in an 14A2-like sequence according to this aspect, the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be IR MG (CDRl); TISSGGNKDYTDAVKD (CDR2); and

EAGTGWATRRGYTY (CDR3). Again, preferably, the combination of CDR's and frameworks present in such a 14A2-Iike ISV are preferably such that the resulting 14A2- like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^'9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

In one particular aspect, any 14A2-like sequence may be a humanized sequence, as further described herein. 15A1-Iike sequences: a "ISAl-lih sequence", "15Al-like ISV" or "15A1- like building block" is defined as an ISV (as described herein) that comprises:

a) a CDRl which comprises or essentially consists of either (i) the amino acid sequence RAAMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence RAAMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence CALSSAGSALTADSVKG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence CALSSAGSALTADSVKG; or (iii) an amino acid sequence that has only 7, 6, 5, 4. 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence CALSSAGSALTADSVKG; and/or

c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence GGYCTRAGVYPY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence GGYCTRAGVYPY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence GGYCTRAGVYPY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the 15Al-like ISV has an IC₅0 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0 lO^"9 M (5.0E-9M or 5.0 nM).

Preferably, in such a 15Al-like sequence, CDRl and CDR2 are as defined under a) and b) , respectively; or CDRl and. CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 15Al-like sequence, CDRl, CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such an 15Al -like sequence, CDRl, CDR2 and CDR3 are preferably such that the 15Al-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-

9M or 5.0 nM).

For example, in. such an 15Al-like sequence: CDRl may comprise or essentially consist of the amino acid sequence RAAMG (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence CALSSAGSALTADSVKG (with CDRl and CDR3 being as defined under a) and c), respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence GGYCTRAGVYPY (with CDRl and CDR2 being as defined under a) and b), respectively). Particularly, when an 15Al-like sequence is according to this aspect: CDRl may comprise or essentially consist of the amino acid sequence RAAMG and CDR2 may comprise or essentially consist of the amino acid sequence CALSSAGSALTADSVKG

(with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence RAAMG and CDR3 may comprise or essentially consist of the amino acid sequence GGYCTRAGVYPY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence CALSSAGSALTADSVKG and CDR3 may comprise or essentially consist of the amino acid sequence GGYCTRAGVYPY (with CDRl being as defined under a) above). Again, in such 15Al-like sequences, CDRl, CDR2 and CDR3 are preferably such that the 15Al-like ISV has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect, a ^JSAl-like sequence", "ISAl-like ISV" or "15AJ-!ike building blocW is an ISV that comprises:

d) a CDRl which is either (i) the amino acid sequence RAAMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence RAAMG; and/or

e) a CDR2 which is either (i) the amino acid sequence

CALSSAGSALTADSVKG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence CALSSAGSALTADSV G; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence CALSSAGSALTADSVKG; and/or

f) a CDR3 which is either (i) the amino acid sequence GGYCTRAGVYPY or

GGYCTRAGVYPY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence

GGYCTRAGVYPY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 are preferably such that the 15A1 -like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

Preferably, in a 15Al-like sequence according to this specifically preferred aspect, CDRl and CDR2 are as defined under d) and e), respectively; or CDRl and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 15Al-like sequence, CDRl, CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an 1 Al -like sequence, CDRl, CDR2 and CDR3 are preferably such that the 15A1-Iike ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^'9 M (7.0E-9M or 7.0 nM), in particular better than 5-OxlO^"9 M (5.0E-9M or 5.0 nM).

For example, in a 15Al-like sequence according to this specifically preferred aspect: CDRl is the amino acid sequence RAAMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

CALSSAGSALTADSVKG (with. CDRl and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence GGYCTRAGVYPY (with CDRl and CDR2 being as defined under d) and e), respectively). Particularly, when an 15A1- like sequence is according to this aspect: CDRl is the amino acid sequence RAAMG and CDR2 is the amino acid sequence CALSSAGSALTADSVKG (with CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence RAAMG and CDR3 is the amino acid sequence GGYCTRAGVYPY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence CALSSAGSALTADSVKG and CDR3 is GGYCTRAGVYPY (with CDR1 being as defined under d) above). Again, in such 15A1 -like sequences, CDR1, CDR2 and CDR3 are preferably such that the 15Al-like ISV has an IC₅₀ in the Jurkat assay of Example 1 , C-4; of better than 7.0 10^"9 M (7.0E- 9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In a particularly preferred 15A1 -like sequence: CDR1 is the amino acid sequence RAAMG, CDR2 is the amino acid sequence CALSSAGSALTADSVKG; and CDR3 is the amino acid sequence GGYCTRAGVYPY.

In all the 15Al-like sequence described in this paragraph G), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%. for example at least 90%, such as at least 95% sequence identity with the framework sequences of 15A1 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 15A1 while disregarding the CDR's in the calculation). Again, the combination of CDR^'s and frameworks present in a given sequence are preferably such that the resulting 1 Al-like ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xlO^"9 M (7.0E-9M. or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 15A1-Iike sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR015A01 (SEQ ID NO: 195). For example, in an 15A1 -like sequence according to this aspect, the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be RAAMG (CDR1); CALSSAGSALTADSVKG (CDR2); and

GGYCTRAGVYPY (CDR3). Again, preferably, the combination of CDR' s and frameworks present in such a 15Al-like ISV are preferably such that the resulting 15A.1- Hke ISV has an IC₅0 in the Jurkat assay of Example L C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl 0^'9M (5.0E-9M or 5.0 nM).

In one particular aspect, any 15A.l-like sequence may be a humanized sequence, as further described herein. 15H3-iike sequences: a "15H3-like sequence", "J5H3-like ISV⁷ or "15 IB- like building block^'" is defined as an ISV (as described herein) that comprises: a) a CDRl which comprises or essentially consists of either (i) the amino acid sequence TYTMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid. difference(s) (as defined herein) with the amino acid sequence TYTMG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence VINWNGDRTNYADSVKG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence VINWNGDRTNYADSVKG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence VINWNGDRTNYADSVKG; and/or c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence RPRFTAEPMTSRRYQY or (ii) an amino acid sequence that has at least 80%. such as at least 85%. for example at least 90% or more than 95%» sequence identity with the amino acid sequence RPRFTAEPMTSRRYQY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence RPRFTAEPMTSRRYQY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl , CDR2 and CDR3 are preferably such that the I 5H3-like ISV has an IC₅₀ in the Jurkat assay of Example 1 , C-4; of better than 7.0xl O^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM).

Preferably, in such a 15H3-like sequence, CDRl and CDR2 are as defined under a) and b), respectively; or CDRl and CDR3 are as defined under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 15H3-like sequence, CDRl , CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such an 15H3-like sequence, CDRl , CDR2 and CDR3 are preferably such that the 15H3-Iike ISV has an IC₅o in the Jurkat assay of Example 1 , C-4; of better than 7.0xl O~⁹ M (7.0E-9M or 7.0 nM), in particular better than 5.0xl O~⁹ M (5.0E- 9M or S.O nM).

For example, in such an 15H3-like sequence: CDRl may comprise or essentially consist of the amino acid sequence TYTMG (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence VINWNGDRTNYADSVKG (with CDRl and CDR3 being as defined under a) and c), respectively); and/or CDR3 may comprise or essentially consist of the amino acid sequence RPRFTAEPMTSRRYQY (with CDRl and CDR2 being as defined under a) and b), respectively). Particularly, when an 15H3-like sequence is according to this aspect: CDRl may comprise or essentially consist of the amino acid sequence TYTMG and CDR2 may comprise or essentially consist of the amino acid sequence

VINWNGDRTNYADSVKG (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence TYTMG and CDR3 may comprise or essentially consist of the amino acid sequence

RPRFTAEPMTSRRYQY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence

VINWNGDRTNYADSVKG and CDR3 may comprise or essentially consist of the amino acid sequence RPRFTAEPMTSRRYQY (with CDRl being as defined under a) above). Again, in such 15H3-like sequences, CDRL CDR2 and CDR3 are preferably such that the 15H3-like ISV has an IC 0 in the Jurkat assay of Example 1, C-4: of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect, a "ISFB-like sequence", "15H3-like ISV or ^ISHS- ke building block^' is an ISV that comprises:

d) a CDRl which is either (i) the amino acid sequence TYTMG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence TYTMG; and/or

e) a CDR2 which is either (i) the amino acid sequence

VINWNGDRTNYADSVKG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90%» or more than 95% sequence identity with the amino acid sequence VINWNGDRTNYADSVKG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence VINWNGDRTNYADSVKG; and/or

f) a CDR3 which is either (i) the amino acid sequence

RPRFTAEPMTSRRYQY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95%o sequence identity with the amino acid sequence RPRFTAEPMTSRRYQY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence RPRFTAEPMTSRRYQY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl , CDR2 and CDR3 are preferably such that the 15H3-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

Preferably, in a 15H3-like sequence according to this specifically preferred aspect, CDRl and CDR2 are as defined under d) and e), respectively; or CDRl and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 15H3-like sequence, CDRl . CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an 15H3-like sequence, CDRl , CDR2 and CDR3 are preferably such that the 15H3-like ISV has an IC₅₀ in the

Jurkat assay of Example 1, C-4; of better than 7.0xlO^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

For example, in a 15H3~iike sequence according to this specifically preferred aspect: CDRl is the amino acid sequence TYTMG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

VI WNGDRTNYADSVKG (with CDRl and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence RPRFTAEPMTSRRYQY (with CDRl and CDR2 being as defined under d) and e). respectively). Particularly, when an 15H3-like sequence is according to this aspect: CDRl is the amino acid sequence TYTMG and CDR2 is the amino acid sequence VINWNGDRTNYADSVKG (with

CDR3 being as defined under f) above); and/or CDRl is the amino acid sequence TYTMG and CDR3 is the amino acid sequence RPRFTAEPMTSRRYQY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence

VINWNGDRTNYADSVKG and CDR3 is RPRFTAEPMTSRRYQY (with CDRl being as defined under d) above). Again, in such 15H3-Iike sequences, CDRl, CDR2 and

CDR3 are preferably such that the 15H3-like ISV has an. IC₅0 in the Jurkat assay of Example 1, C-4; of better than 7.0xlO^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM).

In a particularly preferred 15H3~Hke sequence: CDRl is the amino acid sequence TYTMG, CDR2 is the amino acid sequence VINW GDRTNYADSVKG; and CDR3 is the amino acid sequence RPRFTAEPMTSRRYQY.

In all the 15H3-like sequence described in this paragraph H), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity with the framework sequences of 15H3 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 15H3 while disregarding the CDR's in the calculation). Again, the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 15H3-like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10^'9 M (7.0E-9M or 7.0 nM). in particular better than 5.0xl0^~9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 15H3-like sequence is an ISV that has at least 70%, such at least 80%), for example at least 85%), such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR015H03 (SEQ ID NO: 196). For example, in an 15H3-like sequence according to this aspect, the CDR's may be according to the specifically preferred aspect described above, and. may in particularly (but without limitation) be TYTMG (CDR1); VINWNGDRTNYADSVKG (CDR2); and

RPRFTAEPMTSRRYQY (CDR3). Again, preferably, the combination of CDR's and frameworks present in such a 15H3-like ISV are preferably such that the resulting 15H3- like ISV has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In one particular aspect, any 15H3-like sequence may be a humanized sequence, as further described herein.

283B6-like sequences: a ^{^}283B6-like sequence'', "283B6-like ISV' or ^Li283B6-like building block" is defined as an ISV (as described herein) that comprises: a) a CDR1 which comprises or essentially consists of either (i) the amino acid sequence VATLG or (ii) an amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence VATLG; and/or b) a CDR2 which comprises or essentially consists of either (i) the amino acid sequence DISSGGSTNYADSVRG or (ii) an amino acid sequence that has at least 80%, such as at least 85%o, for example at least 90% or more than 95%o sequence identity with the amino acid sequence DISSGGSTNYADSVRG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the acid sequence DISSGGSTNYADSVRG; and/or c) a CDR3 which comprises or essentially consists of either (i) the amino acid sequence RTSGWRTRSNY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence RTSGWRTRSNY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence RTSGWRTRSNY; in which the framework sequences present in such an ISV are as further described herein, and in which CDRl, CDR2 and CDR3 ar preferably such that the 283B6-like ISV has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0x10^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

Preferably, in such a 283B6-like sequence, CDRl and CDR2 are as defined under a) and b) , respectively; or CDRl and CDR3 are as defined, under a) and c), respectively; or CDR2 and CDR3 are as defined under b) and c), respectively. More preferably, in such a 283B6-like sequence, CDRl, CDR2 and CDR3 are all as defined under a), b) and c), respectively. Again, in such an 283B6-like sequence, CDRl, CDR2 and CDR3 are preferably such that the 283B6-like ISV has an IC50 in the Jurkat assay of Example 1 , C- 4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xl 0^"9 M (5.0E-9M or 5.0 nM).

For example, in such an 283B6-like sequence: CDRl may comprise or essentially consist of the amino acid sequence VATLG (with CDR2 and CDR3 being as defined under b) and c), respectively); and/or CDR2 may comprise or essentially consist of the amino acid sequence DISSGGSTNYADSVRG (with CDRl and CDR3 being as defined under a) and c) , respectively); and/or CDR3 may comprise or essentially consist of the amino acid, sequence RTSGWRTRSNY (with CDRl and CDR2 being as defined under a) and b), respectively). Particularly, when an 283B6-like sequence is according to this aspect:

CDRl may comprise or essentially consist of the amino acid sequence VATLG and CDR2 may comprise or essentially consist of the amino acid sequence

DISSGGSTNYADSVRG (with CDR3 being as defined under c) above); and/or CDRl may comprise or essentially consist of the amino acid sequence VATLG and CDR3 may comprise or essentially consist of the amino acid sequence RTSGWRTRSNY (with CDR2 being as defined under b) above); and/or CDR2 may comprise or essentially consist of the amino acid sequence DISSGGSTNYADSVRG and CDR3 may comprise or essentially consist of the amino acid, sequence RTSGWRTRSNY (with CDR.1 being as defined under a) above). Again, in such 283B6-like sequences, CDR1, CDR2 and CDR3 are preferably such that the 283B6-like ISV has an IC₅o in the Jurkat assay of Example 1, C-4; of better than 7.0xlO^"9M (7.0E-9M or 7.0 nM), in particular better than 5.0xl0^"9 M (5.0E-9M or 5.0 nM).

In a specifically preferred aspect, a "283B6-!ike sequence", "283B6-like ISV^' or " 5556- like building block'^'' is an ISV that comprises:

d) a CDR1 which is either (i) the amino acid sequence VATLG or (ii) an

amino acid sequence that has only 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence VATLG; and/or

e) a CDR2 which is either (i) the amino acid sequence

DISSGGSTNYADSVRG or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence DISSGGSTNYADSVRG; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence DISSGGSTNYADSVRG; and/or

f) a CDR3 which is either (i) the amino acid sequence RTSGWRTRSNY or (ii) an amino acid sequence that has at least 80%, such as at least 85%, for example at least 90% or more than 95% sequence identity with the amino acid sequence

RTSGWRTRSNY; or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein) with the amino acid sequence

RTSGWRTRSNY; in which the framework sequences present in such an ISV are as further described herein, and in which CDR1, CDR2 and CDR3 are preferably such that the 283B6-like ISV has an. IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

Preferably, in a 283B6-iike sequence according to this specifically preferred aspect, CDR1 and CDR2 are as defined under d) and e), respectively; or CDR1 and CDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 are as defined under e) and f), respectively. More preferably, in such a 283B6-like sequence, CDR1, CDR2 and CDR3 are all as defined under d), e) and f), respectively. Again, in such an 283B6-like sequence, CDR1, CDR2 and CDR3 are preferably such that the 283B6-Iike ISV has an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0xl0^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

For example, in a 283B6-like sequence according to this specifically preferred aspect: CDR1 is the amino acid sequence VATLG (with CDR2 and CDR3 being as defined under e) and f), respectively); and/or CDR2 is the amino acid sequence

DISSGGSTNYADSVRG (with CDR1 and CDR3 being as defined under d) and f), respectively); and/or CDR3 is the amino acid sequence RTSGWRTRSNY (with CDR1 and CDR2 being as defined under d) and e), respectively). Particularly, when an 283B6- like sequence is according to this aspect: CDR1 is the amino acid sequence VATLG and CDR2 is the amino acid sequence DISSGGSTNYADSVRG (with CDR3 being as defined under f) above); and/or CDR1 is the amino acid sequence VATLG and CDR3 is the amino acid sequence RTSGWRTRSNY (with CDR2 being as defined under e) above); and/or CDR2 is the amino acid sequence DISSGGSTNYADSVRG and CDR3 is

RTSGWRTRSNY (with CDR1 being as defined under d) above). Again, in such 283B6- like sequences, CDR1 , CDR2 and CDR3 are preferably such that the 283B6-like ISV has an IC5o in the Jurkat assay of Example 1, C-4; of better than 7.0xl0^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0xlO^"9 M (5.0E-9M or 5.0 nM).

In a particularly preferred 283B6-iike sequence: CDR1 is the amino acid sequence VATLG, CDR2 is the amino acid sequence DISSGGSTNYADSVRG; and CDR3 is the amino acid sequence RTSGWRTRSNY.

In all the 283B6-like sequence described in this paragraph I), the framework sequences may be as further described herein. Preferably, the framework sequences are such that the framework sequences have at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity with the framework sequences of 283B6 (which, for example, can be determined by determining the overall degree of sequence identity of a given sequence with the sequence of 283B6 while disregarding the CDR's in the calculation). Again, the combination of CDR's and frameworks present in a given sequence are preferably such that the resulting 283B6-like ISV has an IC₅₀ in the Jurkat assay of Example 1 , C-4; of better than 7.0x10^~9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10^"9 M (5.0E-9M or 5.0 nM).

In one specific aspect, a 283B6-like sequence is an ISV that has at least 70%, such at least 80%, for example at least 85%, such as at least 90% or more than 95% sequence identity with the amino acid sequence 4CXCR283B06 (SEQ ID NO: 197). For example, in an 283B6-like sequence according to this aspect, the CDR's may be according to the specifically preferred aspect described above, and may in particularly (but without limitation) be VATLG (CDR1); DISSGGSTNYADSVRG (CDR2); and

RTSGWRTRSNY (CDR3). Again, preferably, the combination of CDR's and

frameworks present in such a 283B6-Iike ISV are preferably such that the resulting

283B6-like ISV has an IC₅₀ in the Jurkat assay of Example 1 , C-4; of better than 7.0xl0^"9 M (7.0E-9M or 7.0 nM), in particular better than S.OxlO^"9 M (5.0E-9M or 5.0 nM).

In one particular aspect, any 283B6-like sequence may be a humanized sequence, as further described herein.

Constructs, proteins or polypeptides containing one or more building blocks according to the invention may be as further described herein, and form further aspects of the invention. Some specific but-non-limiting examples of constructs, proteins or polypeptides of the invention are given below. In all these constructs, the building blocks may be linked to each other either directly or via one or more suitable linkers and the building blocks may be in any suitable order in respect of each other (i.e., towards the N-terminus or towards the C- terminus).

In each of the constructs, proteins and polypeptides described below (and in the rest of the present specification), each. 10E9-type sequence. 281E10-type sequence, 10E12-type sequence, !OAlO-type sequence, lOGlO-type sequence, 14 A2 -type sequence, 15Al -type sequence, 15H3-type sequence, 283B6-type sequence, 10E9-like sequence, 281E10-like sequence, 10E12-like sequence, lOAlO-like sequence, lOGlO-like sequence, 14 A2 -like- sequence, 15Al.-like sequence, 15H3 -like sequence and 283B6-like sequence (when present in such a construct, protein or and polypeptide) is as defined herein, and the preferences described herein for each such sequence/ISV also apply when such a sequence/ISV is present in a construct, protein or polypeptide described herein). For example, and without limitation, when such a sequence/ISV is present in such a construct, protein or polypeptide, it is preferably such that (i.e., has a combination of CDR's such that and/or has a combination of CDR's and. framework sequences such that) the sequence/ISV, when measured, in the corresponding monovalent format, has an IC₅₀ in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM).

As described herein, a construct, compound, protein or polypeptide of the invention contains at least one ISV of the invention. When a construct, compound, protein or polypeptide comprises two or more ISV's that are directed against CXCR-4, preferably all these ISV's are ISV's of the invention. However, it is also possible that a construct, compound, protein or polypeptide of the invention comprises, besides the at least one ISV of the invention, other ISV's that are directed against CXCR-4. Some non-limiting examples of such ISV's are the Nanobodies® against CXCR-4 that are described in WO 09/138519 and US application 61/358,495, such as 238D2, 238D4 and sequence-optimized and/or humanized variants thereof; and/or the Nanobodies® against CXCR-4 of SEQ ID NO's:371 to 458, or sequence-optimized and/or humanized variants thereof.

Preferably, the compounds, construct, protein or polypeptide of the invention are preferably such that they have IC50 in the Jurkat assay of Example 1, C-4; of better than 20.0 nM, preferably better than ΙΟηΜ, more preferably better than 7.0 nM, even more preferably better than 5.0 nM, such as better than 3.0 nM.

In one aspect, the ISV's, compounds, construct, protein or polypeptide of the invention are such that they are inverse agonists of CXCR-4, in particular in the assay described in Example 1 , D-2 using the mutant CXCR-4 Nl 19S and/or in the assay described in Example 1 , D-2 using the truncated version of the mutant CXCR-4 Nl 19S described by Zhang et al (see Example 1 , D-2). In another aspect, the ISV's, compounds, construct, protein or polypeptide of the invention are such that they are not inverse agonists of CXCR-4.

Some preferred, but non-limiting examples of polypeptides of the invention are the sequences of SEQ ID NO: 198-370, or the corresponding polypeptides in which one or both of the ISV's present therein have been suitably sequence-optimized and/or humanized.

It should also be noted that, from the ISV's of the invention described herein, the following ISV's are preferred: a 10E9-type sequence, 10E12-type sequence. l OG! O-type sequence, 15H3-type sequence, 283B6-type sequence, with the following ISV's of the invention being more preferred: a 10E9-like sequence, 10E12-like sequence, lOGlO-like sequence, 15H3-like sequence or 283B6-like sequence; and the following ISV's of the invention being particularly preferred: 10E9 or (preferably) a humanized (and/or sequence optimized) variant of 10E9; 10E12 or (preferably) a humanized (and/or sequence optimized) variant of 10E12; 10G10 or (preferably) a humanized (and/or sequence optimized) variant of 10G10; 15H3 or (preferably) a humanized (and/or sequence optimized) variant of 15H3; and/or 283B6 or (preferably) a humanized (and/or sequence optimized) variant of 283B6. Accordingly, the use of a 15Al-type sequence, 15Al-like sequence, 10A10-type sequence, lOAlO-like sequence, 14A2-type sequence, 14A2-like sequence, 281E10-type sequence, or 281E10-like sequence will usually be less preferred.

Similarly, in the invention, constructs and polypeptides comprising one or more ISV's of the invention in which each ISV of the invention is a 10E9-type sequence, a 10E12-type sequence, a lOGlO-type sequence, a 15H3-type sequence and/or a 283B6-type sequence; and in particular a 10E9-like sequence, a 10E12-like sequence, a lOGlO-like sequence, a 15H3- like sequence and/or a 283B6-like sequence are particularly preferred. More preferably, all ISV's of the invention that are present in a construct or polypeptide of the invention are a 10E9-type sequence, a 10E12-type sequence, a lOGlO-type sequence, a 15H3-type sequence and/or a 283B6-type sequence; and in particular a 10E9-like sequence, a 10E12-like sequence, a lOGlO-like sequence, a 15H3-like sequence and/or a 283B6-like sequence.

Examples of such constructs and polypeptides of the invention will be clear to the skilled person based on the disclosure herein.

As further described herein, each of the constructs below may optionally also contain one or more further binding domains, binding units, amino acid sequences or other

(functional) groups or moieties, that preferably also confer one or more desired properties to the constructs (some non-limiting examples of the same will become clear from the further description herein).

For example, according to a specific aspect, the constructs, proteins or polypeptides may have been provided with an increased half-life, for example by functionalisation and/or by including in the construct a moiety or binding unit that increases the half- life of the construct. Examples of such functionalisation, moieties or binding units will be clear to the skilled person and may for example be as described herein, and for example may include pegylation, fusion to serum albumin, or fusion to a peptide or binding unit that can bind, to a serum protein such as serum albumin. Specific examples will be given in the further description below, and for example and without limitation include the "monovalent constructs with a serum albumin binding moiety for extending the half-life", "biparatopic constructs with a serum albumin binding moiety for extending the half-life", "preferred biparatopic constructs with a serum albumin binding moiety for extending the half-life" and "bivalent constructs with a serum albumin binding moiety for extending the half-life".

In these constructs, the "serum-albumin binding peptide or binding domain" may be any suitable serum-albumin binding peptide or binding domain capable of increasing the half-life of the construct (compared to the same construct without the serum-albumin binding peptide or binding domain), and may in particular be serum albumin binding peptides as described in WO 2008/068280 by Ablynx N.V. (and in particular WO 2009/127691 and the non- prepublished US application 61/301 ,819, both by Ablynx N.V.), or a serum-albumin binding ISV (such as a serum-albumin binding Nanobody; for example Alb-1 or a humanized version of Alb-1 such as Alb-8, for which reference is for example made to WO 06/122787).

With respect to half-life, it should be noted that in the invention, and by using the various half-life extending techniques described herein (for example, by suitably choosing a serum-albumin binding peptide according to WO 2008/068280, WO 2009/127691 and/or the non-prepublished US application 61/301,819), the half-life of a construct or polypeptide of the invention can (and preferably is) suitably "tailored" for the intended (therapeutic and/or diagnostic) application and/or to obtain the best balance between the desired therapeutic and/or pharmacological effect and possible undesired side-effects. For example and without limitation, for use in stem cell mobilization, it may be preferred to have a relatively short half-life in man (e.g. , 5 to 80 hours). In some embodiments for use in stem cell mobilization, the half-life is between 5 to 70 hours, between 10 to 80 hours, between 10 to 70 hours, between 10 to 60 hours, between 20 to 70 hours, between 20 to 60 hours, between 30 to 60 hours, between 20 to 50 hours, or between 30 to 50 hours, all inclusive of the end values of the range of hours; or approximately 5, 6, 7, 8. 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79 or 80 hours. Also, when a construct or polypeptide of the invention is to be used for the prevention and/or treatment of cancer (as described herein), it will generally be preferred to have a longer half-life (e.g., 7-23 days), but it should be noted that when a construct or polypeptide of the invention is to be used for the prevention and/or treatment of cancer, the construct or polypeptide of the invention may also mobilize stem cells and leukocytes. In some embodiments, the half-life for a construct or polypeptide of the invention to be used for the prevention and/or treatment of cancer is between 7 to 20 days, between 10 to 23 days, between 10 to 20 days, between 10 to 18 days, between 12 to 20 days, between 12 to 18 days, between 12 to 16 days, between 14 to 18 days, or between 14 to 16 days, all inclusive of the end values of the range of days; or approximately 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 or 23 days. In this context, the use of a construct or polypeptide of the invention with a somewhat shorter half-life (e.g., 3-9 days) may allow the clinician to exert more control over the treatment and to set a treatment and/or dosage regimen which provides an optimal balance between treating the cancer to be treated and avoiding undesired or excessive stem cell and leukocyte mobilization. In some embodiments, the half-life for a construct or polypeptide of the invention requiring balancing between treatment of cancer and avoiding undesired or excessive stem cell and leukocyte mobilization is between 3 to 8 days, between 4 to 9 days, between 4 to 8 days, between 4 to 7 days, between 5 to 8 days, between 5 to 7 days, all inclusive of the end values of the range of days; or approximately 3, 4. 5, 6, 7, 8 or 9 days.

All the constructs below are preferred, but non-limiting aspects of the invention. Some specific but non-limiting examples of exemplary constructs according to some of these aspects will become clear from the further description herein.

