CA2290722A1 - Consensus peptide presenting entities - Google Patents

Abstract

The invention provides antigen-binding-fragments specific for tumor cells and effective in treatment and/or diagnosing tumors. Methods of use are also provided as are methods for screening for additional such antigen-binding-fragments and the products obtained thereby.

Description

__ , 1G/U8/88 WI;J) 17: YU b'A~ 4ltf ;ilfL U82;! H1DUU1' ~k mAYB~JI~JUU4 CaNSENS~I-1S PEPTIDE PRESENTING ENTITIES
h CROSS=Et,EFERENGE ~'O RELATED APPLICATIONS
Not applicable.
II. STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH
Not applicable.
III. TECHNICAL FIELD
The invention relates to antigen-binding fragments that are specific for individual or families of PPEs (PPEs}, including stress protein-peptide complexes associated with tumors (SPPCs), particularly human tumors, and compositions tbereo~ The compositions are suitable for diagnostic, palliative and therapeutic use. The invention further provides methods of making and screening for the antigen-binding fragmeztts. 'The invention further encompasses compositions containing peptide-presenting entities, which share antigenic determinants witl tumor-associated SPPC (including derivatives thereof) and methods of use thereof. -fhe tumor-specific SPPC are particularly useful ix~ generating antigen-binding fragments specific for target tumors and in eliciting turrtor-specific immunogenio responses.
V. $,4CI C+RdUND ART
A. Heat Shock Proteins Heat shock protoins ("HSP"s) form a family of highly conserved proteins that are widely distributed throughout the plant and animal kingdoms. On the basis o~ their molecular weights, HSPs are grouped into six diFterent families: small (hsp20-30 IcD); hsp40;
hsp40; hsp70; hsp90;
and hsp100. Although HSPs wire originally identified in tolls subjected to heat stress, they have been found to be associated with many other forms of stress such as infections, and are thus more commonly known as "stress proteins" ("SP"s).

-_ iziusiaa wr:~ i7:ze r~ax 4ia ssz usza Hmuu~r ~ marYSr;r; ~uub Members of the mammalian hsp90 family include cytosolie hsp90 (hsp83) and the endoplasmic reticulum counterparts hsp90 (hsp83), hsp87, Grp94 (lrl~p99) and gp96. See for instance, Gething et al. (1992) Nature 355:33-45. Members of the hsp70 family include Cyto501iC hsp70 (p73) and hsp 70 (p72), the endoplasmic reticulum counterpart BiP (Grp78), and the mitochondria) counterpart hsp 70 (Grp75). Members of the mammalian hsp60 family have only been identified in the mitochondria.
A vat~icty of recent reports eonroborate an association between the presence of SPs on the cell surface and cat~eer. The relationship between SPs and cancer was discovered in the course of efforts to identify cancer-associated antigens by their ability to elicit protective immunity to cancer challenges. The approach typically involved fractionating tumor homogenates into various protein components by conventional chromatographic methods and using these fractions to irnrnunize animals just prior to challenge with live cancer cells. The fractions that elicited protection against the cancer were then repeatedly refractionated until apparently homogeneous preparations were obtained. To the surprise of investigators, all the well-characterized molecules identiF~sd by such methodology turned out to be SPs of the hsp90 or hsp70 family, even from cancers of diverse historic origins. Sce, Srivastava et al., Heat Shock Proteins Comc of Age (supra). Naturally, this finding fostered a focused interest in using such fractions to immunize cancer patients against tumor tissue.
SPs are ubiquitous within cells, ono of the roles of SPs is to chaperone peptides from one cellular compartment to another and to present the peptides to the major histocompatability complex (MHC) molecules for cell surface presentation to the immune system. Tn the case of diseased cells, SPs also chaperone viral or tumor-associated peptides to the cell surface. Li and Srivastava (1994) Behringlnst Mltt 94:37-47; and Suzue et al. (1997) Pros.
Natl. Aead. Srl. USA
94:13146-13151. The chaperone function is accomplished through the formation of complexes between the SPs and proteins and between SPs and viral or tumor-associated peptides in an ATl?-dcpendcnt reaction. 'fhtse complexes are termed "SPPC"s herein. SPs bind a wide spectrum of peptides in an ATP dependent mariner. The bound peptides appear to be a random mix of peptides. The mixtures and exact natures of the peptides have not been deternnined. The association of SPs with various peptides has been observed in normal tissues as well and is not a tumor-specific phenomenon. See Srivastava (1994) Experientla 50:1054.

Iz~u~f~Ha, wlD l7:La mall 41U :fdG u~lz;f HWUUT tk mAYB~I~JUUt3 For instance, expression of hsp26, bsp60, hsp70 and hsp90 on the surface of human chronic myeloid leukemia (CML) cells from patients has been observed. Chant of a1. (1995) Br.
J. Haematol. 90:163-8. Cell surface expression of lisp70 has been detected on normal, premalignant a:nd malignant human oral mucosa. I~aur et al. (1998) Oral Dncol.
34:93-8. A
correlation of hsp70 expression with clinicopathological Features showed a positive association with the severity of dysplasia in oral mucosal epithelium.
Its ct al. (( 1998) .T. Oral. Pathol. Med. 27:18-22.), reported that they examined 24 specimens of squamous cell careinorna of th.e tongue and found that, although SP
immunohistochemistry revealed changes in expression during tumorigenesis of squamous epithelium of the tongue, there was no observed correlation with other clinical features studied (survival period, stage, lymph node metastasis, histalogical grade or p53 immunostauung)-It is C~trc'ently believed that the antigeniclty of $PlsCs results not from the 5P par se, but from the complex of peptide associated with the SP. This conclusion is based on a number of characteristics of the SPPCs. There are no dit>ferences in the structure of SPs derived from normal and tumor cells. Certain SPPCs lose their immunogenicity upon treatment with ATP.
Udono et al. (1993) J. Exp. Med. 178:1391-1396. Such loss of immunogeuicity is due to dissociation of the SPPC into its SP and peptide components.
Vaccination with a mixture of SPPCs can induce a potent immune response. In some instances, the immune response is humoral, as evidenced by the anti-peptide IgG response in BCG-primed mice injected with covalent hspb5 complex or hsp65-oligosaccharide complexes.
Del Guidice Experentia (1994) 50:1061. In contrast, vaccination with mixtures of SPPCs from tumor cehs appears to generatt a strong cell mediated response with little more than a weak humoral response tvcn after prolonged immunization. Srivastava of al. Int. J
Cancer ( 1984) 33:417-422. The explanation for the absence of a strong humors! response is that vaccination of tumor cell-derived SPPCs favors a Thl immune response and would therefore, by deJ:inition, down-regulate any potential antibody response (Th2) directed against the tumor. Srivastava (199t+) Bxperentia 50:1054-1064. This potential for dampening burnoral immu:niry through SPPC vaccination, has led to its proposal as a method for suppressing allergic reactivity to different allergens. PCT/CA97100$97. Further, because the immune response, with, respect to SPPC vaccination, is skewed in the direction of cell mediated immunity it has been suggested that humoral immunity plays but a minor role against neoplasia. ( 1998) Btol.
Chem. 379:295.

--_-12/U8/N8 ,Wr:U 17:;lU b'AIL 4ltf ~ftfG USC:f H1DUU'1' ik 11AYB~1tøJUU7 In a therapeutic context, it has been proposed to use SP-antigen complexes as vaccines.
In particular, U.S. Patent No. 5,750,119 to Srivastava discloses a multi-step, cancer patient-specific method for inhibiting the proliferation of a tumor in a mammal, by (a) removing tumor cells from the mammal; (b) isolating all SPPCs froth the tumor cells; and (c) administering the isolated SPPCa back to the mammal in order to stimulate In the mammal a tumor-specific immune response. Hsp70-peptide, hsp90-peptide and gp9b-peptide complexes are itemized as complexes having particular vaccine utility. Nevertheless, in the practice ofthe method disclosed by Srivastava, it is not considered necessary or even practical to isolate a specific peptide involved or even the particular SPPC involved in eliciting the immune response.
Moreover, Srivastava postulated that, "the prospect of identification of the immunogcnic antigens of individual tumors from cancer patients (or even of 'only' several different typos of imrnunogenic antigens in case the antigens are shared), is daunting to the extent of being impractical." Qn this basis, Srivastava proposes immunizing a mammal harboring a tumor with a mixture of SPPCs derived from the animal's own tumor, without isolating complexes specific to the tumor and without attempting to characterize complexes which are found on more than one tumor in one mammal.
U.S. Patent No. 5,837,251 discloses a method of eliciting an immune response in a mammal comprising administering a specified lvw dose of a SP peptide and an antigenic peptide.
The antigenic peptide can bE provided exogenously, that is, nonc4valently reacted with the Sl' to form a complex, or it can be endogenous i.e_ naturally occurring in a native complex. Again, the native material is a mixture of SPhCs and is not free of complex associated with normal cells.
WO 99/22761 relates to conjugate peptides engineered to non-eovalently bind to heat shock proteins. These peptides 4an be used to link antigenic peptides to heat shock proteins.
B. Anti~idiotypic.9ntibodies The network hypothesis of Lindemann ((1973) Ann. Immunol. 124:171-184) and Jerne ((1974) Ann. Immunol. 125:373-389) offers an elegant approach to transform epitope structures into idiotypic determinants expressed an the surface of antibodies. According to the network concept, immunization with a given tumor-associated antigen will generate production of antibodies against this tumor-associated antigen, termed Abl; this Abl is then used to generate a series of anti-idiotype antibodies against the Abl, termed Ab2. Some of these Ab2 molecules __ iziusiaa ri~y i7:au r~r~ 4ia ;saz usza Hmuu~r ~ mnY~l;~ ~uus can effectively mimic the three-dimensional structure of the tumor-associated antigen identified by the Abl. 'these particular anti-idiotypes called Ab2~ fit into the paratopes of Abl, and express the internal image of the tumor-associated antigen. The Ab2~ can induce specific immune responses similar to those induced by the original tumor-associated antigen and can, kherefore, be used as surrogate tumor-associated antigens. Immunization with Ab2v can lead to the generation of anti-anti-idiotype antibodies (Ab3) that recognize the corresponding original tumor-associated antigen identified by Abl. Because of this Abl-like reactivity, the Ab3 is also called Ab 1' to indicate that it might dif~~r in its other idiotopes from Ab 1.
A potentially promising approach to cancer treatment is immunotherapy employing anti-idiotype antibodies. In this form of therapy, an antibody mimicking an epitope of a tumor-assoeiated protein is administered in an effort to stimulate the patient's immune system against the tumor, via the tumor-associated protein. WO 91111465 describes methods of stimulating an immune response in a human against malignant cells or an infectious agent using primate anti-idiotype antibodies. However, not all anti-idiotype antibodies can be used in therapeutic regimens against tumors. lvloreover, since different cancers have widely varying molecular and clinical characteristics, it has been suggested that anti-idiotype therapy should be evaluated on a case by case basis, in terms of tumor origin and antigens express.
Anti-Id monoclonal antibodies structurally resembling tumor-associated antigens have been used as antigen substitutes in cancer patients. Herlyn et al. (1987) Proc. Natl. Aced. Sci.
USA 84:8055-8059; Mittlcman et al. (1992) Proc. Narl. Aced. Sci. USA 89:466-470; Chatterjec ct al. (1993) Ann. N. Y. Aced. Sei. 690:376-278. It has been proposed that the anti-Id provides a partial anal.o$ of the tumor-associated antigen in an imrnunogenic context.
In contrast t4 the uncharacterized mixtures of SPPCs and artificially engineered SP-antigens previously described, we have now found that en antigen-binding fragment of an antibody that binds specifically to a tunnor-associated SPPC is effective at eliciting a useful anti-tumor response. We have also found that this antibody, hereinafter referred to as "H11 ", recognizes tumor-associated SPPCs containing various different peptides. We have found that tumor-associated SPPCs contain peptides that conform to a consensus peptide motif ("consensus conforming peptides" or "CCPs"), to provide a family of tumor-associated SPPCs. Therefore, we have found that cancer-associated SPPCs share a consensus peptide motif, which is common to many tumors and that, peptides which conform to this consensus motif, cafe be used to s iziusiaa w~;U i7: m r~n~ am auz uaz~ Hmuu~r ~ mAYJiJrlr ~tJUUa generate antigen-binding fragments, which bind to cancer-associated $PPCs, as well as, for making corresponding anti-Ids and for preparing compositions useful for eliciting an anti-tumorigenic response.
VI. S ~' OF THE INVENTION
In one aspect of the invention, we provide a method for identifying specific peptide presenting entities (PPEs) which are immunologically cross-reactive with tumor-associated SPPCs of at least one target riimor by:
1) identifying within a libraKy of ligand-binding raolecul.es, a subset consisting of one or mare of such molecules, that binds to both:
a) SPPCs found on the surface of target tumor cells; and b) one or more PPEs within a population of PPEs, said population containing a suitable representation of PPlrs wherein the peptide portion corresponds to a consensus peptide motif described herein; and, Z) selecting PPEs bound to such fragments.
In some preferred embodiments of this aspect of the invention:
a) the consensus peptide motif is a hydrophobic motif comprising at least 3 hydrophobic amino acids within a span of 7 consecutive amino acids, preferably at least 2 such hydrophobic amino acids being non-contiguous, and more preferably 3;
b) the PPEs are SPPCs, preferably of the HSP70 and HSP90 families;
c) the population of PPEs comprises or is limited to a substantial representation of all potential consensus conforming peptides which correspond to each of the actual consensus conforming peptides fouand within proteins expressed in cells of each such target tumor;
d) the population of ligand-binding molecules is a large naive library of antigen-binding fragments which can optionally be pooled with at least one additional library of ~Cragments derived from a parental binding fragment which binds specifically to an SPPC
(preferably containing SPs of both the HSP70 and HSP90 families); ar e) the population of ligand-binding molecules is derived from a parental binding fragment which bands specifically to an Sl?PC.

iziusiaa wr;~ i7:m rvx 4m aaz usz~ xmuu~r ~ mAY~~;~; Lulu f) the population of ligand-binding molecules are single domain antibody fragments or loop structures as di,~cussed below.
g) the population of antigenybinding fragments competes with H11 far binding to the target tumor h) the consensus motif is designed to establish a strong likelyhood that peptides conforming to the motif would be found in associatiowvith MHC or HSP on the surface at least one (preferably a plurality and more preferably substantially all) cancers cell within a population of cancers cells of different individuals with cancer {preferably of the same cancer typt), when a suitable number of such cells of each sucli individuals are tested.

Another aspect the invenrion is directed to a method of identifying a population of peptides containing a representation of peptides associatod with SPPCs found specifically on tho surface of cells of a target tumor by:
a) using a set of degenerate nucleic acid probes encoding permutations of a consensus peptide motif described herein (i.e. encoding proteins in which a peptide conforming to the motif is present) to identify those mRNA transcripts within cells of the target tumor tliat encode said motif; and, b) manipulating the mRNA transcripts so identified to obtain the peptides.
In some preferred embodiments of this aspect of the invention:
Tumor cell-specific mltNA encoding specific consensus conforming sequences can be identified by any method known in the art. Preferably, an array of oligonucleotidc probes is generated that encodes at least a subset of such consensus sequences. These probes are thin used in a variety of ways to limit the number of poptides that are soreened. In particular, cDNA
complementary to the probes can be identified, cloned and expressed so that the peptides produced thereby can be subject to flat PPE sereetung assay described herein.
Alternatively, the probes are arrayed in a manner such as provided by Affymetrix~ GeneChip technology so that probes of a singlo sequence are at identifiable spots on the chip. The technology is described for instance is US Patent Nos. 5,527,b81; and 5,51 Q,270. Tht chips are hybridized with mRIviA or cDNA and the spots at which hybridization occurs are identified. The sequence for each spot is laiow~n and the peptide encoded thereby can be synthesized and subject to the screening assay described herein. With respect to tumor cells it is desirable to obtain a substantially complete iziusiea wl;~ i7:ai rw 4id :tuz uaza Hmuu~r ~ mAYlir;l; ~ul representation of consensus conforming sequences within the tumor cell using such probes in order to identify substantially all potential CCPs that could be brought to the surface of the tumor cell by an SP.
Methods of making the probes are known in the art. Any suitable DNA
synthesizer can be used. As described in Example t 3, approximately 4x106 different probes are initially obtained. The methods such as identifying cDNA complementary to tlae probes and DNA array screening of mRNA or cDNA narrow these peptides to a subset that represents proteins actually expressed.
In the case of cDNA screening, a cDNA library is Frst obtained, for instance from a cancer or tumor cell line: The single-stranded DNA is isolated. The probes are then annealed to the single-stranded DNA under stringent hybridization conditions. Ai~er an amount Of tulle su'~cient to anneal the probes to the cDNA, the single-stranded regions are digested to yield tile double-stranded region. The remaining double-stranded regions are then ligated (singly or in concatenation) and transfccted into a suitable expression vector. The cloned sequences can be sequenced. Expressed peptides can be isolated, optionally lengthened as described in Example 13, associated with SPs or other peptide presenting portions as described herein and subject to the screening assay to determine which peptides arc found associated with SPs in a canc;er-dependent manner.
In the case of oligonucleotide arrays, the probes are manufactured on the ehip(s) and subject to stringent hybridization to mRNA or cDNA. Preferably the mRNA or cl-3NA arc obtained from a cancer cell. lVtore preferably, they are obtained from a human cancer cell. The spots to which the mRNA or cDNA anneal are then determined, for instance by the Gene~Chip method. As the sequence o~ tack spot is kaown, the peptide encoded thereby can be synthesised, associated with SP and screened by the assays described herein. Optionally, the peptides are lengthened as described in Example 13 or in the course of preparing a set of consensus conforming peptides for screening in the form of PPEs i.e. by randomly adding one of each of the amino acid residues to each of consensus conforming peptides. Each amino acid that is added multiplies the starting population of consensus conforming peptides by a factor of 20, unless cc~tain exclusion or inclusion criteria arc applied as discussed below and in the art. For example, permutations of such added amino acids that do not exist within databases of human iziusiaa wr:~ i7:~z rv~ aia auz usz~ Hl~uu~r 1k mAYBt;Jl~lUlG
proteins can be identified by searching for the occurrence of actual consensus conforming peptides within the literariire and assessing the flanking amino acid residues.
In accordance with another aspect ofthe invention, we identify inclusion and exclusion criteria that can be applied to reduce the number of PPE candidates that are screened for immunological cross-reactivity with tumors associated SPPCs. It is to be understood that those criteria can be applied to one ar more of stages of screening identified herein:
a) to reduce the number of probes used to scxeen for actual representation of the mRNA
encoding the consensus peptide motif of interest;
b) to reduce the number of PPEs tested for immunological crnss-reactivity with SPPCs on the target tumor of interest;
c) to reduce the number of immunologically cross-reactive PPEs used in in vitro, pre-clinical and clinical testing.
It is to lx understood that if more than one such criterion is applied, the broader criterion is to be applied at the earlier stage of screening. In accordance with this aspect of invention the criteria to be applied include:
a) propensity to bind to an SP on the basis of thermodynamic and statistical considerations;
b) the susceptibility of a given CCP to proteolytic cleavage within a proteasome;
c) the propensity to bind to an MIiC of an individual or group of individuals having common HLA, types.
Another aspect the invention is directed to a composition comprising a plurality of peptides, which conform to a consensus peptide motif associated with tumor associated SPPCs.
The peptides can be produced by genetic engineering as described above or by peptide synthesis.
In another embodiment, tha composition oonsists essentially of consensus conforming peptides, which are a subset of the total theoretical set of consensus conforming peptide namely those which are found within known native proteins, preferably (in the case of human tumors) human proteins.
In another aspect the invention is directed to an antigen-binding fragment, which recognizes one or more consensus conforming peptides as presented by SPs or other PPEs which present the peptide in the same fashion as SP {i.e, so as to be irnmunologically cross-reacrive with known tumor-associated SPPCs).

iziusiea wry i7:az r~a~ 4iu saz usz~ xmuu~r ~ mnY~~;la Hula In another aspect, the invention is directed to a composition of matter comprising PPEs, wherein said composition is enriched with at least one PPE having a predetermined peptide portion which shares antigenic determinants with one or more tumor colt-surface associated SPPCs of a tumor target in a mammal, such that said peptide portion renders said PPE potentially immunogenic with respect to said tumor target in such mammal.
In one embodiment it is contemplated that the PPE has a predetermined peptide portion and an associated portion, wherein the peptide portion shares antigenic determinants with one or more tumor cell-surface associated SPPCs of a tunn.or target in a mammas, such that said peptide portion, as presented by said associated portion, renders said PPE
innrounogenic with respect to said tumor target in such mammal.
In another embodiment, the composition substantially comprises at least one PPfi and in another embodiment the composition comprises a plurality of different PPEs, said PP~.s characterized by different predetermined peptides whieli singly andlor collectively render said PPEs immunogenic with respect to said tumor target.
In another embodiment of the invention, the peptide portion of the PPE shares a substantial number of cancer-specific antigenic determinants with the peptide portion of the tumor cell surface associated SPPC.
The peptide portion of said PPE is preferably predetermined in the sense that it conforms to a consensus peptide motif which is common to a plurality of different tumor-associated SPPCs. The remaining or associated portion of the PPE assists in presenting the peptide portion of the PPE in a manner in which it shares antigenic determinants with the geptide portion of the SPPC (c.g. by presenting it in the same configuration as it would be presented by the SP) such that tho peptide portion of the PPE re~o~dera the PpE immunogenic witli respect to the desired tumor target. In one embodiment, the PPE is itself a SPPC. In a preferred emlsodiment, the peptide portion of the PPE is substantially identical to the peptide portion of the tumor cell surface associated SPPC.
ht another aspect of the invention, the invention is directed to methods of generating PPEs having the desired consensus conforming peptides (hereinafter "CCP"), and antigen-binding fragments specific thereto.
In yet another aspect of the invention, the invention is directed to particular CCPs.
to iziusiaa w~ i7:aa rva 4ia ;s~z usz;~ Hmuu~r ~ mnYatrl; ~ulQ
In yet another aspect of the invention, the itwcntion is directed to a pharmaceutical composition comprising PPEs wherein said composition is enriched for, or preferably predominantly comprises, PPEs, wherein the peptide portion renders said PPE
immunogen.ic with respect to a tumor cell surface associated SPPC.
In yet another aspect of the invention, the invention is directed to method of treating a cancer subject comprising administering to the subject an amount of a composition of matter comprising a PPE according to the invention effective to palliate the cancer.
In accordance with another aspect of the invention, we identify one or more subsets of consensus conforming peptides that have, a priori, a better percentage representation of actual 14 tumor-related consensus conforming peptides, relative to the full set of theoretical consensus conforming peptides, as weh as methods of identifying these subsets. In other words, the pcreentags amount of CCPs that render the FFZrs immunolo,~ically cross-reactive with actual tumor-related SFPCs is higher with a smaller well-chosen subset.
For example, the subset of consensus conforming peptides that actually exist within nature is one such s ubset and the utethod of identifying them is to run a search for the consensus motif on the available databases of known proteins. Suitable databases include Genbank NCBI
(e.g, using the rnotif scan function , Stanford University, Stanford Medical Informatics Program). Other databases including well known commercially available databases provided by DIALOG, S?N, etc. include the Chemical Abstracts Registry file. Human proteins are a preferred subset for human tumors proteins.
One can adopt a conservative approach by reducing the number of hits on human proteins, for example, by rationalizing the hits in terms of their potential existence within human tumor cells arid plausible association with the proteasomes of such cells, etc. For example, if a given CCP was found only in proteins expressed in certain types of human cells of limited interest and not related to tumors or tumor genesis, then that CCP could be ignored, since that CCP could not be a product of proteolysis within the tumor eell(s} of interest.
Other subsets:
Peptides which have a preference for binding to one or more types of tumor-surface expressed SPs are suitable for use herein. Preferably the CCPs show a preference for binding to SFs of IiSP70 family, including preferably inducible F~SP72 and the HSP90 family, including pz~eferably I-ISP$5. 1n another embodiment, a smaller subset comprising fhe peptides that are in W~utS~98 wr;D 17:J;f rW 41a ;iUG uSL:! HIDUUT ~C ~IAYBt;l~øJU15 common between those which bind to more than one typt of SP within a f&mily or bind to SPs within more than ono family. The peptides, which are preferred for binding to SPs can be dete»ed directly from the literature and in the manner described the literature. Reference is made to WO 99122761 and the scientific publications on the subject therein referenced as well as, other pertinent references including Flynn et al., Nature, 353:726 (1991);
Fourie of al., J. Biol.
Chcm., 269:30470 (1994); and $lond-Elguindi et al., (1993) Cell 75:717-728, Gething, M.J, et al. Binding sites for Hsp70 Molecular Chaperones in Natural Proteins Cold Spring Harbor Symposia in Quantitative Hialogy Vol. LX p 417; Gething L.M, Current Hiology ( 1994) 4:173;
Gragerov A. et al. J. Mot. Biol. (1994) 241:133; and de Crovy-Chapel A. Gene (1999) 230:163 as well as references citing these references as disclosed in various Science Citation Indices and patent citation indices references cited within those references. All references referred to herein are hereby incorporated herein by reference.
In yet another embodixnent, naturally occurring CCPs or subsets thereof can be ftllrther limited to those which overlap with peptides that have a preference for binding to MHC types within a given individual or group of individuals related by HLA type.
Peptides within the preferred Subset of CCPs can be randomly elongated optionally by one or two residues or more residues to generate additional variability within the population of potential CCPs to be assessed as Iigands for mapping to tumor cells. For example, 7 mars that arc preferred for binding to HSP72 can be generated as 8 mere or 9 mere with the additional 2Q amino acids) (optionally at the carboxy terminus) representing a 20-fold to 400-fold increase in the number of permutations sought to be generated. The choice, for example, of $ mars can be limited to those found in nature or in human protein, or according to any other strategy as discussed herein.
According to another embodiment of the invenkion, candidate CCPs are determined by generating a set of nucleic acid probes corresponding to the starting subset of CCPs of interest, attd sereettirtg of tumor cell mRNA is carried out to determine which of the probes hybridize to the mRNA, using the mRNA from one cell type, preferably at different stages of differentiation, or the combined mRNA of a plurality different tumor types. Probes which hybridize to the selected population of mRNAS can then be analyzed to determine which corresponding CCPs actually exist within the tumor in the Form of proteins and therefore which should be used as the starting population of CCPs. Techniques for generating nucleic acid probes, generating 1~/U8/!~H WhD 17:J4 b'AA 41U St~Y US~J lilJ)Ull'1' ~C mAYB~IløJUlt~
microarrays of such probes, and analyzing the products of hybridization are well known in the art. Some of these are referred to below.
Ia one aspect of the invention, probes, for example, consisting of 27 nucleotides (representing 9 mere), which hybridize to larger segmtnts of mI.~NA from a cancer cell (or S cDNA of tile m~NA,) to .form duplexes can be cleaved or degraded so as to remove the single stranded parts of mRNA (or cDNA) and the remaining duplex material can be used (optionally with Some ~mplificdtion) to generate double stranded DNA for insezfiiort into expressio~.~ vectors to generate a population of PPEs or peptides, which include the set of speei~c peptides within the cell that correspond with a consensus peptide motif.
In a preferred embodiment, the mRNAs are i'irst fractionated on the basis of charge and/or molecular weight. In the case of different molecular weight fractions, it is possible, based on the available databases of proteins and their molecular weight to quax-~tify a priori, khe number and size of such fractions which are most suitable to accommodate the anticipated frequency of binding of probes and thus enhance specific binding.
In a preferred embodiment, for example, where the consensus peptide motif corresponds to the preferred binding motif of an SP found on the surface of tux~r~ors cells (for example HSP72 and HSP$5 bath recognized by H11), the preferred motif can be used so as to have the greatest representation within peptides degraded in proteasomes, for example HyX HyX
HyX Hy, (where Hy is a hydrophobic amino acid and X is any amino acid) to which can be added at least X or XX at one or both ends. This motif is found in many human, proteins.
Without being bound by any one theory, this motif is believed to be widely represented on the surface of a variety of different tumor cells for one ar mare of the following reasons:
a) its prevalence within human proteins (based on the formula provided below it is estimated, a priori, that this motif would occur approximately once within every protein of approximately 500 amino acids in length;
b) its propensity for binding to SP;
c) its propensity in some forms (e.g. perhaps a single aromatic series of aromatic hydrophobic amino acids) to resist proteolytic cleavage;
d) the possiblity that several degraded proteins can be represented within a proteasome so that there is a greater likelihood for the hydrophobic motif to be represented within the population or peptides that surface on SP.

