WO2014124258A2

WO2014124258A2 - Specific sites for modifying antibodies to make immunoconjugates

Info

Publication number: WO2014124258A2
Application number: PCT/US2014/015302
Authority: WO
Inventors: Bernhard Hubert GEIERSTANGER; Weijia Ou; Tetsuo Uno
Original assignee: Irm Llc
Priority date: 2013-02-08
Filing date: 2014-02-07
Publication date: 2014-08-14
Also published as: WO2014124258A3

Abstract

The present invention provides specific sites for modifying antibodies or antibody fragments by replacing at least one amino acid in the constant region of a parental antibody or antibody fragment with a TAG-encoded amino acid such as Pcl, which can be used as a site of attachment for a payload or linker-payload combination. The invention includes modified antibodies or antibody fragments, various conjugates formed therefrom, and uses of the modified antibodies or antibody fragments and their conjugates.

Description

SPECIFIC SITES FOR MODIFYING ANTIBODIES TO

MAKE IMMUNOCONJUGATES

FIELD OF THE INVENTION

[001] The present invention relates to site-specific labeling sites and processes, molecules produced thereby such as antibody drug conjugates, and their uses.

BACKGROUND

[002] The value of methods for modifying antibodies is well known, and many methods for conjugation of antibodies to attach various "payload" moieties have been developed. Many of these methods rely upon the natural occurrence of specific reactive amino acid residues on the antibody, such as lysine and cysteine, which can be used to attach a payload. However, relying on the natural occurrence amino acids is not always desirable, because the location and amount of payload attached depend on the number and position of those reactive amino acids: Too many or too few such residues make it difficult to efficiently control loading of the payload onto the antibody. In addition, placement of the reactive amino acids may make it difficult to get complete conjugation, resulting in heterogeneous products during conjugation. Heterogeneity of a pharmaceutical active ingredient, for example, is typically undesirable: It is far preferable to administer a homogeneous product, and far more difficult to fully characterize a heterogeneous one. Site- specific conjugation of a cytotoxic drug to an antibody through, for example, engineered cysteine residues results in homogenous immunoconjugates that exhibit improved therapeutic index (Junutula et ah, (2008) Nat Biotechnol. 26(8):925-932).

[003] Antibody drug conjugates (ADCs) have been used for the local delivery of cytotoxic agents in the treatment of cancer (see e.g., Lambert, Curr. Opinion in Pharmacology 5:543-549, 2005). ADCs allow targeted delivery of the drug moiety where maximum efficacy with minimal toxicity may be achieved. As more ADCs show promising clinical results, there is an increased need to develop stable engineered antibodies that provide reactive groups capable of conjugation to various agents, especially site-specific conjugations that can generate homogeneous immunoconjugates with a defined drug-to-antibody ratio for use in cancer therapy. More importantly, site-specifically conjugated immunoconjugates exhibit improved therapeutic index, and the attachment sites described in the instant invention provide a means to prepare such site-specific and hence improved immunoconjugates.

SUMMARY OF THE INVENTION

[004] The invention provides specific sites in the constant region of an antibody or antibody fragment at which a native amino acid on a parental antibody or antibody fragment can be replaced with various TAG-encoded amino acids in order to provide a modified antibody or antibody fragment. The TAG sequence in a nucleic acid is normally read as a "stop" codon, but under suitable conditions it can be used to incorporate a number of different amino acids, including pyrroline- carboxy-lysine (Pel), pyrrolysine and unnatural amino acids (Noren et al, (1989) Science 14;244(4901): 182-188; Mendel et al, (1995) Annu Rev Biophys Biomol Struct. 24:435-462; Wang et al., (2001) Science 292 (5516):498-500; Young et al, (2010) J Biol Chem. 285(15): 11039-44). Many of the TAG-encoded amino acids, such as Pel, can be used to attach a payload (drug moiety) to the antibody or antibody fragment to form an immunoconjugate with good efficiency and stability. The invention further provides engineered antibodies or fragments thereof having one or more such TAG-encoded residues in one or more specific sites (Selected TAG sites in Tables 1, 2 and 3), as well as immunoconjugates made from such engineered antibody sequences.

[005] Methods for inserting TAG-encoded amino acids such as Pel at specific locations of an antibody are known in the art. Pel is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pel is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. See, e.g., Ou et al, (2011) Proc Natl Acad Sci U S A. 108: 10437-10442; Cellitti et al, (2011) Nat Chem Biol. 7(8):528-30; Gaston et al, (2011) Nature 471(7340):647-50.

[006] The current invention provides specific sites in the constant region of antibodies where replacing one or more native amino acids on a parental antibody or antibody fragment with Pel or other TAG-encoded amino acids provides one or more advantages as described herein, such as high expression of the corresponding TAG- encoded amino acid containing protein, good conjugation yield, and efficient conjugation loading. Because the sites identified herein are in the constant region of an antibody sequence, they can be used with various antibodies.

[007] In one aspect, the invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with Pel or another TAG-encoded amino acid on its constant region chosen from positions (Selected TAG Sites) identified herein. In certain embodiments, the site for substitution is one of the Selected TAG Sites listed in Table 1, Table 2 or Table 3, or a combination of two or more of those sites. The invention further provides modified antibodies or antibody fragments that are suitable for making these immunoconjugates, methods for making the

immunoconjugates, and methods to use the immunoconjugates for treatment of disorders such as cancer or other cell proliferation disorders.

[008] In one aspect, the invention provides an immunoconjugate of Formula

(I):

wherein Ab represents an antibody or antibody fragment comprising at least one TAG-encoded amino acid residue at one of the substitution sites described herein;

LU is a linker unit as described herein;

X is a payload or drug moiety;

and n is an integer from 1 to 16.

[009] Typically in compounds of Formula (I), LU is attached to a TAG- encoded amino acid such as Pel at one of the specific substitution sites described herein, X is a drug moiety such as an anticancer drug, and n is 2-8 when Ab is an antibody, or n can be 1-8 when Ab is an antibody fragment.

[0010] In another aspect, the invention provides a modified antibody or antibody fragment thereof comprising a substitution of a TAG-encoded amino acid for the native amino acid of a parental antibody or antibody fragment at one or more of the substitution positions (Selected TAG Sites) identified in Table 1, Table 2 and Table 3. In some embodiments, the TAG-encoded amino acid is Pel.

[0011] In another aspect, the invention provides a method to select a site where a TAG-encoded amino acid (e.g., Pel) can be substituted for a native amino acid in a parental antibody or antibody fragment.

[0012] Other aspects and embodiments of the invention are described in greater detail herein.

Definitions

[0013] The term "amino acid" refers to canonical, synthetic, and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the canonical amino acids. Canonical amino acids are

proteinogenous amino acids encoded by the genetic code and include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline serine, threonine, tryptophan, tyrosine, valine, as well as selenocysteine, pyrrolysine and pyrroline- carboxy-lysine. Amino acid analogs refer to compounds that have the same basic chemical structure as a canonical amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a canonical amino acid.

[0014] The term "TAG-encoded amino acid" as used herein refers to any amino acid that can be incorporated into a protein by expression of a TAG codon in a nucleic acid containing a TAG-codon, for example at one of the substitution sites identified herein. Examples of TAG-encoded amino acids include Pyrroline- carboxy-lysine (Pel), pyrrolysine and unnatural amino acids, particularly those

whose incorporation into a protein by expression of a TAG codon is known in the art. In some embodiments, the TAG-encoded amino acid is Pel. Where reference is made herein to a site for Pel substitution, or to an antibody or antibody fragment containing Pel at a substitution site described herein, it is understood that

substitution at that site with other TAG-encoded amino acids is also contemplated.

Non-limiting examples of other TAG-encoded amino acids include reactive

pyrrolysine analogs such as N6-(2-(R)-propargylglycyl)-lysine (Li, Fekner, Chan, Chem Asian J. 2010, 5, 1765-9), N6-[(2-propynyloxy)carbonyl]-L-lysine (Nguyen et al, J Am Chem Soc. 2009, 131(25), 8720-1), N6-[(2-azidoethoxy)carbonyl]-L- lysine (Nguyen et al, J Am Chem Soc. 2009, 131(25), 8720-1) and others described in Fekner and Chan (Fekner and Chan, Curr Opin Chem Biol. 2011, 15(3), 387-91), by Lee et al (Lee et al, Chembiochem 2013, 14 (7), 805-8), by Chin and coworkers (Hancock SM, J Am Chem Soc. 2010 132(42): 14819-24), by Yokoyama and coworkers (for example, Yanagisawa et al. Chem Biol 2008, 15 (11), 1187-97), by Lemke and coworkers (for example, Plass et al, Angew Chem Int Ed Engl. 2011, 50 (17), 3878-81) as well as para-acetyl-phenylalanine, para-azido-phenylalanine and other unnatural amino acids useful for protein conjugation reviewed by Kim et al (Kim, Axup, Schultz, Curr Opin Chem Biol. 2013, 17(3), 412-9).

[0015] The preferred substitution sites of the invention are located in the constant region of an antibody, and are identified herein using standard numbering conventions. It is well known, however, that portions of antibodies can be used for many purposes instead of intact full-length antibodies, and also that antibodies can be modified in various ways that affect numbering of sites in the constant region even though they do not substantially affect the functioning of the constant region. For example, insertion of an S6 tag (a short peptide tag) into a loop region of an antibody has been shown to allow activity of the antibody to be retained, even though it would change the numbering of many sites in the antibody. Accordingly, while the preferred substitution sites described herein are identified by a standard numbering system based on intact antibody numbering, the invention includes the corresponding sites in antigen binding fragments or in antibodies containing other modifications, such as S6 tag insertion. The corresponding sites in those fragments or modified antibodies are thus preferred sites for substitution in fragments or modified antibodies, and references to the substitution sites by number include corresponding sites in modified antibodies or antigen binding fragments that retain the function of the relevant portion of the full-length antibody.

[0016] A corresponding site in a modified antibody or antibody fragment can readily be identified by aligning a segment of the antibody fragment or modified antibody with the parental antibody to identify the site in the antibody fragment or modified antibody that matches one of the preferred substitution sites of the invention. Alignment may be based on a segment long enough to ensure that the segment matches the correct portion of the full-length antibody, such as a segment of at least 20 amino acid residues, or at least 50 residues, or at least 100 residues, or at least 150 residues. Alignment may also take into account other modifications that may have been engineered into the antigen binding fragment or modified antibody, thus differences in sequence due to engineered point mutations in the segment used for alignment, particularly for conservative substitutions, would be allowed. Thus, for example, an Fc domain can be excised from an antibody, and would contain amino acid residues that correspond to the specific substitution sites described herein, despite numbering differences: sites in the Fc domain

corresponding to the substitution sites of the invention would also be expected to be advantageous sites for similar TAG-encoded substitution in the Fc domain, and are included in the scope of the invention.

[0017] Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a canonical amino acid. The term "unnatural amino acid", as used herein, is intended to represent amino acid structures that cannot be generated biosynthetically in any organism using unmodified or modified genes from any organism, whether the same or different. In addition, such

"unnatural amino acids" typically require a modified tRNA and a modified tRNA synthetase (RS) for incorporation into a protein. These "selected" orthogonal tR A/RS pair are specific for the unnatural amino acid and are generated by a selection process as developed by Schultz et al. (see, e.g., Liu et al, (2010) Annu. Rev. Biochem. 79:413-444) or a similar procedure. The term "unnatural amino acid" does not include the natural occurring 22^nd proteinogenic amino acid pyrrolysine (Pyl) as well as its demethylated analog pyrroline-carboxy-lysine (Pel), because incorporation of both residues into proteins is mediated by the unmodified, naturally occurring pyrrolysyl-tRNA/tRNA synthetase pair and because Pyl and Pel are generated biosynthetically (see, e.g., Ou et al, (201 1) Proc. Natl. Acad. Sci. USA. 108: 10437-10442; Cellitti et al, (201 1) Nat. Chem. Biol. 27;7(8):528-30).

[0018] The term "antibody" as used herein refers to a polypeptide of the immunoglobulin family that is capable of binding a corresponding antigen non- covalently, reversibly, and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0019] The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention). The antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).

[0020] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used

functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy -terminal domains of the heavy and light chain, respectively.

[0021] The term "antibody fragment" as used herein refers to a portion of an antibody. For example, an antibody fragment can be the fragment crystallizable region (Fc region), which is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. An antibody fragment can also be an antigen binding fragment, which refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of binding fragments include, but are not limited to, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab') fragments, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al, Nature 341 :544-546, 1989), which consists of a VH domain; and an isolated complementarity determining region (CDR), or other epitope-binding fragments of an antibody.

[0022] Furthermore, although the two domains of the Fv fragment, VL and V¾ are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv ("scFv"); see, e.g., Bird et al, Science 242:423-426, 1988; and Huston et al, Proc. Natl. Acad. Sci. 85:5879-5883, 1988). Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment." These antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0023] Antigen binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature

Biotechnology 23 : 1126-1136, 2005). Antigen binding fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703, 199, which describes fibronectin polypeptide monobodies).

[0024] Antigen binding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al, Protein Eng. 8: 1057-1062, 1995; and U.S. Pat. No. 5,641,870). [0025] The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to polypeptides, including antibodies and antigen binding fragments that have substantially identical amino acid sequence or are derived from the same genetic source. This term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0026] The term "human antibody", as used herein, includes antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000).

[0027] The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing).

[0028] The term "humanized" antibody, as used herein, refers to an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984); Morrison and Oi, Adv. Immunol, 44:65-92 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994).

[0029] The term "recognize" as used herein refers to an antibody or antigen binding fragment thereof that finds and interacts (e.g., binds) with its epitope, whether that epitope is linear or conformational. The term "epitope" refers to a site on an antigen to which an antibody or antigen binding fragment of the invention specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to

denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or 15 amino acids in a unique spatial conformation.

Methods of determining spatial conformation of epitopes include techniques in the art, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

[0030] The term "affinity" as used herein refers to the strength of interaction between antibody and antigen at single antigenic sites. Within each antigenic site, the variable region of the antibody "arm" interacts through weak non-covalent forces with antigen at numerous sites; the more interactions, the stronger the affinity.

[0031] The term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds to one antigen may, however, have cross-reactivity to other antigens. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[0032] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. [0033] For polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitutions providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0034] The term "optimized" as used herein refers to a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a yeast cell, a Pichia cell, a fungal cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence, which is also known as the "parental" sequence.

[0035] The terms "percent identical" or "percent identity," in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 30 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. [0036] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0037] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl.

Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al, Current Protocols in Molecular Biology, 2003).

[0038] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0

algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al, J. Mol. Biol. 215:403-410, 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each

sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for

mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: The cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X

determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0039] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0040] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 1 1-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48:444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0041] Other than percentage of sequence identity noted above, another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is

immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0042] The term "nucleic acid" is used herein interchangeably with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide- nucleic acids (PNAs).

[0043] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, as detailed below, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, (1991) Nucleic Acid Res. 19:5081 ; Ohtsuka et al, (1985) J. Biol. Chem. 260:2605-2608; and Rossolini et al, (1994) Mol. Cell. Probes 8:91-98).

[0044] The term "operably linked" in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0045] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to canonical amino acid polymers as well as to non-canonical amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0046] The term "immunoconjugate" or "antibody conjugate" as used herein refers to the linkage of an antibody or an antigen binding fragment thereof with another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, and the like. The linkage can be covalent bonds or non-covalent interactions and can include chelation. Various linkers, known in the art, can be employed in order to form the immunoconjugate. Additionally, the immunoconjugate can be provided in the form of a fusion protein that may be expressed from a polynucleotide encoding the immunoconjugate. As used herein, "fusion protein" refers to proteins created through the joining of two or more genes or gene fragments which originally coded for separate proteins

(including peptides and polypeptides). Translation of the fusion gene results in a single protein with functional properties derived from each of the original proteins.

[0047] The term "subject" includes human and non-human animals. Non- human animals include all vertebrates, e.g., mammals and non-mammals, such as non- human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms "patient" or "subject" are used herein interchangeably.

[0048] The term "cytotoxin", or "cytotoxic agent" as used herein, refer to any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy.

[0049] The term "anti-cancer agent" as used herein refers to any agent that can be used to treat a cell proliferative disorder such as cancer, including but not limited to, cytotoxic agents, chemotherapeutic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, and immunotherapeutic agents.

[0050] The term "drug moiety" or "payload" as used herein refers to a chemical moiety that is conjugated to the antibody or antigen binding fragment of the invention, and can include any moiety that is useful to attach to an antibody or antigen binding fragment. For example, a drug moiety or payload can be an anticancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent. In certain embodiments a drug moiety is selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a

microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPrV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor. Suitable examples include auristatins such as MMAE and MMAF; calicheamycins such as gamma-calicheamycin; and maytansinoids such as DM1 and DM4. Methods for attaching each of these to a linker compatible with the antibodies and method of the invention are known in the art. See, e.g., Singh et ah, (2009) Therapeutic Antibodies: Methods and Protocols, vol. 525, 445-457. In addition, a payload can be a biophysical probe, a fluorophore, a spin label, an infrared probe, an affinity probe, a chelator, a spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, a surface, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a saccharide or a polysaccharide, a reactive functional group such as those listed in Table 4, or a binding agent that can connect the conjugate to another moiety or surface, etc.

[0051] "Tumor" refers to a neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0052] The term "anti-tumor activity" means a reduction in the rate of tumor cell proliferation, viability, or metastatic activity. A possible way of showing antitumor activity is to show a decline in growth rate of abnormal cells that arises during therapy or tumor size stability or reduction. Such activity can be assessed using accepted in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, MMTV models, and other known models known in the art to investigate anti-tumor activity.

[0053] The term "malignancy" refers to a non-benign tumor or a cancer. As used herein, the term "cancer" includes a malignancy characterized by deregulated or uncontrolled cell growth. Exemplary cancers include: carcinomas, sarcomas, leukemias, and lymphomas.

[0054] The term "cancer" includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from

metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor).

[0055] As used herein, the term "an optical isomer" or "a stereoisomer" refers to any of the various stereo isomeric configurations which may exist for a given compound of the present invention and includes geometric isomers. It is understood that a substituent may be attached at a chiral center of a carbon atom. The term "chiral" refers to molecules which have the property of non- superimposability on their mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner. Therefore, the invention includes enantiomers, diastereomers or racemates of the compound.

"Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1 : 1 mixture of a pair of enantiomers is a "racemic" mixture. The term is used to designate a racemic mixture where appropriate.

"Diastereoisomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror- images of each other. The absolute stereochemistry is specified according to the Cahn-lngold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon may be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain compounds described herein contain one or more asymmetric centers or axes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (5)-. [0056] Depending on the choice of the starting materials and procedures, the compounds can be present in the form of one of the possible isomers or as mixtures thereof, for example as pure optical isomers, or as isomer mixtures, such as racemates and diastereoisomer mixtures, depending on the number of asymmetric carbon atoms. The present invention is meant to include all such possible isomers, including racemic mixtures, diasteriomeric mixtures and optically pure forms. Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or they may be resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have a cis- or trans-configuration. All tautomeric forms are also intended to be included.

[0057] As used herein, the terms "salt" or "salts" refers to an acid addition or base addition salt of a compound of the invention. "Salts" include in particular "pharmaceutical acceptable salts". The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of this invention and, which typically are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

[0058] Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g., acetate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlorotheophyllinate, citrate, ethandisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, laurylsulfate, malate, maleate, malonate, mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, subsalicylate, tartrate, tosylate and trifluoroacetate salts.

[0059] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. [0060] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

[0061] Inorganic bases from which salts can be derived include, for example, ammonium salts and metals from columns I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.

[0062] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Certain organic amines include isopropylamine, benzathine, cholinate,

diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

[0063] The pharmaceutically acceptable salts of the present invention can be synthesized from a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, use of non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable, where practicable. Lists of additional suitable salts can be found, e.g., in "Remington's Pharmaceutical Sciences", 20th ed., Mack Publishing Company, Easton, Pa., (1985); and in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0064] Any formula given herein is also intended to represent unlabeled forms as well as isotopically labeled forms of the compounds. Isotopically labeled compounds have structures depicted by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as ²H, ³H, ^UC, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F ³¹P, ³²P, ³⁵S, ³⁶C1, ¹²⁵I respectively. The invention includes various isotopically labeled compounds as defined herein, for example those into which radioactive isotopes, such as ³H and ¹⁴C, or those into which non-radioactive isotopes, such as ²H and ¹³C are present. Such isotopically labeled compounds are useful in metabolic studies (with ¹⁴C), reaction kinetic studies (with, for example ²H or ³H), detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an ¹⁸F or labeled compound may be particularly desirable for PET or SPECT studies. Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.

[0065] Further, substitution with heavier isotopes, particularly deuterium (i.e., ²H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. It is understood that deuterium in this context is regarded as a substituent of a compound of the formula (I). The concentration of such a heavier isotope, specifically deuterium, may be defined by the isotopic enrichment factor. The term "isotopic enrichment factor" as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope. If a substituent in a compound of this invention is denoted deuterium, such compound has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium

incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium

incorporation), or at least 6633.3 (99.5% deuterium incorporation).

[0066] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0067] The term "a therapeutically effective amount" of a compound of the present invention refers to an amount of the compound of the present invention that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non- limiting embodiment, the term "a therapeutically effective amount" refers to the amount of the compound of the present invention that, when administered to a subject, is effective to at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease, or at least partially inhibit activity of a targeted enzyme or receptor.

[0068] As used herein, the term "inhibit", "inhibition" or "inhibiting" refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0069] As used herein, the term "treat", "treating" or "treatment" of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treat", "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treat", "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, "treat", "treating" or "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder. [0070] As used herein, a subject is "in need of a treatment if such subject would benefit biologically, medically or in quality of life from such treatment.

[0071] As used herein, the term "a," "an," "the" and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0072] The term "ABA compound" as used herein refers to a 2- aminobenzaldehyde compound of the following formula:

wherein R³⁰ is H, LU is a linker unit, X is a payload, and Z is a group selected from H, halo, Ci-4 alkyl, Ci_₄ haloalkyl, C1-4 alkoxy, and a group -LU²-X², where LU² is a second linker unit and X² is a second payload. When Z is -LU²-X², LU and LU² can be the same or different, and X and X² can be the same or different, Similarly, compounds of this formula wherein R³⁰ is methyl are referred to herein as ΆΑΡ compounds' (amino-acetophenones), and compounds of this formula wherein R³⁰ is phenyl are referred to herein as "ABP compounds" (aminobenzophenones).

[0073] "Linker Unit" (LU) as used herein refers to a covalent chemical connection between two moieties, such as an antibody and a payload. Each LU can be comprised of one or more components described herein as Li, L₂, L₃, L₄, L₅ and L₆. The linker unit can be selected to provide suitable spacing between the connected moieties, or to provide certain physicochemical properties, or to allow cleavage of the Linker Unit under certain conditions.

[0074] "Cleavable linker" as used herein refers to a linker or linker unit that connects two moieties by covalent connections, but breaks down to sever the covalent connection between the moieties under physiologically relevant conditions. Cleavage may be enzymatic or non-enzymatic, but generally releases a payload from an antibody without degrading the antibody. Cleavage may leave some portion of a linker or LU attached to the payload, or it may release the payload without any linker-derived residue. [0075] Pel as used herein refers to pyrroline carboxy lysine, e.g.,

where R is H, which has the following formula when incorporated into a peptide:

[0076] The corresponding compound where R is methyl is pyrrolysine.

[0077] "Non-cleavable linker" as used herein refers to a linker or linker unit that is not susceptible to breaking down under physiological conditions, i.e., it is at least as stable as the antibody or antibody fragment portion of the

immunoconjugate. While the linker may be modified physiologically, it keeps the payload connected to the antibody until the antibody is substantial degraded, i.e., the antibody degradation precedes cleavage of the linker. Degradation of the antibody may leave some or all of the linker or LU, and even one or more amino acid groups from the antibody, attached to the payload or drug moiety that is delivered in vivo.

[0078] "Cyclooctyne" as used herein refers to an 8-membered ring containing a carbon-carbon triple bond (acetylene). The ring is optionally fused to one or two phenyl rings, which may be substituted with 1-4 Ci_₄ alkyl, C_1-4 alkoxy, halo, hydroxyl, COOH, COOLi, -C(0)NH-Li, O-Li, or similar groups, and which may contain N, O or S as a ring member. In preferred embodiments, cyclooctyne can be a Cs hydrocarbon ring, particularly an isolated ring that is saturated aside from the triple bond, and may be substituted with F or Hydroxy, and may be linked to a linker or LU via -0-, -C(O), C(0)NH, or C(0)0.

[0079] "Cyclooctene" as used herein refers to an 8-membered ring containing at least one double bond, especially a trans-double bond. The ring is optionally fused to one or two phenyl rings, which may be substituted with 1-4 C_1-4 alkyl, Ci_₄ alkoxy, halo, hydroxyl, COOH, COOL -C(0) H-Li, O-Li, or similar groups, and which may contain N, O or S as a ring member. In preferred embodiments, cyclooctene can be an isolated Cs hydrocarbon ring that is saturated aside from the trans double bond and is optionally substituted with F or Hydroxy, and may be linked to a linker or LU via -0-, -C(O), C(0)NH, or C(0)0.

[0080] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1. Surface accessibility plot of amino acid residues in human IgGl heavy chain (A) and kappa light chain (B). Surface accessibility was calculated using Surface Racer 5.0 and is expressed as Angstrom square [A²].

[0082] FIG. 2. Location of selected 92 TAG mutations in the structure of a human IgGl with a kappa light chain. Selected residues for TAG mutations are shown in black on only one of the two heavy chains and for one of the two kappa light chains (lHZH.pdb). Structures are shown using PyMOL, an open-source molecular modeling package (The PyMOL Molecular Graphics System, Version 1.5.0. Schrodinger, LLC).

[0083] FIG. 3. The amino acid sequence alignment of the heavy chain constant regions of trastuzumab and antibody 14090. Underlined residues in the sequences of trastuzumab antibody and antibody 14090 are the residues that have been mutated into TAG-encoded amino acids. Amino acid residues in the heavy chain are numbered by Eu numbering system (Edelman et al, 1969).

[0084] FIG. 4. Amino acid sequence alignment of constant regions of trastuzumab, human IgGl, IgG2, IgG3 and IgG4.

[0085] FIG. 5. The amino acid sequence alignment of the constant regions of human kappa and lambda light chains. A) Residues mutated to TAG-encoded amino acids in the kappa light chain of trastuzumab and in the lambda light chain of antibody 14090 are underlined. B) Residues selected for mutations to TAG- encoded amino acids are shown in a PyMOL structure model of a human lambda light chain (Protein Databank structure entry 3G6D.pdb).

[0086] FIG. 6. Analysis of trastuzumab Pel antibodies by SDS-PAGE under reducing conditions after Protein A purification.

[0087] FIG. 7. Separation of full-length and truncated form of the trastuzumab HC-T155Pcl antibody. A) Hydrophobic interaction chromatography (HIC). B) SDS-PAGE analysis under reducing conditions before and after HIC separation.

[0088] FIG. 8. Reaction of Pel with a 2-amino-benzaldehyde (ABA) compound.

[0089] FIG. 9. Structure of ABA-MMAF.

[0090] FIG. 10. Analysis of ABA-MMAF conjugation to a trastuzumab Pel antibody by reverse phase high pressure liquid chromatography (HPLC). A) The conjugation mixture of trastuzumab LC-S156Pcl antibody and ABA-MMAF was analyzed by reverse phase HPLC (broken line). The trace of unmodified antibody is shown as solid line. B) The DAR=2 species was purified by HIC from the conjugation mixture and analyzed by reverse phase HPLC (solid line). The trace of the initial reaction mixture is shown as broken line for comparison.

[0091] FIG. 1 1. Analysis of the conjugation mixture of trastuzumab LC- K107Pcl antibody and ABA-MMAF by HIC. Baseline separation allows for efficient purification of DAR=2 LC-K107-Pcl-MMAF ADC molecules.

[0092] FIG. 12. Analysis of HIC-purified trastuzumab LC-T109Pcl-MMAF ADC (dashed line) and unmodified wild-type trastuzumab (solid line) by analytical size exclusion chromatography.

[0093] FIG. 13. Analysis of HIC-purified trastuzumab Pcl-MMAF ADCs by SDS-PAGE.

[0094] FIG. 14. LCMS of HIC-purified trastuzumab LC-K107Pcl-MMAF

ADC.

[0095] FIG. 15. Thermal melting curves of unmodified wild-type trastuzumab (solid line, labeled WT anti-Her2), trastuzumab HC-E152Pcl-MMAF (dotted line) and LC-R108Pcl-MMAF (dashed line) ADCs.

[0096] FIG. 16. Cell proliferation assays for trastuzumab LC-K107Pcl- MMAF ADC with MDA-MB231 clone 16 and clone 40 cells. [0097] FIG. 17. Cell proliferation assays for Antibody 14090 HC-E258Pcl- MMAF ADC with CMK11-5 and Jurkat cells.

[0098] FIG. 18. Structure of the putative toxic metabolite of a Pcl-MMAF ADC (A) and of a Pel ADC with a payload with non-cleavable linker (B).

[0099] FIG. 19. Pharmacokinetic studies of trastuzumab Pcl-MMAF ADCs in mice. Representative plots of plasma concentration vs. sample collection time are shown for (A) unconjugated, wild-type trastuzumab, (B) HC-E258Pcl-MMAF, (C) LC-D122Pcl-MMAF, (D) LC-R142Pcl-MMAF, (E) LC-K169Pcl-MMAF, (F) LC-S1 14Pcl-MMAF, and (G) LC-S156Pcl-MMAF ADCs. ELISA readouts with anti-hlgG antibody are shown as solid symbols while concentrations measured with anti-MMAF-ELISA are represented as open symbols. The standard deviation of measurements in three animals was used as error bars.

[00100] FIG. 20. In vivo efficacy studies of trastuzumab Pcl-MMAF

ADCs in a MDA-MB231 clone 16 xenograft mouse model. (A) Inhibition of MDA-MB231-16 tumor growth in vivo by 6 trastuzumab HC-Pcl-MMAF ADCs. (B) Inhibition of MDA-MB231-16 tumor growth in vivo by 4 trastuzumab LC-Pcl- MMAF ADCs.

[00101] FIG. 21. Location of selected attachment sites in the structure of a human IgGl with a kappa light chain. Selected residues are shown in black on only one of the two heavy chains and for one of the two kappa light chains (lHZH.pdb). Three rotations of the structure are shown using PyMOL, an open- source molecular modeling package (The PyMOL Molecular Graphics System, Version 1.5.0. Schrodinger, LLC).

DETAILED DESCRIPTION

[00102] The present invention provides methods of site-specific labeling of antibodies or antibody fragments by replacing one or more amino acids of a parent antibody or antibody fragment at specific positions with a TAG-encoded amino acid such as pyrroline-carboxy-lysine ("Pel"). The engineered antibodies or antibody fragments are capable of conjugation to various agents (e.g., cytotoxic agents). The present invention also provides antibody drug conjugates that are produced by using the methods described herein.

[00103] TAG is normally recognized as a "STOP" codon during translation of a nucleic acid into a protein, but in some systems, TAG can function as a codon for certain amino acids, including Pel, pyrrolysine, and some unnatural amino acids. It is known, for example, that the pyrrolysyl-tRNA charged with Pel (through the action of the pyrrolysyl-tRNA synthetase (RS)) naturally recognizes TAG and the ribosome inserts Pel at the TAG codon. However, since TAG is also a STOP codon, the release factor RF 1 competes with the pyrrolysyl-tRNA, which may result in either truncation of the nascent polypeptide chain or alternatively in Pel incorporation and continuing protein synthesis. Such competition often reduces protein yield. The extent of truncation depends on the incorporation site and is generally not predictable.

[00104] The present invention describes specific sites on an antibody constant region where a native amino acid of a parental antibody or antibody fragment can be replaced with a TAG-encoded amino acid such as Pel without extensive interference due to truncation. This means that the corresponding nucleic acid containing a TAG codon in place of the codon for the native amino acid of a parental antibody or antibody fragment can be expressed efficiently, without truncation due to the TAG codon, producing a protein in which the native amino acid of the parental antibody or antibody fragment has been replaced by Pel or another TAG-encoded amino acid. Efficient production means the protein production yield is sufficiently high for further conjugation, and that the major product of expression is a protein that has incorporated a TAG-encoded amino acid and has thus not been truncated by the TAG codon. The invention provides methods for selecting advantageous sites for such amino acid substitution in antibody sequences, and identifies advantageous sites that are useful across various antibodies.