Monovalent constructs

- a construct, protein or polypeptide essent ally consisting of a 10E9-type sequence;

a construct, protein or polypeptide essent ally consisting of a 281E10-type sequence;

- a construct. protein or polypeptide essent ally consisting of a 10E12-type sequence;

- a construct, protein or polypeptide essent ally consisting of a lOA!O-type sequence:

-^■ a construct, protein or polypeptide essent ally consisting of a iOGlO-type sequence;

- a construct, protein or polypeptide essent ally consisting of a 14A2-type sequence;

a construct, protein or polypeptide essent ally consisting of a 5Al-type sequence;

- a construct, protein or polypeptide essent ally consisting of a 15H3-type sequence;

- a construct, protein or polypeptide essent ally- consisting of a 283B6-type sequence;

- a construct, protein or polypeptide essent ally consisting of a 10E9-like sequence;

- a construct, protein or polypeptide essent ally consisting of a 281E10-like sequence;

a construct, protein or polypeptide essent ally consisting of a 10E12-like sequence;

- a construct, protein or polypeptide essent ally consisting of a 1 OA 10-like sequence:

- a construct, protein or polypeptide essent ally consisting of a lOGlO-like sequence;

- a construct, protein or polypeptide essent ally consisting of a 14A2-like sequence;

- a construct, protein or polypeptide essent tally consisting of a 15Al-like sequence;

a construct, protein or polypeptide essent tally consisting of a 15H3-like sequence;

- a construct, protein or polypeptide essent tally consisting of a 283B6-like sequence; Monovalent constructs with a serum albumin binding moiety for extended half-life a construct, protein or polypeptide essentially consisting of a 10E9-type sequence and a serum -albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 281E10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 10E12-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 10A10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); - a construct, protein or polypeptide essentially consisting of a 10G10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct protein or polypeptide essentially consisting of a 14A2-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 15Al-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 15H3-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 283B6-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); - a construct, protein or polypeptide essentially consisting of a 10E9-like sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 281E10-like sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 10E12-like sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a lOA!O-like sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a lOGl O-iike sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 14A2-like sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 15A1-Hke sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide essentially consisting of a 15H3-like sequence and a serum-albumin binding peptide or binding domain (as further described herein); - a construct, protein or polypeptide essentially consisting of a 283B6-like sequence and a serum-albumin binding peptide or binding domain (as further described herein);

Biparatopic constructs a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence and at least one more (such as one or two more) binding domain, or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 1 OA 10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

- a construct, protein or polypeptide comprising or essentially consisting of a 10G10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed, against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 15H3- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4; a construct, protein or polypeptide comprising or essentially consisting of a 283B6- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 10A10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 10G10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 15A1 - like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct, protein or polypeptide comprising or essentially consisting of a 15H3- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4;

a construct protein or polypeptide comprising or essentially consisting of a 283B6- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4:

a construct, protein or polypeptide comprising or essentially consisting of two 10E9- type sequences (which may be the same or different); a construct, protein or polypeptide comprising or essentially consisting of two 281E10-type sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 10E12-type sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two l OA!O-type sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two lOGlO-type sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 14A2-type sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 15Al-type sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 15H3-type sequences (which may be the same or different);

a construct protein or polypeptide comprising or essentially consisting of two 283B6-type sequences (which may be the same or different);

a construct, protei or polypeptide comprising or essentially consisting of two 10E9- like sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 281E10-like sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 10EI2-li.ke sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 10A10-like sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two lOGlO-like sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two ] 4A2-like sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 35 Al -like sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of two 15H3-like sequences (which may be the same or different); a -construct, protein or polypeptide comprising or essentially consisting of two 283B6-Iike sequences (which may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of a I0E9- like sequence and a (different) 10E9-type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- Hke sequence and a (different) 281E10-type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 10E 12- like sequence and a (different) 10E12-type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 10A10- like sequence and a (different) 10A10-type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 1GG10- like sequence and a (different) lOGlO-type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- like sequence and a (different) 14A2-type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 15A1- like sequence and a (different) 15Al -type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 15H3- like sequence and a (different) 15H3-type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 283B6- like sequence and a (different) 283B6-type sequence;

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 281E10-type sequence (and preferably a 281E10-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 10E12-type sequence (and preferably a 10E12-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 10A10-type sequence (and preferably a 10A10-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a lOGlO-type sequence (and preferably a lOG!O-like sequence); a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 15Al-type sequence (and preferably a 15A1 -like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and. preferably a 10E9-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 281Ε10· type sequence (and preferably a 281E10-like sequence) and a 10E12-type sequence (and preferably a 10E12-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence (and preferably a 281E10-iike sequence) and a !OAlO-type sequence (and preferably a 10A10-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence (and preferably a 281E10-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-iike sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10 type sequence (and preferably a 281E10-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence (and preferably a 281E10-like sequence) and a 15Al-type sequence (and preferably a 15 A 1 -like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 281E30· type sequence (and preferably a 281E10-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 281Ε10· type sequence (and preferably a 281E10-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence); a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a ! OAlO-type sequence (and preferably a l OAl O-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E 12-Iike sequence) and a 15Al-type sequence (and preferably a 5Al-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-Iike sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a lOAlO-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10A10 type sequence (and preferably a lOAlO-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence):

a construct, protein or polypeptide comprising or essentially consisting of a 10A10 type sequence (and preferably a lOAlO-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10A10 type sequence (and preferably a 10A10-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10Α10· type sequence (and preferably a 10A10-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence); . a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a lOGlO-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence);

a construct, protein, or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a lOGlO-like sequence) and a 15Al-type sequence (and preferably a 15A1-Iike sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a lOGlO-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a lOGlO-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2~Hke sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence (and preferably a 15Al-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence (and preferably a 15Al-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence);

a construct, protein or polypeptide comprising or essentially consisting of a 15H3- type sequence (and preferably a 15H3-like sequence) and a 283B6~type sequence (and preferably a 283B6-like sequence); Preferred biparatopie constructs: a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 281E10 or a humanized variant of 281E10;

a construct protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10EI2 or a humanized variant of 10E12;

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10A10 or a humanized variant of 10A10;

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10G10 or a humanized variant of 10G10;

- a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 14A2 or a humanized variant of 14A2;

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 15A1 or a humanized variant of 15 Al ;

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 15H3 or a humanized variant of 15H3;

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 283B6 or a humanized variant of 283B6;

a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281E10 and 10E12 or a humanized variant of 10E12;

a construct, protein or polypeptide comprising or essentially consisting of 281 E 10 or a humanized variant of 281E10 and 1 OA 10 or a humanized variant of 10A10;

a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281E10 and 10G10 or a humanized variant of 10G10;

a construct, protein or polypeptide comprising or essentially consisting of 281 E 10 or a humanized variant of 281 E 10 and 14 A2 or a humanized variant of 14 A2 ;

a construct, protein or polypeptide comprising or essentially consisting of 281 El 0 or a humanized variant of 281 El 0 and 15A1 or a humanized variant of 15A1 ;

a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281 El 0 and 15H3 or a humanized variant of 15H3 ;

- a construct, protein or polypeptide comprising or essentially consisting of 281 E 10 or a humanized variant of 281E10 and 283B6 or a humanized variant of 283B6; a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 1 OA 10 or a humanized valiant of 10A10;

a construct, protein or polypeptide comprising or essentially consisting of 1.0E12 or a humanized variant of 10E12 and 10G10 or a humanized variant of 10G10;

a construct, protein or polypeptide comprising or essentially consisting of 30E12 or a humanized variant of 10E12 and 14A2 or a humanized, variant of 14A2;

a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 15A1 or a humanized variant of 15A1 ;

a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant, of 10E 12 and 15H3 or a humanized variant of 15H3 ;

a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 283B6 or a humanized variant of 283B6;

a construct, protein or polypeptide comprising or essentially consisting of ί 0A10 or a humanized variant of 10A10 and 10G 10 or a humanized variant of 10G10;

a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 10A10 and 14A2 or a humanized variant of 14A2;

a construct, protein or polypeptide comprising or essentially consisting of 1 OA 10 or a humanized variant of 10A10 and 15A1 or a humanized variant of 15A1 ;

a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 0A10 and 15H3 or a humanized variant of 15H3 ;

a construct, protein or polypeptide comprising or essentially consisting of 1 OA 10 or a humanized variant of 1.0A10 and 283B6 or a humanized variant of 283B6;

a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 14A2 or a humanized variant of 14A2;

a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 15A1 or a humanized variant of 15A1;

a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 1 OG10 and 15H3 or a humanized variant of 15H3;

a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 283B6 or a humanized variant of 283B6;

a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 15A1 or a humanized variant of 15A1; a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 15H3 or a humanized variant of 15H3;

a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 283B6 or a humanized variant of 283B6;

- a construct, protein or polypeptide comprising or essentially consisting of 15A1 or a humanized variant of 15A1 and 15H3 or a humanized, variant of 15H3;

a construct, protein or polypeptide comprising or essentially consisting of 15A1 or a humanized variant of 15A1 and 283B6 or a humanized variant of 283B6;

a construct, protein or polypeptide comprising or essentially consisting of 15H3 or a humanized variant of 15H3 and 283B6 or a humanized variant of 283B6;

Bivalent constructs: a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10E9 or (preferably) a humanized variant of 10E9 and a second ISV which is either 10E9 or (preferably) a humanized variant of 10E9 (in which the first and second ISV may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 281 E10 or (preferably) a humanized, variant of 281E10 and a second ISV which is either 281 El 0 or (preferably) a humanized variant of 281 El 0 (in which the first and second ISV may be the same or different);

- a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10E12 or (preferably) a humanized variant of 10E12 and a second ISV which is either I0E12 or (preferably) a humanized variant of 10E12 (in which the first and second ISV may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10A10 or (preferably) a humanized variant of 1 OA 10 and a second ISV which is either 10A10 or (preferably) a humanized variant of 1 OA 10 (in which the first and second ISV may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10G10 or (preferably) a humanized variant of 10G10 and a second ISV which is either 10G10 or (preferably) a humanized variant of 10G10 (in which the first and second ISV may be the same or different); a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 14A2 or (preferably) a humanized variant of 14A2 and a second ISV which is either 14A2 or (preferably) a humanized variant of 1.4A2 (in which the first and second ISV may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 15A1 or (preferably) a humanized variant of 15A1 and a second ISV which is either 15A1 or (preferably) a humanized variant of 15A1 (in which the first and second ISV may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 15H3 or (preferably) a humanized variant of 15H3 and a second ISV which is either 15H3 or (preferably) a humanized variant of 15H3 (in which the first and second ISV may be the same or different);

a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 283B6 or (preferably) a humanized variant of 283B6 and a second ISV which is either 283B6 or (preferably) a humanized variant of 283B6 (in which the first and second ISV may be the same or different);

Biparatopic constructs with a serum albumin binding moiety for extended half-life a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or pol^ypeptide comprising or essentially consisting of a 10E12- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10G10- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serara-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum- albumin binding peptide or binding domain (as further described herein):

a construct, protein or polypeptide comprising or essentially consisting of a 15H3- type sequence and at least one more (such as one or two more) bmding domain or binding unit (for example, one or more other ISV's) directed, against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 283B6- type sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10A10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct protein or polypeptide comprising or essentially consisting of a 10G10- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 15A1- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum -albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 15H3- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 283B6- like sequence and at least one more (such as one or two more) binding domain or binding unit (for example, one or more other ISV's) directed against (human) CXCR-4 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 10E9- type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 281E10-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of two 10E12-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two lOAlO-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two lOG!O-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 14A2-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 15Ai-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consi sting of two 15H3-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 283B6-type sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 10E9- like sequences (which may be the same or different) and a serum -albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 281E10-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two iOE12-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 10A10-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of two lOGlO-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 14A2-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 15Al-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 15H3-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of two 283B6-like sequences (which may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- like sequence and a (different) 10E9-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10- like sequence and a (different) 281 El 0-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- like sequence and a (different) 10E12-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a lOAiO- like sequence and a (different) 10A10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10G10- like sequence and a (different) 10G10-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- like sequence and a (different) 14A2-type sequence and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 15A1- like sequence and a (different) 15Al-type sequence and a serum -albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 15H3- like sequence and a (different) 15H3-type sequence and a serum-albumin binding peptide^' or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 283B6- like sequence and a (different) 283B6-type sequence and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 281E10-type sequence (and preferably a 281E10-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 10E12-type sequence (and preferably a 10E12-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and. preferably a 10E9-like sequence) and a lOAlO-type sequence (and preferably a 10A10-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a l OGlO-type sequence (and preferably a !OGlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E9- type sequence (and preferably a 10E9 ike sequence) and a 283B6-type sequence (and preferably a 283B6-Iike sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281 El 0- type sequence (and preferably a 281E10-like sequence) and a 10E12-type sequence (and preferably a 10E12-Iike sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10 type sequence (and preferably a 281E10-like sequence) and a lOAlO-type sequence (and preferably a lOAlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281 El 0 type sequence (and preferably a 281E10-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281 El 0 type sequence (and preferably a 281E10-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum -albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281 El 0 type sequence (and preferably a 281E10-like sequence) and a 15Al-type sequence (and preferably a 15A1-Iike sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 281E10 type sequence (and preferably a 281E10-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 281E10- type sequence (and preferably a 281 E10-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a lOAlO-type sequence (and preferably a !OAlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E 12- type sequence (and preferably a 10E12-like sequence) and a lOGlO-type sequence (and preferably a lOGlO-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a consu-uct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum- albumin binding peptide or binding domain (as further described herein);

a construct protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein):

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10E12- type sequence (and preferably a 10E12-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 1 OA 10- type sequence (and preferably a lOAlO-like sequence) and a lOGlO-type sequence (and preferably a lOGl O-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a lOAlO-iike sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a 10A10-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a lOAlO-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10A10- type sequence (and preferably a 10A10-like sequence) and. a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a 1 OGlO-like sequence) and a 14A2-type sequence (and preferably a 14A2-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10G10 type sequence (and preferably a !OGlO-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 10G1O type sequence (and preferably a lOGlG-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a semm-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a lOGlO type sequence (and preferably a lOG!O-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and. a serum-albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 15Al-type sequence (and preferably a 15Al-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

- a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 14A2- type sequence (and preferably a 14A2-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence (and preferably a 15Al-like sequence) and a 15H3-type sequence (and preferably a 15H3-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 15A1- type sequence (and preferably a 15Al-iike sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a 15H3- type sequence (and preferably a 15H3-like sequence) and a 283B6-type sequence (and preferably a 283B6-like sequence) and a serum-albumin binding peptide or binding domain (as further described herein); Preferred biparatopic constructs with a serum albumin binding moiety for extended half-life: a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 281 El 0 or a humanized variant of 281 El 0 and a serum- albumin binding peptide or binding domain (as further described herein);

- a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10E12 or a humanized variant of 10E12 and a serum- albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10A10 or a humanized variant of 10A10 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 10G10 or a humanized variant of 10G10 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E9 or a humanized variant of 10E9 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281 E 10 and 1 OE 12 or a humanized variant of 1 OE 12 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281E10 and 10A10 or a humanized variant of 10A10 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281 El 0 and 10G10 or a humanized variant of 10G10 and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 281E10 or a humanized variant of 281 El 0 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 281 E 10 or a humanized variant of 281E10 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain, (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of 281 El 0 or a humanized variant of 281E10 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 281 El 0 or a humanized variant of 281E10 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein):

a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 10A10 or a humanized variant of 10A10 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E 12 or a humanized variant of 10E12 and 10G10 or a humanized variant of 10G10 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 1.0E12 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10E12 or a humanized variant of 10E12 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 10A10 and 10G10 or a humanized variant of 10G10 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 1 OA 10 or a humanized variant of 10A10 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 1 OA 10 and 15 A 1 or a humanized variant of 15 A 1 and a serum- albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of 10A10 or a humanized variant of 10A10 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 1 OA 10 or a humanized variant of 10A10 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 14A2 or a humanized variant of 14A2 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 30G10 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and. 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 10G10 or a humanized variant of 10G10 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 15A1 or a humanized variant of 15A1 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 14A2 or a humanized variant of 14A2 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 15A1 or a humanized variant of 1.5A1 and 15H3 or a humanized variant of 15H3 and a serum- albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of 15A1 or a humanized variant of 15A1 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein); a construct, protein or polypeptide comprising or essentially consisting of 15H3 or a humanized variant of 15H3 and 283B6 or a humanized variant of 283B6 and a serum- albumin binding peptide or binding domain (as further described herein);

Bivalent constructs with a serum albumin binding moiety for extended half-life: a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10E9 or (preferably) a humanized variant of 10E9 and a second ISV which is either 10E9 or (preferably) a humanized, variant of 10E9 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

- a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 281E10 or (preferably) a humanized variant of 281E10 and a second ISV which is either 281E10 or (preferably) a humanized variant of 281E10 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

- a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10E12 or (preferably) a humanized variant of 10E12 and a second ISV which is either 10E12 or (preferably) a humanized variant of 10E12 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

- a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10A10 or (preferably) a humanized variant of 10A10 and a second ISV which is either 10A10 or (preferably) a humanized variant of 10A10 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

- a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 10G10 or (preferably) a humanized variant of 10G10 and a second ISV which is either 10G10 or (preferably) a humanized variant of lOGlO (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

- a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 14A2 or (preferably) a humanized variant of 14A2 and a second ISV which is either 14A2 or (preferably) a humanized variant of 14A2 (in which the first and second 1SV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a first IS V which is either 15A1 or (preferably) a humanized variant of 15A1 and a second ISV which is either 15A1 or (preferably) a humanized variant of 15A1 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a first ISV which is either 15H3 or (preferably) a humanized variant of 15H3 and a second ISV which is either 1 H3 or (preferably) a humanized variant of 15H3 (in which the first and second ISV may be the same or different) and a serum-albumin binding peptide or binding domain (as further described herein);

a construct, protein or polypeptide comprising or essentially consisting of a first ISV which, is either 283B6 or (preferably) a humanized variant of 283B6 and a second ISV which is either 283B6 or (preferably) a humanized variant of 283B6 (in which the first and second ISV may be the same or different) and a seram-albumin binding peptide or binding domain (as further described herein).

Thus, in some specific but non-limiting aspects, the invention generally relates to: a construct, protein or polypeptide comprising a 10E9-type sequence; and preferably a 10E9-like sequence; and in particular 10E9 or (preferably) a humanized variant of 10E9;

a construct, protein or polypeptide comprising a 281E10-type sequence; and preferably a 281E10-like sequence; and in particular 281E10 or (preferably) a humanized variant of 281 El 0;

a construct, protein or polypeptide comprising a 10E12-type sequence; and preferably a 10E12-Iike sequence; and in particular 10E12 or (preferably) a humanized variant of 10E12;

a construct, protein or polypeptide comprising a !OAlO-type sequence; and preferably a 10A10-like sequence; and in particular 10A10 or (preferably) a humanized variant of 1 OA 10; a construct, protein or polypeptide comprising a lOG!O-type sequence; and preferably a l OGl O-like sequence; and in particular 10G10 or (preferably) a humanized variant of 10G10;

a construct, protein or polypeptide comprising a 14A2-type sequence; and preferably a 14A2-like sequence; and in particular 14A2 or (preferably) a humanized variant of 14A2;

a construct, protein or polypeptide comprising a 35Al-type sequence; and preferably a 15 A 1 -like sequence; and in particular 15A1 or (preferably) a humanized variant of 15A1;

- a construct, protein or polypeptide comprising a 15H3-type sequence; and

preferably a 15H3-like sequence: and in particular 15H3 or (preferably) a humanized variant of 15H3;

a construct, protein or polypeptide comprising a 283B6-type sequence; and preferably a 283B6-Iike sequence; and in particular 283B6 or (preferably) a humanized variant of 283 B6; wherein each of these constructs, proteins or polypeptides optionally have been provided with an increased half-life (as described herein), and may for example for this purpose further comprise a serum-albumin binding peptide or binding domain capable of increasing the half- life of the construct (compared to the same construct without the serum-albumin binding peptide or binding domain), also as described herein.

The constructs, proteins or polypeptides preferably comprise two ISV's against CXC - 4 (at least one of which is an ISV of the invention, i.e., so as to form a bivalent or biparatopic construct of the invention), and preferably two ISV's of the invention, and are optionally half-life extended (e.g., in that they further contain a half-life extending moiety as described herein). However, it should be noted that for use in - for example - stem cell mobilization, either no half-life extension or a very short half-life may be preferred.

For example, some non-limiting examples polypeptides of the invention comprising a first ISV and a second ISV (at least one and preferably both of which are an ISV of the invention) may represented as follows (with the N-terminus of the polypeptide towards the right and the C-terminus towards the left):

[first ISV of the invention ] '-linker- [second ISV of the invention],

- [first ISV of the invention /-linker-/^" non-second ISV of the invention ], - [non-first ISV of the invention ]-\mk x-[ econd ISV of the inventio ],

- [first ISV of the invention y-iinker- ^" econd ISV of the invention ], which construct may optionally be pegylated for increased half-life in circulation;

- [first ISV of the invention) ' -linker- [ Nanobody®/VHH binding to serum albumin, such as Alb-SJ-linker- [second ISV of ^'the invention];

- [first ISV of the invention] '-linker-f Nanobody®/VHH binding to serum albumin, such as Alb-8) '-linker- [non-second ISV of the invention];

[non-first ISV of the

binding to serum albumin, such as Alb-8) '-linker- [second ISV of the invention];

- [first ISV of the invention ] ^'-linker- [second ISV of the inventwn]-\mk r-[Nanobody®/VHH binding to serum albumin, such as Alb-8];

- [first ISV of the invention] -linker- [non-second ISV of the invention) '-linker- [Nanobody®/VHH binding to serum albumin, such as Alb-8];

- [non-first ISV of the invention] '-linker- [second ISV of the invention] Amksx- [Nanobody®/VHH binding to serum albumin, such as Alb-8];

- [serum, albumin) '-linker- [first ISV of the invention) '-linker- [second ISV of the invention];

[first ISV of the

of the invention] '-linker- [serum albumin ] [serum albumin binding peptide (monovalent or in tandem)] -[first ISV of the invention]- linker- [second ISV of the invention];

- [serum albumin binding peptide (monovalent or in tandem)] -[first ISV of the invention]- linker- [non-second ISV of the invention];

[serum albumin binding peptide (monovalent or in tandem)] -[non-first ISV of the invention) '-linker- [second ISV of the invention];

[first ISV of the

invention] -[serum albumin binding peptide (monovalent or in tandem)];

[first ISV of the invention) '-linker- [non-second ISV of the invention] -[serum albumin binding peptide (monovalent or i tandem)];

- [ on-first ISV of the invention ) '-linker- [second ISV of the invention ] -[serum albumin binding peptide (monovalent or in tandem)].

The above polypeptides and/or immunoglobulin single variable domains may optionally be tagged with tags known to the skilled person such as 3xFlag-His6.

Also, the above constructs, proteins or polypeptides may comprise one or more further binding domains or binding units directed against CXCR-4 (and in particular, against human CXCR-4) preferably one or more other ISV's directed against CXCR-4 (and in particular, against human CXCR-4). Some non-limiting examples are the ISV's against human CXCR-4 described in WO 09/138519, US 61/358,495, US 61/358,495 and PCT/EP2010/064766 and/or the amino acid sequences of SEQ ID NO's: 371. to 458 (or humanized and/or sequence-optimized variants thereof).

Thus, in one specific, but non-limiting aspect, the invention relates to an ISV (as defined herein) that has an IC₅o in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM). Such an ISV may be as further described herein, and may for example be a domain antibody, single domain antibody or dAb, but is preferably a Nanobody® (e.g., a VHH, a humanized VHH or a camelized VH such as a camelized human VH).

Such an ISV is preferably an ISV of the invention (as defined herein), and according to a specific aspect of the invention is a 10E9-type sequence, a 281E10-type sequence, a 10E12- type sequence, a lOAlO-type sequence, a lOGlO-type sequence, a 14A2-type sequence, a 15Al-type sequence, a 15H3-type sequence or a 283B6-type sequence; and preferably a

10E9-like sequence, a 281E10-like sequence, a 1.0E12-like sequence, a 10A10-like sequence, a lOGlO-like sequence, a 14A2-like sequence, a 15Al-like sequence, a 15H3-like sequence or a 283B6-like sequence

In another aspect, the invention further relates to a compound, construct, protein or polypeptide that comprises or essentially consists of an ISV (as defined herein) that has an ICso in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM). Again, such an ISV may be as further described herein, and may for example be a domain antibody, single domain antibody or dAb, but is preferably a Nanobody® (e.g., a VHH, a humanized VHH or a camelized VH such as a camelized human VH); and again such an ISV is preferably an ISV of the invention (as defined herein), and according to a specific aspect of the invention may in particular be a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a 10A10-type sequence, a lOGlO-type sequence, a 14A2-type sequence, a 15Al-type sequence, a 1.5H3- type sequence or a 283B6-type sequence; and is preferably a 10E9-like sequence, a 281E10- like sequence, a 10E12-like sequence, a 10A10-like sequence, a lOGlO-like sequence, a

14A2-like sequence, a 15Al -like sequence, a 15H3-like sequence or a 283B6-like sequence. .Another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half-life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers (for example, in the constructs of SEQ ID NO's: 279 to 359, the ISV's of the invention are linked to each other via a 20GS linker and linked to the half-life extending Nanobody© Alb-8 via a 35GS linker. Also, in the construct of SEQ ID NO:370, the ISV's of the invention have been provided with a short C-terminal GGGC sequence to which for example a PEG-group can be linked). In the above constructs, the two or more (and preferably two) ISV's of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain: an illustrative and non-limiting example of such a construct is the construct of SEQ ID NO:369).

The invention further generally relates to a compound, construct, protein or

polypeptide, comprising or essentially consisting of a first ISV that is directed against CXCR- 4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), in which the first and second ISV may be the same or different, and in which at least one of the ISV's present therein have an IC50 in the Jurkat assay of Example 1 , C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM) when measured in the corresponding monovalent format. Preferably, when the compound, construct, protein or polypeptide comprises two (or more) ISV's directed against CXCR-4, both (or all) ISV's present therein have an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM) when measured in the corresponding monovalent format. Again, both the first and second ISV may be as further described, herein, and may for example (each or both) be a domain antibody, single domain antibody or dAb, but is preferably a Nanobody® {e.g., a VHH, a humanized VHH or a camelized VH such as a camelized human VH), and are preferably both ISV's of the invention (as defined herein), and according to a specific aspect of the invention may in particular be a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a 10A10-type sequence, a l OGlO-type sequence, a 14A2-type sequence, a 15Al-type sequence, a 15H3-type sequence or a 283B6-type sequence; and is preferably a 10E9-like sequence, a 281E10-like sequence, a 10E12-like sequence, a 10A10-like sequence, a lOGlO-like sequence, a 14A2-like sequence, a 15Al -like sequence, a 15H3-like sequence or a 283B6- like sequence. Another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half-life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers. In the above constructs, the two or more (and preferably two) ISV s of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain). Also, the ISV's may be the same (so as to provide a bi- or multivalent protein or polypeptide of the invention) or may be different, and when they are different they may be directed against the same epitope, domain or (extracellular) loops on CXCR-4 or against different epitopes, domains or (extracellular) loops on CXCR-4.

The invention further generally relates to a compound, construct, protein or

polypeptide, comprising or essentially consisting of a first ISV that is directed against CXCR- 4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), in which the first and second ISV may be the same or different, and in which the first and second ISV may each independently be a 10E9-type sequence (as defined herein); a 281E10-type sequence (as defined herein); a 10E12-type sequence (as defined herein): a 10A10-type sequence (as defined herein); a lOGlO-type sequence (as defined herein); a 14A2-type sequence (as defined herein); a 15A1- type sequence (as defined herein); a 15H3-type sequence (as defined herein); and or a 283B6- type sequence (as defined herein). Again, another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half- life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers. In the above constructs, the two or more (and preferably two) ISV's of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain).

In particular, according to a specific, but non-limiting aspect, the invention generally relates to a compound, construct, protein or polypeptide, comprising or essentially consisting of a first ISV that is directed against CXCR-4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), wherein the first and second ISV may be the same or different, and in which, the first and second ISV may each independently be a 10E9-like sequence (as defined herein); a 281E10- like sequence (as defined herein); a 10E12-like sequence (as defined herein); a 10A10-like sequence (as defined herein); a lOGlO-like sequence (as defined herein); a 14A2-like sequence (as defined herein); a 15Al-like sequence (as defined herein); a 15H3-like sequence (as defined herein); and/or a 283B6-like sequence (as defined herein). Again, another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half-life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers, in the above constructs, the two or more (and preferably two) ISV's of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain).

More in particular, according to a more specific, but non-limiting aspect, the invention generally relates to a compound, construct, protein or polypeptide, comprising or essentially consisting of a first ISV that is directed against CXCR-4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), wherein the first and second ISV may be the same or different, and in which the first and second ISV may each independently be 10E9 or (preferably) a humanized (and/or sequence optimized) variant of 10E9; 281E10 or (preferably) a humanized (and/or sequence optimized) variant of 281E10; 10E12 or (preferably) a humanized (and/or sequence optimized) variant of 10E12; 1 OA 10 or (preferably) a humanized (and/or sequence optimized) variant of 1 OA 10; 10G10 or (preferably) a humanized (and/or sequence optimized) variant of ί 0G10; 14A2 or (preferably) a humanized (and/or sequence optimized) variant of 14A2; 15A1 or (preferably) a humanized (and/or sequence optimized) variant of 15A1 ; 15H3 or (preferably) a humanized (and/or sequence optimized) variant of 15H3;

and/or 283B6 or (preferably) a humanized (and/or sequence optimized) variant of 283B6. Again, another aspect of the invention relates to such a compound, construct, protein or polypeptide which has been provided with an increased half-life in vivo (as further described herein), for example by linking it to a serum-albumin binding peptide or binding domain (as further described herein), again optionally via one or more suitable linkers. In the above constructs, the two or more (and preferably two) ISV's of the invention may be suitably linked to each other, either directly and/or via one or more suitable linkers, and/or via any half-life extending moiety (such as a serum-albumin binding peptide or binding domain). All the compounds, construct, protein or polypeptide are preferably such that they have IC₅o in the Jurkat assay of Example 1, C-4; of better than 20.0 nM, preferably better than ΙΟηΜ, more preferably better than 7.0 nM, even more preferably better than 5.0 nM, such as better than 3.0 nM. In this respect, it should be noted that, although it is generally expected that bivalent or biparatopic constructs of the invention will generally bind with higher avidity to CXCR-4 than the corresponding monovalent ISV(s) and thus will generally be expected to perform better in the Jurkat assay than the corresponding monovalent ISV(s) (and also show better potency in other suitable assays), half-life extended constructs may, depending on the half-life extension technique used, have a lower potency in the Jurkat assay (and/or in other assays) than the corresponding construct without the half-life extension and even compared to some or all of the building blocks present in the construct. For example, when the construct is pegylated, the bulky PEG groups may interfere with target binding and thus reduce affinity/avidity and/or potency. However, it will be clear to the skilled person that even in these cases, the use of the ISV's of the invention will be of (even) great(er) advantage, as it allows the constructs to be made starting from building blocks with higher potency which may compensate to some extent for any loss of potency due to half-life extension.