icfusiea wry i7:;~4 r~a~ am Paz uaz;~ tcmuu~r a mnYa~;r; ~u17 The HyX HyX HyX H motif broadly represents many different hydrophobic motifs in that X can lx Hy. Because it can be unnecessary for each Hy to be present for binding to SP
(depending on the strength of other intennolecular interactions) this motif accounts for many hydrophobic motifs within a cell. It is noteworthy that if all X are Hy, then Hy is accounttd for and can be any other amino acid.
In another embodiment of the invention, CCPs corresponding to an Zrner of repeating HyX units wherein z is preferably 7 to 21, more preferably 7 to I5, can be used in which a series of 7 mer permutations are systematically represented to generate a rtpcating motif such as I~yX
bIyXHyXHy. Thus, for exatnple, a 9 mer will represent at least 2 series of 7 mers in the NHz -.~
COON orientation and two series 7 mers in the COOH ~ NHz orientation.
Similarly an 11 mer will represent 3 series of 7 mer HyX reputing units in the NHZ ~ COOH
direction and 3 in the opposite dixoctioo. ,Accordictgly, this reduces the number consensus conforming peptides required to create a series of relevant PPEs and several copies of the longer peptide having a series of several 7mers represented, preferably each of comparable affinity to HSP (within a given peptide) can substitute for making each of the 7mers. The relevant set of Zmers required to crcatt the requisite subset of CCPs can be calculated according to well-known mathematical techniques.
According to the invention we providt a set of degenerate nucleic acid probts that aze complementary to rnRNA encoding permutations of 3 hydrophobic amino acid within 7 amino acids, wherein at least 2 and preferably three of such hydrophobic anvno acids are non-contiguous. In a preferred embodiment, probes are designed in accordance with the motif Xm (HyX)" X'°, wherein m is preferably 0, 1, or 2 and n is preferably 3 or 4 and wherein, in the creation of the probes, sonic degeneracy (less that 100% ~Ty) is permitted for Hyi Hyz Hy~ and Hy4 such that the representation ofHy at such positions can be less than 100%.
The permitted degeneracy is a function of whatever biasing occurs at X toward amino acids preferred for intermolecular interaction with SP. 1t is to be understood that X (preferably X,,,, X"z. X,~ or X"4 as opposed to Xm or X~m) can also be biased toward certain amino acids as opposed to being random aad that biasing of X" towards Hy, depending on the degree, permits a greater degree of degeneracy from Hy, in keeping with substantial preservation of an HyXHyXI~IyXX motif.
Where the probes are designed for microarray technology, the motif HyXHyXHyX
or HyXHyXHy substantially reduces the number of permutations of probes required.
The choice of m iziusiea wro m:~a r~Ax am aaz usi~ xmuu~r ~ mAYt;~;~; ,~uls probe motif depends on size and number of micrparrays sought to be employed ,and the preference for larger probes for more accurate hybridization for a given set of stringency conditions. Similar considerations with respect to biasing anti degeneracy from Hy apply.
Accordingly, in another aspect the invention is directed to:
1) a set of probes for determining the set of actual turnQr cell oo~ose~asus conforming peptides corresponding to a motif of 3, and optionally 4, hydrophobic residues within 7 constcutivt amino acids wherein at least two such residues and optionally three such residues are non-contiguous (subject to optional degeneracy from Hy); and, 2) a corresponding set of peptides.
In further embodiments related tp biasing ofHy and Xr~, where less than a full set of permutations of the motif is opted far in the design of the probes (or the selection of peptides as discussed below) it is contemplated Hy and X can be biased towards amino acids that inbibit peptide folding and preclude degradation by protcasomes. It is believed that such peptides can be preferred far survival in proteasomes and interacfiion with SP and will have a greater representation among surface expressed SPPCs. In particular it is contemplated that preferred residues for binding SP are those which have been demonstrated to be preferred iz~ binding experiments, some of which can be classed in terms of steric inhibition of peptide folding (e.g. a series of non-contiguous aromatic residues) ar inhibition of such folding by possible charge repulsion or a combination of steric and charge interactions at several discrete intervals so as to counteract tht tendency for a hydrophobic peptide to fold. In this regard, amino acid compositions that have no predilection towards Folding (weak hydr4phobic interactions, no charge attractions, etc.) arc also preferred. Some of these properties can be demonstrated in the peptides, which have been generated within peptide libraries for preferred SP
binding and in WO
9J1227b1. Peptides that are less likely to be degraded in proteasomes can be ascertained, for example, by transforming a target cancer cell with a polypeptide ((HyX) rIXX)m whEx'c n=4 and rn is preferably 5 to 80 to determine the pattern of degradation within the cell. ~i~ffexent types of hydrophobic residues could be sampled within a plurality of such polypcptidcs to determine which such residues at which one or more of several positions within the motif cause preservation of the motif. The same could be done for the X positions. For example, Atxxxxx,Ax A,rXA.rJ~XXAr and ArXArXArXAr could be sampled with different aromatic hydrophobic iziusiea wrv i7:~5 r~A~ ma aaz usz~ xtvuu~r ~ mnY~~lHula amino aoids and di fFerent X, initially maintaining as many parameters as possible constant for ease of interpretation of results.
As discussed in more detail below, peptides of a length and amino acid compositiozt which are preferred for binding MHC (of the class and HLA type required depending on the mode of administration of the peptide as an immunogtn and the individuals administered to) can also be used in reducing the number of Pl'lrs required for screening. This selection can occur at the level of creation of the probes, or preferably at level of selection of among peptides candidates that have been establislxed to have an ilnmunological cress-reactivity with the surface of the tumor- It is contemplated that the tumors of a series of individuals having common HLA
types can evaluated contemporaneously.
According to various aspects of the invention involving pre-selection among the entire set of thepretieal C~Ps, it is contemplated that a predetermined peptide according to the invention iS
a product of selection at one or more and preferably all of the following levels of screening:
~ identification (via database) of the universe of GCPs that exist witk~ir~ a human cell;
~ design of nucleic acid probes conrespoztdug to a sat of consensus conforming peptides that are actually representative of the proteins being expressed in a tumor cell;
. identification of peptides, among the pool of consensus conforming peptides generated through nucleic acid hybridization screening, which are imrnunologically cross reactive with the surface of a tumor;
~ identification of peptides which are determined to have predisposition far binding to the surface of the tumor based on an analysis of actual SP peptide complexes from various tumors which are idantifia~d by H11 in accordance with the purification procedures outlined below or alternative procedures for identifying the peptide portion of a substantially purified SpPC;
~ HLA typing; or ~ identification among the preceding groups of peptides, in the form of PPEs which cause an in ~umuna response to a target tumor, e.g. in a cytotoxic T~cell assay.
It is also contemplated that screening can be initially carried vut with any of the reduced or more preferred group of CCPs contemplated herein and expanding thereafter as necessary.
It is also contemplated that the starting subset of CCPs of interest can bo systematically broken into mare manageable populations by fixing one or more parameter systematically. For iziusiaa wr~o i7:~s rv~ 4ia adz osz~ Htuuu~r ~ mAYt3r;~ ~uzu example, in the case consensus peptide motif involving a series of hydrophobic amino acids, it can be ascertained (see Fourie et al., 1994) that aromatic hydrophobic amino acids are more important for binding. It can also be ascertained from the literature (see WO
99122761, Sloan lettering) that other amino acids are preferred for binding to SPs.
Accordingly, as discussed above, various crittria can be employed for controlling the variation to obtain a predefined number of starting of CCPs, the number being dictated by the cun:ently available high throughput screening technology and the extent of the effort applitd to each stage of the systematic screening process. For example in the case of the hydrophobic motif HyX HyX HyX Hy such criteria can include excluding certain amino acids from being represented as X, for example negatively charged amino acid and/or amino acids that impart structure such as cysteine, glycine and proline. Another criteria for selection of X can be amino acids that are preferred for intermolecular interactions such as tyrosine, histidine, glutamine, asparagine, lysine, etc-It is also contemplated that standard mathematical techniques can be employed to identify other consensus sequences that have a strong representation witlxiz~ tmnor cells in terms of the frequency of the occurrence within native human protein. It can be first ascertained mathematically how long a proteirx would be required to have the selected ~onotif ocGUr randomly within the prntcin and the motif can be adjusted to correspond to occurrence within 25% to 100%
of all such catalogued proteins, bearing in mind that several proteins might be represented within the proteosame as discussed above. This can be confirmed within a database of human proteins.
Accordingly, the invention contemplates that a motif representing about a 25%
to 1 b0%
occurrence within all catalogued proteins can be selected having regard to number and size of array that can be reasonably screened with the currently available technology.
The number of permutations of the motif float Gan theoretically exist and the well known average protein size allows for the design of a motif that is anticipated to occur within a length of polypeptide which is 1 to 4 times greater than the average protein size. This can be oor~frmed in a database of catalogued human proteins. The levels of screening and inclusion and exclusion critez~a referred to above can also be applied to such motif. The term motif contemplates known categories and sub-categories of amino acids, such as hydropb.obic including hydrophobic aromatic (F, W, Y) ~d othtr hydrnphobic (A, I, L, M, ~ or neutral-weakly hlydrophobic (A, G, P, S, T), uncharged (5, T, ~T, Q), positively charged (K, R, H), negatively charged (l~, D) (sec Lodish et al and the 1G/U8/88 W~ 17:;ft3 r'A~ 4ltf ;itiL USL;f lilDlJU'f !k JIIAYBlrlr ~tJULl brochure protein Sequences on STN Available fxozn CAS), as well as artiixcially created such categories. Artificially grouped amino acids arc grouped with a view to designing a consensus ptptide m4tif that is likely to be represented on the surface a cancer cell, as discussed below.
The percentage representation of each of the categories or sub-categories of amino acids within the total of 20 can be used to calculate the number of permutations required to establish the broadest unreduced set of theoretical CCPs.
It is to be understood tliat there will be a large overlap between the sets of consensus conforming peptides ("CCP") identified, for example, using a DNA microchip, from individual to individual.
This is expected because a given type of cancer cell i5 expected to produce at least for the most part, the same prottitls. Accordingly, even though it might be expected, based on the literature (Srivastava et a1.), that the individual CCPs on the surfaces of cancer cells frozen individual A will be at least somewhat different than those of individual B, the pctmutations of CCPs that need to be represented in the set of CCPs that accommodate individual A, will npt be gubstantially different from the permutations that would be roquired to accommodate individual B and any individual differences will likely represent a small percentage of the total permutations.
Accordingly, the permutations corresponding to these individual differences can readily be added to the total set of CCPs to be tested. These individual differences and differences in frequencies of occurence of cozttmon peptides might also provide some indication of differences between the 24 tumor related biochemistry of individual A and individual B. Further such information could be expanded obtained by determining the nature of the peptides that are cross-reactive with the surface of the cull and the actual cellular proteins from which these are dtrived. Accordingly, such comparisons between different individuals with the same cax~ctr typt afford a method of analyzing differential tumor related protein expression and tumorogenesis.
As previously stated, it is contemplated that the set of relatively unidentifiable ligands are mapped to at least one set of identifiable ligands. As suggested above, the set of identifiable ligands may be designed on the ba$is of the following criteria:
34 1. the probability of occurrence of a permutation of the consensus peptide motif within every given human protein, It is desirable that the probability of occurrence of at least one such izW8W8 wry 17:;ft3 rW 41d ;itfL uBL;f HWUUT tk ~1AYB~~ IgIUZL
permutation exceed 10%, more preferably 20%, more preferably 30%, more preferably 40%, more preferably 50%, more preferably 60%, more preferably 70%, more preferably $0%, more preferably 90%, more preferably 95%, more preferably 99% or greater. From another perspective, the choice of motif can be made on the basis of the predicted number of occurrences of the motif within the target size protein of interest, for example, the average size protein. The predicted number of such occurrences may be less than once in the average size protein {in the sense that the motif will only be predicted to occur within every second, third, fourth, fifth, sixth etc such size protein which would be sufficient, for example, if the corresponding number of proteins were represented within a proteasome) or greater than once, optionally from about 1 to 15 times, preferably 1 to 7 times, more preferably, 1 to 3 times. It is to be understood that a motif design opting for a greater such probability ar number of occurrences is to be counterbalanced by the consequence of having to create a greater nurnbcr of permutations of identifiable ligands for evaluation. It is also contemplated that where the zaotif of interest is one that is selected to occur in a plurality of copies or permutations within an average size protein, that, at Least in the case of a DNA chip, that several passes over a set (on or more) of chips representing substantially all permutation of such motif may be required to identify all such penlnutations. Alternatively, or additionally, it also contemplated that one than one cDTV'A copy of the mI~NA is made available to permit hybridization of the various copies of the cDNA at different locations on the chip, especially if mItNA of a plurality of different cells is used for creating the cDNA for the hybridization.
It is also contemplated that the cDNA can be digested prior to the hybridization with restriction enzymes selected on the basis that they arc not anticipated to cause any significant digestion within the motif of interest, so as to facilitate hybridi2ation under stringent conditions and obviate the need to have several topics of the cDNA to identify all CCPs within a single Such cDNA.
2. the liklihood of preservation of the consensus peptide motif in the course of protcolytic cleavage within the proteasome;
3, the propensity to biztd to stress proteins; and optionally Ig 1G/U8/88 WAD 17:J7 b'AA 41H ;ftfG USLJ H1DULI'1' !k ~IAYBt;J~U~S
4_ the propensity to bind tQ M.I-TC, ,A,s contemplated above, it is possible to determine which amino acid residues at which locations within the consEnsus peptide motif will provide greater assurance that the motif will be preserved on the surface of a cancer cell a$er the protein in which the motif is represented passes through the proteasome. For example, it is contemplated that some hydrophobic amino acid residuts will assist in preserving the motif. It is contemplated that the motif can have at least one hydrophobic amino acid at the beginning or at the end or at both ends, and optionally also one or more hydrophobic residues therebetween; hyrophobie residues optionally including particularly amino acids which are aromatic.
If cleavage sites within the proteasome were assumed to be random, the probability that a gluon motif will not be preserved on the surface of the cancer cell can be calculated as a function of the number o.f annino acid residues within the motif the longer the motif the more likely it would be cleaved. In this connection is contemplated that a motif design based on fewer fixed amino acids (ie. norA-X, fox example just 2 such amino acids eg. HyXHyX " or X "HyXHy where Hy is an aromatic amino acid and n is 3 to 15, preferably 5 to 8, and X is any amino acid and therefore is not fixed) will result in a lower probability of cleavage. From the standpoint of reducing the likclyhood of cleavage, ideally, ignoring momentarily, the rules that might be applied to the propensity to bind to HSP, the fixed positions should be set to occur adjacent td one another eg.
relating back to the previous example HyHyXN+i. In effect the latter strategy contemplates that the numbtr of non-axed positions are reduced and/or made more adjacent to one another, and that the criteria as to how many amino acids are part of the motif at each amino acid position within the motif xre relatively more restricted to compensate for increasing the variability to X
within a greater number of positions. for example rather than choosing a motif where amino acids at positions 1, 3, 5, and 7 arc cacti chosen, for example fmrn groups of eight (eg, barring considerations of propensity for HSP binding, any 8 amino acids) so that (assuming positions 2, 4 and 6 can be any amino acid) the number of probable occurrences of such motif within a stretch of say 280 amino acids is (2$07) X ($~20)ø - approximately one (see formulas below) each of positions l and 2, for example, can be chosen from only 3 or preferably 4 amino acids, while the remaining 5 positions could be any amino acid (i.c. fewer fixed positions with greater 1L/U8/88 W~ 17:~f7 b'AIL 41U :iUG uSG;f ltlDUli'1' ~ ~1AYB~l~IUY4 restnetiQns), so that the number of probable occurrences of such motif within a stretch of say 280 amino acids is (2807) X (3y20)Z in the cast where position 1 and 2 is chosen from 3 amino acids and (280+7) X (4T20)z in the case where position 1 and 2 is chosen from 4 amino acids, both of which will equal approximately one (for the purposes of illustration).
The liktlyhood that two adjacent amino acids would be cleaved in the course of cleavage in, for example, 8 mer lengths, is significantly less. It is also con.ttmplatCd that a motif such as Hy?~3yX ", where N i5 chosen to represent the predicted sizes of the peptides brought to the surface o~ the cell, where N
is equal to or greater than the smallest such size, will be assessed on a database of proteins to determine which species of, for example, Fly is most frequently represented in such motif. rt is also contemplated that a motif wherein X or XX, etc. occurs at both the beginning and end of the motif, e.g. XXXHyHyXXX, is preferred in one aspect of. the invention, sincE
this obviates the consideration of placing certain residues such as frequently occurring hydrophobic amino acids at fhe beginning or and of the motif.
The invention is also directed to a population of probes conforming to the motifs generally and specifically described in the preceding paragraph.
It is also contemplated that a motif design based on the probable occuzzence of several permutations of the motif would make it less likely that all such permutations of the motif will be craved. For example HyldyXN+i occunring twice, 3 times, etc. It is also contemplated that several such motif (hence the phraseology "at least one" can be represented on different respective sets of chips and that using several such sets of chips will assure that a greater number of patients' cancer cells will be able to be accurately mapped to particular CCPs. This prospect would make it less likely for each motif to be lost due to cleavage, an the one hand, and make it possible to design motifs with smaller number of permutations, on the other hand, and to use as many Chips as are incrementally necessary to increase the percentage of patients that are able to be typed to particular CCPs.
As suggested above, it is contemplated that the number of amino acids that are desired to be grouped together as potential candidates for each amino said position within a designed consensus peptide motif, could be chosen to control the probable occurrence of the motif within 1G/Utf/NH W1;11 17:;f7 b'Ax 4lti ;3GL U82;f Htl)UU'f & 11AY13HH ~JUL5 an average size ,protein. Alternatively, to assure occurrence of the motif on the surface of any given cancer cell ox the vast majority of cancers tested, the choice pf grouping could be made on the basis of the controlling the probable occurrence of the motif with the smallest size protein that might be found within the proteasome. This more conservative approach with respect to the choice of the target size protein might be more or less appropriate based on the rigour of the design andlor conservatism of assumptions, in other respects, including 1) the number of different motifs anticipated to be tested; 2) the limitations, if arty, of the current high throughput screening technology as regards the number of permutations of the motif that be practically tested for tumor matches (in accordance with the scale of the effort sought to be applied in the initial &. subsr.,guent stages of screening); and 3) the assumption made about the number of different proteins that might be represented within a proteasome; and 4) the rigor or conservatism applied in choosing th.e number of probable occurrences of permutations of the motif in the target size protein. This latter choice is assessed on the basis of the mathematical relationship:
P = M X (0.05Q~ X 0.05Q2 X 0.05Q3 X 0.05Q4 X O.OSQ3 X O.OSQB X O.OSQ~ X
0.05QN) Where P is the predicted number of occurrences of the motif within a stretch of amino acids of a length which is M X N, where M X N is number of amino acids in the target size protein and N
is the number of amino acid positions in the motif and Q is the number of candidate amino acids within the group for each respective amino acid position Ql , QZ , Q3 , .....QN within the group. "fhe multiplier .05 is an alternative expression for dividing by 20 (see example above).
Fdr ease of reference the expression (O.OSQ, X O.OSQz X 0.05Q3 X 0.05Q, X
0_05Qs X 0.05Qs X
O.OSQ~ X 0-OSQ~) will be represented hereafter by the later L.
In terms of the probability of occurrence of one or more permutations of the motif witliin the target size protein of interest, one formula that may be applied is:
Pr ~ 1 - (1 - >;.)M

iciuaiea wry i7:~s rvx 4ia aaz usz~ xmuu~r ~ mnY>3~~; ~u~a The probability that a given motif will be represented a specific number of times (G
times) is given by:
Pr= (L° x (1- L}tM-off) x M!} T(G! x (M-f'.r)! ) Due to the ability of HSPs and MHCs to bind to diverse different peptides it is contemplated that a variety of different motifs could be designed to obtain a valuable starting set of peptides that are candidates for the methods of mapping defined herein. It is to be understood that the appropriateness of a designed or selected motif can be assessed using a database of human proteins, for example, to determine the number of occurrences o~ that motif in the total number of proteins within the database, preferably also taking inta account the size of such proteins.
It is to be understood that any combination of the above strategies that are not practically or lo~icahy mutually exclusive can be employed for selecting a comprehensive or partially comprehensive (preferably in systematic part) set of CCPs. It is to be understood that the most important criteria according to the invention are:
~ Potential existence of the peptide within nature;
Potential existence of the peptide within the tumor cell determined preferably through nucleic acid hybridization according to well known techniques (sec fox example, Immunological Method Man«al, Ivan tefkovits and references listed including the references listed at p.423;
~ Preferred binding to SP based on thenmadynarnic considerations (including resistance to hydrophobic folding} as evidenced by peptide library panning or other such studies according to technique and/or data published in the art;
~ Conservation of certain types of hydrophobic amino acid sequences within proteasomes; or ~ Those that have a preference for binding to H 11. Method: Peptide library panning.
In accordance with another aspect of the invention, we provide, among others known in tk~e art, a method of identifying one or more candidates For specific peptide antigens that conform to a consensus peptide motif associated with tumor surface expressed peptide antigens, including the steps of ic~us~ea w'J~ 17:'~i rW 41H ;itlL uBLJ HWUUT Ik ~IAYB~Y~ ~JUL7 A) identifying at least one candidate turaor (preferably a population of such tumors) having on its surface at least one specific tumor-associated peptide anrigcn that conforms to said motif;
B) identifying a population of consensus conforming peptides which includes a suitable ,plurality of candidates far said specific tumor-associated peptide antigun;
C) identifying a population of antigen-binding fragments wherein there are a plurality of candidate fragments each characterized in that they bind to at least one specific consensus conforming peptide as presented by a PPE; and D) screening using the candidates identified in steps A, B & C to identify consensus conforming peptides as presented by PPEs (optionally $Ps) that bind to antigen-binding fragments that bind to the candidate tumor(s).
Qne embodiment of the invention provides x subset of a combinatorial library of antigen-binding fragments which bind to SPPCs (but not the SP itself) on one Or more tumor tissues or mioroamay of such tissue. 1~or example, a naive library of antigen-binding fragments can be used to obtain anti-SPPCs by panning against a target tumor and assessing competitive binding with H11 (or an IT11 related antibody), whereupon those fragments can be individually or collectively used to determine which among a population of PPEs representing a desirtd set of CCPs bind to those antibodias_ Therefore, we provide a method to identify a substantially reduced subset of CCPs corresponding to each antigen-binding fragment, which binds to a tumor surface expressed HSSPC found on a given tumor.
Accordiuxg to another embodiment of the invention it is possible to immobilize the aforesaid subset of binding fragment (see for example WO 99/195ph, WC
99/06834, WO
99127$745, WQ 99131509) and screen with a plurality of PPEs representing a large set of CCPs.
It is possible to tnap a population of immobilized CCPs to a population of tumor surface SPPCs by identifying one or more, and preferably a microarray of such tumors which express SPPCs using a population of antibodies which compete with H11 for binding to tumor expressed SPPCs. Preferably the tumor tissues and candidate PPEs are both immobilized and a large na'ivc library is used to map each tumor to one or more PP~.s. Methods of screening proteins immobilized microarrays are referred to for instance in Lucking et al. ( 1999) Anal. Biochem.
270:103-111; Bradbury (1999) Tibtecb 17:137-13$; Mock et al_ (1999) Am. J.
Path. 154:981 98b; Watkins et al. (1998) Anal. Biochem. 256:169-177; and van Dijk-Wolthuis (1999) za iziusiaa wr;~ i7: ~a rw aia aaz us2~ xmuu~r ~ mnlcsr;r; ~uzs Hioconjugate Chem. HSPs can be immobilized in numerous copies and loaded with a set of CCFs selected according to the invention. Techniques for immobilization are well known, in the art. Some of these are described in sources immediately above. Reference is also made to Takenaka ct al. J. Hiol. Chem. 274:19839-19844 (1995) which describes a method of determining when peptides are loaded onto HSPs.
Accordingly, in a general aspect of the invention, we provide a method of mapping a set of relatively unidentifiable ligands to a diverse set of identifiable ligand5, said Set of relatively unidentifiable ligands comprising at least one relatively unidentifiable ligand (preferably a plurality of such relatively unidentifiable ligands), said method comprising the steps of using a 14 combinatorial library of binding molecules to bind to one of said sets of ligands to obtain a subset of binding molecules corresponding to at least one of said relatively identifiable or unidentifiable ligftrtds and pro~ding at least one binding molecule from said subset of binding molecules for use in binding to said other set of ligands in order to identify at least one identifiable ligan.d corresponding to an unidentifiable ligand.
15 The form identifiable in contradistinction to relatively unidentifiable without limiting tb.e generality of: these terms; can be better understood, by way of example, to mean more readily diversified, isolated or characterized, when compared with the relatively unidentifiable l,igand-Without limitation, it is contemplated that isolation, diversification andlor characterization may be relatively facilitated in virtue of mapping the relatively unidentifiable ligand to one or more 20 identifiable ligands, in the sense that:
a) the relatively unidentifiable ligands are of one type and the population of identifiable ligands is simply of another pre-determined type which is readily diversified in the sense tliat the minimum requisite breadth of variations can be created (e.g. facilitates larger scale mimitope Creation). However, it is contemplated that the invention extends beyond high throughput 25 advantages or use of a preferred diversity Set of molecules or a preferred ligand type, for example, where one or more factor contributing to identification are more optimal, as explained in points b) through fj;
b) the identifiable ligands form a relatively precise parameter set of molecules, i.e. in virtue of the identifiable set being predefined in variability according to one or more such parameters, 30 they are, for example, more readily amenable to systematic or rapid analysis, e.g. where the method of analysis is geared to those parameters;
zs 12/U8/8N W~ 17:;18 b'AA 4lti :lHG USYJ HlDUIi'1' Ik mAYBHH ~JUYB
c) the idenrifiable ligands, in contrast to the relatively unidentifiable ligands, are adapted to be identified, for example, in virkue having a component moiety which contributes to a detectable phenomena when bound by the binding molecule, or in virtue of the variable portion being readily separated or isolated;
d) the identifiable ligands are relatively free of associated complexity, for example, a fi-ee standing molecule as opposed to a rn~olecule that is complexed or otherwise associated with other molecules or structures, c.g. cell surface antigens;
e) the identifiable ligand is relatively more isolated in a particular lpeation, e.g. for ease of.
access andlor analysis, for example, in a particular well, or a particular location, on a 14 substrate;
f) the identifiable ligand is not only isolated in a particular location but the ligand at that location is known so that identification readily follows .from binding of the binding molecule at that location.
For examplt, the variable portion of the ligand may be easily separated (e.g, the psptide portion of an SPPC) for analysis-In one embodiment of the invention, the relatively unidentifiable ligands and identifiable tigands are related for example, in the sense that they are the same type of molecule, e.g., PPEs and tumor surface SPPCs.
In another embodiment of the invention, the binding molecules are on the whole, prc-disposed to bind to the identifiable or relatively unidentifiable ligands yr both.
It is contemplated that a given binding moltcule may recognize a population of closely related identifiable ligands corresponding tv any give~t relatively unidentifiable ligand under certain conditions of binding stringency, and may recognize fewer or more such ligands under different such conditions. Accordingly, several round of panning with the population of binding molecules under different conditions of stringency may be desirable depending on the goal of obtaining more yr less candidate identifiable ligands, having regard to the additional levels of screening contemplated, if any. Furthermore, several rounds of panning using one of both sets of ligands may be desired to tackle a larger set relatively unidentifiable ligands (well in excess of two), in order to obtaizt a many to many correspondence in a first pass or set of passes, and then to focus on individual one unidentifiable ligand (mapped to a plurality of identifiable ligands or a minimum number of identifiably ligands), in succeeding passes. Furthermore, it may be 1G/U8/88 WJD 17:40 b'AIl 4ltf ~UL USGJ HlDUIi'1' ~C mAYlit~ ~JU:fU
desirable to use subsets of binding molecules in individual passes as part of a systematic ar partially systematic effort, far exacople, in order to 'use a more pre-determined set of binding molecules (in terms of a given parameter), or in order to use a more conservative approach at the outset, or simply to use different libraries in different passes.
It is also contemplated that one or both sets of the ligands may be immobilized, e.g., in a microarray, for promoting greater binding specificity or autorrtation.
It is also possible to immobilize a full naive set or particular subsets of binding molecules, for example, subsets that have been determined to be tumor specific or ligand-type speciF~e, and to carry vut the mapping in one location, for example, in multiple passes, each time using a full set of both populations of ligands sought to bt compared or relatively fewer passes {possible one) with multiple copies of the different ligands within the respective sets represented in a given round of panning.
In a preferred embodiment of the invention, the relatively unidentifiable ligand is relatively difficult to isolate and characterize in virtue of its association with a cell surface, for 1 ~ example, tumor surface antigens, and the relatively ider~tiftable ligands are a set of potential peptide antigens. In preferred embodiments, the identifiable ligands arc relatively identifiable in the virtue of the greattst number of factors, which contribute to the ease of identification e.g.
precise diversi,ficatioz~ accocdiztg to certain parameters, as well as relative isolation and ease of characterization. Optimally, in the cast of HSPs presenting consensus conforming peptides, each desired permutation is known and assigned to a particular location on a substrate.
Minimally, identifying individual relatively identifiable ligands may be enhanced, for example, in virtue of a threshold amount of diversity and some enhancement in isolation or independence from associated complexity.
The arms binding molecule and ligand are intended to be limited only in terms of the pragmatic import of the mapping method, namely so that the binding molecule is used to identify in a selective manner, identifiable ligands from a diverse sat corresponding to at least one, but advantageously many, relatively unidentifiable ligands, so as to identify candidate identifiable ligands (often the minimal number in excess of one depending on the additional screening contemplated) corresponding to a given individual relatively unidentifiable ligand. With this caveat in mind, certain scope can attempted to be given to these terms.

Zz~uB~Ha wl;D 17:4u rW altf ;iUL u82;f xWUUT tk mAyBl;~ I~JU:Il The term "binding molecule" can refer to a molecule of sufficient size and complexity So as to be capable of selectively binding a ligand. Such molecules are generally macromolecules, such as polypeptides, nucleic acids, carbohydrate or lipid. However, derivatives, analogues and mimetic compounds as well as small organic compounds are also intended to be included within the definition of this term. The size of a binding molecule is not important so long as the molecule exhibits or can be made to exhibit seleckive binding activity to a ligand. For example, a binding molecule can be as little as about one or two, and as many as tens or hundreds of monomer building blocks whidh. constitute a macromolecule-binding molecule.
Similarly, an organic compound can be a simple or complex structure so long as selective binding affinity can be exhibited.
Binding molecules can include, for example, antibodies and other receptor or ligand binding polypeptides of the immune system. Such other molecules of the immune system include for example, T cell receptors (TCR), major histocompatibility complex (MHC), CD4 receptor, and CD8 receptor. Additionally, cell surface receptors such as integrins, growth factor receptors and cytokine receptors, as well as cytoplasmic receptors such as steroid hormone receptors are substantially also included within the definition of the term binding molecule.
furthermore, DNA binding polypeptides such as transcription factors and DNA
replication factors are likewise included within the definition of the teml binding molecule. Finally, polypeptides, nucleic acids and chtmical compbunds such as those stleated from random and combinational libraries are also included within the definition of the term so loztg as such a molecule exhibits or can be made to exhibit selective binding activity toward a ligand.
,As used herein, the term "polypeptidc" when used in reference to a binding molecule or a ligand is intended to refer to peptide, polypeptide or protein of two or more amino acids. The term is similarly intended to refer to derivatives, analogues and functional mimetics thereof.
The term "ligand" can be understood to refer to a molecule that can be selectively bound by a binding rnoleeule. A ligand eambe essentially any type of molecule such as polypepdde, nucleic acid carbohydrate, lipid, or any organic derived compQUnd_ Those skilled in the art know what is meant by the meaning of the term ligand. Specific examples of ligands are the tumor antigens described herein which arc selectively bound by the human antibody binding molecules described in the examples.
2$

iziusiaa wrp m:4u rv~ 4ia saz usca xmuu~r ~ mAY~~;~;
As used herein, the term "diverse population" is intended to minimally zzfor to a group o~
two or maze different molecules.
As used lierein, tile term "selective" or "selectively" whezl referring to tk~e binding of a binding molecule to a ligand is intended to mean that the interaction can be discriminated from unwanted or non-specific interactions. Discrimination can be based on, for example, affinity or avidity and therefore can be derived from multiple low affinity interactions or a small number of high affinity interactions. For example, a binding molecule interaction with a ligand is generally greater than about 10'° M, is preferably greater than about 10-5 M and more preferably greater than about 10~ M. bTigb affinity interactions are generally greater than about 10-s M to 10-y M or greater. Unless otherwise stated, selective binding is implied. It will be appreciated that selective binding is necessary to identify immunologically cross-reactive SPPCs or MHC peptide complexes (i.e. those that Share antigenic determinants with tumor surface species) that will be useful to generate a T-cell response.
As used herein, the term "immobilizing" pr grammatical equivalents thereof, refers to the attachment, as through the binding of a population of binding molecules, to a solid support.
Immobilization can be through specific interactions with the binding molecule and an agent on the solid support. The agent can be, for example, a chemical moiety, which shows covalent or non-covalent interactions sufficient to hold the population of binding molecules to the solid support. Immobilization can also be through tethers or linkers. Such linkers can be covalent linkers, hydrolyzable linkers, photo-labile linkers or other linkers that allow the binding molecules to be selectively attached. Linkers can also be polypeptides or other biomolccular linkers such as antibodies, lipid attachments, streptavidin, receptors, fusion polypeptides, or any biomolecule that can tether the binding molecule to the solid support.
Additionally, domains of polypeptidcs can similarly be linkers. For example, hydrophobic domains which allow direct absorption to a plastic due to specific sequences, which arc molecular tags or recognition sequences can be linkers for binding polypeptides.
As used herein, the term "solid support" refers to a solid medium, which is sufficiently stable so as to allow immobilization of a population of binding molecules.
Solid supports can include, for example, membranes such as nitrocellulose, nylon, polyvinylidene difluoride, plastic, glass, polyacrylamide or a~arose. Solid supports can also be made in essentially any site or shape so long as it supports the immobilization of a population of binding molecules- For iziusiea wr;~ i7:ai r~ax am ~nz u~za xmuu~r ~ mnYtrrr; ~uas example, the solid support cart be a flat planar surface such as a natural or Synth~~c membrane filter or a glass slide. Alternatively, the solid support can be of various spherical shapes, including, for example, bands matte of glass, polyacrylamide or agarose_ Porous mediums can similarly be used as solid supports and such mediums are included within the definition of the tezm as used herein. Additionally, any Of the solid supports can be modified, for example, to include functional chemical groups that can be used di.Iectly or indirectly for attachrnent of binding molecules or linktrs. It is contemplated that multiple HSPs or MHCs can be immobilized on a solid support and later loaded with peptides either at random or pre-defined locations.
The temp "antibody" can be understood to mean a polypeptide, which binds to a ligand and is intended to be used consistently with its meaning within the art. The terror immunoglobulin is similarly intended to fall within the scope of the meaning of the term antibody as it is known and used witliin the art. The polypeptide can be the entire antibody or it can ba any functional fragment thereof which binds to the ligand. The meaning of the term is intended to include minor variations and modifcations ofthe antibody so long as its function remains uncompromiscd. Functional fragments such as Fab, F (ab~, Fv, single chain Fv (scFv) and the like are similarly included within the definition of the term antibody. Such functional fragments are wet l known to those skilled in the art. Accordingly, the use of these terms in describing functional fragments of an antibody are intendtd to cozrespond to tha definitions well lanown to those skilled in the art. Such terms are described in, for example, Harlow aztd ~.ane, Antibodies. A, Lahoratorv 114anual, Cold Spring Harbor Laboratory, New York (1989); Molcc.
Hiolo~y and BiotechnQlog ~ A o nrehensive Desk Reference (Myers, R. A. (ed.), New York:
'VCH Publisher, Ine.); Huston et al., Cell Hiophv i c. 22:1$9-224 (1993);
Pluckthun and Skezra, Wit. >Jnzvmol., 17$:497-515 (1.9$9) and in Day, E. D, Advanced Immunochemistry, Second Ed., Wiley-Liss, Tnc., Ncw York, NY (1990), which arc incorporated herein by reference.
In one embodiment of the invention, said stt of idantifiablt ligands and said set of relatively unidentifiable ligands are antigenically related, for example, PPE
and tanner surface SPPCs. In this case, optionally said library of binding molecules is specific for said set of identifiable ligands as discussed below.

1L/U8/88 W~ 17:41 H'A~ 4lkf ;idL U82;1 H1DUU'1' ~C 1lAYBt;J~U;~4 In a preferred embodiment of the invention, the binding molecules servo to identify identifiable ligands, which are immunogenically cross-reactive with said relatively unidentifiable ligands.
Accordingly, in a more specific general aspect of the invention, we provide a method of mappi~ag a set of identifiable ligxnds (e.g. a population of 1!'PEs) to a set of relatively unidentifiable ligands (e.g. tumor antigens), said set of relatively unidentifiable ligands related to said first set, said set of relatively unidentifiable ligands eornprising at last one relatively unidentifiable ligand, said method comprising the Steps of using a combinatorial libt'ary of binding molecules to bind to one of said sets of ligands to obtain a subset of binding molecules 1 b corresponding to at least one of said relatively identiEable or unidentifiable ligands and providing at least one binding molecule from said subset of binding molecules for use in binding to said other set of ligands in order to identify at least one identi~ablE
li$and corresponding to an unidentifiable ligand. Preferably said set of identifiable ligands and said set of relatively unidentifiable ligands are antigenically related. More preferably said library of binding molecules is specific for said set of identifiable ligands as discussed below.
In another embodiment of the invention, the library of antigen binding fragments used for mapping the relatively unidentifiable ligands to the idenrifiable li~ands is a single variable domai~.~ library, preferably a heavy chain variable domain library. Reference is made to U.S.
patent nos. 5,702,892; 5,759,808; 5,$00,988; 5,840,526; 5,874,541, and to Lauwerey M. et al., EMHO Journal, 17(19), p.3512 (1.998); Rciter Y, J. Mol. Hiol. (1999) 290:3 685-698. In the case of well-known camelid type single domain antibodies disclosed in several of the previously mentioned patents, the candidate binding fragments have loop struotures which are useful for binding into cavities (see also Muyldcrtnans S. ct al. J Mol. Recogtit. (1999) 12(2) 131).
Reference is also made to our co-pertdin.g U.S. patent application filed November 4, 1999, entitled "Enhanced Phage Display Libraries and Methods for Producing the Same", the disclosure of which is hereby incorporated by reference. This application discloses a variable heavy chain domain library based on the heavy chain variable region of the antibody designated Ab which spans from a position upstream of FR1 to a position downstream of F4.
A6 has a CDR3 of 23 amino acids in length, which forms such a loop structure.
l~rthermore, in one embodiment of the A6 libt~ty, positions 44, 45, and 47 (Kabat numbering) may be substituted by non-hydrophobic amino acids, for example, those residues described in the literature on camelid 1L/U8/H8 WJrD 17:46 b'AA 41U ;iHL UEf~J 1L1DUU'1' lk mAYBl;Ir ~p;~b single domain Abs (see Lauwerey of al. 1998, and U.S_ Patent Nos. 5,$4b,526 and 5,$7x,541).
Furthermore, in the absence of those substituted residues, this library surprisingly produces truncated loop structures, which can be advantageously be used for binding to the antigen binding situ of antibodies and may be adapted as described herein to SPPC
binding sites. t'Jther such binding fragments include libraries of cysteine noose peptides (see WQ
99/23222).
Such loop structure based libraries can be varied to present ~t~dom or partially random loop permutations (excluding for example residues that impart configuration [e.g, glycine or proline j or biasing in favor of amino acids such as hydrophobic amino acids in some positions or those that are preferred for intermolecular interaction) that are varied not only in amino acid composition but also in the size of the loop- Alten~atively variations in the sine of the loop can be generated in a second round of panning with candidates that have preferred amino acid compositions determined from a first round of panning. Thcsc libraries and method can bt used to create peptide-specific SPpC binding molecules in the sense that they collectively recognize a substantial diverse population of different SPPCS and individually preferably a minimum number of such SFFCs, preferably less than 15, more preferably less than 10, more preferably less than more preferably less than 3, more preferably 1. Reference is also made to the methods of WO 991120749, which can be adapted to create peptide specific anti-SFFC
binding fragments usiaag H11, It is also contemplated that anti-HSF antibodies can be generated by panning against SPPCs and subtracting from the population of binders those that bind to the SP
alone. Reference is made to our ca.pending PCT application entitled "Antigen Binding Fragxx~ents Specific Far Tumor Associated Antigens" filed November 29, 1999, the contents of which arc hereby incorporated by reference.
In another aspect of the invention, the binding region (amino acids involved in the ligand interaction) of one or more preferred peptide specific anti-SPFCs can be varied randomly or systematically in accordance with a consensus peptide motif while maintaining the flanking regions that impart configuration constant to create a library of peptide specific anti-SPPC
candidates. $ueh libraries can be advantageously used, for example, in mapping tumor-surface SPPCs to PP)GS that conform to a consensus peptide motif as defined herein.
The invention is also directed to a method of creating an HSF binding site mimitope using naive or biased libraries of single domain variable fragments or loop structures by panning against x sub-population of SPPCs from which the pe~ptidc has been released (e.g. by ATP or iziusiea wr;~ i7: az rw aia aaz usza Hmuu~r a mAY~r;~
acid hreatrncnt) to obtain a sub-population of binding fragments which arc HSP
binding site candidates and optionally determining which among such fragments is unable to bind to tht peptide loaded population of SPPCs. Such candidates may be sequenced to determine whether such fragments have sequences, which arc preferred for binding to SPPCs. In a preferred embodiment of this method, the population of binding fragments are engineered to have binding pardons, which contain preferred HSP binding peptide sequences as discussed above and the Elanlting regions of Such binding portions may also be systematically varied in length. to provide different loop geomefies. 'flail method can be used to generate one or more preferred HSP
binding site candidates. In an ensuing step, anti-idiotypes that show preferred affinity binding to such preferred HSP binding site candidates can then be generated, for example, those that have amino acid constitutions or that arc preferred for binding to the same types of peptides that arc preferred for binding to HSP. 'fbese cax~ theca be tested as PPEs, for example by assessing the cross-reactivity of a particular SPPC to a corresponding PPE(s), wherein the peptide portions are identical or substantially identical. In another embodiment of the method, employing the mapping strategy disclosed herein, a population of minor variants of one or more anti-idiotypes that show such cross-reactivity can then be created using teclvtiques such as codon based mutagenesis (see also the technique described in co-pending U.S. application filed November 3, 1999, entitled "Enhanced Phagc Display Libraries and Methods for Producing the Samc" ) such that the anti-idiotype variants are strongly biased to the parental amino acid constitutions. These closely related variants can then be used to create a population of PPEs that are cross-reactive to corresponding SPPCs using for example the anti-SPPC sptci.fio library of single domain fragments or loop structures (as discussed above) as binding fragments for mapping purposes. In this way, a variety of different specific SPPCs could bt tested for cross-reactivity by loading each of the relevant peptides onto each member of a defined set of anti-idiotype variants and testing for matching pairs that bind with high affinity to a given binding fragment. For example, SPPCs pooled from tumor cell extracts o.f a variety of different tumors could be mapped to a set of such anti-idiotypes, preferably in multiple copies, loaded with a suitably large variety of peptides conforming to a consensus peptidE motif and the candidates that map to tine another can then lx assessed for those that have the most closely related , and preferably identical, amino acid constitutions. Amino acid analysis of samples of such anti-idiotypes could be used to create a smaller set of useful :EISP mimics that are each adapted to a specific set of peptides that are 12/U8l88 WJD 17:4 b'A~ 41U ~tfG USG;f lilDUU'1' ~k ~IAYBIW øJUJ7 allocated to a single array or sot of arrays for screening. Moreover amino acid analysis of several such matches or prototype anti-ids could be used to create a universal HSP mimic for the consensus sequence in question, particularly for consensus sequences that are preferred for binding to an HSP, Such as HyXHyXHyXHy.
Accordingly, the invention is also directed to a single variable domain anti-SPPC or a functional fragment thereof when such fragment is expressed in the form of a fusion protein with an outer surface protein residing on a phage. It is also eonternplated that such fragments can be tested for their stability as loop structures (with or without a portion of the outer surface phagt protein e.g. P3, but divorced from the phage particle).
The invention is also directed to a library of single variable domain anti-SPPCs including those of the type adapted to bind to individual peptide specific SPPCs-In a more general aspect of one embodiment of the invention we provide a method of mapping a diverse set of identifiable ligands to a set of relatively unidentifiable ligands, said set of relatively unidentifiable ligands immunogenically related to said set of identifiable ligands through a (preferably SPPC related) consensus peptide motif, said set of relatively unidentifiable ligands comprising at least one and preferably a plurality of relatively unidentifiable ligands, said method comprising the steps of using a combinatorial library of binding molecules to bind to one of said sets of ligands to obtain a subset of binding molecules (at lea.,ct one) corresponding to at least ono of said identifiable or relatively unidentifiable ligands and providing at least one such binding molecule from said subset of binding molecules for use in binding to said the other set of ligands in order to identify at least one identifiable ligand corresponding to an unidentifiable ligand, wherein:
a. said set of binding molecules is collectively adapted to bind to substantial number of permutations of SPPCs b. preferably, a substantial number of such binding molecules are adapted to bind to a minimum number of SPPCs in excess of one, more preferably only one. The same method could be applied to MHC peptide Complexeg or related sets of ligands.
The method of generating the A6 library is detailed in the co-pending patent application filed on November 4, 199 entitled "Enhanced Phage l5isplay Libraries and MEthods for Producing Same."