[00105] In certain embodiments, the invention provides modified antibodies or antibody fragments where Pel or another TAG-encoded amino acid has been substituted for a native amino acid of a parental sequence. It has been found that different substitution sites give different efficiencies for conjugation when the TAG-encoded amino acid is Pel: the sites identified herein often provide higher conjugation yields than non-selected sites for antibodies having Pel in place of at least one native amino acid of a parental sequence.

[00106] In addition, it has been found that substitution of Pel at some sites in an antibody or antibody fragment can lead to dimer formation between inserted Pel groups. Also, some sites which appear to be adequately surface- exposed and otherwise suitable for Pel substitution provide antibodies having reduced binding affinities. Sites described herein for Pel substitution provide antibodies that retain binding affinity and avoid extensive dimerization, thus antibodies or antibody fragments containing Pel in place of a native amino acid of a parental antibody or antibody fragment at any of the sites defined herein are an aspect of the invention.

[00107] The site-specific antibody labeling according to the present invention can be achieved with a variety of chemically accessible labeling reagents, such as anti-cancer agents, fluorophores, peptides, sugars, detergents, polyethylene glycols, immune potentiators, radio-imaging probes, prodrugs, and other molecules.

[00108] Accordingly, the present invention provides methods of preparation of homogeneous immunoconjugates with a defined drug-to-antibody ratio (DAR) for use in cancer therapy, and immunoconjugates prepared thereby, as well as pharmaceutical compositions comprising these immunoconjugates.

[00109] In one aspect, the invention provides immunoconjugates having at least one TAG-encoded amino acid (e.g., Pel) substituted for a native amino acid of a parental antibody or antibody fragment at a substitution site of the invention, e.g., the Selected TAG Sites listed in Tables 1, 2 and 3. In some embodiments, the immunoconjugate comprises an antibody or antibody fragment that comprises a sequence selected from the SEQ ID NOs listed in Table 1, Table 2 and Table 3.

[00110] In one aspect, the immunoconjugate of the invention is of

Formula (I):

wherein Ab represents an antibody or antigen binding fragment comprising at least one TAG-encoded amino acid residue such as Pel at one of the substitution sites described herein;

LU is a linker unit as described herein, and is preferably attached to Pel at one of the substitution sites described herein;

X is a payload or drug moiety; and n is a number from 1 to 16.

The following enumerated embodiments are representative implementations of the invention:

Embodiments:

1. An immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375 and 392 of a heavy chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system.

2. The immunoconjugate of embodiment 1 wherein the TAG encoded amino acid is Pel.

3. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and

165 of a light chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system and wherein the said light chain is a kappa light chain.

4. The immunoconjugate of embodiment 3 wherein the TAG encoded amino acid is Pel.

5. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of a light chain of said antibody or antibody fragment and wherein said positions are numbered according to the EU system, and wherein said light chain is a kappa light chain.

6. The immunoconjugate of embodiment 5, wherein the TAG encoded amino acid is Pel.

7. The immunoconjugate of embodiment 1, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of a light chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system and wherein the said light chain is a kappa light chain.

8. The immunoconjugate embodiment 7, wherein the TAG encoded amino acid is Pel.

9. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region at a position chosen from positions 143, 145, 147, 156, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

10. The immunoconjugate of embodiments 1-9 further comprising a drug moiety.

11. The immunoconjugate of embodiments 10, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with TAG encoded amino acids on its constant region at a site selected from positions 117, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of a heavy chain of said antibody or antibody fragment, and wherein said positions on the heavy chain are numbered according to the EU system.

12. The immunoconjugate of embodiment 11, wherein said drug moiety is connected to said TAG encoded amino acid through a cleavable linker.

13. The immunoconjugate of embodiment 11, wherein said linker is non- cleavable.

14. The immunoconjugates of embodiment 11, 12, or 13 wherein the TAG encoded amino acid is Pel. 15. The immunoconjugate of embodiment 10, wherein said

immunoconjugate comprises a group of the formula (IA) or (IB):

IA IB

where LU is a linker unit;

[X] is the point of attachment for a drug moiety or payload;

R²⁰ is H or methyl;

and R³⁰ H or methyl or phenyl.

16. The immunoconjugate of any of embodiments 1-14, wherein said immunoconjugate comprises a drug moiety that is a cytotoxic agent.

17. The immune conjugate of embodiment 16, wherein said drug moiety is selected from the group consisting of a V-ATPase inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.

18. The immunoconjugate of any of embodiments 1-17, wherein said antibody is a monoclonal antibody.

19. The immunoconjugate of any of embodiments 1-17, wherein said antibody is a chimeric antibody.

20. The immunoconjugate of embodiments 1- 17, wherein said antibody is a humanized or fully human antibody.

21. The immunoconjugate of embodiments 1-17, wherein said antibody is a bispecific or multi-specific antibody.

22. The immunoconjugate of any of embodiments 1-21, wherein said antibody or antibody fragment specifically binds to a cell surface marker

characteristic of a tumor.

23. A pharmaceutical composition comprising the immunoconjugate of any of embodiments 1-22.

24. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with TAG modified amino acid on its constant region chosen from positions 117, 119, 121, 124, 132, 134, 136, 139, 152, 153, 155, 157, 164, 165, 171, 174, 176, 177, 178, 189, 191, 195, 197, 207, 212, 246, 258, 269, 274, 282, 283, 286, 288, 290, 292, 293, 294, 320, 322, 326, 330, 333, 334, 335, 337, 344, 355, 360, 362, 375, 382, 389, 390, 392, 393, 398, 400, 413, 415, and 422 of a heavy chain, and wherein said positions are numbered according to the EU system. 25. The modified antibody or antibody fragment of embodiment 24, wherein said substitution is at a position selected from positions 117, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain.

26. The modified antibody or antibody fragment of embodiment 24 or 25 further comprising a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154 , 161, and 165 of a light chain, and wherein said positions are numbered according to the EU system and wherein the said light chain is a kappa light chain.

27. The modified antibody or antibody fragment of embodiment 24 or 25, further comprising a substitution of one or more amino acids with a TAG encoded amino acid on its constant region at a position chosen from positions 143, 145, 147, 156, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

28. The modified antibody or antibody fragment of embodiments 24-27, wherein said substitution is one to eight TAG encoded amino acids.

29. The modified antibody or antibody fragments of claims 23-28 wherein the TAG encoded amino acid is Pel.

30. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 112, 114, 122, 123, 126, 127, 129, 142, 143, 145, 152, 154, 156, 157, 159, 161, 165, 168, 169, 170, 182, 183, 188, 190, 191, 197, 199, 203, and 206 of a light chain, and wherein the positions are numbered according to the EU system. 31. The modified antibody or antibody fragment of embodiment 30, wherein said substitution is at least TAG encoded amino acid, chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the light chain, and wherein said light chain is a kappa light chain.

32. The modified antibody or antibody fragment of embodiments 30 or 31, wherein said substitution is one to eight TAG encoded amino acids.

33. The modified antibody or antibody fragments of embodiments 30-32 wherein the TAG encoded amino acid is Pel.

34. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with a TAG encoded amino acid at a position selected from on its constant region at a position chosen from positions 143, 145, 147, 156, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

35. The modified antibody or antibody fragment of embodiment 34, wherein said substitution is one to eight TAG encoded amino acids.

36. The modified antibody or antibody fragment of embodiment 34 or 35, wherein the TAG encoded amino acid is Pel.

37. A modified antibody or antibody fragment which comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 5, 8, 9, 10, 12, 16, 17, 28, 33, 34, 36, 44, 51, 55, 63, 64, 65, 73, 75, 76, 77, 81, 82, 96, 97, 98, 99, 100, 101, and 102.

38. The modified antibody or antibody fragment of any of embodiments 25, 26, 27, 28, 30, 31, 32, 34, or 35 wherein the modified antibody or antibody fragment is further attached to a drug moiety, and wherein said drug moiety is attached to the modified antibody or antibody fragment through the TAG encoded amino acid and an optional linker to form an immunoconjugate. 39. The modified antibody or fragment of embodiment 38 wherein the TAG encoded amino acid is Pel.

40. The modified antibody or antibody fragment of embodiment 38, wherein said immunoconjugate comprises a group of the formula:

where LU is a linker unit;

X¹ is a drug moiety or payload;

R²⁰ is H or methyl;

and R³⁰ H or methyl or phenyl.

41. The modified antibody or antibody fragment of any of embodiments 25, 26, 27, 28, 30, 31, or 32, wherein said substitution comprises at least one cysteine substitution, and at least TAG encoded amino acid substitution or a peptide tag for enzyme-mediated conjugation. 42. The modified antibody of embodiment 41 wherein the TAG encoded amino acid is Pel.

43. A nucleic acid encoding the modified antibody or antibody fragment of any of embodiments 25-42.

44. A host cell comprising the nucleic acid of claim 43.

45. A method of producing a modified antibody or antibody fragment comprising incubating the host cell of embodiment 43 under suitable conditions for expressing the antibody or antibody fragment, and isolating said antibody or antibody fragment.

46. A method to select an amino acid of an antibody that is suitable for replacement by TAG encoded amino acid to provide an advantaged site for conjugation, comprising:

identifying residues in the constant region of an antibody that are surface accessible to form an initial set of candidate sites for TAG encoded amino acid substitution;

preparing a set of nucleic acids including one nucleic acid encoding a polypeptide corresponding to TAG encoded amino acid replacement of the native amino acid for each candidate site for TAG encoded amino acid substitution;

expressing each nucleic acid in the set of nucleic acids, and removing from the initial set of candidate sites any site where truncation dominates over full-length polypeptide containing a TAG encoded amino acid substitution to provide a set of advantaged sites for TAG encoded amino acid substitution.

47. The method of embodiments 45 or 46 wherein the TAG encoded amino acid is Pel.

48. A method to prepare an immunoconjugate, comprising providing an antibody or antibody fragment of any of embodiments 25, 26, 27, 28, 30, 31, 34, 35, or 37 comprising contacting the antibody or antibody fragment containing at least one TAG encoded amino acid residue with an ABA compound, or an ABP compound, or an AAP compound.

49. The method of embodiment 48 wherein the TAG encoded amino acid is Pel.

50. The method of embodiment 48 or 49, wherein the immunoconjugate comprises a group of the formula IA or IB:

or

IA IB

where LU is a linker unit;

X¹ is a drug moiety or payload;

R²⁰ is H or methyl;

and R³⁰ H or methyl or phenyl.

51. The method of embodiment 50, wherein the immunoconjugate comprises a group of the formula IA or IB:

or

IA

where LU is a linker unit; X¹ is a drug moiety or payload;

R²⁰ is H or methyl;

and R³⁰ H or methyl or phenyl.

For any of the immunoconjugates in the foregoing embodiment, the Drug- Antibody ration is preferably about 2, about 4, about 6, or about 8.

For any of the immunoconjugates comprising LU, the group LU is typically a group of formula -L1-L2-L3-L4-L5-L6-, wherein Li, L₂, L₃, L₄, L₅ and L₆ are independently selected from -Ai-, -A1X²- and -X²-; wherein:

Ai is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(C(R⁴)₂)_n-, -(0(CH₂)_n)_m- , -(0(C(R⁴)₂)_n)_m-,-((CH₂)_nO)_m-, -((C(R⁴)₂)_nO)_m-, -((CH₂)_nO)_m(CH₂)_n- , -((C(R⁴)₂)_nO)_mC(R⁴)₂)_n-, -(CH₂)_nC(=0)NH-, -(C(R⁴)₂)_nC(=0)NH- , -(CH₂)_nNHC(=0)-, -(C(R⁴)₂)„NHC(=0)-, -NHC(=0)(CH₂)_n-, - NHC(=0)(C(R⁴)₂)_n-, -C(=0)NH(CH₂)_nS-, -C(=0)NH(C(R⁴)₂)_nS-, - S(CH₂)_nC(=0)NH-, -S(C(R⁴)₂)_nC(=0)NH-, - C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-

, -C(=0)NH(C(R⁴)₂)_nNHC(=0)(C(R⁴)₂)_n-, -C(=0)(CH₂)_n-, - C(=0)(C(R⁴)₂)_n-, -(CH₂)_nC(=0)-, -(C(R⁴)₂)_nC(=0)-, - (CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-

, -(C(R⁴)₂)_n(0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)_n-, -(CH₂)_nNHC(=0)(CH₂)_n- , -(C(R⁴)₂)_nNHC(=0)(C(R⁴)₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n- , -(C(R⁴)₂)„NH((C(R⁴)₂)„0)_m(C(R⁴)₂)„-, -(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, or -(0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)_n-;

each X² is independently selected from a bond, R⁸,



, -CHR⁴(CH₂)_nC(=0)NH-, -CHR⁴(CH₂)_nNHC(=0)-,

-C(=0)NH- and -NHC(=0)-;

each R⁴ is independently selected from H, Ci-₄alkyl, side chains of known amino acids, -C(=0)OH and -OH,

each R⁵ is independently selected from H, Ci-₄alkyl, phenyl or Ci-₄alkyl substituted with 1 to 3 -OH groups;

each R⁶ is independently selected from H, fluoro, benzyloxy substituted with -C(=0)OH, benzyl substituted with -C(=0)OH, Ci_₄alkoxy substituted with -C(=0)OH and Ci_₄alkyl substituted with -C(=0)OH; R⁷ is independently selected from H, C_1-4alkyl, phenyl, pyrimidine and pyridine;

independently selected from

, and

R⁹ is independently selected from H and Ci-₆haloalkyl;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

The methods and modified antibodies or antibody fragments of the instant invention can be used in combination with other conjugation methods known in the art.

Site-Specific Labeling

[00111] The antibodies (e.g., a parental antibody, optionally containing one or more non-canonical amino acids) of the present invention are numbered according to the EU numbering system as set forth in Edelman et ah, (1969) Proc. Natl. Acad. USA 63:78-85, except that the lambda light chain is numbered according to the Kabat numbering system as set forth in Kabat et al., Sequences of Proteins of Immunological Interest (1991) Fifth Edition, NIH Publication No. 91-3242. Human IgGl constant region is used as a representative example throughout the application. However, the invention is not limited to human IgGl ; corresponding amino acid positions can be readily deduced by sequence alignment. For example, Figure 4 shows sequence alignment of human IgGl, IgG2, IgG3 and IgG4 heavy chain constant regions, so that an identified TAG-encoded amino acid (e.g., Pel) engineering site in the IgGl constant region can be readily identified for IgG2, IgG3, and IgG4 as shown in Figure 4. For the light chain constant region, IgGl, IgG2, IgG3 and IgG4 are the same. Table 1 below lists the amino acid positions in the constant region of the heavy chain of an antibody that can be replaced by a TAG-encoded amino acid such as Pel. Table 2 lists the amino acid positions in the constant region of the kappa light chain of an antibody that can be replaced by a TAG-encoded amino acid such as Pel. Table 3 lists the amino acid positions in the constant region of the lambda light chain of an antibody that can be replaced by a TAG-encoded amino acid such as Pel.

[00112] Table 1. Identified TAG-encoded amino acid (e.g., Pel) substitution sites in the heavy chain constant region of human IgGl (Sites numbered according to EU numbering system). In addition to Pel incorporation, the TAG sites can be used to incorporate any other TAG-encoded amino acid.

Surface

Eu Selected SEQ ID

Residue accessibility

number TAG sites NO

[A²]

174 LEU 68.1 HC-L174Pcl 17

176 SER 161 .9 HC-S176Pcl 18

177 SER 68.1 HC-S177Pcl 19

187 THR 30.3 HC-T187Pcl 20

189 PRO 86.4 HC-P189Pcl 21

191 SER 126.8 HC-S191 Pcl 22

195 THR 1 1 1 .3 HC-T195Pcl 23

197 THR 89.8 HC-T197Pcl 24

207 SER 50 HC-S207Pcl 25

212 ASP 97 HC-D212Pcl 26

246 LYS 55.1 HC-K246Pcl 27

258 GLU 42.1 HC-E258Pcl 28

269 GLU 189.2 HC-E269Pcl 29

274 LYS 137.8 HC-K274Pcl 30

282 VAL 140.7 HC-V282Pcl 31

283 GLu 80.2 HC-E283Pcl 32

286 ASN 1 19.4 HC-N286Pcl 33

288 LYS 181 .8 HC-K288Pcl 34

290 LYS 177 HC-K290Pcl 35

292 ARG 251 .5 HC-R292Pcl 36

293 GLU 83.3 HC-E293Pcl 37

294 GLU 73.5 HC-E294Pcl 38

320 LYS 55 HC-K320Pcl 39

322 LYS 78.3 HC-K322Pcl 40

326 LYS 212.7 HC-K326Pcl 41

330 ALA 96.3 HC-A330Pcl 42

333 GLU 84.7 HC-E333Pcl 43

334 LYS 49.6 HC-K334Pcl 44

335 THR 70.1 HC-T335Pcl 45

337 SER 15.1 HC-S337Pcl 46

344 ARG 98.2 HC-R344Pcl 47

355 ARG 249.4 HC-R355Pcl 48

360 LYS 1 13.9 HC-K360Pcl 49

362 GLN 40.8 HC-Q362Pcl 50

375 SER 28.9 HC-S375Pcl 51

382 GLU 21 .8 HC-E382Pcl 52 Surface

Eu Selected SEQ ID

Residue accessibility

number TAG sites NO

[A²]

389 ASN 189.5 HC-N389Pcl 53

390 ASN 36.4 HC-N390Pcl 54

392 LYS 81 .8 HC-K392Pcl 55

393 THR 35.8 HC-T393Pcl 56

398 LEU 1 10.9 HC-L398Pcl 57

400 SER 81 .3 HC-S400Pcl 58

413 ASP 79.6 HC-D413Pcl 59

415 SER 69 HC-S415Pcl 60

422 VAL 80.8 HC-V422Pcl 61

[00113] Table 2. Identified TAG-encoded amino acid (e.g., Pel) substitution sites on the kappa light chain constant region of human IgGl (Sites numbered according to EU numbering system). In addition to Pel incorporation, the TAG sites can be used to incorporate any other TAG-encoded amino acid.

Surface

Eu Selected SEQ

Residue accessibility

number TAG sites ID NO

[A²]

161 GLU 66 LC-E161 Pcl 81

165 GLU 74 LC-E165Pcl 82

168 SER 170 LC-S168Pcl 83

169 LYS 241 LC-K169Pcl 84

170 ASP 48 LC-D170Pcl 85

182 SER 59 LC-S182Pcl 86

183 LYS 131 LC-K183Pcl 87

188 LYS 201 LC-K188Pcl 88

190 LYS 167 LC-K190Pcl 89

191 VAL 58 LC-V191 Pcl 90

197 THR 38 LC-T197Pcl 91

199 GLN 127 LC-Q199Pcl 92

203 SER 110 LC-S203Pcl 93

206 THR 70 LC-T206Pcl 94

[00114] Table 3. Identified TAG-encoded amino acid (e.g., Pel) substitution sites on the lambda light chain. In addition to Pel incorporation, the TAG sites can be used to incorporate any other TAG-encoded amino acid.

[00115] Because of the high sequence homology of constant regions of IgGl, IgG2, IgG3 and IgG4 antibodies, findings of the invention are not limited to any specific antibodies. In addition, the findings of the present invention are not limited to using Pel substitutions. The positions in the antibody constant regions identified herein can be used for incorporating other amino acids, especially TAG-encoded amino acids, including non-canonical amino acids and unnatural amino acids.

[00116] In one embodiment, the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids, i.e., a TAG-encoded amino acid such as Pel, on its constant region chosen from the Selected TAG Sites in Table 1, and particularly selected from positions 117, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain of said antibody or antibody fragment. In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof containing at least one TAG-encoded amino acid such as Pel at a site selected from the ones identified herein, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises an amino acid sequence selected from SEQ ID NOs 2, 5, 8, 9, 10, 12, 16, 17, 28, 33, 34, 36, 44, 51 and 55.

[00117] In one embodiment, the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids on its heavy chain constant region chosen from positions identified in Table 1. In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof and a drug moiety, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant region chosen from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain. For example, an

immunoconjugate of the invention, or a modified antibody of the invention, comprises a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with a TAG-encoded amino acid such as Pel on its constant region at a position chosen from positions 117 and 136, 117 and 139, 117 and 152, 1 17 and 155, 1 17 and 171, 117 and 174, 117 and 258, 117 and 286, 1 17 and 188, 1 17 and 191, or 117 and 375; 136 and 139, 136 and 152, 136 and 155, 136 and 171, 136 and 171, 136 and 174, 136 and 258, 136 and 186, 136 and 288, 136 and 292, 136 and 375; 139 and 152, 139 and 155, 139 and 171, 139 and 174, 139 and 258, 139 and 186, 139 and 188, 139 and 292, 139 and 375; 152 and 155, 152 and 171, 152 and 174, 152 and 258, 152 and 286, 152 and 288, 152 and 292, 152 and 375; 155 and 171, 155 and 174, 155 and 258, 155 and 286, 155 and 188, 155 and 292, 155 and 375; 171 and 174, 171 and 258, 171 and 286, 171 and 288, 171 and 292, 171 and 375, 174 and 258, 174 and 286, 174 and 288, 174 and 292, 174 and 374; 258 and 286, 258 and 288, 258 and 292, 258 and 375, 286 and 288, 286 and 292, 286 and 375, 288 and 292, 288 and 375, or 292 and 375 of the heavy chain.

[00118] In another embodiment, an immunoconjugate or modified antibody or antibody fragment of the invention comprises a substitution of three amino acids with a TAG-encoded amino acid such as Pel on its constant region at positions chosen from positions 117, 136 and 139; 1 17, 136 and 152; 1 17, 136 and 155; 1 17, 136 and 171; 1 17, 136 and 174; 1 17, 136 and 258; 1 17, 136 and 286; 117, 136 and 288; 1 17, 136 and 292; 117, 136 and 375; 117, 139 and 152; 117, 139, and 155; 117, 139 and 171 ; 1 17, 139, and 174; 1 17, 139 and 258; 1 17, 139 and 286; 117, 139 and 288; 1 17, 139 and 292; 1 17, 139 and 375; 1 17, 152, and 155; 1 17, 152, and 171; 117, 152, and 174; 1 17, 152 and 258; 117, 152, and 286; 117, 152 and 288; 1 17, 152 and 292; 117, 152, and 375; 117, 155, and 171; 117, 155, and 174; 117, 155, and 258; 1 17, 155, and 286; 1 17, 155, and 288; 117, 155 and 292; 117, 155, and 375; 1 17, 171, and 174; 117, 171, and 258; 117, 171, and 286; 1 17, 171, and 288; 117, 171 and 292; 1 17, 171 and 375; 1 17, 174, and 258; 117, 174, and 286; 1 17, 174 and 288; 117, 174, and 292; 1 17, 174, and 375; 117, 258, and 286; 117, 258, and 288; 1 17, 258, and 292; 1 17, 258, and 375; 117, 286, and 288; 117, 286, and 292; 117, 286, and 375; 1 17, 288, and 292; 117, 288, and 375; 1 17, 292, and 375; 136, 139 and 152; 136, 139, and 155; 136, 139 and 171 ; 136, 139, and 174; 136, 139 and 258; 136, 139 and 286; 136, 139 and 288; 136, 139 and 292; 136, 139 and 375; 136, 152 and 155; 136, 152, and 174; 136, 152, and 258; 136, 152, and 286; 136, 152, and 288; 136, 152 and 292; 136, 152 and 375; 136, 155, and 171; 136, 155 and 174; 136, 155 and 258; 136, 155, and 288; 136, 155, and 292; 136, 155 and 375; 136, 171, and 174; 136, 171 and 258; 136, 171, and 286; 136, 171, and 288; 136, 171, and 292; 136, 171, and 375; 136, 174 and 258; 136, 174 and 286; 136, 174 and 288; 136, 174, and 292; 136, 174 and 375; 136, 258, and 286; 136, 258, and 288; 136, 258 and 292; 136, 158 and 375; 136, 286, and 288; 136, 286, and 292; 136, 286, and 375; 136, 288 and 292; 136, 288 and 375; 136, 292 and 375; 139, 152 and 155; 139, 152 and 171 ; 139, 152, and 174; 139, 152 and 258; 139, 152, and 286; 139, 152, and 288; 139, 152, and 292; 139, 152, and 375; 139, 155, and 171; 139, 155, and 174; 139, 155, and 258; 139, 155, and 286; 139, 155, and 288; 139, 155, and 292; 139, 155 and 375; 139, 171, and 174; 139, 171, and 258; 139, 171 and 286; 139, 171 and 288; 139, 171, and 292; 139, 171 and 375; 139, 174 and 258; 139, 171 and 286; 139, 171, and 288; 139, 171 and 292; 139, 171, and 375; 139, 174 and 258; 139, 174, and 288; 139, 174, and 292; 139, 174, and 375; 139, 258, and 286; 139, 258 and 288; 139, 258, and 292; 139, 258 and 375; 139, 286, and 288; 139, 286, and 292; 139, 286, and 375; 139, 288 and 292; 139, 288, and 375; 139, 292, and 375; 152, 155 and 171 ; 152, 155, and 174; 152, 155, and 258; 152, 155, and 286; 152, 155 and 288; 152, 155, and 292; 152, 155 and 375; 152, 171, and 174; 152, 171 and 258; 152, 171, and 286; 152, 171 and 288; 152, 171 and 292; 152, 171, and 375; 152, 174 and 258; 152, 174, and 286; 152, 174 and 288; 152, 174 and 292; 152, 174, and 375; 152, 258, and 286; 152, 258 and 288; 152, 258 and 292; 152, 258 and 375; 152, 286, and 288; 152, 286 and 292; 152, 286 and 375; 152, 288 and 292; 152, 288, and 375; 152, 292 and 375; 155, 171, and 174; 155, 171 and 258; 155, 171 and 286; 155, 171, and 288; 155, 171, and 292; 155, 171, and 375; 155, 174, and 258; 155, 174, and 286; 155, 174, and 288; 155, 174, and 292; 155, 174, and 375; 155, 258, and 286; 155, 258, and 288; 155, 258, and 292; 155, 258, and 375; 155, 286, and 288; 155, 286, and 292; 155, 286, and 375; 155, 288, and 292; 155, 288, and 375; 155, 292, and 375; 171, 174 and 258; 171, 174, and 286; 171, 174, and 288; 171, 174, and 292; 171, 174, and 375; 171, 258, and 286; 171, 258 and 288; 171, 258 and 292; 171, 258, and 375; 171, 286, and 288; 171, 286, and 292; 171, 286 and 375; 171, 288 and 292; 171, 288 and 375; 171, 292 and 375; 174, 258 and 286; 174, 258 and 288; 174, 258 and 292; 174, 258 and 375; 174, 286, and 288; 174, 286 and 292; 174, 286 and 375; 174, 288 and 292; 174, 288 and 375; 174, 292 and 375; 258, 286 and 288; 258, 286, and 292; 258, 286 and 375; 258, 288 and 292; 258, 288 and 375; 258, 292 and 375; 286, 288 and 292; 286, 288 and 375; 288, 292 and 375. [00119] In another embodiment, the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids on its constant region chosen from the Selected TAG sites in Table 2, particularly positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the light chain of said antibody or antibody fragment, wherein the light chain is a human kappa light chain.

Typically, a TAG-encoded amino acid such as Pel is substituted for the native amino acid of the parental antibody sequence in at least one of the specific substitution sites identified herein. In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NOs: 63, 64, 65, 73, 75, 76,77, 81 and 82. For example, an immunoconjugate of the invention, or a modified antibody of the invention, comprises a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with a TAG-encoded amino acid such as Pel on its constant region at a position chosen from positions 107 and 108; 107 and 109;

107 and 145; 107 and 152; 107 and 154; 108 and 109; 108 and 145; 108 and 152;

108 and 154; 109 and 145; 109 and 152; 109 and 154; 145 and 152; 145 and 154; and 152 and 154 of the human kappa light chain. In another embodiment, an immunoconjugate or modified antibody or antibody fragment of the invention comprises a substitution of three amino acids with a TAG-encoded amino acid such as Pel on its constant region at positions chosen from positions 107, 108 and 109; 107, 108 and 145; 107, 108 and 152; 107, 108 and 154; 107, 109 and 145; 107, 109 and 152; 107, 109 and 154; 107, 145 and 152; 107, 145 and 154; 107, 152 and 154; 108, 109 and 145; 108, 109 and 152; 108, 109, and 154; 109, 145 and 152; 109, 145 and 154; and 109, 152 and 154 of the human kappa light chain.

[00120] In another embodiment, the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of one or more amino acids on its constant region chosen from the Selected TAG sites in Table 3, particularly positions 143, 145, 147, 156, 159, 163 and 168 of the light chain of said antibody or antibody fragment, wherein the light chain is a human lambda light chain, using the Kabat numbering system. Typically, a TAG-encoded amino acid such as Pel is substituted for the native amino acid of the parental antibody sequence in at least one of the specific substitution sites identified herein. In a specific embodiment, the present invention provides an immunoconjugate comprising a modified antibody or antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises SEQ ID NOs: 96, 97, 98, 99, 100, 101 and 102. For example, an immunoconjugate of the invention, or a modified antibody of the invention, comprises a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of two amino acids with a TAG-encoded amino acid such as Pel on its constant region at a position chosen from positions 143 and 145; 143 and 147; 143 and 156; 143 and 156; 143 and 159; 143 and 163; 143 and 168; 145 and 147; 145 and 156; 145 and 159; 145 and 163; 145 and 168; 147 and 156; 147 and 159; 147 and 163; 147 and 168; 156 and 159; 156 and 163; 156 and 168; 159 and 163; 159 and 168; and 163 and 168 of the human lambda light chain. In another embodiment, an immunoconjugate or modified antibody or antibody fragment of the invention comprises a substitution of three amino acids with a TAG-encoded amino acid such as Pel on its constant region at positions chosen from positions 143, 145 and 147; 143, 145 and 156; 143, 145 and 159; 143, 145 and 163; 143, 145 and 168; 144, 147 and 156; 145, 147 and 159; 145, 147 and 163; 145, 147, and 168; 147, 156 and 159; 147, 156 and 163; 147, 156 and 168; 156, 159 and 163; 156, 159 and 168; and 159, 163 and 168 of the human lambda light chain.

[00121] In another embodiment, the present invention provides immunoconjugates comprising a modified antibody or an antibody fragment thereof, and a drug moiety, wherein said modified antibody or antibody fragment thereof comprises a substitution of a TAG-encoded amino acid such as Pel for one or more amino acids on its constant region chosen from the Selected TAG Sites in Table 1, particularly from positions 117, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain of said antibody or antibody fragment, in combination with a second substitution, which may be a substitution of a TAG-encoded amino acid such as Pel for one or more amino acids on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the light chain of said antibody or antibody fragment, or a position chosen from positions 143, 145, 147, 156, 159, 163 and 168 of a lambda light chain. In these embodiments, a TAG-encoded amino acid such as Pel is often substituted for the native amino acid of the parental sequence.

[00122] For example, a modified antibody or antibody fragment according to the present invention, whether alone or as part of an

immunoconjugate, may comprise a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 1 17 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position

107 of a human kappa light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position

108 of a human kappa light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position

107 of a human kappa light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position

108 of a human kappa light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 107 of a human kappa light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 108 of a human kappa light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 109 of a human kappa light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 145 of a human kappa light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 152 of a human kappa light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 154 of a human kappa light chain. Positions on the human kappa light chain are described using the Eu numbering system. Additional embodiments of the invention include any of these antibodies or antibody fragments further including a third Pel substitution selected from the group consisting of: a second Pel substitution in the heavy chain that is different from the one in the foregoing embodiment and is selected from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain; and a second substitution in the light chain that is different from the one in the foregoing embodiment and is selected from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the human kappa light chain.