The invention further relates to the use of a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a lOAlO-type sequence, a lOGlO-type sequence, a 14A2- type sequence, a 15Al-type sequence, a 15H3-type sequence or a 283B6-type sequence as a binding domain or binding unit in a compound, construct, protein or polypeptide that is directed against (as defined herein) CXCR-4, and in particular (at least) against human CXCR-4.

The invention also further relates to the use of a 10E9-like sequence, a 281E10-like sequence, a 10E12-like sequence, a 10A10-like sequence, a 10G10-like sequence, a 14A2- like sequence, a 15Al-like sequence, a 15H3-like sequence or a 283B6-like sequence as a binding domain or binding unit in a compound, construct, protein or polypeptide that is directed against (as defined herein) CXCR-4, and in particular (at least) against human CXCR-4.

The invention also further relates to the use of 10E9 or (preferably) a humanized (and/or sequence optimized) variant of 10E9; 281E10 or (preferably) a humanized (and/or sequence optimized) variant of 281E10; 10E12 or (preferably) a humanized (and/or sequence optimized) variant of 10E12; 1 OA 10 or (preferably) a humanized (and/or sequence optimized) variant of 10A10; 10G10 or (preferably) a humanized (and/or sequence optimized) variant of 10G10; 14A2 or (preferably) a humanized (and/or sequence optimized) variant of 14A2; 15A1 or (preferably) a humanized (and/or sequence optimized) variant of 15A1 ; 15H3 or (preferably) a humanized (and/or sequence optimized) variant of 15H3;

and/or 283B6 or (preferably) a humanized (and/or sequence optimized) variant of 283B6 as a binding domain or binding unit in a compound, construct, protein or polypeptide that is directed against (as defined herein) CXCR-4, and in particular (at least) against human CXCR-4.

Again, when 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a !OAlO-type sequence, a lOGlO-type sequence, a 14A2-type sequence, a 15Al -type sequence, a 15H3-type sequence or a 283B6-type sequence, or when 10E9-like sequence, a 281E10-iike sequence, a 10E12-like sequence, a 10A10-like sequence, a lOGlO-like sequence, a 14A2- like sequence, a 15A1 -like sequence, a 15H3-like sequence or a 283B6-like sequence is present in and/or used in a compound, construct, protein or polypeptide according to any of the preceding aspects, the preferences that have been described above for each of these ISV's of the invention also apply when such an ISV is present in and/or used in such a compound, construct, protein or polypeptide.

Again, in the above aspects, the (use of and/or constructs or polypeptides comprising the) following ISV's are preferred: a 10E9-type sequence, 10E12-type sequence, lOGlO-type sequence, 15H3-type sequence, 283B6-type sequence, with the following ISV's of the invention being more preferred: a 10E9-Hke sequence, 10E12-like sequence, 10G10-like sequence, 15H3-like sequence or 283B6-like sequence; and the following ISV's of the invention being particularly preferred: 3.0E9 or (preferably) a humanized (and/or sequence optimized) variant of 10E9; 10E12 or (preferably) a humanized (and/or sequence optimized) variant of 10E12; 10G10 or (preferably) a humanized (and/or sequence optimized) variant of 10G10; 15H3 or (preferably) a humanized (and/or sequence optimized) variant of 15H3; and/or 283B6 or (preferably) a humanized (and/or sequence optimized) variant of 283B6.

It will be clear to the skilled person from the disclosure herein that the amino acid sequences, compounds, constructs and polypeptides of the invention are directed against human CXCR4. Thus, they can be used for the same purposes, uses and applications as described in WO 09/138519 and the non-prepublished US application 61 /358,495, for example to inhibit signaling that is mediated by human CXCR4 and/or its ligand(s); and/or i the prevention or treatment of diseases associated with an increased signaling of CXCR4 and/or with an undesired proliferation of cells that express or over-express CXCR-4, such as the various diseases in the group of cancer such as hematopoietic cancers like CLL, AML, ALL, MM, Non-Hodgkin lymphoma, (other) solid tumors such as breast cancer, lung cancer (such as SCLC or non-SCLC NSCLC lung cancer), liver cancer (such as HCC), brain tumors (such as glioblastoma), ovarian cancer, stromal chemoresistance of tumors, prostate cancer, leukemia, skin cancer (such as melanoma) and other cancers, chemosensitization in for example AML, CLL or SLL disrupting adhesive stromal interactions that confer tumor cell survival and drug resistance, mobilizing tumor cells form tissue sites and making them better accessible to conventional therapy, inhibiting of migration and dissemination of tumor cells (metastasis), inhibiting or paracrine growth and survival signals, inhibiting pro-angiogenesis effects of SDF-1 , inflammation and inflammatory disorders such as bowel diseases (colitis, Crohn's disease, IBD), infectious diseases, psioriasis, autoimmune diseases (such as MS), sarcoidosis, transplant rejection, cystic fibrosis, asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, viral infection, HIV, West Nile Virus encephalitis, and/or common variable immunodeficiency. Furthermore, the amino acid sequences of the invention can be used for stem cell mobilization in various patients in need of stem cells after X-ray radiation such as e.g., cancer patients after radiation treatment to replenish the stem cell pool after radiation in cancer patients, or in patients in need of more stem cells, e.g. , in patients with ischemic diseases such as myocardial infarction (MI), stroke and/or diabetes (i.e., patients in need of tissue repair) wherein more stem cell would be re-transfused (after mobilization, screening, selection for lineage in need (e.g., cardiac, vascular lineages) and ex- vivo expansion of patient's own stem cells).

For other possible indications for therapeutics against CXCR-4, reference is for example made to EP 1 061 944, EP 1 072 273 and WO99/50461.

In particular, the amino acid sequences of the invention are very potent (i.e., EC₅₀ values as measured, e.g., in the experimental part in the pM range) antagonists of human CXCR4 and some may be inverse agonists in certain continuously active human CXCR4 mutants (see e.g., Example 5 of WO 09/138519).

Such multispecific constructs are preferably proteins and polypeptides (i.e., encoded by a nucleotide sequence and/or capable of being expressed by a host or host cell), as also generally described in WO 09/138519.

The efficacy of the amino acid sequences and polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved.

The in vivo activity of the ISV's and constructs of the invention can be tested in any suitable animal model of the cancer to be treated, for example in a chemically induced model, a xenograft model and/or a transgenic model, and suitable models will be clear to the skilled person, depending on the cancer to be treated. Reference is for example made to Burger and ipps; Dorsam and Gutkind; Kryczek et al; BalkwilL and to Mueller et al., Nervi et al, Redjal et al., Rubin et al., and Zeng et al. (all supra) and the further references therein.

The ability of the ISV's or constructs of the invention to mobilize stem cells can for example be determined according to the methods described in Example 10 of US 61/293, 279.

Depending on the specific ISV or construct, the ISV's and constructs of the invention may be agonists or antagonists in respect of the (desired or undesired) activity of CXCR-4 (i.e., the activity to be obtained/increased or decreased/inhibited). The invention is also not limited to any particular hypothesis, mechanism or explanation as to how the ISV's or constructs of the invention achieve their (desired) biological effect, and may for example do so by competing with the ligand for binding to the receptor, preventing or inhibiting ligand binding, by steric hindrance, by preventing or inhibiting conformational changes that need to occur in order for the ligand to bind to the receptor and/or for receptor-mediated signaling to occur upon binding of the ligand, by preventing, decreasing or conversely increasing receptor internalization or recycling, by promoting or decreasing receptor dimerisation (where required for signaling) or by allosteric interaction; or any combination of one or more of the foregoing, depending on the specific ISV and/or construct, the epitope(s), loop(s) or domains, or confonnation(s) to which it binds, and the interaction of the ISV or construct with the receptor.

Also, according to the invention, amino acid sequences and polypeptides that are directed against CXCR-4 from a first species of warm-blooded animal may or may not show cross-reactivity with CXCR-4 from one or more other species of warm-blooded animal. For example, amino acid sequences and polypeptides directed against human CXCR-4 may or may not show cross reactivity with CXCR-4 from one or more other species of primates (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys {Macaca fascicuiaris) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus)) and/or with CXCR-4 from one or more species of animals that are often used in animal models for diseases (for example mouse, rat, rabbit, pig or dog), and in particular in animal models for diseases and disorders associated with CXCR-4 (such as the species and animal models mentioned herein). In this respect, it will be clear to the skilled person that such cross-reactivity, when present, may have advantages from a drug

development point of view, since it allows the amino acid sequences and polypeptides against human CXCR-4 to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the invention that are cross-reactive with CXCR-4 from multiple species of mammal will usually be advantageous for use in veterinary applications, since it will allow the same amino acid sequence or polypeptide to be used across multiple species. Thus, it is also encompassed within the scope of the invention that amino acid sequences and polypeptides directed against CXCR-4 from one species of animal (such as amino acid sequences and polypeptides against human CXCR- 4) can be used in the treatment of another species of animal, as long as the use of the amino acid sequences and/or polypeptides provide the desired effects in the species to be treated.

The ISV's and constructs of the invention are preferably (at least) cross-reactive with CXCR-4 from cynomolgus monkeys {Macaca fascicularis).

The present invention is in its broadest sense also not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of CXCR-4 against which the amino acid sequences and polypeptides of the invention are directed (and as shown herein, the invention advantageously provides a number of ISV's binding to different epitopes, which may be an advantage whe the ISV's are to be used as building blocks for making - for example - biparatopic constructs). For example, the amino acid sequences and polypeptides may or may not be directed against an "interaction site" (as defined herein).However, it is generally assumed and preferred that the amino acid sequences and polypeptides of the invention are preferably directed against an interaction site (as defined herein).

As further described herein, a polypeptide of the invention may contain two or more amino acid sequences of the invention that are directed against CXCR-4. Generally, such polypeptides will bind to CXCR-4 with increased avidity compared to a single amino acid sequence of the invention. Such a polypeptide may for example comprise two amino acid sequences of the invention that are directed against the same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of CXCR-4 (which may or may not be an interaction site); or comprise at least one "first" amino acid sequence of the invention that is directed against a first same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of CXCR-4 (which may or may not be an interaction site); and at least one "second" amino acid sequence of the invention that is directed against a second antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) different from the first (and which again may or may not be an interaction site). Preferably, in such "biparatopic" polypeptides of the invention, at least one amino acid sequence of the invention is directed against an interaction site (as defined herein), although the invention in its broadest sense is not limited thereto.

Also, when the target is part of a binding pair (for example, a receptor-ligand binding pair), the amino acid sequences and polypeptides may be such that they compete with the cognate binding partner (e.g., the ligand, receptor or other binding partner, as applicable) for binding to the target. It is also within the scope of the invention that, where applicable, an amino acid sequence of the invention can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or confimiations of CXCR-4. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of CXCR-4 to which the amino acid sequences and/or polypeptides of the invention bind may be essentially the same (for example, if CXCR-4 contains repeated structural motifs or occurs in a multimeric form) or may be different (and in the latter case, the amino acid sequences and polypeptides of the invention may bind to such different antigenic determinants, epitopes, parts, domains, subunits of CXCR-4 with an affinity and/or specificity which may be the same or different). Also, for example, when CXCR-4 exists in an activated conformation and in an inactive conformation, the amino acid sequences and polypeptides of the invention may bind to either one of these confirmation, or may bind to both these confirmations (i.e., with an affinity and/or specificity which may be the same or different). Also, for example, the amino acid sequences and polypeptides of the invention may bind to a conformation of CXCR-4 in which it is bound to a pertinent ligand, may bind to a conformation of CXCR-4 in which it is not bound to a pertinent ligand, or may bind to both such conformations (again with an affinity and/or specificity which may be the same or different).

It is also expected that the amino acid sequences and polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of CXCR-4; or at least to those analogs, variants, mutants, alleles, parts and fragments of CXCR-4 that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the amino acid sequences and polypeptides of the invention bind in CXCR-4 (e.g., in wild-type CXCR-4). Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and/or specificity that are the same as, or that are different from (i.e., higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to (wild- type) CXCR-4. It is also included within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to some analogs, variants, mutants, alleles, parts and fragments of CXCR-4, but not to others.

Also, as will be clear to the skilled person, proteins or polypeptides that contain two or more amino acid sequences directed against CXCR-4 may bind with higher avidity to CXCR-4 than the corresponding monomeric amino acid sequence(s). For example, and without limitation, proteins or polypeptides that contain two or more amino acid sequences directed against different epitopes of CXCR-4 may (and usually will) bind with higher avidity than each of the different monomers, and proteins or polypeptides that contain two or more amino acid sequences directed against CXCR-4 may (and usually will) bind also with higher avidity to a multimer of CXCR-4.

As mentioned, an ISV as described and used herein may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence) or a suitable fragment thereof. When the amino acid sequence of the invention is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a VH sequence that is derived from a human antibody) or be a so-called VHH-sequence (as defined herein) that is derived from a so-called "heavy chain antibody" (as defined herein).

In particular, any ISV described herein may be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® (as defined herein, and including but not limited to a VHH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term "dAb's", reference is for example made to Ward et al. (Nature 1989 Oct 12; 341 (6242): 544- 6), to Holt et al., Trends Biotechnol., 2003, 21(1 1 ):484-490; as well as to for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629).

However, it should be noted that the invention is not limited as to the origin of the amino acid sequence of the invention (or of the nucleotide sequence of the invention used to express it), nor as to the way that the amino acid sequence or nucleotide sequence of the invention is (or has been) generated or obtained. Thus, the amino acid sequences of the invention may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences. In a specific but non-limiting aspect of the invention, the amino acid sequence is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VRH sequences or Nanobodies©), "camelized" (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences. PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person: or any suitable combination of any of the foregoing.

Reference is for example made to the standard handbooks, as well as to the further description and prior art mentioned herein,

Similarly, the nucleotide sequences of the invention may be naturally occurring nucleotide sequences or synthetic or semi- synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequences that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se. As mentioned, the ISV's used herein are preferably Nanobodies® and may as such for example be naturally occurring VHH's, humanized VHH's or camelized VH's (such as camelized human VH's). Again, suitable Nanobodies® may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring VHH sequences (i.e., from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to "humanized" (as defined herein) Nanobodies®, "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as Nanobodies® that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PGR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. Also, when a Nanobody® comprises a V_HH sequence, said Nanobody® may be suitably humanized, as further described herein, so as to provide one or more further

(partially or fully) humanized Nanobodies® of the invention . Similarly, when a Nanobody® comprises a synthetic or semi -synthetic sequence (such as a partially humanized sequence), said Nanobody® may optionally be further suitably humanized, again as described herein, again so as to provide one or more further (partially or fully) humanized Nanobodies® of the invention.

In particular, humanized Nanobodies® may be amino acid sequences that are as generally defined for Nanobodies® in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanizing substitution (as defined herein). Some preferred, but non-limiting humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human V_H sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized V_HH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a

Nanobody® may be partially humanized or fully humanized.

DETAILED DESCRIPTION OF THE INVENTION

In another aspect, the invention relates to a compound or construct, and in particular a protein or polypeptide (also referred to herein as a "compound of (he invention" or

"polypeptide of the invention", respectively) that comprises or essentially consists of one or more amino acid sequences of the invention (or suitable fragments thereof), and optionally further comprises one or more other groups, residues, moieties or binding units. As will become clear to the skilled person from the further disclosure herein, such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the amino acid sequence of the invention (and/or to the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence of the invention.

For example, such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain. antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb's", amino acid sequences that are suitable for use as a dAb, or Nanobodies®.

Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more amino acid sequences of the invention so as to provide a

"derivative" of an amino acid sequence or polypeptide of the invention, as further described herein.

Also within the scope of the present invention are compounds or constructs, that comprises or essentially consists of one or more derivatives as described herein, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more amino acid sequences of the invention and the one or more groups, residues, moieties or binding units may be linked directly to each other and/or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be amino acid sequences, so that the resulting compound or construct is a fusion (protein) or fusion (polypeptide).

As will be clear from the further description above and herein, this means that the amino acid sequences of the invention can be used as "building blocks" to form polypeptides of the invention, i.e., by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or constructs as described herein (such as, without limitations, the biparatopic. bi/multivalent and bi/multispecific polypeptides of the invention described herein) which combine within one molecule one or more desired properties or biological functions.

The compounds or polypeptides of the invention can generally be prepared by a method which comprises at least one step of suitably linking the one or more amino acid sequences of the invention to the one or more further groups, residues, moieties or binding units, optionally via the one or more suitable linkers, so as to provide the compound or polypeptide of the invention. Polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.

The process of designing/selecting and/or preparing a compound or polypeptide of the invention, starting from an amino acid sequence of the invention, is also referred to herein as "formatting'^'' said amino acid sequence of the invention; and an amino acid of the invention that is made part of a compound or polypeptide of the invention is said to be "formatted" or to be "in the format of said compound or polypeptide of the invention. Examples of ways in which, an amino acid sequence of the invention can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted amino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention or a polypeptide of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention that comprise at least one amino acid sequence of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half- life of the amino acid sequence of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, "dAb's", amino acid sequences that are suitable for use as a dAb, or Nanobodies® that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrine; reference is made to the further description and. references mentioned herein); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, witliout limitation, the proteins and peptides described in WO 2008/068280, WO 2009/127691 or the non-prepublished US application 61/301,819 by applicant), or a serum-albumin binding ISV.

For example, in one preferred but non-limiting aspect, the half-life extending moiety is an albumin-binding TSianobody® (see for example WO 2004/041863), and in particular Alb-1 or a humanized version of Alb- 1 such as Alb-8 ( for which reference is for example made to WO 06/122787).

Generally, the compounds or polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention er se. For example, the compounds or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention have a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half- life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 1 1 days (such as about 1 1 to 16 days), more preferably at least about 12 days (such as about 12 to 1.8 days or more), or more than 14 days (such as about 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodes an amino acid sequence of the invention or a polypeptide of the invention (or a suitable fragment thereof). Such a nucleic acid will also be referred to herein as a "nucleic acid of the invention" and may for example be in the form of a genetic construct, as further described herein.

In another aspect, the invention relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) an amino acid sequence of the invention and/or a polypeptide of the invention; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein. The invention further relates to a product or composition containing or comprising at least one amino acid sequen ce of the invention, at least one polypeptide of the invention (or a suitable fragment thereof) and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e., depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

The invention also relates to the use of an amino acid sequence, Nanobody© or polypeptide of the invention, or of a composition comprising the sam e, in (methods or compositions for) modulating CXCR-4, either in vitro (e.g., in an in vitro or cellular assay) or in vivo (e.g., in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a CXCR-4 related disease or disorder (such as one of the disease mentioned herein, for example a cancer that can be treated using the ISV's or constructs of the invention) or in need of stem cell mobilisation).

The invention also relates to methods for modulating CXCR-4, either in vitro (e.g., in an in vitro or cellular assay) or in vivo (e.g., in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a CXCR-4 related disease or disorder (such as one of the disease mentioned herein, for example a cancer that can be treated using the ISV's or constructs of the invention) or in need of stem cell mobilisation), which method comprises at least the step of contacting CXCR-4 with at least one amino acid sequence, Nanobody® or polypeptide of the invention, or with a composition comprising the same, in a manner and in an amount suitable to modulate CXCR-4, with at least one amino acid sequence, Nanobody® or polypeptide of the invention.

The invention also relates to the use of an one amino acid sequence, Nanobody® or polypeptide of the invention in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating CXCR-4, either in vitro (e.g.. in an in vitro or cellular assay) or in vivo (e.g., in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a CXCR-4 related disease or disorder (such as one of the disease mentioned herein, for example a cancer that can be treated using the ISV's or constructs of the invention) or in need of stem cell mobilisation).

In the context of the present invention, "modulating" or "to modulate" generally means either reducing or inhibiting the activity of, or alternatively increasing the activity of, CXCR-4, as measured using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein). In particular, "modulating" or "to modulate" may mean either reducing or inhibiting the activity of, or alternatively increasing the activity of CXCR-4, as measured in vitro using a suitable in vitro, cellular or in vivo assay (such as those mentioned herein), by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of CXCR-4 in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.

As will be clear to the skilled person, "modulating" may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of CXCR-4 for one or more of its targets, ligands or substrates; and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of CXCR-4 for one or more conditions in the medium or surroundings in which CXCR-4 is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention. As will be clear to the skilled person, this may again be determined, in any suitable manner and/or using any suitable assay known per se, such as the assays described herein or in the prior art cited herein.

"Modulating" may also mean effecting a change {i.e., an activity as an agonist or as an antagonist, respectively) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which CXCR-4 (or in which its substrate(s), Iigand(s) or pathway(s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved. Again, as will be clear to the skilled person, such an action as an agonist or an antagonist may be

determined in any suitable manner and/or using any suitable (in vitro and usually cellular or in assay) assay known per se, such as the assays described herein or in the prior art cited herein. In particular, an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the amino acid sequence, Nanobody® or polypeptide of the invention.

Modulating may for example involve reducing or inhibiting the binding of CXCR-4 to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to CXCR-4. Modulating may also involve activating CXCR-4 or the mechanism or pathway in which it is involved. Modulating may be reversible or irreversible, but for pharmaceutical and pharmacological purposes will usually be in a reversible manner.

The invention further relates to appli cations and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host ceils, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with CXCR-4. Some preferred but non-limiting applications and uses will become clear from the further description herein. The invention also relates to the amino acid, sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy.

In particular, the invention also relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of a disease or disorder that can be prevented or treated by administering, to a subject in need thereof, of (a pharmaceutically effective amount of) an amino acid sequence, compound, construct or polypeptide as described herein.

More in particular, the invention relates to the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein for use in therapy of various forms of cancer (as listed above) and/or in stem cell mobilisation.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description herein, in which the invention will be described and discussed in more detail with reference to the Nanobodies® of the invention and polypeptides of the invention comprising the same, which form some of the preferred aspects of the invention. As will become clear from the further description herein, Nanobodies® generally offer certain advantages (outlined herein) compared to "dAb's" or similar (single) domain antibodies or immunoglobulin sequences, which advantages are also provided by the

Nanobodies® of the invention. However, it will be clear to the skilled person that the more general aspects of the teaching below can also be applied (either directly or analogously) to other amino acid sequences of the invention.

As generally described herein for the amino acid sequences of the invention, the Nanobodies® of the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more Nanobodies® of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or

polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e., against one or more other targets than CXCR-4), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. In particular, such a protein or polypeptide may comprise or essentially consist of one or more Nanobodies® of the invention and optionally one or more (other) Nanobodies® (i.e., directed against other targets than CXCR-4), all optionally linked via one or more suitable linkers, so as to provide a monovalent, multivalent or multispecific

Nanobody® construct, respectively, as further described herein. Such proteins or

polypeptides may also be in essentially isolated form (as defined herein).

In a Nanobody® of the invention, the binding site for binding against CXCR-4 is preferably formed by the CDR sequences. Optionally, a Nanobody® of the invention may also, and in addition to the at least one binding site for binding against CXCR-4, contain one or more further binding sites for binding against other antigens, proteins or targets. For methods and positions for introducing such second binding sites, reference is for example made to Keck and Huston, Biophysical Journal, 71 , October 1996, 2002-2011; EP 0 640 130; and WO 06/07260.

As generally described herein for the amino acid sequences of the invention, when a

Nanobody® of the invention (or a polypeptide of the invention comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably directed against human CXCR-4; whereas for veterinary purposes, it is preferably directed against CXCR-4 from the. species to be treated. Also, as with the amino acid sequences of the invention, a Nanobody® of the invention may or may not be cross-reactive (i.e., directed against CXCR-4 from, two or more species of mammal, such as against human CXCR-4 and CXCR-4 from at least one of the species of mammal mentioned herein).

As already described herein, the amino acid sequence and structure of a Nanobody® can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's" (or sometimes also referred to as "FW's"), which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary detenriining regions or "CDR's", which are referred to in the art as "Complementarity Determining Region For "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively. Some preferred framework sequences and CDR's (and combinations thereof) that are present in the

Nanobodies® of the invention are as described herein. Other suitable CDR sequences can be obtained by the methods described herein.

According to a non-limiting but preferred aspect of the invention, (the CDR sequences present in) the Nanobodies® of the invention are such that:

- the Nanobodies® can bind to CXCR-4 with a dissociation constant (K_D) of 10^"5 to 10^{" 12} moles/liter or less, and preferably 30^" 7 to 10^* 1 ^ " moles/hter or less and more preferably 10^"8 to 10^~12 moles/liter (i.e., with an association constant ( A) of 10^ to 10¹² liter/ moles or more, and preferably 10 to 10 liter/moles or more and more preferably 10 to 10° liter/moles);

and/or such that:

the Nanobodies® can bind to CXCR-4 with a k_on-rate of between 10² IvfV¹ to about 10⁷ MV, preferably between 10³ M ¹ and 10⁷ M^~ V¹, more preferably between 10 M^' V and 10⁷ IvlV, such as between 10⁵ M^'V¹ and 10⁷ VP^' s^"1 :

and/or such that they:

- the Nanobodies® can bind to CXCR-4 with a k_off rate between 1 s^"1 (t_1/2 ^:=0.69 s) and 10^' ⁶ s^"1 (providing a near irreversible complex with a t]_/2 of multiple days), preferably between 1.0^'2 s^"1 and 10^"6 s^"1, more preferably between 10^"3 s^"1 and 10^"6 s^"1, such as between 10^"4 s^"1 and 10^"6 s^"1. Preferably, (the CDR sequences present in) the Nanobodies® of the invention are such that: a monovalent Nanobody® of the invention (or a polypeptide that contains only one Nanobody® of the invention) is preferably such that it will bind to CXCR-4 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

The affinity of the Nanobody® of the invention against CXCR-4 can be determined in a manner known per se for example using the general techniques for measuring KD. KA, _f or ko„ mentioned herein, as well as some of the specific assays described herein.

Some preferred IC50 values for binding of the Nanobodies® of the invention (and of polypeptides comprising the same) to CXCR-4 will become clear from the further description and examples herein.

In another one aspect of the invention, a polypeptide of the invention, comprises one or more (such as two or preferably one) ISV's of the invention linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that allow the resulting polypeptide of the invention to cross the blood brain barrier. In particular, said one or more amino acid sequences that allow the resulting polypeptides of the invention to cross the blood brain barrier may be one or more (such as two and preferably one) Nanobodies®, such as the Nanobodies® described in WO

02/057445, of which FC44 (SEQ ID NO: 1 89 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In particular, polypeptides comprising one or more ISV's of the invention are preferably such that they:

bind to CXCR-4 with a dissociation constant (KD) of 10^~5 to 10^~12 moles/liter or less, and preferably 10^"7 to 10^~!2 moles/liter or less and more preferably 10^"s to 10^"12 moles/liter (i.e., with an association constant (K_A) of lO³ to 10¹² liter/ moles or more, and preferably 10 to 10 " liter/moles or more and more preferably 10 to 10 liter/moles);

and/or such that they:

bind to CXCR-4 with a k^-rate of between 10² M^'V¹ to about 10⁷ M^'V¹, preferably between 10³ M^'V and 10⁷ NT's^"1, more preferably between 10⁴ M^'V¹ and 10⁷ M^'V¹, such as between 10⁵ M^'V¹ and 10⁷ M^"V^!;

and/or such that they: bind to CXCR-4 with a ka rate between 1 s^"! (t]_/2=0.69 s) and 10^"6 s^"1 (providing a near irreversible complex with a t_\ of multiple days), preferably between 10^" s^" and 10^" s^" more preferably between 10^"3 s^"1 and 10^"6 s^"s, such as between 10^"4 s^"1 and 10^"6 s^"1. Preferably, a polypeptide that contains only one amino acid sequence of the invention is preferably such that it will bind to CXCR-4 with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. In this respect, it will be clear to the skilled person that a polypeptide that contains two or more

Nanobodies® of the invention may bind to CXCR-4 with an increased avidity, compared to a polypeptide that contains only one amino acid sequence of the invention.

Another aspect of this invention relates to a nucleic acid that encodes an amino acid sequence of the invention (such as a Nanobody® of the invention) or a polypeptide of the invention comprising the same. Again, as generally described herein for the nucleic acids of the invention, such a nucleic acid may be in the form of a genetic construct, as defined herein.

In another aspect, the invention relates to host or host cell that expresses or that is capable of expressing an amino acid sequence (such as a Nanobody® of the invention) and/or a polypeptide of the invention compri sing the same; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

Another aspect of the invention relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e., depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

The invention further relates to methods for preparing or generating the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and.

compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

The invention further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with CXCR-4. Some preferred but non-limiting applications and uses will become clear from the further description herein.