iziuniaa w~a i7:sa rw am aaz usca Hmuu~r rx mnY~~t; Boas Without limiting the invention herein defmcd or being bound by any theory, it is postulated that parts of the peptides that are actually bound to the SP
peptide-binding site are directly immunogenic with respect to the tumor. Thus, according to one embodiment of the invention, the entire consensus conforming peptide, or a part thereof corresponds to the cpnsensus motif HyXHyXHyXHy.
According to another embodiment of the invention, the consensus conforming peptides con cspond to peptides, which prefer to bind to HI l, and con.scnsus motifs, which are derived from these peptides.
The population of candidate consensus conforming peptides, can optionally be generated using a peptide display library. Optionahy, the candidate consen$us conforming peptides are presented on the surface of PPEs, preferably human tumor surface associate SPs such as SPs of 70 and 90 families. This oan be accpmplished according to methods known in the art (see, W) 99/22761) including such as that described in WO 99/29834. Optionally, the population of candidate consensus conforming peptides is presented on the surface of a population of professional APCs according to methods known in the art.
The population of antigen-binding fragments can be generated, by screening for CandldatC5 that bind to the population of co~a5ensus conforming peptides and creating one or more populations of variants related to one or more of such candidates according to the scheme dei:ined below. Alternatively, the population of antigen binding-fragments can be generated by using H11 as a parental binding molecule using the scheme and methods defined below. The populations generated according to either of the preceding approaches can be pooled. H11 itself which is believed to bind to a variety of different specific consensus conforming peptides, can be used as control (e.g. via a competition binding assay) in selecting candidates for specific tumor-associated peptides antigens which conform to the consensus motif of choice, in the manner discussed herein. These candidates can be used to identify peptide antigens according to the scheztie and methods defined below, as probes for various diagnostic applications, to identify new antigen-binding fragments and variants thereof for the use in the last described screening method (for isolating specific tumor-associated SPPCs by mapping CCPs to tumor using a suitable antigen-binding-fragment library), to identify different and possibly more specific consensus motifs, and to create antigenic compositions enriched for potentially relevant SPPCs, as discussed below.

iziusiaa wry i7:4;~ rva am ;snz usza xmuu~r s: maY~r;~ ~u~e The prefeiTed library of antigen-binding fragments (as discussed below) can also be used to generate other antibodies that bind to a plurality of different SPPCs in viriuc of the associated CCPs by the using the scheme described herein. These antibodies could also be used as controls 1~y way of competition assay to ensure appropriate tumor localization to SPPCs.
In another aspect the invention is directed a plaage display library which displays a plurality of antigen-binding-fragments that recognize one or more consensus conforming peptides, In one embodiment this library is created using Hl 1 as a parcntxl binding fragment, in the manner described below.
The invention is also directed to a population of genetic packages having a genetically determined outer surface protein, which collectively display a plurality of potential binding fragm,enty in association with said outer surface protein, each package, including a nucleic acid construct coding for a fusion protein, which encodes at least a potion of said outer surface protein and a variant of at least one parental binding fragment, wherein said parental binding fragment binds specifically to one or more tumor-associated consensus conforming peptides as presented by a SP, wherein at least part of said construct, including preferably at least a portion of the CDR3, is only partly randomized in that it is biased in Favor of encoding the amino acid constitution of said parental binding fragment such that said plurality of different potential heavy chain binding domains are on the whole adapted to be or are better capable of binding to tumor-associated consensus conforming peptides as presented by SPs. Preferably said parental binding 24 fragment is H 11. Preferably said genetic package is a phage and said soluble parental binding fragment is selected from the group consisting of an seFv, Fab, 'VH, Fd, Fabe, F(ab')2.
In another embodiment, the population of genetic packages or phage, comprising a plurality of libraries, which are pooled, wherein at least a first and second of said pooled libraries differ in the degree of biasing to wild-type amino acids. 1n one embodiment, libraries wherein the CDR3 is biased 95%, 90%, 85%, 80%, 75% and ?0% in favor of the wild-type (e.g. H11) constitution are pooled.
A SPPCs.
The invention also encompasses a substantially isolated, tumor-associated SPpC
designated C-antigen. The cozttpositions can also include physiologically acceptable excipients >_ziusiea wr~u i7:aa rw aia adz uaza Hmuu~r a mnYS>;1; Luau andlor adjuvants. The invention further encompasses compositions containing substantially purified SPPC peptides and immunogenic Gagments thereof.
Any of the SIyJ?C or SPPC peptide compositions can be formulated in therapeutically or immunogenically e~'ective amounts. These compositions can also be provided in dried or concentrated form for rehydration or dilution prior to use.
The invention is further directed to a method Qf isolating an intact SPPC by:
fractionating a tumor cell extract to obtain a hydrophobic fraction; identifying an antigenically active fraction, thereof using an antigen-binding fragment which binds specifically to the SPPC; applying the antigenically active fraction to an ADP chromatographic media; applying the active fraction eluted from the A.~1' chromatographic media to a strong anionic medium;
Collecting active fractions eluted from the strong anionic medium where activity is determined by specific reactivity with the antigen-binding fragment; and, prtferably, purifying the active fractions under non-denaturing conditions, preferably electrophoretic extraction.
The invention is also diroated to a method of isolating an antigenic SPPC by:
fractionating a tumor cell extract on an affinity medium, such as an immunoaffinity column, to bind the complex; eluting the complex to obtain an eluate; applying the eluate to a molecular sieve capable of separating the SP from the peptido; isolating, (and if necessary, sequencing,) the peptide; and re-associating the SP with the isolated peptide. The invention is also directed to C-antigen peptide isolated by said method and as described in more general terms below.
24 B Antigen-binding-Fragments.
The invention encompasses a composition of matter comprising an isolated antigen-binding-fragment specific for a SPPC. These antigen-binding fragments are termed "anti-SPPCs."
The invention further encompasses a composition of matter comprising an isolated 2S antigen-binding fragment of an antibody specilie for a tumor-associated SPPC and a physiologically acceptable excipient.
The invention also encompasses a method of obtaining antigen-binding fragments specific for a tumor-associated SPPC by generating a population of antigen-binding fragments;
generating tumor-associated SPPC; screening the antigen-binding fragments with the complex to 30 obtain antigen-binding fragments that bind Specifically to tumor-associated SPPC; and screening the antigen-binding fragments obtained for ce11 surface tumor-associated reactivity.

iziusiea wr:~ i7:aa r~a~ sm asz usz' ttmuu~r a mnY~r;r; ~u4i As discussed in greater detail below, p~xrtacularly with reference to specific Examples heroin, the final scrttning step is preferably accomplished by screening the antigun-binding fragments obtained with at least one and preferably several cell lines derived from ono or more cancer types anal at least one and preferably several normal non-cancerous cell types. Suitable screening methods and parameters are known in the art and art also described in the Examples with respect to antibody H I 1.
C. Polynucleotides The invention encompasses compositions containing polynucleotides encoding the antigen-binding fragments. Recombinant vectors containing the polynucleotidcs and host cells transfected with the vectors are also encompassed by the invention.
The invention encompasses compositions containing poiynucleotides encoding the peptide portion of an SPPC. In the case of C-antigen, the invention encompasses polynueleotides encoding the peptide portion of the complex.
D. Kits.
The invention encompasses kits comprising tbie antigen-binding fragments of the invention and buffers, labeling agtnts, toxins and radioisotopes necessary for the diagnostic or therapeutic use of the antigen-binding iiagments-Tlae invention further trtcompasses kits comprising the SPPC or peptide portion thereof of the invEntion and buffers, adjuvants etc, for the therapeutic and/or immunogenic use of the compositions.
E. Compositions.
The invention encompasses therapeutic or pharmaceutically or physiologically acceptable compositions of matter. These compositions include an active eornponent connprised of the SPPCs, peptides, antigen-binding fragments and polynucleotides described herein and a physiologically acceptable buffer, vehicle or excipiez~t thereof. Preferably, the active component is prGSent in an "effective amount," that is, an amount to effect the desired result such as amelioration or palliation of symptoms or imaging. In particular, the antigen-binding fragments are suitable for inhibiting metastases. Methods of use therein and compositions for use therefor are further encompassed by the invention.

1L/U8/88 WJD 17:45 b'AA 41U ;fkiL USL:1 H1DUU'1' !k mAYBIJ~øJU4L
F. lVtethods of Treatment.
The invention encompasses methods of treating cancer patients. The methods comprise administering to the patient a therapeutically effective amount of an antigen-binding fragment of the invention. The methods further comprise administering to the patient an immunogeni.c amount of an SPPC or SPPC peptide of the invention.
Tlle invention also encompasses a method of treating a cancer subject compzising administering to the subject an amount of a composition of matter comprising an isolated antigen-binding fragment specific for a tumor-associated SPPC and a physiologically acceptable excipicnt effective to elicit a cancer-specific immune response.
The invention further encompasses a method of treating a cancer subject comprising administering to the subject an amount of a composition of manor comprising an isolated antigen-binding fragment specific for a tumor-assveiated SPPC and a physiologically acceptable excipient efl:eetive to ameliorate the cancer.
G. Additional Methods of Use.
The invention encdmpasscs methods of inducing a tumor-specific immune response in a subject. The methods can be used fvr cancer treatment as above, or as a preventative measure, particularly in a subject at risk for cancer. The methods include administering to the subject an amount of an active effective to induct a cancer-specific immune response in the subject. The active can be tumor-associated SPPC or an antigenic fragment thereof or an anti-idiotype anti-SPPC antibody.
The invention also encompasses methods of detecting or imaging cancer cells.
In the case of in vitro detection, labeled anti-SPPCs are incubated with biological samples under conditions and for a time sufficient to allow specific binding of the anti-SPPCs to cancer cells.
Unbound anti-SPPCs are then removed and bound label measured or detected. In the case of imaging, labeled anti-SPPCs are administered to a patient (either having or suspected of having cancer), or animal model system in an amount and under conditions sufficient for the anti-SPPCs specifically binding to cancer cells. Excess ar non-specifically bound anti-SPPCs are removed, if necessary, and bound anti-SPPCs are detected.
The invention also encompasses compositions and methods of use thereof in diagnostic ~0 antibody clearance. Anti-SPPC can be administered to an individual who has received a labeled anti-SPPC the course of radioscintigraphy or radiotherapy to remove the label.
Effective 1G/U~f/H8 Wll) 17:45 b'Ax 41H :fUL USGJ HlDUIi'1' ~ ~tAYB~IIø1U43 imaging using radiolabeled antibodies is hampered due to excess circulating radiolabclod antibody, which often takes several days to clear. Accordingly, the SPPC
recognized by the anti-SPPC is administered to the individual at a specified time after administration of the labeled anti-SFPC. Antigen that is complexes with the antigen-binding fragments at sites other than the tumor, such as in the circulation and interstitial spaces, promotes clearance of non-bound antibody and decreases background radiation. As a result, the level of label in unaffected tissues is reduced, and the image of the tumor (in comparison to neighboring tissues) is enhanced.
The invention further encompasses methods of monitoriung progress and efficiency of anti-cancer therapy. In this case, cancer patients undergoing chemotherapy or other form of anti-cancer therapy are treated as described far diagnostic imaging but repeatedly and at defined intervals. A decrease in tumor burden as indicated by decreased antigen-binding fragment binding is indicative of successful chemotherapy.
In another aspect the invention is directed to a kit, which comprises a CCP
library as pzesented by PpEs, and a antigen-binding fragment library for screening such CCP library.
According to another aspect of the invention, the invention is directed to antigen-binding fragments, which bind to at least one tumor-associated SPPC, and preferably to a plurality of such SPPCs that share a common consensus peptide motif. Such antigen-binding fragonents arc screened against a panel of different tumor types to identify positive clones which are specific for one or more tumor-associated SP peptide complexes and a correspondingly wide variety of tumor single and multi-tumor speciftcities. In a preferred embodiment of the invention such variants of H11 antigen-binding fragments are mufti-carcinomic anti-SPPC, which bind to consensus peptide motif which is enriched for hydrophobic residues. Various embodiments of the preferred hydrophobic motifs art presented in the claims.
Alternatives to generating $P-CCP complex libraries arc described herein.
According to one such alternative, suitable cells are caused to uptake one or more selected CCPs transfected with nucleic acid constructs comprising a polynucleoddc encoding one or selected CCPs. In the case of professional APCs, the cells are then directly screened with antigen-binding fragments gs defined in the preceding aspect of the invention. Alternatively, Sl?PCs are then axtractcd from such cells, partially purified and screened with tumor-associated antigen-binding fragments according to a preceding aspect of the invention is order to identify antigtn-binding fragments and corresponding SPPCs which are tumor-associated. The positive clone wbieh shows strong 1L/U8/HN W~ 17:4tf b'AIL 41B :flit UZSGJ HlDUIi'1' Ik mAYBlrI~IU44 reactivity identify the 5ClGCted consensus conforming peptide and link these CGPs to one or more particular tumors.
As an alternative to generating a library of SP-GCP complexes, selected consensus confa~n»ing peptides can be generated in a pepride display library or presented in the form of 3 PPEs and Screened with tumor-associated antigen-binding fragments representing a wide variety of single and mufti-tumor specificities.
Preferred consensus conforming peptides are identifitd according to one or more of the following criteria:
( 1 ) strongest representation in databases of human proteins;
(2) strongest representation in preferred SP binding peptides (see W4 99/22761 );
strongest conformity to peptides motifs which bind to H 11 _ Vu. BRIEF D .S;E~-R'~PT10N OF THE D AWIN t~
Figure 1 depicts flow cytometric analysis of cells recognized by H11.
Figure 2 depicts a flow cytomctric analysis of cells recognized by H11.
Figure 3 depicts binding of H11 to tumor cell extracts.
Figure 4 depicts binding of H11 to tumor cell extracts.
Figure 5 depicts binding of H 11 to human tumor cell lines.
Figure 6 depicts a schematic of the expression vector pSJF 1.
Figure 7 is a graph depicting rneatt tumor volume per day after treatment with H11 seFv (closed circles) or PBS (open circles).
VIII. DETAIt.RD D)~ C ~TIOhI OF TH PREF .RRF.n :MT~ODIMENTS
Antigen-binding fragments that bind to SPPCs are more generally described in our co-pending application No. 601149,587.
The terns "associated portion" as used herein with reference to a Pl?B refers to the portion of the PPE other than the predetermined peptide portion and includes a polypcptidc (or other moiety) to which the predetermined peptide portion is terminally or otherwise, linked two non-contiguous portions of a polypeptide in which the predetermined peptide portion is peptide segment, an MHCI, a heat shock protein, which forms a complex with said predetermined peptide portion, etc. The associated portion can actively (by imparting conformation to) or .41 W~uB~eH wry 17:9a rv~ 41a Paz UriG;1 xWUrr !k ~tAYBt;r~ ~øJU95 passively ("linkage" without imparting conformation) present the prcdetcrmined peptide portion in which it shares antigenic determinants with an Sk'I'C, for example in the same conformation which the SP imparts to the SPPC peptide.
Tire term "pz~sdetermined peptide portion" refers to a peptide, for example, a 7 mcr, which is selected on the basis that it has a particular amino acid sequence or constitution or a particular consensus nnotif. The predetermined peptidt parrion can be linked to any other suitable ligand provided the ligand does not interfere with antigen presentation. Ligands that enhance binding to SPs are described for instance, in WO 97/06821; and WO 99/22741.
Methods of making "DNA vaccines" using polynueleotides encoding various subjects of consensus conforming peptides according to the invention have been described for various application. Reference is xxxade to US Patent Nos. 5,580,859 and 5,589,466, which describe the generation of nucleic acids that can by used in "DNA vaccine" applications as well as US Patent Nos. 5,843,913, 5,814,617, 5,811,406, 5,736,524, 5,676,954, 5,620,896, 5,593,972, 5,589,466 and 5,580,859. The disclosures of all of which are hereby incorporated by reference.
Methods of loading consensus conforming peptides onto antigen presenting cells, particularly dendritic cells (DCs), are described in references provided herein. Methods to isolate DCs from blood and the expansion of these in vitro to yield APCs for clinical use in immunotherapy is described in references provided herein.
All references disclosed in this application are hereby incorporated by reftret~ce 1 ) Pardoll B.M. (1998) Cancer vaccines. Nat. Med., 4:525-531; 2) Young J.W. and Inaba I~. (1996) Dextdritic cells as adjuvants for class I major histocompatibility complex-rtstzicted anti-tumor immunity..l. Exp. Med-, 183:7-11; 3) Hart I. and Colaco C. (1997). Fusion induces tumor rejection. Nature, 388:627-628; 4) Banchereau J. and Steinman R.M. (1998).
DendritiC cells and the control of immunity. Nature, 392:245-252; 5) Hakkar A.B.H, et al_ (1995).
C3eneratian of anti-melanoma cytotoxic T lymphocytes from healthy donors after presentation of melanoma-associated antigen-derived epitopes by dendritic cells in vitro. Cancer Res., 55'5330-5334; b) PogadorA. and Gilboa E. (1995). Bone marrow-generated dcndritic cells pulsed with class-I
restricted peptides are potant inducers of cytotoxic 'f-lymphocytes. J Exp_ ~Ied., 182:255-260;
7) Hsu F.J. et al. (1996). Vaccination of patients with B-cell lymphoma using autoiogous antigen-pulsed dendritic cells. Nat. Med., 2.52-58; 8) Boc~kowski D. et al.
(1996). Dendritic cells pulsed with ltiVA are potent antigen-presenting cells in vitro and in vivo. J. Faep. Med., >_ziusiea w» i7: a~ r~a~ am adz usca xmuu~r ~ mnYt;~~
184:465-472; 9} Gong J, et al. (1997). Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. Nat. Med_, 3.558-5~1; 10) l~lurphy Gr. et al. (1996).
Phase I clinical trial: T-cell therapy for prostate cancer using autologous dendritic cells pulsed with HLA-A0201-specific peptides from prostate-specific membrane antigen.
Prostate, 29:371-380; and, 11 ). Nestle F.O. et al. ( 1998). Vaccination of melanoma patients with peptide- or tumor lysatt-pulsed dtndritic cells- Nat. Med., 4:328-332.
A. SPPCs The invention encompasses compositions comprising tumor-associated SPPCs. The invention also encompasses compositions comprising substantially isolated SPPC
peptide.
1. Characterization It has now been found that an antigen designated "C-antigen" which is found on a variety of cancer cells but only at low levels or not at all on normal, non-cancerous cells, consists of a SIpPC. This antigun was previously dtsCribtd by its immunologic reactivity with an antibody designated H11, but was not previously isolated or characterized. H11 is described in detail in W097/44461. Tht antigen comprises the complex of SP and a peptide; H11 binding is lost when the complex is disgociated. H11 Specifically recognizes a broad range of many, but not a11, neoplastic cells. The specificity of H11 includes, but is not limited to, glioblastoma, neuxoblastoma, malignant melanoma, breast adenocarcinoma, lung adenocarcinoma, small cell lung carcinoma, colon adenocarcinoma and prostate adenocarcinom:~.
The term "polypepti.de", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues of any length. ?he polymer can be linear or branched, it can comprise modih.ed amino acids or amino acid analogs, and it can be interrupted by chemical rnoieties other than amino acids. The terms also encorttpass an amino acid polymer that has been modified naturahy or by intervention; including, but not limited to, disulfide bored formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioaetive component.
Unless stated or implied otherwise, the term aatig$n-binding fragment includes any polypeptidc monomer or polymer with immunologic specificity, including the intact antibody, and smaller and larger functionally equivalent polypeptides, as described herein. With respect to "stress protein-peptide complex," "peptide" refers to the peptide moiety non-coval.ently 4omplexed specifically to SP.

1G/U8/N8 WJU 17:47 b'AA 41B ;iifG U~1Y;! ltIDUU'1' ~C mAYBI;JløJU47 PVlaintcnancc of the complex is typically ATP dependent and can be dis$Qciate~
by the removal of ATP. Dissociation also occurs under denaturing conditions.
The eomplexed pepride is endogenous. Endogenous peptides are native peptides complexed with SPs in vivo. Native peptides can be those associated with SPs in vivo or modifications thereof including those made by associating a peptide with a SP
in vitro to form a complex which is antigenically similar to that found in vivo, particularly so as to be specifically reactive with the same antigen-binding fragment. Native peptidos and modified peptides can be made by recombinant DNA techniques, peptide synthesis and other methods known in the art. In vitro complex association can be obtained with peptides either isolated from a mammalian source or obtained by recombinant means. Such peptides can be isolated and sequenced by any method known in the art. These methods include, but are not limited to, those of Kassel et al.
(1994) Anal. Chem. 6G:23G-243; and Kistlar et al. (1999) Anal. Chem.
71:1792.1801.
The invention also encompasses compositions comprising at least one SPPC, which is specifically immunagenieally crass-reactive with one or more cell surface-associated SPPCs specifiic to a target cancer. In particular the SPPC contains a non-covalently bound peptide, which confers the specific immunogenicity.
The invention also encompasses compositions corr~pzising a plurality of SPPCs which arc specifically immunogenically cross-reactive with one or more cell surface-associated S>PPCs specific to a target cancer. In particular the SI~PCs Contain different non-covalently bound peptides, which confer the specific imrnunogenicity.
Preferably, for the purposes of tumor-specific treatment, SPPCs are "tumor-associated."
The use of disease-associated SPPCs for treatment is also encompassed by the invention. The SPPCs of interest might n.ot be found exclusively on cancer cells but might also be found on other cells. To the extent SlSPCs are on normal ells not found associated with tumors, it is at a level of detection below that of the invention. Therefore, as used herein, "not on normal cells"
indicates that the SPPCs have not yet been detected on normal cells. However, non~ial cells could expross SPPCs if diseased. In this sense, the tcnan "tumor-associated"
complexes encompasses both tumar.specifie and disease-associated. Accordingly, it is contemplated that a SPPC and/or an antigen-binding fragment specifically reactive therewith, obtained according to the methods defined herein, can be useful therapeutically against other diseases, particularly, virally or otherwise infected cells or tissues.

iziuaiea wrl 17:47 rw 4ia ;tnz usz~ xmuu~r ~ m~tY~~~ ,~~4s "Stxcss protein"("SP", "hsp") refers to any member of the various families of heat shock proteins. These families include, but are not limited to, hsp26, hsp44, hsp60, hsp70, hsp94, and hsp100. Preferably, the SPs are hsp72, hsp$5 and hsp96- Most preferably the SP
is hsp72, 2. 1 lat'on of SP .s. ~1'P~~ntide and C-a_n~g~;
Exemplary methods for isolating SPPCS in general and C-antigen in particular fpllOw. It is understood that the isolation methods can be modiFed by the addition or deletion of steps and changes in the steps within functional parameters. Provided the result of the isolation of at Icast ono SPPC as defined and described herein and as detected or measured as described herein, the isolation method is encompassed by the invention- C-antigen is best characterized by obtaining the antigen following such a procedure and, particularly, the procedtues more specifically provided in the Examples.
By way of example, tumor-aggociated SPPC oan be purified t4 suitable homogeneity with the following protocol:
Tumor cells grown in tissue culture had their n-~embr2:ne5s disrupted, and an extract made by freeze-thaw. In particular detail, after cell harvest, cells are centrifuged at 1500 rpm for 10 min. The cells are washed twice in a PBS/1 mM phenylmcthyl-sulfonyl fluoride (PMSE)110yg/ml aprotinin solution. After washing, the pellet is resuspended in the wash solution and the cell concentration is adjusted to LO-24 x 106 cells/mL. This suspension is then subjected to ~vc freeze-thaw sequences consisting of froezing in a dry-ice-acetone solution, followed immediately by thawing in a 37°C water bath. After the freeze-thaw treatments, the extract mixture is centrifuged at 1000 rpm to obtain a pellet of cellular debris and a supernatant.
The supernatant is combined with 3M ammonium sulfate buffer in a 2:1 ratio.
This sample is then loaded onto a general-purpose hydrophobic chromatographic meduun (preferably Phenyl Sepharose) at a rate of 4.5 ml/min using a pump. The column is connected to an FPLC
system. Once loaded, the column is washed with 15 column volutrtes (C~ of Buffer A (50 mM
sodium phosphate and 11~ ammonium sulfate pH 7.0). The bound proteins arc eluted step-wise with Huffer B (50 tnM sodltun phosphate pH 7.0). Activo fractions are determined by immunolagical methods. During elution, the bulk of the bound proteins are eluted with 30%
Buffer A170% Buffer B. The 70% Buffer B elution is followed by 100% Buffer B.
SPPC is eluted in the latter fraction. The positive fraction is concentrated on a membrane concentrator with a MW cut-off of 10 kl~, preferably a Centriprep 10. The concentrated sample is passed iziusiaa wxo i7:as rvx am sac usz;~ xmuu~r ~ mnY~r;x ~uaa through a buffer exchange medium (preferably G-25) to the ADP-agarost chromatographic Buffer A (20 mM Tris-acetate, 20 mM NaCI, 3 mM MgClz,pH 7.5).
Six mL of the buffer exchanged material is incubated overnight with an additional 4 mL
Buffer A and 5 mL ADP-agarose at 4°C on a platform shaker. Following incubation, the mixture is poured into a 716 x 40 column. The column is washed with the ADP-agarose chromatographic Buffer A until the OD at 280 reaches baseline. The column is further washed with O.S1VI NaCI in chromatographic-Buffer A and re-equilibrated with Buffer A. The bound protein is then eluted with 3 n~lVl ADP in the ADP $ufler A and fractions collected. The active fraction is cottcentrated on a membrane concentrator with a MW cut-off of 10 kD (preferably Amicon).
The concentrated, eluted sample is diluted with anionic chromatographic Buffer A (2Q
mM Tris pH 7.8) at 1:10. One mL of diluted sample is loaded onto a strong anionic column (preferably a Mono Q Sepbarose) attached to an FPLC. The flow raft is set at 1 ml/nzin.
Fractions are collected and the antigenic fraction identified as outlined above. This three-step procedure gives a suitable, substantially homogeneous, active, SFPC.
ADP chromatographic media are media to which ADP is bound, and includes, but is not limited to, AI~P bound to Sepharose and agarose. Preftzably the medium is ADP
agarose.
Although the preceding method applies most aptly to hsp70 (particularly as detailed below) and (with limited routine modification, if any such modification is required) to hsp60, it can be used for tumor-associated SPPCs that are determined to be of the hsp20-30 and hsp40 families (with necessary modifications according to routine skill in the art). Additionally, in the case of the hsp90 family, a lectiun oolumn, preferably a Concanavalin A column, can substitute for the ADP
chromatagxaphie media described above.
Optionally, i~o a preferred method, C-antigen and other such SPPCs can be further purified under non-denaturing conditions, preferably in an electrophoretic extraction step_ For example, after final concentration from the anionic column (particularly, in the case of C-antigen, the SPPC is already substantially purified), lSpL ofthc complex is mixed SO150 with 2X Laemmli's buffer. The sample is separated on a suitable polyacrylamide gel electrophoresis apparatus under native, non-denaturing conditions (no SDS, mercaptoethanol or boiling). After completion of electrophoresis, the gel is blotted onto a membrane (PVDF or nitrocellulose) again under non-denaturing conditions. Identification of the SPPC location on the blotted membrane is iziuaiue wru i7:as rv~ aia aaz uaz~ xmuu~r ~ mAY~r;r~ ~u~u confirmed by treatzztent with an anti-SPPC antigen-binding fragment followed by binding an appropriately labeled secondary antibody. The SPPG oan be "cut"' from the membrane and the bound SPPC can be treated to cause the release of the peptide from the eornplex and subjected to further analysis. For example; the membrane can be treated to cause the release of the complex and the subsequently released peptide can be analyzed, for instance. by capillary electrophoresis and sequentially applied to a MALDI mass spectrograph.
An alternate method for the purification of SPs is developed from the creation of affinity chmmatographie media of SPPC specific IgG antibodies or fragments thereof, for example th.e recombinant H11 IgG described in WO 97144461. A 5 mL sample from a hydrophobic column (preferably Phenyl Sephaxose) is incubated with 2 mL of SPPC-specific IgG
Sepliarose. The IgG-Sepharoselsample is incubated over-night at A~°C on a rotary shaker. Ai~er incubation, the mixture is poured into a small chroroatagraphie eoluzrin (preferably Bioltad 10 mr, lrcono-Column). The column is washed with ten column volumes (CV) of PBS (pH 7.4) followed by throe CV of 4.5 M NaC1 in pl3S. The affinity column is then re-equilibrated with QBS.
Following equilibration, SPPC is eluted using a glycine buffer pH 2.8. The eluate is concentrated on a micro-pare concentrator (preferably Centriprep 3). The acid elution results in the dissociation of the SP from its peptide. The small molecular weight fraction (peptide) is concentrated with a peptide concentrator (preferably Microcan SCX). The purified SP is retained on the micro-pore concentrator.
After concentration, the eluted mixture of peptide and SP dissociated complex is passed through a peptide concentrator (preferably Microcon-SCX). The resultant material is freeze dried, and re-dissolved in 0.1% TFA. After re-constitution the material is fractionated on a reverse-phase PLC column. Fractions are analyzed directly on a MALDI mass spectrometer.
Reconstitution of the peptide with the SP can be effected by any method known in the art such as mixing fhe affinity column purified SP with the peptide (purified native, recombuoant or synthesized peptide) in P13S in the presence of 1 mM ADP and 1 mM MgCl2 and incubating at 37°G for 30 min. Other suitable methods are described for instance in Davis et al. (1999) Proc.
Natl. Acad. 5ci. US,A 96:9296.
3. Isolation of SPPCs using an gG Aff-it 'tv Column sing Alkaline Elution Buffer 100 mL of A-375 cell extract is centrifuged at 1400g far 30 min and the supernatant collected. The supernatant is then diluted five times with I~iTrap Q buffer A
(Tris 20 mM, pH
d7 5~ 08/12/1888 x17:50 X416 362 OB23 received 1C/U8/88 W1':U 17:51 t'A~, 41U aGL U~il~! lilDUU'1' tk ~fAYBI~;JløJUUL
8.2). Diisopropyl fluorophosphate (DFP), a protease inhibitor, is added to a final concentration of 1 nzM. The sample is loaded on a 10 mL anionic HiTrap Q column at a rate of 3 Sl~ mLlmin.
The column is then washed with 15 CV of 50 SP mM NaCI in HiTrap Q buffer A.
Hound protein is eluted with 20 CV of a 50 mM to 600 SP mM NaCI gradient and 10 SP
mT. fractions collected. Fractions are concentrated using Centriprep 10. The SPPC-containizlg fractions are identified by western blot analysis using H11 IgG as the primary antibody and an appropriately labeled second antibody.
SPPC, partially purified tlsrough the HiTrap Q anionio column, is applied to the IgG
affinity column and incubated for 2 hours at room temperature with gentle rotation. Following l0 incubation, the column is washed with 20 SP mL TBS (iris 20 SP mM, NaCI 150 nnM, pT~i 7.4).
The bound S15PC is eluted with 50 SP mM diothylamine pH 11. The eluted material is cax~centratod and the purity determined using Western blot analysis. ?he results show that, under these conditions, the SPPC is eluted intact.
4. ,6- pecifc Purification In lieu of the ADP agarose purificatiatt step, hsp9G (Grp9b) complexes can be purified as described by ~lahore et al. J. Exp. Med. (1997) 186:1315-1322. Cancer cell extract is applied to a lectin column, specifically concanavalin A and incubated over-night at 4°C. The SP is eluted from the column with 10% a-methylmannoside. The h5p90 active fractions are concentrated on a micm-pore filter (preferably Centriprep 10).
SPPCs can be isolated from other diseased cells in a manner analogous to the methods described herein for isolarion of such complexes from tumar cell extracts, for example cells which are virally or otherwise infected, with the result that the screening protocols described herein for differentiating between tumor and nan-tumor cells could be analogously applied, by persons skilled in the art, according to methods within the skill of those in the art, to identify: 1) complexes that are found on tile surface of infected but not tumor cells; and 2) antibodies which react specifically with such complexes.
5. ~ to ated P~ tn ide The invention fbrther encompasses compositions comprising the isolated, disassociated SpPC peptides of the invention and fl~nctionally equivalent fragments and derivatives thereof. In the case of C-antigen, the invention encompasses peptides containing at ).east 5-10 amino acid residues of the peptide sequence.
4$

iiiusiaa wr;~ i7 _5i rw aia :taz uaz;~ Hmuu~r ~ mdrYar;r; ~uus cit 1. i a - nt her This invention encompasses antigen-binding fragments that specifically recognize SPPCs in a tumor-or disease-assQCiated manner. That is, in the case of tumors, the SPPC is predominantly found an tumor ceps such that antigen-binding fragments that recognize the complexes preferentially t~ecognize or bind to cancer cells. The team ''disease-associated" means associated with cancer as well as one or snore other patk~ologic conditions that induce cell surface expression of $PPC.
The invention further encompasses a composition of matter comprising an isolated antigen-binding fragment of an antibody which binds specifically to at least one SPPC which is specifically immunogenically cross-reactive with one or morn cell surface-associated SpPCs specific to a target cancer. In particular, the at least one SPPC contains a non-covalently bound peptide which confers the specific immunogenicity.
The invention fuzther encompasses a composition comprising an antigen-binding fragment of an antibody which binds specifically to a plurality of SPPCs which is specifically immunogenically cross-reactive with one or more cell surface-associated SPPCs specific to a target cancer. In particular, the SPPCs contain different non-covalently bound peptides, which confer the specific immunogcnicity.
The term "atrtigcn-binding fragment" includes any peptide that binds to the Cancer-specific SPPCs in a ca~acer cell-specific manner. Typically, these fragments include such immunoglobulin fragments as Fab, F(ab')z, Fob', seFv (both monomer and polymeric forms) and isolated H and L chains. An antigen-binding fragment retai~as specificity of the intact imtttunoglobulin, although avidity and/or affinity can be altered- First generation therapies are those based on such compounds and compositions. lrspecially preferred are the anti-C and anti-SPPC scFvs.
">'I11." the exemplary anti-SPPC antibody is an antibody obtained from the fusion of peripheral blood lymphocytes of a 64 year old male with a law grade glioma and-fused to a human myeloma cell line to produce a hybridoma designated NBGIvtI IH1 i. The generation and characterization o~Hl l is described in Example 1. "Anti-C" represents any antibody, or antigen-binding fragment thereof, either monoclonal, polyclvnal or derivative thereof tl.~at recognizes iziusiaa _w~ i7:5i b'A~ 41f1 ;ftiL Ot~CJ ltlyUli'1' tk mnYBr;Jt~JUUa specifically the C-antigen and distinguishes between cancer and nOncancer calls. Anti-C, as defined herein, does not include H11 and its derivatives.
Certain compounds, compositions and rnetliods described in this application relate generally to anti-C and derivatives thereof which can be generated routinely by standard im~munochcmical technidues. This includes, but is not limited to, anti-C
coupled to another compound by chemical conjugation, or associated with by mixing with an excipient or an adjuvant. Specific conjugation partners and methods of making them arc described herein and well known in the art. More preferred are anti-C and anti-SppC scFvs that are not coupled to a chemical agent.
Antigen-binding fragments (also encompassing "derivatives" thereof) are typically generated by genetic engineering, although they can be obtained alternatively by other methods az~d combinations of methods. This classification includes, but is not limited to, cnganeercd peptide fragments and fusion peptides. Preferred compounds include polypopride fragments containing the anti-Sl?-peptide CDRs, antibody fusion proteins contai~oing cytokine cfif'ectar components, antibody fusion protei~os containing adjuvants or drugs, and, single-chain V region proteins. Antigen-binding fragments are considered to be of human origia if they are isolated from a human source, and used directly or cloned (either intact genes or portions thereof) and expressed in athcr cell types and derivatives thereof.
A "fusion polypeptide" is a polypeptidc comprising contiguous peptide regions ini a different position than would be found in nature. The regions can normally exist in separate proteins and are brought together in the fusion polypeptide; thoy can normally exist in the same protein but are placid in a new arrangement in the fhsion polypeptide; or they can be synthetically arranged. Fox instance, as described below, the invention encompasses recombinant proteins (and the polynucleotides encoding the proteins or complementary thereto) Z5 that are comprised of a functional portion of an antigen-binding fragment and a toxin. Methods of making these i~sion proteins are known in the art and are described far instance in W093/07286.
A "functionally equivalent fragment" of a polypeptidc varies from the native sequence by any combination of additions, deletions, yr substitutions while preserving at least one .functional property of the fragment relevant to the context in which it is being used.
The antigen-binding fragments are designated anti-SP-peptide.