[00123] In further examples, a modified antibody or antibody fragment according to the present invention, whether alone or as part of an immunoconjugate, may comprise a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 1 17 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 1 17 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 117 of a heavy chain, and a Pcl substitution on position 159 of a human lambda light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 117 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 136 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 139 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 152 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 155 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 171 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 174 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 258 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 286 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 288 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 292 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 143 of a human lambda light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 145 of a human lambda light chain; a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 147 of a human lambda light chain; a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 156 of a human lambda light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 159 of a human lambda light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 163 of a human lambda light chain; or a Pel substitution on position 375 of a heavy chain, and a Pel substitution on position 168 of a human lambda light chain; wherein the lambda light chain positions are numbered according to the Kabat numbering system. Additional embodiments of the invention include any of these antibodies or antibody fragments further including a third Pel substitution selected from the group consisting of: a second Pel substitution in the heavy chain that is different from the one in the foregoing embodiment and is selected from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain; and a second substitution in the light chain that is different from the one in the foregoing embodiment and is selected from positions 143, 145, 147, 156, 159, 163, and 168 of the human lambda light chain.

[00124] In some aspects of the invention, the amino acid substitution described herein is Pel, though other TAG-encoded amino acid can be used as well. Where Pel is introduced, it is typically used as the site of conjugation to which a drug moiety is attached. Conjugates of Formula IA or IB are embodiments of this aspect of the invention. In some embodiments, the immunoconjugates of the invention comprise a drug moiety selected from the group consisting of a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRMl, a DPPIV inhibitor, proteasome inhibitors, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor. In some embodiments, the immunoconjugates of the invention comprise a drug moiety that is an anti-cancer agent. The modified antibody or antibody fragments of the present invention can be any formats known in the art, such as a monoclonal, chimeric, humanized, fully human, bispecific, multispecific antibody or antibody fragment thereof.

[00125] According to the present invention, the modified antibody heavy chain and/or light chain (or antibody fragment thereof) may contain 1, 2, 3, 4, 5, 6, 7, 8, or more Pel substitutions in its constant regions. In one embodiment, the modified antibodies or antibody fragments contain 2, 4, 6, 8, or more Pel substitutions in its constant regions.

[00126] In one embodiment, the parental antibody (antibody without Pel substitution) is an IgG, IgM, IgE, or IgA antibody. In a specific embodiment, the parental antibody is an IgGl antibody. In another specific embodiment, the parental antibody is an IgG2, IgG3, or IgG4 antibody.

[00127] The present invention also provides modified antibodies or antibody fragments thereof comprising a substitution of a TAG-encoded amino acid such as Pel for one or more amino acids on its constant region chosen from the Selected TAG sites in Table 1, particularly selected from positions 117, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain of said antibody or antibody fragment. In a specific embodiment, the modified antibody or antibody fragment of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs 2, 5, 8, 9, 10, 12, 16, 17, 28, 33, 34, 36, 44, 51 and 55.

[00128] In some embodiments, the present invention provides modified antibodies or antibody fragments thereof comprising a substitution of at least one TAG-encoded amino acid such as Pel for one or more amino acids on its constant region chosen from the Selected TAG Sites in Table 2, especially positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of the light chain of said antibody or antibody fragment. In a specific embodiment, the modified antibody or antibody fragment of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 63, 64, 65, 73, 75, 76, 77, 81, and 82.

[00129] In some embodiments, the present invention provides modified antibodies or antibody fragments thereof comprising a substitution of at least one TAG-encoded amino acid such as Pel for one or more amino acids on the constant region of a lambda light chain chosen from the Selected TAG sites in Table 3, especially positions 143, 145, 147, 156, 159, 163, and 168 of the lambda light chain of said antibody or antibody fragment, using the Kabat numbering system. In a specific embodiment, the modified antibody or antibody fragment of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 96, 97, 98, 99, 100, 101, and 102.

[00130] In certain embodiments, the modified antibodies provided herein are labeled using the methods of the invention in combination with other conjugation methods known in the art including, but not limited to, chemos elective conjugation through lysine, cysteine, histidine, tyrosine, formyl-glycine, and protein tags for enzyme-mediated conjugation (e.g., S6 tags).

2. Pyrroline-carboxy-lysine ("Pel")

[00131] Pyrrolysine (Pyl) is the 22nd natural, genetically encoded amino acid found in certain methanogenic Archaea of the family

Methanosarcinaceae and two unrelated bacterial species. Specifically, pyrrolysine is found in MtmBl, the monomethylamine (MMA) methyltransferase which initiates methane formation in such Archaea bacteria, (see Srinivasan et ah, (2002), Science, 296, 1459-62; Soares, et al., (2005) Journal of Biological Chemistry, 280, 36962-9; Hao et al, (2002) Science, 296, 1462-6; Krzycki (2005) Current Opinion in Microbiology, 8, 706-12; Krzycki (2004) Current Opinion in Chemical Biology, 8, 484-91, and Ambrogelly et ah, (2007) Nature Chemical Biology 3, 29-35). Pyrrolysine is considered a dipeptide wherein the ξ- amine of lysine is linked to the D-isomer of 4-methyl-pyrroline-5-carboxylate via an amide bond (see, Polycarpo et ah, (2006) FEBS Letters, 580, 6695-700). The structure of pyrrolysine was deduced from the crystal structure of MtmBl and from the residue's mass (see, J. Biol. Chem. 2005, 44, 36962-36969; PNAS, 2007, 104, 1021-1026).

Structure of Pel Incorporated into a Peptide.

where IC^U = H (R^zu = Me is Pyl).

The mtmBl gene encoding MtmBl possesses an in-frame amber (TAG) codon, which is normally a canonical stop codon. However, in mtmBl mRNA the UAG codon, encoded as TAG on the DNA level, does not terminate translation during production of the MtmB 1 protein, but instead the UAG codon encodes pyrrolysine which is incorporated into the protein. Pyrrolysine is endogenously synthesized and is co-translationally incorporated at such in-frame UAG codons as the free amino acid.

[00132] The biosynthesis and incorporation of pyrrolysine are facilitated by the natural genes pylT, pylS, pylB, pylC and pylD. pylT encodes pyrrolysyl-tRNA, pylS encodes pyrrolysyl-tRNA synthetase while pylB, pylC and pylD encode proteins required for the biosynthesis of pyrrolysine. These genes have been derived from Methanosarcina mazei, (see, Longstaff et ah, (2007) Proceedings of the National Academy of Sciences of the United States of America, 104, 1021-6, and Namy et ah, (2007) FEBS Letters, 581 , 5282-8). The pylT and pylS genes along with pylB, pylC and pylD genes form a pylTSBCD gene cluster which is a natural genetic code expansion cassette whose transfer allows the UAG codon to be translated as pyrrolysine, which is incorporated into a protein at the UAG site.

[00133] Although various precursors for pyrrolysine have been proposed, such as D-glutamate, D-isoleucine, D-proline and D-ornithine, D- ornithine was stated to be the most effective precursor for pyrrolysine biosynthesis in Escherichia coli transformed with a plasmid carrying the natural genes pylT, pylS, pylB, pylC and pylD. Incorporation of pyrrolysine was not verified directly by mass spectrometry but the conclusion was supported by previous mass spectrometry data that in the absence of added D-ornithine, pyrrolysine is biosynthesized and incorporated, albeit at low levels, into proteins produced within Escherichia coli transformed with a plasmid the natural genes pylT, pylS, pylB, pylC and pylD (see, Longstaff e? al, (2007) Proceedings of the National Academy of Sciences of the United States of America, 104, 1021-6).

[00134] However, it has been shown that introduction of the pylT, pylS, pylB, pylC and pylD genes into Escherichia coli or mammalian cells, and the addition of D-ornithine into the growth media resulted in the biosynthesis and incorporation of a "demethylated pyrrolysine" (Pcl-A and Pcl-B), as identified using mass spectrometry (see WO2010/048582):

[00135] Accordingly, the modified Pel-containing antibodies of the invention can be prepared by using a pyrrolysine analogue, pyrroline-carboxy-lysine (Pel) that is naturally encoded, biosynthetically generated and incorporated into antibodies or antibody fragments using the natural genes, pylT, pylS, pylB, pylC and pylD, and D-ornithine as a precursor. In other embodiments, as D-arginine is a precursor to D-ornithine, is the modified Pel-containing antibodies of the invention can be prepared by using a pyrrolysine analogue, Pel, that is naturally encoded, biosynthetically generated and incorporated into proteins using the natural genes, pylT, pylS, pylB, pylC and pylD, and D-arginine as a precursor. Alternatively, Pel (or certain synthetic Pel analogs) can be added to the antibody expression reaction.

[00136] As shown previously, attempts to biosynthesize pyrrolysine in Escherichia coli and 293 Freestyle™ F cells using D-ornithine as a precursor did not result in the formation of pyrrolysine, but rather a "demethylated pyrrolysine" referred to herein as pyrroline-carboxy-lysine (Pel) (Pcl-A or Pcl-B). The biosynthesis of Pcl-A or Pcl-B does not require the presence of the pylB gene. The observation that both PylD and PylC are required for Pel incorporation and the inability of PylS to accept as substrate pyrrolysine analogues with a sp2 carbon at positions equivalent to the C-5 position of the 1 -pyrroline ring of Pel, initially suggested that Pel is likely incorporated into proteins primarily in the form of Pcl-A rather than as Pcl-B that is equal in molecular mass.

[00137] Recent studies (see Cellitti et al, (2011) Nat Chem Biol.

7(8):528-30; and Gaston et al, (2011) Nature 471(7340):647-50) suggest that D- ornithine is ligated to the epsilon amine of L-lysine by the enzyme PylC and ATP resulting in (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid (L-lysine-N- ε-D-ornithine dipeptide). PylD is involved in the activation of (2S)-2-amino-6-((R)- 2,5- diaminopentanamido)hexanoic acid into the semialdehyde ((S)-2-amino-6-((R)- 2-amino-5- oxopentanamido)hexanoic acid) that spontaneously cyclizes to Pcl-A as suggested by in vitro NMR measurements with PylD, L-lysine-N-e-D-ornithine dipeptide, ATP and NAD⁺ (Cellitti et al. (201 1) Nat Chem Biol. 7(8):528-30).

Addition of 3-methyl-D-ornithine to the growth media in the absence oipylB but in the presence of pylC, pylD, pylS and pylT resulted in the incorporation of pyrrolysine into TAG containing proteins. Addition of L-lysine- N-e-D -ornithine to the growth media in the absence oipylB and pylC but in the presence oipylD, pylS and pylT resulted in the incorporation of pyrrolysine and Pel into TAG containing proteins. Furthermore, MS studies recently suggested that PylB converts L-lysine to D-3- methyl-ornithine (Gaston MA, Zhang L, Green-Church KB, Krzycki JA. (201 1) Nature 471(7340):647-50). Hence pyrrolysine appears to be generated from two L- lysine molecules through the sequential action of the PylB, PylC and PylD enzymes. Without being held to any particular theory, it is thought that the demethylated Pcl-A or Pcl-B are obtained in the presence of the added D-ornithine as a result of either the deactivation of PylB, the absence of required cofactor(s), or combinations thereof. However, this does not preclude the presence of an alternative mechanism.

[00138] Accordingly, the present invention provides conjugation methods encompassing site specific incorporation of biosynthetically generated pyrrolysine and/or pyrroline-carboxy-lysine ((S)-2-amino-6-(3,4-dihydro-2H-pyrrole- 2-carboxamido) hexanoic acid (Pcl-A) or (S)-2-amino-6-(3,4-dihydro-2H-pyrrole-5- carboxamido)hexanoic acid (Pcl-B)), where the Pcl-A or Pcl-B is incorporated at one of the substitution sites identified herein, e.g., at one of the sites listed in Table 1, Table 2 or Table 3. The pyrrolysine analogues Pcl-A and Pcl-B, both referred to herein as Pel, lack the methyl group of pyrrolysine (Pyl). In certain embodiments of such methods, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell. In other embodiments, the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney (293 Freestyle™) cells, human epitheloid carcinoma (HeLa and GH3) cells, monkey kidney (COS) cells, rat C6 glioma cells, baby hamster kidney (BHK-21) cells and Chinese hamster ovary (CHO) cells. In certain embodiments, the yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include, but are not limited to, Spodoptera frugiperda (sf9 and sf21) cells, Trichoplusia ni (BTI TN-5B 1-4 or High-Five(TM)) cells and Mammestra brassicae cells. In certain embodiments, the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis.

[00139] In certain embodiments such methods for the site specific incorporation of biosynthetically generated pyrrolysine and Pel involves introducing the genes pylT, pylS, pylB, pylC and pylD, and the gene for the desired protein (e.g., antibody), into prokaryotic cells and/or eukaryotic cells, and optionally adding a precursor for pyrrolysine or Pel to the growth media of the transfected cells. In certain embodiments, the precursor is D-ornithine, while in other embodiments the precursor is L-ornithine. In certain embodiments, the precursor is D,L- ornithine. In certain embodiments, the precursor is D-arginine, while in other embodiments the precursor is L-arginine. In certain embodiments, the precursor is D,L-arginine. In certain embodiments, the precursor is (2S)-2-amino-6-(2,5- diaminopentanamido)hexanoic acid.

[00140] In certain embodiments, the precursor is (2S)-2-amino-6-((R)-

2,5-diaminopentanamido)hexanoic acid.

[00141] In certain embodiments, the precursor is 2,5-diamino-3- methylpentanoic acid.

[00142] In certain embodiments, the precursor is (2R,3R)-2,5-diamino-3- methylpentanoic acid.

[00143] In certain embodiments, the eukaryotic cell is a mammalian cell, a yeast cell, an insect cell, a fungal cell or a plant cell. In other embodiments, the mammalian cells used in the methods provided herein include, but are not limited to, human embryonic kidney 293 Freestyle™ cells, human epitheloid carcinoma HeLa and GH3 cells, monkey kidney COS cells, rat C6 glioma cells, baby hamster kidney BHK- 21 cells and Chinese hamster ovary CHO cells. In certain embodiments, the yeast cells used in the methods provided herein include, but are not limited to, Saccharomyces cerevisiae and Pichia pastoris cells. In other embodiments, the insect cells used in the methods provided herein include, but are not limited to, Spodoptera frugiperda sf9 and sf21 cells, Trichoplusia ni (BTI TN-5B 1-4 or High-Five(TM)) cells and Mammestra brassicae cells. In certain embodiments, the prokaryotic cell is a bacterium, while in other embodiments, the bacterium used in the methods provided herein include, but are not limited to, Escherichia coli, Mycobacterium smegmatis, Lactococcus lactis and Bacillus subtilis.

[00144] The biosynthesis of Pel requires the presence of biosynthetic genes pylC and pylD, but not pylB, to the host cells. In the biosynthesis of pyrro lysine within methanosarcina mazei, it has been suggested that PylD contains the NADH- binding domain of dehydrogenases and thereby generates D-l-pyrroline-5-carboxylate from D-proline. However, adding D-proline to the growth media does not result in significant Pel incorporation. Addition of 3,4-dihydro-2H-pyrrole-5-carboxylate (also referred to herein as l-pyrroline-2-carboxylate; P2C) and D-l-pyrroline-5-carboxylate (P5C) to the growth media also fails to produce full-length proteins while (2S)-2- amino-6-((R)-2,5-diaminopentanamido)hexanoic acid yields Pel containing protein. PylC has sequence homology with D-alanyl-D-alanine ligases and in the biosynthesis of Pel or pyrro lysine, could catalyze the attachment of D-omithine to the epsilon-amino group of lysine to give (2S)-2-amino-6-((R)-2,5-diaminopentanamido)hexanoic acid. Thus, it is postulated that the biosynthesis of Pel from D-ornithine within mammalian or Escherichia coli cells likely involves conversion of D-ornithine to (2S)-2-amino-6- ((R)-2,5-diaminopentanamido)hexanoic acid by PylC. PylD is likely involved in the activation of (2S)-2-amino-6-((R)-2,5- diaminopentanamido)hexanoic acid into the semialdehyde ((S)-2-amino-6-((R)-2-amino-5- oxopentanamido)hexanoic acid) that spontaneously cyclizes to Pcl-A. This suggestion is supported by recent studies (see Cellitti et al. (201 1) Nat Chem Biol. 7(8):528-30; and Gaston et al. (201 1) Nature 471(7340):647-50) describing the formation of Pcl-A from D-ornithine and L-lysine through the sequential action of PylC and PylD. Alternatively, Pcl-B would form spontaneously if PylD has D-amino-acid transaminase or D- ornithine oxygen oxidoreductase like activity.

[00145] PylB is the iron-sulfur SAM enzyme believed to be required to generate 3 -methyl-D -ornithine from L-lysine in the biosynthesis of pyrrolysine (Quitterer et al. (2012) Angew Chem Int Ed Engl. 51(6): 1339-42; Gaston et al. (2011) Nature 471(7340):647-50)). Even in the presence of the pylB gene, the relative amounts of Pel and pyrrolysine containing proteins varied from fermentation to fermentation with Pel protein typically being more prominent. These observations suggest that PylB's activity or the required co-factors are limiting for efficient pyrrolysine biosynthesis in Escherichia coli and mammalian cells. As an iron-sulfur cluster containing enzyme, PylB is likely deactivated during fermentation with good oxygenation (see Cellitti et al. (201 1) Nat Chem Biol. 7(8):528-30; Quitterer et al. (2012) Angew Chem Int Ed Engl. 51(6): 1339-42).

[00146] In addition, the gene product oipylB may not be expressed efficiently. Therefore, in certain embodiments, modified pylB genes are used in the biosynthesis of pyrrolysine or other pyrrolysine analogues. For the biosynthesis of pyrrolysine, Pel and other pyrrolysine analogues in Escherichia coli, mammalian and other host cells, one or more of the pylB, pylC and pylD genes may be modified. Such modifications may include using homologous genes from other organisms, including but not limited to other species of Methanosarcinae, or mutated genes. In certain embodiments, site-directed mutagenesis is used, while in other embodiments random mutagenesis combined with selection is used. Such methods also include the addition of the DNA of the desired protein and the inclusion of the pylT and pylS genes to incorporate the pyrrolysine, Pel or pyrrolysine analogues into the protein. [00147] In addition, for certain embodiments, the formation of intermediates in the biosynthesis of pyrrolysine, Pel and/or other pyrrolysine analogues from D-ornithine or the biosynthesis of pyrrolysine may be limited by the function of host enzymes and proteins. In certain embodiments, low activity or concentration of one or more host enzymes may be limiting the formation of intermediates required in the biosynthesis of pyrrolysine, Pel or other pyrrolysine analogues. In certain embodiments, the activity of host enzymes may divert the intermediates from the pathway leading to pyrrolysine, Pel or other pyrrolysine analogues to other metabolic pathways, or may be inhibiting the formation of such intermediates. Thus, for the biosynthesis of pyrrolysine, Pel and other pyrrolysine analogues in Escherichia coli, mammalian or other host cells, one or more host enzyme may be modified. Such modification include, but are not limited to, the overexpression, activation, suppression or inhibition of such host enzyme by genetic or chemical means, the addition of the DNA encoding such host enzymes, the addition of silencing RNA (siRNA) to suppress mRNA translation, and the addition of cofactors required for the formation of said intermediates from D-ornithine.

3. Conjugation Chemistry

The modified antibody or antibody fragment thereof provided herein is site- specifically labeled by post-translational modification of a pyrrolysine and/or desmethyl pyrrolysine (Pel) residue that has been incorporated into the antibody or antibody fragment thereof. Methods for this modification are known in the art, see, e.g., WO2010/048582. The modified antibody or antibody fragments can be converted into conjugates by known methods, also, providing a conjugate having the general formula:

or

IA IB where LU is a linker unit and X¹ is a drug moiety or payload; and R²⁰ and R³⁰ are as defined herein. Note that IA can be reduced as described herein to form IB. In some embodiments, R²⁰ is H or Me, and R³⁰ is H or Me or phenyl. LU can be a group of the formula -L2-L3-L4-L5-L6 where these linker components are as defined herein. Immunoconjugates comprising one or more, e.g., 1-8, modified Pel residues of Formula IA or IB at one or more of the Pel substitution sites identified herein are embodiments of the invention. In some embodiments, of these immunoconjugates, R²⁰ is H and R³⁰ is H or Methyl.

[00148] Upon conjugation, the modified antibodies of the invention typically contain 1-12, frequently 2-8, and preferably 2, 4 or 6 -LU-X¹ (Linker Unit- Payload) moieties. In some embodiments, an antibody light or heavy chain is modified to incorporate two Pel residues at two of the specific sites identified herein for substitutions (or alternatively one Pel is incorporated in the light chain and one in the heavy chain), so the tetrameric antibody ultimately contains four conjugation sites. Similarly the antibody can be modified by replacement of 3 or 4 of its native amino acids with Pel at the specific sites identified herein, in light chain or heavy chain or a combination thereof, resulting in 6 or 8 conjugation sites in the tetrameric antibody.

[00149] X¹ in these conjugates represents a payload, which can be any chemical moiety that is useful to attach to an antibody. In some embodiments, X¹ is a drug moiety selected from a cytotoxin, an anti-cancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, an immune potentiator, and an anesthetic agent or any other therapeutic, or biologically active moiety or drug moiety. In other embodiments, X¹ is a label such as a biophysical probe, a fluorophore, an affinity probe, a spectroscopic probe, a radioactive probe, a spin label, or a quantum dot. In other embodiments, X¹ is a chemical moiety that modifies the antibody's physicochemical properties such as a lipid molecule, a polyethylene glycol, a polymer, a polysaccharide, a liposome, or a chelator. In other embodiments, X¹ is a functional or detectable biomolecule such as a nucleic acid, a ribonucleic acid, a protein, a peptide (e.g., an enzyme or receptor), a sugar or polysaccharide, an antibody, or an antibody fragment. In other embodiments, X¹ is an anchoring moiety such as a nanoparticle, a PLGA particle, or a surface, or any binding moiety for specifically binding the conjugate to another moiety, such as a histidine tag, poly-G, biotin, avidin, streptavidin, and the like. In other embodiments, X¹ is a reactive functional group that can be used to attach the antibody conjugate to another chemical moiety, such as a drug moiety, a label, another antibody, another chemical moiety, or a surface.

[00150] The Linker Unit (LU) can be any suitable chemical moiety that covalently attaches the Pcl-derived cyclic group to a payload. Many suitable LUs are known in the art. For example, LU can be comprised of one, two, three, four, five, six, or more than six linker components referred to herein as L_1; L₂, L₃, L₄, L₅ and L₆. In certain embodiments, LU comprises a non-enzymatically cleavable linker, a non- cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo- cleavable linker or any combination thereof, and the LU optionally contains a self- immolative spacer. Where one of the components of LU is a bond, that component represents a direct bond between the components flanking it, so the groups on either side of that component are directly bonded to each other.

[00151] In some embodiments, LU is a group of the formula -Li-L-L₃-

L₄- or -Li-L-L₃-L₄-L₅-L₆- . Linkers Li, L₂, L₃, L₄, L₅ and L₆ for use in LU include alkylene groups -(CH₂)_n- (where n is 1-20, typically 1-10 or 1-6), ethylene glycol units (-CH₂CH₂0-)_n (where n is 1-20, typically 1-10 or 1-6), -0-, -S-, carbonyl (- C(=0)-), amides -C(=0)-NH- or -NH-C(=0)-, esters -C(=0)-0- or -0-C(=0)-, rings having two available points of attachment such as divalent phenyl, C5-6 heteroaryl, C₃_ s cycloalkyl or C₄_s heterocyclyl groups, amino acids -NH-CHR*-C=0- or -C(=0)- CHR*-NH-, where R* is the side chain of a known amino acid (frequently one of the canonical amino acids, but also including e.g. norvaline, norleucine, homoserine, homocysteine, phenylglycine, citrulline, and other named alpha-amino acids), polypeptides of known amino acids (e.g., dipeptides, tripeptides, tetrapeptides, etc.), thiol-maleimide linkages (from addition of-SH to maleimide), -S-CR2- and other thiol ethers such as -S-CR2-C(=0)- or -C(=0)-CR2-S-,where R is independently at each occurrence H or Ci_₄ alkyl, -CH₂-C(=0)-, and disulfides (-S-S-), as well as combinations of any of these with other linkers described below, e.g., a bond, a non- enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo-stable linker, a photo-cleavable linker or a linker that comprises a self- immolative spacer. Suitable examples of LU include -0-(CH₂)i-6-C(=0)- and -S- (CH₂)_!.₆-C(=0)- .

[00152] In some embodiments when LU is -Li-L-L₃-L₄-L₅-L₆-, Li, L₂,

L₃, L₄, L₅ and L₆ can be selected from:

-Ai-, -A1X²- and -X²-; wherein: Ai is a bond, O, S, NH,-C(=0)-, -C(=0)NH-, -C(=0)NH(CH₂)_n-, - C(=0)NH(C(R⁴)₂)_n-, -(0(CH₂)_n)_m-, -(0(C(R⁴)₂)_n)_m-,-((CH₂)_nO)_m-, - (((C(R⁴)₂)_nO)_m-, -((CH₂)_nO)_m(CH₂)_n-, -(((C(R⁴)₂)_nO)_mC(R⁴)₂)_n -, - (CH₂)_nC(=0)NH-, -(C(R⁴)₂)_nC(=0)NH-, -(CH₂)_nNHC(=0)-, - (C(R⁴)₂)_nNHC(=0)-, -NHC(=0)(CH₂)_n-, -NHC(=0)(C(R⁴)₂)_n- , -C(=0)NH(CH₂)_nS-, -C(=0)NH(C(R⁴)₂)_nS-, -S(CH₂)_nC(=0)NH- , -S(C(R⁴)₂)_nC(=0)NH-, -C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n- , -C(=0)NH(C(R⁴)₂)_nNHC(=0)(C(R⁴)₂)_n-, -C(=0)(CH₂)_n-, - C(=0)(C(R⁴)₂)_n-, -(CH₂)_nC(=0)-, -(C(R⁴)₂)_nC(=0)-, - (CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-

, -(C(R⁴)₂)_n(0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)_n-, -(CH₂)_nNHC(=0)(CH₂)_n- , -(C(R⁴)₂)„NHC(=0)(C(R⁴)₂)„-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n- , -(C(R⁴)₂)_nNH((C(R⁴)₂)_nO)_m(C(R⁴)₂)_n-, -(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, or -(0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)_n-;

each X² is independently selected from a bond, R⁸,

71

, -CHR⁴(CH₂)_nC(=0)NH-, -CHR⁴(CH₂)_nNHC(=0)-, -

C(=0)NH- and -NHC(=0)-;

each R⁶ is independently selected from H, fluoro, benzyloxy substituted with -C(=0)OH, benzyl substituted with -C(=0)OH, Ci_₄alkoxy substituted with -C(=0)OH and Ci_₄alkyl substituted with -C(=0)OH; R⁷ is independently selected from H,

phenyl, pyrimidine and

pyridine;

R⁸ is independently selected

, and

R⁹ is independently selected from H and

each n is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1 , 2, 3, 4, 5, 6, 7, 8 and 9.

[00153] In some embodiments, at least one of Li, L₂, L₃, L₄, L₅ and

L6 is a stable, or non-cleavable, linker. In some embodiments, at least one of Li, L2, L3, L₄, L5 and L6 is a cleavable linker, which may be chemically cleavable (hydrazones, disulfides) or enzymatically cleavable. In some embodiments, the enzymatically cleavable linker is one readily cleaved by a peptidase: the Val-Cit linker (valine-citrulline) , a dipeptide of two known amino acids, is one such linker. In other embodiments, the enzymatically cleavable linker is one that is triggered by activity of a glucuronidase. Here is an example of such a linker, which comprises a self-immolative spacer that falls apart spontaneously under physiological conditions once glucuronidase cleaves the glycosidic linkage:

[00154] In certain embodiments the modified antibodies or antibody fragment thereof provided herein are labeled by a one-step method wherein the post-trans lational modification occurs by reacting a pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue) with either an aminobenzaldehyde (ABA) analogue linked to an X¹ group, an amino-acetophenone (AAP) analogue linked to an X¹ group or an amino-benzophenone (ABP) analogue linked to an X¹ group. The X¹ group is defined herein, and such one-step methods are shown in Schemes (Ia)-(Ib) below.

[00155] Alternatively, in other embodiments of the post-translational modification of the modified antibodies or antibody fragment thereof provided herein, the modified antibodies or antibody fragment thereof are labeled by a two- step method wherein the post-translational modification involves first reacting a pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue) with either an aminobenzaldehyde (ABA) analogue linked to an X^a group, an amino- acetophenone (AAP) analogue linked to an X^a group or an amino-benzophenone (ABP) analogue linked to an X^a group, followed by coupling the X^a group with an X^b group that is directly attached or linked to an X¹. The X^a and X^b groups are complementary reactive or coupling groups such as those illustrated herein that combine covalently so that X^a and X^b together form a linker selected from the options described herein for linkers Li, L₂, L₃, L₄, L₅ and L₆: exemplary two-step methods are shown in Schemes (Ila)-(IId) below.

[00156] Alternatively, in other embodiments of the post-translational modification of the modified antibodies or antibody fragment thereof provided herein, the modified antibodies or antibody fragment thereof are labeled by a two-step method wherein the post-translational modification involves reducing the labeled antibodies or antibody fragment thereof obtained by the one-step method. Such two- step methods are shown in Schemes (Ile)-(IIf) below.

[00157] Alternatively, in other embodiments of the post-translational modification of the modified antibodies or antibody fragment thereof provided herein, the modified antibodies or antibody fragment thereof are labeled by a three- step method wherein the post-translational modification involves reducing the labeled antibodies or antibody fragment thereof obtained by the two-step method. Such three-step methods are shown in Schemes (Illa)-(IIId) below.

One-Step Method

[00158] By way of example only, such posttranslational

modifications are illustrated in Schemes (Ia)-(Ib) below. Scheme (la)

where:

when R²⁰ = H, then a PCL residue incorporated into an antibody

when R²⁰ = CH₃, then a Pyrrolysine residue incorporated into an antibody where:

R²⁰ is H or CH₃;

R³⁰ is H, CH₃ or phenyl;

LU is a Linker Unit (LU), and

X¹ is a drug moiety selected from an anti-cancer agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, and an anesthetic agent,

or X¹ is a biophysical probe, a fluorophore, an affinity probe, a chelator, a

spectroscopic probe, a radioactive probe, a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA, RNA, a protein, a peptide, an antibody, an antibody fragment, a nanoparticle, a quantum dot, a liposome, a PLGA particle, a polysaccharide, or a surface.

In certain embodiments the Linker Unit (LU) comprises a linker selected from a non- enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo stable linker, a photo-cleavable linker or any combination thereof, and the Linker Unit (LU) optionally contains a linker that comprises a self- immolative spacer.

In certain embodiments the Linker Unit (LU) is -L₁-L₂-L3-L4-, wherein

Li is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker;

L₂ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker; L3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker, and

L₄ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

enzymatically cleavable linker, a photo stable linker, a photo-cleavable linker or a linker that comprises a self-immolative spacer.

In certain embodiments the Linker Unit (LU) is -L1-L2-L₃-L4-, wherein

Li is a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker;

L2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker; L₃ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker, and

In certain embodiments the Linker Unit (LU) is -L1-L2-L₃-L4-, wherein

Li is a bond, -Ai-, -A1X²- or -X²-; where Ai and X² are as defined above;

L2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

L4 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

In certain embodiments, Li is C(=0)-CH₂CH2-NH-C(=0)-CH₂CH2-S- , so LU is -

C(=0)-CH₂CH2-NH-C(=0)-CH2CH2-S-L2-L3-L₄-.