As mentioned herein, an ISV of the invention is preferably a Nanobody®, e.g., a V_H¾ a humanized VHH o a camelized V_H (such as a cameiized human VH). Accordingly, herein, the invention is described with particular reference to such Nanobodies® (which are also referred to herein as "Nanobodies® of the invention". It will however be clear to the skilled person that the teaching herein can also be applied to other ISV's of the invention.

According to one preferred, but non-limiting aspect of the invention, a Nanobody® in its broadest sense can be generally defined as a polypeptide comprising:

a) an amino acid sequence that is comprised of four framework regions/sequences

interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 108 according to the Kabat numbering is Q;

and/or:

b) an amino acid sequence that is comprised of four framework regions/sequences

interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E;

and/or:

c) an amino acid sequence that is comprised of four framework regions/sequences

interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody® of the invention may have the structure

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4

in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q;

and/or in which: b) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid or a cysteine and the amino acid residue at position 44 according to the Kabat numbering is preferably E;

and/or in which:

c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S. and is in. particular chosen from the group consisting of R and S;

and in which:

d) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In particular, a Nanobody® in its broadest sense can be generally defined as a polypeptide comprising:

a) an amino acid sequence that is comprised of four framework regions/sequences

and/or:

b) an amino acid sequence that is comprised of four framework regions/sequences

interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R;

and/or:

c) an amino acid sequence that is comprised of four framework regions/sequences

interrupted by three complementarity determining regions/sequences, in which the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting ofR and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody® of the invention may have the structure

FR1 - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4. respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q;

and/or in which:

b) the amino acid residue at position 44 according to the Kabat numbering is E and in which the amino acid residue at position 45 according to the Kabat numbering is an R; and/or in which:

c) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S, and is in particular chosen from the group consisting of R and S;

and in which:

In particular, a Nanobody® against CXCR-4 according to the invention may have the structure:

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which a) the amino acid residue at position 108 according to the Kabat numbering is Q;

and/or in which:

and in which:

d) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein. In particular, according to one preferred, but non-limiting aspect of the invention, a Nanobody® can generally be defined as a polypeptide comprising an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which;

a- 1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, G, Q, R, S, L; and is preferably chosen from the group consisting of G, E or Q; and

a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L. R or C; and is preferably chosen from the group consisting of L or R; and

a-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R or S; and is preferably W or R, and is most preferably W;

a-4) the amino acid residue at position 108 according to the Kabat numbering is Q;

or in which:

b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q; and

b-2) the amino acid residue at position 45 according to the Kabat numbering is R; and b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W;

b-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; and is preferably Q;

or in which:

c-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E. D, Q, R, S and L; and is preferably chosen from the group consisting of G, E and Q; and

c-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R; and

c-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S; and c-4) the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q;

and in which

Thus, in another preferred, but non-limiting aspect, a Nanobody® of the invention may have the structure FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: a-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D. G, Q, R, S, L; and is preferably chosen from the group consisting of G. E or Q;

and in which:

a-2) the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R or C; and is preferably chosen from the group consisting of L or R;

and in which:

and in whi ch

and in which:

d) CD l , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody® of the invention may have the structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4. respectively, and in which CDR1 to CDR3 refer to the complementaiity determining regions 1 to 3, respectively, and in which: b-1) the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of E and Q;

and in which:

b-2) the amino acid residue at position 45 according to the Kabat numbering is R;

and in which:

b-3) the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of W, R and S; and is preferably W;

and in which:

d) CDR1 , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody® of the invention may have the structure

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: 1. the amino acid residue at position 44 according to the Kabat numbering is chosen from the group consisting of A, G, E, D, Q, R, S and L; and is preferably chosen from the group consisting of G. E and Q;

and in which:

2. the amino acid residue at position 45 according to the Kabat numbering is chosen from the group consisting of L, R and C; and is preferably chosen from the group consisting of L and R;

and in which: 3. the amino acid residue at position 103 according to the Kabat numbering is chosen from the group consisting of P, R and S; and is in particular chosen from the group consisting of R and S;

and in which:

4. the amino acid residue at position 108 according to the Kabat numbering is chosen from the group consisting of Q and L; is preferably Q;

and in which:

Two particularly preferred, but non-limiting groups of the Nanobodies® of the invention are those according to a) above; according to (a-1) to (a-4) above; according to b) above; according to (b-1 ) to (b-4) above; according to (c) above; and/or according to (c-1) to (c-4) above, in which either:

i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as described herein) and the amino acid residue at position 108 is Q;

or in which:

ii) the amino acid, residues at positions 43-46 according to the Kabat numbering form the sequence KERE or KQRE (or a KERE-iike sequence as described) and the amino acid residue at position 108 is Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody® of the invention may have the structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein) and the amino acid residue at position 108 is Q;

and in which: ii) CD 1 , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: i) the amino acid residues at positions 43-46 according to the abat numbering form the sequence KERE or QRE (or a KERE-Hke sequence) and the amino acid residue at position 108 is Q or L, and is preferably Q;

and in which:

ii) CDR1 , CDR2 and CDR3 are as defined herein, and. are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In the Nanobodies® of the invention in which the amino acid residues at positions 43- 46 according to the Kabat numbering form the sequence KERE or KQRE, the amino acid residue at position 37 is most preferably F. In the Nanobodies® of the invention in which the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW, the amino acid residue at position 37 is chosen from the group consisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the amino acid residues present on the positions mentioned above, the Nanobodies® of the invention can generally be classified on the basis of the following three groups:

i) The "GLE -group": Nanobodies® with the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering and Q at position 108 according to the Kabat numbering. As further described herein, Nanobodies® within this group usually have a V at position 37, and can have a W, P, R or S at position 103, and preferably have a W at position 103. The GLEW group also comprises some GLEW-like sequences such as those mentioned in Table B-2 below. More generally, and without limitation,

Nanobodies® belonging to the GLEW-group can be defined as Nanobodies® with a G at position 44 and/or with a W at position 47, in which position 46 is usually E and in which preferably position 45 is not a charged amino acid residue and not cysteine; ii) The ^uKERE-group": Nanobodies® with the amino acid sequence KERB or KQRE (or another KERE-like sequence) at positions 43-46 according to the abat numbering and Q or L at position 108 according to the Kabat numbering. As further described herein, Nanobodies® within this group usually have a F at position 37, an L or F at position 47; and caii have a W, P. R or S at position 103, and preferably have a W at position 103. More generally, and without limitation, Nanobodies® belonging to the KERE-group can be defined as Nanobodies® with a K, Q or R at position 44 (usually K) in which position 45 is a charged amino acid residue or cysteine, and position 47 is as further defined herein;

iii) The "103 P, R, S-group": Nanobodies® with a P, R or S at position 103. These

Nanobodies® can have either the amino acid sequence GLEW at positions 44-47 according to the Kabat numbering or the amino acid sequence KERB or KQRE at positions 43-46 according to the Kabat numbering, the latter most preferably in combination with an F at position 37 and an L or an F at position 47 (as defined for the KERE-group); and can have Q or L at position 108 according to the Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies® may belong to {i.e., have characteristics of) two or more of these classes. For example, one specifically preferred group of Nanobodies® has GLEW or a GLEW- like sequence at positions 44-47; P,R. or S (and in particular R) at position 103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred to above describe and apply to Nanobodies® in the form of a native (i.e., non-humanized) VHH sequence, and that humanized variants of these Nanobodies® may contain other amino acid residues than those indicated above (i.e., one or more humanizing substitutions as defined herein). For example, and without limitation, in some humanized Nanobodies® of the GLEW-group or the 103 P, R, S-group, Q at position 108 may be humanized to 108L. As already mentioned herein, other humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human V_H sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and. the resulting humanized V_HH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework, regions of) a Nanobody® may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody® of the invention may be a Nanobody® belonging to the GLEW-group (as defined herein), and in which CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody® of the invention may be a Nanobody® belonging to the KERE-group (as defined herein), and CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody® of the invention may be a Nanobody® belonging to the 103 P, R, S-group (as defined herein), and in which CDRl , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,S residues mentioned above, the Nanobodies® of the invention can contain, at one or more positions that in a conventional VR domain wou!d form (part of) the VH/VL interface, one or more amino acid residues that are more highly charged than the amino acid residues that naturally occur at the same position(s) in the corresponding naturally occurring V_H sequence, and in particular one or more charged amino acid residues (as mentioned in Table A-2 on page 48 of the international application WO 08/020079). Such substitutions include, but are not limited to, the GLEW-like sequences mentioned in Table B-2 below; as well as the substitutions that are described in the International Application WO 00/29004 for so-called "microbodies", e.g., so as to obtain a Nanobody® with Q at position 108 in combination with KLEW at positions 44-47, Other possible substitutions at these positions will be clear to the skilled person based upon the disclosure herein.

In one aspect of the Nanobodies® of the invention, the amino acid residue at position 83 is chosen from the group consisting of L, M, S, V and W; and is preferably L.

Also, in. one aspect of the Nanobodies® of the invention, the amino acid residue at position 83 is chosen from the group consisting of R, , N, E, G, I, T and Q; and is most preferably either or E (for Nanobodies® corresponding to naturally occurring V_HH domains) or R (for "humanized" Nanobodies®, as described herein). The amino acid residue at position 84 is chosen from the group consisting of P, A, R, S, D T, and V in one aspect, and is most preferably P (for Nanobodies® corresponding to naturally occurring V_HH domains) or R (for "humanized" Nanobodies®; as described herein).

Furthermore, in one aspect of the Nanobodies® of the invention, the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108, which, in the Nanobodies® are as mentioned above, will also be referred to herein as the "Hallmark Residues". The Hallmark Residues and the amino acid residues at the corresponding positions of the most closely related human VH domain, VH3, are summarized in Table B-2.

Some especially preferred but non-limiting combinations of these Hallmark Residues as occur in naturally occurring V_HH domains are mentioned in Table B-3. For comparison, the corresponding amino acid residues of the human VH3 called DP-47 have been indicated in italics.

Table B-2: Hallmark Residues in Nanobodies®

Table B-3: Some preferred but non-limiting combinations of Hallmark Residues in naturally occurring Nanobodies®.

For humanization of these combinations, reference is made to the specification.

11 37 44 45 47 83 84 103 104 1 8

DP -47 (human) M V G L W R A G I

"KERB" group L F E R L K P w G Q

L F E R F E P w G Q

L F E R F P w G Q

L Y Q R L K P w G Q

L F L R V K. P Q G Q

L F 0 R L P w G Q

L F E R F K P w G Q

"GLEW" group L V G L W K s w G Q

M V G L W P R G Q

IIS

In the Nanobodies®, each amino acid residue at any other position than the Hallmark Residues can be any amino acid residue that naturally occurs at the corresponding position (according to the abat numbering) of a naturally occurring VHH domain.

Such amino acid residues will be clear to the skilled person. Tables B-4 to B-7 mention some non-limiting residues that can be present at each position (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4 of naturally occurring VHH domains. For each position, the amino acid residue that most frequently occurs at each position of a naturally occurring VHH domain (and which is the most preferred amino acid residue for said position in a Nanobody®) is indicated in bold; and other preferred amino acid residues for each position have been underlined (note: the number of amino acid residues that are found at positions 26-30 of naturally occurring V_HH domains supports the hypothesis underlying the numbering by Chothia (supra) that the residues at these positions already form part of CDR1).

In Tables B-4 - B-7, some of the non-limiting residues that can be present at each position of a human V_H3 domain have also been mentioned. Again, for each position, the amino acid residue that most frequently occurs at each position of a naturally occurring human VH3 domain is indicated in bold; and other preferred amino acid residues have been underlined.

For reference only, Tables B-4 - B-7 also contain data on the VHH entropy (" VHH Ent") and VHH variability ("VHH Var ") at each amino acid position for a representative sample of 7732 VHH sequences (including data kindly provided by David Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The values for the V_HH entropy and the V_HH variability provide a measure for the variability and degree of conservation of amino acid, residues between the 7732 V_HH sequences analyzed: low values (i.e., <1, such as < 0.5) indicate that an amino acid residue is highly conserved between the V _HH sequences ( . e., little variability). For example, the G at position 9 and the W at position 36 have values for the VH_H entropy of 0.01 and 0 respectively, indicating that these residues are highly conserved and have little variability (and in case of position 36 is W in all 7732 sequences analysed), whereas for residues that form part of the CDR's generally values of 1.5 or more are found (data not shown). Note that the data represented below support the hypothesis that the amino acid residues at positions 27-30 and maybe even also at positions 93 and 94 already form part of the CDR's (although the invention is not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat is used). For a general explanation of sequence entropy, sequence variability and the methodology for determining the same, see Oliveira et al.₅ PROTEINS: Structure, Function and Genetics, 52: 544-552 (2003). Table B-4: Non-limiting examples of amino acid residues in FR1 (for the footnotes, see the footnotes to Table B-2)

Pos. Amino acid residue(s): V_HH VHH

Ent.

Human. V_H3 Camelid VHH 'S Var.

1 E, Q E, Q, K, D, A, G, R 0,47 5

2 V V, M, A, E, L 0,04 1

3 Q Q, K, P, H, F, R 0,04 1

4 L 0,02 1.

5 V, L V, Q, M, E, A, L, P, K, R 0,35 3

6 E, A, Q, D, K, H 0,21 5

^E

7 S, T S. F, L, W, T 0,05 2

8 G, R G, R, E, V 0,04 1

9 * G, R, V, A 0,01 1

10 G, V G, D, R, S, K, E, A, Q, N, T, V 0,22 4

1 1 Hallmark residue: L. S. V. M. W. F. T, O, E. A, R, G, , Y, N, P, 1; 0,35 4 preferably L

12 V, l V, A, L, M, E, G, T 0, 1 1 2

13 Q, K, R Q, L, R, H, P, E, , T, S, V, D, G, A, N, M 0,46 j

14 P A, P, T, V, S, D, F, N, I, E, L, R, G, Y, Q, H 0,92 5

15 G G, E 0 1

16 G, R G, D, E, A, S, N, V, R, K, T, P, C, L 0,47 4

17 S S, F, P, Y, T, A, C, R, N 0, 14 2

18 L L, V, R, M, P, Q, S, A, T, K, H 0,06 1

19 R, K R, T, , S, N, G, A, I, L, Q, F, E, V, M 0,36 4

20 L. F, V, 1, P, H, S 0, 18 3

^L

21 S S, A, T, P, F, V, H, D, R, L, I, G 0, 13 3 22 C c, w 0 1

23 A, T A, V, T, E, S, L, G, I, , Q, R, D, F, N, P, M 0,88 5

24 A A, D, V, T, H, Y, P, G, S, F, L, I, N, Q, E, R 0,78 9

Table B-4: Non-limiting examples of amino acid residues in FR1 (continued)

Pos. Amino acid residue(s): VHH. V™ .

Ent.

Human VH3 Camelid VHH 'S Var.

25 S S. P. T, A, F, L, N, Y, R, H, D, V, I, W, G, K, Q, C 0,2 2

26 G G, E, R, V, T, A, S, K, D, L, I, Q, N, F, Y_} M, W, P, H 0,45 6

27 F R, F, S, P, L, G, I, N, T, D, H, V, E, A, Y, K, M, Q, W, 1 ,89 1.2

C

28 T T, L S, A, P, F, D, N, V, R, M, L, G, Y, , E, H, W, Q 1 ,29 12

29 F, V F, L, S, V, I, A, W, Y, G, D_S R, T, P, N, E, M, PL Q, K, 1,23 11

C

30 S, D, G S. D, N, G, R, T, A, E, L Y, , V, H, L, F, W, M, P. C, 1 ,55 12

Q

Table B-5: Non-limiting examples of amino acid residues in FR2 (for the footnotes, see the footnotes to Table B-2)

Tabic B-6: Non-limiting examples of amino acid residues in FR3 (for the footnotes, see the footnotes to Table B-2)

Pos. Amino acid residue(s): VHH VHH

Ent.

Human Vn3 Camelid VHH 'S Var.

66 R R 0 1

67 F F, S, L, V, I, C, A, Y, M, G 0, 1 1

68 T T. A, S, I, F, V, P, N, G, R, K, M, D, L, W, Q 0,34 4

69 I I, V, M, T, L, A, F, P, S, G, N 0,5 5

70 s S, T, A, F, P. V, Y, L, D, G, N, H, W, E, C 0,22 4

71 R S, K, G, T, I, W, A, N, V, E, L, M, F, D, Q, C 0,61 7

72 D, E D, N. E, G, V, A, H, L, S, T, I. Q, F, P, Y, R 0,34 4

73 N. ! ⁾. G N, D, S, K, I, Y_f G, T, H, R, A, V, F, L, E, M, P, C 0,65 9

74 A, S A, T, V, S, F, G, D, P, N, Ϊ, R, L, Y, H, E, Q, K, W, 0,8 8

M

75 K K, N, E, R, Q, A, G, T, M, S, L, D, V, W, Y, I 0,7 ] 6

76 N. S N, , S, R, D, T, H, G, K A, Y, I, M, Q₉ L, W, P, F, 0,66 7

V

77 S, T, I T, A, M, S, R, I, V. L, P, E, N, K, G, W, Q 0,72 7

78 L, A V, L, A, M, 1, G, T, F, W, Q, S, E, N, H 1 ,1 1 6

79 Y, H Y, F, D, S, H, N, T_} A, L, W, V, C, G, E, 1, P, R 0,68 8

80 L L, M, V, P, F 0,05 2

81 Q Q, E, R, H, L, D, T, G, , P, A, 1, S, N, Y, V, M 0,38 4

82 M M, I, L, V, A, T, S, K 0, 12

82a N, G N, S, D, T, E, H, K, 1, A, G, R, Y, L, V, F, Q 0,77 5

82b S S, N, T, G, H, D, R, A, K, I, M, V, F, E, P_S Y, C, L 0,72 8

82c L L, V, M, P, A, T, G 0,08 2

83 Hallmark residue: R. K^w, T. E⁽ⁱ⁾. O, N. S. I. V, G, M. L, A, D, Y, H; 0,66 6 preferably K or R; most preferably K

84 Hallmark residae: P⁽⁵⁾. S. H. L. A, V, I, T, F, D. R, Y, N, O, G. E; 0,85 7 preferably P 85 E, G E, D, G, A, Q, V, S, N, K, T, R, L 0,27 3

86 D D, E, G, N 0,02 1

87 T. M T, S, A, M, R, P. K, E 0,15 3

Table B-6: Non-limiting examples of amino acid residues in FR3 (continued)

Pos. Amino acid residue(s): VHH

Ent.

Human V_H3 Camelid VHH 'S Var.

88 A A, G, S, D, N, T, P, V 0,23 2

89 V, L V, L L, E, A, R, T, D, F, M, N, S, K, G, Q, H 0,71 7

90 Y Y, H, F, N 0 1

91 Y, H Y, F, R, S, H, T, 1, V, L, N, D, C, Q, W, A, E, M 0,6 7

92 C C, R, P 0 1

93 A, K, T A, N, 1, . G, V, R. Y. S. H, W, I., F. Q, M, I, E, C, 1 ,JJ 10

D

94 K, R, T A, K, V, T, R, L, G, S, D, 0, 1, M, F, Y, N, E, H, P, C, 1,55 12

W

Table B-7: Non-limiting examples of amino acid residues in FR4 (for the footnotes, see the footnotes to Table B-2)

Thus, in another preferred, but not limiting aspect, a Nanobody® of the invention can be defined as an amino acid sequence with the (general) structure FR1 - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:

i) one or more of the amino acid residues at positions 1 1, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2;

and in which:

ii) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

The above Nanobodies® may for example be VHH sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are VHH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein.

In particular, a Nanobody® of the invention can be an amino acid sequence with the (general) structure FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FRl to FR4 refer to framework regions 1 to 4, respectively, and in which CDRl to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:

i) (preferably) one or more of the amino acid residues at positions 11 , 37, 44, 45, 47, 83, 84, 103, 104 and. 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table B-2 (it being understood that VHH sequences will contain one or more Hallmark residues; and that partially humanized Nanobodies® will usually, and preferably, [still] contain one or more Hallmark residues [although it is also within the scope of the invention to provide - where suitable in accordance with the invention - partially humanized Nanobodies® in which all Hallmark residues, but not one or more of the other amino acid residues, have been humanized]; and that in fully humanized Nanobodies®, where suitable in accordance with the invention, all amino acid residues at the positi ons of the Hallmark residues will be amino acid residues that occur in a human VH3 sequence. As will be clear to the skilled person based on the disclosure herein that such VHH sequences, such partially humanized Nanobodies® with at least one Hallmark residue, such partially humanized Nanobodies® without

Hallmark residues and such, fully humanized Nanobodies® all form aspects of this invention);

and in which:

ii) said amino acid sequence has at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO's: 1 to 22, in which for the purposes of determining the degree of amino acid identity, the amino acid residues that form the CDR sequences (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are disregarded;

and in which:

iii) CDR1 , CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

The above Nanobodies® may for example be V_HH sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are VHH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein. Table B-8: Representative amino acid sequences for Nanobodtes® of the KERE, GLEW and P,R,S 103 group.

The CDR's are indicated with XXXX

KERB sequence no. 1 SEQ TD NO: 1 EVQLVESGCJGLVQPCJGSLRESCAASGIPFSXXXXXWERQAPG Q DSVAXXXXXRFTIS

RDNA NTVYEQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS

KERE sequence no. 2 SEQ ID NO:2 QVKLE_^ESGGGEVQAGGSERLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTIS

RDTASNROYLHMNNLTPEDT A V Y YC AAXXXXX WGQGTQ V TV S S

KJERE sequence no. 3 SEQ ID NO:3 AVQLVDSGGGLVQAGDSLKLSCALTGGAETXXXXXWFRQTPGREREFVAXXXXXRFn

SRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS

KERE sequence no. 4 SEQ ID NO:4 QVQEVESGGGEVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTIS

RJ3DAKNTVWLHGSTLKPED TAV YYCAAXXXXXWGQGTQVTVSS

KERE sequence no. 5 SEQ ID NO:5 AVQLVESGGGLVQGGGSEREACAASERIFDXXXXXWYRQGPGNEREEVAXXXXXRFTI

SMDYTKQTVYLHMNSLRPEDTGLYYCK1XXXXXWGQGTQVTVSS

KERE sequence no. 6 SEQ ID NO:6 DVKI ^'V ESGC;GLVQAGGSI,RI.,SCVASGFNFDXXXXX FRQAPGKEREEVAXXXXXRFTI

SSEKDKNSVYLQ NSLKPEDTALYICAGXXXXXWGRGTQVTVSS

KERE sequence no. 7 SEQ ID NO:7 QVREAESGC;GLVQSGGSLRI,SCVASGS'I Y I'XXXXXWYRQYPGKQRALVAXXXXXRFTI

ARDST DTl-^'Cl.Q NNLKPED^'rAVYYCYAXXXXXWGQGTQVTVSS

KERE sequence tio. 8 SEQ ID NO:8 EVQLVESGGGLVQAGGSLRESCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTIS

TDNAKNI^'VHELMMRVNAEDTALYYCAVXXXXXWGRGI RVTVSS

KERE sequence no. 9 SEQ ID NO:9 QVQEVESGGGLVQPGGSERESCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTIS

GDN AKRAI YLQM NL PDDTA VY YCN RXXXXX WGQGTQ VTVSP

KERE sequence no. 10 SEQ ID NO: 10 QVPVVESGGGLVQAGDSERi.,FCAVPSF^'rSTXXXXXWFRQAPGKEREFVAXXXXXRFTIS

RNAT NTLTLRMDSEKPEDTAVYYCAAXXXXXWGQGTQVTVSS

KE E sequence no. 1 1 SEQ ID NO: 1 1 EVQLVESGGGEVQAGDSLRLFCTVSGGTASXXXXXWF^'RQAPGEKREEVAXXXXXRF^'ri

ARENAGNMVYEQ NNI .KPDDTALY^'FCAAXXXXXWGRG 1 QV^'FVSS

Table -8 (continued):

KERE sequence no. 12 SEQ ID NO: 12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTI

SRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQG^'FQVTVSS

KERB sequence no. 13 SEQ ID NO: 13 AVQLVESGGGLVQAiiGSLRLSCVVSGETSSXXXXXWFRQ'FPWQERDFVAXXXXXRFTI

SRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS

KERE sequence no. 14 SEQ ID NO: 14 AVQLVESGGGLVQAGASLRLSCATS TRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTV

SRDSAENTVALQMNSLKPED^'S^'AVYYCAAXXXXXWGQGTRVTVSS

KERE sequence no. 15 SEQ ID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTIS

RDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQG r^'QVTVSS

KER.E sequence no. 16 SEQ ID NO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFTV

SRDNGKNTAYLR NSLKPEDTADYYCAVXXXXXLGSGTQVTVSS

GLEW sequence no. 1 SEQ ID NO: 17 AVQLVESGG(iLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTI

SRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQV^'FVSS

GLEW sequence no. 2 SEQ ID NO: ! 8 EVQLVESGGGLVQPGGSERLSCVCVSSGCTXXXXX VRQAPGKAEEWVSXXXXXRFKI

SRDNAKKf LYLQMNSLGPEDTAMYYCQRXXXXXRGQG^'FQVTVSS

GLEW sequence no. 3 SEQ ID NO: 19 EVQLVESGOGEALPGGSLTLSCVFSGSTFSXXXXXWVRIFFPGKAEEWVSXXXXXRF^'riS

RDNAKNTLYLE NSLSFEDTAMYYCGRXXXXXRSKGIQVTVSS

P,R,S 103 sequence no. 1 SEQ ID NO:20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRl'TI

SRDNAKNTVYLQMNSLKPED^'FAVYYCAAXXXXXRGQGTQVTVSS

P,R,S 103 sequence no. 2 SEQ ID NO:21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRETi

SRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQOTQVTVSS

P,R,S 103 sequence no. 3 SEQ ID NO:22 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRFKI

S RON AKKTLYLQMNSLGPEDT AM Y Y^'CQRXXXXXRG QGTQ VTV S S

In particular, a Nanobody® of the invention of the KERE group can be an amino acid sequence with the (general) structure

FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

in which:

i) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an and in which:

H) FRl is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table B-9: Representative FW1 sequences for Nanobodies® of the KERE-gro p.

and in which:

iii) FR2 is an amino acid sequence that has at least 80%> amino acid identity with at least one of the following amino acid sequences: Table B-10: Representative FW2 sequences for Nanobodies® of the KERE-group.

and in which:

iv) FR3 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table B-l 1 : Representative FW3 sequences for Nanobodies® of the KERE-group.

KERE FW3 sequence no. 1 SEQ ID O:50 RFTI SRDNAKNTV YLQMN S LKPEDTAV YRCYF

KERE FW3 sequence no. 2 SEQ ID O:51 RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAA

KERE FW3 sequence no. 3 SEQ ID NO:52 RFTVAR NAKNTVNLEMNSLKPEDTAVYYCAA

KERE FW3 sequence no. 4 SEQ ID NO:53 RFTISRDIAKNTVDLL NNLEPEDTAVYYCAA

KER FW3 sequence no. 5 SEQ ID NO:54 RLTISRDNAVDTMYLQ NSLKPEDTAVYYCAA

KERE FW3 sequence no. 6 SEQ ID NO:55 RFTI SRDNAKNTV YLQMDN VKPEDTAIY YC AA

KERE FW3 sequence no. 7 SEQ ID NO:56 RFTISKDSGKNTVYLQMTSLKPEDTAVYYCAT

KERE FW3 sequence no. 8 SEQ ID NO:57 RFTISRDSAKNMMYLQ NNLKPQDTAVYYCAA

KERE FW3 sequence no. 9 SEQ ID NO:58 RFTISRENDKSTVYLQLNSLKPEDTAVYYCAA

KFRE FW3 sequence no. 10 SEQ ID NO:59 RFTISRDYAGNTAYLQMNSLKPEDTGVYYCAT and in which:

v) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table B-12: Representative FW4 sequences for Nanobodies® of the KERE-group.

and in which:

vi) CDR.L CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_H.H sequences or partially humanized Nanobodies®).

Also, the above Nanobodies® may for example be VHH sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are VHH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein.