iziusiea wru i7:5z rv~ aia ~snz usza xmuu~r ~ mnYxr;>; ~uus The antigen-binding fragments provided herein arc useful in palliating the clinical conditions related to a wide variety of cancers. The invention encompasses antigen-binding fragments (excluding fIl l) recognizing C-antigen. Those are designated anti-C_ The invention further comprises polypeptide derivatives of the antigen-binding fragments and methods for using these compositions in diagnosis, treatment, and manufacture of novel reagents.
The invention also encompasses antigen-binding fragments conjugated to a chemically functional moiety. Typically, the moiety is a label capable of producing a detectable signal.
These conjugated aatigen-binding fragments are useful, for example, in detection systems such as quantitation of tumor burden, and imaging of metastatic foci and tumor imaging. Such labels are known in the art and include, but arc not limited to, radioisotopes, enzymes, fluorescent compounds, chemilumincsccnt compounds, bioluminescent compounds, substrate cofactors and inhibitors. For examples of patents teaching the use of such labels, sec, for instance Lf.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. T'he moieties can be covalently linked, rccombinantly linked, or conjugated (covalently or non-1 ~ covalently) through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex.
Other functional moieties include signal peptides, agents that enhance immunologic reactivity, agents that facilitate coupling to a solid support, vaccine carritrs, bioresponse modifiers, paramagnetic labels and drugs. Signal peptides art described above and include prokaryotic and eukaryotic forms. ~0.gents that enhance immunologic reactivity include, but are not limited to, bacterial superantige~s and adjuvants. Agents that facilitate coupling to a solid support include, but arc not limited to, biotin, avidin or derivatives thereof. Immunogcn carriers include, but are not limited to, physiologically acceptable buffers.
Bioresponse modifiers include, but are nit limited to, cytokines, e.g. tumor necrosis factor (TNP'), IL-2, interleulcin-4 (IL,-4), GM-CSF; and certain interferons. See also, U51'atent 5,750,119; and WO patent publications: 96/10411; 98134b41; 98123735; 98/34642; 87/10000; 97110001; and 97106821.
A "signal peptidd' or "leader sequence" is a short amino acid sequence that directs a newly synthesized protein through a cellular membrane, usually the endoplasmie rcticulum in cukaryotic cells, and zither the inner membrane or both inner and outer membranes of bacteria.
Signal peptides aze typically at the N tet~ninus o~a polypeptide and are removed enzymatically _. l~/U~i/88 W~ 17:5G b'A11 4ltl ~UL USZJ ltlDUl~'1' & ~tAYBJrJløJUUt3 between biosynthesis and secretion of the polypeptide from the cell. Thus, the signal peptide is not present in the secreted protein but is present only duriu~g protein production.
Suitable drug moieties include antineoplastic agents. These include, but are not limited to, fSdlolSOtopCS, immunotoxins, vireo alkaloids such as the vinblastine, vincristine and vindesine sulfates, adtYamycin., bleomycin sulfate, carboplatin, cisplatin, cyelophosphamide, cytarabinc, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, lomustine, mechlorotthamine hydrochlori~te, melpbalan, mezcaptopurine, methotroxate, mitomycin, mitotane, pentostatin, pipobroman, procarbaze hydrochloride, streptoxotocin, taxol and analogs thereof, thioguauine, and uracil mustard.
Immunotoxins, including single chain conjugates, can be produced by recombinant means. Production of various im,znunotoxins is well known in the art, and methods can be found, for examgle, in "Monoclonal Antibody-toxin Conjugates: Aiming the Magic Bultct," Thorpe nt al. {198Z) Monoclonal flnttbodi~s in Clinical Medicine, Academic Press, pp.
168--190; Vitatta (1987) Science 238:1098-1104; and Winter and Milstein (1991) Natr~re 349:293-299. Suitable toxins include, but are not limited to, ricin, radionuclides, ,pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins suah as fbngal ribosome inactivating proteins such as gelonin, restrictocin and phospholipase enzymes.
See, generally, "Chimeric Toxins," Dlsnes and Pihl, ,t'harmac. Ther. 15:355-381 ( 1981); and "Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Hyers, pp. 159-179, 224-266, Academic Press (1985).
The chemically functional moieties can be made recombinantly for instance by creating a fusion gene encoding the antigen-binding fragment and functional regions from other genes (c.g.
enzymes). in the case of gent fusions, the two components are present within the same gene.
Alternatively, antigen-binding fragments can be chemically bonded to the moiety by any of a variety of well known chemical procedures. For example, when the moiety is a protein, the linkage can be by way of hetcro-bifllnetional cross linkers, e.g., SPDP, carbodiimide glutaraldehyde, or tl~o lika. The moieties can be covalently linked, or conjugated, through a secondary reasent, including, but not limited to a second antibody, protein A, or a biotin-avidin complex. Paramagnetic moieties and the conljugation thereof to antibodies are well-known in the art. See, e.g., Miltcnyi et al. (1990) Cytometry 1 t:231-238.

iziusiaa wru i7:~s rw am 'nz usz' xmuu~r ~ mnYxxx ~uu7 Methods of antibody production and isolation arc well lmown in the art. See, for example, Harlow and Lane ( 1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The H11 antibody is a human immunoglobulin of tha IgM
subclass, and can be isolated by any technique suitable for inomunoglobulins of this isotype such as by salt fractionation coupled with size exclusion to obtain a crude isolate- Antibody purification methods include, but are not limited to, salt precipitation (for example, with arrunonium sulfate);
ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength); gel laltration chromatography (including gel ~ltratidn HPLC); and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin. Antigen-binding fragments can also be purified on affvoity columns comprising the C-antigen or an antigenic portion thereof.
Preferably fragments are purified using Protein-,A-CL-SepharoscT"" 48 chromatography followed by chromatography on a DEAI;-SepharoseTM 4B ion exchange column.
The invention also encompasses hybrid antibodies, for instance xn which one pair of H
anti L chains is obtained from a first antibody, while the other pair of I-I
and L chains is obtained from a different second antibody. For purposes of this invention, one pair of L and H chains is from anti-SP-peptide. bn one example, each L-H chain pain binds different epitopes of the C-antigen. Such hybrids can also be formed using humanized H or I~ chains. The invention also encompasses other bispecific antibodies such as those containing two separate antibodies covalently linked through thtir constant regiozts.
Other antigen-binding fragments encompassed by this invention are antibodies in which the H or L chain has been modified to provide additional properties. For instance, a change in amino acid sequence can result in reduced immunogcnicity of the resultant polypeptide. The changes range from changing one or more amino acids to the complete redesign of a region such as a C region domairf. Typical changes include, but are not limited to, those related to complement fixation, inkeraction with membrane receptors, and other effector functions. A
recombinant antibody can also be designed to aid the specific delivery of a substance (such as a cytokinc) to a tumor cell. Also encompassed by the invention are peptides in which various immunoglobulin domains have been placed in an order othar than that which occurs in nature.
The size of the antigen-binding fragments can be only the minimum size required to provide a desired function. It can optionally comprise additional amino acid sequence, either lY/U8/88 Wr:U 17:5;f r'A~ 4ltf ;flit U~iY~! HlDUIi'1' Ik ~tAYBHJ~øJUUEi native to the antigen-binding fragment, or from a heterologous source, as dasired. Anti-SPPCs can contain only 5 consecutive amino acids .from an anti-SP-peptide V region sequence.
Polypeptides comprising 7 amino acids, morn preferably about 10 amino acids, mare preferably about 15 amino acids, mare preferably about 25 amino acids, more preferably about 50 amino acids, more preferably about 75 amino acids from the anti-SP-peptide L or H
chain V region art also included. Even more preferred are polypeptides, comprising the entire anti-SP-peptide L or hI chain V region.
Substitutions can range from changing or modifying one or more amino acid residue to complete redesign of a region, such as the V region. Amino acid substitutions, if present, are preferably conservative substitutions that do not deleteriously affect folding or functional properties of the peptide. Groups of functionally related amino acids within which conservative substitutions can be made are glycinc/alaninc; valinelisolsucinc/Icucine;
asparagine/glutamine;
aspartic acid/glutamic acid; serine/threoninelmethionine; lysine/arginine; and phenylalanine/tryosine/tryptophan. Antigen-binding fragments of this invention can be in glycosylated or unglycosylated form, can be modif.td post-translationally (e.g., aeetylation, and phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group}.
Polypeptida derivatives comprising both an L chain arid an H chain can be formed as separate L and H chains and then assembled, or assembled in situ by an expression system for both chains. Such expression systems can be created by transfecting with a plasmid comprising separate transeribable regions for the L and H chain, or by co-transfecting the same cell with plasmids for each chain. In a third method, a suitable plasmid with an H chain encoding region is transfccted into an H chain loss mutant.
I~ chain loss mutax~ks can bo obtained by treating anti-SP-peptide producing cells with fluorescein-labeled rabbit anti-mouse IgIJ (H chain specific. DAKO
Corporation, Carpinteria, CA) according to the supplier's instruction. 'The stained and unstained cell populations are analyzed by flow cytometry. Unstained cells arc collected in a sterilized tube and placed in 96-vvell plates at l cell/well by limiting dilution. Culture supernatants are then assayed by ELISA
using goat anti-mouse IgG (H chain specific) and goat anti-rnou.se kappa.
Clones having a kappa-positive, IgG-negative phenotype are subcloned at least 3 times to obtain stable anti-SP-peptidtl'u~ mutants. mRNA from putative H chain loss ntutants can be isolated and the sequence of the L chain V region cDNA determined. Reverse PCR of the mRNA for the VH is performed 1L/U~i/H8 WHJ) 17:54 b'A~ 41B ~tiL US~;! HlDUI~'1' tk ~IAYBHH I
with 2 sets of 5'- and 3'- primers, and used for cloning of a~nri-SP-peptide-H~ cDNA. An H chain lose mutant yields no detectable DNA band. Transfection of the cells proceeds with a suitable H
chain plasmid.
Another antigen-binding fragment derivative encompassed by this invention is an antibody in which the constant region of the H or L chain has been modified to provide additional properties. For instance, a change in amino acid sequence can result in altered immunogenicity of the resultant polypeptide. The changes range from one or more amino acids to the complete redesign of constant region domain. Changes contemplated affect complement fixation, interaction with membrane receptors, and other cffector functions. A
recombinant antibody can also be designed to aid the specific delivery of a substance (such as a lymphokine) to an effector cell. Also encompassed by the invention are proteins in which various immunaglobulin domains have been placed in an order other. than that which occurs in nature.
The invention also encompasses single chain V region fragments ("scFv") of anti-SP-pcptides. Single ehaitt V region fragments are made by linking I. andlor H
chain V regions by using a short liking peptide. Bird et al. (19$$) Science 242:423-426. Any peptide having sufficient t7exibility and length can be used as a linker in a scFv. Usually the linker is selected to have little to no immunogenicity- An example of a linking peptide is {CiGGGS)3, which bridges approximately 3.5 nm between the carboxy terminus of one V region and the amino terminus of another V region. Other linker sequences can also be used, and can provide additional functions, such as a means for attaching to a drug or solid support. Frefcrably, for therapeutic use, the scFvs are not coupled to a chemically functional moiety.
Ah or any portion of the H or L chain can be used in any combination.
Typically, the entire V regions ors included in the seFv. For instance, the L chain V region can be linked to the H chain V region. Alternatively, a portion of the L chain V region can be lir>iccd to the H chain V region, or portion thereof. Also contemplated are sc~'vs in rrrhich the H
chain V region is from H11, and the L chain V region is from another immunoglobulin. It is also possible to construct a biphasic, scFv in which one component is an antigen-binding fragment and another component is a different polypeptide, such as a T cell epitope.
The scPvs can be assembled in any order, for example, V,.r~(linkerr--v~ or 3O V~-(llnkC?)-VH. There cari be a difference in the levtl of expression of these two con~guratxons in particular expression systems, in which case oxie of thest forms can be ss iziusiea wr;U i7:5a rv~ am ;saz usz' Hmuu~r a mnYerar; emu preferred. Tandem scFvs can also be made, such as (X}-{linkerr-{Xr(linker)---(X), in which X are scFvs , or combinations thereof with other polypeptides. In another embodiment, single chain antibody polypeptidcs have no linker polypeptide, or just a short, inflexible linker.
Possible configurations are V~ VH and VH---V~. The linkage is too shoat to permit interaction between VL and V~ within the chain, and the chains fat~rt homodimers with a Vt/VH antigen-binding site at each end. Such molecules are referred to in the art as "diabodies".
ScFvs can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. Fvr recombinant production of scFv, a suitable plasmid-containing polynucleotidt that encodes the scFv ca~o be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as Escheri~hid colt, and the expressed protein can be isolated using standard protein purification techniques. ScFv can also be obtained iionn a phagc display library as described in more detail herein and in Example 10.
A particularly useful system for the production of scFvs is plasmdd pET-22b(+) (Novagen, Madison, WI) in E. coli pET-22b(-~) contains a nickel ion binding domain consisting of 6 sequential histidine residues, which allows the expressed protein to be purified on a suitable affinity resin. Another example of a suitable vector is pcDNA3 (Invitrogen, San Diego, CA), described above.
Conditions of gene expression should ensure that the scFv a55umcs optimal tertiary structure. Depending on the plasmid used (especially the activity of tht promoter), and the host ceU, it can be necessary to modulate the rate ofproduction. For instance, use of a weaker promoter, or expression at lower temperatures, can be Necessary to optimize production of properly folded scFv in prokaryotic systems; or, it can be preferably to express scFv in eukaryotic Cells.
The invention also encompasses polymeric forms of antigen-binding fragments, containing a plurality of anti-SP-peptide. One ez~nbodiment is a linear polymer of antigen-binding iiagments, optionally conjugated to carrier. These linear polymers can comprise multiple copies of a single antigen-binding fragment polypcptide, or combinations of different polypeptidcs, and can have tandem polypeptides, or polypcptides separated by otb,er amino acid sequences. Another embodiment is multiple antigen peptides (MAPs). MAPS have a small immunalogically inert core having radially branching lysine dendrites, onto which a number of iziusiaa wr:~ i7:54 r~a~ am aaz ooze xmuu~r ~ maY~~;~ gull antigen-binding fragment polypcptides are covalently attached. See for instance, Posnctt et al.
(1988) .I. Bdod. Cltem. 263:1719-1725; and Tam (1989) Met. Enz. 1.68:7-15. The result is a large macromolecule having a high molar ratio of antigen-binding fragment polypeptides to core.
MAPS are efficient immunogens and useful antigens for immunoassays. The cone for creating MAPS can be made by standard peptide synthesis techniques, or obtained cornmercially (Quality Controlled Biochemicals, Inc., Hopkinton, MA). A typical core matrix is made up of three levels of lysine and eight amino acids.
The invention further includes anti-~idiotypic-antigen-binding fragments to anti-C-antigen-specific antibodies. Such anti-idiotypes can be made by any method known in the art.
1.0 Specifically, the invention encompasses ariri-Hi l anti-idiotype antigen-binding fragments. Anti-idiotypes are particularly suitable for use as vaccines.
Cancer patients are often immunosupprcased and tolerant to some tumor-associated antigens (TAA). Triggering an active immune response to such TAA represents an important challenge in cancer therapy. Immunization with a given antigen generates the production of antibodies against the antigen. The invention encompasses anti-tumor monoclonal antibodies;
anti-idiotypic monoclonal antibodies; and anti-anti-idiotypic monoclonal antibodies. See also, P~,"T/L1S95117103.
While vaccines are generally designed for asymptomatie individuals, vaccines can also be used to treat those with advanced cases of disease. For example, a vaccine therapy of 16 patients with advanced epithelial ovarian cancer or recurrences involved ACA125. ACA125 is an immunoglobulin C31 (IgG 1) marine monoclonal anti-idiotype antibody that mimics a specific epitope on CA125, an antigen that is expressed by most malignant ovarian tumors. Nine of 16 patients developed a CA125-specific cellular immune response by their peripheral blood lymphocytes (PHL). Wagner et al. (1997) Hybridoma 16:33-40. Far work related to the use of anti-idiotype antibodies in cancer vaccines, see Durrant tt al. (1997) Hybridoma 16: 23-b.
Any carrier can be used which is not harmhhl to the host. Suitablt carriers are typically large, slowly metabolized macromolecules such as protons; polysaccharides (such as latex functionali2ed Sepharose, agarose, cellulose, cellulose beads and the like);
polymeric amino acids (such as polyglutamie acid, polylysine, and the like); amino acid copolymers; and inactive virus particles or attenuated bacteria, such as Salmonedlo. Especially useful carrier proteins arc iziusiea wr:u i7:~~ rva~ aia Paz usz;~ Hmuu~r ~ mAY~~r; ~mz scrum albumins, keyhole limpet hemacyanin (KLH), certain Ig molecules, thyroglobulin, ovalbumin, and tetanus toxoid. 1CLH is especially preferred.
2. ~t f The invention encotnpasses methods of obtaining anti-SPPCs. Anti-SPPCs can be obtained and isolated in a number of ways.
Methods of generating new antigen-binding fragments to C-antigen or other such tumor-associated SPPCs, as detailed below, include: 1 ) employing phagc display techniques (see, generally, Hoogenboom et al. (199$) Immurtotechnology 4:1-20) by which cDNA
encoding antibody repertoires arc preferably atnplifed from lymphocyte or spleen RNA
using PCR and oligonueleotide primers specific for species-specific V regions; 2) immunizing mammals with the antigen and generating polyclonal ox monoclonal antibodies (blabs); 3) generating hybridomas from cancer patients including human:humar hybridomas; and 4) employing phage display to rnako antibodies without prior immunization by displaying on phage, very large and diverse V gene repertoires. Preferably, for therapeutic purposes, if non-htuna~ antibodies are to be used, can be humanized by any method known in the art.
With respect to the first of these techniques, the method of Medez et al. ( 1997) Nature r~xenetics 1$:410 can be used. Briefly, purified SPPC (such as C-antigen), is used to immunize transgenic mice lacking the native marine antibody repertoire and instead having most of the human antibody V-genes in the germ. line configuration. Human antibodies are subsequently produced by the marine B cells. The antibody genes are recovered from the ~
cells by PCR
library selection or classic hybridoma technology.
Alternatively, by using the second of these techniques, antibodies can be obtained from mice (such as BAL.B~c) after injection with puriFled Sh-peptide. blabs are generated using standard liybridoma technology. See for instance, Maiti et al. ( 199' Biotechnology International 1-$5-93 (human hybridomas); and Kohler and Milstein (1975) Nature 256:495 -497 (mouse hybridomas). Marine antibodies can be subsequently humanized for instance by the method of RQSok et al. (1996) J. Biol. Chem. 271:22611-22618; Baca et a1.
(1997) ,I. Biol. Chem.
272:10678-10684; Radar et al. Prnc. Natl. Acad. Scl. USA 95:$910-8915; and Winter and Milstein (1991) Natz<re 349:293-299.
According to the third technique, a phage display approach can be used to rapidly generate human antibody against C-antigen or other SPPCs. "this approach can employ the ss 1L/U8/88 W~ l7:bb r'A~ 41a ;itlL USZJ ltlDUU'1' ~C ~IAYBIJIøJU13 method outlined by Henderikx ct al. (1998) Cancer Res. 58:4324-32. Antibody fragments displaydd on phage are selected from a large naive phage antibody/fragrnent library containing different single chain antibodies by separating those which bind to immobilized antigen. As regards the antigen, preferably the entire SPPC is used. lauman antibody fragments are selected from naive repertoires constructed either from gozmline V-domains or synthesized with many mutations (mutations axe tar~cted either by homologous gene re-assortments or error prone PCR) in both the framework and CDR regions. .Antigen-binding fragments specif.~cally reactive with sPpCs can be screened against tumor and normal tissues as described herein in order to identify tumor-specific antigen-binding fragments.
The invention also encompasses methods of identifying antigen-binding fragments specific for a tumor-associated SPPCs by generating a suitable phage display library; isolating SPPCs from a tumor or recombinant host; screening the phag~ display library with the complexes according to standard immuz~ochcmical techniques to obtain phage that display an a~tigcn-binding fragment that binds specifically to SPPC; and screening the phage obtained for specific cell surface tumor-associated reactivity, by screening against tumor and normal cells and selecting flee phage that bind preferentially to tumor but not norms! cells.
Methods of generating antigen-binding fragments by phage display are well known in the art. See, Hoogenboom et al.
(1998) fmmunotechnology 4:1-20.
Lymphocyte (PBL) or spleen RNA is typically used to make antibody fragment repertoires. Mutagenesis using homologous reassortment or error prone PCR
increases diversity.
Phagc display libraries created from human lymphocytes of cancer patients are expected to be enriched in anribodies specific for tumor-associated SPPCs. Also, antibody phage display libraries have been prepared from B-cells of patients undergoing active specific immunotherapy (,ABI) with autologous tanner cells. Hall et al. (199$} Immunotechnology 4:
i27-140.
Repertoires of antibody genes can be amplified from inomunized rnicc or humans using PCR and seFv or Fab antibody fragments obtained thereby can be cloned and expressed on the surface of baeteriophagc. The antibody gene repertoires are amplified from lymphocyte or spleen RNA using PCR and oligonucleotide primers specific for host animal-sptcific V regions.
Phage display can also be used to make antibodies without prior immunization by displaying very large and diverse V gene repertoires on phage. The natural V gene repertoires present in PBL (periphcra! blood lymphocytes) are isolated by 1?CR amplification and the VH and VL

lY/US/88 WJU 17:5tf b'A2. 4ltf ;itlL USCJ lill)l)U'1' ~t ~tAYBIrJløJU14 regions are spliced together at random using PGR. Mutations can be targeted to the V-domain genes by homologous gene reassortments (Zhao et al. Nat. Biotechnol. (1998) 15:258) or error-prone PCR. ~oogenboom et al. Immurtotechnology (1998) 4:1-24. Tatally synthetic human libraries can also be created and used to screen for SPPC-specific antibody fragments.
Regardless of the method used to operate the phage display library, each resulting phage has a functional antibody fragment displayed on its surface and contains the gene encoding the antibody fragment in the phage genome. See, e.g. W09'1~02342.
Affinity chromatography in which binding antibodies can be subtracted from non-binding antibodies has bean established for sorae time. Nissim et al. (1994) EMBO J.
13:692-698; and Vaughan of al. (199b) Nat- Biotechnol. 14:309-314. Critical parameters affecting success are the number and affinity of antibody fragments generated against a particular antigen. Until recently, the production of loge, diverso libraries rcmainod somewhat dif~ault.
Historically, scFv repertoires have been assembled directly from VH and VL RT-PCR products. RNA
availability and the efficiency of RT-PCR were limiting factors of the nunnber of V genes available. Also, assembly was based on ligating three fragments, namtly VH and VL and the linker regions.
Marks et al. (1991 ) J. Mol. Biol. 222:581-597.
An improved library construction method (Sheets ct al. (1998) Pros. Natl.
Acid. Sci. U,SA
95:6175-6162) uses cloned VH and VL gene repertoires in separate plasmid vectors to provide a stable and limitless supply of material for seFv assembly. Also, the efficiency is increased by having DNA encoding the linker region at the 5' End of tht VL library.
Therefore there are only two fragments to be ligated instead of three.
The improved strategies (Sheets et a1.) for generating phage antibody libraries have been demonstrated to cf~ciently and rapidly produce high affinity antibodies td a wide variety of protein antigens. Thus, a large library (> b.0 x 10 9) of phagc displayed antibody fragments (e.g.
scFv), panned against SpPCs can result in the selection of a panel of high affinity antibodies.
See, Example 10 for a method overview.
Anti-SPPCs can also be derived or manipulated using genetic recambination. For example, the immunogenic activity of the V regions of the L and H chains can be screened by preparing a series of short polypeptides that togethtr span the entire V
region amino acid sequence. Using a series of polypcptidcS of 20 or 50 amino acids in length, each V region can be surveyed for useful functional properties. It is also possible to carry out a computer analysis of a 1L/U~i/88 Wr:U 17:513 b'AI1 4113 :3UL USC;! H1DUU'1' ~C ~IAYBHH ~RJU16 prottln sequence to identify potentially interesting polypeptides. Such peptides can then be synthesized and tested for irnrnunogenic activity.
The invention further encompasses various adaptations of antigen-binding fragments described in this section combined in various fashions to yield other anti-SP-peptides with desirable properties. For instance, anti-SPPCs with modified residues can be comprised in a MAP. In another example, an cad-SPPC scFv is tLsed to a cytokine, such as IL-2. All such combinations are contemplated in this invention.
The antigen-binding fragments of this invention can be made by any suitable procedure, including proteolysis of the antibody, by recombinant methods or by chemical syntheses. These methods arc known in the art and need not be described its detail herein.
Examples of protcolytic enzymes include, but are not limited to, trypsin, chymotrypsin, pepsin, papain, V8 protease, subtilisin, plasmin, and thrombin. intact anti-SPPCs can be incubated with one or mare proteinases simultaneously or sequentially. Alternatively, yr in addition, intact an.tibady can be treated with disulfide reducing agents. Peptides cae than be separated from each other by techniques known in the art, including but not limited, to gel filtration chromatography, gel electrophoresis, and reverse-phase I-iPlrC.
Anti-SPPCs can also be made by expression from a polynucleotide encoding the peptide, in a suitable expression system by any method kmown in the art. Typically, polynucleotides encoding a suitable polypcptide are ligated into an expression vector undtr control of a suitable 2Q promoter and used to genetically alter the intended host cell. Both eukaryotic and prokaryotic host systems can be used. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Examples of prokaryotic host cells appropriate for use with this invention include E. coli. Examples of eukaxyotic host cells include avian, insect, plant, arad animal cells such as COS7, l~eLa, and CFIO
cells.
Optionally, matrix-coated channels ar hoads and cell co-cultures can be included to enhance growth of antibody-producing cells. For the production of large amounts of antibody, it is generally more convenient to obtain ascitic fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naYve, histocomopatible or immunotolerant mammal, ~spccially a mouse. The mammal can be primed for ascites production by prior administration of a suitable composition; e.g., Pristane.
The ascitic fluid is then removed from the animal and processed to isolate the antibodies.

ieiusiaa wtsu 17:57 rv~ aia s~z us~;~ xmuu~r a mnYar;r; ~ul~
Alternatively, antigen-binding fragments can be chemically synthesized using amino acid sequence data and other information provided in this disclosure, in conjunction with standard methods of protein synthesis. A suitable method is the solid-phase Merrificld technique.
Automated pepride synthesizers are commercially available, such as those manufactured by Applied Biosysterns, Inc. (Foster City, CA).
Another method of obtaining anti-SPPCs is to immunize suitable host animals with tumor- ar disease-associated SpPCs and to follow standard procedures for polyclonal or Mab production and isolation. Mobs thus produced cam be "humanized" by methods known in the art.
3'he invention thus encompasses humanized Mabs.
"Humanized" antibodies are those in which at least part of the sequence has been altered from its initial form to render it more like human itnmunoglobulins. In one version, the H chain and L ehaiun C regions are replaced with human saquence. This is a fusioa polypeptide comprising an anti-SPPC V region and a hcterologous immunoglobulin (C) region.
In another version, the CDR regions comprise anti-SPPC amino acid sequences, while the V
framework regions have also been converted to human sequences. EP 0329400. In a third version, Y
regions are humanized by designing consensus sequences of human and mouse V
regions, and converting residues outside the CDRs that are different between the consensus sequences.
In making humanized antibodies, the choice of framework residues help in retaining high binding affinity. In principle, a framework sequence from any human antibody can serve as the template for CDR graftiung; however, straight CDR replacement into such a framework can lead to signi~oant loss of binding affinity to the antigen. Glaser et al. (1992) J.
Immunol. 149:2606;
Tempest et al. ( 1992) Biotechnology 9:266; and 5halaby et al. ( 1992) J.
F,xp. Med. 17:2 L 7. The more homologous a human antibody is to the original marine antibody, the less likely that the human framework will introduce distortions into the marine Cl7Rs that could reduce affinity.
Based on a sequence homology search against an antibody sequence database, the human antibody 1C4 provides good framework homology to muM4TS.22, although other highly homologous human antibodies would be suitable as well, especially kappa L
chains from human subgroup I or H chains from human subgroup HI. Kabat et al. ( 1987). Various computer programs such as ENCAD (Levitt et al. (1983) J. Mol. ,8iol. 1b8:595) are available to predict the ideal sequence for the V region. The invention thus encompasses human antibodies with different V regions. It is within the skill of one in the art to determine suitable V region b2 lzWB~aa wr:J) 17:67 rvu 41G :ltlL USZJ XWUUT NC D1AYB~h ~øJU17 sequences and to optimize these sequences. Methods for obtaining antibodies with reduced immunogenicity are also described in U.S. Patent No. 5,270,202 and pP 699,755.
In certain applications, such as when an antigen-binding-fragment is expressed in a suitable storage medium such as a plant seed, the antigen-binding-fragment can be stored without purification. Fiedler et al. (1995) Biotechnology 13:1490-1093. For most applications, it is generally preferable that the polypeptide is at least partially purified from other cellular constituents. Preferably, the antigen-binding fragment is at least about 50%
pure as a weight perctnt of total protein. More preferably, the antigen-binding fragment is at least about 50-75%
pure. For clinical use, the antigen-binding fragment is preferably at least about 80% pure.
If the compositions of the invention are to be administered to an individual, the antigen-binding &~agn~ent is preferably at least 80% pure, more preferably it is at least 90% pure, oven more preferably it is at least 95% pure and free of pyrogens and other contaminants. In this context, the percent purity is calculated as a weight percent of the total protein content of the preparation, and does not include constituents which are deliberately added to the composition after the antigen-binding fragment is purified.
The invention also encompasses methods of detecting caztcer or disease-associated SPPCs in a biological sample. The methods include obtaining a biological sample, contacting the sample with an anti-S'pisC under conditions that allow antibody-antigen-binding and detecting binding, if arty, of the antibody to the sample as compared to a control, non-cancerous or non-diseased biological sample.
The invention also encompasses methods of detecting anti-SPPCs in. a biological sample.
These methods are applicable in a clinical setting, ;for example, for monitoring antibody levels in an individual, as well as an industrial sating, as in cotnmereial production of anti-SPPCs.
After a biological sample is suitably prepared, for instance by enriching for antibody ZS concentration, it is mixed with excess and-SPPC wader conditions that permit formation of a complex between SPPC and any target antibody that can be present. The amount of complex is then deternnined, and compared with Complexes fornned with standard samples containing known amounts of target antibody in the range expected. Complex formation can be observed by immunoprecipitation or nephelometry, but it is generally more sensitive to employ a reagent 34 labeled with such labels as radioisotopes like ~~I, enzymes like peroxidase and p-galactosidase, ar fluorochromes like fluorescein.