In certain embodiments the Linker Unit (LU) is -L1-L2-L₃-L4-, wherein

Li is a bond, -Ai-, -A1X²- or -X²-; where Ai and X² are as defined above;

L2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker; L3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker; L₄ is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

In certain embodiments the Linker Unit (LU) is -Lx-I^-Ls-I^-, wherein

Li is a bond, -Ai-, -A₁X²- or -X²-; where:

Ai is -0-, -S-, -NH-, -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(CH₂)_nS-, - (0(CH₂)_n)_m-, -((CH₂)_nO)_m(CH₂)_n-, -NHC(=0)(CH₂)_n-, - C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n- or - (0(CH₂)_n)_mNHC(=0)(CH₂)_n-;

each X² is independently selected from a bond, R⁸,



, -CHR⁴(CH₂)_nC(=0)NH-, -CHR⁴(CH₂)_nNHC(=0)-, -

C(=0)NH- and -NHC(=0)-;

each R⁴ is independently selected from H, Ci-₄alkyl, -C(=0)OH and -OH, each R⁵ is independently selected from H, Ci-₄alkyl, phenyl or

substituted with 1 to 3 -OH groups;

each R⁶ is independently selected from H, fluoro, benzyloxy substituted with -

C(=0)OH, benzyl substituted with -C(=0)OH, Ci-₄alkoxy substituted with

-C(=0)OH and Ci_₄alkyl substituted with -C(=0)OH;

R⁷ is independently selected from H, phenyl and pyridine;

R⁸ is independently selected

independently selected from H and Ci^haloalkyl; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9;

L2 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

enzymatically cleavable linker, a photo stable linker or a photo-cleavable linker; L3 is a bond, a non-enzymatically cleavable linker, a non-cleavable linker, an

In certain embodiments the Linker Unit (LU) is -L1-L2-L3-L4-, wherein

Li is a bond, -Ai-, -A1X²- or -X²-; where:

Ai is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(CH₂)_nS-, -(0(CH₂)_n)_m-, - ((CH₂)_nO)_m(CH₂)_n-, -NHC(=0)(CH₂)_n-, -

C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n- or- (0(CH₂)_n)_mNHC(=0)(CH₂)_n-;

each X² is independently selected from a bond, R⁸,



, -CHR⁴(CH₂)_nC(=0)NH-, -CHR⁴(CH₂)_nNHC(=0)-, -

C(=0)NH- and -NHC(=0)-;

substituted with 1 to 3 -OH groups;

C(=0)OH, benzyl substituted with -C(=0)OH, Ci-₄alkoxy substituted with

-C(=0)OH and Ci_₄alkyl substituted with -C(=0)OH;

R⁷ is independently selected from H, phenyl, pyrimidine, and pyridine;

R⁸ is independently selected

, and

R⁹ is independently selected from H and

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9;

L₂ is a bond, a non-enzymatically cleavable linker or a non-cleavable linker;

L3 is a bond, a non-enzymatically cleavable linker or a non-cleavable linker;

L₄ is a bond, an enzymatically cleavable linker or a linker that comprises a linker that comprises a self-immolative spacer.

In certain embodiments the Linker Unit (LU) is -L₁-L₂-L3-L₄-, wherein

Li is a bond, -Ai-, -A₁X²- or -X²-;

L₂ is a bond, -A₂-, or -A₂X²-;

L₃ is a bond, -A3-, or -A₃X²-;

L₄ is a bond, -A₄-, -A₄X²-,

where Ai and X² are as defined above;

A₂ is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(C(R⁴)₂)_n-, -(0(CH₂)_n)_m-, - (0(C(R⁴)₂)_n)_m-,-((CH₂)_nO)_m-, -(((C(R⁴)₂)_nO)_m-, -((CH₂)_nO)_m(CH₂)_n-, - (((C(R⁴)₂)_nO)_mC(R⁴)₂)_n -, -(CH₂)_nC(=0)NH-, -(C(R⁴)₂)_nC(=0)NR⁴-, - (CH₂)_nNHC(=0)-, -(C(R⁴)₂)_nNHC(=0)-, -NHC(=0)(CH₂)_n-, -NHC(=0)(C(R⁴)₂)_n-, -C(=0)NH(CH₂)_nS-, -C(=0)NH(C(R⁴)₂)_nS-, -S(CH₂)_nC(=0)NH-, - S(C(R⁴)₂)„C(=0)NH-,-(CH₂)_nS-, -(C(R⁴)₂)„S-, -S(CH₂)_n-, -S(C(R⁴)₂)„-, - (CH₂)_nNH-, -(C(R⁴)₂)_nNH-, -C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, - C(=0)NH(C(R⁴)₂)_nNHC(=0)(C(R⁴)₂)_n-, -C(=0)(CH₂)_n-, -C(=0)(C(R⁴)₂)_n-, - (CH₂)_nC(=0)-, -(C(R⁴)₂)_nC(=0)-, -(CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, - (C(R⁴)₂)_n(0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)_n-, - (CH₂)_n(0(CH₂)_n)_mOC(=0)NH(CH₂)_n-, -

(C(R⁴)₂)_n(0(C(R⁴)₂)_n)_mOC(=0)NH(C(R⁴)₂)_n-, -(CH₂)_nNHC(=0)(CH₂)_n-, - (C(R⁴)₂)_nNHC(=0)(C(R⁴)₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n-, - (C(R⁴)₂)_nNH((C(R⁴)₂)_nO)_m(C(R⁴)₂)_n-, -(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, -

A₃ is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(C(R⁴)₂)_n-, -(0(CH₂)_n)_m-, - (0(C(R⁴)₂)_n)_m-, -((CH₂)_nO)_m-, -(((C(R⁴)₂)_nO)_m-, -((CH₂)_nO)_m(CH₂)_n-, - (((C(R⁴)₂)_nO)_mC(R⁴)₂)_n -, -(CH₂)_nC(=0)NH-, -(C(R⁴)₂)_nC(=0)NH-, - (CH₂)_nNHC(=0)-, -(C(R⁴)₂)_nNHC(=0)-, -NHC(=0)(CH₂)_n-, -NHC(=0)(C(R⁴)₂)_n-, -C(=0)NH(CH₂)_nS-, -C(=0)NH(C(R⁴)₂)_nS-, -S(CH₂)_nC(=0)NH-, - S(C(R⁴)₂)_nC(=0)NH-, -(CH₂)_nS-, -(C(R⁴)₂)_nS-, -S(CH₂)_n-, -S(C(R⁴)₂)_n-, - C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, -C(=0)NH(C(R⁴)₂)_nNHC(=0)(C(R⁴)₂)_n-, - C(=0)(CH₂)_n-, -C(=0)(C(R⁴)₂)_n-, -(CH₂)_nC(=0)-, -(C(R⁴)₂)_nC(=0)-, - (CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, -(C(R⁴)₂)_n(0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)_n- ,-(CH₂)_n(0(CH₂)_n)_mOC(=0)NH(CH₂)_n-, -

(C(R⁴)₂)_n(0(C(R⁴)₂)_n)_mOC(=0)NH(C(R⁴)₂)_n-, -(CH₂)_n(0(CH₂)_n)_mOC(=0)-, - (C(R⁴)₂)„(0(C(R⁴)₂)„)_mOC(=0)-, -(CH₂)_n(0(CH₂)_n)_mC(=0)-, - (C(R⁴)₂)_n(0(C(R⁴)₂)_n)_mC(=0)-, -(CH₂)_nNHC(=0)(CH₂)_n-, - (C(R⁴)₂)_nNHC(=0)(C(R⁴)₂)_n-, -(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, -

(0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)„-,

A₄ is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(C(R⁴)₂)_n-, -(0(CH₂)_n)_m-, - (0(C(R⁴)₂)_n)_m-,-((CH₂)_nO)_m-, -(((C(R⁴)₂)_nO)_m-, -((CH₂)_nO)_m(CH₂)_n-, - (((C(R⁴)₂)„0)_mC(R⁴)₂)„ -, -(CH₂)_nC(=0)NH-, -(C(R⁴)₂)„C(=0)NH-, - (CH₂)_nNHC(=0)-, -(C(R⁴)₂)_nNHC(=0)-, -NHC(=0)(CH₂)_n-, -NHC(=0)(C(R⁴)₂)_n-, -C(=0)NH(CH₂)_nS-, -C(=0)NH(C(R⁴)₂)_nS-, -S(CH₂)_nC(=0)NH-, - S(C(R⁴)₂)_nC(=0)NH-, -C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, - C(=0)NH(C(R⁴)₂)_nNHC(=0)(C(R⁴)₂)_n-, -C(=0)(CH₂)_n-, -C(=0)(C(R⁴)₂)_n-, - (CH₂)_nC(=0)-, -(C(R⁴)₂)_nC(=0)-, -(CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, - (C(R⁴)₂)_n(0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)_n-, -(CH₂)_nNHC(=0)(CH₂)_n-, - (C(R⁴)₂)„NHC(=0)(C(R⁴)₂)„-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n-, - (C(R⁴)₂)_nNH((C(R⁴)₂)_nO)_m(C(R⁴)₂)_n-, -(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, or - (0(C(R⁴)₂)_n)_mNHC(=0)(C(R⁴)₂)_n-; each R⁴ is independently selected from H, Ci_₄alkyl, -C(=0)OH and -OH,

each R⁵ is independently selected from H, Ci_₄alkyl, phenyl or Ci_₄alkyl substituted with 1 to 3 -OH groups;

C(=0)OH, benzyl substituted with -C(=0)OH, Ci_₄alkoxy substituted with -

C(=0)OH and Ci_₄alkyl substituted with -C(=0)OH;

R⁷ is independently selected from H, phenyl and pyridine;

independently selected from

R⁹ is independently selected from H and Ci-₆haloalkyl;

In certain embodiments the Linker Unit (LU) is -L1-L2-L3-L4-, wherein

Li is a bond, -Ai-, -A1X²- or -X²-;

L₂ is a bond, -A₂-, or -A₂X²-;

L₃ is a bond, -A3-, or -A₃X²-;

88

Ai is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -(0(CH₂)_n)_m-, -((CH₂)_nO)_m-, -

((CH₂)_nO)_m(CH₂)_n-, -(CH₂)_nC(=0)NH-, -NHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)-, - C(=0)NH(CH₂)_nS-, -S(CH₂)_nC(=0)NH-, -C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, - C(=0)(CH₂)_n-, -(CH₂)_nC(=0)-, -(CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, - (CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n- or - (0(CH₂)_n)_mNHC(=0)(CH₂)_n-;

A₂ is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -(0(CH₂)_n)_m-, -((CH₂)_nO)_m-, -

((CH₂)_nO)_m(CH₂)_n-, -(CH₂)_nC(=0)NH-, -NHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)-, - C(=0)NH(CH₂)_nS-, -S(CH₂)_nC(=0)NH-, -C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, - C(=0)(CH₂)_n-, -(CH₂)_nC(=0)-, -(CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, - (CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n-, -

A₃ is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -(0(CH₂)_n)_m-, -((CH₂)_nO)_m-, -

((CH₂)_nO)_m(CH₂)_n-, -(CH₂)_nC(=0)NH-, -NHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)-, - C(=0)NH(CH₂)_nS-, -S(CH₂)_nC(=0)NH-, -C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, - C(=0)(CH₂)_n-, -(CH₂)_nC(=0)-, -(CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, - (CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n-,

A₄ -C(=0)NH-, -C(=0)NH(CH₂)_n-, -(0(CH₂)_n)_m-, -((CH₂)_nO)_m-, -((CH₂)_nO)_m(CH₂)_n-, -(CH₂)_nC(=0)NH-, -NHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)-, -C(=0)NH(CH₂)_nS-, - S(CH₂)_nC(=0)NH-, -C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, -C(=0)(CH₂)_n-, - (CH₂)_nC(=0)-, -(CH₂)_n(0(CH₂)_n)_mNHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)(CH₂)_n-, - (CH₂)_nNH((CH₂)_nO)_m(CH₂)_n- or -(0(CH₂)_n)_mNHC(=0)(CH₂)_n-;

each X² is independently selected from a bond,

CHR⁴(CH₂)_nC(=0)NH-, -CHR⁴(CH₂)_nNHC(=0)-, -C(=0)NH- and -NHC(=0)-; each R⁴ is independently selected from H, Ci_₄alkyl, -C(=0)OH and -OH, each R⁵ is independently selected from H, Ci_₄alkyl, phenyl or Ci_₄alkyl substituted with 1 to 3 -OH groups;

In certain embodiments the Linker Unit (LU) is -L1-L2-L3-L4-, wherein Li is a bond, -Ai-, -A1X²- or -X²-;

L₂ is a bond, -A₂-, or -A₂X²-;

L₃ is a bond, -A3-, or -A₃X²-;

L₄ is a bond,

-A₄-, -A₄X

Ai is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(CH₂)_nS-, -(0(CH₂)_n)_m-, - ((CH₂)_nO)_m(CH₂)_n-, -NHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)-, - C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n- or - (0(CH₂)_n)_mNHC(=0)(CH₂)_n-;

A₂ is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(CH₂)_nS-, -(0(CH₂)_n)_m-,- ((CH₂)_nO)_m(CH₂)_n-, -NHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)-, - C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n-, -

A₃ is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(CH₂)_nS-, -(0(CH₂)_n)_m-, - ((CH₂)_nO)_m(CH₂)_n-, -NHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)-, - C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n-, -

A₄ is -C(=0)NH-, -C(=0)NH(CH₂)_n-, -C(=0)NH(CH₂)_nS-, -(0(CH₂)_n)_m-, - ((CH₂)_nO)_m(CH₂)_n-, -NHC(=0)(CH₂)_n-, -(CH₂)_nNHC(=0)-, - C(=0)NH(CH₂)_nNHC(=0)(CH₂)_n-, -(CH₂)_nNH((CH₂)_nO)_m(CH₂)_n- or - (0(CH₂)_n)_mNHC(=0)(CH₂)_n-;

each X² is independently selected from a bond, R⁸,

93

, -CHR⁴(CH₂)_nC(=0)NH-, -CHR⁴(CH₂)_nNHC(=0)-, - C(=0)NH- and -NHC(=0)-;

substituted with 1 to 3 -OH groups;

each R⁶ is independently selected from H, fluoro, benzyloxy substituted with - C(=0)OH, benzyl substituted with -C(=0)OH, Ci-₄alkoxy substituted with - C(=0)OH and Ci_₄alkyl substituted with -C(=0)OH;

R⁷ is independently selected from H, phenyl and pyridine;

independently selected from

, and

R⁹ is independently selected from H and Ci-₆haloalkyl;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9, and

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[00159] In certain embodiments L₄ is a bond or a val-cit linker of this formula:

where [X] indicates the point of attachment to a payload.

When L₄ is a val-cit linker, L₃ is preferably -(CH₂)2-6-C(=0)-.

[00160] In some embodiments of any of the compounds described above, R²⁰ is H and R³⁰ is H or Methyl. [00161] In certain embodiments the X¹ group is a maytansinoid such as

DM1 or DM4, or a dolostatin 10 compound, e.g. auristatins MMAF or MMAE, or a calicheamicin such as N-acetyl-y-calicheamicin, or a label or dye such as rhodamine or tetramethylrhodamine.

[00162] As used herein, a "linker" is any chemical moiety that is capable of linking an antibody or a fragment thereof to an X¹ group. Linkers can be susceptible to cleavage, such as, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active.

Alternatively, linkers can be substantially resistant to cleavage. A linker may or may not include a self-immolative spacer.

[00163] Non-limiting examples of the non-enzymatically cleavable linkers as used herein to conjugate an X¹ group to the modified antibodies or antibody fragment thereof provided herein include, acid-labile linkers, linkers containing a disulfide moiety, linkers containing a triazole moiety, linkers containing a hydrazine moiety, linkers containing a thioether moiety, linkers containing a diazo moiety, linkers containing an oxime moiety, linkers containing an amide moiety and linkers containing an acetamide moiety.

[00164] Non-limiting examples of the enzymatically cleavable linkers as used herein to conjugate an X¹ group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, linkers that are cleaved by a protease, linkers that are cleaved by an amidase, and linkers that are cleaved by -glucuronidase.

[00165] In certain embodiments, such enzyme cleavable linkers are linkers which are cleaved by cathepsin, including cathepsin Z, cathepsin B, cathepsin H and cathepsin C. In certain embodiments the enzymatically cleavable linker is a dipeptide cleaved by cathepsin, including dipeptides cleaved by cathepsin Z, cathepsin B, cathepsin H or cathepsin C. In certain embodiments the enzymatically cleavable linker is a cathepsin B-cleavable peptide linker. In certain embodiments the enzymatically cleavable linker is a cathepsin B-cleavable dipeptide linker. In certain embodiments the enzymatically cleavable linker is valine-citrulline or phenylalanine- lysine. Other non-limiting examples of the enzymatically cleavable linkers as used herein conjugate an X¹ group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, linkers which are cleaved by β-

glucuronidase, e.g.,

See Ducry et al, Bioconjugate Chem, vol. 21(1), 5-13 (2010).

[00166] "Self-immolative spacers" are bifunctional chemical moieties covalently linked at one terminus to a first chemical moiety and at the other terminus to a second chemical moiety, thereby forming a stable tripartate molecule. A linker can comprise a self-immolative spacer bonded to a third chemical moiety that is cleavable from the spacer either chemically or enzymatically. Upon cleavage of a bond between the self-immolative spacer and the first or third chemical moiety, self- immolative spacers undergo rapid and spontaneous intramolecular reactions and thereby separate from the second chemical moiety. These intramolecular reactions generally involve electronic rearrangements such as 1,4, or 1,6, or 1,8 elimination reactions or cyclizations to form highly favored five- or six-membered rings. In certain embodiments of the present invention, the first or third moiety is an enzyme cleavable group and this cleavage results from an enzymatic reaction, while in other embodiments the first or third moiety is an acid labile linker and this cleavage occurs due to a change in local pH. As applied to the present invention, the second moiety is the "Payload" group as defined herein. In certain embodiments, cleavage of the first or third moiety from the self-immolative spacer results from cleavage by a proteolytic enzyme, while in other embodiments it results from cleaved by a hydrolase or glucosidase. In certain embodiments, cleavage of the first or third moiety from the self-immolative spacer results from cleavage by a cathepsin enzyme or a

glucuronidase.

[00167] In certain embodiments, the enzyme cleavable linker is a peptide linker and the self-immolative spacer is covalently linked at one of its ends to the peptide linker and covalently linked at its other end to a drug moiety. This tripartite molecule is stable and pharmacologically inactive in the absence of an enzyme, but which is enzymatically cleavable by enzyme at the bond covalently linking the spacer moiety and the peptide moiety. The peptide moiety is cleaved from the tripartate molecule which initiates the self-immolating character of the spacer moiety, resulting in spontaneous cleavage of the bond covalently linking the spacer moiety to the drug moiety, to thereby effect release of the drug in pharmacologically active form.

[00168] In other embodiments, a linker comprises a self-immolative spacer that connects to the peptide, either directly or indirectly at one end, and to a payload at the other end; and the spacer is attached to a third moiety that can be cleaved from the spacer enzymatically, such as by a glucuronidase. Upon cleavage of the third moiety, the spacer degrades or rearranges in a way that causes the payload to be released. An example of a linker with this type of self-immolative spacer is this glucuronidase-cleavable linker:

[00169] Non-limiting examples of the self-immolative spacer optionally used in the conjugation of an X¹ group to the modified antibodies or antibody fragment thereof provided herein include, but are not limited to, moieties which include a benzyl carbonyl moiety, a benzyl ether moiety, a 4-aminobutyrate moiety, a hemithioaminal moiety or a N-acylhemithioaminal moiety.

[00170] Other examples of self-immolative spacers include, but are not limited to, p-aminobenzyloxycarbonyl groups, aromatic compounds that are electronically similar to the p-aminobenzyloxycarbonyl group, such as 2- aminoimidazol-5-methanol derivatives and ortho or para-aminobenzylacetals. In certain embodiments, self-immolative spacers used herein which undergo cyclization upon amide bond hydrolysis, include substituted and unsubstituted 4-aminobutyric acid amides and 2-aminophenylpropionic acid amides. [00171] In certain embodiments, the self-immolative spacer is

or . n ot er em o ments t e se -mmoatve spacer s

, where n is 1 or 2. In other embodiments the self-immolative spacer is

embodiments the self-immolative spacer is , where n is 1 or

2. Schemes (lb) illustrates the post-translational modification of the modified antibodies or antibody fragment thereof provided herein wherein the Linker Unit (LU) is -L1-L2-L3-L4-.

Scheme (lb)

where:

when R²⁰ = H, then a PCL residue incorporated into an antibody

20 30 1 20

IT , R , U, L₂, , and X¹ are as defined herein. In some embodiments, R is H.

In some embodiments, R³⁰ is H or Me.

In certain embodiments, the modified antibodies or antibody fragment thereof provided herein are site-specifically labeled at one (or more) of the substitution sites identified herein by a one-step method as shown in Scheme (la) or Scheme (lb), wherein an X¹ group linked to either an aminobenzaldehyde (ABA) analogue, an amino-acetophenone (AAP) analogue or an amino-benzophenone (ABP) analogue reacts with a pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue) engineered into the antibody or antibody fragment thereof.

The one step method includes the steps of:

(a) providing a modified antibody or antibody fragment thereof which has been engineered to contain one or more pyrrolysine residues or a desmethyl pyrrolysine (Pel) residues and

(b) labeling the modified antibody or antibody fragment thereof with an X¹ group by reacting the modified antibody or antibody fragment thereof with a compound having the structure of Formula (A):

Formula (A)

wherein:

R³⁰, Linker Unit (LU) and X¹ are as described herein.

In certain embodiments, the one step method includes the steps of:

(b) labeling the modified antibody or antibody fragment thereof with an X¹ group by reacting the modified antibody or antibody fragment thereof with a compound having the structure of Formula (B):

Formula (B)

where R³⁰, Li, L₂, L₃, L₄ and X¹ are as defined herein.

In some embodiments of any of the compounds described above, R²⁰ is H and R³⁰ is H or Methyl.

Two-Step Method

[00172] Alternatively, the modified antibodies or antibody fragment thereof provided herein are site-specifically labeled by a two-step method, wherein, in the first step a pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue) which has been incorporated into the antibody or antibody fragment thereof at one (or more) of the substitution sites identified herein is reacted with either an aminobenzaldehyde (ABA) analogue linked to an X^a group, an amino-acetophenone (AAP) analogue linked to an X^a group or an amino-benzophenone (ABP) analogue linked to an X^a group. In the second step an X^b group, which is directly attached or linked to an X¹, is reacted with the X^a group on the modified pyrrolysine residue (or a desmethyl pyrrolysine (Pel) residue), thereby directly attaching the X¹ group to the modified antibody or antibody fragment thereof or attaching the X¹ group to the modified antibody or antibody fragment thereof via a Linker Unit (LU).

[00173] One embodiment of the Two-Step Method is shown in Scheme

(Ila):

Scheme (Ila)

where:

when R²⁰ = H, then a PCL residue incorporated into an antibody

when R²⁰ = CH₃, then a Pyrrolysine residue incorporated into an antibody wherein X^a and a corresponding X^b are as given in Table 4, and where R²⁰, R X², L2, L3, L₄ and X¹ are as defined herein.

Table 4. Reactive Functional Groups useful in the Conjugates of the Invention.

a cyclooctyne or

a diaryl tetrazine

cyclooctene

a diaryl tetrazine a cyclooctyne or cyclooctene

a monoaryl

a norbornene

tetrazine

a norbornene a monoaryl tetrazine

a hydroxylamine or a hydrazine or NH₂-NH- an aldehyde

C(=0)- a hydroxylamine or a hydrazine or NH₂-NH- a ketone

C(=0)- a hydroxylamine an aldehyde or a ketone

a hydrazine an aldehyde or a ketone

NH₂-NH-C(=0)- an aldehyde or a ketone

a haloacetamide a thiol

a maleimide a thiol

[00174] The alkene, alkyne, triaryl phosphine, cyclooctyne, oxanobornadiene, diaryl tetrazine, monoaryl tetrazine and norbornene of X^a and X^b are optionally substituted.

[00175] The Two-Step Method of Scheme (Ha) includes the steps of:

(a) providing a modified antibody or antibody fragment thereof which has been engineered to contain one or more pyrrolysine residues and/or one or more desmethyl pyrrolysine (Pel) residues at one (or more) of the substitution sites identified herein;

(b) labeling the modified antibody or antibody fragment thereof by:

reacting a pyrrolysine residue and/or desmethyl pyrrolysine (Pel) residue with a compound having the structure of Formula (B),

Formula (B) thereby linking a reactive X^a group to the modified antibody or antibody fragment thereof;

and

reacting the reactive X^a group with a compound of Formula (Ila) having a reactive X^b group that is complementary to X^a:

X^b— L2-L3-L4-X¹

Formula (Il-a),

where R³⁰, X^a, X^b, A_h L₂, L₃, and X¹ are as defined herein.

[00176] Another embodiment of the Two-Step Method is shown in Scheme (lib).

Scheme (lib)

where:

when R²⁰ = H, then a PCL residue incorporated into an antibody

when R²⁰ = CH₃, then a Pyrrolysine residue incorporated into an antibody wherein X^a and a corresponding X^b are a complementary pair of reactive functional groups selected from those in Table 4, and where R , R , Li, A₂, X , L₃, L₄ and X are as defined herein.

[00177] The Two-Step Method of Scheme (lib) includes the steps of: (a) providing a modified antibody or antibody fragment thereof which has been engineered to contain one or more pyrrolysine residues and/or one or more desmethyl pyrrolysine (Pel) residues at one (or more) of the substitution sites identified herein;

(b) labeling the modified antibody or antibody fragment thereof by: reacting a pyrrolysine residue and/or desmethyl pyrrolysine (Pel) residue with a compound having the structure of Formula (C), ^a

Formula (C)

thereby linking a reactive X^a group to the modified antibody or antibody fragment thereof

and

reacting the reactive X^a group with a compound of Formula (lib) containing complementary reactive group X^b to react with X^a:

X^b-L₃-L₄-X¹

Formula (Il-b),

where R³⁰, X^a, X^b, , A₂, , and X¹ are as defined herein.

[00178] Another embodiment of the Two-Step Method is shown in

Scheme (lie).

Scheme (lie)

where:

when R²⁰ = H, then a PCL residue incorporated into an antibody

when R²⁰ = CH₃, then a Pyrrolysine residue incorporated into an antibody wherein X^a and a corresponding X are as given in Table 4, and where R²⁰, R³⁰, , L₂, A₃, X², L₄ and X¹ are as defined herein.

[00179] The Two-Step Method of Scheme (lie) includes the steps of: (a) providing a modified antibody or antibody fragment thereof which has been engineered to contain one or more pyrrolysine residues and/or one or more desmethyl pyrrolysine (Pel) residues at one (or more) of the substitution sites identified herein;

(b) labeling the modified antibody or antibody fragment thereof by: reacting a pyrrolysine residue and/or desmethyl pyrrolysine (Pel) residue with a compound having the structure of Formula (D), a

Formula (D)

and

reacting the reactive X^a group with a complementary reactive group X^b in a compound of Formula (lie): x^b— L₄-X¹

Formula (II-c),

where R³⁰, X^a, X^b, , , A₃, and X¹ are as defined herein.

Another embodiment of the Two-Step Method is shown in Scheme (lid).

Scheme (lid)

where:

when R²⁰ = H, then a PCL residue incorporated into an antibody

[00180] The Two-Step Method of Scheme (lid) includes the steps of:

(b) labeling the modified antibody or antibody fragment thereof by:

reacting a pyrrolysine residue and/or desmethyl pyrrolysine (Pel) residue with a compound having the structure of Formula (E), -X^a

Formula (E)

and

reacting the reactive X^a group with a complementary reactive group X^b in a compound of Formula (Il-d): x^b-x¹

Formula (Il-d),

where R³⁰, X^a, X^b, , , , A₄ and X¹ are as defined herein. Suitable reactive functional groups for Xa, and complementary reactive functional groups for Xb, include those in Table 4.

[00181] Other embodiments of a two-step method are shown in

Schemes (He) and (Ilf), wherein the tricyclic group formed from the modified pyrrolysine residue and/or desmethyl pyrrolysine (Pel) residue obtained using the one-step method are reduced.

Scheme (He)

where

when R²⁰ = H, then PCL residue incorporated into an antibody

when R²⁰ = CH₃, then Pyrrolysine residue incorporated into an antibody where:

R²⁰ , R³⁰, LU and X¹ are as defined herein. In certain embodiments the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.

Scheme (Hi)

where

when R²⁰ = H, then PCL residue incorporated into an antibody

R²⁰ , R³⁰, , , , and X¹ are as defined herein. In certain embodiments the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.

Three-Step Method

[00182] Alternatively, the modified antibodies or antibody fragment thereof provided herein are site-specifically labeled by a three-step method, wherein the modified antibodies or antibody fragment thereof obtained using the two-step methods shown in Schemes (Ila)-(IId) are reduced. Certain embodiments of such three-step methods are shown in Schemes (Ilia)- (Hid) below.

Scheme (Ilia)

wherein X^a and a corresponding X^b are as given in Table 4, and where R²⁰, R³⁰, A_\, X², L₂, L3, L₄ and X¹ are as defined herein. In certain embodiments the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.

Scheme (Illb)

wherein X^a and a corresponding X^b are as given in Table 4, and where R²⁰, R³⁰, Ai, X², L₂, L3, L₄ and X¹ are as defined herein. In certain embodiments the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.

Scheme (lllc)

Scheme (Hid)

wherein X^a and a corresponding X^b are as given in Table 4, and where R²⁰, R³⁰, A_\, X², L₂, L₃, L₄ and X¹ are as defined herein. In certain embodiments the reducing agent is sodium borohydride, while in other embodiments the reducing agent is sodium cyanoborohydride.

[00183] Table 4 shows certain embodiments of compounds of Formula

(Il-a) which are used in the Two-step methods or the Three-step methods described herein to react with an R^a group coupled to at least one pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue incorporated into a modified antibody or antibody fragment thereof. The resulting modified pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue located in the modified antibody or antibody fragment thereof are also shown. Note Ai, L₂, L₃, L₄,

R²⁰, R³⁰, X^a, X^b and X¹ are as defined herein, and Y¹ is

Table 5

[00184] Table 6 shows certain embodiments of compounds of Formula

(Il-b) which are used in the Two-step methods or the Three-step methods described herein to react with an R^a group coupled to at least one pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue incorporated into a modified antibody or antibody fragment thereof. The resulting modified pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue located in the modified antibody or antibody fragment thereof are also shown. Note , A₂, , , R²⁰, R³⁰, X^a, X^b and X¹ are as

Table 6

X^b-L₃-L₄-X¹

Y-L -A₂-X^A Y-L_RA₂-X²-L₃-L4-X¹

Formula (II- b)

Y-L₁-A₂-N₃ HC≡C-L₃-L₄-X¹

Y-L₁-A₂-N₃ HC≡C-L₃-L₄-X^{1 ~ "Χ1}

Y^L,-A₂-N~N

X^b-L₃-L₄-X¹

Y-L₁-A₂-X^a Y-L_rA2-X²-L₃-L4-X¹

Formula (II- b)

[00185] Table 7 shows certain embodiments of compounds of Formula (II-c) which are used in the Two-step methods or the Three-step methods described herein to react with an R^a group coupled to at least one pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue incorporated into a modified antibody or antibody fragment thereof. The resulting modified pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue located in the modified antibody or antibody fragment thereof are also shown. Note L_l5 A₂, L₃, L₄, R²⁰, R³⁰, X^a, X^b and X¹ are as defined herein,

where * indicates attachment site to Li.

Table 7

[00186] Table 8 shows certain embodiments of compounds of Formula

(Il-d) which are used in the Two-step methods or the Three-step methods described herein to react with an R^a group coupled to at least one pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue incorporated into a modified antibody or antibody fragment thereof. The resulting modified pyrrolysine residue and/or or desmethyl pyrrolysine (Pel) residue located in the modified antibody or antibody fragment thereof are also shown. Note L_l5 A₂, L₃, L₄, R²⁰, R³⁰, X^a, X^b and X¹ are as defined herein,

Table 8

x^b-x¹

Y-L,-L₂-L3-A4-X^a Y-L₁-L₂-L₃-A₄-X²-X¹

Formula (I I- d)

[00187] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (F):

Formula (F)

where R²⁰, R³⁰, LU and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X¹ group.

[00188] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (G):

*

Formula (G)

[00189] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (H):

Formula (H)

where R²⁰, R³⁰, , , , and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X¹ group.

[00190] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (J):

_*

Formula (J)

[00191] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (K):

Formula (K)

where R²⁰, R³⁰, A_h X², L₂, L₃, and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X¹ group.