With regard to framework 1 , it will be clear to the skilled person that, when an amino acid sequence as outlined above is generated by expression of a nucleotide sequence, the first four amino acid sequences (i.e., amino acid residues 1-4 according to the Kabat numbering) may often be determined by the primer(s) that have been used to generate said nucleic acid. Thus, for determining the degree of amino acid identity, the first four amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27 to 30 are according to the Kabat numbering considered to be part of the framework regions (and not the CDR's), it has been found by analysis of a database of more than 1000 V_HH sequences that the positions 27 to 30 have a variability (expressed in terms of VHH entropy and VRH variability - see Tables B-4 to B-7) that is much greater than the variability on positions 1 to 26. Because of this, for determining the degree of amino acid identity, the amino acid residues at positions 27 to 30 are preferably also disregarded.

in view of this, a Nanobody® of the KERE class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

i) the amino acid residue at position 45 according to the Kabat numbering is a charged amino acid (as defined herein) or a cysteine residue, and position 44 is preferably an E; and in which:

ii) FRl is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table B-13: Representative FWl sequences (amino acid residues 5 to 26) for Nanobodies® of the KERE-group.

and in which:

iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies® of the KERE-class;

and in which: iv) CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

A Nanobody® of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which

i) preferably, when the Nanobody® of the GLEW-class is a non-humanized Nanobody®, the amino acid residue in position 108 is Q;

ii) FR1 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table B-14: Representative FW1 sequences for Nanobodies® of the GLEW-group.

and in which:

hi) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-15: Representative FW2 sequences for Nanobodies® of the GLEW-group.

and in which:

Table B-16: Representative FW3 sequences for Nanobodies© of the GLEW-group.

and in which:

v) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-17: Representative FW4 sequences for anobodies® of the GLEW-group.

and in which:

vi) CDRL CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are VHH sequences or partially humanized Nanobodies®),

With regard to framework 1 , it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody® of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

and in which:

ii) FRl is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences: Table B-18: Representative FWl sequences (amino acid residues 5 to 26) for Nanobodies® of the KERE-grou .

and in which:

iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies® of the GLEW-class;

and in which:

iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

The above Nanobodies® may for example be VHH sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are VHH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanobodies®, they may optionally be further suitably humanized, again as described herein. In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_HH sequences or partially humanized Nanobodies®).

A Nanobody® of the P, R, S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which

i) the amino acid residue at position 103 according to the Kabat numbering is different from W;

and in which:

ii) preferably the amino acid residue at position 103 according to the Kabat numbering is P, R or S, and more preferably R;

and in which:

iii) FR1 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following acid sequences: Table B-19: Representative FW1 sequences for Nanobodies® of the P,R,S 103-group.

and in which

iv) FR2 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table B-20: Representative FW2 sequences for Nanobodies® of the P,R,S 103-group.

and in which: FR3 is an amino acid sequence that has at least 80% amino acid identity with one of the following amino acid sequences:

Table B-21 : Representative FW3 sequences for Nano bodies® of the P,R,S 103-group.

and in which:

vi) FR4 is an amino acid sequence that has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table B-22: Representative FW4 sequences for anobodies® of the P,R,S 103-group.

in which:

CDRl, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein. In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are VHH sequences or partially humanized Nanobodies®).

In view of this, a Nanobody® of the P,R,S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

i) the amino acid residue at position 103 according to the abat numbering is different and in which:

and in which:

iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the Kabat numbering, has at least 80% amino acid identity with at least one of the following amino acid sequences:

Table B-23: Representative FWl sequences (amino acid residues 5 to 26) for Nanobodies® the P,R,S 103-group.

and in which:

iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of Nanobodies® of the P,R,S 103 class;

and in which:

v) CDRL CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

The above Nanobodies® may for example be VHH sequences or may be humanized Nanobodies®. When the above Nanobody® sequences are VHH sequences, they may be suitably humanized, as further described herein. When the Nanobodies® are partially humanized Nanohodies®, they may optionally be further suitably humanized, again as described herein.

In the above Nanobodies®, one or more of the further Hallmark residues are preferably as described herein (for example, when they are VHH sequences or partially humanized Nanobodies®).

According to one non-limiting aspect of the invention, a Nanobody® may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring human VH domain, and in particular compared to the corresponding framework region of DP -47. More specifically, according to one non- limiting aspect of the invention, a Nanobody® may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring human VH domain, and in particular compared to the corresponding framework region of DP -47. Usually, a Nanobody® will have at least one such amino acid difference with a naturally occurring V_H domain in at least one of FR2 and/or FR4. and in particular at at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

Also, a humanized Nanobody® of the invention may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) in at least one of the framework regions compared, to the corresponding framework region of a naturally occurring V_HH domain. More specifically, according to one non-limiting aspect of the invention, a humanized Nanobody® may be as defined herein, but with the proviso that it has at least "one amino acid difference" (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring VHH domain. Usually, a humanized Nanobody® will have at least one such amino acid difference with a naturally occurring VHH domain in at least one of FR2 and/or FR4, and in particular at at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as analogs") of the ISVs Nanobodies® of the invention as defined herein. Generally, in such analogs, one or more amino acid residues may have been, replaced, deleted and/or added, compared to the Nanobodies® of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDR's. When such substitutions, insertions or deletions are made in one or more of the framework regions, they may be made at one or more of the Hallmark residues and/or at one or more of the other positions in the framework residues, although substitutions, insertions or deletions at the Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).

By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another VHH domain (see Tables B-4 to B-7 for some non-limiting examples of such substitutions), although the invention is generally not limited thereto. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the Nanobody® of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Nanobody® of the invention (i.e., to the extent that the Nanobody® is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the Nanobodies® thus obtained.

For example, and depending on the host organism used to express the Nanobody® or polypeptide of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation (again as described herein).

As can be seen from the data on the VHH entropy and VHH variability given in Tables

B-4 to B-7 above, some amino acid residues in the framework regions are more conserved than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to CXC -4 with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a KA-value (actual or apparent), a

or alternatively as an IC50 value, as further described herein) that is as defined herein for the Nanobodies® of the invention.

The analogs are preferably also such that they retain the favourable properties the Nanobodies®, as described herein.

Also, the framework sequences and CDR's of the analogs are preferably such that they are in accordance with the preferred aspects defined herein. More generally, as described herein, the analogs will have (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103.

One preferred class of analogs of the Nanobodies® of the invention comprise

Nanobodies® that have been humanized {i.e., compared to the sequence of a naturally occurring Nanobody® of the invention). As mentioned in the background art cited herein, such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring VHH with the amino acid residues that occur at the same position in a human VH domain, such as a human V_H3 domain. Examples of possible humanizing substitutions or combinations of humanizing substitutions will be clear to the skilled person, for example from the Tables herein, from the possible humanizing

substitutions mentioned in the background art cited herein, and/or from a comparision between the sequence of a Nanobody® and the sequence of a naturally occurring human VH domain.

The humanizing substitutions should be chosen such that the resulting humanized

Nanobodies® still retain the favourable properties of Nanobodies® as defined herein, and more preferably such that they are as described for analogs in the preceding paragraphs. A skilled person will generally be able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible humanizing substitutions and determining their influence on the properties of the Nanobodies® thus obtained. Generally, as a result of humanization, the Nanobodies® of the invention may become more "human-like", while still retaining the favorable properties of the Nanobodies® of the invention as described herein. As a result, such humanized Nanobodies® may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. Again, based on the disclosure herein and optionally after a limited degree of routine experimentation, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring VHH domains on the other hand.

The Nanobodies® of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined herein) or at one or more other framework residues (i.e., non-Hallmark residues) or any suitable combination thereof. One preferred humanizing substitution for Nanobodies® of the "P,R,S-103 group" or the " ERE group" is Q 108 into L 108. Nanobodies® of the "GLEW class" may also be humanized by a Q108 into L I 08 substitution, provided at least one of the other Hallmark residues contains a cameiid (camelizing) substitution (as defined herein). For example, as mentioned above, one particularly preferred class of humanized Nanobodies® has GLEW or a GLEW-like sequence at positions 44-47; P, R or S (and in particular R) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding the same, can be provided in any manner known per se,. for example using one or more of the techniques mentioned on pages 103 and 104 of WO 08/020079.

Also, in addition to humanizing substitutions as described herein, the amino acid sequences of the invention may contain one or more other/further substitutions. Again, some preferred, but non-limiting examples of such other/further substitutions will become clear from the further description herein, and for example may include (and preferably essentially consist of) one or more of the following substitutions:

(a) one or more conservative amino acid substitutions; and/or

(b) one or more substitutions in which a "cameiid" amino acid residue at a certain position is replaced by a different "cameiid" amino acid residue that occurs at said position, for which reference is for example made to Tables A-6 to A-9 from PCT/EP2008/066365 (published on June 4, 2009 as WO 09/068627), which mention the various Camelid residues that occur as each amino acid position in wild-type VHFTs. Such substitutions may even comprise suitable substitutions of an amino acid residue that occurs at a Hallmark position with another amino acid residue that occurs at a Hallmark position in a wild-type VHH (for which reference is for example made to Tables A-6 to A-9 from PCT/EP2008/066365); and/or

(c) one or more substitutions that improve the (other) properties of the protein, such as substitutions that improve the long-term stability and/or properties under storage of the protein. These may for example and without limitation be substitutions that prevent or reduce oxidation events (for example, of methionine residues); that prevent or reduce pyroglutamate formation; and/or that prevent or reduce isomerisation or deamidation of aspartic acids or asparagines (for example, of DG, DS, NG or NS motifs). For such substitutions, reference is for example made to the International application WO 09/095235, which is generally directed to methods for stabilizing single immunoglobulin variable domains by means of such substitutions, and also gives some specific example of suitable substitutions (see for example pages 4 and 5 and pages 10 to 15). One example of such substitution may be to replace an NS motif at positions 82a and 82b with an NN motif.

As mentioned there, it will be also be clear to the skilled person that the Nanobodies® of the invention (including their analogs) can be designed and/or prepared starting from human V_H sequences (i.e., amino acid sequences or the corresponding nucleotide sequences), such as for example from human V_H3 sequences such as DP -47, DP-51 or DP -29, i.e., by introducing one or more camelizing substitutions (i.e., changing one or more amino acid residues in the amino acid sequence of said human VH domain into the amino acid, residues that occur at the corresponding position in a V_HH domain), so as to provide the sequence of a Nanobody® of the invention and/or so as to confer the favourable properties of a Nanobody® to the sequence thus obtained. Again, this can generally be performed using the various methods and techniques referred to in the previous paragraph, using an amino acid sequence and/or nucleotide sequence for a human V_H domain as a starting point.

Some preferred, but non-limiting camelizing substitutions can be derived from Tables B-4 - B-7. It will also be clear that camelizing substitutions at one or more of the Hallmark residues will generally have a greater influence on the desired properties than substitutions at one or more of the other amino acid positions, although both and any suitable combination thereof are included within the scope of the invention. For example, it is possible to introduce one or more camelizing substitutions that already confer at least some the desired properties, and then to introduce further camelizing substitutions that either further improve said properties and/or confer additional favourable properties. Again, the skilled person will generall be able to determine and select suitable camelizing substitutions or suitable combinations of camelizing substitutions, based on the disclosure herein and optionally after a limited, degree of routine experimentation, which may for example involve introducing a limited number of possible camelizing substitutions and determining whether the favourable properties of Nanobodies® are obtained or improved (i.e., compared to the original V_H domain).

Generally, however, such camelizing substitutions are preferably such that the resulting an amino acid sequence at least contains (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably also an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103; and optionally one or more further camelizing substitutions. More preferably, the camelizing substitutions are such that they result in a Nanobody® of the invention and/or in an analog thereof (as defined herein), such as in a humanized analog and/or preferably in an analog that is as defined in the preceding paragraphs.

Nanobodies® can also be derived from V_H domains by the incorporation of substitutions that are rare in nature, but nonetheless, structurally compatible with the VH domain fold. For example, but without being limiting, these substitutions may include on or more of the following: Gly at position 35, Ser, Val or Tlir at position 37, Ser, Thr, Arg, Lys, His, Asp or Glu at position 39, Glu or His at position 45, Trp, Leu, Val, Ala, Thr, or Glu at position 47, S or R at position 50. (Barthelemy et al. J Biol Chem. 2008 Feb 8;283(6):3639- 54. Epub 2007 Nov 28)

As will also be clear from the disclosure herein, it is also within the scope of the invention to use parts or fragments, or combinations of two or more parts or fragments, of the Nanobodies® of the invention as defined herein, and in particular parts or fragments of an ISV of the invention. Thus, according to one aspect of the invention, the term "Nanobody® of the invention" in its broadest sense also covers such parts or fragments. Generally, such parts or fragments of the Nanobodies® of the invention (including analogs thereof) have amino acid sequences in which, compared to the amino acid sequence of the corresponding full length Nanobody® of the invention (or analog thereof), one or more of the amino acid residues at the N-terminal end. one or more amino acid residues at the C- terminal end, one or more contiguous internal amino acid residues, or any combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to CXCR-4 with an affinity (suitably measured and/or expressed as a o-vaiue (actual or apparent), a K_A-value (actual or apparent), a k_on-rate and/or a k₀fyJ"ate, or alternatively as an IC50 value, as further described herein) that is as defined herein for the Nanobodies® of the invention.

Any part or fragment is preferably such that it comprises at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, more preferably at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues, of the amino acid sequence of the corresponding full length Nanobody® of the invention.

Also, any part or fragment is such preferably that it comprises at least one of CDR1 ,

CDR2 and/or CDR3 or at least part thereof (and in particular at least CDR3 or at least part thereof). More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least one other CDR (i.e., CDR1 or CDR2) or at least part thereof, preferably connected, by suitable framework sequence(s) or at least part thereof. More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least part of the two remaining CDR's, again preferably connected by suitable framework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect, such a part or fragment comprises at least CDR3, such as FR3, CDR3 and FR4 of the corresponding full length Nanobody® of the invention, i.e., as for example described in the international application WO 03/050531 (Lasters et ah).

As already mentioned above, it is also possible to combine two or more of such parts or fragments (i.e., from the same or different Nanobodies® of the invention), i.e., to provide an analog (as defined herein) and/or to provide further parts or fragments (as defined herein) of a Nanobody® of the invention. It is for example also possible to combine one or more parts or fragments of a Nanobody® of the invention with one or more parts or fragments of a human V_H domain. According to one preferred aspect, the parts or fragments have a degree of sequence identity of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, such as at least 90%, 95% or 99% or more with one of the ISV's of the invention.

The parts and fragments, and nucleic acid sequences encoding the same, can be provided and optionally combined in any manner known per se. For example, such parts or fragments can be obtained by inserting a stop codon in a nucleic acid that encodes a full-sized Nanobody® of the invention, and then expressing the nucleic acid thus obtained in a manner known per se (e.g., as described herein). Alternativel , nucleic acids encoding such parts or fragments can be obtained by suitably restricting a nucleic acid that encodes a full-sized Nanobody® of the invention or by synthesizing such a nucleic acid in a manner known per se. Parts or fragments may also be provided using techniques for peptide synthesis known per se.

The invention in its broadest sense also comprises derivatives of the Nanobodies® of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g., enzymatical) modification, of the Nanobodies® of the invention and/or of one or more of the amino acid residues that form the Nanobodies® of the invention.

Examples of such modifications, as well as examples of amino acid residues within the Nanobody® sequence that can be modified in such a manner (i.e., either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g., by covalent linking or in an other suitable manner) of one or more functional groups, residues or moieties into or onto the Nanobody® of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the Nanobody® of the invention. Example of such functional groups will be clear to the skilled person,

For example, such modification may comprise the introduction (e.g., by covalent binding or in any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or the absorption of the Nanobody® of the invention, that reduce the immunogenicity and/or the toxicity of the Nanobody® of the invention, that eliminate or attenuate any undesirable side effects of the Nanobody® of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties of the Nanobodies® and/or polypeptides of the invention; or any combination of two or more of the foregoing. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv's and single domain antibodies), for which reference is for example made to Remington's

Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may for example be linked directly (for example covalently) to a Nanobody® of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv's); reference is made to for example Chapman, Nat. BiotechnoL, 54, 531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA.

Preferably, site-directed pegylation is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a Nanobody® of the invention, a Nanobody® of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attacliment of PEG may be fused to the N- and/or C-terminus of a Nanobody® of the invention, all using techniques of protein engineering known, per se to the skilled person. Preferably, for the Nanobodies® and proteins of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example in the range of 20,000-80,000.

Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-transktional and/or post-translational modification, depending on the host cell used for expressing the Nanobody® or polypeptide of the invention.

Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled Nanobody®. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, the fluorescent labels, phosphorescent labels, chemiluminescent labels, bioluminescent labels, radio-isotopes, metals, metal chelates, metallic cations, chroraophores and enzymes, such as those mentioned on page 109 of WO 08/020079. Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy.

Such labelled Nanobodies® and polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, diethyl- enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the Nanobody® of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e., through formation of the binding pair. For example, a Nanobody® of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated Nanobody® may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the Nanobody® of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposomal formulations described by Cao and Suresh, Journal of Drug Targeting, 8, 4. 257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the Nanobody® of the invention.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies® of the invention are directed (e.g., in the treatment of cancer), or to reduce or slow the growth and/or proliferation such a ceil, the Nanobodies® of the invention may also be linked to a toxin or to a toxic residue or moiety. Examples of toxic moieties, compounds or residues which can be linked to a Nanobody® of the invention to provide - for example - a cytotoxic compound will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g., to study function-activity relationships). Reference is for example made to Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143- 151 (1997).

Preferably, the derivatives are such that they bind to CXCR-4 with an affinity (suitably measured and/or expressed as a Ko-value (actual or apparent), a KA- value (actual or apparent), a k_on-rate and/or a k₀ff-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein for the Nanobodies® of the invention.

As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of or comprise at least one Nanobody® of the invention. By "essentially consist of is meant that the amino acid sequence of the polypeptide of the invention either is exactly the same as the amino acid sequence of a Nanobody® of the invention or corresponds to the amino acid sequence of a Nanobody® of the invention which has a limited number of amino acid residues, such as 1 -20 amino acid residues, for example 1 -10 amino acid residues and preferably 1 -6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the amino acid sequence of the Nanobody®.

Said amino acid residues may or may not change, alter or otherwise influence the (biological) properties of the Nanobody® and may or may not add further functionality to the Nanobody®. For example, such amino acid residues: can comprise an N-terminal Met residue, for example as result of expression in a heterologous host cell or host organism.

may form a signal sequence or leader sequence that directs secretion of the Nanobody® from a host cell upon synthesis. Suitable secretory leader peptides will be clear to the skilled person, and may be as further described herein. Usually, such a leader sequence will be linked to the N-terminus of the Nanobody©, although the invention in its broadest sense is not limited thereto;

may form a sequence or signal that allows the Nanobody® to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody® to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such amino acid sequences will be clear to the skilled person and include those mentioned in paragraph c) on page 112 of WO 08/020079.

may form a "tag", for example an amino acid sequence or residue that allows or facilitates the purification of the Nanobody®. for example using affinity techniques directed against said sequence or residue. Thereafter, said sequence or residue may be removed (e.g., by chemical or enzymatical. cleavage) to provide the Nanobody® sequence (for this purpose, the tag may optionally be linked to the Nanobody® sequence via a cleavable linker sequence or contain a cleavable motif). Some preferred, but non-limiting examples of such residues are multiple histidine residues, glutatione residues and a myc-tag (see for example SEQ ID NO:31 of WO 06/12282).

may be one or more amino acid residues that have been functionalized and/or that can serve as a site for attachment of functional groups. Suitable amino acid residues and functional groups will be clear to the skilled person and include, but are not limited to, the amino acid residues and functional groups mentioned herein for the derivatives of the Nanobodies® of the invention.

According to another aspect, a polypeptide of the invention comprises a Nanobody® of the invention, which is fused at its amino terminal end, at its carboxy terminal end, or both at its amino terminal end and at its carboxy terminal end to at least one further amino acid sequence, i.e., so as to provide a fusion protein comprising said Nanobody® of the invention and the one or more further amino acid sequences. Such a fusion will also be referred to herein as a "Nanobody® fusion". The one or more further amino acid sequence may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the Nanobody®. and may or may not add further functionality to the Nanobody® or the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the Nanobody® or the polypeptide of the invention.

For example, the further amino acid sequence may also provide a second binding site, which binding site may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including but not limited to the same protein, polypeptide, antigen, antigenic determinant or epitope against which the Nanobody® of the invention is directed, or a different protein, polypeptide, antigen, antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv's and single domain antibodies). Reference is for example made to the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 1 126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptides of the invention, compared to the Nanobody® of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).

In particular, it has been described in the art that linking fragments of

immunoglobulins (such as VH domains) to serum albumin or to fragments thereof can be used to increase the half-life. Reference is for made to WO 00/27435 and WO 01/077137).

According to the invention, the Nanobody® of the invention is preferably either directly linked to serum albumin (or to a suitable fragment thereof) or via a suitable linker, and in particular via a suitable pepti de linked so that the polypeptide of the invention can be expressed as a genetic fusion (protein). According to one specific aspect, the Nanobody® of the invention may be linked to a fragment of serum albumin that at least comprises the domain III of serum albumin or part thereof. Reference is for example made to WO

07/1 12940 of Ablynx N.V.

Alternatively, the further amino acid sequence may provide a second binding site or binding unit that is directed against a serum, protein (such as, for example, human serum albumin or another serum protein such as IgG), so as to provide increased half-life in serum. Such amino acid sequences for example include the Nanobodies® described below, as well as the small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO 04/00301.9. Reference is also made to Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, as well as to EP 0 368 684, as well as to WO 08/028977, WO 08/043821, WO 08/043822 by Ablynx N.V. and US provisional application of Ablynx N.V. entitled "Peptides capable of binding to serum proteins " filed on December 5, 2006 ((see also PCT/EP2007/063348).

Such, amino acid sequences may in particular be directed against serum albumin (and more in particular human serum albumin) and/or against IgG (and more in particular human IgG). For example, such amino acid sequences may be amino acid sequences that are directed against (human) serum albumin and amino acid sequences that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787) and/or amino acid sequences that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see again for example WO 06/0122787); amino acid sequences that have or can provide an increased half-life (see for example WO 08/028977 by Ablynx N.V.); amino acid sequences against human serum albumin that are cross-reactive with, serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaco (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ur sinus), reference is again made to WO 08/028977; amino acid sequences that can bind to serum albumin in a pH independent manner (see for example WO

08/043821 by Ablynx N.V. entitled "Amino acid sequences thai bind to serum proteins in a manner that is essentially independent of the pH, compounds comprising the same, and uses thereof ) and/or amino acid sequences that are conditional binders (see for example WO

08/043822 by Ablynx N.V. entitled ''Amino acid sequences that bind to a desired molecule in a conditional manner"). According to another aspect, the one or more further amino acid sequences may comprise one or more parts, fragments or domains of conventional 4-chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, although usually less preferred, a Nanobody® of the invention may be linked to a conventional (preferably human) \½ or VL domain or to a natural or synthetic analog of a VH or V_L domain, again optionally via a linker sequence (including but not limited to other (single) domain antibodies, such as the dAb's described by Ward et al.).

The at least one Nanobody® may also be linked to one or more (preferably human) CHI , CH2 and/or CH_.3 domains, optionally via a linker sequence. For instance, a Nanobody® linked to a suitable CRI domain could for example be used - together with suitable light chains - to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab')₂ fragments, but in which one or (in case of an F(ab')₂ fragment) one or both of the conventional VH domains have been replaced by a Nanobody® of the invention. Also, two Nanobodies® could be linked to a C¾3 domain (optionally via a linker) to provide a construct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, one or more Nanobodies® of the invention may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more CH2 and/or CH3 domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and/or may form (part of) and Fc region, for example from IgG (e.g., from IgGl, IgG2, IgG3 or IgG4). from IgE or from another human Ig such as IgA, IgD or IgM. For example, WO 94/04678 describes heavy chain antibodies comprising a Camelid VHH domain or a humanized derivative thereof (i.e., a Nanobody®), in which the Camelidae CH2 and/or CH3 domain have been replaced by human CH and CH3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a Nanobody® and human CH2 and CR3 domains (but no CH I domain), which immunoglobulin has the effector function provided by the CH and (¾3 domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the Nanobodies® of the invention so as to provide an effector function will be clear to the skilled person, and may be chosen on the basis of the desired effector function(s). Reference i s for example made to WO 04/058820, WO 99/42077, WO 02/056910 and WO 05/017148, as well as the review by Holliger and Hudson, supra; and to the non-prepublished US provisional application by Ablynx N.V. entitled "Constructs comprising single variable domains and an Fc portion derived from IgE" which has a filing date of December 4, 2007. Coupling of a Nanobody® of the invention to an Fc portion may also lead to an increased half-life, compared to the corresponding Nanobody® of the invention. For some applications, the use of an Fc portion and/or of constant domains {i.e., CH2 and/or CH3 domains) that confer increased half-life without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more Nanobodies® and one or more constant domains with increased half-life in vivo will be clear to the skilled person, and may for example comprise two Nanobodies® linked to a C_H3 domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weight of more than 50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form a polypeptide of the invention, one or more amino acid sequences of the invention may be linked (optionally via a suitable linker or hinge region) to naturally occurring, synthetic or semisynthetic constant domains (or analogs, variants, mutants, parts or fragments thereof) that have a reduced (or essentially no) tendency to self-associate into dimers {i.e., compared to constant domains that naturally occur in conventional 4-chain antibodies). Such monomeric {i.e., not self- associating) Fc chain variants, or fragments thereof, will be clear to the skilled person. For example, Helm et al., J Biol Chem 1996 271 7494, describe monomeric FcD. chain variants that can be used in the polypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they are still capable of binding to the complement or the relevant Fc receptor(s) (depending on the Fc portion from which they are derived), and/or such that they still have some or all of the effector functions of the Fc portion from which they are derived (or at a reduced level still suitable for the intended use). Alternatively, in such a polypeptide chain of the invention, the monomeric Fc chain may be used to confer increased half-life upon the polypeptide chain, in which case the monomeric Fc chain may also have no or essentially no effector functions.

Bivalent/multivalent, bispecific/multispeciiic or biparatopic/multiparatopic polypeptides of the invention may also be linked to Fc portions, in order to provide polypeptide constructs of the type that is described in the non-prepublished US provisional application US 61/005,331 entitled immunoglobulin constructs¹' filed on December 4, 2007.

The further amino acid sequences may also form a signal sequence or leader sequence that directs secretion of the Nanobody® or the polypeptide of the invention from a host cell upon synthesis (for example to provide a pre-, pro- or prepro- form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention).

The further amino acid sequence may also form a sequence or signal that allows the Nanobody® or polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody® or polypeptide of the invention to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Suitable examples of such amino acid sequences will be clear to the skilled person, and for example include, but are not limited to, those mentioned on page 1 18 of WO 08/020079. For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies® of the invention are directed {e.g., in the treatment of cancer), or to reduce or slow the growth and/or proliferation of such a cell, the Nanobodies® of the invention may also be linked to a (cyto)toxic protein or polypeptide. Examples of such toxic proteins and polypeptides which can be linked to a Nanobody® of the invention to provide - for example - a cytotoxic polypeptide of the invention will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so- called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or more further amino acid, sequences comprise at least one further Nanobody®, so as to provide a polypeptide of the invention that comprises at least two, such as three, four, five or more Nanobodies®, in which said Nanobodies® may optionally be linked via one or more linker sequences (as defined herein). As described on pages 1 19 and 120 of WO 08/020079, polypeptides of the invention that comprise two or more Nanobodies®, of which at least one is a Nanobody® of the invention, will also be referred to herein as "multivalent" polypeptides of the invention, and the Nanobodies® present in such polypeptides will also be referred to herein as being in a "multivalent format". For example, "bivalent" and "tri valent" polypeptides of the invention may be as further described on pages 1 19 and 120 of WO 08/020079. Polypeptides of the invention that contain at least two Nanobodies®, in which at least one Nanobody® is directed against a first antigen (i.e., against CXCR-4,) and at least one Nanobody® is directed against a second antigen (i.e., different from CXCR-4,), will also be referred to as "multispecific" polypeptides of the invention, and the Nanobodies® present in such polypeptides will also be referred to herein as being in a "multispecific format". Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide that comprises at least one Nanobody® directed against a first antigen (i.e., CXCR-4,) and at least one further Nanobody® directed against a second antigen (i.e., different from CXCR-4,), whereas a "trispeciflc" polypeptide of the invention is a polypeptide that comprises at least one

Nanobody® directed against a first antigen (i. e., CXCR-4,), at least one further Nanobody® directed against a second antigen (i.e., different from CXCR-4,) and at least one further Nanobody® directed against a third antigen (i.e., different from both CXCR-4, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of the invention is a bivalent polypeptide of the invention (as defined herein), comprising a first Nanobody® directed against CXCR-4, and a second Nanobody® directed against a second antigen, in which said first and second Nanobody® may optionally be linked via a linker sequence (as defined herein); whereas a trispeciflc polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined herein), comprising a first Nanobody® directed against CXCR-4, a second Nanobody® directed against a second antigen and a third Nanobody® directed against a third antigen, in which said first, second and third Nanobody® may optionally be linked via one or more, and in particular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, the invention is not limited thereto, in the sense that a multispecific polypeptide of the invention may comprise at least one Nanobody® against CXCR-4, and any number of Nanobodies® directed against one or more antigens different from CXCR-4.

Furthermore, although it is encompassed within the scope of the invention that the specific order or arrangement of the various Nanobodies® in the polypeptides of the invention may have some influence on the properties of the final polypeptide of the invention (including but not limited to the affinity, specificity or avidity for CXCR-4, or against the one or more other antigens), said order or arrangement is usually not critical and may be suitably chosen by the skilled person, optionally after some limited routine experiments based on the disclosure herein. Thus, when reference is made to a specific multivalent or multispecific polypeptide of the invention, it should be noted that this encompasses any order or arrangements of the relevant Nanobodies®, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that the polypeptides of the invention contain two or more Nanobodies® and one or more further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001 ; Muyldermans, Reviews in Molecular Biotechnology 74 (2001 ), 277-302; as well as to for example WO 96/34103 and WO 99/23221. Some other examples of some specific multispecific and/or multivalent polypeptide of the invention can be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody® of the invention and at least one Nanobody® that provides for an increased half-life. Such Nanobodies® may for example be Nanobodies® that are directed against a serum protein, and in particular a human serum protein, such as human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one of the serum proteins listed in WO 04/003019. Of these, Nanobodies® that can bind to serum albumin (and in particular human serum albumin) or to IgG (and in particular human IgG, see for example Nanobody® VH-1 described in the review by Muyldermans, supra) are particularly preferred (although for example, for experiments in mice or primates, Nanobodies® against or cross-reactive with mouse serum albumin (MSA) or serum albumin from said primate, respectively, can be used. However, for phannaceutical use, Nanobodies® against human serum albumin or human IgG will usually be preferred). Nanobodies® that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies® directed against serum albumin that are described in WO 04/041865, in WO 06/122787 and in the further patent applications by Ablynx N.V., such as those mentioned above.