1G/U8/88 WJ~ 17:57 P'AX 41a ;lUL UBC;f ltlyUli'1' ~c bIAYBt;Jr ~øJU18 Anti-SPPC can be characterized by any method known in the art. For instance, by the ability to bind speci~catly to tumors, cancer cell lines, C-antigen or a tumor-or disease-associated SPPC. An antigen-binding fragment can also be testtd for the ability to specifically inhibit the binding between antigen and intact antibody either competitively or non-S competitively. Anti-SPPCs can also be tested for tbtir ability to palliate or ameliorate neoplastic disease, such as carcinomas. It is understood that only one of these properties need be present in order far a polypeptide to come within the scope of this invention, although preferably more than one of these properties is present.
The ability of an anti-SPPC to bind antigen can be tested by any immunoassay known in lU the art. Any farm of direct binding assay is suitable. In one such assay, one of the binding partners, the antigen or the putative anti-SPPC, is labeled. Suitable labels include, but are net limited to, radioisotopes such as ~zsl, enzymes such as pcroxida3e, fluorescent labels such as fluorescein, and chenniluminescent labels. Typically, the other binding partner is insolubilized (for example, by coating onto a solid phase such as a microtiter plate) to facilitate removal of 1 S unbound soluble binding partner. After combining the labeled binding partner with the unlabeled binding partner, the solid phase is washed and the amount of bound label is determined. Another such assay is a sandwich assay, in which the putative anti~SPPC is captured by a first anti-imtzaunoglobulin on a solid phase, the anti-SPPC is added and any resultant captured complex is labeled and with an antibody that binds to anti-SPPC. The anti-20 innmunoglobulin can be sgecx~c, for instance, an antibody constant region such as by mouse anti-human IgG. In eithtr of these examples, the extent of binding of aztti-SPPC is related to the amount of label bound to the solid phase.
When used far immunotherapy, anti-SPPCs can be unlabeled or labeled with a therapeutic agent as described herein and as known in tk~a art. These agents can be coupled 25 either directly or indirectly to the antigen-binding fragments of the invention- One example of indirect coupling is by use of a spacer moiety. These spacer moietits, in turn, can be either insoluble or soluble (Diener et al. (198ti) Science 231:148} and can be selected to enable drug release at the target site. Examples of therapeutic agents that can bt coupled to antigen-binding fragments for immunotherapy includt, but arc not limited to bioresponse modif ers, drugs, 30 radioisotopes, lcctins, and toxins. Bioresponse modifiers include lymphokines which include, but arc not limited to, TNF-a, IL-1, -2, arid 3, lymphotoxin, macrophage activating factor, 6a iziusiua wr:~ i7:5a rw 4ia ~nz usea Hmuu~r ~ mnY~~;r; Mme migration inhibition factor, colony stimulating factor, and IFs. Intcrferons with which autigcn-binding fragments can be labeled include IFN-a,1FN-(3, and IFN-'y and their subtypes.
In using radioisotopically coyugated antigen-binding fragments for immunotherapy, certain isotypes can be more preferable than others depending on such factors as leukocyte distribution as well as isotype stability and emission. rf desired, the malignant cell distribution can be evaluated by the in vtvo diagnostic techniques described below.
Depending on the malignancy, some emitters are preferable. In general, alpha and beta particle-emitting radioisotopes art preferred in immunotherapy. For example, if a subject has solid tumor foci, as in a carcinoma, a high energy beta emitter capable of penetrating several millimeters of tissue, such as ~°Y, can be preferable. On the other hand, if the malignancy consists of simple target cells, as in the case of leukemia, a short range, high energy alpha emitter, such as Z'ZBi, can be preferable. Bxampl.cs of radioisotopes which can be bound to the antigen-binding fragments of the izxvention for therapeutic purposes include, but are not limited to,'2sI, ~3~I, 9°y, 6~~, ilz'gi, anAt~ mxpb~ a~SC~ iosPd~ and ~ssRe.
Lectins are proteins, usually isolated front plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. However, ricin is a toxic lectin Which has been used immunotherapeutically. This is preferably accomplished by binding the alpha-peptide chain of ricin, which is responsible for toxicity, to the antibody molecule to enable site specific delivery of the toxic effect.
Toxins are poisonous substances produced by plants, animals, or mieroorgani.sms that, in sufficient dose, are often lethal. Diphtheria toxin is a substance produced by Corynebacterium diphtheria which can be used therapeutically. This toxin consists of an alpha and beta subunit which under proper conditions can be separated. The toxic A chain component can be bound to an anti-SPPC and used for site specific delivery to a neoplastic cell.
Thus, far example, anti-SP-peptide can be used in combination with IFN-a. This treatment modality enhances lVlab targeting of melanomas by increasing the expression of Mab reactive antigun by the molanot~na cells. Greiner et al. (1987) Science 235:895. Alternatively, anti-S1~FC can be used, for example, io combination with Il~'N-y to thereby activate and increase the expression of Fc receptors by effector cells which, izt turn, results in azt enhanced binding of the antigen~binding fragments to the effector cell and killing of target malignant cells. Those of iziusiea wr~u i7:5s rw aia anz usr~ xmuu~r a mnYli~;h skill in the art will be able to select f3rom the various biological response modifiers to create a desired offector flmction that enhances the efficacy of anti-SPPC.
C. Po vnucleotides 1. Compositions of Matter A "polynucleotide" is a polymeric form of nucleotides of any length that contains deoxyribanucleotides, ribonucleotides, and analogs thereof in any combination.
Polynucleotides can knave any three-dimensional structure, and can perfornn any polynueleotide-specif c function, known or unknown. The term "polynucieotidc" includes double-, siztgle~stranded, and tniple-helieal molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompassos bath the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form of either the DNA, RNA or hybrid molecules.
A functionally equivalent fragment of a polynucleotide either encodes a polypoptide that is functionally equivalent to the original polypeptide when produced by an expression system, or has similar hybridization specificity as the original polynucleotide when used in a hybridization assay. A functionally equivalent fragment of a native antigen-binding fragment described herein typically has one or more of the following properties: ability to bind tumor-or diseasc-associated SPPCs; ability to bind at least arc type of cancer cell in a specific manner; and an ability to elicit a cancer-specific immune response.
The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRN'A, ribozyrnes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, viruses, isolated DNA of any sequence, isolated RNA of any soquenee, nucleic acid probes, and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fYuororibose and thioatc, and nucleotide branches. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynuclcotide can be further modified , such as by conjugation with a labeling cornponent_ Other types o.f modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to otb,er compounds or supports, including, without limitation, proteins, metal ions, laboling components, other polynucleotidcs, or a solid support.

ieiusiae wry i7:se rva~ 4ia aaz usza Hmuu~r ~ mAYt;~~ ~uzi 'Y'ho term "recombinant" polynuclcotidc mcaris a polynucleotide of genomic, cDNA, semisynthetic, or Synthetic origin that either does not occur in nature or is covalently linked to another polynucleotidc in an atzangement not found in nature. Recombinant nnethods are well known in the art. 'The practice of the invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, Cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, Much as, "Molecular Cloning:
A, Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonuclcotide Synthesis"
{Gait, ed., 1984);
"Animal Cell Culture" (Freshney, ed., 1987); "Methods in Enzyrrtology"
(Academic Press, Inc.);
"handbook of Experimental Immunology" (Wci & Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (Miller & Calos, eds., 1987); "Current Protocols in Moltcular Biology"
(Ausubel et al., cds., 1987); "PCR: The Polymerasc Chain Reaction", (Mullis et al., eds., 194);
"Current Protocols in Immunology" (Coligan et al., eds., 1991). These techttiqucs arc applicable to the production of the polynuelcotidcs and polypeptides, and as such, can be considered in making and practicing the invention. Particularly useful techniques for particular embodiments are discussed in the sections that follow.
A "vector" refers to a recombinant plasmid or vizus that comprises a hetexotogous polynucleotidc to be delivered, either in vitro or in vivo, into a target cell. The hctcrologous polynucleotide can comprise a sequence of interest for purposes of therapy, and cant be optionally in the form of an expression cassette. As used herein, a vector need not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors for the replication of a polynueleotide, and expression vectors for translation of a polynueleotide encoding sequence. Also included are viral vectors, which comprise a polynuclGOtide cncapsidated or enveloped in a viral particle.
A "cell line" or "eel! culture" denotes bacterial, plant, insect or higher eukaryotie cells grown or maintained in vitro. 'the progeny of a cell can not be completely identical (either morphologically, genotypically, or pbenotypically) to the parent cell. A
hybridoma refers to a cell line that produces a Mab. Methods of making hybridomas, both marine and human, arc known in the art. Particular methods of producing human hybridomas are described and referenced throughout the spec~cation.

iziusiea wr:~ i7:su r~ax am adz usza xmuu~r a mAYtital, Buzz A "host colt" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell, and to the progeny thereof.
"Heterologous" refers to are entity genotypically distract from the entity to wlxi.ch it is being compared. For example, a polynucleotide can be placed by genetic engineoang techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotidc. A
promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
An '"isolated" polynucleotidc, polypeptide or SP-peptide complex is one that is øubstantially free of the materials with which it is associated in its native environment. Hy substantially free is meant that at least 50%, preferably at least 70%, more preferably at least $0%, and even mare preferably at least 95% free of these materials, and even more preferably to clinically acceptable standards of purity. The "native environment" is the call in which it is synthesized whether in vitro or in vivo.
A "stable duplex" of polynucleotides refers to a duplex that is sufficiently long-lasting to persist between the formation of the duplex or complex and subsequent detection, including any optional washing steps or other manipulation that can take place in the interim.
The invention also encompasses polynuelcotides encoding for functionally equivalent variants and derivatives of the native peptide and functionally equivalent fragments thereof which can enhance, decrease or not significantly affect properties of the polypeptides encoded thereby. These functionally equivalent variants, derivatives, and fragments display the ability to specifically recognize disease and tumor-associated SPPCs. For instance, changes in a 17NA
sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amine acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide.
The polynucleotides of the invention can comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, and polyadenylation sites, additional tianscriptian units under control of the same or a different promoter, sequences that permit cloning, expression, and l.ziusisa w~;u is:uu 1'w 4m auz uaza xmuu~r ~ mnY~~;r; ~oza transformation of a host cell, and any such construct as can be desirable to provide tznbodimcnts of this invention.
The invention encompasses a polynuclaotida of at least about 9,15 consecutive nucleotides, preferably at Least about 20 nucleotides, more preferably at least about 25 consecutive nucleotides, more preferably at least about 35 con$ecutive nucleotides, more preferably at least about 50 consecutive nucleotides, even more preferably at least about 75 nucleotides, still more preferably at least about 100 nucleotides, still more preferably at least about 200 nucleotides, and even more preferably at least about 300 nucleotides that forms a stable hybrid with a polynucleotide encoding the L chain or I~ chain V region of anti-Sp-peptide, but not with other immunoglobulin encoding regions known at the time of filing of this application. Any set of conditions can be used for this test, as long as at least one set of conditions exist wherein the test polynucleotide demonstrates the required specificity.
Hybridization reactions can be pcnformed under conditions of different "stringency."
Conditions that increase stringency of a hybridization reaction are known.
See, for example, Sambrook ct al. Examples of relevant conditions include (in order of increasing stringency):
incubation temperatures of 25°C, 37°C, 50°C and 68°C; buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x SSC (whore SSC is 0.1 S M NaCI and 15 mM citrate but~'er) and their equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%;
incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of l, 2, or 15 minutes; and wash solutions of 6 x SSC, 1 x SSC, 0.1 x SSC, or deionized water.
The polynucleotides of this invention psn be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail heroin.
One of skill in the art can use the sequence data provided heroin to obtain a desired pOlynucleotidc by employing a fINA synthesizer or ordering from a commercial service.
Alternatively, nucleotides can be obtained from cal) lines producing the peptide, cloning vectors, or expression vectors. RNA or DNA encoding the desired sequence can be isolated, amplified, and processed by standard recombinant techniques. Such techniques include digesrian with restriction endonuclcascs, and amplification by polymerase chain reactiozt (PCR), or a suitable combination thereof. PCI~ technology is described in U.S. Patent Nos. 4,683,195, iziusiue w~ is:uu rv~ am ;adz uaz' xmuu~r ~ mnY~r;~ ~uza 4,800,159, 4,754,065 and 4,683,202, as wall as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, $oston (1994).
(a) SPPC peptide encoding polynueleotides.
The invention encompasses compositions comprising polynucleotides that encode SPPC
peptides. In the case of C-antigen, the polynucleotides encode at least three consecutive amino acid residues an SFPC.
(b~Polynueleottdes encoding antigen-binding, fragments-The invention encompasses polynuclcotides encoding anti-C, derivatives thereof and complementary polynucleotides therefor. Methods of use of the polynucleotides are also encompassed by the invention. Methods of obtaining polynucleotides encoding anti-SPPC and methods oFuse thereof are the same as for and-C. As used herein anti-SPPC
encompasses and-C. As used herein, anti-C and anti-SPPC specifically exclude H11 end derivatives thereof.
The invention further comprises polynucleotides encoding the SPPC-specific antibody V
regions and derivatives thereof. These include isolated polynucleotide fragments, recombinant polynucleotides, and therapeutic plasmids and vectors, such as vaccinia vectors, comprising the polynucleotides.
Included in all these embodiracnts are polyoucleotides with eacoding regions for anti-SP-peptides, fusiozt proteins, humanized immunoglobulins, single-chain V regions, and other particular polypcptides of interest. These palypeptides are described above.
The invention alsp provides polynucleotides covalently linked with a detectable label.
Such polynucleotides are useful, far example, as probes for detection of related nucleotide sequences.
2_ Recombinant Expression Vectors.
polynueleotides comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification.
j7olynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by any method known in the art, including, but not limited to, direct uptake, endocytosis, transfeotion, f mating or electroporation.
Once introduced, the exogenous polynucleaiydc can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome.
Amplified DNA
can be isolated from the host cell by standard methods: see, e.g., Sambrook et al. {19$9). RhTA

iziusiea w~ is:uu r~nx ais Paz usz~ Hmuu~r ~ mnY~l;~ ~uzs can also be obtained from transformed host cell, or it can be obtained by using a DNA-dependent ItNA polyrnerase.
The invention fttrther includes a variety of vectors comprising a polynucleotide encoding anti-SPPC. These vectors can be used for expression of recombinant polypcptides as well as a source of anti-SPPC polynucleoti.des. Cloning vectors can be used to obtain replicate copies of the polynucleoddes they contain, or as a means of storing the polynucleotides in a depository for future recovery. Expression vectors (and host cells cozttaining these expression vectors) can be used to obtain polypeptides produced from the polynueleoddcs they contain.
They can also be used where it is desirable to express anti-SPPC in an individual and thus have intact cells capable of synthesizing the polypeptidc, such as in gene therapy. Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are laiown in the art and arc not described in detail herein. See e.g. Gacesa and Ramji, (1994) Vectors, John Wiley & Sons.
Cloning and expression vectors typically cozttain a selectable marker (for example, a gene encoding a protein necessary far the survival or growth of a host cell transformed with the vxtor), although such a marker gene can be carried on another polynucleotidc sequence co-introduced into the host cell. Only those host cells into which a selectable sere has been introduced will grow under selective conditians_ Typical selection genes either: (a) confer resistance to antibiotics or other toxic substatrces, e.g., ampicillin, neomycin, methotrexate; (b) complement suxotropb.ic deficiencies; or (c) supply critical nutrients not available from a defined medium. The choice of the proper marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art. Vectors also typically contain a replication system recognized by the host.
Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large nunnbar of cloning vectors available in the art. While the cloning vector selected can vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self replicate, can possess a single target for a particular restriction cndonuclease, or can carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g_, pUClB, mpl8, mpl9, pBR322, pMB9, ColEl, pCRI, RP4, phase DNAs, and shuttle vectors such as pSA3 and pAT28.
7t iciusiea wrla is:ui r~aa am ;snz usz~ Hmuu~r a mnY~~;~ ~uza Thcsc and many other cloning vectors arc available from commercial vendors such as HioRad, Stratagone, and Invitrogen.
Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide encoding an anti-SP-peptide of interest. The polynucleotide encoding the anti-s SPPC is operatively linked to suitable transcriptional controlling elemeztts, such as promoters, cnhancers and terminators. For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sitos, and stop codons. These controlling elements (transcriptional and translational) can be derived from a gene encoding an anti-SPPC, or they can be heterologous (i.e., derived from other genes or other organisms). A polynucleotide sequence encoding a signal peptide can also be included to allow an anti-SPpC to cross or lodge in ctll mtmbra~es or be secreted frnrn the cell. A number of expression vectors suitable fox expression. in eukaryotio cells including yeast, avian, and mammalian cclis arc known in the art. Onc example of an expression vector is pcDNA3 (lnvitrogen, San :Diego, C.~, in which transcription is driven by the cytomegalovirus (CMS early promotcr/enhanecr. This vector also contains recognition sites for multiple restriction enzymes for insertion of the polynuclcotide of interest. Another cxamplo of an expression vector (system) is the baculovirus/insect system.
Also encompassed herein are expression systems suitablt for use in anribody-targeted gene therapy comprising a polynucleotide encoding an anti-SPPC. Suitable systems are 2Q described for instance by Brown et al. ( 1994) Yirol. 198:477--488; and Miyamura at al. ( 1994) Proc. Natl. Aced. Sci. USA 91:$5078511.
The vectors containing the polynucleotides of intertst can be introduced into the host cell by any of a number of appropriate means, including electroporation, transftction employing calcium chloride, rubidium chlonido, calcium phosphate, DFrAE-dextran, or other substances;
?5 Ani~oprojeetile bombardment; lipofection; and infection. The choice of means of introducing vectors or polynueleotides encoding anti-SPPCs will ofte~a depend on features of the host cell.
bnce introduced into a suitable host cell, expression of an anti-SPPC can be determined using any assay known in the art. For example, the presence thereof can bo detecttd by RIA or IrLISA of the culture supernatant (if the polypcptide is secreted) or cell lysates.
30 A vector of this invention can contain one or more polynucleotides encoding an anti-SPPC. It can also contain polynueleotide sequences encoding other polypeptidcs that enhance, Zz~uB~aa Wr:U 18:U1 rv~ 4lti ;fell US2;f xIDUUT & DIAYBIJJ~øJU~7 facilitate, or modulate the desired result, such as lymphokines, including, but not limited to, IL-2, 1L-4, GM-CSF, ThTF-a and IFN-'y. A preferred iymphokine is GM-CSF. Preferred GM-CSF
constructs are those which have been deleted for the AU-rich elements from the 3' untranslated regions and sequences in the 5' untranalatcd region that are capable of forming a hairpin loop.
Also embodied in this invention arc vaccinia vectors encoding for recombinant anti-SPPCs, such as scFvs and other antigen-binding fiagrrtents, chimeras, and polymers. The nuvetttion fuzther encompasses the generation of antigen-binding fragments from phage display libraries that have bran selected by at Icast one round of screening with C-antigen or other disease- or cancer-associated SP-peptide- This includes use of phage display to humanize murine antibodies/antibody fragments to SPPCs. See, far example, (1996) J. Biol.
Chem. 13:2'71; (1997) J. ,8tol. Chem. 18:272, and 10678-10684; and (1998) Proc. Natl. Acad. Scl. USA
95:$910-8915.
Isolated phage and the anti-SPPCs encoded therein obtained by such a screening process are also included in the invention.
3. Host Cells.
Other embodiments of this invention encompass host cells transformed with polynucleotides encoding anti-SPPCs and vectors comprising anti-SPPCs polynucleotide sequences, as described abave. Hoth prokaryotic and eulcaryatic host cells can be used.
Prokaryotic hosts include bacterial cells, for example t'. cell and mycobacteria. Among eulcaryotic hosts are yeast, insect, avian, plant and mammalian cells. Host systems are known in the art and need not be described in detail herein. Exaxxxples of a rnamrnalian host cells include CHO and NSO, obtainable from the European Collection of Ccll Cultures (England).
Transfection ofNSO cells with a plasmid, for example, which is driven by a CMV
promoter, followed by amplification of this plasmid in using glutamint synthttase provides a useful system f0~ protein production. Cockttt et al. ( i 990) BiolTechnology $:662-667.
The host cells of this invention can be used, inter olio, as repositories of polynucleotides encoding anti-SPPCs, or as vehicles far production thereof. They can be used also as vehicles fvr iln vivp expresgion of anti-SPPCs. The polynucleotides of this invention can to used in expression systems to produce polypeptides, intact antigen-binding fragments, or recombinant forms thereof.

1G/U8/88 WHJ) lki:UL b'A~ 41t1 ~tlL USY;f HlDUII'f tk mAYI3HH ~øJUGS
4. Methods of use of the polynucleotides.
The polynucleokides of this invention have several uses. For example, in expression systems for the production of anti-SPPC. They are also useful as hybridization probes to assay for the presence of polynucleotides encoding anti-SPPC or related sequences in a sample using methods well known to those in the art. Further, the polynucleotides are also useful as primers to effect amplification of desired polynueleotides. The polynucleotides of this invention are also usei~Zl in pharmaceutical compositions including vaccines and for gene therapy.
Tht polynucleotides can also be used as hybridization probes for detectiozz of anti-SPPC
encoding sequences. Suitable hybridization samples include cells transformed ox vivo for use in gene therapy. In one illustration, DNA or RNA is extracted from a sample, and optionally run on a gel andlor digested with restriction endonucleases. The processed sample polynucleotide is typically transferred to a medium suitable for washing. The sample polynucleotide is then contacted with the anti-SP>?C polynucleotide probe under conditions that permit a stable duplex to form if the sample contains a matching polynucleotide sequence. Any stable duplexes formed are detected by any suitable means. For exannple, the polynucleotide probe can be supplied in labeled form, and label remaining with the sample after washing will directly reflect the amount of stable duplex formed. In a second illustration, hybridization is performed in Situ. A suitably prepared tissue sample is overlaid with a labeled probe to indicate the location anti-SPPC
encoding sequences.
A short polynucleotide can also be used as a primer for a PCR reaction, particularly to amplify a longer sequcnc~ comprising a region hybridizing with the primer.
This can be conducted preparatively, in order to produce polynucleotide for further genetic manipulation. It can also be conducted analytically, to determine whether an anti-SPPC encoding polynueleotidc is present, for example, in a sample of diagnostic interest.
Another use of the polynucleotides is in vaccines and gene therapy. The general principle is to administer the polynucleotide sv that it either promotes or attenuates the expression of the polyptptide encoded therein. Thus, the invention includes methods of inducing an immuna response and methods of treatment comprising administration of an effective amount polynucleotides encoding anti-SPPC or an SPPC to an individual. In. these methods, a polynuclcotide encoding an anti-SPPC or SPPC is administered to an individual, either directly or via cells transfected with the polynucleotide. Preferably, the polynucleotide is in the form of a iziusiea wro is:uz rvx am 'nz usz~ Hmuu~r ~ mnYt;~;~; Fuze circular plasm~id, preferably in a supercoiled co~igucatior~. Preferably, the polynuclcotidc is replicated inside a cell. Thus, the polynuclcotidc is operatively linked to a suitable promoter, such aS a heterologous promoter that i9 intrinsically active in cells of the target tissue type.
Preferably, ones in cell nuclei, plasmids persist as circular non-replicating episomal molecules.
In vitro mutation can be carried out with plasmid constructs to encode, .for exan~plo, molecults with greater affinity and/or avidity.
To determine whether plasmids containing polynucleotidcs encoding anti-SPPC
arc capable of expression in eukaryotic cells, cells such as COS-7, CHO, or HeLa can be transfected with the plasmids. Expression is then determined by immtmoassay; for example, by Western blot. Smaller SPPCs can be detected, for example, by constructing the plasmid so that the resultant polypeptide is flised with a tag, such as a target cpitope or enzyme label. Further characterization Qf the expressed polypeptide can be achieved by purifying the peptide and then conducting one of the functional assays described herein.
D. j The invention encompasses kits containing anti-SPPC. Diagnostic procedures using the kits can be performed by diagnostic laboratories, dxperirnental laboratories, practitioners, or private individuals. The clinical sample is optionally pre-treated for enrichment of the target being tested for. The user thin applies a reagent contained in the kit in order to detect the changed level yr alteration in the diagnostic component.
Each kit comprises antigen-binding fragments used for detecting cancer-associated SPPC
in the sample. Optionally, tlae reagent can be conjugated with a label to permit detection. of any complex formed with the target in the sample. In another option, a second reagent is provided that is capable of combining with the first reagent after it has found its target and thereby supplying the detectable label. For example, labeled anti-human IgG can be provided as a secondary reagent for use with intact anti-SP-peptide. Labeled avidin is a secondary reagent when the primary reagent has been conjugated to biotin.
'The kits can be employed on a variety of biological samples including, both liquid samples cell suspensions and tissue samples. Suitable assays using anti-C that can be supplied in kit form include those described herein.
Eaoh reagent is supplied in a solid form or dissolved/suspended in a liquid buffer suitable for inventory storage and later for exchange or addition into the reaction medium when the test is iziusiea wry is:ua r~aa aia asz uaz~ xmuu~r ~ mnY~~~ ~ u;~u performad- Suitable packaging is provided. Tho kit can optionally provide additional components that arc useful in the procedure. These optional components include, but are not limited to, buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.
jr. 'fhera ~ i .om~ Si ions 1. Compositions of Matter.
The preparation of pharmaceutical compositions dexribed herein is conducted in accordance with generally accepted procedures for the preparation of pharmaceutical preparations. Sec, for example, Remtngton's Pharmaceactical fciences 18th Edition (1990), E.W.
1~!lartin ed., Mack Publishing C4., PA. Depending on the intended use and mode of administration, it can be desirable to process the active ingredient further in the preparation of pharmaceutical compositions. Appropriate processing can include sterilizing, mixing with appropriate non-toxic and non-interfering components, dividing into dose units, and enclosing in a delivery device.
I 5 (a) General modes of administration Pharmaceutical compositions of the invention are administered by a mode appropriate for the form of composition. Typical routes include intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal, oral, intranasal, intradermal, and intrapulmonary (i.c., by aerosol).
Pharmaceutical compositiozts oftbis invention for human use are typically administered by a parenteral route, most typically intravenous, subcutaneous, intramuscular.
Although not required, pharmaceutical compositions are preferably supplied in unit dosage form suitable for administration of a precise amount. Also contemplated by this invention are slow release or sustained release forms, whtreby a relatively consistent level of the active compound arc provided over an extended period.
(b) Liquid formulations Liquid pharmaceutically acceptable compositions can, for example, be prepared by dissolving or dispersing a polypeptidc or polynucleotide embodied herein in a liquid excipicnt, such as water, saline, aqueous dextrose, glycerol, or ethanol. The composition can optionally also contain other medicinal agents, pharmaceutical agents, carriers, and auxiliary substances such as wetting or emulsifying agents, and pH buffering agents. Compositions for injeetioa can WuB~Ha wry lB:Ua tW 41U aaC uSGa xtUUUT !k mAYBt;It:zJUal be Supplied as liquid solutions or suspensions, as emulsions, or as solid forms suita>ale for dissolution or suspension iri liquid prior to injection_ Pharmaceutical compositions for oral, intranasal, or topical administration can be supplied in solid, semi-solid or liquid farms, including tablets, capsules, powders, liquids, and suspensions. For administration via the respiratory tract, a preferred composition is one that provides a solid, powder, or liquid aerosol when used with an appropziate aerosolizer device.
The invention also encompasses compositions comprising liposomes with membrane bound peptide to specifically deliver the liposome to the area of the tumor or neoplastic cells or to the immune system. These liposomes can be produced such that they contain, in addition to peptide, immunotherapcutic agents such as those described above which would then be released at the site of malignancy. Wolff et al. (1984) Biochem. Biophys. Acta 802:259.
Another such delivery system described by Brown et al. ((1994) virology 198:477-488; and Miyamura et al.
(1994) Froc. Natl. Aced. Sci. USA 91:8507-8511) utilizes chimeric paxvovirus B19 capsids for presentation of the antigen-binding fragments. Such chimeric systems arc encompassed for use in the claimed methods.
Compositions embodied in this invention can be assessed for their effcacy in a number of ways. Accordingly, test compounds are prepared as a suitable pharmaceutical composition and administered to test subjects. Initial studies arc preferably done in small animals such as mice or rabbits, optionally next in non-human primates and then ultimately in humans.
Immunogenicity is preferably tasted in individuals without a previous antibody response. A test composition in an appropriate test dose is administered on an appropriate treatment schedule. It can be appropriate to compare dil~'ere~nt doses and schedules within the predicted range. The dosage ranges for the administration of anti-SPpC are those large enough to produce the desired effect in which the symptoms of the malignant disease are ameliorated without causing undue side effects such as unwanted cross-reactions, anaphylactic reactions, and the like. (areneral[y, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication. dosage can vary from about 0.1 mg/kg to about 2000 mg/kg, preferably about 0.1 mglkg to about 500 m.g/kg, in one or more dose administrations daily, for ~0 one or several days. Generally, when the compositions are administered conjugated with iziuttiaa wr:~ is:ua rw am adz uaza Hmuu~r a mnY>il;r; ~uaz therapeutic agents, lower dosages, oomparablc to those used for in vivo immunodiagnostic imaging, can be uged_ 2. Antigen-binding Fragments The invention encompasses pharmaceutical compositions containing anti-SPPC.
Such pharmaceutical compositions are useful for inducing or aiding an immune response and treating neoplastic diseases, either alone or in conjunction with other forms of therapy, such as chemotherapy, radiotherapy or immune therapies described in W098/23735;
W098/34642;
W097/10000; W097/10001; and WO97/06821.
Compositions containing antigen-binding fragments specific far disease-associated SPPCs and methods of use thereof, as described for cancer treatment, are also encompassed by the invention.
3. lmmunogenic Compositions.
The SPPC compositions and SPPC peptide compositions of the invention can be used as cancer immunogens including vaccines. These compositions can comprise a cancer-specific 1~ antigen or epitope (e.g. one found on cancer cells but not on non-ca~tcerous oells), which can be in the form of native peptides, artificial proteins, for example multiantigcn peptides, branched polypeptides, fusion and recombinant peptides, as well as single T cell epitopes and tumor antigen peptides. Ben-Yedidia et al. ( 1997) Curr. Opin. Bloteehnol. 8:442-8;
and Hellstrom et al. (1997) Mol. Med. Today 3:286-90. A cancer vaccine can alternatively comprise a polynucleotide encoding an antigen, which is directly injected into rztuscle or skin to cause an immune response against the encoded antigen. Moclling (1997) Cytokines Cell.
Mol. They.
3:127-35; and Moling (1997) J. Mol. Med. 75:242-d. Cancer vaccines can also comprise tumor cells. Mackensen et al. (1997),1. Mvl. Med. 75:290-6. Anti-idiotypc antigen-binding fragments are also suitable for use as vaccines.
23 A new method for generatitxg useful tumor cell populations for such vaccines from tumor biopsies has been described. Latin et al. (1997) Earn Surg. Res. Z9:292~302.
Whole tumor cells used fQr this purpose can be lethally irradiated and transformed to produce a eytpkine such as grartulocytc-macrophage colony stimulating factor (GM-CSF). Mahvi et al.
(1997) Hum. Gene Tleer. 8:875-891; Stingl et al. (1997) J. Mol. Med. 75:297-9; and toffee et al. (1997) Methods 12:143-53. While both whole cells and cell lysates can be used as vaccines, whole cell vaccines 7d iziusie~ wry ia: ua r~aa~ aia aaz uaz' xmuu~r & ~tAYBh~r~
exn induce a better immune response against cell-surfae~ antigens.
ltavindranath ct a1. (1997) Anticancer Drugs 8:21.7-24.
In a marine breast canr~ model, plt3-Ligand (FIt3-L), a stimulatory cytokine for a variety of hematopoietic lineagcs, including dendritic cells and B cells, has been used in conjunction with marine breast cancer cells as a vaccine. Chen et al. (1997) Cancer Res.
57:3511-6. Dcndritic cells (DCs) can also be loaded with or transdueed to express tumor antigens; these cells are then used as adjuvants to tumor vaccination. DCs present tumor-assooiated antigens endogenously to the afferent lymphatic system in the appropriate histocompatibility corr~plex (MFiC)-restricted context. Wan et al. (1997) Hum.
Gene Ther.
8:1355-63; Peiper et al. (1997) Surgery 122:235-41; and Smith et al. (1997) Int. Immunol.
9:1085-93. Current melanoma vaccines manipulate antigen presentation networks and combine the best cellular and antibody antitumor immune response effective in mediating tumor protective immunity. T'hcsc therapies have caused regression, delayed disease progression or an improvement in survival in some cases, with a paucity of side effects. Kahn et al. ( 1997) Dermatol. Surg. 23:649-54. Melanoma vaccines arc also reviewed in Conforti ct al. ( 1997) J.
Surg. Oncol. 66:55-64.
Vaccines can be packaged in pharmaceutically acceptable carriers or admixed with adjuvants or other components (such as eytokincs) as is known in the art_ More specifically, an SPPC for use in a vaccine can comprise at least one polypeptidc, which is an antigenic fragment, anti-idiotype of anti-SPPC, derivative, or variant of C-antigen or C-antigen peptide. Preferably the SPPC comprises an epitvge of C-antigen. As used herein, the SPPCs arc considered to be derived from a cellular membrane fraction of at least one cancer cell population. That is to say that the $PPC or epitope tktereof can be found preferentially in the membrane fraction of disrupted and separated cells but the SPPC or portion thereof can be obtained in any manner including recombinant genetics. Thus, the SPPC or epitope thereof can be derived directly or indirectly from such a fraction. By "preferentially" in the membrane fraction, it is meant that more than 50%, preferably more than 75% and, even more preferably, more than 90°/a is found in the membrane fraction with corresponding ampur~ts in the cytosolic or non-membrane fractions.
An epitope typically includes 5-10 amino acid residues. The C-antigen polypeptide comprises derivatives of C-antigen which preferably retain at least one epitope present on native, 1G/US/88 W~ 1S:U4 r'A~ 4ltf :1BG USG;f H1DUU'1' b'C ~IAyBHH tølU;l4 whale C-antigen. This polypop~ide can be administered as a vaccine in the form of free C-antigen polypeptide, C-antigen present on a cell expressing C_~tigen; C-antigen in the context of mufti-antigen peptides, branched polypeptides, fttsion peptides, recombinant peptides; or C-antigens loaded onto dendritic culls (DCs). The cell expressing C-antigen can be a tumor cell naturally expressing C-antigen or a cull, which does not normally express C-antigen, which has been transformed with the C-antigen polynucleotide in order to express C-antige~a. The cell can be irradiated or otherwise rendered non-viable. ~"he C-antigen-expressing cell can also be altered (e.g. by transduction) to express a cytokinc.
Vaccines for veterinarian use are substantially similar to that in humans with the exception that adjuvants containing bacteria and bacterial components such as Freund's complete or incomplete adjuvants, arc allowed in the formulations.
4. Ccnc Therapy The invention further encompasses methods of gent therapy and compositions for use therein. In one mode of gene therapy, the polynuoleotides are used for ,genetically altering cells ex vivo. In this strategy, tolls removed from a donor or obtained from a cell line arc trsnsfected or transduced with vectors encoding an anti-SPPC, and then. administered to a recipient. Suitable cells for transfeetion include peripheral blood mononuclear cells.
In another mode of gene therapy, the palynuclcotides of this invention are used for genetically altering polls in vivo. ~'he purpose can include, but is not limited to, treating various types of cancer.
F_ Methods of Treatment Also included in this invention arc methods for treating cancer. The rr~ethods comprise administering an amount of a pharmaceutical composition containing a composition of the invention in an amount effective to achieve the desired effect, be it palliation of an existing tumor mass or prevention of recurrence. For treatment of cancer, the amount of a pharmaceutical composition administered is an amount elective in producing the desired effect.
An affective amount can be provided in uric 8r a strit$ of administrations. An effective amount can be provided in a bolus or by continuous perfusion. Suitable active agents include the anti-neoplastic drugs and bioresponse modifiers described above and cffector cells such as those ~0 described by l5ouillard et al. (19$6) !i'ybridornas (Supp. 1:5139).
So iziusiaa wru is:us r~aa~ aia aaz uaz~ Hmurr tx mAY~~~ ~uab Pharmaceutical compositions and treatrrlent modalities of this invention are suitable for treating a patient by either directly or indirectly eliciting an immune response against neoplasia.
An "individual", "patient" or "subject" is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to: humans, wild anivnals, feral animals, farm animals, sport animals, and pits. A "cancer subject" is a mammal, preferably a human, diagnosed as having a malignancy or neoplasia or at risk thereof As used herein, "trearix~ent" refers to clinical uitervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include arithout limitation, preventing pccttrt~ence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progmssion, amelioration or palliation of thr disease state, and remission or improved prognosis.
The "pathology" associated with a disease condition is any condition that compromises the well-being, normal physiology, or quality of life of the affected individual. This can involve, but is not limited to, destructive invasion of affected tissues into previously unaffCCtcd areas, growth at the expense of, normal tissue ftmction, in;egular or suppressed biological activity, aggravation or suppression of an inflammatory or immunologie response, increased susceptibility to other pathogenic organisms or agents, and undesirable clinical symptoms such as pain, fever, nausea, fatigue, mood alterations, and such other disease-related features as can be determined by an attending physician.
An "effective amount" is an amount sufFcient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a patient in one or more doses. In terms of treatment, an effective amount is an amount that is suffeient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within tht skill of one in the art.
Several factors arc typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the patient, the condition being treated, the severity of the condition and tht farm and effective concentration of the antigen-binding fragment administered.

iziusiea tvru ia:us rw 4ia 'az uaz~ xmuu~r ~ mnYSr;r; ~uaa Suitable s~rbje~ts include those who are suspected of being at risk of a pathological effect of any neaplasia, particularly carcinoma, are suitable for treatment with the phamtaceutical.
compositions of this invention. Those with a history of cancer are especially suitable.
Suitable human subjects for therapy fitrther comprise two treatment groups, which can be distinguished by clinical criteria. Patients with "advanced disease" or "high tumor burden" are those who bear a clinically measurable tumor. A clinically measurable tumor is one that can be detected on the basis of tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram or X-ray; positive biochemical or histopathologic markers on their own are insufficient to identify this population). A pharmaceutical composition embodied in this invention is administered to these patients td elicit an anti-tumor response, with the objective of palliating their condition. Ideally, reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit.
Clinical improvement includes decreased risk or rate of progression or reduction in pathological consequences of the tumor.
t~ second group of suitable subjects is known irx the art as the "adjuvant group." These are individuals who have had a history of cancer, but have been responsive to another mode of therapy. The prior therapy can have included (but is not restricted to, surgical resection, radiotherapy, and traditional chemotherapy. As a result, these individuals have no clinically measurable tumor. However, they arc suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases.
"Adjuvant" as used herein has several meanings, all of which will be clear depending on the context in which the term is used. Ln the context of a pharmaceutical preparation, an adjuvant is a chemical or biological agent given in combination (whether simultaneously or otherwise) with, or recombinantly fustd to, an antigen to enhance immunogenicity of the antigen. In the conkext of caztcer diagnosis or treatment, adjuvant refers to a class of cancer patients with no clinically detectable tumor mass, but who are may be at risk of recurrence.
This group can be further subdivided into high.-risk and low-risk individuals.
The subdivision is made on the basis of features observed btfore or after tho initial treatment. Those features are irnown in the Clinical arts, and are suitably defined for each different oancer.
peatures typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.