[00192] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (L):

Formula (L)

where R²⁰, R³⁰, , A₂, X², , and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X¹ group. [00193] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof

by a linking group of Formula (M):

Formula (M)

20 30 2 1

where IT , R , _h , A₃, X , and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**)

indicates site of attachment to the X¹ group.

[00194] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (N):

Formula (N)

20 30 2 1

where IT , R , _h , , A₄, X and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**)

indicates site of attachment to the X¹ group.

[00195] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (O):

Formula (O)

[00196] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (P):

Formula (P)

20 30 2 1

where R , R , U, A₂, X , , and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X¹ group.

[00197] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (Q):

Formula (Q)

20 30 2 1

where IT , R , _h , A₃, X , and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X¹ group.

[00198] In certain embodiments of the modified antibody or antibody fragment thereof provided herein the X¹ group is linked to the antibody or antibody fragment thereof by a linking group of Formula (R):

*

Formula (R)

20 30 2 1

where R , R , U, , , A₄, X and X¹ are as defined herein and the (*) indicates site of attachment to the antibody or antibody fragment and the (**) indicates site of attachment to the X¹ group.

[00199] In certain embodiments, the modified antibody or antibody fragment thereof provided herein are labeled with an "X¹ group-to-antibody" ratio of 1, 2, 3, 4, 5, 6, 7, or 8, wherein the modified antibody or antibody fragment thereof contains 1, 2, 3, 4, 5, 6, 7, or 8 pyrrolysine and/or or desmethyl pyrrolysine (Pel) residues. For example, a "X¹ group-to-antibody" ratio of 4 is achieved by incorporating a Pel residue into the heavy chains and a Pel residue into the light chains of an antibody resulting in 4 conjugation sites, two in the two heavy chains and two in the two light chains. A ratio greater than about 8 is typically best achieved by combining the Pel substitution methods of the

invention with other protein conjugation methods. Thus for loading more than 8 payload groups onto one antibody molecule, the methods of the invention can be combined with methods such as reactions at cysteine sulfur, acylations at lysine, or conjugation via S6 tags.

[00200] While the payload to antibody ratio has an exact value for a specific conjugate molecule, it is understood that the value will often be an average value when used to describe a sample containing many molecules, due to some degree of inhomogeneity, typically in the conjugation step. The average loading for a sample of an immunoconjugate is referred to herein as the drug to antibody ratio, or DAR. In some embodiments, the DAR is between about 1 and about 16, and typically is about 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, at least 50% of a sample by weight is compound having the average ratio plus or minus 2, and preferably at least 50% of the sample is a conjugate that contains the average ratio plus or minus 1. Preferred embodiments include immunoconjugates wherein the DAR is about 2 or about 8, e.g., about 2, about 4, about 6 or about 8. In some embodiments, a DAR of 'about n' means the measured value for DAR is within 10% of n.

4. Further Alteration of the Framework of Fc Region

[00201] The present invention provides site-specific labeled immunoconjugates. The immunoconjugates of the invention may comprise modified antibodies or antibody fragments thereof that further comprise modifications to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "back-mutated" to the germline sequence by, for example, site- directed mutagenesis. Such "back-mutated" antibodies are also intended to be encompassed by the invention. [00202] Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[00203] In addition or alternative to modifications made within the framework or CDR regions, antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

[00204] In one embodiment, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[00205] In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6, 165,745 by Ward et al.

[00206] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in, e.g., U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al. [00207] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in, e.g., U.S. Patent Nos. 6, 194,551 by Idusogie et al.

[00208] In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described in, e.g., the PCT Publication WO 94/29351 by Bodmer et al. In a specific embodiment, one or more amino acids of an antibody or antibody fragment thereof of the present invention are replaced by one or more allotypic amino acid residues, such as those shown in Figure 4 for the IgGl subclass and the kappa isotype. Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of the IgGl, IgG2, and IgG3 subclasses as well as the constant region of the light chain of the kappa isotype as described by Jefferis et a/., MAbs. 1 :332-338 (2009).

[00209] In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids. This approach is described in, e.g., the PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGl for FcyRl, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al, J. Biol. Chem. 276:6591- 6604, 2001).

[00210] In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for "antigen." Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al. [00211] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered

glycosylation. For example, EP 1, 176, 195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields et al, (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al, Nat. Biotech. 17: 176-180, 1999).

[00212] In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, and T256F, as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6, 121,022 by Presta et al.

5. Antibody Conjugates

[00213] The present invention provides site-specific labeling methods for incorporating a TAG-encoded amino acid such as Pel at one (or more) of the substitution sites identified herein, modified antibodies and antibody fragments thereof, and immunoconjugates prepared accordingly, comprising a TAG-encoded amino acid such as Pel at one (or more) of the substitution sites identified herein. Using the methods of the invention, a modified antibody or antibody fragments thereof can be conjugated to any moiety that is useful to connect to an antibody. Some of the payloads to which the antibody can be conjugated include a label, a biophysical probe, immunopotentiator, enzyme, RNA, DNA, saccharide or polysaccharide, reactive functional group, or a drug moiety, e.g., an anti-cancer agent, an autoimmune treatment agent, an anti-inflammatory agent, an antifungal agent, an antibacterial agent, an anti-parasitic agent, an anti-viral agent, or an anesthetic agent, or an imaging reagent. An antibody or antibody fragments can also be conjugated using several identical or different labeling moieties combining the methods of the invention with other conjugation methods.

[00214] In certain embodiments, the immunoconjugates of the present invention comprise a drug moiety selected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizers, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPrV inhibitor, proteasome inhibitors, an inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.

[00215] Further, the modified antibodies or antibody fragments of the present invention may be conjugated to a payload such as a drug moiety that modifies a given biological response. Drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be an immune potentiator, such as an immune potentiator, a small molecule immune potentiator, a TLR agonist, a CpG oligomer, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope peptide or a like. The drug moiety may also be an oligonucleotide, a siRNA, a shRNA, a cDNA or a like. Alternatively, the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a cytokine, an apoptotic agent, an anti-angiogenic agent, or, a biological response modifier such as, for example, a lymphokine.

[00216] In one embodiment, the modified antibodies or antibody fragments of the present invention are conjugated to a drug moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Examples of cytotoxin include but not limited to, taxanes (see, e.g., International (PCT) Patent Application Nos. WO 01/38318 and PCT/US03/02675), DNA-alkylating agents (e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycin analogs, auristatin E, auristatin F, maytansinoids, and cytotoxic agents comprising a reactive polyethylene glycol moiety (see, e.g., Sasse et al., J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa et ah, Bioorg. Med. Chem., 8, 2175-84 (2000), Ichimura et ah, J. Antibiot. (Tokyo), 44, 1045-53 (1991), Francisco et al, Blood (2003) (electronic publication prior to print publication), U.S. Pat. Nos. 5,475,092, 6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application Publication No. 2001/0036923 Al, Pending U.S. patent application Ser. Nos. 10/024,290 and 10/1 16,053, and International (PCT) Patent Application No. WO 01/49698), taxon, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t. colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine), ablating agents (e.g., mechlorethamine, thiotepa chlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU),

cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). (See e.g., Seattle Genetics

US20090304721).

[00217] Other examples of therapeutic cytotoxins that can be conjugated to the modified antibodies or antibody fragments of the invention include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available (Mylotarg^Tm; Wyeth-Ayerst).

[00218] For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents to antibodies, see also Saito et al, (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail et al, (2003) Cancer Immunol.

Immunother. 52:328-337; Payne, (2003) Cancer Cell 3 :207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan and Kreitman, (2002) Curr. Opin. Investig. Drugs 3: 1089-1091 ; Senter and Springer, (2001) Adv. Drug Deliv. Rev. 53 :247-264.

[00219] Also within the present invention, modified antibodies or antibody fragments thereof can be conjugated to a payload comprising a radioactive isotope to generate cytotoxic radiopharmaceuticals, referred to as

radioimmunoconjugates. Examples of radioactive isotope payloads that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹, yttrium⁹⁰, and lutetium¹⁷⁷. Methods for preparing radioimmunoconjugates are established in the art. Examples of

radioimmunoconjugates are commercially available, including Zevalin™ (DEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention. In certain embodiments, the macrocyclic chelator is 1,4,7, 10-tetraazacyclododecane- N,N',N",N" '-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule. Such linker molecules are commonly known in the art and described in Denardo et al, (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al, (1999) Bioconjug. Chem. 10(4):553-7; and Zimmerman et al, (1999) Nucl. Med. Biol. 26(8):943-50, each incorporated by reference in their entireties.

[00220] The present invention further provides modified antibodies or fragments thereof that specifically bind to an antigen. The modified antibodies or fragments may be conjugated or fused to a payload such as a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins. In particular, the invention provides fusion proteins comprising an antibody fragment described herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide, or peptide. [00221] In some embodiments, modified antibody fragments without antibody specificity, such as but not limited to, modified Fc domains with engineered Pcl(s) according to the present invention, are used to generate fusion proteins comprising such an antibody fragment (e.g., engineered Fc) and a heterologous protein, polypeptide, or peptide.

[00222] Additional fusion proteins may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon- shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof (e.g., antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al, (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) Trends Biotechnol. 16(2):76-82; Hansson et al, (1999) J. Mol. Biol.

287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24(2):308- 313 (each of these patents and publications are hereby incorporated by reference in its entirety). Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A

polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigen may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

[00223] Moreover, the modified antibodies or antibody fragments thereof of the present invention can be conjugated to payloads such as marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1), among others, many of which are commercially available. As described in Gentz et al, (1989) Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, (1984) Cell 37:767), and the "FLAG" tag (A. Einhauer et al, J. Biochem. Biophys. Methods 49: 455-465, 2001). According to the present invention, antibodies or antibody fragments can also be conjugated to tumor-penetrating peptides in order to enhance their efficacy.

[00224] In other embodiments, modified antibodies or antibody fragments of the present invention are conjugated to a diagnostic or detectable agent as a payload. Such immunoconjugates can be useful for monitoring or prognosing the onset, development, progression and/or severity of a disease or disorder as part of a clinical testing procedure, such as determining the efficacy of a particular therapy. Such diagnosis and detection can accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to,

streptavidin/biotin and avidin/biotin; fluorescent materials, such as, but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,

dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as, but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹I,), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ^mIn,), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, 47Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴² Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ⁶⁴Cu, ¹¹³Sn, and ¹¹⁷Sn; and positron emitting metals using various positron emission tomographies, and non-radioactive paramagnetic metal ions.

[00225] Modified antibodies or antibody fragments of the invention may also be attached to solid supports, which are particularly useful for

immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

6. Pharmaceutical Composition To prepare pharmaceutical or sterile compositions including immunoconjugates, the immunoconjugates of the invention are mixed with a pharmaceutically acceptable carrier or excipient. The compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing cancer (breast cancer, colorectal cancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer, gastric cancer, pancreatic cancer, acute myeloid leukemia, chronic myeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheral nerve sheath tumors schwannoma, head and neck cancer, bladder cancer, esophageal cancer, Barretts esophageal cancer, glioblastoma, clear cell sarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renal cancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynacomastica, and

endometriosis).

[00226] Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman et al., Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y., 2001 ; Gennaro, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis, et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY, 1993; Lieberman, et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY, 1990; Lieberman, et al. (eds.) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY, 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y., 2000). Typically, antibodies or conjugates of the invention would be formulated for parenteral administration.

[00227] Selecting an administration regimen for a therapeutic depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, an administration regimen maximizes the amount of therapeutic delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of biologic delivered depends in part on the particular entity and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available (see, e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996; Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y., 1991 ; Bach (ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y., 1993; Baert et al, New Engl. J. Med. 348:601-608, 2003; Milgrom et al, New Engl. J. Med. 341 : 1966-1973, 1999; Slamon et al, New Engl. J. Med. 344:783-792, 2001 ; Beniaminovitz et al, New Engl. J. Med. 342:613-619, 2000; Ghosh et al, New Engl. J. Med. 348:24-32, 2003; Lipsky et al, New Engl. J. Med. 343: 1594-1602, 2000).

[00228] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced.

[00229] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of

administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the medical arts.

[00230] Compositions comprising antibodies or fragments thereof of the invention can be provided by continuous infusion, or by doses at intervals of, e.g., one day, one week, or 1-7 times per week. Doses may be provided

intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, or by inhalation. A specific dose protocol is one involving the maximal dose or dose frequency that avoids significant undesirable side effects. [00231] For the immunoconjugates of the invention, the dosage administered to a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight. The dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight. In some embodiments, the dosage is between 0.1 and 10 mg/kg, or between 1 and 10 mg/kg. The dosage of the antibodies or fragments thereof of the invention may be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg/kg.

[00232] Doses of the immunoconjugates the invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In a specific embodiment, does of the immunoconjugates of the invention are repeated every 3 weeks.

[00233] An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects (see, e.g., Maynard et ah, A Handbook of SOPs for Good Clinical Practice,

Interpharm Press, Boca Raton, Fla., 1996; Dent, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK, 2001).

[00234] The route of administration may be by, e.g., topical or cutaneous application, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional, or by sustained release systems or an implant (see, e.g., Sidman et ah, Biopolymers 22:547-556, 1983; Langer et ah, J. Biomed. Mater. Res. 15: 167-277, 1981 ; Langer, Chem. Tech. 12:98-105, 1982; Epstein et ah, Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985; Hwang et ah, Proc. Natl. Acad. Sci. USA 77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO

99/66903, each of which is incorporated herein by reference their entirety.

[00235] A composition of the present invention may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.

Selected routes of administration for the immunoconjugates of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Parenteral administration may represent modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, a composition of the invention can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In one embodiment, the immunoconjugates of the invention is administered by infusion. In another embodiment, the

immunoconjugates of the invention is administered subcutaneously.

[00236] If the immunoconjugates of the invention are administered in a controlled release or sustained release system, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al, Surgery 88:507, 1980; Saudek et al, N. Engl. J. Med. 321 :574, 1989). Polymeric materials can be used to achieve controlled or sustained release of the therapies of the invention (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23 :61, 1983; see also Levy et al, Science 228: 190, 1985; During et al, Ann. Neurol. 25:351, 1989; Howard et al, J. Neurosurg. 7 1 : 105, 1989; U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5, 128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2 -hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. A controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138, 1984).

[00237] Controlled release systems are discussed in the review by

Langer, Science 249: 1527-1533, 1990). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more immunoconjugates of the invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al, Radiotherapy & Oncology 39: 179-189, 1996; Song et al, PDA Journal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleek ei a/., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, 1997; and Lam et al, Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, 1997, each of which is incorporated herein by reference in their entirety.

[00238] If the compositions comprising the immunoconjugates are administered intranasally, it can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[00239] Methods for co-administration or treatment with a second therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, or radiation, are known in the art (see, e.g., Hardman et al, (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, lO.sup.th ed., McGraw- Hill, New York, N.Y.; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice:A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.). An effective amount of therapeutic may decrease the symptoms by at least 10%; by at least 20%; at least about 30%; at least 40%, or at least 50%.

[00240] Additional therapies (e.g., prophylactic or therapeutic agents), which can be administered in combination with the immunoconjugates of the invention may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart from the immunoconjugates of the invention. The two or more therapies may be administered within one same patient visit.

[00241] In certain embodiments, the immunoconjugates of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,81 1; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade, (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153 : 1038); antibodies (Bloeman et al, (1995) FEBS Lett. 357: 140; Owais et al, (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al, (1995) Am. J. Physiol. 1233: 134); p 120 (Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

[00242] The invention provides protocols for the administration of pharmaceutical composition comprising immunoconjugates of the invention alone or in combination with other therapies to a subject in need thereof. The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the present invention can be administered concomitantly or sequentially to a subject. The therapy (e.g., prophylactic or therapeutic agents) of the combination therapies of the present invention can also be cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time and repeating this sequential administration, i.e., the cycle, in order to reduce the development of resistance to one of the therapies (e.g., agents) to avoid or reduce the side effects of one of the therapies (e.g., agents), and/or to improve, the efficacy of the therapies.

[00243] The therapies (e.g., prophylactic or therapeutic agents) of the combination therapies of the invention can be administered to a subject concurrently.

[00244] The term "concurrently" is not limited to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather it is meant that a pharmaceutical composition comprising antibodies or fragments thereof the invention are administered to a subject in a sequence and within a time interval such that the antibodies of the invention can act together with the other therapy(ies) to provide an increased benefit than if they were administered otherwise. For example, each therapy may be administered to a subject at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapy can be administered to a subject separately, in any appropriate form and by any suitable route. In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 15 minutes, less than 30 minutes, less than 1 hour apart, at about 1 hour apart, at about

I hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about

I I hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart. In other embodiments, two or more therapies (e.g., prophylactic or therapeutic agents) are administered to a within the same patient visit.

[00245] The prophylactic or therapeutic agents of the combination therapies can be administered to a subject in the same pharmaceutical

composition. Alternatively, the prophylactic or therapeutic agents of the combination therapies can be administered concurrently to a subject in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration.

[00246] The invention having been fully described, it is further illustrated by the following examples and claims, which are illustrative and are not meant to be further limiting.

EXAMPLES

Example 1. Selection of 92 sites for Pel mutation in human IgGl heavy chain and kappa light chain.

[00247] Surface exposed residues in the constant region of human

IgGl heavy and human kappa light chains were identified in a crystal structure of an hlgGl/kappa antibody (Protein Databank structure entry lHZH.pdb, Table 9, Table 10, Figure 1) using the computer program Surface Racer 5.0, as described by Tsodikov et al (2002) J. Comput. Chem. 23 :600-609. 92 residues were selected for a single TAG substitution; 60 residues in the hlgG heavy chain and 32 in the human kappa light chain, based on the following criteria: 1) select residues in CHI, CH2 and CH3 domains of the constant regions of heavy chain and the constant regions of light chain; 2) select surface exposed residues; 3) focus on polar or charged residues such as Ser, Thr, Lys, Arg, Glu, and Asp; and 4) exclude residues in FcRn binding domain, Protein A binding domain and heavy chain hinge region. [00248] Criterion 1), namely the selection of Pel substitution sites in the constant region of the antibody, assures transferability of the conjugation sites to many different antibodies. Criterion 2) is based on observation of inter-antibody dimer formation for Pel substitutions of prominently exposed residues (residues excluded based on this criteria are listed in Table 8). Based on the IgG crystal structure, the putative orientation of the Pel side chain was taking into consideration: Residues for which the Pel side chain may be partially shielded from interactions with another antibody but may still react with a small molecular payload, were favored over residues with larger surface accessibility but with an orientation that may enable interactions with a large macromolecule, such as dimer formation.

Criterion 3) was implemented to favor conservative mutations in order to minimize destabilizing effects of the mutations on the antibody. Similarly, criterion 4) was used to avoid functional changes to the antibody such as effects on FcRn and Protein A binding, which may affect the antibody's pharmacokinetic properties or may result in the loss of a purification handle, respectively. Residues excluded based on criterion 4 are listed in Table 9.

[00249] Inspection of the 92 selected mutation sites in the structure model of hlgGl/kappa verified that all selected sites are surface accessible (Figure 2). In Tables 9 and 10, Pel was used as an example for a TAG-encoded amino acid at a certain residue position. TAG-encoded amino acids include but are not limited to unnatural amino acids such as ara-acetyl-phenylalanine, para-azido- phenylalanine and reactive pyrrolysine analogs (Liu and Schultz, 2010; Neumann, 2012), but also include the natural amino acids pyrrolysine (O) and pyrroline- carboxy-lysine (Pel; Z). It is hence important to realize that the incorporation sites of the invention can be used for the site-specific incorporation of many different TAG-encoded amino acids. For all proteins prepared for this study, however, Pel was used as the TAG-encoded amino acid.

[00250] Table 9. Surface accessibility of amino acid residues in human IgGl heavy chain. Surface accessibility was calculated using Surface Racer 5.0 and is expressed as Angstrom square [A²]. Reasons for exclusion" indicate the sites that are excluded from selection due to the reasons mentioned in the table. In addition to Pel incorporation, the TAG sites can be used to incorporate any other TAG-encoded amino acid. Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

117 SER 128 HC-S117Pcl

118 ALA 2

119 SER 79 HC-S119Pcl

120 THR 71

121 LYS 136 HC-K121Pcl

122 GLY 21

123 PRO 2

124 SER 40 HC-S124Pcl

125 VAL 0

126 PHE 1

127 PRO 0

128 LEU 0

129 ALA 0

130 PRO 0

131 SER 0

132 SER 34 HC-S132Pcl

133 LYS 87

134 SER 123 HC-S134Pcl

135 THR 1

136 SER 183 HC-S136Pcl

137 GLY 84

138 GLY 40

139 THR 33 HC-T139Pcl

140 ALA 0

141 ALA 0

142 LEU 0

143 GLY 0

144 CYS 0

145 LEU 0

146 VAL 0 Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

147 LYS 0

148 ASP 1

149 TY 0

150 PHE 0

151 PRO 0

152 GLU 52 HC-E152Pcl

153 PRO 89 HC-P153Pcl

154 VAL 10

155 THR 69 HC-T155Pcl

156 VAL 0

157 SER 39 HC-S157Pcl

158 TRP 0

159 ASN 4

160 SER 164 Dimer

161 GLY 35 Dimer

162 ALA 115 Dimer

163 LEU 17

164 THR 125 HC-T164Pcl

165 SER 183 HC-S165Pcl

166 GLY 20

167 VAL 12

168 HIS 5

169 THR 60

170 PHE 0

171 PRO 33 HC-P171Pcl

172 ALA 9

173 VAL 0

174 LEU 68 HC-L174Pcl

175 GLN 0

176 SER 162 HC-S176Pcl Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

177 SER 68 HC-S177Pcl

178 GLY 8

179 LEU 0

180 TYR 6

181 SER 0

182 LEU 2

183 SER 0

184 SER 0

185 VAL 0

186 VAL 0

187 THR 30 HC-T187Pcl

188 VAL 0

189 PRO 86 HC-P189Pcl

190 SER 21

191 SER 127 HC-S191Pcl

192 SER 17

193 LEU 0

194 GLY 18

195 THR 111 HC-T195Pcl

196 GLN 79

197 THR 90 HC-T197Pcl

198 TYR 0

199 ILE 25

200 CYS 0

201 ASN 8

202 VAL 0

203 ASN 22

204 HIS 0

205 LYS 217

206 PRO 66 Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

207 SER 50 HC-S207Pcl

208 ASN 91

209 THR 24

210 LYS 234 Dimer

211 VAL 30

212 ASP 97 HC-D212Pcl

213 LYS 70

214 LYS 146

215 ALA 0

216 GLU 79

217 PRO 0

218 LYS 4

219 SER 149

220 CYS 7

221 ASP 0 Hinge

222 LYS 208 Hinge

223 THR 112 Hinge

224 HIS 1 Hinge

225 THR 22 Hinge

226 CYS 12 Hinge

227 PRO 22 Hinge

228 PRO 133 Hinge

229 CYS 7 Hinge

230 PRO 84 Hinge

231 ALA 114 Hinge

232 PRO 49 Hinge

233 GLU 114 Hinge

234 LEU 90

235 LEU 88

236 GLY 9 Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

237 GLY 46

238 PRO 14

239 SER 9

240 VAL 0

241 PHE 0

242 LEU 0

243 PHE 1

244 PRO 34

245 PRO 0

246 LYS 55 HC-K246Pcl

247 PRO 18

248 LYS 47

249 ASP 1

250 THR 0 FcRn binding

251 LEU 0

Protein A, FcRn

252 MET 53

binding

253 ILE 155 Protein A binding

Protein A, FcRn

254 SER 157

binding

255 ARG 103

256 THR 86 FcRn binding

257 PRO 0 FcRn binding

258 GLU 42 HC-E258Pcl

259 VAL 0 FcRn binding

260 THR 0

261 CYS 0

262 VAL 0

263 VAL 0

264 VAL 0 Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

265 ASP 11 FcRn binding

266 VAL 0

267 SER 10

268 HIS 79

269 GLU 189 HC-E269Pcl

270 ASP 23

271 PRO 20

272 GLU 152

273 VAL 19

274 LYS 138 HC-K274Pcl

275 PHE 2

276 ASN 1

277 TRP 0

278 TYR 14

279 VAL 0

280 ASP 66

281 GLY 72

282 VAL 141 HC-V282Pcl

283 GLU 80 HC-E283Pcl

284 VAL 25

285 HIS 133

286 ASN 119 HC-N286Pcl

287 ALA 67

288 LYS 182 HC-K288Pcl

289 THR 5

290 LYS 177 HC-K290Pcl

291 PRO 51

292 ARG 252 HC-R292Pcl

293 GLU 83 HC-E293Pcl

294 GLU 73 HC-E294Pcl Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

295 GLN 170

296 TY 29

297 ASN 61 Glycosylation

298 SER 125 Glycosylation

299 THR 2 Glycosylation

300 TYR 28

301 ARG 18

302 VAL 0

303 VAL 10

304 SER 0

305 VAL 17

306 LEU 0

307 THR 27 FcRn binding

308 VAL 0 FcRn binding

309 LEU 122

310 HIS 4 Protein A binding

Protein A, FcRn

311 GLN 145

binding

312 ASP 14

313 TRP 0

314 LEU 6 Protein A binding

315 ASN 151 Protein A binding

316 GLY 12

317 LYS 81

318 GLU 49

319 TYR 0

320 LYS 55 HC-K320Pcl

321 CYS 0

322 LYS 78 HC-K322Pcl

323 VAL 0 Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

324 SER 0

325 ASN 0

326 LYS 213 HC-K326Pcl

327 ALA 10

328 LEU 9

329 PRO 158

330 ALA 96 HC-A330Pcl

331 PRO 44

332 ILE 32

333 GLU 85 HC-E333Pcl

334 LYS 50 HC-K334Pcl

335 THR 70 HC-T335Pcl

336 ILE 13

337 SER 15 HC-S337Pcl

338 LYS 0

339 ALA 37

340 LYS 217 Protein A binding

341 GLY 37

342 GLN 235

343 PRO 42

344 ARG 98 HC-R344Pcl

345 GLU 105

346 PRO 0

347 GLN 24

348 VAL 3

349 TYR 3

350 THR 0

351 LEU 0

352 PRO 38

353 PRO 0 Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

354 SER 0

355 ARG 249 HC-R355Pcl

356 ASP 53

357 GLU 0

358 LEU 36

359 THR 144 Dimer

360 LYS 114 HC-K360Pcl

361 ASN 155

362 GLN 41 HC-Q362Pcl

363 VAL 0

364 SER 0

365 LEU 0

366 THR 0

367 CYS 0

368 LEU 0

369 VAL 0

370 LYS 1

371 GLY 0

372 PHE 0

373 TYR 23

374 PRO 0

375 SER 29 HC-S375Pcl

376 ASP 9

377 ILE 11

378 ALA 11

379 VAL 4

380 GLU 18 FcRn binding

381 TRP 0

382 GLU 22 HC-E382Pcl

383 SER 1 Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

384 ASN 147

385 GLY 102 Dimer

386 GLN 161

387 PRO 99

388 GLU 4

389 ASN 189 HC-N389Pcl

390 ASN 36 HC-N390Pcl

391 TYR 44

392 LYS 82 HC-K392Pcl

393 THR 36 HC-T393Pcl

394 THR 0

395 PRO 72

396 PRO 47

397 VAL 9

398 LEU 111 HC-L398Pcl

399 ASP 0

400 SER 81 HC-S400Pcl

401 ASP 68

402 GLY 29

403 SER 0

404 PHE 22

405 PHE 0

406 LEU 0

407 TYR 0

408 SER 0

409 LYS 0

410 LEU 0

411 THR 0

412 VAL 0

413 ASP 80 HC-D413Pcl Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

414 LYS 83

415 SER 69 HC-S415Pcl

416 ARG 53

417 TRP 0

418 GLN 108

419 GLN 177

420 GLY 39

421 ASN 35

422 VAL 81 HC-V422Pcl

423 PHE 0

424 SER 2

425 CYS 0

426 SER 0

427 VAL 0

428 MET 0 FcRn binding

429 HIS 0

430 GLU 14

431 ALA 22

432 LEU 1

433 HIS 227 Protein A binding

Protein A, FcRn

434 ASN 126

binding

435 HIS 28

436 TYR 54

437 THR 36

438 GLN 82

439 LYS 12

440 SER 62

441 LEU 2

442 SER 34 Surface Reason for

Eu number Residue accessibility exclusion (if Selected TAG sites

[A²] applicable)

443 LEU 101

444 SER 70 Dimer

Table 10. Surface accessibility of amino acid residues in human kappa light chain. Surface accessibility was calculated using Surface Racer 5.0 and is expressed as Angstrom square [A²]. In addition to Pel incorporation, the TAG sites can be used to incorporate any other TAG-encoded amino acid.

Surface Surface

Eu Selected Eu Selected

Residue accessibility Residue accessibilit number TAG sites number TAG sites

[A²] y [A²]

LC-

122 ASP 90 176 SER 0

D122Pcl

LC-

123 GLU 51 177 SER 0

E123Pcl

124 GLN 0 178 THR 0

125 LEU 21 179 LEU 0

LC-

126 LYS 230 180 THR 13

K126Pcl

LC-

127 SER 101 181 LEU 21

S127Pcl

LC-

128 GLY 12 182 SER 59

S182Pcl

LC- LC-

129 THR 41 183 LYS 131

T129Pcl K183Pcl

130 ALA 0 184 ALA 32

131 SER 0 185 ASP 52

132 VAL 0 186 TYR 0

133 VAL 0 187 GLU 77

LC-

134 CYS 0 188 LYS 201

K188Pcl

135 LEU 0 189 HIS 42

LC-

136 LEU 0 190 LYS 167

K190Pcl

LC-

137 ASN 5 191 VAL 58

V191 PCI

138 ASN 18 192 TYR 0

139 PHE 0 193 ALA 0

140 TYR 0 194 CYS 0

141 PRO 3 195 GLU 12

LC-

142 ARG 55 196 VAL 0

R142Pcl

LC- LC-

143 GLU 1 17 197 THR 38

E143Pcl T197Pcl

144 ALA 7 198 HIS 4

LC- LC-

145 LYS 160 199 GLN 127

K145Pcl Q199Pcl

146 VAL 1 1 200 GLY 11

147 GLN 22 201 LEU 17

148 TRP 0 202 ARG 343

149 LYS 48 203 SER 110 LC- Surface Surface

Eu Selected Eu Selected

Residue accessibility Residue accessibilit number TAG sites number TAG sites

[A²] y [A²]

S203Pcl

150 VAL 0 204 PRO 69

151 ASP 59 205 VAL 30

LC- LC-

152 ASN 157 206 THR 70

N 152Pcl T206Pcl

153 ALA 51 207 LYS 44

LC-

154 LEU 1 17 208 SER 47

L154Pcl

155 GLN 26 209 PHE 5

LC-

156 SER 122 210 ASN 44

S156Pcl

LC-

157 GLY 1 14 211 ARG 89

G157Pcl

158 ASN 19 212 GLY 15

LC-

159 SER 22 213 GLU 107

S159Pcl

160 GLN 36 214 CYS 58

Example 2. Preparation of trastuzumab TAG mutant constructs.