For example, the some preferred Nanobodies® that provide for increased half-life for use in the present invention include Nanobodies® that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787); Nanobodies® that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see for example WO 06/0122787); Nanobodies® that have or can provide an increased half-life (see for example WO 08/028977 by Ablynx N.V mentioned herein); Nanobodies® against human serum albumin that are cross -reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) (see for example WO 08/028977 by Ablynx N.V)); Nanobodies® that can bind to serum albumin in a pH independent manner (see for example WO2008/043821. by Ablynx N.V. mentioned herein) and/or Nanobodies® that are conditional binders (see for example WO 08/043822by Ablynx N.V.).

Some particularly preferred Nanobodies® that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies® ALB-1 to ALB-10 disclosed in WO 06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) is particularly preferred.

According to a specific, but non-limiting aspect of the invention, the polypeptides of the invention contain, besides the one or more Nanobodies® of the invention, at least one Nanobody® against human serum albumin.

Generally, any polypeptides of the invention with increased half-life that contain one or more Nanobodies® of the invention, and any derivatives of Nanobodies® of the invention or of such polypeptides that have an increased half-life, preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding Nanobody® of the invention per se. For example, such a derivative or polypeptides with increased half-life may have a half- life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding Nanobody® of the invention per se.

in a preferred, but non-limiting aspect of the invention, such derivatives or polypeptides may exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, such derivatives or polypeptides may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 1 1 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable of binding to one or more molecules which can increase the half-life of the polypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and their half-life increased by binding to molecules which resist degradation and/or clearance or sequestration. Typically, such molecules are naturally occurring proteins which themselves have a long half-life in vivo.

Another preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody® of the invention and at least one Nanobody® that directs the polypeptide of the invention towards, and/or that allows the polypeptide of the invention to penetrate or to enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody® to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such Nanobodies® include Nanobodies® that are directed towards specific cell-surface proteins, markers or epitopes of the desired organ, tissue or cell (for example cell-surface markers associated with tumor cells), and the single- domain brain targeting antibody fragments described in WO 02/057445 and WO 06/040153, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies® and the one or more polypeptides may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be clear to the skilled person, and may generally be any linlier or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing proteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers that are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, its should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent VH and VL domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each Nanobody® by itself forms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (gly_xser_y);,, such as (for example (gly ser)3 or (glysse^s, as described in WO 99/42077 and the GS30, GS15, GS9 and GS7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/0401.53 and WO

06/122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678 ).

Some other particularly preferred linkers are poly-alanine (such as AAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance,

poly(ethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for CXCR-4, or for one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

For example, in multivalent polypeptides of the invention that comprise Nanobodies® directed against a multimeric antigen (such as a multimeric receptor or other protein), the length and flexibility of the linker are preferably such that it allows each Nanobody® of the invention present in the polypeptide to bind to the antigenic determinant on each of the subunits of the multimer. Similarly, in a multispecific polypeptide of the invention that comprises Nanobodies® directed against two or more different antigenic determinants on the same antigen (for example against different epitopes of an antigen and/or against different subunits of a multimeric receptor, channel or protein), the length and flexibility of the linker are preferably such that it allows each Nanobody® to bind to its intended antigenic determinant. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) used confer one or more other favourable properties or functionality to the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g., as described herein for the derivatives of the Nanobodies® of the invention). For example, linkers containing one or more charged amino acid residues (see Table A-2 on page 48 of the International application WO 08/020079) can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of the invention will be a linear polypeptide. However, the invention in its broadest sense is not limited thereto. For example, when a polypeptide of the invention comprises three of more Nanobodies®, it is possible to link them, by use of a linker with three or more "arms", which each "arm" being linked to a Nanobody®, so as to provide a "star-shaped" construct. It is also possible, although usually less preferred, to use circular constructs.

The invention also comprises derivatives of the polypeptides of the invention, which may be essentially analogous to the derivatives of the Nanobodies® of the invention, i.e., as described herein.

The invention also comprises proteins or polypeptides that "essentially consist" of a polypeptide of the invention (in which the wording "essentially consist of has essentially the same meaning as indicated hereinabove).

According to one aspect of the invention, the polypeptide of the invention is in essentially isolated from, as defined herein. The amino acid sequences, Nanobodies®, polypeptides and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from the further description herein. For example, the Nanobodies® and polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for preparing the amino acid sequences, Nanobodies®, polypeptides and nucleic acids include the methods and techniques described herein.

As will be clear to the skilled person, one particularly useful method for preparing an amino acid sequence, Nanobody® and/or a polypeptide of the invention generally comprises the steps of:

i) the expression, in a suitable host cell or host organism (also referred to herein as a "host of the invention") or in another suitable expression system of a nucleic acid that encodes said amino acid sequence, Nanobody® or polypeptide of the invention (also referred to herein as a "nucleic acid of the invention^'"), optionally followed by:

ii) isolating and/or purifying the amino acid sequence, Nanobody® or polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

i) cultivating and/or maintaining a host of the invention under conditions that are such that said host of the invention expresses and/or produces at least one amino acid sequence, Nanobody® and/or polypeptide of the invention; optionally followed by: ii) isolating and/or purifying the amino acid sequence. Nanobody® or polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or double stranded DNA or RNA, and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA. cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism).

According to one aspect of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form. The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. To provide analogs, nucleotide sequences encoding naturally occurring VHH domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analog. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least one nucleotide sequence encoding a Nanobody® and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site- directed mutagenesis; combimng two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated, expression product; introduction of one or more restriction sites (e.g., to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PGR reaction using one or more "mismatched" primers, using for example a sequence of a naturally occurring form of CXCR-4 as a template. These and other techniques will, be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art and as described on pages 131 -134 of WO 08/020079 (incorporated herein by reference). Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such, as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), tenninator(s), etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the invention".

The genetic constructs of the invention may be DNA or RN A, and are preferably double-stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e., a vector that can provide for expression in vitro and/or in vivo (e.g., in a suitable host cell, host organism and/or expression system).

In a preferred but non-limiting aspect, a genetic construct of the invention comprises i) at least one nucleic acid of the invention; operably connected to

ii) one or more regulatory elements, such as a promoter and optionally a suitable

terminator;

and optionally also

iii) one or more further elements of genetic constructs known per se

in which the terms "operably connected" and "operably linked" have the meaning given on pages 131-134 of WO 08/020079; and in which the "regulatory elements",

"promoter", "terminator" and "further elements" are as described on pages 131-134 of WO 08/020079; and. in which the genetic constructs may further be as described on pages 131 -134 of WO 08/020079.

The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e., for expression and/or production of the amino acid sequence, Nanobody® or polypeptide of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, for example those described on pages 134 and 135 of WO 08/020079.; as well as all other hosts or host cells known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments), which will be clear to the skilled person. Reference is also made to the general background art cited hereinabove, as well as to for example WO 94/29457; WO 96/34103; WO 99/42077; Frenken et al, (1998), supra; Riechmann and Muyldermans, (1999), supra; van der Linden, (2000), supra; Thomassen et al., (2002), supra; Joosten et al, (2003), supra; Joosten et al., (2005), supra; and the further references cited herein.

The amino acid sequences, Nanobodies® and polypeptides of the invention can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g., as a gene therapy), as further described on pages 135 and 136 of in WO 08/020079and in the further references cited in WO 08/020079.

For expression of the Nanobodies® in a cell, they may also be expressed as so-called "intrabodies", as for example described in WO 94/02610, WO 95/22618 and US-A-7004940; WO 03/014960; in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer- Verlag; and in Kontermann, Methods 34, (2004), 163-170.

The amino acid sequences, Nanobodies® and polypeptides of the invention can for example also be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep (see for example US-A-6,741,957, US-A-6,304,489 and US-A- 6,849,992 for general techniques for introducing transgenes into mammals), in plants or parts of plants including but not limited to their leaves, flowers, fruits, seed, roots or turbers (for example in tobacco, maize, soybean or alfalfa) or in for example pupae of the silkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies® and polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear to the skilled person. Some preferred, but non-limiting examples include expression in the wheat germ system; in rabbit reticulocyte lysates; or in the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies® is that the polypeptides based thereon can be prepared through expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, host cells, regulatory elements, etc., will be clear to the skilled person, for example from the references cited above. It should however be noted that the invention in its broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an {in vivo or in vitro) expression system, such as a bacterial expression system, is used that provides the polypeptides of the invention in a form that is suitable for pharmaceutical use, and such expression systems will again be clear to the skilled person. As also will be clear to the skilled person, polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the (industrial) production of Nanobodies® or Nanobody®-containing protein therapeutics include strains of E. coli, Pichia pastoris, S. cerevisiae that are suitable for large scale expressiort production/fermentation, and in particular for large scale pharmaceutical (i.e., GMP grade) expression/production/fermentation. Suitable examples of such strains will be clear to the skilled person. Such strains and production/expression systems are also made available by companies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, can be used for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also made available by some of the companies mentioned above.

The choice of the specific expression system would depend in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a Nanobody® -containing recombinant protein for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e., the kind, number and position of residues attached) will depend on the cell or cell line that is used for the expression. Preferably, either a human cell or cell line is used (i.e., leading to a protein that essentially has a human glycosylation pattern) or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same as human glycosylation or at least mimics human glycosylation. Generally, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotes such as yeast usually leads to a glycosylation pattern that differs from human glycosylation. Nevertheless, it should be understood that all the foregoing host cells and expression systems can be used in the invention, depending on the desired amino acid sequence, Nanobody® or polypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is glycosylated. According to another non- limiting aspect of the invention, the amino acid sequence, Nanobody® or polypepti de of the invention is non-glycosylated.

According to one preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale pharmaceutical production, such as cells of the strains mentioned above. According to another preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is produced in a mammalian cell, in particular in a human cell or in a cell of a human cell line, and more in particular in a human cell or in a cell of a human cell line that is suitable for large scale pharmaceutical production, such as the cell lines mentioned hereinabove,

As further described on pages 138 and 139 of WO 08/020079, when expression in a host cell is used to produce the amino acid sequences, Nanobodies® and the polypeptides of the invention, the amino acid sequences, Nanobodies® and polypeptides of the invention can be produced either intracellullarly (e.g., in the cytosol, in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or can be produced extracellularly (e.g., in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. Thus, according to one non- limiting aspect of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is an amino acid sequence, Nanobody® or polypeptide that has been produced intracellularly and that has been isolated from the host cell, and in particular from, a bacterial cell or from an inclusion body in a bacterial cell. According to another non-limiting aspect of the invention, the amino acid sequence, Nanobody® or polypeptide of the invention is an amino acid sequence, Nanobody® or polypeptide that has been produced extracellularly, and that has been isolated from the medium in which the host ceil is cultivated.

Some preferred, but non-limiting promoters for use with these host cells include those mentioned on pages 139 and 140 of WO 08/020079.

Some preferred, but non-limiting secretory sequences for use with these host cells include those mentioned on page 140 of WO 08/020079.

Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above.

After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the amino acid sequence of the invention, e.g., using specific antibodies.

The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g., under suitable conditions), an amino acid sequence, Nanobody® or polypeptide of the invention (and in case of a host organi sm: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention, that may for instance be obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of the invention, the transformed host cell or transformed host organism may generally be kept, maintained and/or cultured under conditions such that the (desired) amino acid sequence, Nanobody® or polypeptide of the invention is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium, the presence of a suitable source of food and/or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g., when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person. Again, under such conditions, the amino acid sequences of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acid sequence, Nanobody® or polypeptide of the invention may (first) be generated in an immature form (as mentioned above), which may then be subjected to post-translational modification, depending on the host cell/host organism used. Also, the amino acid sequence, Nanobody® or polypeptide of the invention may be glycosylated, again depending on the host cell/host organism used. The amino acid sequence, Nanobody® or polypeptide of the invention may then be isolated from the host ceil/host organism and/or from the medium in which said host cell or host organism was cultivated, using protein isolation and/or purification techniques known per se. such as (preparative) chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g., using a specific, cleavable amino acid sequence fused with the amino acid sequence, Nanobody® or polypeptide of the invention) and/or preparative immunological techniques (i.e., using antibodies against the amino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention may be formulated as a pharmaceutical preparation or compositions comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein.

Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one amino acid of the invention, at least one Nanobody® of the invention or at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances.

Generally, the amino acid sequences, Nanobodies® and polypeptides of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020079) as well as to the standard handbooks, such as Remington's Pharmaceutical Sciences, 18^Λ Ed., Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21^st Edition, Lippincott Williams and Wilkins (2005): or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see for example pages 252-255).

For example, the amino acid sequences, Nanobodies® and polypeptides of the invention may be formulated and administered, in any manner known per se for conventional antibodies and antibody fragments (including ScFv's and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (i.e., transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, those mentioned on page 143 of WO 08/020079. Usually, aqueous solutions or suspensions will be preferred.

The amino acid sequences, Nanobodies® and polypeptides of the invention can also be administered using gene therapy methods of delivery. See, e.g.,, U.S. Patent No.

5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene encoding an amino acid sequence.

Nanobody® or polypeptide of the invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells and can additionally be transfected with signal and stabilization sequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies® and polypeptides of the invention may be systemically administered, e.g.,, orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the amino acid sequences, Nanobodies® and polypeptides of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the amino acid sequence, Nanobody® or polypeptide of the invention. Their percentage in the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the amino acid sequence, Nanobody® or polypeptide of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain binders, excipients, disintegrating agents, lubricants and sweetening or flavouring agents, for example those mentioned on pages 143-144 of WO 08/020079. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the amino acid sequences, Nanobodies® and polypeptides of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the amino acid sequences, Nanobodies® and polypeptides of the invention may be incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also be provided with an enteric coating that will allow the constructs of the invention to resist the gastric environment and pass into the intestines. More generally, preparations and formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies® and polypeptides of the invention may also be administered intravenously or intraperitoneally by infusion or injection, as further described on pages 144 and 145 of WO 08/020079.

For topical administration, the amino acid sequences, Nanobodies® and polypeptides of the invention may be applied in pure form, i.e.,, when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid, as further described on page 145 of WO 08/020079.

Generally, the concentration of the amino acid sequences, Nanobodies® and polypeptides of the invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration i a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-

%. The amount of the amino acid sequences, Nanobodies® and polypeptides of the invention required for use in treatment will vary not only with the particular amino acid sequence, Nanobody® or polypeptide selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the amino acid sequences, Nanobodies® and polypeptides of the invention varies depending on the target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication.

In another aspect, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be treated using a compound or construct of the invention (such as cancer) or for stem cell mobilisation, said method comprising

administering, to a subject in need, thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term "prevention and/or treatment" not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated. The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

The invention relates to a method for the prevention and/or treatment of at least one disease or disorder that is associated with CXC -4, with its biological or pharmacological activity, and/or with the biological pathways or signalling in which CXCR-4 is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be treated by modulating CXCR-4, its biological or

pharmacological activity, and/or the biological pathways or signalling in which CXCR-4 is involved, said method comprising administering, to a subject in need thereof, a

pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, said pharmaceutically effective amount may be an amount that is sufficient to modulate CXCR-4, its biological or pharmacological activity, and/or the biological pathways or signalling in which CXCR-4 is involved; and/or an amount that provides a level of the amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention in the circulation that is sufficient to modulate CXCR-4, its biological or pharmacological activity, and/or the biological pathways or signalling in which CXCR-4 is involved.

The invention furthermore relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence of the invention, a Nanobody® of the invention or a polypeptide of the invention to a patient, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder chosen from the group consisting of the diseases and disorders listed herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises administering, to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody® of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In the above methods, the amino acid sequences, Nanobodies® and/or polypeptides of the invention and/or the compositions comprising the same can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, the amino acid sequences, Nanobodies® and/or polypeptides of the invention and/or the compositions comprising the same can for example be administered orally, intraperitoneally (e.g., intravenously, subcutaneously, intramuscularly, or via any other route of administration that circumvents the gastrointestinal tract), intranasally. transdermally, topically, by means of a suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used. The clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration, depending on the disease or disorder to be prevented or treated and other factors well known to the clinician.

The amino acid sequences, Nanobodies® and/or polypeptides of the invention and/or the compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific amino acid sequence, Nanobody® or polypeptide of the invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of one or more amino acid sequences, Nanobodies® and/or polypeptides of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to administered can be determined by the clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or disorder to be treated, the potency of the specific amino acid sequence, Nanobody® and polypeptide of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the amino acid sequences, Nanobodies® and polypeptides of the invention will generally be administered in an amount between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1 , 10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g., by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. Generally, some guidance on the amounts to be administered can be obtained from the amounts usually administered for comparable conventional antibodies or antibody fragments against the same target administered via essentially the same route, taking into account however differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person.

Usually, in. the above method, a single amino acid sequence, Nanobody® or polypeptide of the invention will be used. It is however within the scope of the invention to use two or more amino acid sequences, Nanobodies® and/or polypeptides of the invention in combination.

The Nanobodies®, amino acid sequences and polypeptides of the invention may also be used in combination with one or more further pharmaceutically active compounds or principles, i. e., as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies® and polypeptides of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.

When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g., essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.

Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect.

However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side- effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. The clinician will also be able, where appropriate and on a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

In another aspect, the invention relates to the use of an amino acid sequence,

Nanobody® or polypeptide of the invention in the preparation of a pharmaceutical composition for prevention and/or treatment at least one disease or disorder that can be treated using a compound or construct of the invention (such as cancer) or for stem cell mobilisation; and/or for use in one or more of the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

The invention also relates to the use of an amino acid sequence, Nanobody® or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence, Nanobody® or polypeptide of the invention to a patient.

More in particular, the invention relates to the use of an amino acid sequence, Nanobody® or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment at least one disease or disorder that can be treated using a compound or construct of the invention (such as cancer) or for stem cell mobilisation, and in particular for the prevention and treatment of one or more of the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acid sequences, Nanobodies® or polypeptides of the invention may also be suitably combined with one or more other active principles, such as those mentioned herein.

Finally, although the use of the Nanobodies® of the invention (as defined herein) and of the polypeptides of the invention is much preferred, it will be clear that on the basis of the description herein, the skilled person will also be able to design and/or generate, in an analogous manner, other amino acid sequences and in particular (single) domain antibodies against CXCR-4, as well as polypeptides comprising such (single) domain antibodies.

For example, it will also be clear to the skilled person that it may be possible to "graft" one or more of the CDR's mentioned above for the Nanobodies® of the invention onto such (single) domain antibodies or other protein scaffolds, including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting will be clear to the skilled person and are well blown in the art, see for example those mentioned in WO 08/020079. For example, techniques known per se for grafting mouse or rat CDR's onto human frameworks and scaffolds can be used in an analogous manner to provide chimeric proteins comprising one or more of the CDR's of the Nanobodies® of the invention and one or more human framework regions or sequences.

It should also be noted that, when the Nanobodies® of the inventions contain one or more other CDR sequences than the preferred CDR sequences mentioned above, these CDR sequences can be obtained in any manner known per se, for example using one or more of the techniques described in WO 08/020079.

Further uses of the amino acid sequences. Nanobodies®, polypeptides, nucleic acids, genetic constructs and hosts and host cells of the invention will be clear to the skilled person based on the disclosure herein. For example, and without limitation, the amino acid sequences of the invention can be linked to a suitable carrier or solid support so as to provide a medium than can be used in a manner known per se to purify CXCR-4 from compositions and preparations comprising the same. Derivatives of the amino acid sequences of the invention that comprise a suitable detectable label can also be used as markers to determine (qualitatively or quantitatively) the presence of CXCR-4 in a composition or preparation or as a marker to selectively detect the presence of CXCR-4 on the surface of a cell or tissue (for example, in combination with suitable cell sorting techniques).

When compounds or constructs of the invention are to be used for treatment, in one aspect, a single contiguous polypeptide of the invention will be used. In one embodiment two or more polypeptides of the invention are provided in combination.

The polypeptides of the invention may be used in combination with one or more further pharmaceutically active compounds or principles, i.e., as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgment.

In particular, the polypeptides of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician, and generally include the cytostatic active principles usually applied for the treatment of the tumor to be treated.

Specific contemplated combinations for use with the polypeptides of the invention for oncology include, but are not limited to, e.g., RON antagonists, chemokine receptor antagonists, taxol; gemcitobine; cisplatin; cIAP inhibitors (such as inhibitors to cIAP 1 , cIAP2 and/or XIAP); MEK inhibitors including but not limited to, e.g., U0126, PD0325901 ; bRaf inhibitors including but not limited to, e.g. , RAF265; and mTOR inhibitors including but not limited to, e.g., RAD001 ; VEGF inhibitors including but not limited to e.g., bevacizumab, sutinib and sorafenib; Her 2 inhibitors including but not limited to e.g., trastuzumab and lapatinib; EGFR, Her3, Her4, PDGFR, FGFR, src, JAK, STAT and/or GSK3 inhibitors; selecti ve estrogen receptor modulators including but not limited to tamoxifen; estrogen receptor downregulators including but not limited to fulvestrant. Specific contemplated combinations for use with the polypeptides of the invention for inflammatory conditions include, but are not limited to, e.g., interferon beta 1 alpha and beta, natalizumab; TNF alpha antagonists including but not limited to e.g., infliximab, adalimumab, certolizumab pegol, etanercept: disease-modifying antirheumatic drugs such as e.g., Methotrexate (MTX);

glucocortioids including but not limited to e.g., hydrocortisone; Nonsteroidal antiinflammatory drugs including but not limited to e.g., ibuprofen, sulindac.

The invention will now be further described by means of the following non- limiting preferred aspects, examples and figures,

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure.

The figures are illustrative only and are not required for enablement of the disclosure.

- Figure 1 gives the sequences of the monovalent building blocks of the invention.

- Figure 2 gives the sequence of a number of non-limiting examples of

constructs/polypep tides of the invention.

- Figure 3 gives the sequences of a number of monovalent Nanobodies® against CXCR-4, useful as building blocks (either per se or in humanized and/or sequence-optimized form) in constructs and polypeptides of the invention. - Figures 4A and 4B illustrate the sequence diversity of the CXCR4 Nanobody® families obtained in Example 1. The family number, the number of different family members, and the library from which families were identified are indicated. - Figure 5 A shows the radiolabeled SDF-1 displacement of a number of purified

Nanobodies® against CXCR-4 from Example 1 on HEK-CXCR4 membrane extracts; Figure 5B shows the potencies of a number of purified Nanobodies® against CXCR-4 from

Example 1 in antagonizing infection of different HIV strains. - Figure 6A shows antagonism of SDF-1 activation by a number of purified Nanobodies® against CXCR-4 from Example 1 in the cAMP assay (see Example 1 for further experimental details)Figure 6B shows the influence of SDF-1 concentration on potency of MDX-1338 and 12A10 in the cAMP assay (see Example 1 for further experimental details). - Figure 7 shows the competition of some of the Nanobodies® of Example 1 with AMD3100 for binding to CXCR4 VLPs (see Example 1 for further experimental details).

- Figures 8 A and 8B show competition with Alexa647-!abelled 10G10 (Figure 8 A) and 238D4 (Figure 8B) for CXCR4 binding in FACS (see Example 1 for further experimental details).

- Figures 9A to 9C show that the Nanobodies® mentioned in these Figures show cross- reactivity to cynomolgus CXCR4 but not to mouse CXCR4 in binding FACS (see Example 1 for further experimental details). Figure 9D shows the estimated EC50 values of the selected constructs for binding to transiently transfected HEK293T cells with human and cynomolgus CXCR4.

- Figure 10A shows, and Figure 10B lists, the Tm values for a number of purified

Nanobodies® against CXCR-4 from Example 1 over broad pH range.

- Figures 11 A to C: data showing inverse agonism of a number of Nanobodies® against CXCR-4 from Example 1 : assay validation with Nl 19S CAM for control samples and obtained data for the monovalent lead panel. - Figures 12A and 12B show graphs obtained from the Chemotaxis assay on Jurkat cells for the benchmark, controls and Immunoglobulin Single Variable Domain constructs of the invention in the presence of HSA.

- Figures 13: Inverse agonism on cells expressing the CXCR4-N1 19S valiant or a truncated thereof. Figures 13A to F show data obtained with constructs of the invention (in some cases, compared to monovalent references or other reference compounds); Figures 13G to J show data obtained with ISV's of the invention (in some cases, reference compounds).

- Figure 14: Potencies of the HLE Nanobody® constructs in the SDF-1 competition reporter assay in absence and presence of HSA.

- Figures 15A to 15C: constructs of the invention (Table C-l 1) show cross-reactivity to cynomolgus CXCR4 but not to mouse CXCR4 in binding FACS (QC of BAF-3 cells using the a-mCXCR4 was performed during a previous FACS analysis, cells have been frozen.) Figure 15D gives the estimated EC50 values of the constructs for binding to transiently transfected HEK293T cells with human and cynomolgus CXCR4. - Figure 16: melting temperature profiles of the constructs from Table C-l 1 (Figure 16A) with the corresponding Tm at pH7 in °C (Figure 16B).

EXAMPLES

Experimental Part:

The following examples describe some embodiments of making and practicing the methods and compositions of the disclosure. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the disclosure.

The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

Example 1: Identification, optimization and characterization of Immunoglobulin single variable domains of the invention A: Isolation of parental Immunoglobulin single variable domains

A-l : Immunizations

Two llamas were immunized with frozen HEK293 cells (human embryonic kidney) transiently transfected with human CXCR4, according to standard, protocols with 6 boosts of cells (lxlOE7 cells) at weekly intervals. Blood was collected from these animals at 4 and 8 days after the 6th boost. For the DNA immunization, human chemokine receptor CXCR4 encoding cDNA (NM_003467) was purchased from Open Biosystems and introduced into the pVAX-1 expression vector (Invitrogen). Functional CXCR4 expression was confirmed by transiently transfection of HEK293 cells. In addition, four other llamas were intradermally immunized with pVAXl-hCXCR4 (2mg/llama/ immunization) using needle-free Jet injection for four times at day 0, 14, 28 and 42. Blood was collected 3 and. 9 days (PBL1, PBL2) after the last DNA administration. Three weeks after the final DNA administration, all llamas were subcutaneously injected with a single dose Caki-CXCR4 cells (2 10E7 cells), after which PBL3 and PBL4 were collected after 4 and 8 days. A biopsy of the palpable bow lymph node (LN) was collected from each llama via local surgery three to four days after the cell boost.

After completion of the immunization protocol, the immune response in each animal was analyzed by flow cytometry. To this end, Caki-CXCR4 and Caki wt cells were incubated with serial dilutions of serum samples collected at days 0, after completion of the genetic immunization (PBL2), after the Caki-CXCR4 boost (PBL4), or after the final Hek-CXCR4 boost (PBL2) (starting 1/250). Detection of cell bound llama total IgG (conventional + heavy- chain antibody) was detected via goat anti -llama IgG followed by secondary staining with PE-conjugated donkey anti-goat IgG. In three of the four llamas (with exception of llama 403) a clear increase in mean cell fluorescence (MCF) was observed in sera from the animals immunized with DNA only, compared with the pre-immune level, as measured on CXCR4- transfected CAKI cells vs. Caki control. The cell boost enhanced the serum response magnitude in all four llamas (data not shown).

For the Hek-CXCR4 immunized llamas, only one animal showed a strong serum response to CXCR4-expressing cells, compared to the untransfected control cells (the pre- immune sample gave extremely high results, and cannot be trusted).

A-2: Construction of Nanobody® Libraries

RNA extracted from peripheral blood mononuclear cells and LN were used as starting material for RT-PCR to amplify Nanobody® encoding gene fragments. For each genetic immunized llama, two separate libraries were generated: one library derived from pooled PBLl +2 cDNA ('DNA' library) and a second one derived from pooled PBL3+4 and LN (post boost or 'PB' library). These fragments were cloned into phagemid vector pAX50 derived from pUCl 19 which contains the LacZ promoter, a coliphage pill protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multicloning site and the gen3 leader sequence. In frame with the Nanobody® coding sequence, the vector codes for a C-terminal c~myc tag and a (His)6 tag. All library construction work was done at Ablynx' facilities in Porto (Portugal). Phage was prepared according to standard methods and stored after filter sterilization at 4°C for further use. A-3: Selection and screening

Selections of CXCR4 specific Nanobodies® were done using two consecutive selection rounds on CXCR4 expressing Viral-Like Lipoparticles (VLPs) using empty VLPs as negative control.