Zz~uB~sa w~ 18:U0 rW 41U ;iBG u8Y;1 HWUUT tk 1lAYBlW øJUJ7 Another suitable group of subjects is those with a genetic predisposition to cancer but who have trot yet evidenced clinical signs of cancer. For instance, women testing positive for a genetic mutation associated with breast cancer, but still of childbearing age, can wish to receive anti-SPPC treatmeilt prophylactically to prevent the occurrence of cancer until it is suitable to perform preventive surgery.
A pharmaceutical composition embodied in this invention is administered to patients in the adjuvant group, ar in either of these subgroups, in order to elicit an anti-cancer response.
Ideally, the composition delays recurrence of the cancer, or even better, reduces the risk of recurrence (i.c., improves the cure rate). Such parameters can be determined in comparison with other patient populations arid other modes of therapy.
Of course, crossovers between those two patient groups occur, and the pharmaceutical compositions of this invention can be administered at any time that is appropriate. p'or example, anti-SPPC therapy can be conducted before or during traditional therapy of a patient with high tumor burden, and continued after the tumor becomes clinically undetectable.
Anti-SFPC
therapy can be continued in a patient who initially fell in the adjuvant group, but is showing signs of recurrence. The physician has the discretion to determine how or when the compositions are to be used.
Various compounds and co~ttpositions of this invention have other clinical indications, of which the following section provides only a survey.
One indication is the treatment of cells ex vivo. This can be desirable for experimental purposes, or for treatment of an individual with a neoplastic disease. In one example, anti-SPPC
is admitustered to a culture of Cells, such as peripheral blood cells obtained from a donor, or a suitable cell line. About 0.5 to 2 pg/mL of anti-C can be an effective dose for this purpose. In a second example, donor cells are genetical ly altered with an expression vector of this invention, to provide for ongoing secretion of anti-SPPC after administration of the cells to the recipient.
Human cancer patients, including, but not limited to, glioblastoma, melanoma, ncuroblastoma, adcnocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (includi.ng small cell lung cancer) are especially appropriate subjects- Suitable carcinomas Further include any known in the field of oncology, including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural cctodcrmal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal i2iusiea wru is:ua r~e~ 4iu anz usz~ xmuu~r ~ mnY~rr; vu's adenocarcinoma, small and large cell lung adenocarcinomas, chordoma, angiogarcQma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hcpatoma, cholangiocarcinoma, synovionna, mesothelioma, Ewing's tumor, rhabdomyosareoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcir~oma, cystadonocarcinoma, madullary carcinoma, bronchogenic carcinoma, renal toll carcinoma, bileduct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, epcndymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastottta, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenoeareinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell carcinoma of the bladder, 8 ar~,d T cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukcmias, malignant melanoma, soft tissue sarcomas and leiomyosarcomas.
The patients cats have an advanced form of disease, in which ease the treatment objective can include mitigation or reversal of disease progression, and/or amelioration of side effects.
The patients can have a history of the condition, for which they have already boon treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of reC'utrCItGt.
"Immunologie activity" of an antigen-binding fragment refers to the ability to specifically bind the antigen which the intact antibody recognizes. Such binding Can or can not elicit an imrnuae response. A specific immune response can elicit antibody, B cell responses, T cell responses, any combination thereof, and effector functions resulting therefrom. Included, without limitation, are the antibody-mediated functions ADCC and complement-mediated cytolysis (CDC). The T cell response includes, without limitation, T helper cell function, cytotoxic T cell function, inflammation/inducer T cell function, and T cell mediated immune suppression. A compound (either alone or in combination with a carrier or adjuvant) able to elicit eitber directly ox indirectly, a specific immune response aeeordin~ to any of these criteria is referred to as "immunogeni.c." Antigen-binding fragment "activity" or "function" refers to any iziusiaa wl,u is:ua rw 4iu :tnz usz~ ltmuu~r ~ mnY>it;>5 ~u~a of the immunologic activities of an antibody, including the detection, amelioration or palliation of canper.
An "immune response" refers to induction ar enhancement of an immunologic response to malignant or diseased tissue, disease-causing agents and other foreign agents to which the body is exposed. Inunune responses can be humoral, as evidenced by antibody production;
and/or cell-mediated, as evidenced by cytolytic responses demonstrated by such cells as natural killer cells or cytotoxic T lymphocytes (C'fL.s) and the cytokines produced thereby. Immune responses can be monitored by a mouonuelear cell infiltrate at the site of infection or malignancy. Typically, such monitoring is by histopathology. A "cancer-specific immune response" is one that occurs against the tnaligaancy but not against non-cancerous cells. The treatments described herein typically induce or augment an antibody-mediated response but can also induce or augment a cell-mediated immune response.
When anti-SPPC is used in combination with various therapeutic agents, such as those described herein, the administration of both usually occurs substantially contemporaneously.
The term "substantially contemporaneously" means that they are administered reasonably close together with respect to time. Usually, it is preferred to administer tt~e therapeutic agent before anti-SPPC, For example, the therapeutic agent can be administered 1 to 6 days before anti-SPPC. The administration of the therapeutic agent can be daily, or at any other suitable iztterval, depending upon such factors, for example, as the nature of the malignancy, the condition of the patient and half life of the agent.
Anti-SPPC enables therapies combining all of the characteristics described herein. For example, in a given situation it can be desirable to adminiata a therapeutic agent, or agents, prior to the admlt~istratiot~ of anti-SPPC in combination with effeetor cells and the same, or different, therapeutic agent or agents. For example, patients can be treated by first administering IFN-Y
and IL-2 daily for 3 to 5 days, and on day 5 administering anti-SPPC in cornbinadon with effector cells, IFN-y, and IL-Z.
'I"hcrapcutic compositions can be administered by injection or gradual perfusion. Anti-SPPCs can lx administered intravenously, m~peritoneally, infra-muscularly, subcutancously, intraeavity, intrathecally or transdcrmally, alone or in combination with effector cells.
Another method of administration is intralesionally, for instance by injection directly into the tumor. Intralesional administration of various forms of immunothcrapy to cancer patients Zz~uWaa wr:J) 18:07 rv.~ 41H Paz UBYa HtUUUT 1k IIAYBt;r~ ~JU4U
does not cause the toxicity seen with systemic administration of immunologic agents. Flctchcr ct al. (19$7) ,Gymphoklne Res. 6:45; Rabinowich ct al. ( 1987) Cancer Res.
47:173; Rosenberg et al.
(1989) Science 233:1318; and pizz et al. (1984) J. Int. Cancer 34:359.
Anti-$PPC is suitable for use in treating and imaging brain cancer. vVhen the site of delivery is the brain, the therapeutic agent must be capable of being delivered to the brain. The blood-brain burner limits the uptake of many therapeutic agents into the brain and spinal cord from the general circulation. Molecules that cross the blood-brain barrier use two main mechanisms: free diffusion; and facilitated transport. Because of the presence of the blood-brain barrier, attaining beneficial concentrations of a given therapeutic agent in the CNS can 1 d require the use of drug delivery strategies. Delivery of therapeutic agents to the CNS can be achieved by several methods.
One method relies on neurosurgical techniques. In the case of gravely ill patients, surgical intervention is warranted despite its attendant risks. )ior instance, therapeutic agents can be delivered by direct physical introduction into the GNS, such as intraventricular, intralesional, or inrrathecal injection, lntraventricular injection can be facilitated by an intraventrIcular cathettr, for example, attached to a reservoir, such as an Omaha reservoir.
Methods of introduction can also be provided by rechargeable or biodegradable devices.
Another approach is the disruption of the blood-brain barrier by substances which increase tlae permeability of the blood-brain barrier. Examples include intro-arterial infusion of poorly diffusible agents such as mannitol, pharmaceuticals which increase cerebrovascular permeability such as etaposide, or vasoactive agents such as leukotrienes. N'euwelt and Rappoport { 19$4) ,Fed.
Pros. 43:214-219;
Baba et al. (1991 ) J Cereb. Blood Flow Metab. 11:638-643; and Crcnnuso ct al.
( 1993) Cancer Invest. 11:638-643.
further, it can be desirable to administer the romposltions locally to the area in need of treatment; this can be achieved by, for example, local infusion during surgery, by injection, by means of a ~theter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers. A suitable such mtmbrane is Gliadol~ provided by Guilford sciences.
Another method involves pharmacological techniques such as na.odification or selection of the anti-Sl'PC to provide an analog which will crass the blood-brain barrier. Examples include increasing the hydrophobieity of the molecule, decreasing net charge or molecular iziusiea wlsu m:u~ rvx 4m aaz uaca xmuu~r ~ mAYt3~;~ ~uai weight of the molecule, or modifying the molecule, such as to resemble one normally transported across the blood-brain barrier. Levin (1980) J. Med. Chem. 23:682-b$4;
Pardridge (1991) in:
Peptide Drug Delivery to the Brain; and Kostis et al. ( 1994) J. Clin.
Pharmacol. 34:989~99b_ Encapsulation of anti-SPPC in a hydrophobic environment such as liposomes is also 3 ei~'ecdve in delivering drugs to the CNS. For example, WO 91/04014 describes a liposomal delivery system in which the drug is encapsulated within liposomes to which molecules have been added that are normally transported across the blood-brain barrier.
Yet another method takes advantage of physiological techniques such as conjugation of anti-SP-peptide to a transportable agent to yield a new chimeric, transportable, molecule. For 1.0 example, vasoactive intestinal peptide analog (VIPs) exerts its vasoactive efrects only after conjugation to a Mab to the specific can;ier molecule transferrin receptor, which facilitates the uptake of the VIPs-Mab conjugate through the blood-brain barrier. pardridge (1991); and Bi.ckel et al. (1993) Proc. Natl. Aced. Sci. USA 90:261$-2622. Several other specific transport systems have been identified, these include, but arc not limited ta, those far transferring insulin, or 15 insulin-like growth factors T and II. Other suitable, non-specific carriers include, but arc not limited to, pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives.
G. Additional Methods ofUsd 1. Dia~nostie Antibody Clearance The invention also encompasses compositions and methods of use thereof in diagnostic 20 antibody clearance. Anti-SPPC can be administered to an individual who has received a labeled anti-SPPC the course of radioscintigraphy or radiotherapy to remove the label.
Effective imaging using radiolabeled antibodies is hampered due to excess circulating radioiabeled antibody, which often takes several days to clear. Accordingly, the SPPC
recognized by the anti-SPPC is administered to the individual at a specified time after administration of the labeled anti-25 SPPC_ Antigen that is complexed with the antigen-binding fragments at sites other than the tumor, such as in the circulation and interstitial spaces, promotes clearance of non-bound antibody and decreases background radiation. As a result, the level of label in unaffected tissues is reduced, and the image of the tumor (in comparison to neighboring tiasues) is enhanced.
Similarly, when radionucleotides are given to subjects for irradiation of a tumor site, it is 30 desirable t4 reduce collateral exposure of unaffected tissue. This invention this includes iziusiea wry is:us r~ax am adz uszs kmurr ~ mnYSr;r; ~lu4z methods of treatment in which a radiplabeled anti-SPPC is administered in a t>atrapcutic dose, a~ad followed by administration of a molar excess of SPPC.
2. ImaginglISiagnostic, in vitro 'fhe invention liirther encompasses methods for in vivo detection of antigen.
A
diagnostically effecrive amount of detectably labeled anti-SPPC is given to the subject in need of tumor imaging. The kerm "diagnostically effective" means that the amount of detectably labeled anti-SPPC is administered in sufficient quantity to enable detection of the neoplasia.
The concentration of detectably labeled anti-SPPC which is administered should be sufficient such that the binding to those cells having tumor-associated SPPC
is detectable compared to the background. )~urther, it is desirable that the non-bound labeled antigen-binding frag~nuent be rapidly cleared from the circulatory system in order to give tht best target-to background signal ratio.
As a rule, the dosage of delectably labeled antigen-binding fragment far tn vivo diagnosis is somewhat patient-specific and depends on such factors as age, sex, and extent of disease. The dosage can vary from about Q.p1 mglr~2 to about 504 mglmZ, preferably 0.1 mglm2 to about 2Q0 mg/mz, most preferably about 0.1 mgfm2 to about 10 mg/m2. Such dosages can vary, for example, depending on number of injections given, tumor burden, and other factors known to those of skill in tht art.
Fdr in vivo diagnostic imaging, the type of detection instrument available is a zztajor factor in selecting a given radioisotope. The radioisotope chosen must have a type of decay, which is detectable for a given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the balf life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the individual is minimized. Ideally, a radioisotope used for in vivo imaging lacks a particle tmission, but produces a large number of photons in the l40-250 keV range, to be readily detected by conventional gamma cameras.
For in viva diagnosis, radioisotopes can be bound to anti-5P-peptide either directly or indirectly by using an intermediate fimctional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic iozts to immunoglobulins are the bifunctional chelating agents such as dicthylenc triamincpentacctic acid (DTPA) and 1L/U8/U8 W!':U 1S:U~i fAA 41U :lkfG USLJ lilDUU'1' !k mAY131;JtølU4;f Ethylenediaminetetraacetio acid (EpTA) and similar molecules. Typical examples of metallic ions which can be bound are "'In, 9'Itu, 6'Ga, 68Ga,'zAs, s9Zr, ~°Y, 99m.Lc and 2°'TI.
Antigen-binding fi-agments can also be labeled with a paramagnetic isotope for purposes of in viVq diagnosis, as in ma~metie resonance imaging (MItI) or electron spin resonance (ESR).
In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually, gamma and positron emitting radioisotopes arc used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful. in such techniques includc'S'Gd, ssMn, iszpY, ssCr, and s6Fe_ 3. Imagingldiagnostic, in vitro Antigen-binding fragments can also be used to detect neoplasias using in vitro assays.
Biological samples are talon from the patient and subject to any suitable immunoassay with anti-SPPC to detect the presence of tumor-associated SPPCs. This is particularly useful in detecting lymphomas and leukemias where the tumor cells are circulating in the patient's bloodstream.
A "biological sample" encompasses a variety oaf sample types, including blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimens or tissue cultures, or tolls derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, suc;b as proteins or polynucleotides. The term encompasses various kinds of clinical samples obtained from any species, and also includes cells in culture, cell supernatants, cell lysates and fractions thereof.
Particuarly, for the purposes described herein, biological samples comprise tumor tissue or tissue thought tv be tumorous and are obtained for instance by surgical resection, biopsy, aspiration or any method latown in the art.
4. Therapeutic ~ooxto~iz~g.
Antigen-binding fragments can also be used to monitor the course of amelioration of malignancy in an individual. Thus, by measuring the increase or decrease in the number of cells expressing tumor-associated SPPC or changes in the concentration of the complex present in various biological samples, it is possible to dcternune whether a particular therapeutic regimen aimed at ameliorating the malignancy is effective.

iziusida w~u is:uu rv~ am anz usza xmuu~r & ~tAYBIIløJ044 Phage Display Library Recognizing Consensus Conforming Peptides It is to be understood that potentially suitable genetic packages include cells, spores and viruses (sec UB Patent No. 5,571,698), namely replicable genetic packages.
Preferably, the replicable genetic package is a recombinant phagc and said heterogeneous population of replicable genetic packages collectively constitute a phage display library.
~a~
Unless otherwise implied or stated, the term "biasing" and related forms of this term, are generally intended to refer to weighting in the course of introducing variation in the parental binding-fragment.
It is to be understood, for txample, that 90% biasing in favor of wild-type ar~,ino acids at a given amino acid position is to be approximated by controlling the percentage amounts of each of the three relevant nucleotides (so that, for example, the product of the probabilities of occurrence of the three desired nucleotides in sequence in the growing chain is 90%) so as to supply 90% of correct coding triplets) and a total of 10% of random coding triplets, having regard to the degeneracy of the genetic code (for example if two different coding triplets result in a given amino acid, then the sum of the probabilities of achieving those two triplets will have to equal 90%). This is preferably accomplished on an amino acid by amino acid basis so that, for example the probability of achieving two and three wild-type amino acids in sequence, in the case of 90% biasirjg is 0.81 and 0.73, respectively, etc.
It is to be understood that this high level of biasing can be suitable only for part of the coding sequence into which variability is introduced and that higher levels of biasing arc acceptable, when for example substantially all of the amino acids o;f a long CDlt3 are biased, as disclosed in ono of embodiments heroin. Accordingly there is a balance to be struck between a large diverse library and biasing for maintaining parental binding fragment characteristics.
Nevertheless it is contemplated, in another aspect of invention that the final library can be a pooled library in which several libraries each having varying degrees of biasing to wild-type, for example, b0%, SO%, 4Q% and 3Q%, are pooled together to obtain the both desired variability and similarity.
1t is to be understood that biasing of a percentage less than 100% implies unless otherwise implied or Stated that the remaining percentage is fully randomized.

iziuaiaa wro ia:ue rvx aia aaz uax~ muuu~r !k ~IAYB~g løJU46 Unless otherwise specified, a given "% biasing" or "% of biniding-fragments"
(or "biasing 10-100%", ttc.) refers to biasing on an individual amino acid basis (though other techniques to accomplish the same effect might apparent to those skilled in the art).
Similarly, the specification that wild-type amino acids occur at a specified position or ser. ies of positions in, for example, at least approximately 50% of potential binding-fragments is intended to mean both that 50% biasing is sought at a given such position or that a total of 50%
of the correct nucleotide triplets are represented. The use of the term "approximately" in reference to percentages is intended to accommodate attrition of various desired potential binding-fragments, the inaccuracy of the assumption that the probabilistic outcomes will be 1Q achieved in practice and that certain variation in methods to accomplish the specified results is deemed to be suitable.
The practice of the invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual.", second edition (Sambrook ct al., 1989); "Oligonuclcotide Synthesis" (M.J. Gait, td., 1984); "Animal Cell Culture (R.I. Freshncy, ed., 1987); "Mtthads in Enrymology" (Academic Press, Ine.);
"handbook of Experimental Immunology" (~.M. Wei & C.C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J.M. Miller & M.1'. Calos, cds., 1987); "Current Protocols in 24 Molecular Hialagy (F.M. Ausubel et al., eds., 1987); "FCR: The Polymerase Chain Reaction", (Mullis et al., eds" 1994); "Current Protocols in Immunology" (J.E. Coligan et al., eds., 1991).
These references are incorporated herein by reference. ?hose techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, can be considered in malting and practicing the ittve>atian. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
Recombinant genetic techniques have allowed cloning and expression of antibodies, functional fragments thereof and th.t antigens recognized. These engineered antibodies provide novel methods of production and treatment modalities. For instance, functional immunoglobulin fragments have been expressed in bacteria and transgenie tobacco seeds and plants. Skerra (1993) Curr.ppin. ~Cmmunol. 5:256:262; Fiedler and Conrad (1995) BiolTechnology 13:1090-1093; Zhang ct al. (1993) Cancer Res. 55:3384-3591; Ma et al. (1995) Science 268:916; and, for iiiuaiaa wry is:ua rw 4ia aaz usz~ xmuu~r ~ mnlcBr;x ~uaa a review of synthetic antibodies, see Barbas ( 1995) Nature Med. 1:836-839.
These and more current references describing these techniques, which these references, particularly those well known to persons practicing in the relevant arts, are hereby incorporated herein by reference.
Nucleotide sequences can be isolated, amplified, and processed by standard recombinant techniques. Standard techniques in the art include digestion with restriction endonucleases, and amplification by PCR, or a suitable combination thereof. PCR technology is described in 1rT_S_ Patent 1'Jos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, as well as PCR:
The Polymerase Ciaain Reaction, Mullis et al., eds., Birl~auswer Press, Boston (1994).
Polynucleotides comprising a desired sequence can be inserted into a'sui,table vector, and the vector in turn can be introduced into a suitable host cell far replication and amplification.
Polyr~uolevtides can be introduced into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, f mating or electroporation. Once introduced, the exogenous polynucleatide can be maintained within the cull as a non-integrated vector (such as a plasmid) or integrated into the host cell by standard methods. See, e.g., Sambrook et al. (1989). RNA can also be obtained from transformed host cell, or it can be obtained directly from the DNA by using a DNA-dependent RNA polymerasc.
Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific ve~ors and suitable host cells are known in the art and are n.ot described in detail herein. See e.g.
Gaccsa and Ramji, Vectors, John Wilcy & Sons (1994).
Phage display techniques are generally described or referenced in some of the preceding general references, as well as in U.S. Patent Nos. 4,593,002; 5,403,484;
5,837,500; 5,571,698;
5,750,373; 5,$21,047; 5,223,409 and 5,702,892. "Phage I]isplay of Peptides and Proteins", (Kay, Brian IC. et al., 1996}; "Methods in En2ymology", Vol. 267 (Abclson, John N., 1996);
"immunology Methods Manual", (Lefkovits, Ivan, 1997); "Antibody phage display technology and its applications", (Hoogenbo4m, Hennie R. et al., 1998). Immuaotechnology 4 p.l-20.
Generally, DNA encoding millions of variants of a parental binding-fragment can be batch-cloned into the phage genome as a fusion to the gene encoding one of the phage coat proteins (pIII, pVI ox pVIII). Upon expression, the coat protein fusion will be incorporated into new phagt particles that are assembled in the bacterium. Expression of the fusion product and iziusiaa wru is:iu rv~ aia anz usza xmuu~r ~ marYxxx ~ua7 its subsequent incprppration into the mature phage coat results in tho ligand being presented on the phage surface, while its genetic material resides within the phage particle. This connection between ligand genotype and phenotype allows the enrichment of specific phage, e.g. using selection on immobilized target. Phage that display a relevant ligand will be retained, while non.
adherent phage will be washed away. Bound phage can be recovered from the surface, reinfected into bacteria and re-grown for further enrichment, and eventually for analysis of binding. The success of ligand phage display hinges on the combination of this display and enrichment methad, with the synthesis of large combinatorial repertoires on phage. Creation of single chain Fv's and other important methods are generally described in U.S.
Patent Nos.
4,946,778; 5,260,203; 5,482,858; 5,258,498 and 5,525,491.
PPEs SPpCs, wherein the peptide portion is a consensus conforming peptide, can be used to create effective treatment against tumor in the manner described, for example, in use Patent Nos.
5,750,119; 5,$30,464; 5,$37,251; 5,94$,646; 5,935,$76; WO 97/Ob685 and W4 991227fi1, the disclosures of which are hereby incorporated by reference. In one embodiment it is contemplated that a composition enriched for consensus conforming peptides as presented by PPEs. Preferably the composition at least predominately comprising such PPEs.
PPEs present the consensus conforming peptides in a manner in wliich they share antigenic determinants with SPs e.g. HSP7Qg and F~SP90 so that for a given peptide the PPE will be immunogenic with respect to that peptide as presented by a SP. It follows that PPEs are preferably SP - CCP complexes wherein, the Sly is preferably the same $P as tlae SPPC targeted.
PPEs include APCs and other well known peptide presenting scaffolds (sec c.g.
Hoogenboom 1998 rGferenccd above, which discusses and refers to several of these). It is contemplated that the non-CCP parti,on of the PPE can be a substrate presents difFerent CCPs on different locations on the substrate preferably within a grid in which each individual CCP is preferably characterized by its specific amino acid sequence and location on the grid.
Alternatively, with respeots to aspects of the invention calling for a library of CCPs, individual CCPs can synthesized, reconstituted with commercially available SPs (e.g. soureed from Stressgen) and plated out in wells in a nnanner conducive for high through put screening. For example, peptide mieroamays for antibody screening can be accomplished in the following manner=
peptides candidate conforming to the consensus sequence for MAb H11 and SP binding are expres$ed as iziusiea wry ia:iu rw am aaz uaz;t xmuu~r ~C DIAYBI;yøJU48 baeteriophage fusion proteins, other fusion proteins or synthetically synthesized and tethered to tile surface of microchips, polymer beads or conventional plastic ware (e.g.
96-well plate) or similar display systems. A human combinatorial antibody (antibody fragment) phagc display library is generated and used to screen against the array of consensus conforming peptides.
Positive binding antibodies and their corresponding peptide antigens are identii'ied by the sequence of the peptide to which they bind. For recombinant peptide protein fusions, control antibody screening is performed against the bacteriophage without peptide.
Positive binding is considered to be two times the signal to noise ratio above the control.
Screening of tumor cells or tumor ccl l surface extracts (fresh, frozen or fixed or other method of preparing tumor cells or tumor ctll sur-.faCe extracts), either individually or in a display system described above.
Correlation of the cancer type with the peptide typo is used to establish the relationship between individual peptides and cancer types. Ultimately, the cancer specific peptides) could be used as candidate itnmunogens to induce an anti-tumor response specific for the specific tumor type.
Various methods for high through put screening are described in the art.
Reference is zztade to Spacer, R. W. Btoteehnol Btoeng 1998 Winter 61:1 61-7; MeDonald, O.B. ct al.
Anal Btochem 1999 Mar 15 268:2 31$-29; Crameri, R. et al. Comb Chem Htgh Throughput Screen 1999 Apr 2:2 63-72; Platonova, G.A, et al. J. Chromatogr A. 1999 Aug 6 852(1) 129-40;
Crameri, R. et al.
Comb Chem High Throughput Screen 1999 Apr 2(2) 63-72; LuekW g, A. et al. Anal Biochem x 999 Can 15 270( 1 ) 103,11; Schullel., :l.R. et al. Anal Biochem 1997 Mar 1 24b:1 20-9; Kay, B.K. ct al. Mod Divers 1996 Fcb 1:2 139-40.
Alternatively, individual CCP-SP complexes can be generated by creating, via PCR., a variety of polypcptidcs that have a repeating consensus motif e.g.[(HyX HyX
HyX Hy~, wherein n = 2 to 50] and individually translating into separate populations of cells so that they arc naturally associated with SPs after proteolysis, within the cells, and later extracted for use a's a source of SP complexes according mall known method (see far example U.S.
Pate~~t Nos.
3,750,119; 5,830,464; 5,837,251; 5,948,66 and 5,435,376 and references disclosed therein).
For "cEllular loading" of endogenous SPs with exogenous-derived CCPsv one optional host cell line is vne that down-regulates MHC gxp~sgiQn, i,e., most human cells. 1n one embodiment, where the exogenous CCP is not expressed in. cuff cicnt abundance to be identifiable by its preponderance (e.g. evaluating the degree of binding to a given antigen-binding fragment specifc for HS-CCP complexes) strategies can be employed to decrease lc~us~99 wrlJ 15:11 rW 41a ;iaY USLJ xWUUT ~C mAYBl;ItøJU48 competition for Sps by host-derived proteins/peptides the cells arc infected by a virus for example, which takes over the host cell's pz~otein synthesis machinery such that almost all of the protein produced by the cell is viral proteins. One can modify the mammalian virus outer coat structural protein by introducing a CCP sequence in a location of the viral protein that does not adversely affect virus assembly and cell infection. Individual CCP sequence virus libraries can be produced in this manner. To create a library one can transfcct each individual wells/plateslflasks containing the appropriate host ceh line (e.g. Daudi) with a specific recombinant-CCP-containing virus and incubate for an appropriate period anal then isolate SPPCs from each specific infection and analyze for CCF content.
According to another tmbodimcnt of the invention nucleic acids encoding CCPs can be used for immunization in the form of a "DNA vaccine", according to well-known methods.
SP-CCP Complexes: Other Methods of Production In another embodiment of the invention heat shock proteins bvuod to consensus conforming peptides (CCPs) can be generated intracellularly by synthetically generating a polynucleotide encoding a plurality of such CCPs as segments within larger peptide. Optionally, enzymatic peptide cleavage sites can be introduced between the various CCP
segments to ensure intracellular cleavage of intact segments. These polynucleotides are optionally amplified prior to introduction into a host cell for expression. The polynucleotides are then inserted into an expression vector or intraehromosomally integrated, operatively linked to regulatory elemcnt(s) such as a promoter, for purposes of expressing the encoded proteins in suitable host cells in vitro.
Reference is made to US Patent No. 5,94$,646, which describes an.alagous methods using cancer cell DNA, the disclosure of which is hereby incorporated by reference.
The polynuclcotides are introduced into host cells where they are expressed by tht host cells, thereby producing intracellularly noncovalent complexes of SPs and peptides (including those peptides encoded by the polynuclcotides). The recombinant host calls can be cultured on a large scale for production of large amounts of the immunogenic complexes. The polynucleotide library can be stored for ftrture use (e.g. by lyophillzation or freezing), or expanded by replication in a cloning vector in suitable host cells to meet increased demand for the subject immunogenic complexes.

iziusiaa w~~ ia: ii r~a~ 4ia adz uaza xmuu~r ~ mAYti~;~
Optionally, the host cell is a cancer cell, optionally which expresses SPPCs on its surface, optionally the same type of cancer as the tumor target, optionally cancer cells of individual sought to be treated by the SP-CCP complexes.
Methods of Purifying SPPCs S A variety of methods have been proposed in the literature for purifying SPPCs.
Reference is made to W095/24923 and to more recent. US Patent No. 5,948,646 (Srivastava et al.) and W0991291$2, the disclosures of which are hereby incorporated by reference.
Consensus Sequences Aecordi~ng to one embodiment of the invention, the consensus sequence is Hy~HyXHyXH, as described in Blond-Elguindi at al-, 19 Nov. 1993, Vol. 75 pages 717-728.
Consensus conforming peptides arc preferably those that are represented within nature.
According to another embodiment of the invention, with respect to human tumors, consensus conforming peptides that arc represented within human cells are preferred, inasmuch as these proteins are the potential source of consensus conforming peptide that are picked up by SPs and brought to the surface of the cell.
According to another embodiment of the invention, the consensus sequences were generated by panning against H11 antibody described in published PCT
Application Ivlo.
PCT/US97/0$942, an antibody that recognizes heat-shock protein peptide complexes on a number of different tumors, and to SP peptide complexes before treatment with ATP but not after. Other preferred consensus peptide motifs are described throughout the application.
Suitable phage display libraries including, but not limited to the Ph.D. Phage Display 12-mer peptide library (NEB) were panned against the H11 antibody (including and not limited to the IgM, IgC3I, seFv and other antibody fragments) exactly as described in the relevant NEB
technical bulletin. See page 11 Sloan Kettering Patent WO 99/22761 for full details. Phage particles were prepared from individual clones and DNA was extracted and sequenced using the Applied Biosystems automatic scquenecr and the deduced amino sequences were obtained.
hollowing three rounds of panning the library was enriched for the following sequences:
7-rner peptide library 0 2. Consensus: [H] [+l (+7 [H] (ul IH~-1 f~1 s a 08/12/1888 18:13 X416 362 0823 i0receivecJ

iziusiea wr;~ is:m rw am svz use; xmurr ~ mnY~r;x ~uuz 412 H R 'Y S L P

F H F. Y S D Y

F H R Y S P T

F H R Y T P G

F l1 R Y S L P

M H R Y T P L

12-mer peptide library 14 Consensus: [H] L+] [HIU] [UIHI+] [HIU][HIUI-]LU/HJ

8 $ 6/2 413/1 6/2 5/2/14/4 V R L Q P G A

M H P 'W P T Q

I H W S L v P

W I~ W S W I Q

F H W P T L Y

M H G L N A N

'V' R G H L D P

W H W T W P N

Where (H) = Hydrophobic amino acid (+) = Positively charged amino acid (~!) - Uncharged amino acid ( -) i Negatively charged .
amino acid 3. SEQUENCES : FHRYSLP

2$ FHRYSDY

FHRYSPT

FHRYTPG

F'HRYSLP

MHRYTPL

YHVRLQPGAA.A.A

AQSMHPWPTQSL

IHWSLVkW SNRS

WHW S WIQNA,APN

FHWPTLYNMYtP

QLQMHGLNANRQ

VRGHLDPPEAWP

WHWTWPNMTIPQ

Amino acids were grouped aeeordintgto olec. ed. 1990; Darnell M Cell ct al.
Biol., W.H. Freeman and Co.