[00251] DNA encoding variable regions of trastuzumab heavy and light chains (Carter et al, (1992) Proc. Natl. Acad. Sci. USA, 89, 4285-4289) were chemically synthesized and cloned into two mammalian expression vectors, pOG- HC and pOG-LC that contain constant regions of human IgGl and human kappa light chain, resulting in two wild-type constructs, pOG-trastuzumab-HC and pOG- trastuzumab-LC, respectively. In the vectors the expression of antibody heavy and light chain constructs in mammalian cells is driven by a CMV promoter. The vectors contain a synthetic 24 amino acid signal sequence:

MKTFILLLWVLLLWVIFLLPGATA, in the N-terminal of heavy chain or light chain to guide their secretion from mammalian cells. The signal sequence has been validated to be efficient in directing protein secretion in hundreds of mammalian proteins in HEK293 cells (Gonzalez et al, (2010) Proc Natl Acad Sci U S A. 107:3552-3557). Oligonucleotide directed mutagenesis (Higuchi et al (1988) Nucleic Acids Res. 16(15):7351-67) was employed to prepare TAG mutant constructs in trastuzumab wild-type constructs, pOG-trastuzumab-HC and pOG- trastuzumab-LC. 92 pairs of mutation primers (Table 11) were chemically synthesized that correspond to the 92 TAG mutation sites selected in the constant regions of human IgGl heavy chain and kappa light chain as described in Example 1. The sense and anti-sense mutation primer pairs were mixed prior to PCR amplification. PCR reactions were performed by using PfuUltra II Fusion HS DNA Polymerase (Stratagene) with pOG-trastuzumab-HC and pOG-trastuzumab-LC as the templates. After PCR reactions, the PCR products were confirmed on agarose gels, and treated with DP I followed by transformation in DH5a cells (Klock and Lesley, 2009). Sequences of 92 TAG mutant constructs were confirmed by DNA sequencing. The amino acid sequence of full length wild type trastuzumab heavy chain is shown as SEQ ID NO: l (Table 12) and light chain as SEQ ID NO:62 (Table 13). The encoded protein sequence of the constant regions for 60 trastuzumab-HC TAG mutant constructs (SEQ ID NO:2 through SEQ ID NO:61) and 32 trastuzumab-LC TAG mutant constructs (SEQ ID NO:63 to SEQ ID NO:94) are shown in Table 12 and Table 13, respectively. Amino acid residues in human IgGl heavy chain and human kappa light chain are numbered by Eu numbering system (Edelman et al, (1969) Proc Natl Acad Sci U S A. 63(l):78-85). Since TAG codon can be used to encode Pel, pyrrolysine and unnatural amino acids (Noren ei a/., (1989) Science 14;244(4901): 182-188; Mendel et al, (1995) Annu Rev Biophys Biomol Struct. 24:435-462), the TAG constructs described in the invention can be extended to the incorporation of all TAG-encoded amino acids.

Table 1 1. DNA sequences of mutation primers used to prepare 92 TAG mutations in trastuzumab heavy and light chains. In addition to Pel incorporation, the TAG sites can be used to incorporate any other TAG-encoded amino acid.

Primer SEQ ID

Antibody Sequence

name NO

LC-TAG- CACGCTGGGCTAGGCCACCGTTCGTTTGATC 1 10 A4 TCCACCTTG

LC-TAG- GCCGCTCCCTAGGTGTTCATCTTCCCCCCCA 1 1 1

LC- S5 GCGACGAGCA

S114Pcl LC-TAG- ATGAACACCTAGGGAGCGGCCACCGTTCGTT 1 12

A5 TGATCTCCA

LC-TAG- CCCCCAGCTAGGAGCAGCTGAAGAGCGGCAC 1 13

LC- S6 CGCCAGCGT

D122Pcl LC-TAG- CAGCTGCTCCTAGCTGGGGGGGAAGATGAAC 1 14

A6 ACGCTGGGA

LC-TAG- CCCAG CG ACTAG CAG CTG AAG AG CGG CACCG 1 15

LC- S7 CCAGCGTG

E123Pcl LC-TAG- TTCAGCTGCTAGTCGCTGGGGGGGAAGATGA 1 16

A7 ACACGCTG

LC-TAG- AGCAGCTGTAGAGCGGCACCGCCAGCGTGGT 1 17

LC- S8 GTGCCTGCT

K126Pcl LC-TAG- GTGCCGCTCTACAGCTGCTCGTCGCTGGGGG 1 18

A8 GGAAGATGA

LC-TAG- AGCTGAAGTAGGGCACCGCCAGCGTGGTGTG 1 19

LC- S9 CCTGCTGAA

S127Pcl LC-TAG- GCGGTGCCCTACTTCAGCTGCTCGTCGCTGG 120

A9 GGGGGAAGA

LC-TAG- AGAGCGGCTAGGCCAGCGTGGTGTGCCTGCT 121

LC- S10 GAACAACTT

T129Pcl LC-TAG- CACGCTGGCCTAGCCGCTCTTCAGCTGCTCG 122

A10 TCGCTGGGG

LC-TAG- TCTACCCCTAGGAGGCCAAGGTGCAGTGGAA 123

LC- S1 1 GGTGGACAA

R142Pcl LC-TAG- TTGGCCTCCTAGGGGTAGAAGTTGTTCAGCA 124

A1 1 GGCACACCA

LC-TAG- TACCCCCGGTAGGCCAAGGTGCAGTGGAAGG 125

LC- S12 TGGACAAC

E143Pcl LC-TAG- ACCTTGGCCTACCGGGGGTAGAAGTTGTTCA 126

A12 GCAGGCACA

LC-TAG- CGGGAGGCCTAGGTGCAGTGGAAGGTGGAC 127

LC- S13 AACGCCCTGC

K145Pcl LC-TAG- CACTGCACCTAGGCCTCCCGGGGGTAGAAGT 128

A13 TGTTCAGCA

LC-TAG- AAGGTGGACTAGGCCCTGCAGAGCGGCAACA 129

LC- S14 GCCAGGAGA

N152Pcl LC-TAG- TGCAGGGCCTAGTCCACCTTCCACTGCACCTT 130

A14 GGCCTCCC

LC-TAG- ACAACGCCTAGCAGAGCGGCAACAGCCAGGA 131

LC- S15 GAGCGTCA

L154Pcl LC-TAG- GCCGCTCTGCTAGGCGTTGTCCACCTTCCAC 132

A15 TGCACCTTG

LC-TAG- GCCCTGCAGTAGGGCAACAGCCAGGAGAGC 133

LC- S16 GTCACCGAGCA

S156Pcl LC-TAG- GCTGTTGCCCTACTGCAGGGCGTTGTCCACC 134

A16 TTCCACTGCA

LC-TAG- CTG CAG AG CTAG AACAG CCAGG AG AG CGTCA 135

LC- S17 CCGAGCAGGA

G157Pcl LC-TAG- TGGCTGTTCTAGCTCTGCAGGGCGTTGTCCA 136

A17 CCTTCCACT

LC- LC-TAG- AG CGG CAACTAG CAG G AG AGCGTCACCG AG C 137 Primer SEQ ID

Antibody Sequence

name NO

S159Pcl S18 AGGACAGCAA

LC-TAG- CTCTCCTGCTAGTTGCCGCTCTGCAGGGCGT 138 A18 TGTCCACCT

LC-TAG- AACAGCCAGTAGAGCGTCACCGAGCAGGACA 139

LC- S19 GCAAGGACT

E161 Pcl LC-TAG- GTGACGCTCTACTGGCTGTTGCCGCTCTGCA 140

A19 GGGCGTTGT

LC-TAG- GAGCGTCACCTAGCAGGACAGCAAGGACTCC 141

LC- S20 ACCTACAGC

E165Pcl LC-TAG- CTGTCCTGCTAGGTGACGCTCTCCTGGCTGTT 142

A20 GCCGCTCT

LC-TAG- GAGCAGGACTAGAAGGACTCCACCTACAGCC 143

LC- S21 TGAGCAGCA

S168Pcl LC-TAG- GAGTCCTTCTAGTCCTGCTCGGTGACGCTCTC 144

A21 CTGGCTGT

LC-TAG- CAGGACAGCTAGGACTCCACCTACAGCCTGA 145

LC- S22 GCAGCACC

K169Pcl LC-TAG- GTGGAGTCCTAGCTGTCCTGCTCGGTGACGC 146

A22 TCTCCTGG

LC-TAG- ACAGCAAGTAGTCCACCTACAGCCTGAGCAG 147

LC- S23 CACCCTGAC

D170Pcl LC-TAG- TAGGTGGACTACTTGCTGTCCTGCTCGGTGA 148

A23 CGCTCTCCT

LC-TAG- TGACCCTGTAGAAGGCCGACTACGAGAAGCA 149

LC- S24 TAAGGTGTA

S182Pcl LC-TAG- GTCGGCCTTCTACAGGGTCAGGGTGCTGCTC 150

A24 AGGCTGTAG

LC-TAG- GACCCTGAGCTAGGCCGACTACGAGAAGCAT 151

LC- S25 AAGGTGTAC

K183Pcl LC-TAG- TAGTCGGCCTAGCTCAGGGTCAGGGTGCTGC 152

A25 TCAGGCTGT

LC-TAG- GACTACGAGTAGCATAAGGTGTACGCCTGCG 153

LC- S26 AGGTGAC

K188Pcl LC-TAG- ACCTTATGCTACTCGTAGTCGGCCTTGCTCAG 154

A26 GGTCAGG

LC-TAG- G AG AAG CATTAG GTGTACG CCTG CG AGGTG A 155

LC- S27 CCCACCAG

K190Pcl LC-TAG- G GCGTACACCTAATG CTTCTCGTAGTCG G CCT 156

A27 TGCTCAGG

LC-TAG- AGCATAAGTAGTACGCCTGCGAGGTGACCCA 157

LC- S28 CCAGGGCCT

VI 91 Pel LC-TAG- CAGGCGTACTACTTATGCTTCTCGTAGTCGGC 158

A28 CTTGCTCA

LC-TAG- GCGAGGTGTAGCACCAGGGCCTGTCCAGCCC 159

LC- S29 CGTGACCAA

T197Pcl LC-TAG- CCCTGGTGCTACACCTCGCAGGCGTACACCT 160

A29 TATGCTTCT

LC-TAG- GTGACCCACTAGGGCCTGTCCAGCCCCGTGA 161

LC- S30 CCAAGAGCT

Q199Pcl LC-TAG- G ACAG G CCCTAGTG G GTCACCTCG CAG GCGT 162

A30 ACACCTTAT

LC-TAG- GGCCTGTCCTAGCCCGTGACCAAGAGCTTCA 163

LC- S31 ACAGGGGCGA

S203Pcl LC-TAG- GTCACGGGCTAGGACAGGCCCTGGTGGGTCA 164

A31 CCTCGCAGG Primer SEQ ID

Antibody Sequence

name NO

LC-TAG- AGCCCCGTGTAGAAGAGCTTCAACAGGGGCG 165

LC- S32 AGTGCTAA

T206Pcl LC-TAG- AAGCTCTTCTACACGGGGCTGGACAGGCCCT 166

A32 GGTGGGTC

HC-TAG- ACCGTCTCCTAGGCTAGCACCAAGGGCCCCA 167

HC- S1 GCGTGTT

S117Pcl HC-TAG- TGGTGCTAGCCTAGGAGACGGTGACCAGGGT 168

A1 TCCTTGA

HC-TAG- TCCTCGGCTTAGACCAAGGGCCCCAGCGTGT 169

HC- S2 TCCCCCTGG

S119Pcl HC-TAG- CCCTTGGTCTAAGCCGAGGAGACGGTGACCA 170

A2 GGGTTCCTT

HC-TAG- CTAGCACCTAGGGCCCCAGCGTGTTCCCCCT 171

HC- S3 GGCCCCCA

K121 Pcl HC-TAG- GCTGGGGCCCTAGGTGCTAGCCGAGGAGAC 172

A3 GGTGACCAG

HC-TAG- AGGGCCCCTAGGTGTTCCCCCTGGCCCCCAG 173

HC- S4 CAGCAAGA

S124Pcl HC-TAG- GGGGAACACCTAGGGGCCCTTGGTGCTAGCC 174

A4 GAGGAGACG

HC-TAG- CCCCCAGCTAGAAGAGCACCAGCGGCGGCA 175

HC- S5 CAGCCGCCCT

S132Pcl HC-TAG- GGTGCTCTTCTAGCTGGGGGCCAGGGGGAAC 176

A5 ACGCTGGGG

HC-TAG- AGCAGCAAGTAGACCAGCGGCGGCACAGCC 177

HC- S6 GCCCTGGGCT

S134Pcl HC-TAG- CCGCTGGTCTACTTGCTGCTGGGGGCCAGGG 178

A6 GGAACACG

HC-TAG- AGAGCACCTAGGGCGGCACAGCCGCCCTGG 179

HC- S7 GCTGCCTGGT

S136Pcl HC-TAG- GTGCCGCCCTAGGTGCTCTTGCTGCTGGGGG 180

A7 CCAGGGGGA

HC-TAG- AGCGGCGGCTAGGCCGCCCTGGGCTGCCTG 181

HC- S8 GTGAAGGACT

T139Pcl HC-TAG- CAGGGCGGCCTAGCCGCCGCTGGTGCTCTTG 182

A8 CTGCTGGGG

HC-TAG- TACTTCCCCTAGCCCGTGACCGTGTCCTGGA 183

HC- S9 ACAGCGGA

E152Pcl HC-TAG- GGTCACGGGCTAGGGGAAGTAGTCCTTCACC 184

A9 AGGCAGC

HC-TAG- TCCCCGAGTAGGTGACCGTGTCCTGGAACAG 185

HC- S10 CGGAGCCCT

P153Pcl HC-TAG- CACGGTCACCTACTCGGGGAAGTAGTCCTTC 186

A10 ACCAGGCAG

HC-TAG- GAGCCCGTGTAGGTGTCCTGGAACAGCGGAG 187

HC- S1 1 CCCTGACCT

T155Pcl HC-TAG- CAGGACACCTACACGGGCTCGGGGAAGTAGT 188

A1 1 CCTTCACCA

HC-TAG- TGACCGTGTAGTGGAACAGCGGAGCCCTGAC 189

HC- S12 CTCCGGCGT

S157Pcl HC-TAG- CTGTTCCACTACACGGTCACGGGCTCGGGGA 190

A12 AGTAGTCCT

HC-TAG- GGAGCCCTGTAGTCCGGCGTGCACACCTTCC 191

HC- S13 CCGCCGTGCT

T164Pcl

HC-TAG- ACGCCGGACTACAGGGCTCCGCTGTTCCAGG 192 Primer SEQ ID

Antibody Sequence

name NO

A13 ACACGGTCA

HC-TAG- CCCTGACCTAGGGCGTGCACACCTTCCCCGC 193

HC- S14 CGTGCTGCA

S165Pcl HC-TAG- TGTGCACGCCCTAGGTCAGGGCTCCGCTGTT 194

A14 CCAGGACAC

HC-TAG- CACACCTTCTAGGCCGTGCTGCAGAGCAGCG 195

HC- S16 GCCTGTACA

P171 Pcl HC-TAG- CAGCACGGCCTAGAAGGTGTGCACGCCGGA 196

A16 GGTCAGGGCT

HC-TAG- CCGCCGTGTAGCAGAGCAGCGGCCTGTACAG 197

HC- S17 CCTGTCCA

L174Pcl HC-TAG- GCTGCTCTGCTACACGGCGGGGAAGGTGTGC 198

A17 ACGCCGGAG

HC-TAG- TGCTGCAGTAGAGCGGCCTGTACAGCCTGTC 199

HC- S18 CAGCGTGGT

S176Pcl HC-TAG- ACAGGCCGCTCTACTGCAGCACGGCGGGGAA 200

A18 GGTGTGCACG

HC-TAG- CTGCAGAGCTAGGGCCTGTACAGCCTGTCCA 201

HC- S19 GCGTGGTGA

S177Pcl HC-TAG- TACAGGCCCTAGCTCTGCAGCACGGCGGGGA 202

A19 AGGTGTGCA

HC-TAG- AGCGTGGTGTAGGTGCCCAGCAGCAGCCTGG 203

HC- S20 GCACCCAGA

T187Pcl HC-TAG- G CTGG GCACCTAC ACCACG CTG G ACAG GCTG 204

A20 TACAGGCCG

HC-TAG- TGACAGTGTAGAGCAGCAGCCTGGGCACCCA 205

HC- S21 GACCTACAT

P189Pcl HC-TAG- CTG CTG CTCTACACTGTC ACCACG CTGG ACA 206

A21 GGCTGTACA

HC-TAG- TGCCCAGCTAGAGCCTGGGCACCCAGACCTA 207

HC- S22 CATCTGCAA

S191 Pcl HC-TAG- CCCAGGCTCTAGCTGGGCACTGTCACCACGC 208

A22 TGGACAGGCT

HC-TAG- G CCTGG G CTAG CAG ACCTACATCTGCAACGT 209

HC- S23 GAACCACAA

T195Pcl HC-TAG- GTAGGTCTGCTAGCCCAGGCTGCTGCTGGGC 210

A23 ACTGTCACCA

HC-TAG- GCACCCAGTAGTACATCTGCAACGTGAACCA 21 1

HC- S24 CAAGCCCA

T197Pcl HC-TAG- GCAGATGTACTACTGGGTGCCCAGGCTGCTG 212

A24 CTGGGCACT

HC-TAG- CACAAGCCCTAGAACACCAAGGTGGACAAGA 213

HC- S26 GAGTGGAGC

S207Pcl HC-TAG- CCTTGGTGTTCTAGGGCTTGTGGTTCACGTTG 214

A26 CAGATGTAG

HC-TAG- ACCAAG GTGTAG AAG AG AGTG GAG CCCAAG A 215

HC- S27 GCTGCGACA

D212Pcl HC-TAG- CACTCTCTTCTACACCTTGGTGTTGCTGGGCT 216

A27 TGTGGTTCA

HC-TAG- TCCCCCCCTAGCCCAAGGACACCCTGATGAT 217

HC- S28 CAGCAGGA

K246Pcl HC-TAG- GTCCTTGGGCTAGGGGGGGAACAGGAACAC 218

A28 GGAGGGTCCG

HC- HC-TAG- AGGACCCCCTAGGTGACCTGCGTGGTGGTGG 219

E258Pcl S29 ACGTGAG Primer SEQ ID

Antibody Sequence

name NO

HC-TAG- CAGGTCACCTAGGGGGTCCTGCTGATCATCA 220 A29 GGGTGTCCT

HC-TAG- TGAGCCACTAGGACCCAGAGGTGAAGTTCAA 221

HC- S30 CTGGTACG

E269Pcl HC-TAG- CTCTGGGTCCTAGTGGCTCACGTCCACCACC 222

A30 ACGCAGGTC

HC-TAG- CCAGAGGTGTAGTTCAACTGGTACGTGGACG 223

HC- S32 GCGTGGAGG

K274Pcl HC-TAG- CCAGTTGAACTACACCTCTGGGTCCTCGTGG 224

A32 CTCACGTCCA

HC-TAG- GTGGACGGCTAGGAGGTGCACAACGCCAAGA 225

HC- S33 CCAAGCCCA

V282Pcl HC-TAG- TGCACCTCCTAGCCGTCCACGTACCAGTTGAA 226

A33 CTTCACCT

HC-TAG- G ACG GCGTGTAG GTG CACAACG CCAAG ACCA 227

HC- S34 AGCCCAGA

E283Pcl HC-TAG- TTGTGCACCTACACGCCGTCCACGTACCAGTT 228

A34 GAACTTCA

HC-TAG- GAGGTGCACTAGGCCAAGACCAAGCCCAGAG 229

HC- S35 AGGAGCAGT

N286Pcl HC-TAG- GGTCTTGGCCTAGTGCACCTCCACGCCGTCC 230

A35 ACGTACCAGT

HC-TAG- CACAACGCCTAG ACCAAG CCC AG AG AG GAG C 231

HC- S36 AGTACAACA

K288Pcl HC-TAG- GGCTTGGTCTAGGCGTTGTGCACCTCCACGC 232

A36 CGTCCACGT

HC-TAG- GCCAAGACCTAGCCCAGAGAGGAGCAGTACA 233

HC- S37 ACAGCACCT

K290Pcl HC-TAG- CTCTCTGGGCTAGGTCTTGGCGTTGTGCACC 234

A37 TCCACGCCGT

HC-TAG- ACCAAGCCCTAGGAGGAGCAGTACAACAGCA 235

HC- S38 CCTACAGGGT

R292Pcl HC-TAG- CTGCTCCTCCTAGGGCTTGGTCTTGGCGTTGT 236

A38 GCACCTCCA

HC-TAG- AAGCCCAGATAGGAGCAGTACAACAGCACCT 237

HC- S39 ACAGGGTG

E293Pcl HC-TAG- TACTGCTCCTATCTGGGCTTGGTCTTGGCGTT 238

A39 GTGCACCT

HC-TAG- G CCCAG AG AGTAG CAGTACAACAG CACCTAC 239

HC- S40 AGGGTGGT

E294Pcl HC-TAG- TTGTACTGCTACTCTCTGGGCTTGGTCTTGGC 240

A40 GTTGTGCA

HC-TAG- AAGGAATACTAGTGCAAGGTCTCCAACAAGG 241

HC- S41 CCCTGCCA

K320Pcl HC-TAG- ACCTTG CACTAGTATTCCTTG CCGTTCAG CCA 242

A41 GTCCTGGT

HC-TAG- TACAAGTGCTAGGTCTCCAACAAGGCCCTGC 243

HC- S42 CAGCCCCCA

K322Pcl HC-TAG- GTTGGAGACCTAGCACTTGTATTCCTTGCCGT 244

A42 TCAGCCAGT

HC-TAG- GGTCTCCAACTAGGCCCTGCCAGCCCCCATC 245

HC- S43 GAAAAGACC

K326Pcl HC-TAG- GGCAGGGCCTAGTTGGAGACCTTGCACTTGT 246

A43 ATTCCTTGC Primer SEQ ID

Antibody Sequence

name NO

HC-TAG- G CCCTG CC ATAG CCC ATCG AAAAG ACCATCA 247

HC- S44 GCAAGGCCA

A330Pcl HC-TAG- TTCGATGGGCTATGGCAGGGCCTTGTTGGAG 248

A44 ACCTTGCACT

HC-TAG- GCCCCCATCTAGAAGACCATCAGCAAGGCCA 249

HC- S45 AGGGCCAGC

E333Pcl HC-TAG- GATGGTCTTCTAGATGGGGGCTGGCAGGGCC 250

A45 TTGTTGGAGA

HC-TAG- CCCATCGAATAGACCATCAGCAAGGCCAAGG 251

HC- S46 GCCAGCCA

K334Pcl HC-TAG- G CTG ATG GTCTATTCG ATG GG GG CTG GC AGG 252

A46 GCCTTGTTG

HC-TAG- TCGAAAAGTAGATCAGCAAGGCCAAGGGCCA 253

HC- S47 GCCACGGGA

T335Pcl HC-TAG- CTTGCTGATCTACTTTTCGATGGGGGCTGGCA 254

A47 GGGCCTTGT

HC-TAG- AGACCATCTAGAAGGCCAAGGGCCAGCCACG 255

HC- S48 GGAGCCCCA

S337Pcl HC-TAG- CCTTGGCCTTCTAGATGGTCTTTTCGATGGGG 256

A48 GCTGGCAGG

HC-TAG- GGCCAGCCATAGGAGCCCCAGGTGTACACCC 257

HC- S50 TGCCTCCAT

R344Pcl HC-TAG- CTGGGGCTCCTATGGCTGGCCCTTGGCCTTG 258

A50 CTGATGGTCT

HC-TAG- CTCCATCCTAGGACGAGCTGACCAAGAACCA 259

HC- S51 GGTGTCCCT

R355Pcl HC-TAG- CAGCTCGTCCTAGGATGGAGGCAGGGTGTAC 260

A51 ACCTGGGGCT

HC-TAG- AGCTGACCTAGAACCAGGTGTCCCTGACCTG 261

HC- S52 TCTGGTGA

K360Pcl HC-TAG- CACCTGGTTCTAGGTCAGCTCGTCCCGGGAT 262

A52 GGAGGCAGG

HC-TAG- CCAAGAACTAGGTGTCCCTGACCTGTCTGGT 263

HC- S53 GAAGGGCTT

Q362Pcl HC-TAG- TCAGGGACACCTAGTTCTTGGTCAGCTCGTCC 264

A53 CGGGATGGA

HC-TAG- TTCTACCCCTAGGACATCGCCGTGGAGTGGG 265

HC- S54 AGAGCAACG

S375Pcl HC-TAG- GGCGATGTCCTAGGGGTAGAAGCCCTTCACC 266

A54 AGACAGGTCA

HC-TAG- TGGAGTGGTAGAGCAACGGCCAGCCCGAGAA 267

HC- S55 CAACTACA

E382Pcl HC-TAG- GGCCGTTGCTCTACCACTCCACGGCGATGTC 268

A55 GCTGGGGTAG

HC-TAG- AGCCCGAGTAGAACTACAAGACCACCCCCCC 269

HC- S56 AGTGCTGGA

N389Pcl HC-TAG- CTTGTAGTTCTACTCGGGCTGGCCGTTGCTCT 270

A56 CCCACTCCA

HC-TAG- CCCGAGAACTAGTACAAGACCACCCCCCCAG 271

HC- S57 TGCTGGACA

N390Pcl HC-TAG- GGTCTTGTACTAGTTCTCGGGCTGGCCGTTG 272

A57 CTCTCCCACT

HC-TAG- GAACAACTACTAGACCACCCCCCCAGTGCTG 273

HC- S58 GACAGCGAC

K392Pcl

HC-TAG- GGGGTGGTCTAGTAGTTGTTCTCGGGCTGGC 274 Primer SEQ ID

Antibody Sequence

name NO

A58 CGTTGCTCT

HC-TAG- AACTACAAGTAGACCCCCCCAGTGCTGGACA 275

HC- S59 GCGACGGCA

T393Pcl HC-TAG- TGGGGGGGTCTACTTGTAGTTGTTCTCGGGC 276

A59 TGGCCGTTG

HC-TAG- CCCCAGTGTAG G AC AG CG ACG GCAG CTTCTT 277

HC- S60 CCTGTACA

L398Pcl HC-TAG- GTCGCTGTCCTACACTGGGGGGGTGGTCTTG 278

A60 TAGTTGTTCT

HC-TAG- TG CTGG ACTAGG ACG G CAG CTTCTTCCTGTA 279

HC- S61 CAGCAAGCT

S400Pcl HC-TAG- GCTGCCGTCCTAGTCCAGCACTGGGGGGGTG 280

A61 GTCTTGTAGT

HC-TAG- TGACCGTGTAGAAGTCCAGGTGGCAGCAGGG 281

HC- S62 CAACGTGTT

D413Pcl HC-TAG- ACCTGGACTTCTACACGGTCAGCTTGCTGTAC 282

A62 AGGAAGAAG

HC-TAG- TGGACAAGTAGAGGTGGCAGCAGGGCAACGT 283

HC- S63 GTTCAGCT

S415Pcl HC-TAG- CTGCCACCTCTACTTGTCCACGGTCAGCTTGC 284

A63 TGTACAGG

HC-TAG- AGGG CAACTAGTTCAG CTG CAG CGTG ATG CA 285

HC- S64 CGAGGCCCT

V422Pcl HC-TAG- G CAG CTG AACTAGTTG CCCTGCTGCCACCTG 286

A64 GACTTGTCCA

[00252] Table 12. Amino acid sequences of the constant region of 60

TAG mutant constructs in human IgGl heavy chain of trastuzumab. SEQ ID NO: 1 is the sequence for full length trastuzumab heavy chain wild-type. SEQ ID NO:2 to SEQ ID NO:61 indicate the sequence ID numbers for 60 TAG mutant constructs in human IgGl heavy chain constant region. In the table, Z is used as the single letter code for Pel (same position can also be used to incorporate other TAG-encoded amino acids.

[00253] Table 13. Amino acid sequences of the constant region of

32 human kappa light chain TAG mutant constructs. SEQ ID NO: 62 is the sequence of the constant region of the wild-type, full length trastuzumab light chain. SEQ ID NO: 63 to SEQ ID NO:94 indicate the sequence ID numbers for 32 TAG mutant constructs in the constant region of human kappa light chain. In the table, Z is used as the single letter code for Pel (same position can also be used for any other TAG-encoded amino acid).

SEQ ID NO:62

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFL YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:63

ZRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:64

KZTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:65

KRZVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:66

KRTVAZPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:67

KRTVAAPZVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:68

KRTVAAPSVFIFPPSZEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:69

KRTVAAPSVFIFPPSDZQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:70

KRTVAAPSVFIFPPSDEQLZSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID N0:71

KRTVAAPSVFIFPPSDEQLKZGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID NO:72

KRTVAAPSVFIFPPSDEQLKSGZASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:73

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPZEAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID NO:74

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRZAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID NO:75

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAZVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC SEQ ID NO:76

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDZALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID NO:79

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNAZQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:80

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQZGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID N0:81

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSZNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID NO:80

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNZ QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID N0:81

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQZSVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:82

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTZQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:83

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDZKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:84

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSZDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:85

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKZSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC SEQ ID NO:86

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLZKADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:87

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSZADYEKHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:88

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEZHKVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:89

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHZVYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID NO:90

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKZYACEVTHQGLSSPVTKSFNR

GEC

SEQ ID N0:91

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVZHQGLSSPVTKSFNR

GEC

SEQ ID NO:92

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHZGLSSPVTKSFNR

GEC

SEQ ID NO:93

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSZPVTKSFNR

GEC

SEQ ID NO:94

KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVZKSFNR

GEC Example 3. Transfer of the trastuzumab heavy chain and light chain TAG mutations to different antibodies.

[00254] For trastuzumab, all TAG mutations were chosen to be made in the constant region of its human IgGl heavy and human kappa light chain and used for Pel incorporation. Because constant regions of antibodies are highly conserved in primary sequence and structure, residues that are identified as good payload attachment sites in the context of trastuzumab should also serve as preferred attachment residues in other antibodies. To demonstrate the

transferability of these generic conjugation sites to other antibodies, we cloned a set of TAG mutations into antibody 14090. Antibody 14090 is an antibody with a human IgGl heavy chain and a human lambda light chain. The DNA encoding variable region of antibody 14090 was cloned into nine selected pOG trastuzumab HC TAG mutant plasmid constructs (SEQ ID NO: listed in Table 14) to replace the variable regions of trastuzumab constructs in the plasmids as described in Example 2. As the results, the nine TAG mutant constructs of antibody 14090 contain the same sequences in the constant region of trastuzumab TAG mutant constructs shown in Table 14 (Figure 3). In subsequent examples, we will show that these sites can be conjugated readily. Conversely, due to a high degree of similarity in primary sequences and in tertiary structures for different human IgG isotypes (Figure 4), TAG mutations on the kappa light chain of trastuzumab can readily be transferred to equivalent light chains on human antibodies containing different isotype heavy chains. In the same way, the sites identified in the constant region of IgGl may be transferred to IgG2, IgG3 and IgG4.

[00255] Table 14. TAG mutant constructs generated in the constant region of the heavy chain of antibody 14090. 9 TAG constructs were prepared in the IgGl heavy chain constant region of antibody 14090. SEQ ID numbers equivalent to those in the heavy chain of trastuzumab are shown. In addition to Pel incorporation, the identified sites can be used to incorporate any other TAG- encoded amino acid. Eu number SEQ ID NO

HC-S1 17Pcl 2

HC-S124Pcl 5

HC-S136Pcl 8

HC-T139Pcl 9

HC-E152Pcl 10

HC-L174Pcl 17

HC-E258Pcl 28

HC-K360Pcl 49

HC-S375Pcl 51

Example 4. TAG mutations in human lambda light chains.

[00256] Human Lambda and kappa light chains have little amino acid sequence similarity (Figure 5A). Mutations in the lambda light chain of antibody 14090 were selected based on the approximate similarity of the residues' location in a protein crystal structure model (Protein Databank structure entry 3G6D.pdb) of a Fab containing the human lambda light chain in reference to the desirable residues in the kappa light chain of trastuzumab antibody (Figure 5 A and B). Seven additional TAG mutant constructs were generated in antibody 14090-lambda light chain plasmid using oligonucleotide directed mutagenesis (Higuchi et al. (1988) Nucleic Acids Res. 16(15):7351-67) in combination with PIPE cloning strategy (Klock and Lesley, (2009) Methods Mol Biol. 498:91-103). The mutation primers used to generate TAG point mutations in the lambda light chain are listed in Table 15. The secretion of antibody 14090 is also directed by the synthetic 24 amino acid signal sequence: MKTFILLLWVLLLWVIFLLPGATA. Sequences of antibody 14090 TAG constructs were confirmed by DNA sequencing. The amino acid sequence of the constant region of wild type human lambda light chain is shown as SEQ ID NO: 95, and the constant region of 7 TAG mutant constructs in human lambda light chain are shown as SEQ ID NO:96 to SEQ ID NO: 102 in Table 16.