Initial selections were carried out with the libraries from the first two animals used under A). Phages were eluted with trypsin, and outputs from round 1 using lOU/well were rescued and used as input for a second round on lU/well or 10 U/well. Each selection output was analysed for enrichment factor (# phage present in eluate relative to control), and both libraries yielded strong enrichments after 2 rounds. For the other four llama's, parallel selections were performed on each of the eight DNA and PB libraries and on the two aforementioned libraries in parallel for comparison. Eight out of ten round 2 outputs showed large enrichments over controls. From each output 46 individual colonies were picked for systematic screening and comparison of titrates. Clones were grown in 96 deep well plates (1 mL volume) and induced by adding IPTG for Nanobody® expression. Periplasmic extracts were prepared according to standard methods (volume: ~ 80 μΐ). Periplasmic extracts of single clones were screened for binding to CXCR4 in ELISA using CXCR4 VLP and null VLPs (lU/well). Clones that showed a 5-fold higher signal to CXCR4 VLPs over control VLPs were considered as CXCR4 specific.

All CXCR4 specific binders identified in the screening were sequenced and redundant Nanobodies® were removed. The initial selections identified ten unique sequences from five different Nanobody® families from the first two animals. The Nanobodies® 238D2 and 238D4 (WO 09/138519) were again selected (See Table C-2).

The panel of ten Nanobodies® was recloned into the pAX51 expression vector for expression and further characterization. In frame with the Nanobody® coding sequence, the vector codes for a C-terminal c-myc tag and a (His)6 tag. Figure 4A summarizes the families obtained from the first two animals.

From the second set of four animals (389, 401, 402 and 403) 501 clones were obtained and sequenced, and 1 71 unique sequences were identified, belonging to 70 distinct

Nanobody® B-ceil lineages. Three of the five families from the first two animals (217 and 218) were found: 238D2 (Fam 34), 10 family members of 238D4 (Fam 17), and 5 family members of 283E2 (Fam 54). The results are summarized in Figure 4B.

B: Screening for SDF-1 displacement

All 171 unique sequences were screened in a radioligand displacement assay.

Membrane extracts of HEK293 cells transfected with CXCR4 were incubated with 1/10 dilutions of periplasmic extracts and 75 pM of [ ^~ FJ-SDF-1. Aspecific binding was determined in presence of 100 nM cold SDF-1. As plate controls 238D4 (HI 2), irrelevant control NB (F12) and blank (H12) were included, while the internal duplicates and the 238D4 family members divided over the plates also served as internal controls. The screen was performed three times, and average percentages of SDF-1 inhibition were calculated. Based on the results it was decided to select the Nanobodies® based on all three screening assays: (i) the 16 best in SDF-1 displacement; (ii) the 5 with highest binding signal in CXCR4 VLP ELISA, that also show ligand displacement; and (iii) the 10 with highest binding signal in Caki-CXCR4 FACS. In addition, a further nine Nanobodies® considered of interest for further characterization were added.

Figure 3 lists the sequences of a number of monovalent Nanobodies® identified in the screen described in this Example 1. The sequences of SEQ ID NO: 374, 377, 379, 381, 383, 385 to 390, 397, 398, 400, 405, 406, 408, 414, 416 to 424, 427, 430 to 432, 434, 440 to 442, 444, 445, 451, 453 and 456 were selected for further characterization (in addition to

238D2/SEQ ID NO: 447 and 238D4/SEQ ID NO: 449).

C: In vitro performance of monovalent parental Nanobodies®

C-l : Expression and, purification

A number of the Nanobodies® obtained under A) and B) were selected for purification, based on the sequence, the CXCR4 binding and the displacement results. Thereto the encoding sequences were recloned in the production vector pAX300, and expressed in E. coli as c-myc, His6-tagged proteins in a culture volume of 0.5L. Expression was induced by addition of 1 niM IPTG and allowed to continue for 3 hours at 37°C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets and resuspension in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using Histrap FF crude columns (GE healthcare). Nanobodies® were eluted from the column with 250 niM imidazole and subsequently desalted towards dPBS. The purity and integrity of Nanobodies® was verified by SDS-PAGE and western blot using anti-Myc and anti-VHH detection. C-2: Radiolabeled SDF-1 displacement

A number of the selected Nanobodies® were tested in a radioligand displacement assay using membrane extracts of HEK-CXCR4 cells. The p i values are presented in Figure 5 A. All new family members of 238D4 were confirmed as potent ligand competitors in the low nanomolar range, with clone 281E10 as the most potent. In addition, both family IV members 283B6 and 283E2 were both potent displacers. Figure 5B depicts the potencies of Nanobodies® in neutralization of infection of different HIV strains (see Example 3 of WO 09/138519).

C-3: cAMP inhibition assay: Inhibition of CXCR4-mediated signal transduction: cAMP assay The ability of 24 Nanobodies® identified in the Example 1 to inhibit G-protein signaling was assessed using a cAMP assay (DiscoverX PathHunter assay, see also Example 43 of US 61/358,495). In this assay, SDF-1 activation of the receptor reduces intracellular cAMP levels (induced by forskolin), which is subsequently detected by an enzyme complementation assay using competition between labeled cAMP and a capturing antibody. Based on data obtained during assay optimization (data not shown), a high SDF-1.

concentration of 125 nM SDF-1 (37 x EC50, 4.6 x EC90) was used for competition with the Nanobodies®.

For the cAMP assay, cells were seeded in a 96-well plate at a density of 30,000 cells/well in OCC2 medium and incubated overnight in a 37°C, 5% C0₂, humidified incubator. The medium was aspirated the next day and 45 μίΛνεΙΙ HBSS/10 mM

Hepes/ Antibody Reagent mix was added. This mix was composed of 1/3 Antibody Reagent and 2/3 HBSS/10 mM Hepes. A ½ serial dilution of Nanobody®, together with 25 nM SDF-1 (R&D Systems) and 20 μΜ forskolin (provided with kit) was added to the plate and incubated for 30 minutes at 37°C. Afterwards, 60 μΕ cAMP Detection Reagent/cAMP Solution D mixture, composed of 1 part Substrate Reagent 2, 5 parts Substrate Reagent 1 and 19 parts cAMP lysis buffer, was transferred to the wells and incubated for I hour at room temperature protected from light. Finally, 60 μΕ cAMP solution A was added to each well and incubated for 3 hours at room temperature protected from light. The plates were read with Tecan Infinite F200 using the luminescence program.

IC50 values resulting from, screening the Nanobody® panel in one or two assay runs are listed in Table C- 1. In both assays 15 Al was the most potent Nanobody®, followed by

281E10, 10E9, 10E12 and 10G10. The results obtained are further illustrated by Figures 6A and 6B.

It was found that while Nanobodies® could efficiently block the CXCL12 -induced cAMP inhibition at high ligand concentration (125 nM SDF-1), the benchmark MDX-1338 (An anti-CXCR4 monoclonal antibody; Medarex) was showing more strongly shifted potency and not full competition at the concentrations used (Figure 6B). Using lower SDF-1 concentrations of 25 nM and 5 nM, respectively, shifted the potency and restored the efficacy of MDX-1338. This suggests that MDX-1338 competes with SDF-1 in at least a partial allosteric manner and with potential differences in fine epitope specificity, as compared with the monovalent Nanobodi.es®, which appear to show true orthosteric competition

The results also suggests that a number of the monovalent Nanobodies® have significantly better potency in the cAMP assay at physiologically relevant ligaiid

concentrations, as compared with the bivalent benchmark^

C-4: Jurkat assay: inhibition of CXCR4-mediated chemotaxis

The panel of 24 Nanobodies® tested in the cAMP assay were also analysed for inhibition of CXCL 12 -induced chemotaxis of Jurkat cells endogenously expressing CXCR4 (see WO 09/1385] 9, the paragraph bridging pages 193-914 and US 61/358,495, Example 4).

To measure functional activity of the ISV's/constructs, cells from the Jurkat ceil line (Jurkat E6-1; ATCC) were seeded 1 day before the experiment at a cell concentration of 0.5x10⁶ cell/ml in complete medium (RPMI1640 + 10% FBS). The following day, SDF-1 (200pM final concentration, R&D Systems) and serially diluted ISV's/constructs were added to the bottom of a small chemotaxis plate (Neuprobe 106-5) in a total volume of 29ul. A chemoiaxis filter membrane (ChemoTx® Disposibla, pore size 5μη ) was placed on top of the wells, ensuring that the membrane was in contact with the solution in the wells below. A Nanobody® dilution (lOul at 5X the serially diluted final concentration as below the membrane in each well) was added on top of the membrane, followed by 40ul of Jurkat cell suspension (6.25xl0⁶ cell/ml). The plates were incubated for 3 hours at 37°C in a humidified incubator (5% CO₂). After incubation, the filters were carefully removed and the cells in the well below were resuspended in the existing solution. The complete cell suspension was transferred to the corresponding wells of white polystyrene Costar plates. After this, 30ul of Cell Titer Glo reagent (Promega G7571) was added, to each well, followed by a 10 minute incubation, with shaking in the dark. Luminescence was measured (1 sec/well) using Envision 2103 Multilabel Reader with emission filter 700 (Perkin Elmer).

In the present assay, a concentration of 200 pM SDF-1 was used as chemoattractant, corresponding to the EC 75 value. The results are also depicted in Table C-l . Table C-1: Potencies of CXCR4 Nanobodies®: (i) in competition with SDF-1 (125 n!Vl) in inhibition of c AMP levels and (ii) in inhibitio of SDF-1 induced migration of Jurkat cells (Note: 4CXCR238D02, 4CXCR238D02 and MDX-1338 are reference compounds. Also, where no value is mentioned, the value was not determined).

Table C-1 (continued):

4CXCR 7.897E-7 to 5.69E-9 to

45 1.0E-06 1.1E-08 86

01ΘΒ03 1 .295E-6 2.27E-8

4CXCR 1.394E-7 to 9.6E- 7.648E-7 to 1.16E-9 to 2.3E- 1.250E-9 1O 4.1E- 2.4I6E-9 to

49 1.6E-07 2.1E-09 93

010G10 I .940E-7 07 1.200E-6 3.68E-9 09 4.294E-9 09 6.980E-9

Table C-1 (continued):

Nb cAMP inhibition assay Chemotaxis Jurkat cells

IC5 IC₅₀ ICso

Clone 1C₅₀ (M) I so (M) % CI ICso

Family CI ICso 95% (M) CI ICso 95% CI I so 95% (M) CI I so 95% (M)

ID n=l Inhib. 95%

n=2 n=2 H=3

4CXCR 7.549E-7 to 5.1E- 4.090E-6 to 1.20E-9 to 3.1E- 2.037E-9 to 1.2E- 6.1 15E-9 to

53 9.3E-07 1.8E-09 90

01 OA 10 1.135E-6 06 6.364E-6 2.65E-9 09 4.787E-9 08 2.199E-8

4CXCR 6.682E-7 to 2.3E- 1.358E-6 to 1.41 E-9 to 3.4E- 2.257E-9 to 3.8E- 2.273E-9 to

54 7.5E-07 2.3E-09 90

283B06 8.440E-7 06 3.943E-6 3.91E-9 09 5.025E-9 09 6.405E-9

4CXCR 3.405E-7 to 2.5E- 2.104E-6 to 2.03E-9 to 8.1E- 5.478E-10 to

57 4.0E-07 3.3E-09 88

014A02 4.588E-7 06 2.944E-6 5.43E-9 10 L204E-9

4CXCR 9.350E-7 to 6.86E-9 to

64 1.1E-06 L3E-08 80

015A12 1.215E-6 2.37E-8

4CXCR 7.641E-7 to 3.99E-9 to

66 9.5E-07 7.0E-09 85

014A04 1 .171 E-6 1.23E-8

M.DX- 7.8E- J .1 12E-7 to 2.13E-9 to

7.5E-07 3.0E-09 87

1338 07 5.509E-6 4.35E-9

D: Epitope binning of Nanobodies®

In the non-prepublished PCX application PCT/EP2010/064766, the binding epitope of the Nanobodies® 238D2 and 238D4 was identified using shotgun mutagenesis (Integral Molecular). This analysis suggested that 238D2 and 238D4 both bind to ECL2 of CXCR4, with overlapping but distinct binding modes. Both Nanobodies® compete with AMD3100 (A bicyclam compound that binds SDF-1 ; See e.g., Antimicrob Agents Chemother 2000, 44: 1.667-11673). which also binds to residues at the base of ECL2, but which has a smaller footprint than the Nanobodies®.

Similar techniques as used in PCT/EP2010/064766 were used to determine the epitope binding of the Nanobodies® identified in Example 1.

D-l : Competition ELISA using VLP's

As a first approach for epitope binning, a competition ELISA between AMD3100 and representatives of different Nanobody® families was set up. Maxisorp plates were coated with 0.5U/wel3 CXCR4 VLPs, after which a non- saturating concentration of Nanobody® (5 nM) was incubated in presence or absence of 25 uM of AMD3100. Nanobody® binding was measured by detection of the Myc tag. While the vast majority of Nanobodies® were outcompeted from binding to CXCR4 by the excess AMD31G0, the binding of 8

Nanobodies® was disrupted by less than 50% (data not shown). These partially competing Nanobodies® were next analysed for dose-dependent inhibition by AMD3100, using the

ECgo concentrations of each Nanobody®. As positive control, Nanobody® 281D4 (Fam 17) was included. Using this assay format, as shown in Figure 7, one Nanobody® lOGlO showed no competition with AMD3100, while four other Nanobodies® (2B7, 13D8, 15A1, 15H3) showed partial competition with AMD3100.

D-2: Competition assay using CXCR-4 expressing cells

To confirm the results of D-l, Nanobodies® 10G10 and 238D4 were labeled by Alexa- 647 to allow direct competition of the Nanobody® with AMD3100. Labeled Nanobodies® were titrated on Caki-CXCR4 cells to assess the EC30 value, after which the competition with each of the unlabelled Nanobodies® or AMD3100 was determined. Figures 8A an 8B show that in this setup Alexa-IOGIO is partially competed with for binding to CXCR4 by

AMD3100 (41%), while the control 238D4 is fully displaced by increasing concentrations of AMD3100. By contrast, Nanobody® 238D4 can fully compete with 10G10, and vice versa, suggesting that despite the different competition behavior with AMDS 100, both Nanobodies® bind in close proximity with each causing sterical hindrance. An alternative interpretation of this data could be that 10G10 (and the other Nanobodies® showing partial competition with AMD3100) are having an allosteric effect on CXCR4, changing

conformation such that the binding of AMDS 100 is reduced.

D-3: Competition assay for ISV's

To determine whether a given ("first") ISV against CXCR-4 competes with another ("second") ISV against CXCR-4 (for example, with one of the ISV's of the invention) for binding to CXCR-4, either the competition ELISA using VLP's described under D-l and/or the competition assay using CXCR-4 expressing cells described under D-2 can be used, in each case using the second ISV (for example, an ISV of the invention) as a reference rather than AMD3100. The first ISV is said to compete with the second ISV if it competes with the second ISV for binding to CXCR-4 in either the assay of D-l, or the assay of D2, or both.

D-4: Epitope mapping and footprint analysis (PCT/EP2Q 10/064766, Example 1.9)

It was also investigated, by an epitope mapping effort, which of the anti-CXCR4 Nanobodies® from Example 1 could be characterized as being "238D2-like" (group I) and/or "238D4-like" (group II) in binding behavior to CXCR-4, by a epitope mapping effort.

Thereto three CXCR4 variants were generated containing a single point-mutation in ECL2 of CXCR4 that allowed for discrimination between 238D2 and 238D4-like epitopes: V196E (specifically disturbing D2 binding), Dl 87V (specifically disturbing D4 binding), F189V (disturbing binding of both D2 and D4). In addition, the cynomolgus monkey ortholog was included, which differs from human CXCR4 only at four positions: three mutations in the lineai- N-terminus (M24I, N35H, 38R) and one position in the ECL (N176S).

For the epitope mapping, transient transfections of the three CXCR4 mutants and wild- type human CXCR4 in the pCDNA3.1 vector were done in HEK293T cells, after which Nanobody® binding of different families was assessed by flow cytometry using detection of the Myc-tag, followed by secondary anti-mouse PE. Two concentrations of Nanobody® were tested, 10 nM and 100 nM, and the experiment was repeated with 30 nM Nanobodies® with essentially the same results. The mab 12G5 bound to all point-mutants and thus served as a control to normalize for different membrane expression levels. Binding of the Nanobodies® to HEK293T hCXCR4 cells was used to calculate the percentage of binding to the mutant receptors for each Nanobody® concentration, and a position was considered critical when less than 25% residual binding was observed, while if > 70% of binding was retained the position was not considered critical for binding. For some Nanobodies® only partial loss of binding was observed, which may indicate that the introduced mutation was tolerated but still positioned within the footprint.

Again, based on to their different binding patterns or footprints, Nanobodies® could be grouped according to their different binding patterns, or footprints, not only in "238D2-like" and "238D4-like" but also into other new groups (Table C-2). Most Nanobodies® showed loss of binding to at least one of the three point mutants, while only a small number of Nanobodies® showed no binding to the cynomoigus CXCR4. Only one Nanobody® tested (15H3) was insensitive to any of the introduced mutations for its interaction with CXCR4, showing a similar pattern as MDX38 antibody. This suggests that the epitope of 15H3 may be considerably different from the other Nanobodies® and located outside the ECL.

Table C-2: Footprint analysis of different CXCR4-specific Nanobodies® for binding to mutant CXCR4 receptors and the cyno CXCR4 orthologue expressed on Hek293T cells.

Table C-2 (continued):

Note:

Binding was calculated according to the formula: [Ratio 15G5 mAb] = [Binding mutant CXCR-4] I [Binding human CXCR-4]

The percentage binding was calculated by (i) calculating {binding hCXCR-4] x [ratio 12G5 mAb]; (ii) subtracting from the value thus obtained the [Binding mutant CXCR4] (iii) dividing the value thus obtained by [Binding hCXCR4] x [ratio 12G5 mAb]; (iv) subtracting the value thus obtained from 1.000; and (v) multiplying the value thus obtained with 100%.

E: Species Cross-Reactivity Analysis {binding to mouse and Cynomolgus CXCR4)

Ten Nanobodies® from Example 1 were evaluated for cross-reactivity between human CXC -4, mouse and cynomolgus CXCR-4 in a binding FACS assay. Nanobody® binding to HEK293 parental cells and HEK293 cells transfected with either the human or Cynomolgus CXCR4 plasmid was detected using anti-myc antibodies, followed by subsequent anti-mouse- PE secondary staining. Binding to mouse CXCR4 was assessed by using murine BaF-3 cells (pre B cell line) that endogenously express CXCR4. Results are shown in Figures 9A to 9D.

None of the tested Nanobodies® is able to bind mouse CXCR4. This was not considered surprising considering the significant sequence diversity between mouse and human CXCR4 in EL 2 and the N-terminal region, involved in ligand binding (alignment not shown). In line with the high sequence conservation between human and cynomolgus CXCR4. most Nanobodies® show similar binding affinities for human and cynomolgus CXCR4, with the exception of 10E12 and 10G10 (which showed a reduced affinity for cynomolgus CXCR4 receptor and, moreover, EC50 values that were not of the same order as their EC₅0 for human CXCR-4).

F: Thermal shift assay

The temperature stability of individual Nanobodies® was measured in a thermal shift assay. Nanobodies® were diluted to a concentration of 0.2 mg/ml in buffers of different pFI, Melting temperature (Tm) was determined by stepwise increase in temperature in presence of Sypro Orange, a dye that binds to Trp residues that become exposed upon unfolding of the protein, using the Lightcycler for detection (Roche). The melting temperature profiles of the ten Nanobodies® for which Tm was determined are shown in Figure 10A and listed in Figure 10B. All these Nanobodies® have Tm above 60 °C at pH7.0, ranging to those with Tm close to 75 °C (10E12).

G: Selection of panel of the preferred Nanobodies®

Based on the results in the different characterization assays described above, the following panel of nine preferred (lead) Nanobodies® (see Table C-3) was selected for further use (for example, as building blocks for making multivalent, multispecific and/or biparatopic constructs/polypeptides of the invention). P4CXCR3E12 was not chosen because its footprint is similar to that of 10E9, and because it is only a partial competitor for SDF-1 in the competition FACS (see Table 5). P4CXCR15H3 was added because it showed binding to all generated CXCR4 mutants during the epitope mapping experiments (Table C-2). Table C-3: preferred ISV's of the invention:

SDF-1 Binding Binding

cAMP

displaceTm Cynomulgus Chemotaxis

ΛΜ 1) 100 inverse

Clone Family Caki- VLP Footprint ICso (nM) #

ment iC₅₀ IC_S„ (nM) # agonise)

CXCR4 EC50 oss-react. com p. (%)

CO cr Jurkat

(nM) 125 nM SDF

EC₅₀ (nM ) (nM)

15A 1 32 5.1 nd 0.20 69.8 ++ 85 F189 95, 399 2.7, 0.8 -

D 187 F189

*

281E10 17 3.1 3.1 nd 71.1 100 102, 773 3.5, 4.7 weak

(238D4)

F189

10G 10 49 7.9 3.3 0.29 65.3 ^•f 0 164, 958 2.1, 2.3 - -V196

10E12 2 3 nd 0.22 63.2 + 100 VI 96 191, 945 3.5, 1 .4 weak

F189 V 196

10E9 44 4.7 2.4 0.52 65.7 ^— 100 232, 71 1 3.5, 1.2 - (238.D2)

D187 F 1 89

14A2 57 4.1 4.0 0.73 62.8 ++ 100 395, 2490 3.3, 0.81. -

V I 96

Table C-3 (continued):

* Footprint cyno X -react result not obtained on actual NB, but on family member and anticipated to be the same (see table epitope binning results) ** Has been determined for family member 281D4, here competition with AMD31 0 was 95%

*** 15H3 showed high efficacy, but low potency

(") Used and mentioned as a reference.

H: Sequence Analysis

Analysis of the protein sequences of the lead panel for manufacturability and stability red flags (identification of sites of potential post translational modification sites and chemical degradation, for example of potential methionine oxidation sites or potential N- deamidation sites/D-isomerisation sites such as NG or NS sites) was undertaken (results not shown - see the general description for an explanation of possible sites for post-iranslational modification and how ISV's Nanobodies® can be sequence-optimized to exclude such sites).

I: Analytical SEC lead panel

The nine Nanobodies® of Table C-2 and the Nanobodies® 6G9 and 3E12 were analysed for the potential occurrence of aggregates by analytical size exclusion

chromatography (SEC) using the Phenomenex column. None of the Nanobodies® showed aggregation (data not shown). J: Screenin for inverse agonist activity

The mode of action of the monovalent lead panel was determined using the

constitutively active CXCR4 mutant (CAM.) Nl 19S. This mutant is described in literature as having a better response to CXCL12 and reversible conformation compared to the Nl 19A CAM. (Zhang et al, 2007, Mol Pharmacol. , 72(5):1310-21). For assay optimization, both CHO and HEKT cells were tested with the SRE, CRE and NFAT-luciferase response. Best read-out was obtained using CHO cells transfected with the Nl 19S CAM and the pCRE- luciferase reporter gene. Transfected cells were treated with a serial dilution of the compounds and 2μΜ Forskolin in order to upload the cells with cAMP. A decrease of the CXCR4 CAM activity is monitored by an increase of luminescence. Figures 1 1 A to 1 1C show the data obtained for the relevant control samples and the monovalent lead panel.

Three monovalent Nanobodies® - 281 El 0 (weak inverse agonist), 10E12 (weak inverse agonist) and 15H3 (high efficacy) - exhibit inverse agonist activity on the CXCR4 Nl 19S variant. Although 10E12 and 281E10 have a better IC₅o, the efficacy for 15H3 is higher, even comparable with the multivalent control samples (Note: the ISV of the invention. 281E10 is a family member of 238D4, but with higher potency in the Jurkat assay). 281E10, 10E12 and 15F13 were also tested on a cells expressing a truncated version of the CXCR4N1 19S mutant. Reference is made to Figures 13G to 13 J. Example 2: Formatting of ISV's of the invention

A: Formatting of non-humanized anti-CXCR4 Nano bodies® A-l : Monovalent Half-Life Extension (HLE and bivalent constructs

Some of the nine anti-CXCR4 Nanobodies® selected Example 1 were formatted for HLE, with the anti-CXCR4 Nanobody® N-terminal3y linked to ALB8 (Also referred to herein as Alb-8 or ALB-8; See also WO06/122787 via a BOGS linker. In parallel, constructs were generated for a bivalent format with a 20GS linker in between. All constructs were generated in E.coli. The formatted anti-CXCR4 constructs thus obtained are listed in Table C-4.

Table C-4: non-limiting examples of constructs/polypeptides of the invention

Format

ID ID (monovalent HLE

(bivalent construct)

construct)

P4CXCR1001 15A1-20GS-15A1 P4CXCR1010 15A1-30GS-ALB8

P4CXCR1002 281E10-20GS-281E10 P4CXCR101 1 281 E10-30GS-ALB8

P4CXCR1003 1 GG10-20GS-10G10 P4CXCR1012 10G10-30GS-ALB8

P4CXCR1004 10E12-20GS-10E12 P4CXCR1013 10E12-30GS-ALB8

P4CXCR1005 10E9-20GS-10E9 P4CXCR1014 10E9-30GS-ALB8

P4CXCR1006 14A2-20GS- 14A2 P4CXCR1015 14A2-30GS-ALB8

P4CXCR1007 10A10-20GS-10A10 P4CXCR1016 10A10-30GS-ALB8

P4CXCR1008 283B6-20GS-283B6 P4CXCR1017 283B6-30GS-ALB8

P4CXCR1009 3E12-20GS-3E12 P4CXCR1018 3E12-30GS-ALB8

Notes:

P4CXCR1001 ( 15A1-20GS-15A1 ) and P4CXCR1006 (34A2-20GS-14A2) were found to be less preferred because they showed some protein degradation upon expression.

Similarly, P4CXCR1005 (10E9-20GS-10E9) and P4CXCR1015 (14A2-30GS-ALB8) were found to be less preferred because they showed some protein degradation upon expression. A-2: Expression and purification

The constructs were cloned in the production vector pAXlOO and expressed in E, coli as c-myc, His6-tagged proteins in a culture volume of 0.5L. Expression was induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37°C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets and resuspension in dPBS. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC) using Histrap FF crude columns (GE healthcare). Nanobodies® were eluted from the column with 250 mM imidazole and subsequently desalted towards dPBS. The purity and integrity of Nanobodies® was verified by SDS-PAGE and western blot using anti-Myc and anti-VHH detection.

B: Characterization of constructs

B-l : Competition with biotinylated SDF-1 on HEK293T-CXCR4 cells

The constructs were analysed for their binding to HEK293T-CXCR4 cells, and their ability to compete with SDF-1 for receptor binding in flow cytometry. To this end serial dilutions of Nanobodies® were incubated with 30 nM of biotinylated SDF-1 (R&D Systems Fluorokine kit) on cells, after which ligand binding was visualised using extravidin-PE. In parallel the HLE constructs were tested in presence of 50μΜ HSA. IC₅o values were determined in the SDF-1 displacement assay (data not shown) and compared with data obtained for the corresponding monovalent ISV's of the invention (data not shown). The results suggest that the addition of a serum albumin binding moiety appears to have a negative effect on the IC50 for the HLE constructs of 10A10 and 10G10, and appears to negatively impact on SDF-1 competition for 10E12 and 281 E10.

B-2: Inhibition of CXCR4-mediated chemotaxis

The constructs were analyzed for inhibition of CXCL12-induced chemotaxis of Jurkat cells endogenously expressing CXCR4. A concentration of 200pM SDF-1 was used as chemo-attractant, corresponding to the EC₇5 value. To investigate the binding of ALB8 to HSA, the samples were also tested in the presence of 15μΜ HSA. In Table C-5 the IC50 values (ranked according to the most potent HLE constructs in the chemotaxis assay with addition of HSA) are shown of a single experiment. ALB -formatting of 10A10 seems to negatively influence the chemotaxis. Upon addition of HSA, the biggest impact is observed for the P4CXCR1013 and P4CXCR1018 (I 0E12-ALB8 and 3E12-ALB8 respectively).

Table C-5: potency of constructs/polypeptides of the invention in the Jurkat assay.

Chemotaxis relative chemotaxis relative

IC₅() to MDX ICso to MDX

Monovalent ISV's

(M) (M)

-HSA +HSA

MDX (average) 2,01 E-09 I.78E-09

P4CXCR281E10 2,08E-09 1,10 2,20E-09 1 ,17

P4CXCR10E09 3,29E-09 1,38 1 ,93 E-09 0,81

P4CXCR283B06 3,82E-09 1 ,81 2,79E-09 1,32

P4CXCR 15.401 1.70E-09 0,74 l,18E-09 0,52

P4CXCR10G1 4, 11 E-09 1 ,36 7,74E-10 0,26

P4CXCR10E12 2,78E-09 1,24 l, 12E-09 0,5

P4CXCR03EI2 2,80E-09 1,07 9.10E-10 0,35

P4CXCR10A10 l , 36E-08 5,43 9, 1 1 E-09 4,27

Table C-5 (Continuation)

The assay was repeated for the constructs 4CXCR1012 (10G10-Alb8), 4CXCR1013 (10E12-Alb8) and 4CXCR1017 (283B6-A3b8). The results are shown in Table C-6 below.