7-nner Peptide library analysis Residue 1: INVARIABLE - Hydrophobic [FsM]

iziusiaa whu m :i4 rvx am sat usza muuu~r ~ mAY~~h ~ uus Residue 2: INVARIABLE - Basic [H6]
Residue 3: INVARIABLE - $asic [R4]
Residue 4: 1NVARIA)3LE - Hydrophobic [Y~]
A. Residue 5: YARIABl.E- Uncharged ~S4T=J
B. Residue d: YARIABLE - Predominantly hydrophobic ~P3l.~lDJ
C. ~4'S~d~E 7: YARIABLE- Hydrophobic/ Uncharged ~PaZYTC'xJ
12-mer peptide library analysis Residue 1: INVARIABLE - Hydrophobic [VzMzIWiF]
Residue 2: INVARIABLE - Basic [HGRz]
Residue 3: VARIABLE - Predominantly Hydrophobic [WøI,PIG2]
Residue ~: VARIABLE - Predom. UnclaargedlHydrophobie [Sa'~'~IWPIH]
17. Residue 5: ~ARIABZE- Fredorn. HydrophobicliJncharged (PlLZYYz~TNJ
E. Residue 6: VARIABLE - Predominantly Hydrophobic fYlLsl.PlGTlDJ
F. Residue 7: VARIABLE- Predom. Hydrophobic or Uncharged (Q3YN~lAP?J
Phagc isolated from the panning were also panned against the I~sc 70 and bound the same peptides. 1. Biol. Chem. Vol 270; 19839 (1995). Panning a 15-mer peptide phage display library with Hsc70 resulted in the following observations:
Binding peptides require:
1. Peptide contains large hydrophobic internal. residues.
2. Enrichment for basic residues.
3. Substitution of basic residues results in reduction of binding affinity.
4. Comparison of poptide binding to Hsc 70, BiP and bnal~ indicated that some residues were common whilst others were exclusive and binding can be fine-tuned.
See also Proc. Nail. Acad. Sci USA 87:6378 (1990); and Cell Vol. 75:717 (1993).
Panned peptide $-mer and 20-mer peptide display library; BiP 70 preferentially binds:
1. Peptides captaining aromatic and hydrophobic amino acids ire alternating positions, 2. Peptides bind in extended conformation with side chains of alternating amino acids pointing iota the cleft of the BiP 70 molecule.

1L/U8l88 Will 18:14 1~'A~ 4ltf ;fUG USl;f HlDUIi'1' !k mAYBt;J~IUU4 3. Synthetic peptides could be reassociatcd with BiP in vitro and Shown to stimulate its ATPase activity.
Exa Peptide-SP Reconstitution protocols SP 90 or SP 70 and associated peptides are mixed in appropriate concentrations For example (but not limited to) 1 u$ of peptide to 9 ug of Sp or 1 molar ratio of 5 peptide molecules to I molecule of SP.
The mixture is incubated for Z h at 30°C with an incubation bufl;'er consisting of 3 mM
MgCl2, 1 mM ISMSF, at pH 7.2. Excess or unbound peptides are removed by centrifugation through a Centrieon 10 concentrator. Altenaatively, SP 90 and associated peptides can be incubated in NaxP04 bu~'er at 50°C for 10 min followed by incubation at room temperature for 30 minutes.
HSP70 and associated peptides arc incubated at 37°C in sodium phosphate buffer containing 1 mM ADP and 1 mM MgClz. Free peptides are t~emovcd as above.
(Reference, Blachcre, N.E et al; J. F,xp. Med. 186, 1315 (1997) for details).
Introducing genetic variation into the sequence corresponding to the H 11 heavy chain CDR3 region: Oligonucleotides comprising randomly mutated CDR3 regions were prepared on an Applied Biosystems 394 DNA synthesizer as described above.
This anti-colon formula [(AIC)NN], is used and results in a reduction in possible colon usage from 64 to 32 and reduces the number of possible stop colons. Position one, therefore, comprises only A and C in the synthetic reaetipn mixture. For complete randomization of the second and third positions of the colons the dN?P mixture comprises 25% A,G,C
and T.
The 3' oligonucleotide randomizing primer was designed such that the last 15 nucleotides of framework 3 and the first 16 nucleotides of framework 4 were kept constant for hybridization.
The nucleotides encoding the intervening amino acids, namely all the amino acids of the CDR3 region except 101 and 102 (ICabat numbering system, Kabat of al. 1991 ) were randomized.
ti n f a bin i : MBthods of generating scFv and binding fragment libraries are well (mown in the art.

iziusiaa w~ is:ia rva am avz usza Hmuu~r Ik ~IAYB~J~JUU6 For maintaining various peroentagcs of wild type amino acid residues: This is achieved by creating residue substitutions by using different spiking levels of the various dN'TPs as described below.
To achieve approximately 50% biasing to wild type at any one position in the region during antisense synthesis using the DNA synthesizer , the following example would be used. In the case of tyrosine, which is encoded by TAC or TAT (antisense strand GTA or A.TA) the nucleotides would be spiked as follows for the antisense strand.
The nucleotide spiking levels would be as follows:
First anticodon nucleotide position: Only 80°fo of A and 20% of C is added, G and T are 14 not added to reduce colon degeneracy.
Compositions of CCPs Suitable compositions of CCPs for use in a conservative approach (starting with a smaller number of CCPs to deterrruine their representation on tumor calls, as opposed to using some of the larger subset disclosed herein) to screening CCPs for tumor-relatedness include evaluation of the following two subsets of consensus conforming peptides generated $om 7 mer and 12 mer peptide library panning ofHl l binding sit, respectively, arc as follows:
This was generated on Genbank with the 7 amino acid consensus sequence [PAILMFW'VY][~H][KRHI[PAILMFVVVY~[STNQG]jPAILIvIFW'VYED~[PAILMFWVYST
NQG), which corresponds to the consensus sequence of H + f H ~T H/- H/U
loo iziu~iea wru is:is r~ax am auz usz~ Hmuu~r ~ mn1t13r;r; ~uua Conacnsus Conformln~
Seqaonce:

PHHPGFL YKKWQEN 110 FKHVSPA AKKLQpM LKHVNAL

LKKYGFS WKRYNIL VKRVSEN 165 AKI~LQAM MKRI~5YI

VHRPGFY AKHVQFV ARKIQVP AKKL,QAM22O PKItWSVI

VHRPGFY AKHVQFV ARKIQVp MKRLGMF pKRWSV1 VHRPGFY 60 IKF1PNLV FICK,AQEL LHHx,TIP PKItWSVI

VHIi.PGFY IKHPNLY 11S FKICISEG ARRLTLA PKRWSVI

VHRPGFY AHRLGLF FKKI$$G 17O ARRLTLA PKRWSVI

VHRPGFY YKRVGAL 'FKKISEG VHRL.SML225 AKKFGVL

IOVHRPGFY YKRVGAL FKKISEG IKICVSYN PRKAGVF

AKKMGLV 65 YKRVGAL FKK1SEG iKKVSYN PRKAGVP' MHKAQLV YKrtV~fAL120 FKKAQIrI, LKKLSFY F~iPTYG

AKi~,SMY AKRFQAQ AKHLTPV 1KKVS~'N23O 1RRYSDA

15A~tLGYI AKRFQAQ AKHLTPV VRKANDI IRHFTDS

FKRVGLA 70 AT~RFQAQ AKHLTPV YRICANDI IRRYSDA

AHHLGPA AKRFQAQ 125 AKT~,TpV LRHISPQ YRRLQYL

AHHLGpA AKRFQAQ LRRISLS 180 LRIiISPQ YRRLQYL

20FHRPSEL PRRVGIA AKICLQpI LRH1SPQ YRYtLQYL

FHRpSEL 75 VKKVNBS AKYCLQPA IKKANEV PKRISPV

LRRLQDV AKRPGIY 130 AKKLQPA 11CKAN$V PKRWSLI

LRRLQDV ARRLTVS AKKLQPA 1$5 1KKANEV AKKPTAA

LRRLQDV LItKPGLP AKKLQPA 1KKANEV 240 1ICKVQ17L

25LRRLQAV LKHPGLA IHIiWNAN LRHISPQ IKKYQDL

VKKVNAV 80 YHRINEG LHHLTIp LRI~SPQ IKKVQDL

3OLRRLQAQ VRHMQEW LHKIGAL VRR.AQPP AKRLGYI

VRKFQIP $5 FKRLNIN LHK1GAL VRRAQPP AKRLGYI

VKITVTVI FKRLNIN LKKLSI~Y195 VRRPSLQ VKKIQES

IK,KtSPF FKRLNIN AKKASAF VRRPSLQ 2SO VKKIQES

35IKKISPF FKRLNIN AKKASAF VRF~SLQ VKK1QE5 IKKISPF 90 Al3IiA5EN LRRANLT VRRFSLQ LRHYSWA

FIifLLSDQ AHHASEN IIZ.~tIVEN200 VRRPSLQ VKKPGAS

4OFRRLSDQ LRKIQES MKRLCMF VRIi.PSLQ VKKPGA$

Alili.PTVG VRHITEQ 15O WKKLQLG MKKVTIS VKKPGES

VRHATVT Pt-RiPGFL IfiRLSLMZOS WKHI,Sr31 LKKPGES

vKKLGFN pl~IPGFL MKRLGMF wKHI.SDI260 VKKPGES

pFIHYSEPlO0 LRRLQLL MKRLGMF AKKASAF LKKpGBS

YHKPQFA LRRLQLL 155 LH>:ILTLP AKKASAF LKKPGES

YHKPQFA AI~MGVM LHHLTIP 21Q LRRAT1S LKKPGES

bO

PRRLSLG MKHMQVN AKKLQAM 21$ pIiKVSAG VKKPGES

WHKLGIS LHHPSEA AKKLQA.M PHKVSAG 270 MKKPGES

iziuaiea wr;u is: i6 rw aia aaz uaz:f ltmuu~r ~ mnYli>;r;

MKKPGES LHKFNEF LRRLSAC3170 FHR.TNIY FKRLGEL

MKKPGES LHKFNEF fi~L,QDw PKKAGDI AKKLQAM

MKKPGE$ LHKFNEF IHKLQDW AI~KVNWI230 LRRIQpP

MKKI'GES LIWEF 1HKLQDW 17S AKKVNWI LRRIQPP

MKKPGES LHK>:;NEF1 VKRAGLN VHR1ST.F LRRIQPP
ZO

MKKPGES 6S LHKFNEF VKKVQAF TI11~LQDW LRRIQPP

MKKPGES LHKFNBF VKIUNES lriKLQDW FItHFIYPG

LKT~PGLS LHKFNEF PRRAGPI IHKI,QDW23$ LRRIQPF

VKKPGES LHT~FNEP PRKPSAp 1.80 IHKLQDW YRKLSMQ

VKKYGES LIZKYTYN 125 ARRpGAA IHKLQDW YRKLSMQ

VKKPGE$ 70 VKKMTVT ARRPGAA IflItLQDW IKRWQAI

LKSCPGAS vKKMTVT ARRPGAA LRRMSVI LAHLNFT

VKKPGES VItKMTVT ARRPGAA AKRANAN 240 LRHLNFT

PKKPSAG VKKATVV AItR.PGAA1$5 AKRANAN AKKLG~.T

AKRLQVN VKKATVV 130 PRHISVA AKRANAN VKi~IQEN

AKRLQVN PHRAQPA LKKPSET ARRLGWL FKKANMA

FRKLTPS PKRATEM LKKPTET ARRI,.GWL245 MRKLQLG

FRKLTPS VKKWG1N 135 YRHWNEW AKKYTBF WICIt.PTEL

FRKLTPS $0 1KHPNLV FKRISEQ FRKPSYI VRHLGIV

VKKPGE$ LRKAGLL Pl~Hi5YA F'KKFNIrI250 FRKLNFN

VKKPC,A$$$ VKHLQVF PItHISVA FK1~FNE1 FRKLNFN

IZCHLSVN VKHLQVF PRH1SVA FKKFNEI AI:,ICLSEQ

IKHLSVN VKHLQVF IRHLGWL FKKFNEI 255 AIttCLSEQ

LHI(F1YEF90 YRHWQ1P LKKVTMN ViII4LSYY LRRANPS

LHKFNEF LHHPSEA VKKATV VHKLSYY L~RA1VPS
V

LHKFNEF 1F~KVQFA VKKATW VFiKLSYY26O )aKRMTAL

LHKFNEF VRKVQFA 150 IRKVQYA WKR.AS1Q ARHFQL[, LRTCMGAP95 VRRPQAV IRKVQYA LKKLGVN LI~LNFT

LRKMGAP PRKVNLG IRKVQyA LKKLQVN LRHWGWG

LKKLTI$ IHfIFNEG IRICVQYA Vl-1KLSYY26S LRHWGWG

LHKFNEF VKRLTDA II-ECYNAY PRRVGIF ARHPSFF

LHtCFN$F VKRLTDA LKKPT81' FRHLGES 27b ARHPSFF

L1~KFIVEF VKRLTDA FKRISEQ 215 yKKFGAT ARHP$FF

LHKFNEF VKRLTpA 1~O FKRISFQ LKRLSLG LRTC,FGVP

LHKFNEF 105 LRRI,TPS FKRISEQ LKRLSLG LRKFGVP

LHKFNEF LRRLTFS FKRISEQ LKHAQDS LRKFGVP

LHKk'NEF FHHLTVA LKRVSEQ MKHI,TVQ275 LRKFGVP

I.HKFNEF FHHL'I'VA LKRVSEQ 220 MKHLTVQ VRRLTVG

LHKFNEF AI~LN1A 165 PHRFNIaT M.KHL,TVQ L1IKVTYL

LHKPNEF 110 ARRLSLM k"ICKVSLL LRHPQPG LKKLGIL

LT~pNEP AKKFSAV LKRATVQ LRNPQPG LKKLGIL

LIIKFNEF AKKF$AV IKHVGPS LRI~PTAL280 LKKLGT,L

iziusiea wru is:ia rw am Paz uaza Itmuu~r a~ m~rY~>r>; ~uo~

AKRLQVN AKRPSAA LRKPNVA 170 M1~'NBN BCKt~TFG

MHRLQLS LI'IHLQPF113 PHHVSPQ M1CRFNEN LHHpNLG

Mt-IR.LQLS6O WRRLGVQ PHHVSPQ ARRF$TN FRKVTLT

S MHRLQLS FKIUSEG PHHVSPQ ARRYGDV PRHLSLA

PKKMSVL AKKVGYT PHHVSPQ LRKAQLQ 230 IIiFiWTPP

LRHI'NIV AKKV[',y~'120 LRRPSDQ PKHVGFA PRKAGVF

LRHPNIV 65 Vf?,ItAQLV FHKVTVQ PKHVGFA LRR1QPP

lO YRRMSLA LRKIQFN FHIKVTVQ LRIxFSLM LRRIQPP

LRHPNIV PHHAGLN FKKATVT AfiRYGDV235 AKIZLTES

IKHFG1~IL LKKYGDi PHHIOVA 1$0 FKKLGIP AHRVSAL

IKI~IFGML LKKFNEL 125 PKKMSVL FKRLG1P AHRVSAL

PKRVGLI 70 AKRLTLG PKK:MSYL FKKLGl'P VHKPGPI

15 PKRVGLI PKRWSVI WKKYQFP FKKLG1P MIiKYQMT

LKHPNVI PKRWSVI WKKYQFp PKHVGFP 240 MRKYQMT

LRRMQEM PKItWSVI N~HI.TEN185 IRRFSLM LHKPQES

LHHFNLC~ PKRWSVI 130 MHHLTBN IIi.ItFSLM FRKLQDA

20 LkHVQLA LKKATAY MHHI.TIrN MKRFNEN YRKYSDY

LKRYSEM LKKATAY MFIHI.'Y'EN FTCKL(',Ip245 YRKYSDY

LKRYSE14I LKKATAY 135 LHRLSLP MKRFNEN Lt;RpGLL

25 LKRYSfiM LKKATAY LKRpSVL MKRFN~S1 AKRAQLQ

FKKPSAA YRRLQPS LIUiVGPG IRRFSLM 250 IHKYNAY

>~IZHVQLA iKKITA1 LKHYGPG 195 1RRFSLM IHICYNAY

LRHVQLA tT~ITAi 140 LKIiYQPG IRRFSLM LRRFGPP

YRHFNAS S3 1KKTTAI LKH'YG1G PKl-IVGFA LRRFGPP

30 LHHLQEQ IKKITAI VRKYSES PKHVGFA VRHiSPT

YHKYSLI MKIILTPS LKKIGDT 200 PKHVGFA VRRASDp LHHMSLQ IIiKLQDW145 LKKIGDT LRRFSVT PRRLSES

VRKFQIP 90 iHKLQDW LKICIGDT LRRFSVT LHRAGLL

3$ AKKIGFG IHKLQDW ARRVTFS IRRFSLT LHRAGLL.

LHItLGII IHKLQDW LHH1GMQ IRRFSLT 260 WR105SPF

AKK1GFG VKKISAA LHHIC~MQ205 FHRVGPI LRKATTP

FRKFSPF 95 wHKLGIS rRKFStv LR~LGiV MRKATAS

LRKLNPP VKKiSAA IRKFSIV LKRATVA 265 YRHWQiP

VKRLTYP ARK1GVL 1RRFSLM zlo IiI;KASDV NIKRPNPP

FRKAQ1G 104 ARKIGVL MRKINPL FRRIQDP LRRP'NAG

43 VRRAQLY A~GVL MRKINPL ARRAQEL ARRAQEL

LRK1QFN ARKIGVL P1~YSDY ARRAQ$L 27O ARRAQ$L

LRKIQFN LRHPTWP VKHLSAS 215 LFIRLSC..P ARRAQEL

LItKIQFN LKKFQDS 16O uKHLSAS PHKVQVN ARRAQEL

IRRFQEG 145 LKi~QDS VKHL$AS FRKMTEA LItRFNAG

SO IHRFQEG LKKFTEY MRKp,GtF AKRIQLS VRKIGEL

FKRLNt,V WKKVTVT IyRRYSDY P~,TDL 275 LKRMGMS

PIZRINLT WKRYGAL PRRYSDY 22O FHi3ANFp LK$MGMS

PRRiNLT WKRYGAL 165 VKHLSAS MKKLSYI ARLCIQYP

ARRIQDP 110 PKKINLN ARRFSIT1 MKKLSYI VKRITF,S

55 IKKPCVV WKRFSVP RILI~SIN MRKATAS ARKPGY1 IKKPGW MRKLQAT MKRFNEN YKICLTDP2$O ARKPGYI

iziusiaa wru is:ia rv~ am :~nz usza xmuu~r ~ mnYl3r;r; ~uua FRRLNFA PHKINPL LHKVTYL LKKAC~YV225 IKKAGAA

k~RRLNFA ARRWGIQ WKRVSDi 170 LKKISIP IK.KAGAA

FRRLNFA LKKLQEA 115 pRKI,QDV LRRVTDL IKIAGAA

FRRLNFA 60 WKHLSDI ARKLQDV yRKpGLp ~GA,~

S LHRVTIA WKF3LSDI LRKMGAP YRRYQIaW AKKFGVL

FHKATAN AIKKi LRKMGAP YRRYQDW 230 FKHPQpF
SMY

LRRFSIW 6S LKKL.QTY LRKMGAP LRKVSVS VKKIQPL

iKKIGY1V VRYtLQAL LRKMGAP LRKVSVS AKRLQDY

YH,1(pSVF ARRWGIQ 125 PRRVSEA MKKtSDL VKHLSLG

VHKLTtA YKKFGAT PRKLTLM 185 YKRFQLL VKHLSLG

VHKLTIA FRKLGLY 130 PRKLTLM AKRFSPP VKHI,SLG

VKRFSPM WKRASVV PHKINPL LKKLGVN VKHLSLG

IKKIGYN LRHLNF'T LIC.RFTVV'1' AKKVGEI 245 VKHLSLQ

IKKIGYN PI3RASDG LKR'FTWT 1.90AKKYGEI VKHI,SLG

IKKIGYN LRRLTPS 133 ARHATLS AKKVGEI VKxlr gLG

VI

I1QQGYN LKHISEL vR1-IFQFL PKRVQVL LRRMgLI

IKKLQIQ LKIiISEL YRRPSVA ARHLQEY 250 VKRINMA

VRRANE.4 LKHISEL 140 LRHLGLS FKKLGTZ' VKRINMA

LRRYNIP IK.K,AGDG PHRFQYP LHRLQVS IRRIGLF

LRRYN1P FRKLSFT VRKAGIA LHRLQVS 255 IR.RIGLF

LRKLQEL IKKITA1 VRKAGIA 2OO VKKVQAF ARHL'~

LRKLQEL IKICITAI145 VRK,AGIA VRRLTpS ARHLTLS

MRHLTAS 90 MKHLTPS VR1GAGIA VRRE,TPS ARHLTLS

VRRYQvL MKHLTPS VRKAGIA VRRLTPS AHHFSEP

VRRYQVL MKHLTPS LRHLGLS 2O5 VHRYC~ES AHHFSI;P

VRRYQVL IKKFQFL 15O ARKI,QDV MKItVGFQ AHHFSEP

YHKVTAA AHHYSVA MKHPQFL MKRVGFQ AHHFSEP

VRHYNYT IRRPSPF VKRVNIL MKRV.r,FQ265 VHRFTVP

YHKVTAA IRRPSPF FKRLGAG 210 PRKVGYW VHRFTVp YHKVTAA LRKMGAM 155 MKRVGFQ 1KXAGpp ~T-Vp PHRYNIL LRKA,QLQ MKRLTAG IKKAGAA AHHFSEP

LKRMNPN LRKLQEL MRHVSIS 215 PRIUGYW VHI~'TVP

LKRLQAN LRKLQEL 160 FRKLSDS WKKIGIW VHRFTVp AKKMGLV lO5 LRKLQEL YRKAGLP AKHVGYS VHRFTVP

5O VHKJrSIN LRKAQLQ YRKAG~.p AKHVGYS VHRFTVP

IHRLTIG VKKYQAV PRKASVG 220 AKI1VGYS VHI~'TVp VRKANDI AKHLTPV 165 IKKAGAA MRRPNFQ AHIiFSEP

SS ARRWGIQ ~.IIXVTYL PRHISVA AKHVGYS IRKYNLS

>,ziuaiaa wru hs:i7 rva~ am ;saz usz~ xll~uu~r ~ mnY~~r; emu PRkPC3PT VKKDVES ITIRLTEA IRTCYGLN225 IKKF(3LT

YKRWQDY EO IRKFQIL PRHLQLA PHICINPL rRRITAA

YKRWQDV M>~KPGLW PRHLQLA VARL,QAL FRKVGDA

YKRWQDV LKKFNEP PRHLQLA IItHVSFS230 LKI~FNDP

YKRWQDV LKKFNEP PRHLQLA 175 AKRLQEA ARIiPTPN

YKRWQbV LKKFNEP I20 PRI-rLQLA AKRLQEA YHKVNF$

YKRWQDV 65 WKKVTVT PRHLQLA A~LQEA AAi~GDY

YKRLQDS WKKVTVT PRHLQLA YKHINEV IKItFSAS

LKHLTLA FRKLGLY PRFiLQLA LKKFNEP 235 LRKLSAV

FRKLQLS IRRLQLY PRHLQLA I$O LKKrNEP LRKLSAV

MRKFQEQ VK~PGES 125 PRHLQLA LKK.FIVEP LRRVQDL

AHKIQVQ 70 MKKVGVT PItHLQLA AI~tIQFL AFTRVT'DS

LKHLTLA AKItIGEV AKItVTIM LRRPSDQ AriRVTDS

LKHLTLA ARRLSFT AKRV~'1M185 IrHII'PSAV MRRISLF

LKHLTLA LKKLGIL 130 AKRVT1M LRkASAY MRR1SLF

LKHLTLA 75 LKRIS1A AKRVTIM PRK)JQAA MRRISPF

IRICYNLS LRICpSLQ AKRV'I'IM PRKLQAA FRRITLY

MKRLTAG LRKFSLQ AKkVTIM VKRFSPI 245 MItRTSPF

VKRLGIP WHRVTAL AKRVTIM 19O MRKISLF FRItTY'LY

AHHF$EP WHRVTA>; 13S AKRVTIM MRKISLF VKKFGAS

ARHPSPS SO WFHtV'fAL AKRVTiM WHRVTAI. MKKPGES

VRRPSES AIiRLSAS AKRVTIM WI-1RVTA1. VKKPGAS

PRF1LQLL AHRLSAS AKRVTIM WI~tVTAL250 VKKPGAS

TKHFQIT LIaKPGIry140 pRRAGPT AxRISAY VKKPGAS

PRHLQYV $S PHKPQFT PRRAGPl ARKLSEL VKKPGAS

PRHLQYV PHKPQFT VRRVGDP ARKL$EL VKKpGAS

LKT~,SLL LKRYSIP VRRVGDP ARKLSEL 25$ VKKPGAS

FKRLQFT WKRASVV AKRAGVT 200 ARKLSL~L LKKPGAS

LKKVGFN PHHPNPP 145 YHKVNFS LRItMQEM VKKPGAS

YKHLGVF 9O PHHPNPP AKKLGEM TRxr.QTQ VKKPGAT

k'ISRIGYP PI3IIPNPP AKKLGEM FHKWSLS VKKPGAS

YRKVQPS LKTxWSAA AKKLGEM AKKVGrEI2GO VKKPGAS

YRKVQFS LIiKPGVY AKT~LGjrM205 AKKVGEI VKKPGES

YRKVQPS PRHLQLL I5p AKKLGEM AKKVGEi VKKPGAS

PRKISYS 95 AKKLGEM FHRASVL VIi.RVSVA VKICPGAS

PRKISYS YKKVQLV FHRASVL AKRVGDT VKKPGAS

PRKISYS LKKMQAN LRKLGLG FHRASVL 2b5 ARRANLI

PRKTSYS WKRATYI AKi~LQEA210 FHRASVL VRKPGAS

PRKxSYS WKRATYI 155 ARRVNLT MRRISPF VI~pGAS

AhFCLTAQ100 WKItATYI AKRLQE,A, PKKATEL VKKPGAS

YRKVQPS VI~iCVGYL AI~RLQEA ARHLTLS VKKPGES

LKKLGTQ PRHPNVF PRRFGFW ARI~,TLS270 VKKPGp$

PRx~INVS YKRWQDV AKRLQEA 215 ARHLTLS VKKpGAS

II~RLQLY YKRWQDV X60 LKHVNAL PRriISVA VKKPGAS

LKKFSLG 105 YKItVNQDV LRKLNLS PRHISVA VKKFGAS

VKRLTDA PRHLQLA LFtKLNLS M'RKLQAT275 VKKPGAS

LRRPGIrG PRHLQLA LRKLNLS 220 LKRATNPN VK1~GAS

LRRPGLG FHICYGiry165 ITCRLQIA WKRIT1Q VICKPQAS

AKKAQAQ AKKAGAA FHHLTYL WKRITTQ VKKPGAS

I~ziusiaa w~ is:i7 r~ax am ~nz usz~ ltlUUUr c~ mnYl3>r1; gum VKKPGAS VI~KFGAS VKKPGAS I70 VKKJ'G$S MRRISpF

VICKPGES VKKPGAS 115 VKKPOAP VIGIGPGES MRItISpF

S VKKPGVS VKKPGA$ VKKPGAS VKKPGES FICKISBL

1.ICKPGAS LKKPGAS VKKpGES VKKPGES 230 PKKISEL

VKKLNEI LKKPGAS 120 LKKPGA$ VKIGPGES MRR1SPF

VKKI'GAS65 L1CKPGAS LKKpGAS VKKPGES MRKISLF

LIIKVTYL LKKPGAS ARHPSMV YKKPGES Z35 FItKAQTG

VKKPGAS 7O LKKPGAS VICICIyCIAS VKKpGES FRRITLY

15 VKKPGAS LKKPGAS VKKPGAS Vj~KPGES MRRISpF

VKKPGES AHItPGIA LHRYGYN YKKPGES 240 FRKAQIG

VKKFGAS IKI~PGAS 130 VKICpGI~S VKKPGES IKKINPL

MKRFNEN 75 VKKPGF.S VKKFGAS VKI~I'GE5 IKICTNPL

20 M~tFNIrN VKKPGE$ VKKPGAS VKKPGES FRYCAQTG

ARRAQEA Vl.~j(pG$S VKKPGAS VKKPGfiS245 PKKATEL

25 ARI~FTYY VKKPGES VRR1QEP VKKPGES LTiKI,SES

VKKPGAS VKKPGES VK~pGAS VKKPQD$ 25O LHKLSF"S

VKKPGAS VKKPGAS MKKPGAS 195 VKKpGEA AHItAGVL

VKKPGES vKKPGES 140 LKKPGAS VKIceGAS LRRx,QLL

VKKPGES $S VKKPGES VKKPGE$ LRRATEY LRItLQLL

VKKPGES AKKPGES VKKPGES AR,)ZFGVP255 PHKVSAG

VKK1'C3E5 ARHLGYS VKKPGES 204 ARRFGVP PKKATPJ..

35 IKKpGAS VKKPGAS VKKPGES VHR~QEY WRHPTMG

VICR,PGAS VKKPC~A~S VKKPGES YHRFQEY Z60 WRHPTMG

VKKPGAS uKICt'GAS VKKPGES ZOS FHRPSLI WRHPTMG

VKKPGAS 95 VKTCPGAS VKIGPGES VKttLTDA WRHPTMG

4O VKKPGAS VKKPGAS YKKFGES FRIH,SP'Y PKKATEL

V'KKPGAS VKKPGAS VKKPGES PRRPQEP 265 WRI~'MG

VKKPGAS VICKIyGAS VKKPGES 210 PRRPQEP PKKATEL

VK~:PGASlOO VKKPGAS VKKPGES PRRI'QEP FKKPSPF

45 VKKPGAS VKKPGAS VKKPGES LRHFTEY LHKI,SES

VKKPGAS VKKPGAS VICICPGES21S YItKLGVY hRHPNN

VKKPGAS VKKPGAS 1.60VKKPGLS VRHPNYL F1~KL,QEI

V1C1C,PGAS10S VKKPGAS VKKPGES VKKFGEN AKKLQPI

LKKPGAS VICKpGAS VKKPGES MHRYQVN 275 M)~RY'Z
LN

VKKPGAS VKKPGAS VICKpGES220 VKKFGEN MRRYTLN

LKKPGAS VKKPOAS 145 VKKPGES IRKYGDp VHRLSAT

LICKPGAS110 VKKPGAS VKKPGES LIIRLNIP IKKA['.AA

VKK,I'GAS VKKPGAS YKKpGES LHItLNtP2g0 VRKVTLS

musiez~ w>;u is:i7 rv~ mu :Inz uaz' xli~uu~r a~ mnYli>;r; ~mz VRKVTLS YRKPSPQ VEiHPSFL170 ARI~pGbY AKHFSAL

IKKFTFG AHKMQLP 115 LR.ItLSAV MItHLSFT AKHFSAL

IKKFTFG MRKYTvL MH>:,YQYN LRICIT1G230 1KT~LTLQ

MtIRYQVN LKKASVI MI~RISPF175 LIZKITIG LRHVSVY

AKKLQFY ARHYSLS 1,20MRR1SPF ARRITEV VRRLTVG

AKKLQFY f>S PRHFGPV 1KRMQEV ARRITEV PRRYSEG

AKKLQFY AKHASDN IKRMQEV VKRVGli LHIC~QEQ

MKIci.QPT F'fLRPSEG TKItMQBV VKItVGIS235 VKICVQAA

MKKLQPT FRRFSEG iKRMQEV 180 IRKYSI1 VKKVQAA

MKKLQPT 70 AKKTQVP IKRMQEV PRKA.GDF PRRINLT

MKKLQPT THI~PGAF FKH1TPL iTCiCr IRRL.$LG
T.LQ

MKKLQPT PKKAQLl 130 LKRVQLY IRKWNVY' AHRI~(GMG

M1CKLQPT75 TKKISPF I,KI~VQLY TRKWNVT LKICINEL

LRHLGAV 1AGPG 135 YRRVSDM MRR,LGVV TRRYSDA

FHK1GVG $O PRKASLQ AKRAGVT MRRLGVV 1RRYSDA

YHKIGVG LHHVNFS AKRAGVT MRRLGVV PRRLQEG

YHKIGVG PRRFSAL LHIGVTYL195 MRRLGVV VKKP(3A5 WRRFSDQ H5 PRRFSA1. LHHVNFS MRRLGVV VKKPGAS

3U AKHLGFQ IRTCTGbT MRRYTLN MRRLGLA LRRLGAW

AKHLGFQ uRHPNYL YKRLNLT M'ItIxLGLA2S5 LRHINVT

AKHLGFQ iRKIGDT YKRLNLT 200 MRRLGLA LRH11WT

ARHiSFG 90 MKRLSYI LRRIQPP LRRLGLG LRHPQPG

LHIiVGWF PHL~YSPY LRRpNPS LRRLGLG AKKVGYT

LKHIQAP WHRITVI LI~RANPS WKKAGLN 260 LKK1NEA

LE;H/~TVL WHR1TVI 150 LRRIQPP VKKLGFN TRRIGLF

LKiCLTEL95 WHRITV1 FKRFNAL VKKLGFN LR13LGIyV

PRHTNLP WHKVTAN FKIi.FNAL VK1CLGFN AI~HL,NYQ

MKKYQEQ WHRITVI IRKPQW VKKLGFN 2b5 PKRWSVI

AICRPNYV WHRITVI FRRLSDQ 21O FIiIiMNFT FKRWSVI

AICRPNYV W$RTfV1 1SS PRI~'SAL LKRLGMW LKKATAY

IHRYGEG 100 WHRTTVI PRRFSAL LKI~LQPO LKKVNEA

i1-IKLS MKRLSl'i LRRVTIA 215 FKKPTAI IRKPSPF
VS

VKRPQLM WHIcVTAN 160 VKRLGAG PKKpTAI AKKIGEL

VKRPQLM 1O5 VKKLNEI YKRLGAG YHKYSLl AKI~1GBL

SO VKRPQLM WRHLTPT IRKASDV WRRFSDQ VRRAGFA

VRKYQAQ FKKAGWT PKRAGIIvI LKKAGLS 275 AKKIGEL

LRRATVL LRRITYV 165 IRKMSLL AKItLNVT VRHI,T17W

LRRLNFV 110 YKRLNLT VRHATAA AK.RLN VIiHLTDW
VT

SS iRKPGES YKRLNLT MKKLQPT AKRLNVT VRHLTDW

izm~iea wru ia:is rvx 4iv :adz uaz;l itmurr ~ mAY~>rr; ~ma IRRAGYp FKKLTDS FRK~,SPT FRKLSFT 225 IK~iPNLV

Fi:RLSLL FKKLTDS YRRVTET 170 AKRVGLP LKRANEF

VRRAC3YA FKKLTDS 11 IRKYSII AKKPTAA pRKF~T
S

VRRAGfA 6O FKKLT>aS MKItLSYI LKFII,QDI PRI~IJET

$ IRRAGYP FKICLTDS AK1~IQ1S LRRASMQ F'KHLNBI

WRRPCrAA FKKLTDS 12O MiQtPSVV LRR,ASMQ PIiHVSPQ

lO WRRPGAA FKKLTDS MKRPSW LKRLQMQ LKKLN'LS

WRRPQAA MRKATpT IHRVSAA 1$O LKRFQFV PKHVGFA

W:tRI'GAA MRKATPT 125 ZHItVSAA IKKIGYN PKHVGFA

WRRPGAA MRKATPT PIIItYNIL tICHLNVS240 VRRLGIF

WItRPGAA MRKATPT PHRYML 1$S FHKMGVG VRRLGIP

WRRPGAA MFiKATPT PRI-IASLP YkXLSLI 245 YItRATVF

VHRLCrYV MRKATPT FHHLSVV 1~O PRRA'fiS VKKMTFS

LHIiLSVLSO MRKATPT FHHLSW AKK1GLV LRRVTAA

25 AKKIGEL MRKATPT LKxCAT'EY AIZKLQDV LR~VTAA

AKKIGEL MRKATPT LKKATEY ptCKAQLI250 LKKATAY

WKRVHEP LKKYTEY I40 LKI~ATEY ARKWGYT PRKLQAA

WKRVNEP $S PKtfLTDA LRKPSYA AlucWGYT PRKLQAA

VRRASEp IKRP(~DQ AKKASAF ARKWQYT 255 1HHAQbL

iIiKYSAr ARRLGEA AKKASAF 200 LRxLQLQ PRKT.QAA

pRHISVA PI-H~PNPp145 AI~iLNIA 1KRPQDQ PI~LQp,A

LKKAGW 9O PHHFNPP AKKLGEM I.KRPQDQ AKK,WTAY

VHKA,NIM pHHPNPP LKKANLQ VRKLQAN 260 VHRPQPI.