[00257] Table 15. Nucleotide sequences of primers used in

mutagenesis of seven TAG mutant constructs in antibody 14090 lambda light chain. In addition to Pel incorporation, the TAG sites can be used to incorporate any other TAG-encoded amino acid. SEQ

Sequence ID

Antibody name Sequence NO

LC- Seq-0047 CCGGGATAGGTGACAGTGGCCTGGAAGGCAGATAGC 287

A143Pcl Seq-0048 TGTCACCTATCCCGGGTAGAAGTCACTTATGAGACA 288

LC- Seq-0049 GCCGTGTAGGTGGCCTGGAAGGCAGATAGCAGCCCC 289

T145Pcl Seq-0050 GGCCACCTACACGGCTCCCGGGTAGAAGTCACTTAT 290

LC- Seq-0051 ACAGTGTAGTGGAAGGCAGATAGCAGCCCCGTCAAG 291

A147Pcl Seq-0052 CTTCCACTACACTGTCACGGCTCCCGGGTAGAAGTC 292

LC- Seq-0053 CCCGTCTAGGCGGGAGTGGAGACCACCACACCCTCC 293

K156Pcl Seq-0054 TCCCGCCTAGACGGGGCTGCTATCTGCCTTCCAGGC 294

LC- Seq-0055 G CG GG ATAG G AG ACCACCACACCCTCCAAACAAAG C 295 VI 59Pcl Seq-0056 GGTCTCCTATCCCGCCTTGACGGGGCTGCTATCTGC 296

LC- Seq-0057 ACCACCTAGCCCTCCAAACAAAGCAACAACAAGTAC 297

T163Pcl Seq-0058 GGAGGGCTAGGTGGTCTCCACTCCCGCCTTGACGGG 298

LC- Seq-0059 AAACAATAG AACAACAAGTACG CG GCC AG CAG CTAT 299

S168Pcl Seq-0060 GTTGTTCTATTGTTTGGAGGGTGTGGTGGTCTCCAC 300

[00258] Table 16. Amino acid sequence of the constant region of 7

TAG mutant constructs in antibody 14090 lambda light chain. SEQ ID NO: 96 to SEQ ID NO: 102 indicate the 7 TAG mutants in the constant region of human lambda light chain of antibody 14090. SEQ ID NO:95 is the sequence for the constant region of wild type human lambda light chain. In the table, Z is used as the single letter code for Pel (the same site can also be used for any other TAG-encoded amino acid).

SEQ ID NO:95

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS

KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:96

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGZVTVAWKADSSPVKAGVETTTPS

KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:97

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVZVAWKADSSPVKAGVETTTPS

KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:98

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVZWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO:99

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVZAGVETTTPS

KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:100

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGZETTTPS

KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:101

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTZPS

KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO:102

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQZNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Example 5. Expression and purification of Pel mutant antibodies

[00259] Pyrroline-carboxy-lysine (Pel) was identified as a novel amino acid that is generated and incorporated into proteins at TAG codons by the unmodified biosynthetic machinery of the 22^nd proteinogenic amino acid, pyrrolysine (Celliti et al, (2011) Nat Chem Biol. 7:528-530). Pel can be incorporated into a variety of proteins at TAG codons in both mammalian cells and E. coli (Ou et al, (201 1) Proc Natl Acad Sci U S A. 108: 10437-10442; Celliti et al, 2011). Biosynthesis of Pel from D-omithine requires co-expression of gene products of pylC and pylD, two of the three biosynthetic genes in the pyrrolysine operon identified in methanogenic archaea, Methanosarcina. In addition, incorporation of Pel at TAG codons in a target protein gene requires a specific tRNA synthetase encoded by pylS and its corresponding tRNA, encoded by pylT (Ou et al, 2011 ; Celliti et al., 201 1). It has been demonstrated that co-expression of pylC, pylD, pylS an pylT with a protein gene containing an engineered TAG codon in either mammalian cells or E. coli, in the presence of D-omithine, leads to exclusive incorporation of Pel at the TAG codon (Ou et al, 201 1; Celliti et al, 201 1). Coding regions of pylC, pylD, pylS, and pylT were amplified by PCR from genomic DNA of Methanosarcina mazei (ATCC). pylC, pylD, and pylS were separately cloned into a pRS vector under the control of a CMV promoter, and pylT was cloned into each of pRS-pylD, pRS-pylC, and pRS-pylS vectors under the control of a U6 promoter. pRS-pylD, pRS-pylC, and pRS-pylS were co-transfected with heavy chain or light chain constructs of Trastuzumab antibody, containing a TAG codon in selected positions, into 293 Freestyle™ cells (Ou et al., 2011 ; Celliti et al., 201 1). The plasmids DNA used in co-transfection were prepared using Qiagen plasmid preparation kit according to manufacturer's protocol. 293 Freestyle™ cells were cultured in suspension in Freestyle™ expression media (Invitrogen) at 37°C under 5% C02. On the day before transfection, cells were split to 0.7 x 10⁶ cells /ml in fresh media. The cells were transfected with a mixture of pRS-pylD, pRS-pylC, and pRS-pylS, heavy chain and light chain plasmids of trastuzumab antibody in a ratio of 1 : 1 : 1 :2:2 (Celliti et al, 201 1) using the PEI method (Meissner et al, 2001). D- ornithine was added to the culture media at 5 mM 8 hours post- transfection. The transfected cells were further cultured for five days. The media from the culture was harvested by centrifugation of the culture at 2000 x g for 20 min and filtered through 0.2 micrometer filters. The expressed antibodies were purified from the filtered media using Protein A-Sepharose (GE Healthcare Life Sciences). Antibody IgGs were eluded from the Protein A-Sepharose column by the elution buffer (pH3.0) and immediately neutralized with 1 M Tris-HCl (pH 8.0) followed by a buffer exchange to PBS.

[00260] Expression levels of Pel containing trastuzumab antibodies in transiently transfected 293 Freestyle™ cells varied widely depending on the location of the TAG codon (Table 9, Protein A purified). Since there is a competition between Pel-charged tRNA and the release factor of translation termination at TAG codons, an expected by-product of the Pel-incorporation is the truncated form of the target protein, truncated at the engineered TAG codon. Analysis of the Protein-A purified trastuzumab antibodies on SDS PAGE revealed that this is the case. In addition to protein bands corresponding to the full length heavy chain (55 kDa) and light chain (25 kDa), another dominant protein band was observed with the molecular weight matching to the truncated product at the engineered TAG (Figure 6). To prevent the interference of the truncated products with downstream applications (conjugation, purification), a purification step was added that employed a hydrophobic interaction column (HIC) to purify full length antibodies from the truncated products. Ammonium sulfate was added to each Protein-A-purified trastuzumab Pel construct to a final concentration of 1 M. The samples were loaded to a HIC column (TSKgel Phenyl-5PW, 21.5x150 mm;

Tosoh Bioscience LLC) at a flow rate of 5 ml/min operated in a HPLC (Waters 600E). The bound proteins were eluted from the column by a gradient of 20% A solution (1.5 M ammonium sulfate in 20 mM sodium phosphate, pH7.4) to 100% B solution (20 mM sodium phosphate, pH7.4 and 20% 2-isopropanol). A typical elution chromatography profile is shown in Figure 7A. Under the conditions the truncated product was separated well in HIC from the full length trastuzumab antibody (Figure 7A). SDS-PAGE of the HIC purified IgG fractions show that the purified IgG was devoid of truncated products (Figure 7 B). After HIC purification, the yield of the full-length, Pel containing IgG for each trastuzumab construct are shown in Table 17 (HIC purified).

[00261] Incorporation of Pel into TAG mutants of antibody 14090 antibodies was also performed in 293 Freestyle™ cells. As shown in Table 18, the expression level of antibody 14090 Pel antibodies ranges from 4 mg/L to 13 mg/L. Table 17. Yield of trastuzumab Pel antibodies transiently expressed in 293 Freestyle™ cells.

Protein Protein A trastuzumab A and HIC- antibody purified purified

(mg/L) (mg/L)

HC-S191 Pcl 17 13

HC-T195Pcl 15 11

HC-T197Pcl 6 3

HC-S207Pcl 21 14

HC-D212Pcl 12 3

HC-K246Pcl 14 6

HC-E258Pcl 27 15

HC-E269Pcl 33 15

HC-K274Pcl 22 6

HC-V282Pcl 23 11

HC-E283Pcl 9 4

HC-N286Pcl 7 4

HC-K288Pcl 10 5

HC-K290Pcl 7 4

HC-R292Pcl 23 12

HC-E293Pcl 13 4

HC-E294Pcl 9 2

HC-K320Pcl 31 16

HC-K322Pcl 6 4

HC-K326Pcl 25 7

HC-A330Pcl 37 9

HC-E333Pcl 24 2

HC-K334Pcl 8 2

HC-T335Pcl 15 2

HC-S337Pcl 26 2

HC-R344Pcl 45 14

HC-R355Pcl 40 9

HC-K360Pcl 51 8

HC-Q362Pcl 32 9

HC-S375Pcl 47 22

HC-E382Pcl 34 5

HC-N389Pcl 9 3

HC-N390Pcl 31 10

HC-K392Pcl 30 8

HC-T393Pcl 25 4 Protein Protein A trastuzumab A and HIC- antibody purified purified

(mg/L) (mg/L)

HC-L398Pcl 28 3

HC-S400Pcl 26 7

HC-D413Pcl 27 6

HC-S415Pcl 27 2

HC-V422Pcl 23 2

LC-K107Pcl 12 11

LC-R108Pcl 12 11

LC-T109Pcl 12 11

LC-A112Pcl 15 15

LC-S114Pcl 9 8

LC-D122Pcl 10 9

LC-E123Pcl 10 9

LC-K126Pcl 6 5

LC-S127Pcl 11 10

LC-T129Pcl 12 11

LC-R142Pcl 11 10

LC-E143Pcl 10 9

LC-K145Pcl 27 10

LC-N152Pcl 14 18

LC-L154Pcl 85 20

LC-S156Pcl 6 3

LC-G157Pcl 10 9

LC-S159Pcl 12 11

LC-E161 Pcl 9 8

LC-E165Pcl 4 3

LC-S168Pcl 8 7

LC-K169Pcl 8 8

LC-D170Pcl 8 7

LC-S182Pcl 6 5

LC-K183Pcl 6 5

LC-K188Pcl 6 5.5

LC-K190Pcl 4 4

LC-V191 Pcl 4 3

LC-T197Pcl 6 5

LC-Q199Pcl 7 6 Protein Protein A

trastuzumab A and HIC- antibody purified purified

(mg/L) (mg/L)

LC-S203Pcl 8 7

LC-T206Pcl 5 4

Table 18. Yield of antibody 14090 Pel proteins transiently expressed in 293 Freestyle™ cells.

Example 6. Conjugation of trastuzumab Pel antibodies with ABA-MMAF.

[00262] Previously, it has been shown that Pel incorporated into proteins can be derivatized with 2-amino-benzaldehyde (ABA) and 2-amino- acetophenone (AAP) moieties, resulting in stable conjugates (Figure 8. Ou et al, 2011). The ABA- and AAP-Pcl reactions are specific and efficient at pH 5 and pH 7 in aqueous solutions. A compound containing ABA linked to a cytotoxic compound MMAF through a linker was synthesized (ABA-MMAF; Figure 9). ABA-MMAF was specifically conjugated to the Pel-containing trastuzumab (Figure 10A, B). Trastuzumab Pel constructs were incubated at room temperature with ABA-MMAF at a molar ratio of 20: 1 (ABA-MMAF :Ab) in 0.2 M sodium acetate (pH 5.0). After 20 hours incubation, sodium cyanoborohydride was added to conjugation mixtures at a final concentration of 20 mM and the incubations were carried out for two more hours. The conjugation process was then monitored by reverse phase HPLC, which allows separation of conjugated antibodies from non-conjugated ones. The conjugation reaction mixture were analyzed on a PRLP-S 4000A column (50 mm x 2. lmm, Agilent) heated to 80° C and elution of the column was carried out by a linear gradient of 30-60% acetonitrile in water containing 0.1% TFA at a flow rate of 1.5 ml/min. The elution of proteins from the column was monitored at 280 nm, 254 nm and 215 nm. Figure 10A shows a typical conjugation mixture of a trastuzumab Pel construct and ABA-MMAF analyzed by reverse phase HPLC.

[00263] Drug antibody ratio (DAR) is used to measure the extent of a drug conjugation of an antibody. Since each antibody contains two Pel residues, a fully ABA-MMAF conjugated antibody will have a DAR of 2. An incomplete conjugation reaction will yield a mixture of DAR=0, DAR=1 and DAR=2 species, that can be separated by reverse phase HPLC. To enhance the separation, reverse phase HPLC was conducted at 80° C. As shown in Figure 10A, a conjugation reaction with trastuzumab-LC-S 156Pcl and ABA-MMAF produced three products (peaks) on reverse phase HPLC, representing DAR=0, DAR=1 and DAR=2 species, respectively. Reverse phase HPLC was used to quantitate the conjugation efficiency for all trastuzumab Pel constructs in this study. The conjugation efficiency was calculated as the total conjugation efficiency, quantitated from percentage of MMAF conjugation of all Pel sites including all species (total conjugation efficiency = [fraction of DAR=2+0.5 x fraction of DAR=l]/[fraction of DAR=0+fraction of DAR=l+fraction of DAR=0]). DAR=2 efficiency was calculated from percentage of DAR2 product (DAR=2 efficiency = [fraction of DAR=2]/[fraction of DAR=0+fraction of DAR=l+fraction of DAR=2]). As shown in Table 19, conjugation efficiencies varied depending on location of the Pel site. The total conjugation efficiency for many Pel sites reached a level of > 80% while some sites exhibited low efficiency at 20-30% (Table 19). DAR=2 efficiency for different Pel constructs also varied depending on the Pel sites. The best efficiency for many sites exceeded 60% (Table 19).

[00264] For most trastuzumab Pel antibodies the three conjugation products were separated well by a non-denaturing separation, a hydrophobic interaction column chromatography (Figure 1 1). HIC chromatography was used to purify the different DAR species. For the majority of the Pcl-MMAF trastuzumab conjugates (ADC), the purification by HIC yielded a dominant DAR=2 Pcl-MMAF ADC conjugates as revealed in reverse phase HPLC analysis (Figure 10B). The purified DAR=2 Pcl-MMAF ADCs were further analyzed by size exclusion chromatography. All purified DAR=2 Pcl-MMAF ADCs were monomeric (Figure 12) with a negligible level of oligomer (below detection limit) in all cases, suggesting that incorporation of a Pel residue in selected sites and conjugation of the MMAF payload to the sites did not cause aggregation. SDS-PAGE analysis of the purified DAR=2 ADCs showed a homogeneous single band under non-reducing and HC and LC bands under reducing conditions, demonstrating that the conjugation with MMAF through Pel maintains the integrity of the antibodies (Figure 13). LCMS analysis further confirmed the successful conjugation of ABA- MMAF to the expected Pel site (Figure 14).

[00265] The yield of the final purified DAR=2 ADC for each trastuzumab Pel construct is shown in Table 20. Because production of DAR=2 ADCs from each Pel trastuzumab antibody involves multiple processes, including Pel incorporation, drug conjugation, and HIC purification, the yield of the purified DAR=2 ADC products differs widely among different Pel sites with the best sites producing more than 10 mg/ml and worst sites generating less than 1 mg/L (Table 20). Yield of Pcl-MMAF ADC produced for antibody 14090 ranged from 0.7 mg/L to 6 mg/L (Table 21).

[00266] Conjugation mixtures were analyzed in reverse phase HPLC as shown in Figure 10. The area percentages from DAR=0, DAR=1 and DAR=2 peaks were quantitated separately. Conjugation efficiencies are expressed as the total Pel efficiency and the DAR=2 efficiency in percentages. The total Pel efficiency is calculated by the formula: total conjugation efficiency = [fraction of DAR=2+0.5 x fraction of DAR= Infraction of DAR=0+fraction of DAR=l+fraction of DAR=0]. DAR=2 efficiency is calculated by the formula: DAR=2 efficiency = [fraction of DAR=2]/ [fraction of DAR=0+fraction of DAR=l+fraction of DAR=0].

[00267] Table 19. Conjugation efficiencies of trastuzumab Pel constructs with ABA-MMAF. Conjugation of ABA-MMAF to anti-Her2 Pel constructs were performed as described in Example 6. Conjugation mixtures were analyzed in reverse phase HPLC as shown in Figure 10. The area percentages from DAR=0, DAR=1 and DAR=2 peaks were quantitated separately. Conjugation efficiencies are expressed as the total Pel efficiency and the DAR=2 efficiency in percentages. The total Pel efficiency is calculated by the formula: total conjugation efficiency = [fraction of DAR=2+0.5 x fraction of DAR=l]/[fraction of

DAR=0+fraction of DAR=l+fraction of DAR=0]. DAR=2 efficiency is calculated by the formula: DAR=2 efficiency = [fraction of DAR=2]/[fraction of

DAR=0+fraction of DAR=l+fraction of DAR=0].

Total Pel DAR=2 trastuzumab ADC efficiency efficiency

(%) (%)

HC-E283Pcl-MMAF 62 35

HC-N286Pcl-MMAF 78 61

HC-K288Pcl-MMAF 75 57

HC-K290Pcl-MMAF 70 50

HC-R292Pcl-MMAF 78 61

HC-E293Pcl-MMAF 74 55

HC-K320Pcl-MMAF 61 37

HC-K326Pcl-MMAF 74 55

HC-A330Pcl-MMAF 65 30

HC-E333Pcl-MMAF 70 50

HC-K334Pcl-MMAF 80 65

HC-T335Pcl-MMAF 74 55

HC-S337Pcl-MMAF 64 43

HC-R344Pcl-MMAF 45 26

HC-K360Pcl-MMAF 51 36

HC-Q362Pcl-MMAF 58 39

HC-S375Pcl-MMAF 72 56

HC-E382Pcl-MMAF 70 50

HC-N389Pcl-MMAF 76 60

HC-N390Pcl-MMAF 68 47

HC-K392Pcl-MMAF 75 57

HC-T393Pcl-MMAF 68 46

HC-L398Pcl-MMAF 63 41

HC-S400Pcl-MMAF 65 43

HC-D413Pcl-MMAF 64 41

HC-S415Pcl-MMAF 56 30

HC-V422Pcl-MMAF 70 49

LC-K107Pcl-MMAF 62 41

LC-R108Pcl-MMAF 64 44

LC-T109Pcl-MMAF 63 37

LC-A1 12Pcl-MMAF 73 56

LC-S1 14Pcl-MMAF 66 44

LC-D122Pcl-MMAF 54 37

LC-E123Pcl-MMAF 36 54

LC-K126Pcl-MMAF 29 15

LC-S127Pcl-MMAF 59 44 Total Pel DAR=2

trastuzumab ADC efficiency efficiency

(%) (%)

LC-T129Pcl-MMAF 73 39

LC-R142Pcl-MMAF 75 57

LC-E143Pcl-MMAF 67 45

LC-K145Pcl-MMAF 74 61

LC-N152Pcl-MMAF 71 53

LC-L154Pcl-MMAF 73 55

LC-S156Pcl-MMAF 69 52

LC-G157Pcl-MMAF 74 55

LC-S159Pcl-MMAF 43 26

LC-E161 Pcl-MMAF 71 50

LC-E165Pcl-MMAF 54 34

LC-S168Pcl-MMAF 75 57

LC-K169Pcl-MMAF 65 50

LC-D170Pcl-MMAF 74 54

LC-S182Pcl-MMAF 72 53

LC-K183Pcl-MMAF 44 33

LC-K188Pcl-MMAF 62 43

LC-K190Pcl-MMAF 40 0

LC-V191 Pcl-MMAF 59 41

LC-T197Pcl-MMAF 71 55

LC-Q199Pcl-MMAF 64 51

LC-S203Pcl-MMAF 69 51

LC-T206Pcl-MMAF 76 58

Table 20. Yield of DAR=2 Pcl-MMAF ADC with trastuzumab Pel constructs.

trastuzumab ADC Yield (mg/L)

HC-P153Pcl-MMAF 2.2

HC-T155Pcl-MMAF 2.0

HC-S157Pcl-MMAF 1 .7

HC-T164Pcl-MMAF 3.4

HC-S165Pcl-MMAF 2.9

HC-P171 Pcl-MMAF 10.3

HC-L174Pcl-MMAF 9.8

HC-S176Pcl-MMAF 2.5

HC-S177Pcl-MMAF 0.3

HC-T187Pcl-MMAF 2.2

HC-P189Pcl-MMAF 3.7

HC-S191 Pcl-MMAF 2.2

HC-T195Pcl-MMAF 3.1

HC-T197Pcl-MMAF 0.3

HC-S207Pcl-MMAF 3.2

HC-D212Pcl-MMAF 0.5

HC-K246Pcl-MMAF 3.6

HC-E258Pcl-MMAF 5.5

HC-E269Pcl-MMAF 4.7

HC-K274Pcl-MMAF 1 .5

HC-V282Pcl-MMAF 1 .5

HC-E283Pcl-MMAF 0.8

HC-N286Pcl-MMAF 0.8

HC-K288Pcl-MMAF 0.6

HC-K290Pcl-MMAF 2.7

HC-R292Pcl-MMAF 3.4

HC-E293Pcl-MMAF 0.5

HC-E294Pcl-MMAF 1 .5

HC-K320Pcl-MMAF 2.7

HC-K322Pcl-MMAF 2.5

HC-K326Pcl-MMAF 2.8

HC-A330Pcl-MMAF 1 .0

HC-E333Pcl-MMAF 0.7

HC-K334Pcl-MMAF 1 .2

HC-T335Pcl-MMAF 0.7

HC-S337Pcl-MMAF 0.7

HC-R344Pcl-MMAF 1.3 trastuzumab ADC Yield (mg/L)

HC-Q362Pcl-MMAF 1 .8

HC-S375Pcl-MMAF 6.2

HC-E382Pcl-MMAF 0.5

HC-N389Pcl-MMAF 1.4

HC-N390Pcl-MMAF 1.6

HC-K392Pcl-MMAF 1 .3

HC-T393Pcl-MMAF 0.9

HC-L398Pcl-MMAF 0.5

HC-S400Pcl-MMAF 1 .2

HC-D413Pcl-MMAF 0.5

HC-S415Pcl-MMAF 0.5

HC-V422Pcl-MMAF 0.5

LC-K107Pcl-MMAF 5.9

LC-R108Pcl-MMAF 9.8

LC-T109Pcl-MMAF 11 .3

LC-A1 12Pcl-MMAF 2.1

LC-S1 14Pcl-MMAF 3.0

LC-D122Pcl-MMAF 3.1

LC-E123Pcl-MMAF 2.5

LC-K126Pcl-MMAF 0.4

LC-S127Pcl-MMAF 3.0

LC-T129Pcl-MMAF 2.0

LC-R142Pcl-MMAF 1 .4

LC-E143Pcl-MMAF 6.0

LC-K145Pcl-MMAF 5.6

LC-N152Pcl-MMAF 8.6

LC-L154Pcl-MMAF 10.5

LC-S156Pcl-MMAF 2.2

LC-G157Pcl-MMAF 4.4

LC-S159Pcl-MMAF 4.6

LC-E161 Pcl-MMAF 1 .6

LC-E165Pcl-MMAF 1 .2

LC-S168Pcl-MMAF 2.7

LC-K169Pcl-MMAF 2.3

LC-D170Pcl-MMAF 2.3

LC-S182Pcl-MMAF 1 .8

LC-K183Pcl-MMAF 0.4 trastuzumab ADC Yield (mg/L)

LC-K188Pcl-MMAF 4.2

LC-K190Pcl-MMAF 0.3

LC-V191 Pcl-MMAF 1 .9

LC-T197Pcl-MMAF 1 .9

LC-Q199Pcl-MMAF 2.0

LC-S203Pcl-MMAF 5.3

LC-T206Pcl-M MAF 1 .2

Table 21. Yield of DAR=2 Pcl-MMAF ADCs with antibody 14090 Pel constructs.

Example 7. Thermal stability assay of trastuzumab Pcl-MMAF ADCs.

[00268] Conjugation of the MMAF payload to trastuzumab may stabilize or destabilize the antibody, leading to changes in melting temperature of the antibody. The melting temperature of the antibody can be determined by differential scanning fluorimetry (DSF) that is based on temperature induced denaturation monitored by an environmentally sensitive dye, such as sypro orange. ADC samples were aliquoted in triplicate into 384-well plates into PBS (6.7 mM sodium phosphate pH 7.2; 150 mM NaCl). In each well, 8 μΐ of 0.25 mg/ml antibody was mixed with 2 μΐ 25x sypro orange dye (Invitrogen). Plates were sealed and analyzed in a Roche LightCycler 480 system with a temperature ramp from 30 to 85°C with 20 fluorescence scans recorded per degree C. Melting temperatures were manually fit to the peaks in the first derivative of the fluorescence intensity vs. time curves.

[00269] A typical thermal shift assay for wild-type trastuzumab revealed as expected two melting transitions (Tm), Tml at 69.7° C and Tm2 at 81.2° C, respectively (Table 22) (Ionescu RM, Vlasak J, Price C, Kirchmeier M. (2008) J Pharm Sci. 97: 1414-1426). When trastuzumab Pcl-MMAF ADCs were subjected to the thermal stability assays, the majority of the ADCs showed a similar pattern to that of wild-type trastuzumab with little changes in either Tml or Tm2. As an example, the thermogram of anti-Her2-HC-E152Pcl-MMAF ADC is shown in Figure 15. Only for one ADC (LC-R108Pcl-MMAF) was a Tml decrease of more than 10°C observed (Table 22).

Table 22. Melting temperatures Tml and Tm2 of trastuzumab Pcl-MMAF ADCs observed by differential scanning fluorimetry. n.d.: Not determined. Broad: A broad transition prevented extraction of a transition temperature.

trastuzumab ADC Tm1 [°C] Tm2 [°C]

HC-P171 Pcl-MMAF 69.5 81 .4

HC-L174Pcl-MMAF 69.5 78.3

HC-S176Pcl-MMAF 68.7 80.5

HC-S177Pcl-MMAF 69.6 80.0

HC-T187Pcl-MMAF 69.2 79.6

HC-P189Pcl-MMAF 69.3 81 .5

HC-S191 Pcl-MMAF 68.5 81 .3

HC-T195Pcl-MMAF 68.7 81 .4

HC-T197Pcl-MMAF 69.1 81 .2

HC-S207Pcl-MMAF 68.8 Broad

HC-D212Pcl-MMAF 68.8 80.6

HC-K246Pcl-MMAF 64.5 80.8

HC-E258Pcl-MMAF 66.4 81 .1

HC-E269Pcl-MMAF 62.9 81 .3

HC-K274Pcl-MMAF 66.7 80.9

HC-V282Pcl-MMAF 64.5 80.8

HC-K290Pcl-MMAF 67.6 80.9

HC-R292Pcl-MMAF 67.0 81 .0

HC-E294Pcl-MMAF 68.1 81 .4

HC-K320Pcl-MMAF 61 .2 80.5

HC-K322Pcl-MMAF 63.0 80.8

HC-K326Pcl-MMAF 64.5 80.9

HC-A330Pcl-MMAF 66.2 81 .3

HC-E333Pcl-MMAF 59.1 81 .3

HC-S337Pcl-MMAF 62.1 81 .4

HC-R344Pcl-MMAF 68.5 81 .3

HC-K360Pcl-MMAF n.d. n.d.

HC-Q362Pcl-MMAF 69.1 80.9

HC-S375Pcl-MMAF 67.2 81 .6

HC-E382Pcl-MMAF 67.9 81 .2

HC-N390Pcl-MMAF 68.5 81 .2

HC-K392Pcl-MMAF 68.1 81 .4

HC-T393Pcl-MMAF 67.1 81 .6

HC-L398Pcl-MMAF 69.7 80.9

HC-S400Pcl-MMAF 68.5 81 .4

HC-S415Pcl-MMAF 69.5 80.8

HC-V422Pcl-MMAF 69.9 75.5 and 81 .4 trastuzumab ADC Tm1 [°C] Tm2 [°C]

LC-K107Pcl-MMAF 69.6 80.9

LC-R108Pcl-MMAF 57.4 Broad peak

LC-T109Pcl-MMAF 69.6 80.8

LC-S1 14Pcl-MMAF 69.3 81 .3

LC-D122Pcl-MMAF 68.9 81 .2

LC-E123Pcl-MMAF 69.0 79.8

LC-K126Pcl-MMAF 68.7 80.8

LC-S127Pcl-MMAF 69.1 80.5

LC-T129Pcl-MMAF 70.0 80.4

LC-R142Pcl-MMAF 69.7 80.1

LC-E143Pcl-MMAF 69.7 80.5

LC-K145Pcl-MMAF 69.5 80.7

LC-N152Pcl-MMAF 68.4 81 .5

LC-L154Pcl-MMAF 69.2 80.7

LC-S156Pcl-MMAF 69.3 80.5

LC-G157Pcl-MMAF 68.7 80.9

LC-S159Pcl-MMAF 69.7 79.3

LC-E161 Pcl-MMAF 69.5 80.5

LC-S168Pcl-MMAF 69.6 79.7

LC-K169Pcl-MMAF 69.5 80.5

LC-D170Pcl-MMAF 69.4 80.6

LC-S182Pcl-MMAF 69.3 80.3

LC-K183Pcl-MMAF 69.1 80.6

LC-K188Pcl-MMAF 68.8 80.8

LC-K190Pcl-MMAF 68.7 81 .5

LC-V191 Pcl-MMAF 68.9 81 .1

LC-T197Pcl-MMAF 69.7 81 .1

LC-Q199Pcl-MMAF 69.6 80.9

LC-S203Pcl-MMAF 68.9 81 .2

LC-T206Pcl-M MAF 68.8 81 .0

Example 8. Cell proliferation assays to measure In vitro cell killing potency of Pel ADCs.

[00270] Cells that naturally express target antigens or cell lines engineered to express target antigens are frequently used to assay the activity and potency of ADCs. For evaluation of trastuzumab ADCs' in vitro cell killing potency, two engineered cell lines, MDA-MB231 clone 16 and clone 40, and HCC1954 cells were employed. MDA-MB231 clone 16 cells stably express high copy numbers (~5xl0⁵ copies /cell) of recombinant human Her2 while clone 40 express low copy numbers (~5xl0³ copies /cell) of human Her2. HCC1954 cells endogenously express high level (~5xl0⁵ copies /cell) of human Her2 in the surface (Clinchy et al., (2000) Breast Cancer Res Treat. 61(3):217-228). For determination of the cell killing potency of antibody 14090 ADCs, CMK1 1-5 and Jurkat cells were used. While CMK1 1-5 cells express a high level of the antigen for antibody 14090 in the cell surface there is no detectable antigen expression in Jurkat cells. An antigen dependent cell killing should only occur for cells that express sufficient antigen in the cell surface but not cells lacking the antigen. The cell proliferation assays were conducted with Cell-Titer-Glo™ (Promega) five days after cells were incubated with various concentrations of ADCs. (Riss TL, Moravec RA. Assay Drug Dev Technol. (2004) 2:51-62). In some studies, the cell based assays are high throughput and conducted in an automated system (Melnick et al., (2006) Proc Natl Acad Sci U S A. 103 :3153-3158).