Table C-6: potency of constructs/polypeptides of the invention in the Jurkat assay.

C: Bivalent and biparatopic HLE constructs

The nine monovalent anti-CXCR4 Nanobodies® selected in Example 1 were formatted into bivalent and biparatopic constructs with HLE (ALBS). The ALB 8 is in the C-terminal position, linked to the bivalent/biparatopic construct with a 35 GS linker. A 20GS-linker is used between the anti-CXCR4 building blocks. The formatted constructs thus obtained are listed in Table C-7 by number/code. The sequences of the corresponding formatted constructs can be found in Figure 2. The vertical column indicates the ISV at the N-terminus and the horizontal column indicates the ISV between the N-terminal ISV and Alb-8. For example, construct 2009 (4CXCR2009) is [10A10]-20GS~[10E9]-35GS-[Alb~8] (SEQ ID NO: 288). Table C-7: Bivalent and biparatopic HLE constructs comprising ISV's of the invention

C-l : Expression and purification

The constructs 4CXCR2000, 4CXCR2001 , 4CXCR2002, 4CXCR2003, 4CXCR2004, 4CXCR2005, 4CXCR2006, 4CXCR2007, 4CXCR2009, 4CXCR2021, 4CXCR2023, 4CXCR2029, 4CXCR2030, 4CXCR2031, 4CXCR2033, 4CXCR2034, 4CXCR2036, 4CXCR2038, 4CXCR2039, 4CXCR2041, 4CXCR2042, 4CXCR2047, 4CXCR2048, 4CXCR2055, 4CXCR2058, 4CXCR2060, 4CXCR2061, 4CXCR2066, 4CXCR2068,

4CXCR2073 and 4CXCR2078 were successfully cloned and expressed in Pichia pasloris. For this purpose, these constructs were cloned in Pichia vector pAXl 66 via a library approach. For missing building blocks, the constructs were picked up via mini libraries in E.coli and subsequently cloned into P. pastoris.

All proteins were expressed in Pichia pastoris as tagless proteins in a culture volume of

16mL BMCM. Yeast was grown in YPD, followed by BGCM and after 2 days, expression was induced by performing a medium switch to BMCM and further induction with methanol at several time points during the following 48 hrs. Medium was purified via affinity chromatography, using MabCapA resin (Poros). Nanobody® constructs were eluted from the column using glycine buffer at low pH and the fractions were directly neutralized by the addition of Tris pH8.8. Subsequently samples were desalted towards dPBS using Zeba Spin columns (Perbio). The purity and integrity of Nanobodies® was verified, by SDS-PAGE (data not shown).

D: Characterization of constructs

D-l : Inhibition of CXCR4-mediated chemotaxis

A panel of 31 HLE biparatopic/bivalent Nanobodies® expressed under C) was analyzed for inhibition of CXCL12-induced chemotaxis of Jurkat cells endogenously expressing CXCR4. To have a better assay window and to prevent ligand depletion, the concentration of SDF-1 was augmented till InM. To investigate the binding of ALB 8 to HSA, the samples were also tested in the presence of 15μΜ HSA. Some representative graphs obtained during this screen are given in Figure 12A and 12B. In Table C-8 the IC50 values are shown of a single experiment, ranked according to most potent HLE multivalent chemotaxis inhibitors in presence of HSA.

Even though some-interplate variation for the benchmark MDX-1338 was observed (which somewhat hampered ranking of the Nanobodies® with respect to potencies and comparison to the benchmark MDX-1338), the data shows that formatting improves the potency of the Nanobodies® to inhibit SDF-1 induced chemotaxis of Jurkat cells. Moreover, all constructs with curves suitable for calculation of an IC50 value seem to be better or comparable to the benchmark MDX-1338. The impact of HSA on the inhibition of chemotaxis seems to be minimal for most of the constructs.

Table C-8: Potencies of HLE biv alent and biparatopic Nanobodies® for inhibition of migration of Jurkat cells in the presence and absence of HSA

Note: (*) indicates that IC50 determination was not possible due to incomplete titration or flattened curves)

Chemotaxis Chemotaxis ratio

Construct ICso (M) CI IC50 95% IC50 (M) CI IC₅₀ 95% +HSA

-HSA +HSA /-HSA

2.000e-010 to 7.836e-01 1 to

P4CXCR2039 2,56E-30 1,45E-10 0,56

3.286e-010 2.673e-010

1.372e-010 to 9.031e-0i l to

P4CXCR2038 2,88E-10 1.52E-10 0,53

6.032e-010 2.547e-010

1.485e-010 to 1.072e-010 to

P4CXCR2047 2,10E-10 1.68E-10 0,80

2.963e-010 2.620e-010

2.828e-010 to 7.858e-011 to

P4CXCR2060 3.62E-10 1.72E-10 0,48

4.643e-010 3.784e-010

3.168e-010 to 1.261e-010 to

P4CXCR2034 4,05E-10 1.79E-10 0,44

5.163e-010 2.540e-010

6.579e-01 1 to 9.902e-01 1 to

P4CXCR2041 1,34E-10 1,83E-10 1,37

2.715e-010 3.390e-010

1.661 e-010 to 1.103e-010 to

P4CXCR2042 2,36E-10 2,04E-10 0,86

3.362e-010 3.783e-010

1.303e-010 to 5.926e-01 1 to

P4CXCR2066 1,88E-10 2.12E-10 1,13

2.726e-010 7.581e-010

3.582e-010 to 1.549e-010 to

P4CXCR2055 5,00E-10 2.21E-10 0,44

6.977e-010 3.158e-010

1.328e-010 to 1.403e-010 to

P4CXCR2068 1.93E-10 2,22E-10 1 ,15

2.805e-010 3.499e-010

Table C-8 (continued):

Chemotaxis Chemotaxis ratio

Construct IC50 (M) CI IC₅₀95% rC₅₀(M) CI IQo 95% +HSA

-HSA +HSA /-HSA

1.589e-010to L563e-010to

P4CXCR2048 2.31E-10 2,27E-10 0,98

3.355e-010 3.302e-010

2.759e-010to 1.874e-010to

P4CXCR2061 4_;43E-]0 2J2E-10 0,62

7.099e-010 3.954e-010

2.287e-010to 2.738e-010to

P4CXCR2021 3,39E-10 4J8E-10 1,23

5.025e-010 6.372e-010

4.620e-010to 3.808e-010to

P4CXCR2036 7,68E-10 6,62E-10 0,86

1.276e-009 1.150e-009

1.511e-010to 4.747e-010 to

P4CXCR2023 1,95E-10 7,05E-10 3,62

2.504e-010 1.047e-009

1.175e-010to 4.826e-010to

P4CXCR2029 L55E-10 8,47E-10 5,45

2.055e-010 L485e-009

2.265e-010to 6.727e-010to

P4CXCR1023 3,82E-10 8,66E-10 2,27

6.437e-010 U16e-009

2.847e-010to 6.619e-010 to

P4CXCR2078 3,99E-10 9,85E-10 2,47

5.593e-0J0 1.466e-009

MDX

61E-09 l,70E-09 1,06 (average)

6.625e-011 to

P4CXCR2001 8.94Ε-Π

1.207e-010 (*)

1.078e-010to

P4CXCR2005 1.43E-10

1.886e-010 (*)

1.067e-010to

P4CXCR2004 1.53E-10

2.200e-010 (*) Table C-8 (continued):

Chemotaxis Chemotaxis ratio

Construct IC₅₀ (M) CI ICso 95% ICso (M) CI IC₅o 95% +HSA

-HSA /-HSA

L239e-010 to

P4CXCR2030 1,97E-10

3.117e-010 (*)

1.940e-010 to

P4CXCR2006 2,38E-10

2.925e-010 (*)

2.121e~010 to

P4CXCR2007 2 «.73 J^- 10

3.512e-010 (*)

2.825e-010 to

P4CXCR2009 3,58E-10

4.527e-010 (*)

3.456e-010 to

P4CXCR2002 4,39E-10

5.582e-010 (*)

3.160e-010 to

P4CXCR2003 4,46E-10

6.289e-010

3.805e-010 to

P4CXCR2000 5,03E-10

6.640e-010 (*)

3.727e-010 to

P4CXCR2058 5,59E-10

8.386e-010 (*)

5.795e-010 to

P4CXCR2031 7,57E-10

9.893e-010 (*)

P4CXCR2073 (*) (*)

P4CXCR2033 (*) (*) Ό-2 Screening for inverse agonist activity

The mode of action of the bivalent/biparatopic HLE constructs was investigated using the constitutively active CXCR4 mutant (CAM) Nl 19S. This mutant is described in literature (Zhang et ah, 2007, Mol Pharmacol. , 72(5):1310-21) as having a better response to CXCL12 and reversible conformation compared to the Nl 19 A CAM, which was confirmed by comparing assays with the same set-up, but using a different CAM (data not shown). For assay optimization, both CHO and HEKT cells were tested with the SRE and CRE and NFAT-luciferase response. Best read-out was obtained using CHO cells transfected with the Nl 19S CAM and the pCRE-luciferase reporter gene. Transfected cells were treated with, a serial dilution of the compounds and 2μΜ Forskolin in order to upload the cells with cAMP. A decrease of the CXCR4 CAM activity is monitored by an increase of luminescence.

Surprisingly, all formatted Nanobodies® - regardless of their composition - seemed to show inverse agonist activity using this assay set-up, whereas only three monovalent building blocks (10E12 - 281E10 - ] 5H3) could interfere with the cAMP pathway using the CAM. Some representative curves are shown in Figures 13A to 13E, and obtained IC₅0 values are listed in Table C-9. Moreover, all tested mAbs were found in this assay not to significantly inhibit cAMP signaling. Although the invention is not limited to an specific mechanism or hypothesis, this may indicate that the biparatopic/bivalent Nanobody® constructs and the mAbs possibly have a different mode of action.

To further investigate the possible mechanisms that might be involved in/contribute to the inverse agonism observed for the ISV's and constructs of the invention, the above assay was repeated for a number of ISV's/constructs, but using a truncated version of the CXCR-4- l 19S mutant (Zhang et al, supra). The results are shown in Figure 13F.

Most Nanobodies® display reduced inverse agonistic activity using the truncated CXCR4 variant, except for ALX-0651 (a construct based the 238D2 and 238D4 building blocks; which is accordance with US 61/358,495) and 4CXCR2060 Nanobodies®, where internalization seems to play a minimal/no role.

Also, the monovalent ISV's 15H3, 10E12 and 4CXCR1024 (a construct in which ALX- ^' 0651 is linked to Aib-8) were tested on cells expressing the truncated CXCR-4 variant. The results are shown in Figure 13G (non-truncated, without HSA), 13H (non-truncated, with HSA), 131 (truncated, without HSA) and 13 J (truncated, with HSA). Although the invention is not limited to any specific mechanism or hypothesis, the following specific observations can be derived from these results:

15H3 has inverse agonist activity to CXCR4 Nl 19S CAM, which likely may be driven (at least to some extent, if not to a large extent) by internalisation;

10E12 lhas a weaker inverse agonist activity to CXCR4 Nl 19S CAM, which likely is not (or at least not to a significant extent) driven by internalisation;

10G10 and 238B6 essentially show little or no inverse agonist activity to CXCR4 N1 19S CAM

The constructs 4CXCR20384 and CXCR2048 of the invention show inverse agonist activity to CXCR4 Nl 19S CAM, which likely may be driven (at least to some extent, if not to a large extent, and probably to a larger extent than for the reference construct 4CXCR1024) by internalisation

Table C-9: Potenc of the HLE constructs for inverse agonism (CXCR4-M 19S) in the absence of HSA

Inverse

Construct agonism CI ICso 95% Bottom Top

IC_So (M)

P4CXCR2039 6,628E-10 4.981e-010 to 8.820e-010 6697 12785

P4CXCR2048 6_;866E-10 5.313e-010 to 8.873e-010 6735 14028

P4CXCR2042 9,505E-10 6.695e-010 to 1.349e-009 6736 13840

P4CXCR2029 L027E-09 6.295e-010 to 1.676e-009 641 1 10182

P4CXCR2066 L08E-09 6.235e-010 to 1.872e-009 7180 12717

P4CXCR104 l,1 9E-09 8.671e-010 to 1.523e-009 5980 13001

P4CXCR2005 l,215E-09 8.219e-010 to 1.795e-009 641 1 10917

P4CXCR2034 1.354E-09 1.005e-009 to 1.822e-009 7126 13394

P4CXCR2047 E623E-09 1.282e-009 to 2.056e-009 6484 12651

P4CXCR100 l,775E-09 1.276e-009 to 2.470e-009 7143 14196

P4CXCR2061 2,03E-09 0 7365 11538

P4CXCR2001 2,064E-09 1.603e-009 to 2.658e-009 641 1 12065

P4CXCR2007 2.071E-09 1.763e-009 to 2.434e-009 6435 12862

P4CXCR2055 2,3E-09 1.852e-009 to 2.856e-009 7063 13693

P4CXCR2031 3,061E-09 2.268e-009 to 4.133e-009 6411 12240

P4CXCR2058 3,643E-09 2.306e-009 to 5.756e-009 7419 14126

P4CXCR2023 4,049E-09 2.834e-009 to 5.783e-009 641 1 1 1792

P4CXCR2004 l,212E-08 8.676e-009 to 1.692e-008 641 1 11910 P-3: Competition reporter assay

The purified Nanbodody constructs were screened for competition with SDF-1 in a CRE-reporter assay using CHO-CXCR4 cells. For assay optimization, both CHO and HE T cells were compared with SRE and CRE-luciferase response. An alternative read-out, using the NFAT response element with cotransfection of Gqi5, was tested in parallel. Best read-out was obtained using CHO cells with the CRE-luciferase respons. Transfected cells were treated with lOOnM SDF-1, a serial dilution of the compounds and 2μΜ of forskolin. In parallel the assay was also run in the presence of 1 ΟμΜ HSA. Figure 14 depicts a

representative graph obtained in this assay, with results give in Table C-10.

All constructs compete with SDF-1. with IC50 values ranging from 3.9 IE- 10 to 7.12E- 09 when the assay is performed in the absence of HSA. HSA seems to slightly impact the SDF-1 competition. Obtained IC50 values in presence of HSA range between 1.27E-09 and 4.30E-08.

Based on the results in the different characterization assays and characteristics of the building blocks, it was concluded that the constructs in Table C-l 1 are some of the preferred constructs of the invention, and accordingly, these were further characterized. It was also concluded that constructs comprising the building blocks 15A1, lOAiO. 14A2 and 281E10 may in certain instances be less preferred (i.e., compared to constructs based on the building blocks 10E9, 10E12, 10G10, 15H3 and/or 283 B6) for the following reasons: 15A1 because it might be limited in terms of scope for further optimization (for example, for improved production/ expression); 281E10 because its sequence-optimization may lead to an ISV which, although functional, may be close in sequence to the known ISV 283D4; 14A2 may have a negatively influence the competition reporter assay; and constructs with 1 OA 10 are not amongst best in ranking in both assays

Table C-10: Potencies of HLE bivalent and biparatopic anobodies® for ligand competition via the CRE-reporter assay in the presence and absence of HSA

Note: (*) indicates that IC50 determination was not possible due to incomplete titration flattened curves)

Competition Competition ratio

Construct ICso (M) CI ICso 95% ICso (M) CI ICso 95% +HSA

-HSA +IISA /-USA

1.747e-010 to 4.138e-010 to

P4CXCR2048 3,91E-10 L27E-09 3,25

8.770e-010 3.913e-009

4.330e-010 to 4.891e-010 to

P4CXCR2047 8,89E-10 l,55E-09 1,74

1.823e-009 4. 13e-009

5.190e-010 to 1.586e-009 to

P4CXCR2001 8,27E-10 2,55E~09 3.08

1.317e-009 4.099e-009

3.414e-010 to 1.467e-009 to

P4CXCR2030 5,96E-10 2.56E-09 4,29

1.041e-009 4.452e-009

1.063e-009 to 1.304e-009 to

P4CXCR2038 l,59E-09 2,58E-09 L62

2.385e-009 5.087e-009

2.525e-010 to 1.650e-009 to

P4CXCR2060 6,41E-10 2,70E-09 4,21

1.628e-009 4.415e-009

9.454e-010 to 1.473e-009 to

P4CXCR2006 1.57E-09 3,00E-09 1 ,90

2.616e-009 6.089e-009

1.358e-009 to 2.178e-009 to

P4CXCR2068 2,06E-09 3,78E-09 1,83

3.132e-009 6.566e-009

6.714e-010 to 2.512e-009 to

P4CXCR2023 1.06E-09 3,97E-09 3,75

1.671 e-009 6.266e-0Q9

8.860e-010 to 2.055e-009 to

P4CXCR2007 l,41E-09 4,08E-09 2,89

2.240e-009 8.078e-009 Table C-10 (continued):

Competition Competition ratio J

Construct IC₅₀ (M) CI IC₅₀ 95% ICso (M) CI ICso 95% +HSA 1

-HSA +HSA /-HSA

1.212e-010 to 2.391e-009 to

P4CXCR2061 839E-10 4,09E-09 4,87

5.807e-009 6.986e-009

9.091e-010 to 2.634e-009 to

P4CXCR2066 1.30E-09 4,35E-09 3,35

1.858e-009 7.197e-009

3.441e-010 to 2.878e-009 to

P4CXCR2021 5,34E-10 4,54E-09 8,49

8.299e-010 7.152e-009

9.219e-010 to 2.972e-009 to

P4CXCR2073 1 _f34E-09 5,07E-09 3,80

1.933e-009 8.642e-009

9.746e-010 to 3.01 1e-009 to

P4CXCR2009 l,56E-09 5,23E-09 3,35

2.491e-009 9.068e-009

8.824e-010 to 4.063e-009 to

P4CXCR2034 l,26E-09 5,94E-09 4,71

1.806e-009 8.692e-009

7.421e-010 to 4.419e-009 to

P4CXCR2029 U7E-09 6,87E-09 5,88

1.840e-009 1.068e-008

3.919e-010 to 1.676e-009 to η

P4CXCR2003 8.91E-10 6,94E-09

2.026e-009 2.878e-008

2.995e-010 to 1.432e-009 to

P4CXCR2055 8.54E-10 7,23E-09 8,47

2.433e-009 3.647e-008

1.511e-00 to 5.146e-009 to

P4CXCR2002 3,49E-09 8,94E-09 2,57

8.036e-009 1.554e-008

9.734e-010 to 6.199e-009 to

P4CXCR2033 1,70E-09 1.07E-08 6,33

2.960e~009 1.860e-008

6.887e-010 to 8.270e-009 to

P4CXCR2000 l,36E-09 1.57E-08 1 1,52

2.680e-009 2.966e~008 Table C-10 (continued):

Competition Competition ratio

Construct IC50 (M) CI ICso 95% ICso ( ) CI IC₅₀ 95% +HSA

-HSA ÷HSA /-HSA

4.353e-010 to 3.883e-010 to

P4CXCR2005 1.00E-09 L88E-08 18,74

2.305e-009 9.079e-007

P4CXCR2004 (*) 1.95E-08

4.076e-009 to 2.376e-008 to

P4CXCR2036 7,12E-09 4J9E-08 5,88

1.243e-008 7.377e-008

3.862e-009 to 2.461 e-008 to

P4CXCR2031 6,70E-09 4J0E-08 6,42

1.163e-008 7.514e-008

2.65 e-009 to

P4CXCR2058 4₅20E-09

6.655e-009 (*)

6.275e-011 to

P4CXCR2042 3,58E-10

2.037e-009 (*)

5.946e-010 to

P4CXCR2041 E09E-09

2.012e-009 (*)

5.786e-010 to

P4CXCR2039 l,06E-09

1.934e-009 (*)

P4CXCR1023 (*) (*)

P4CXCR2078 (*) (*)

Table C-11. Non-limiting examples of some preferred constructs of the invention based on the preferred building blocks 10E9, 10E12, 10G10, 15H3 and/or 283B6

D-4: Further characterization of constructs from Table C-3 1

The constructs of Table C-11 were subjected to a second, round of characterization in the chemotaxis and reporter assays, essentially as described above. The results are shown in Table C-12.

Table C-12: further characterization of the constructs of Table C-l l.

Chemotaxis Jurkat cells Reporter assay: competition

Chemotaxis Competition Competition ratio IC₅₀ (M) CI ICso 95% % inhib. ICso (M) IC50 +HSA/- +HSA -USA +.HSA HSA

2.419e-010 to 8.166e-010 to

P4CXCR2001 6.533E-10 96,2 1.32E-09

1 .764e-009 2.062e-009

3.634e-010 to 1.319e-009 o

P4CXCR2002 6,097E-10 101 ,5 2,30E-09

1.023e-009 4.007e-009

7.536e-010 to

P4CXCR20U6 2,216E-09 39,0

6.513e-009

4.175e-010 to 1.385e-009 to

P4CXCR2030 6,871 E-10 92,9 2,36E-09

1.131 e-009 4.032e-009

1.646e-010 to 1.277e-009 to

P4CXCR2038 2,658E-10 101,3 2,24E-09

4.292e-010 3.931e-009

8.725e-012 to 2.167e-010 to 9.606e-010 to

P4CXCR2042 4,602E-1 1 55,8 5,05E-10 2,85E-09 5,65

2.427e-010 1.281e-009 8.476e-009

1.953e-010 to 1.833e-010 to 1.175e-009 to

P4CXCR2048 3,61 1 E-10 101,7 3.93E-10 2.30E-09 5,86

6.678e-010 7.906e-010 4.501 e-009

Table C-12 (continued):

Chemotaxis Jurkat cells Reporter assay: competition

Chemotaxis Competition Competition ratio IC₅₀ (M) CI IC₅₀ 95% % inhib. IC50 (M) IC50 +HSA7- +HSA -HSA +I1SA HSA

6.814e-010 to 1.346e-009 to

P4CXCR2060 1.076E-09 68,8 2,71 E-09

1.700e-009 5.441e-009

2.598e-010 to 3.71 5e-010 to 1.354e-009 to

P4CXCR2066 7.300E-10 93,5 8,00E-10 2.85E-09 3,56

2.051e-009 1.668e-009 6.000e-009

MDX (average) 1.522E-09 94,1 5.46E-09 3.73E-09 0,68

2.078e-010 to 7.691e-010 to

P4CXCR1023 4,123E-10 91 ,5 2,15E-09

8.182e-010 5.988e-009

E: Species Cross-Reactivity Analysis (binding to mouse and cynomolgus CXCR4) To determine whether the ISV's from Table C-11 were able to bind to CXCR4 of mouse and cynomolgus origin, a binding FACS was performed. Nanobody® binding to HEK293 parental cells and HEK93 cells transfected with either the human or Cynomolgus CXCR4 plasmid was detected using an anti-ALB8 biotinylated antibody (2F10-biot), followed by Streptavidin-PE detection. Binding to mouse CXCR4 was assessed using murine BaF-3 cells (pre B cell line) that endogenously express CXCR4. Results are shown in Figures 15 A to 15C, and the Table in Figure 15D contains the estimated EC50 values for binding to cynomolgus and human CXCR4 transfected HEK293T cells.

F: Selectivity analysis (binding to CXCR7)

To determine whether the final lead panel of nine Nanobody® constructs was selective for CXCR4 and not for the CXCR7 GPCR, Nanobody® binding to HEK93 cells transfected with the human CXCR4 plasmid and to Caki cells transfected with human CXCR7 plasmid was detected using an anti-ALB8 biotinylated antibody (2F10-biot), followed by

Streptavidin-PE detection (data not shown). It was found that only mAb 8F1 1 shows a good dose-response curve to hCXCR7 expressed on Caki cells. All the constructs from Table C-1 1 and mAb 12G5 show some aspecific background binding at the highest concentrations tested (ΙΟΟηΜ), but no dose-response curves of the Nanobodies® to hCXCR7 are detected,

G: Protein stability tests

G-l : Temperature stability (thermal shift assay)

The temperature stability of the constructs from Table C-11 was measured in a thermal shift assay. The constructs were diluted to a concentration of 0.05 mg/ml in buffers of different pH, and melting temperature (Tm) was determined by stepwise increase in temperature in presence of Sypro Orange, a dye that binds to Trp residues that become exposed upon unfolding of the protein, using the Lightcycler for detection (Roche). The melting temperature profiles of the constructs from Table C-11 are shown in Figure 16 A, with the corresponding Tm at pH7 (°C) being mentioned in Figure 16B.

In general, all tested constructs show a drop in melting temperature if compared with the monovalent building blocks (see Figure 16). All leads have a similar Tm, ranging between 59 °C and 61.5 °C at pH7.0. The Tm drop to a value around 60 °C has also been observed for other constructs (not of the invention) comprising the Alb-8 building block. G-2: Analytical SEC lead panel

The constructs from Table C-l 1 were analyzed for the potential occurrence of aggregates by analytical size exclusion chromatography (SEC) using the BioSep-SEC-S2000 column (Phenomenex) column. None of the constructs showed aggregation (data not shown).

G-3: Freeze-thaw stability

The constructs from Table C-l 1 were subjected to 5 freeze thaw (FT) cycles (RT to - 70°C). In order to evaluate its effect on protein recovery, SEC analysis was performed. In parallel samples were monitored for turbidity, using visual inspection and OD280/320/340 measurements.

During visual inspection, no turbidity could be observed after 5 FT cycles. However, an increase of the 320/280 ratio (data not shown) and a loss in area with analytical SEC (data not shown) were observed after 5 FT cycles, which indicates that all CXCR4 constmcts may be susceptible to freeze thaw stress.

H: Initial expression feasibility studies

An initial expression feasibility study was performed for the constructs from Table C- 1 1. Results suggest that P4CXCR2048 and -2002 are expected to reach expression levels of > lg/L in fermentation (even before further sequence-optimization), while the other constructs will probably reach lower expression levels of about 0.5g/L.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from ti e foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

What is claimed is:

Claims

C L A I M S

1. ISV that has an IC50 in the Jurkat assay of Example 1, C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM).

2. ISV according to claim 1, that is a domain antibody, single domain antibody or dAb, but is preferably a Nanobody (e.g., a VHH, a humanized VHH or a camelized VH such as a camelized human VH).

3. ISV according to claim 1 or 2, that is a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a lOAlO-type sequence, a lOGlO-type sequence, a 14A2-type sequence, a 15Al-type sequence, a 15H3-type sequence or a 283B6-type sequence.

4. ISV according to any of the preceding claims, that is a 10E9-like sequence, a 281E10- like sequence, a ί 0E 12-Iike sequence, a 10A10-like sequence, a 10G10-like sequence, a 14A2-like sequence, a 15Al-like sequence, a 15H3-Iike sequence or a 283B6-Iike sequence

5. Compound, construct, protein or polypeptide, comprising or essentially consisting of a first ISV that is directed against CXCR-4 (and in particular against human CXCR-4) and a second ISV that is directed against CXCR-4 (and in particular against human CXCR-4), in which the first and second ISV may be the same or different, and in which at least one of the ISV's present therein have an IC₅₀ in the Jurkat assay of Example 1 , C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9

M (5.0E-9M or 5.0 nM) when measured in the corresponding monovalent format.

6. Compound, construct, protein or polypeptide according to claim 5, comprising at least one a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a 10A10- type sequence, a lOGlO-type sequence, a 14A2-type sequence, a 15Al -type sequence, a

15H3-type sequence or a 283B6-type sequence. Compound, construct, protein or polypeptide according to claim 6, comprising at least one a 10E9-like sequence, a 281E10-like sequence, a 10E12-like sequence, a 1 OA 10- like sequence, a l OGl O-like sequence, a 14A2-like sequence, a 15Al -like sequence, a 15H3-like sequence or a 283B6-like sequence.

Compound, construct, protein or polypeptide according to claim 5, which comprises two (or more) ISV's directed against CXCR-4, and in which both (or all) ISV's present therein have an IC₅₀ in the Jurkat assay of Example 1 , C-4; of better than 7.0x10-9 M (7.0E-9M or 7.0 nM), in particular better than 5.0x10-9 M (5.0E-9M or 5.0 nM) when measured in the corresponding monovalent format.

Compound, construct, protein or polypeptide according to claim 5 or 6, which comprises two (or more) ISV's directed against CXCR-4, and in which both (or all) ISV's are a domain antibody, single domain antibody or dAb, but is preferably a Nanobody (e.g., a VHH, a humanized VHH or a camelized VH such as a camelized human VH), and are preferably both ISV's of the invention (as defined herein).

Compound, construct, protein or polypeptide according to claim 8 or 9, which comprises two (or more) ISV's directed against CXCR-4, and in which both (or all) ISV's are a 10E9-type sequence, a 281E10-type sequence, a 10E12-type sequence, a 10A10-type sequence, a l OGl O-type sequence, a 14A2-type sequence, a 15Al -type sequence, a 15H3-type sequence or a 283B6-type sequence.

Compound, construct, protein or polypeptide according to claim 8, 9 or 10, which comprises two (or more) ISV's directed against CXCR-4, and in which both (or all) ISV's are a 10E9-like sequence, a 281E10-like sequence, a 10E124ike sequence, a 10A10-like sequence, a 10G10~like sequence, a 14A2-like sequence, a 15Al-like sequence, a 15H3-like sequence or a 283B6-like sequence.