[00271] 89 trastuzumab Pcl-MMAF ADCs were assayed in both

MDA-MB231 clone 16 and MDA-MB231 clone 40 cells. All ADCs specifically inhibited proliferation of MDA-MB231 clone 16 but not MDA-MB231 clone 40 cells. A typical example is shown in Figure 16. IC₅₀ of the trastuzumab Pcl- MMAF ADCs in MDA-MB231 clone 16 cell assays ranges from 100 pM to 400 pM (Table 23). The trastuzumab Pcl-MMAF ADCs (as demonstrated for a set of selected ADCs) also killed three other cell lines that express high levels of human Her2, namely HCC1954, PC3-9, and SKOV3-ip cells (Clinchy et al, 2000), and a cell line that express a medium level of human Her2, Jim-Tl cells. The sensitivity of the cell lines to trastuzumab Pcl-MMAF ADCs is proportional to the expression level of human Her2 on the cell surface. HCC1954 cells and SKOV3-ip cells are the most sensitive and JimTl cells are least sensitive to trastuzumab-MMAF ADCs. Similarly, antibody 14090-Pcl-MMAF ADC displayed an antigen dependent cell killing in cell proliferation assays. The antibody 14090 Pcl-MMAF ADCs killed antigen expressing CMK1 1-5 cells but not antigen negative Jurkat cells (Figure 17). The IC₅₀ of the antibody 14090 Pcl-MMAF ADC in CMK1 1-5 proliferation assay is in the range of 300 pM to 2 nM (Table 24). [00272] After binding to target cells, ADCs are quickly internalized into endosomes, which are subsequently fused to lysosomes where the antibody portion of the ADCs is degraded by lysosomal proteolytic enzymes (Chari, (2008) Acc Chem Res.41 :98-107; Jaracz et al. (2005) Bioorg Med Chem. 13 :5043-5054; Sanderson et al. (2005) Clin Cancer Res. 11 :843-852). The final metabolites generated by lysosomal degradation for ADCs with a non-cleavable linker, have been reported as the drug payload-linker conjugated to the amino acid residue of the conjugation site (Erickson et al. (2006) Cancer Res. 66(8):4426-4433; Erickson et al., (2010) Bioconjug Chem. 21 :84-92; Sanderson et al. 2005). Therefore for ADCs prepared based on maleimide and NHS conjugation technologies, the final metabolites would be drug-linker-Cys and drug-linker-Lys molecules, respectively. These two metabolites are able to pass through the lysosomal membrane to the cytoplasm to carry out their actions (Chari, 2008; Jaracz et al. 2005; Sanderson et al. 2005; Erickson et al. 2006, 2010; Sanderson et al. 2005). Similarly, for a non- cleavable linker, the ADCs prepared by the Pel conjugation method will generate a drug-linker-Pcl metabolite in the lysosomes (Figure 18A). As shown in this invention all trastuzumab Pcl-MMAF ADCs effectively inhibited proliferation of multiple antigen-expressing cells regardless the sites of the conjugation, suggesting that: 1) The degradation of Pcl-MMAF ADCs in lysosomes is a collectively efficient process leading toxic metabolites comparable in potency to metabolites generated from ADCs prepared by cysteine and lysine conjugation technologies; 2) the putative metabolite, MMAF-linker-Pcl, is able to effectively pass to the cytoplasm to exert its role as a tubulin inhibitor; 3) the mechanism of action for Pcl-MMAF ADCs to kill cells works for various cells types. Similarly, other drug payloads can be conjugated to Pel antibodies through the Pel conjugation process resulted in ADCs that likely will produce a cellular metabolite as illustrated in Figure 18B.

Table 23. IC₅₀ of trastuzumab Pcl-MMAF ADCs in MDA-MB231-16 Her2⁺ cell proliferation assay.

trastuzumab ADC ICso (μΜ)

HC-S124Pcl-MMAF 2.04E-04

HC-S132Pcl-MMAF 3.85E-04

HC-S134Pcl-MMAF 2.48E-04

HC-S136Pcl-MMAF 2.82E-04

HC-T139Pcl-MMAF 2.28E-04

HC-E152Pcl-MMAF 1 .94E-04

HC-P153Pcl-MMAF 3.80E-04

HC-T155Pcl-MMAF 3.57E-04

HC-S157Pcl-MMAF 2.25E-04

HC-T164Pcl-MMAF 2.85E-04

HC-S165Pcl-MMAF 3.06E-04

HC-P171 Pcl-MMAF 1 .99E-04

HC-L174Pcl-MMAF 1 .88E-04

HC-S176Pcl-MMAF 1 .89E-04

HC-S177Pcl-MMAF 1 .80E-04

HC-T187Pcl-MMAF 1 .90E-04

HC-P189Pcl-MMAF 2.98E-04

HC-S191 Pcl-MMAF 2.51 E-04

HC-T195Pcl-MMAF 2.30E-04

HC-T197Pcl-MMAF 2.68E-04

HC-S207Pcl-MMAF 3.44E-04

HC-D212Pcl-MMAF 2.34E-04

HC-K246Pcl-MMAF 1 .19E-04

HC-E258Pcl-MMAF 2.06E-04

HC-E269Pcl-MMAF 1 .97E-04

HC-K274Pcl-MMAF 2.04E-04

HC-V282Pcl-MMAF 1 .99E-04

HC-E283Pcl-MMAF 1 .69E-04

HC-N286Pcl-MMAF 1 .29E-04

HC-K288Pcl-MMAF 1 .20E-04

HC-K290Pcl-MMAF 1 .58E-04

HC-R292Pcl-MMAF 1 .78E-04

HC-E293Pcl-MMAF 1 .25E-04

HC-E294Pcl-MMAF 2.51 E-04

HC-K320Pcl-MMAF 1 .66E-04

HC-K322Pcl-MMAF 1 .96E-04

HC-K326Pcl-MMAF 1 .89E-04 trastuzumab ADC ICso (μΜ)

HC-A330Pcl-MMAF 2.95E-04

HC-E333Pcl-MMAF 1 .65E-04

HC-K334Pcl-MMAF 1 .27E-04

HC-T335Pcl-MMAF 1 .30E-04

HC-S337Pcl-MMAF 3.91 E-04

HC-R344Pcl-MMAF 1 .94E-04

HC-Q362Pcl-MMAF 2.94E-04

HC-S375Pcl-MMAF 2.12E-04

HC-E382Pcl-MMAF 5.76E-04

HC-N389Pcl-MMAF 1 .11 E-04

HC-N390Pcl-MMAF 1 .86E-04

HC-K392Pcl-MMAF 1 .51 E-04

HC-T393Pcl-MMAF 2.43E-04

HC-L398Pcl-MMAF 8.88E-04

HC-S400Pcl-MMAF 1 .63E-04

HC-D413Pcl-MMAF 8.73E-04

HC-S415Pcl-MMAF 6.52E-04

HC-V422Pcl-MMAF 7.31 E-04

LC-K107Pcl-MMAF 2.19E-04

LC-R108Pcl-MMAF 2.18E-04

LC-T109Pcl-MMAF 2.45E-04

LC-A1 12Pcl-MMAF 2.13E-04

LC-S1 14Pcl-MMAF 1 .85E-04

LC-D122Pcl-MMAF 2.77E-04

LC-E123Pcl-MMAF 1 .10E-04

LC-K126Pcl-MMAF 1 .43E-04

LC-S127Pcl-MMAF 1 .99E-04

LC-T129Pcl-MMAF 2.13E-04

LC-R142Pcl-MMAF 2.73E-04

LC-E143Pcl-MMAF 1 .81 E-04

LC-K145Pcl-MMAF 1 .74E-04

LC-N152Pcl-MMAF 2.55E-04

LC-L154Pcl-MMAF 1 .66E-04

LC-S156Pcl-MMAF 1 .84E-04

LC-G157Pcl-MMAF 2.88E-04

LC-S159Pcl-MMAF 2.97E-04

LC-E161 Pcl-MMAF 2.03E-04 trastuzumab ADC IC50 (μΜ)

LC-E165Pcl-MMAF 1 .34E-04

LC-S168Pcl-MMAF 2.40E-04

LC-K169Pcl-MMAF 3.47E-04

LC-D170Pcl-MMAF 3.33E-04

LC-S182Pcl-MMAF 2.15E-04

LC-K183Pcl-MMAF 8.06E-05

LC-K188Pcl-MMAF 2.12E-04

LC-V191 Pcl-MMAF 2.90E-04

LC-T197Pcl-MMAF 4.18E-04

LC-Q199Pcl-MMAF 2.48E-04

LC-S203Pcl-MMAF 2.13E-04

LC-T206Pcl-M MAF 3.95E-04

Table 24. IC₅₀ of antibody 14090 Pcl-MMAF ADCs in CMK11-5 cell proliferation assay.

Example 9. Pharmacokinetic studies of trastuzumab Pcl-MMAF ADCs.

[00273] It has been demonstrated that a long serum half-life is critical for high in vivo efficacy of ADCs (Hamblett et al., (2004) Clin Cancer Res. 10:7063- 7070; Alley et al, (2008) Bioconjug Chem. 19(3):759-765). Attaching an usually hydrophobic drug payload to an antibody could significantly affect the properties of an antibody which may potentially lead to a fast clearance of the ADCs in vivo (Hamblett et al., 2004). To evaluate the effects of different conjugation site on MMAF ADCs' clearance in vivo, pharmacokinetic studies in non-tumor bearing mice were carried out with 84 trastuzumab Pcl-MMAF ADCs. To detect MMAF containing ADCs in mice plasma, an anti-MMAF antibody was generated. ELISA assays for the detection of ADCs were developed using the extracellular domain of human HER2 to capture trastuzumab IgG molecules from the plasma and an anti- human IgG (anti-hlgG) antibody and the anti-MMAF antibody for signal generation in two separate assays. The two ELISA assays measure the serum concentration of the trastuzumab antibody and the "intact" ADC respectively as discussed in more detail below.

[00274] Three mice per group were administered with a single dose of a trastuzumab Pcl-MMAF ADC at 1 mg/kg. Ten plasma samples were collected over the course of two weeks and assayed by ELISA using the extracellular domain of human HER2 to capture all trastuzumab IgG molecules including trastuzumab Pcl-MMAF ADCs and trastuzumab lacking MMAF. An anti-MMAF and an anti- hlgG antibody were then used for detection in two separate assays. The anti- MMAF antibody ELISA measures the concentration of trastuzumab MMAF conjugates only and the anti-hlgG ELISA quantitates both trastuzumab Pcl-MMAF conjugates and trastuzumab antibodies that lack MMAF. Standard curves were generated for each ADC separately using the same material as injected into the mice. The assays with anti-MMAF and anti-hlgG should therefore yield identical concentration readouts if no changes to the drug loading of the trastuzumab Pcl- MMAF ADC occur after injection into mice. For trastuzumab Pcl-MMAF ADCs that lost some of the MMAF payload, the ELISA assay with the anti-MMAF antibody will measure a lower concentration than the anti-hlgG ELISA. A comparison of the two concentration readouts therefore allows to measure drug- release from trastuzumab Pcl-MMAF ADCs during in vivo incubation in the mouse.

[00275] As measured by anti-hlgG ELISA, all 84 ADCs displayed a pharmacokinetic profile similar to unconjugated wild-type trastuzumab antibody, indicating that ABA-MMAF payload conjugation to these sites did not affect the antibody's clearance significantly. As examples, the pharmacokinetic profile of six trastuzumab Pcl-MMAF ADCs (B to G) are compared to that of trastuzumab (WT) (Figure 19 A). To determine the chemical stability of linkage between the MMAF payload and the antibody at the various Pel sites, the concentrations of trastuzumab Pcl-MMAF ADC and total trastuzumab hlgG from the same plasma samples were compared to each other. All trastuzumab Pcl-MMAF ADCs, within the error of the measurements, displayed a good overlap between the two concentrations over the course of two weeks, suggesting that Pcl-MMAF conjugates at these Pel sites were stable during circulation in mice over this period (six examples are shown in Figure 19B to G).

[00276] In pharmacokinetic studies, the area-under-the-plasma- concentration-versus-time-curve (AUC) is an important parameter in estimating total clearance and bioavailability of an administered drug. In our pharmacokinetic studies, for each trastuzumab Pcl-MMAF ADC two AUC values, AUC-MMAF and AUC-hIgG, were calculated separately from measurements with the anti-MMAF and the anti-hlgG ELISA. The ratios of AUC-MMAF to AUC-hIgG for all trastuzumab Pcl-MMAF ADCs varied from 0.7 to 1.6 (Table 25). Figure 19 includes PK curves for six ADCs over the full range of observed AUC-MMAF/AUC-hlgG ratios and illustrates the variability and uncertainty of the measurements. All but one trastuzumab Pcl-MMAF ADCs show a ratio of AUC-MMAF/AUC-hlgG of >0.8. This suggests, that within the accuracy of the measurement, little drug loss is observed for the Pel conjugation sites tested because of a stable linkage between the MMAF moiety and the Pel amino acid in trastuzumab Pcl-MMAF ADCs.

[00277] In contrast, ADCs with drug payloads conjugated to free thiol groups in Cys residues of antibodies via maleimide chemistry display varied degrees of instability due to reversibility of the maleimide-thiol reaction (Alley, S.C. et al. (2008) Bioconjug. Chem. 19, 759-765). When these ADCs were injected into mice or incubated with serum in vitro for an extended time, a significant drug loss from the ADCs has been observed (Shen et al, (2012) Nat Biotechnol. 22;30(2): 184-9). The higher stability observed for Pcl-MMAF ADCs compared to Cys-MMAF ADCs conjugated at the same site, suggests that the Pel conjugation chemistry described herein is a general improvement over current state-of-the-art site-specific ADC conjugation methodologies such as Thiomab™ drug conjugates (Junutula et al, (2008) Nat Biotechnol. 26(8):925-32). Table 25. AUC from PK studies with trastuzumab Pcl-MMAF ADCs in mice.

AUC-

AUC- AUC- MMAF

trastuzumab ADC MMAF hlgG

/ AUC- (hr*nM) (hr*nM)

hlgG

HC-P153Pcl-MMAF 24261 25448 1.0

HC-S337Pcl-MMAF 17952 18798 1.0

HC-R344Pcl-MMAF 17230 17952 1.0

HC-R292Pcl-MMAF 20830 21627 1.0

HC-S134Pcl-MMAF 14826 15378 1.0

LC-S203Pcl-MMAF 14718 15167 1.0

LC-L154Pcl-MMAF 18068 18267 1.0

HC-T187Pcl-MMAF 26496 26471 1.0

HC-S157Pcl-MMAF 27712 27667 1.0

LC-Q199Pcl-MMAF 26412 26316 1.0

LC-A112Pcl-MMAF 14428 14366 1.0

HC-S191Pcl-MMAF 24385 24258 1.0

HC-K274Pcl-MMAF 16397 16167 1.0

LC-T109Pcl-MMAF 14352 14119 1.0

LC-K169Pcl-MMAF 20417 19988 1.0

HC-E258Pcl-MMAF 24516 23988 1.0

LC-D122Pcl-MMAF 19114 18658 1.0

HC-K322Pcl-MMAF 20631 19980 1.0

LC-T129Pcl-MMAF 16056 15498 1.0

LC-R142Pcl-MMAF 16979 16378 1.0

HC-K326Pcl-MMAF 21340 20471 1.0

HC-V282Pcl-MMAF 21121 20028 1.1

HC-S400Pcl-MMAF 15008 14213 1.1

LC-K145Pcl-MMAF 17433 16296 1.1

HC-P189Pcl-MMAF 21569 20138 1.1

LC-S168Pcl-MMAF 25737 24024 1.1

LC-T197Pcl-MMAF 25737 24024 1.1

HC-S177Pcl-MMAF 19896 18500 1.1

HC-S132Pcl-MMAF 13000 12080 1.1

LC-S127Pcl-MMAF 23661 21822 1.1

HC-T155Pcl-MMAF 18412 16947 1.1 AUC-

AUC- AUC- MMAF

trastuzumab ADC MMAF hlgG

/ AUC- (hr*nM) (hr*nM)

hlgG

LC-T206Pcl-MMAF 14628 13305 1.1

HC-S375Pcl-MMAF 16617 14982 1.1

HC-N389Pcl-MMAF 18464 16581 1.1

HC-K334Pcl-MMAF 18464 16192 1.1

HC-S136Pcl-MMAF 24353 21135 1.2

HC-S207Pcl-MMAF 23517 20098 1.2

HC-S124Pcl-MMAF 22967 19521 1.2

LC-D170Pcl-MMAF 28906 24345 1.2

HC-A330Pcl-MMAF 21971 18391 1.2

HC-E152Pcl-MMAF 22904 19167 1.2

HC-E283Pcl-MMAF 21397 17850 1.2

HC-A320Pcl-MMAF 25686 21133 1.2

HC-E269Pcl-MMAF 21022 17075 1.2

HC-N286Pcl-MMAF 16284 13183 1.2

LC- 132Pcl-MMAF 16407 13235 1.2

HC-T335Pcl-MMAF 17668 14228 1.2

HC-T393Pcl-MMAF 23457 18196 1.3

HC-E293Pcl-MMAF 23489 18178 1.3

HC-E294Pcl-MMAF 23950 17410 1.4

LC-S156Pcl-MMAF 35953 26061 1.4

LC-E165Pcl-MMAF 17797 12874 1.4

LC-K126Pcl-MMAF 21136 14419 1.5

HC-K392Pcl-MMAF 25419 15933 1.6

Example 10. In vivo efficacy studies of trastuzumab Pcl-MMAF ADCs.

[00278] In vivo xenograft tumor models simulate biological activity observed in humans by grafting relevant and well characterized human primary tumors or tumor cell lines into immune-deficient nude mice. Studies on treatment of tumor xenograft mice with anti-cancer reagents have provided valuable information regarding in vivo efficacy of the tested reagents (Sausville and Burger, 2006). Since MDA-MB231 clone 16 cells were sensitive to trastuzumab Pcl- MMAF ADCs in antigen dependent manner (Figure 16), the cell line was chosen as the in vivo model to evaluate the trastuzumab Pcl-MMAF ADCs. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication; National Academy Press, 8^th edition, 2001). MDA— MB231 clone 16 cells were implanted in nu/nu mice subcutaneous ly (Morton and Houghton, 2007). After the tumor size reached -200 mm³, trastuzumab Pcl-MMAF ADCs were administered into the mice by IV injection in a single dose at 5 mg kg. The tumor growth was measured weekly after ADC injection. Each treatment group included 9 mice. Treatment of mice with trastuzumab Pcl-MMAF ADCs not only inhibited tumor growth, but also caused tumor regression in 9 out of 10 mice of the ADC treated groups (Figure 20A and B). No weight loss was observed associated with the ADC treatment. The results confirmed that with a single dose treatment at 5 mg kg, trastuzumab Pcl-MMAF ADCs effectively caused regression of MDA-MB231 clone 16 tumors.

Example 11. Hydrophobic interactions of site-specifically conjugated ADCs vary with attachment sites.

[00279] Trastuzumab Pel antibodies were conjugated with ABA-

MMAF as described in Example 6. As mentioned in Example 6, different DAR species of the ADC preparations were purified by hydrophobic interaction chromatography (HIC).

[00280] Specifically, reaction mixtures were HIC purified using a

TSKgel Phenyl-5PW column (Tosoh Bioscience, TSKgel Phenyl-5PW, 13 μιη, 21x150 mm, stainless steel, Cat# 07656) as follows: 10 ml of each sample were diluted with 10 ml 2 M ammonium sulfate buffer and then filtered through a 0.2 μιη filter. 1.5 M Ammonium sulfate in 20 mM NaPi (pH7.4) and 20% isopropanol in 20 mM NaPi (pH7.4) were used as running buffer A and B, respectively. Samples were then injected onto the HIC column at a flow rate of 5 ml/min and subjected to a linear gradient from 20% to 80% running buffer B over 90 minutes. Sample elution was monitored by UV absorbance at 280 nm and 5 ml fractions were collected, concentrated and characterized further.

[00281] Surprisingly, it was observed that retention times of the DAR

2 species varied greatly among ADCs although the only difference is the site of ABA-MMAF attachment (Table 26). HIC separates molecules on the basis of the hydrophobicity. All DAR 2 ADCs have a HIC retention time larger than that of unconjugated antibody (WT = 45 min, Table 26) which is to be expected when a hydrophobic drug molecule such as ABA-MMAF is attached to an antibody.

However, attaching the payload at different sites increases the hydrophobicity of the ADC to various extents.

[00282] The surprisingly large differences in retention times can be rationalized from the inspection of location of the attachment sites on the structure of an antibody (Figure 21): The retention times are higher if the drug payload is attached at an exposed site on the outside of an antibody, for example at HC- K288Pcl, HC-N286Pcl, HC-V422Pcl, HC-L398Pcl and HC-S415Pcl where retention time between 87 and 94 min were measured for the respective ADCs (Table 24). Conversely, if the payload is attached at an interior site such as the cavity formed between the variable and CHI domains (for examples, HC-P153Pcl, HC-E152Pcl, HC-L174Pcl, HC-P171Pcl, LC-R142Pcl, LC-E161Pcl, LC-E165Pcl, LC-S 159Pcl) or the large opening between the CH2 and CH3 domains of the antibody (for examples, HC-K246C, HC-S375Pcl, HC-T393Pcl, HC-K334Pcl), the HIC retention time increased to only 47 to 57 mins because the payload is partially sequestered from interacting with solvent and the HIC column. For other sites, for example, the relatively exposed sites, LC-K107Pcl and HC-K360Pcl, intermediate retention times of 70 and 83 min were measured.

[00283] Reducing hydrophobicity of a protein drug is generally considered beneficial because it may reduce aggregation and clearance from circulation. We propose that the HIC data presented herein enables selection of preferred payload attachment sites. Carefully selecting attachment sites that result in minimal changes in hydrophobicity may be particularly beneficial when 4, 6 or 8 drugs are attached per antibody, or when particularly hydrophobic payloads are used.

Table 26: Retention times of trastuzumab Pel MMAF ADCs as measured by Hydrophobic Interaction Chromotography.

WT refers to the unconjugated antibody. Conditions are described in Example 11. Trastuzumab ADC DAR2

Trastuzumab ADC DAR2 HIC

HIC retention retention (min)

(min) HC-N286PCI-MMAF 91

HC-S1 17Pcl-MMAF 67 HC-K288PCI-MMAF 87

HC-S1 19Pcl-MMAF 69 HC-K290PCI-MMAF 62

HC-K121 Pcl-MMAF 63 HC-R292Pcl-MMAF 69

HC-S124Pcl-MMAF 59 HC-E293PCI-MMAF 78

HC-S132Pcl-MMAF 61 HC-E294Pcl-MMAF 61

HC-S134Pcl-MMAF 65 HC-K320PCI-MMAF 63

HC-S136Pcl-MMAF 64 HC-K322Pcl-MMAF 61

HC-T139Pcl-MMAF 67 HC-K326PCI-MMAF 72

HC-E152Pcl-MMAF 50 HC-A330Pcl-MMAF 62

HC-P153Pcl-MMAF 47 HC-E333Pcl-MMAF 63

HC-T155Pcl-MMAF 62 HC-K334Pcl-MMAF 56

HC-S157Pcl-MMAF 61 HC-T335PCI-MMAF 61

HC-T164Pcl-MMAF 64 HC-S337Pcl-MMAF 60

HC-S165Pcl-MMAF 67 HC-R344Pcl-MMAF 67

HC-P171 Pcl-MMAF 51 HC-K360Pcl-MMAF 83

HC-L174Pcl-MMAF 50 HC-Q362Pcl-MMAF 73

HC-S176Pcl-MMAF 65 HC-S375PCI-MMAF 52

HC-S177Pcl-MMAF 60 HC-E382Pcl-MMAF 81

HC-T187Pcl-MMAF 63 HC-N389Pcl-MMAF 67

HC-P189Pcl-MMAF 68 HC-N390Pcl-MMAF 70

HC-S191 Pcl-MMAF 68 HC-K392Pcl-MMAF 60

HC-T195Pcl-MMAF 70 HC-T393Pcl-MMAF 53

HC-T197Pcl-MMAF 71 HC-L398Pcl-MMAF 94

HC-S207Pcl-MMAF 63 HC-S400PCI-MMAF 69

HC-K246PCI-MMAF 51 HC-D413Pcl-MMAF 81

HC-E258PCI-MMAF 69 HC-S415Pcl-MMAF 94

HC-E269PCI-MMAF 70 HC-V422Pcl-MMAF 91

HC-K274Pcl-MMAF 72 LC-K107Pcl-MMAF 70

HC-V282Pcl-MMAF 76 LC-R108Pcl-MMAF 66

HC-E283PCI-MMAF 80 LC-T109Pcl-MMAF 70 Trastuzumab ADC DAR2

HIC

retention

(min)

LC-A1 12Pcl-MMAF 71

LC-S1 14Pcl-MMAF 64

LC-D122Pcl-MMAF 54

LC-E 123Pcl-MMAF 63

LC-K126Pcl-MMAF 61

LC-S 127Pcl-MMAF 60

LC-T129Pcl-MMAF 60

LC-R142Pcl-MMAF 51

LC-E143PCI-MMAF 61

LC-K145Pcl-MMAF 66

LC-N 152Pcl-MMAF 66

LC-L154Pcl-MMAF 66

LC-S 156Pcl-MMAF 69

LC-G157Pcl-MMAF 63

LC-S 159Pcl-MMAF 57

LC-E161 Pcl-MMAF 55

LC-E165PCI-MMAF 55

LC-S 168Pcl-MMAF 58

LC-K169Pcl-MMAF 68

LC-D170Pcl-MMAF 75

LC-S 182Pcl-MMAF 66

LC-K188Pcl-MMAF 71

LC-K190Pcl-MMAF 60

LC-T197Pcl-MMAF 71

LC-S203Pcl-MMAF 69

LC-T206Pcl-MMAF 68

WT 45

Claims

2. The immunoconjugate of claim 1 wherein the TAG encoded amino acid is Pel.

165 of a light chain of said antibody or antibody fragment, and wherein said positions are numbered according to the EU system, and wherein said light chain is a kappa light chain.

4. The immunoconjugate of claim 3 wherein the TAG encoded amino acid is Pel.

6. The immunoconjugate of claim 5, wherein the TAG encoded amino acid is Pel.

7. The immunoconjugate of claim 1, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of a light chain of said antibody or antibody fragment and wherein said positions are numbered according to the EU system, and wherein said light chain is a kappa light chain.

8. The immunoconjugate of claim 7, wherein the TAG encoded amino acid is Pel.

9. An immunoconjugate comprising a modified antibody or antibody fragment thereof, wherein said modified antibody or antibody fragment comprises a substitution of one or more amino acids with a TAG encoded amino acid on its constant region at a position chosen from positions 143, 145, 147, 156, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is human lambda light chain.

10. The immunoconjugate of claims 1-9 further comprising a drug moiety.

11. The immunoconjugate of claim 10, wherein said modified antibody or antibody fragment further comprises a substitution of one or more amino acids with TAG encoded amino acids on its constant region at a site selected from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of a heavy chain of said antibody or antibody fragment, and wherein said positions on the heavy chain are numbered according to the EU system.

12. The immunoconjugate of claim 11, wherein said drug moiety is connected to said TAG encoded amino acid through a cleavable linker.

13. The immunoconjugate of claim 11, wherein said linker is non- cleavable.

14. The immunoconjugates of claims 11, 12, or 13 wherein the TAG encoded amino acid is Pel.

15. The immunoconjugate of claim 10, wherein said immunoconjugate comprises a group of the formula (IA) or (IB):

IA IB

where LU is a linker unit;

[X] is the point of attachment for a drug moiety or payload;

R²⁰ is H or methyl;

and R³⁰ H or methyl or phenyl.

16. The immunoconjugate of any of claims 1-14, wherein said immunoconjugate comprises a drug moiety that is a cytotoxic agent.

17. The immune conjugate of claim 16, wherein said drug moiety is selected from the group consisting of a V-ATPase inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, an inhibitor of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.

18. The immunoconjugate of any of claims 1-17, wherein said antibody is a monoclonal antibody.

19. The immunoconjugate of any of claims 1-17, wherein said antibody is a chimeric antibody.

20. The immunoconjugate of claims 1- 17, wherein said antibody is a humanized or fully human antibody.

21. The immunoconjugate of claims 1-17, wherein said antibody is a bispecific or multi-specific antibody.

22. The immunoconjugate of any of claims 1-21, wherein said antibody or antibody fragment specifically binds to a cell surface marker characteristic of a tumor.

23. A pharmaceutical composition comprising the immunoconjugate of any of claims 1-22.

24. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with TAG modified amino acid on its constant region chosen from positions 117, 119, 121, 124, 132, 134, 136, 139, 152, 153, 155, 157, 164, 165, 171, 174, 176, 177, 178, 189, 191, 195, 197, 207, 212, 246, 258, 269, 274, 282, 283, 286, 288, 290, 292, 293, 294, 320, 322, 326, 330, 333, 334, 335, 337, 344, 355, 360, 362, 375, 382, 389, 390, 392, 393, 398, 400, 413, 415, and 422 of a heavy chain, and wherein said positions are numbered according to the EU system.

25. The modified antibody or antibody fragment of claim 24, wherein said substitution is at a position selected from positions 1 17, 124, 136, 139, 152, 155, 171, 174, 258, 286, 288, 292, 334, 375, and 392 of the heavy chain.

26. The modified antibody or antibody fragment of claim 24 or 25 further comprising a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of a kappa light chain.

27. The modified antibody or antibody fragment of claim 24 or 25, further comprising a substitution of one or more amino acids with a TAG encoded amino acid on its constant region at a position chosen from positions 143, 145, 147, 156, 159, 163, and 168 of a light chain of said antibody or antibody fragment, wherein said positions are numbered according to the Kabat system, and wherein said light chain is a human lambda light chain.

28. The modified antibody or antibody fragment of claims 24-27, wherein said substitution is one to eight TAG encoded amino acids.

30. A modified antibody or antibody fragment thereof comprising a substitution of one or more amino acids with a TAG encoded amino acid on its constant region chosen from positions 107, 108, 109, 112, 1 14, 122, 123, 126, 127, 129, 142, 143, 145, 152, 154, 156, 157, 159, 161, 165, 168, 169, 170, 182, 183, 188, 190, 191, 197, 199, 203, and 206 of a light chain, and wherein said positions are numbered according to the EU system.

31. The modified antibody or antibody fragment of claim 30, wherein said substitution is at least TAG encoded amino acid, chosen from positions 107, 108, 109, 142, 145, 152, 154, 161, and 165 of a light chain, and wherein said light chain is a kappa light chain.

32. The modified antibody or antibody fragment of claims 30 or 31, wherein said substitution is one to eight TAG encoded amino acids.

33. The modified antibody or antibody fragments of claims 30-32 wherein the TAG encoded amino acid is Pel.

35. The modified antibody or antibody fragment of claim 34, wherein said substitution is one to eight TAG encoded amino acids.

36. The modified antibody or antibody fragment of claims 34 or 35, wherein the TAG encoded amino acid is Pel.

38. The modified antibody or antibody fragment of any of claims 25, 26, 27, 28, 30, 31, 32, 34, or 35 wherein the modified antibody or antibody fragment is further attached to a drug moiety, and wherein said drug moiety is attached to the modified antibody or antibody fragment through the TAG encoded amino acid and an optional linker to form an immunoconjugate.

39. The modified antibody or fragment of claim 38 wherein the TAG encoded amino acid is Pel.

40. The modified antibody or antibody fragment of claim 38, wherein said immunoconjugate comprises a group of the formula:

IA IB

where LU is a linker unit;

X¹ is a drug moiety or payload;

R²⁰ is H or methyl;

and R³⁰ H or methyl or phenyl.

41. The modified antibody or antibody fragment of any of claims 25, 26, 27, 28, 30, 31, or 32, wherein said substitution comprises at least one cysteine substitution, and at least TAG encoded amino acid substitution or a peptide tag for enzyme-mediated conjugation.

42. The modified antibody of claim 41 wherein the TAG encoded amino acid is Pel.

43. A nucleic acid encoding the modified antibody or antibody fragment of any of claims 25-42.

44. A host cell comprising the nucleic acid of claim 43.

45. A method of producing a modified antibody or antibody fragment comprising incubating the host cell of claim 43 under suitable conditions for expressing the antibody or antibody fragment, and isolating said antibody or antibody fragment.

47. The method of claims 45 or 46 wherein the TAG encoded amino acid is Pel.

48. A method to prepare an immunoconjugate, comprising providing an antibody or antibody fragment of any of claims 25, 26, 27, 28, 30, 31, 34, 35, or 37 comprising contacting the antibody or antibody fragment containing at least one TAG encoded amino acid residue with an ABA compound, or an ABP compound, or an AAP compound.

49. The method of claim 48 wherein the TAG encoded amino acid is Pel.

50. The method of claim 48 or 49, wherein the immunoconjugate comprises a group of the formula IA or IB:

or

IA IB

where LU is a linker unit;

X¹ is a drug moiety or payload;

R²⁰ is H or methyl;

and R³⁰ H or methyl or phenyl.