LRHMGLF LItICVSVS LRKLQAQ 2O5 YHHI,TYI YHKPSVF

40 AICRIQIs LRKVsv9 LKRLQLQ WRKLGAp LRHVNI:Q

AKRIQIS LRKVSVS k'HKATAN MKHLSTA 265 LttHLNFT

VKKLTVN LRKVSVS LKHMSPP 21O MKHLS1A LTiRAQAQ

MHR$GPI lO0 LRKVSVS LKHM$PP IKKFGLT LHRAQAQ

45 YItICYTEQ MKR1'SVY LKHMSPP IKKFGLT LHRAQAQ

YRKYTEQ F~IMSLL AKR1TEW Li~LQDV 270 LKf~'YQ

ARKASIV FRHMSLL PRRTSAV 215 LKRLQDV VIiR.PQPL

PHI;WQpS105 LHKIQEQ PHHITVS PKKASFQ VHRpQPL

5O LICRLSEF VKRFSPI PHHIGVT PRK~.QAA VHRPQPL

YRKVQEL IKRWQAI PIiHIGVT P1~KLQAA275 k~CKATVT

YRKVQ$.L WKRPTHI PI3HIGVT220 LRRLQDV PKKPSPT

AKKAG131 YRRFTEI 165 PHH1GVT LRRLQDV VHKyNPT

AKKAGEI 110 IKICpQAQ PHHITVS LRRLQDV IKKVQISL

55 AKKAGE1 PHH1GVA PHHITVS YRxCIGEL IKKVQDL

FKKLTDS IHHAQD.L PHHIGVT IK.HPNLV280 xItKVQDL

iziuniea w~ m:m rv~ 4ia aaz usza ltmuu~r ~ mnY~>Jr; ~u14 PHHFNAS AKKLQFA LIiKFNEF MKKITLL 225 VKHANET

Pl-1HFNAS LRRPQLP 115 LHlCFNEF WRRPGAA LI:~LNFQ

YHItINEG 60 AKKl.QPA LHKFNEF WRRPGAA MKKLSYt YHRINEG LRKIQES LHKFNEF WRRI'GAA MKKLSYI

YRHFQIP YHRPGEG MKKLQES WRRPGpA 230 LKKPGAA

LRItLQEG YHItpGIrG IKRPGAS 175 LKHPNIV LKKPQAA

YHRTNEG PHHLTVI 120 FItRFQMI MKKLQPT IKNLNVS

FKHITPL b5 PHHLTVI FRRFQMI MKKLQPT IKHPNLV

lOFKHITPL PHHLTVI PRRWSWI MKKLQPT IKHPNLV

FKHtTPL VKRPGAA AHHFSEP LHHPGVV 235 IK.HPNLV

FKHiTPL F(fKATVT ll~IiP'IFT1$0 VFIK.PGPI IICHIPNLV

LRRLQEG l:KKATVT 12$ iRHFTFT ARHPQLL ARRVNLS

LRRLQI;G 70 MKKVTIS IRHFTFT VKRAGLN PRRYSPV

15WKtCACLt~r FKxATV'r IRFIFTFT PRKtQFT IKRLQVA

WICK.AGLN MKKVTIS IRI~"TFT AKKLQPA 240 1Kk r QVA

WKKAGLN FKKATVT IRHFI'FT185 AHRPGWL IRHFTFT

AKKLNVQ VKKPC~.~S130 IRHFTFT AHRPGWL LKKYGVT

IKRLNVQ 7S LRKASD$ 1Rt-IFTPT AHItpGWL M11~FQ

20AKKLNVQ LIiKASDS IT AHRPGWL IKRiQAM

SO

vIIRLSAT MxICITL 135 PKKPSAG VRxASLS FHKVTVQ
L

1KJCLSMT 80 MICKITLL AICR.PGIY VRI~ASLS LRRI,TPS

wKKAGLN vKKLTVN Mxt~L,TPS195 IHI~SEP PItRwSWI

MHHLTI'sN VKKLTVN 140 MKHLTPS IHH1SEP ARKLQpV

MHHLTEN $5 LRRLSAG LRRLGAW 1HHISEP PHKFTVS

AKRYQEF LREI,SAG LKKPT'VN IHHtSEP 255 LKRAGIS

AKRYQEF AKRYNVS LKKPTVN X00 IHIiISEP L~ATEY

AKRYQLF AKRYNVG 145 IRKWNVT iHHISEP VKKPGAS

AKRYQEF 9O IHKYNAY 1RKWNVT ITiHISEP LRKIQFN

35A~YQEF PHHYTPI AKR.PGIY IHHISEP ARRIQDP

AKRYQEF PHHYTPI lR~INEN LI~yGVT 2GO ARRIQDP

MRItYTLN 95 VKKWGIN ARHLQ?[', VRHVTIQ FKRFTPP

44FItRLSIS LKICLGLI PKKASFQ 1HI~ISEP LRRPTET

FRRLSIS VRRLQAA PKKASFQ IHIIZSEP265 LRR.pr'ET

FRR1,SIS VRRLQAA LRRATIS 210 IHHISEP LRRFTET

FRRLS1S YHICIGVG 155 PHKYTIG LRItLNFQ LRRPT'ET

YRHIGVL 100 PHI-II,GAM FHICYTIO WHRp'I'EL LltRPTET

45LHKLNIV IKRATLN FHKYTIG LKKYGVT LRRp'1'$T

YRHIGVL FKH1TPL FHKYTIG LKKYGVT 270 1.RRPTET

L'R,Z~Y:IIV VHHVTVS MKHPQ>~M215 VRHFQFL LRRPTET

VKKMTFS PRRYTL4 1 IKRWQAI MR1It11~'Q LRRPTET
GU

VKKMTFS 1 PR7tYTIA Fl~.Ri,TFT AKKIGPL LliltpTET

50VKKMTFS PRRYTIA LHKI,NAV IIiHISEP LHHFNLG

VICICMTFS PRRYTIA PRRLSLG z20 IHHISEP ARHLQEN

VICKMTFS PRItYTIA 1.65IKIQSpF MK1CMQLP FKRLG/~G

VKKMTFS 110 LHHLQEQ MKHPNIV LR'RLGAA AFI~,,QAV

AKKLQPA AHHFSBP MKKTTLL IHIiPGYL280 MHKAQLV

iziu~iue wr;I~ m:m rva aia anz usz~ xll~uu~r rx mnYI;r;Ii ~ul~
y'RHFQIP FRHAQLL FHRIQDQ FIiRPSLI225 LKKLQIY

LKRLSLS LKEiMSVS VFiHPQLV170 PRRMSDP LKKL.QIY

WRRI~QEL IRRLSLG 11S FHKYGEY PKKAC3DI VI~KINES

WRItLQEL60 tRRl.$LG VItICYQEQ PHRPSLT Luxr.QAQ

WRRLQEL ARKFNVG AKKVTVT FHRPSLI LRKLQAQ

PRRASWV ARKFNVG AKKVTVT PFiRPSLI230 LRKLQAQ

FKt-IYQMS VKHLNV$ AKKVTVT 175 MKRYGLL LIcI-IASFL

FKHl'QMS VkHLNVS 120 AiCKVTVT MKRYGLL VKKLSDV

VRKASDV LRKASVT AKKV'1'VT LRRFSIL VKKLSDV

VRItASDV VHKYGLA PKRISPV VRI~VTEN235 AliKWSLP

VRKASDV VIiKYGLA MKHASDS 1$O LRKATTF YRRPSVA

VRKpSDV IHHTSVM AI~ISLA IKRLNIQ FI~KPSVA

VRKASDV IfIHISVM AHHISLA FKKVSPW 240 FKRLTDL

VRKASDV IHHiSVM LKRLNPQ 185 WHHAQI,A PKRLTDL

YKRFQLL IHHISVM 130 IKKLTLQ VKI~V P_K_KT_'I'pL

LRItITLP7S VI~PGAS AKRA$VF ARRIQDP ' VFTRYSVL

VKKLQLM LRKFGAS LRHLGLT ARRIQDP LRRYNVA

1HKPGYL LKKTQEL WRKAGYQ VKKIQAS Z45 vRRAGLv LKKLTEL AKKITAA LKHYGPG 190 FHIiWQIV YKKPGYN

IHTQ~GFS PRKVTAA 13S LKfEvIQFP ARKI,QDV AHRWGIQ

IRKFQEQ 8Q PRKVTAA WKHx,QEN YHRLSEL YRRVGDV

YRRMSLA TRRASAI AHKISYP LHRPNLL VKKLGDF

LRRLTTL LKKMQAN AHKISYP LHRPNLL 250 VKICLGIaF

LRRLQDV LKKMQAN AHKISYP 195 LFIRpNLL VKKLGDF

3O MRKISLF IRKYGLN IRKLGWG LIiHATit" 1RHYQLL

MItRISFF VRIClTVG IRKLGWG 1KKFQDL 255 II'tIiYQLL

MRKlSLF VHKATDT YHRFSPL 2QO PKKAGLS LRKPGEI

MRRISPF 90 ARKI~GFT LRHLGIV PKKAGLS LRKPGDT

FRRITLY ARKLCrFT AKRISIN FRRINMV LI~AGIL

MRRISPF LRRAQDI 15O MKKITL,Z, IKHPNLV FRHPGYS

AItHWGDG95 PRRISAV LKF~N7,V FK1:,IGDL PRRATDW

ARHWGDG LWCVTAN LIGHPNIV YIiICPTVT AKKWTAV

' (' VKHIGEL LRKVTAN 155 LKHANIV FRI-IPQWT LKHVNpI, VRHFQFL 1 LRKVTAN LKHANIV AKKWNLF LI~VNAL
QQ

VRHFQFL LRKVTAN LKHANIV YKRFSEA LKHVNAL

LKI;p,NVQ LRKVTAN YKHINEV 215 PRI~LQLL IRKIGEA

VKHTGEL LRKVTAN 16O PKHAGIM WF~SVF LRHITVV

PRRLNVA 105 LRKVTAN PKHAGIM AKKVTVT VKI~PGDL

L~,SPT MRRYTIS LKHLSLL 220 VKKLSpV VKKVQEA

MKRLTLG VRKFQTP 165 t:.RRLSAV AKItIGEV LRHVSVY

MKRLTLG 110 VRKFQIP LKRLQLQ IItKPSPY LRHWSDM

MKRLTLG AHRPGLQ PHRPSLI IRKPSPY LKRLTFQ

FRHAQLL AHRpGLQ PRHLQLL LKKLSTF Z80 VKHLSLQ

Ilo izm~iaa wry i:l:ia rv~ am adz usza limuu~r ~ mnYl3>;1;
VKHLSLQ WHRAQAS MRHLGAF PRR~,NVw225 M~,SDL

LICKPSAL WIZRLNVA AIIRIQEL170 pI~LNVA ~QEI, PRRWSWx PHHVSLA LKKMSPQ FKKIQAL TRKANTp PR~WSWI LKRANEF YHHPNDM FKKIQAL 230 VICKPGES

IRRLSPA AKItPSVS 120 VKKYSIV FKKIQAL VKHLQVF

LItRI,TMN PRKPSAS VI~KASYL MKRFNPP LRZrISEF

LIZRLTVQ Alts-IATYG VKKASYL PRKVSEL 235 WICRPQMS

LKKIQFP LRRPSLV VKKASYL 1$O PRKVSEL LRHVSVY

LKKIQFp LRRVQYL 125 VKKASYL LHRL'CMM VRHVSVY

LRKAOPS 7O LRRVQYL PRRIQLS ARHI'GDY VRHVSVY

PKHLTDA LRRVQYL MKHYTEA PKRPGPA LRHVSVY

AKKATVN AKRPSAA AKRLSFV 185 IHHISEI' LKI1LSLL

LRICVGAP LICKYNLF 130 VFtHVSFV IvIKRFNPP PRKLNFQ

PRRWSWI LKKATAY 1K1KT.NEI PKKMTFP LRRIQPP

LKRI,TFQ PKRLGPL TRKFSIV LRHLQVT 245 LRRIQPP

LKRLTFQ PKItLGPL TRKFSTV 190 IK1~TFG MKx3LTVQ

LKKPSAL YRRLQPS LHHPNfV ARHFSAA LRRIQPP

LKIcPSAL YRRI,QPS LKKPQDS V1CKFSAM250 LRRIQPp LKRLTFQ LNRPQVG LKKPQDS 195 FKKYSFM LIiRIQPP

LI~pSVQ 85 VRRFQIA WRRFSDQ FRKATPY LRRIQPP

LKRLTFQ FKN.ASPI PRRYTIA FRT~ATI'Y LRRxQPF

AKHFSLQ PRRVSLA pRRYTIA II;HLNAT255 LI~tQPP

VKHLSLQ iRRPGAT 145 AKRPSAA MKKVQE$ LRRIQPP

LICICPSAL90 LRRLQAQ AKRpSAA MKKLQES LRRIQPP

33 1RHVGAT PTiRYTIA MKRLNEI MKICLQES LRRIQPP

PRRWSWI MKKMGEM MKRLNEI IRHI.NAT260 1.RRIQFF

PARW5W1 MKKMGEM MHHWSIp 205 IRHLNAT T~R~t,e,NpS

PI~RWSWI P1I~LSDS 150 VKi~F'SIV VK7~LQEQ LRRANpS

LRKPSVQ 95 PHHLTVI IIi.RYSDA LK1LF'SYT LI~QPP

LKKPSAL PAHLTVI PKKISDL VKRLSVS IRRMQYF

PRRpGPT PHHLTV1 PKKISDL MKHINLS 265 VKKLSDV

LKKPSAL PH'FH,TVI MHRVSVI 210 WRKPGPS ARRVGLV

VKHLSLQ PIiHLTVI 155 LRHFSLS VRRVSFA ARRVGLv YHRPGLG 100 AHRPGWL LRHFSIS PKRLSAV LIrICAQMA

LRRVQYL AHRPGWL PRKMSVl IKRYNLQ LKKAQMA

MKRLGMN VRRLGLS LHRLGLP LHRLSDp 270 LKKAQMA

VItICYGEG VREtLGLS LHRLGLP 215 LKICAT'FA LiCKAQMA

VRKYGEG Pi~iLTVI I60 FKRISEQ LKKATFA LKKAQMA

SO IKKVQDL LKKLGLI VRRLGL$ LKKATFA LKKAQMA

LRru.GLV LKKLGLI vRRLGLS LxxATFA 275 LKKAQMA

YKKPSWL LKKLGLI VR1~LGLS220 Lf~K,ATFA PRKVQVA

LRKPGIN LRKANDQ a65 VRRLGLS LKKFQVT VTI;KVQFA

PKRVSDS MluILGAF LKKFTEY PRRAGPG WKKAGLN

IKKIQEA MRI~LGAF WIQ~pTYI MKKI~SDL280 AKKPSFP

lzWB~ea wr;I) 1S: Zu rv~ 41t! ;faL ua~;f liIDUUT b'C ~IAYBIrI; ~øIU17 PRRATIS FRGTLDP VRGLLVN WRpSLVP 225 FHLTTTN

PI~RATIS MRLLPLA FRLPPDp 170 VRGTLAY WRPSLVP

T'RRATIS IHP1'PPA11 FRLPPDP VRGTLAY FRLSPTA
S

PRRATIS 6U VRGLTGP MRPSNPP FHLQPVP VRGLTT,p PRRATTS MHGLPGP FRGPPLA FHLQPVP VHLSNGP

FHRPSEL MRGPPGP FHPPpLP MRGPTpN 230 VRGLTGP

FHRPSEL TRGPPGP MRPSNPP 175 FlZCi5LDY IHGHPGP

FHRPSBL IRGPPGP 120 MRLLTLA VRLFiPTA IHGHPGP

10FHRpSEL FHPTLVA VRGSLOA VRLHPTA WHGTNbN

FHRFSL~'L FIiPPPLP VILPPFFP FRGPLVF Z3$ f_F~I'r~r.I,LQ

IKKANEV FHPPPLP VRGSNGA 1$0 VHGLPTP VRGPNGA

IK~CANEV 70 FHPPPLP VRLPPPA vI~.LTLA VHLLLAA

15IKKANEV FHPFPLP VRLPFFP IHGLLFP WItLTPFA

FHRPSEL IRGQLGP VHLLLAA 1$5 MRLLTLA FRGPNGA

FHRPSEL MItWLLLY13O MRPPPpA VRLLLGA FRGPNGA

IRKVTVS 75 FHPSLGP IRLPTGP VRLLLGA FRGpNGA

2OPKItLNFS IRGPPGP IRWLLAA VRLLLGA FRGPNGA

JaRl3PPGF VHGLPTP 135 IRWLLAA VHLLTPQ VRGSNGA

VRr,I-LL,LN VHLLPGQ VRLPTGY VRGSNGA 2SO VRGSNGA

vRLHLLN IRGPPGP VRLwNPQ 195 vRGSNGA MRwLLLY

FRGPPGP IItGPPGP140 WRGLLVP VRGTFVP FRLLPYQ

IHpTTGQ $S IRPPPPP VRGPPGP IRWTPIQ VRLTPAQ

30IHPTTGQ TRGQPGP VRGPPGP IRGPPpp VRLTPAQ

VI~.pNIQ FRLQTDP 145 VRPLPPA MHGLNAA YfILSNGP

VRLHLLN 90 IRGQPGP IHGPNI,,Y MHGLNAA IR.PSTLN

FRLSWTA VRLLLGA lHGPNLY FItLSPAP 260 VHj,pNIQ

VRPPpAP VRLPTPN WRLTPPA 205 FRLPPDP VRLTPAQ

VRPPPAP VRLpTPN 150 WRLTPPA FRLPPDP IRGPPPP

FRLSWTA 95 VRGLTGP FHPHPGP FRLppDP MRGPPpp 40VHLSTGA IRGQPGP VRGSLGA FRLPPDP IItGpPPP

VHLS'T'GA VHLSPDA WRGTNVN IRGPPGP ,265IRGPPPP

VHPPPYP VI-IZ,SpDA WRGTNDQ 21O FRLSPAP MRGPPPP

VHPPPVP YRGPPGP 155 FRppPPP WI~GTT1VN MRWTPAA

FHLTPDQ 100 1RGSTPY VRPLPPA ufIGSPPA MRWTpAA

45FHLTP17Q iRGSTPY VRGWLLA iRPQLAA VRGPPGF

IHLSLAP IRGSTPY IHLLpGQ 215 FHLTTTN VRGLTLP

FRLS WTA l05 MRGLLAQ VHLLPPP IHPSLLP IHGLPVP

SOFRGLTpp VRLSPLA VHLLPPP FHLTTTN IkLPTGA

IRLTLAQ IHGLI,Pp VRGSLGA FHLTTTN 27S IRLPTGA

VRGSNGA FHPSLGP 14S VHl.LPPP FHI,TTTN MRPLWVQ

IRGppGP 11O FHPSLGP IHGLNAA FHLTTTN VRLLLCiA

SSIRGPPGP FRGPNGA WRLTPPA FHLTTTN VRLLLGA

VRPLFPA VHpSTVN VRLWLDN FHLTTTN 280 VRLLLGA

ll2 iziusiea wrv is: zu rw 4ia auz uaza xmuu~r ~ marY~~;r; Hula VRLLLGA VRLPTPN IHLTPTQ 20 VRGPPGP MAG$PGp VRLLLGA VRLPTPN 15 IIiLT'PTQfHP'fLVA FItPpPPy VRLLLGA 10 VRLLLaA TIlLTPTQ FHFTLVA FHPTLVA

S VRLLLGA VRLLLGA 1HLTPTQ VHGPTTp VRLLNPN

VRLLLGA VTIGpTTP tHGLPvP VRLLLOA 30 FRLLTGQ

Exannple 5 35 Suitable phagc display libraries including, but not limited to the Ph.D.
Phage Display 12-mer peptide library (NEB) were panned against the H 11 antibody (including arid not limited to the 1gM , IgGI, scFv and other antibody fragments) exactly as described in the relevant NEB technical bulletin. See page 1 I
Sloan If ettering Patent WO 99/22761 for full details. Fhage particles were prepared from 40 individual clones and DhTA was extracted and sequenced using the Applied Biosystems automatic scquEncer and t$e deduced amino sequences were obtained.

MET[~QD OF OBTg t MAH H ~
Mab NBGM 1 /H 11 ("H 1 I "), i s a human monoclonal IgM antibody reactive 45 against the following human tumor tissues and con;csponding tumor cell lines:
glio»a, malignant melanoma, colon adcnocarcinoma and breast adenocarcinom~a.
In vitro characterization of Mab NBGM1/H 11 is shown in Example 4.
Fusion of HI 1 was accomplished by fusing 8 x 106 peripheral blood lymphocytes obtained from a 64 year old male with a low grade glioma with tlae TM-50 H2-SP2 human myeloma cell line. TM-I~2-SP2 toll line is the im~munoglobulin non-secreting subline of the parental cell line TM-H2, a hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.$)-deficient derivative of an unknown human myeloma-like line selected in 0.$% mcthylcellulose for its resistance to 6-thio~uanine (6 ug/mL) and failure to grow in hypvxanthinc-aminoptcrin-tlaymidine medium.
The 55 karyotypc of T"M-H2-SP2 is 462, XX.
The resultant viable hybridoma cells were plated (0.2 mL/well) into 40 microwells at a density of 2 x 105 cells/mL. '1~hc frequency of outgrowth frorr~ fusion H11 was 12 of40 (30%) potential hybridoma-containing wells. Qutgrowtb resulting fronn sustained growth is defined as prolonged growth with culture expansion for 1L/Utf/88 Wr:U 1S:C1 b'AA 41U ;itlG USLJ lilyUU'1' & ~tAYBt;I~øJU1H
periods longer than 3 months; instances of hybridoma growth failure occurring later than 3 months post-fusion ware not observed.
Screening of hybridoma clones was performed by antigen-capture cnzyme-linked irnmunosorbent assay (EL15A) in microtiter plates using polyclonal anti-.
S human IgM or IgG as coating antigen. A hybridoma culture supernatant was positive if the measured optical density (O.IS.) value exceeded the moan background level of a control culture supernatant by greater than two standard deviations (S.D.).
Selection of a hybridoma clone was performed by cell-fixed ELISA. Culture supernatants from b microtiter wells, which tasttd high for IgM or IgG
secretion, wore screened against the following previously attached and fixed human tumor cell lines: glioblastoma (SKMG-1 and D-54MG); melanoma (A-37i); and colon adenocarcinoma (SK-CG-1). A hybridoma supernatant was considered to be positive if the measured o.D. value exceeded the mean background level of control culture supernatants by greater than two S.D. Mabs produced by hybridoma NBGIV11/H 11, obtained in this manner, continues to be reactive against these tunnor cell lines. The "I~i l" antibodies arc IgM~I.
The nacthods used for the characterization of Mab N$G1VI1/H 11 include:
antigen-capture ELISA, antigen $LISA, cell-fixed ELISA, flow cytornetry, immun.opcroxidase staiaaing of human tumor cell lines and immunohistochemistry of human tumor and normal tissues (see following examples).
Binding characteristics of this human lvlab to human tumor cell lines as determined by flow cytometry, immunoperoxidase staining, cell-fixed ELISA and antigen >YLISA (i.c., tumor cell freeze-thaw extracts) are presented below.
oy-Z37953 ma iziuniaa wr~u is:zi rva~ am aaz usza xmuu~r ~ mnYSr;x ~uzo ~xAMPLE 7 FLOW CYTOMETR1C ANALYS[$~ OF '~ 11 B~D)NC TO HUMAN
GLTOBLA~TOMA ISKM ~-1) AND Iy>;~'LANOMA ~ -X37 ~ _F.r.l~ rnrF~
In order to determine HI 1 binding to intact tumor cells, anchorage-dependent tumor cells growing in T-flasks were detached by incubation with PBS-EDTA and examined by flow cytometry. Cells were collected by low speed centrifugation, washed with ice-cold PHS-1% FBS, centrifuged and the supernatant aspirated.
The cell pellet was resuspended in culture medium spiked with one of the following: a control human melanoma IgM; hybridoma NBGM1/H11 culture supernatant; or PHS
containing purified Mab Hl 1; and incubated on ice for 30 cainutes. After incubation, the cells were collected by centrifugation, washed by rcsuspension in PBS-FHS
and centrifuged. The cell pellet was then incubated for 30 min with FITC-conjugated goat anti-human IgM. After incubation, the cells were washed with PBS-FBS.
Finally, the ells were resuspended in PBS-FBS followed by addition of propidit~m iodide (PI) and washed. lyI-positive and FITC-positive cells were analyzed by flow cytomctry.
The results of the flow cytometric analyses are shown in Figures 1 and 2.
These results indicate that crude and purified forms of Mab H11 bind to a cell surface-associated antigens) expressed on gliobla,~toma (SKMG-1) and melanoma (A-375) live human tumor cell lines.
F"LE 8 ANALYSIS OF MA~~T11 BINDING TO FRRE~E-THAW EXT Ac'T~ OF
HUMAN TUMOR ELL LINP,S RY R1.15,~
?5 In order to determine the ability of H11 to bind spsGifically to human tumor samples, ELISA plates were coated with human tumor cell extracts prepared by repeated freezing and thawing of glioblastoma (SKMG~1), breast adenocarcinoma (BT-2Q, MB-468 and MH-453), colon adcnocarcinoma (SK-CO-1 and HT-29) cells.
ny-237953 iziusiea wr;u is:zi rw 4m ~~z usz~ Hmuu~r ~ mAYtit;r; ~uei 'The coated EL1SA plates were incubated for 1(-18 hours at 2-8°C. The plates were blocked with PBS-3% BSA for 1 hr at room temperature. Then the plates were incubated with either biotinylated Mab HI 1 in PBS or biotinylated eontrpl IgM
in PBS ar culture medium for 2 hrs at room temperature. The plates were washed an,d incubated with strtptavidirt-conjugated alkaline phosph.atasc far 2 hrs_ After washing, p-nitrophenyl phosphate substrate was added to each plate and, after incubation, the plates were read at 405 nm in an EL,1SA plate reader.
The binding of Mab H11 to the tumor cell extracts is shown in Figures 3 and 4. These results indicate that Mab Hl l binds to tumor cell extracts prepared from glioblastoma, breast adenocarcinoma and colon adenocarcinama cells in a dose-deptndent mariner.
G OF Mss H11 TD 1MAN TUMORs'.ELL~~R~D BY
IMMLTNOPERO?CIDASI ;,~;;CAINI~G
In order to determine immunortactivity of H11, the following experiment was perfornied. TSumor calls gown in 24-well plates on coverslips for 48-9fi hrs.
were washed. The cells were washed with PISS, fixed with formaldehyde and incubated with 5% normal goat serum on PAS for 30 min. After washing, the cells were incubated for 2 hrs with either hybridama NBGMIlHI l culture supernatants or purified Mab H 11 in PBS of Culture medium spiked with control human myeloma I$M. The cells wcr~ then washed and incubated with anti-human IgM conjugated to HRP. Finally, the cells were washed, incubated with DAB substrate to visualize Mab I~11 binding, counter-stained with hematoxylin and mounted in GVA.
The results o.f the immunoreactivity oflvlab HI1 are shown in Table 1 whore reactivity is indicated as negative (--), weak positive (+), positive (++), strong positive (+++). Those results indicate that, as determined by immunopcraxidase ny-237953 iziusiaa wr:~ ia:zz r'A~ 4lff ~tiL USZ;f HlDUI~'1' & IfAYB~I~o«
staining, the epitope recognizedexpressed by a number ol'difFerent by Mab Hl l is types of human tumor cell lines.

Table 1 CELL
~,iNESIT1'pE BEA~TIVrTy OF T~TMQR

Control 1gM M$b H1 i ~A_ N r .
(' ~LASTOMA
, SKMG --U- I __ MG

MG

~"~AN ICiNANT MRI.q,~'~MA
M

__ SK-MEL-5 ._ n R
~r O n L N ~A_ _ CINOMA __ g~ ' STC-CO-1' MAN $

~j c MG-468 Q~,g, __ REA~ AD .NO A

MB-453 _ +

BT-20 _ BT-474 ,_ HUMAN IDNEY ADE~jOC~~tCTIVOMA
K

SW-839 ,_ L~(UMAN STFOCEIvIIC ~ RCOj,'~A
O

SAOS-2 __ A

sK-ov-~ __ ++

ny-287953 iziusiaa wr;U is:zz rva~ aia auz usz~ xmuu~r c~ mnY>ilJr; ~uzs EXAMPLE. 10 BI1~1DING OF MAH H1 l 'rd HUMAN TI~ZOR GEL LTNES
DETER_M1N D HY~ELL-FI FD FLISA
The binding of HI 1 to human. tumor cells and cell lines was also determined by cell-fixed ELISA. Growing tumor cells were detached from the T-flask surface by incubating with EDTA-PHS. Cells were collected by centrifugation, washed with PBS, resuspended in culture medium, counted, and aliquots of 5-10 x 106 cells placed in each well of 96-well ELISA plates. After allowing the cells to attach to the plates, the culture supernatants were removed and the plates were blocked with PAS-BSA. The cells were then incubated with different concentrations (1-20 pg/mL) of either Mab H1 I or control human myeloma IgM for 2 hrs. After incubation, the plates were washed, incubated with biotin-conjugated goat antihuman lgM, washed again and incubated with streptavidin-conjugated alkaline phosphatase. Finally, the plates were washed, incubated with p-nitrophenyl phosphate substrate and read at 445 nm in an IyLISA plats reader.
l~.esults of the reactivity of Mab H11 to human tumor cell lines by cell-fixed ELISA are sliown in Table ~ and Figure S. In Table 2, Control IgM lOpgIroL w a re used for testing the reactivity, and values arc given as ~ S.». These results indicate that: 1) Mab H11 reacts strongly with gliobl.astoma cells (SKMG-1), even at a low concentration of 1 yg/mL, whereas cotxtrol IgM at 20 pglmL does not react with SKMG-1 tolls; and 2) Mab FI11 recognizes the tumor ankigen(s) present on numerous tumor cell lines (breast adenocarcinoma, colon adenocarcinoma, malignant melanoma, neuroblastoma, glioblastoma, lung adcnocarcinoma, small cell lung carcinoma and prostate adenocarcinoma). The degree for Mab reactivity varies both with the type of cancer and the tumor cell lines.
ny-237953 izmneu wru ls:ez rv~ am Paz usza xmuu~r ~x mAYt;r;r; ~uza TA,)3LE 2 Cell lineslTumor TypeReactivity (O.D.
at 405 nm) Control IgM Mab H11 Human Glioblastoma SKMG-1 0.21 t 0.01 0.95 f 0.06 D-54-MG 0.13 ~ 0.02 0.43 f 0.07 U-87MG 0.13 t 0.02 0.60 ~ 0.01 Neuroblastoma SK-IY-SH 0.14 t 0.42 0.96 ~ 0.06 SK-N-MC 0.17 ~ 0.03 1.00 t 0.05 Malignant Melanoma A-375 0.25 ~ 0.04 1.25 t 0.04 SK-M)JL-5 0.18 ~ 0.03 1.42 ~ 0.04 SIC-MEL-28 0.19 t 0.03 1.79 t 0.05 Breast adenoCarcinoma MB-453 0.68 ~ 0.18 2.85 ~ 0.14 MB-468 4.60 t 0.03 2.39 f 0,10 SK-BR-3 0.60 t 0.03 2.14 ~ 0.13 T47D 0.58 ~ O.OI 2.13 t 0.04 BT-zo o.s7 ~ o.oa z.o7 ~ 0.13 BT-474 0.61 ~ 0.03 2.20 ~ 0.17 Lung aden~ocarcinoma SW-900 0.20 ~ 0.02 0.6$ f 0.10 SK-L1:J-I 0.19 ~ 0.02 0.57 ~ 4.47 A-427 0.22 ~ 0.01 0.88 ~ 0.07 Small cell lung carcinoma NCI-H69 0.25 ~ 0.04 1.42 ~ 0.20 NCI-~I$2 0.20 ~ 0.09 1.16 t 0.13 Colon adenocarcinoma SK-Co-1 0.27 ~ 0.03 0.98 t 0.11 TAT-29 0.37 t 0.02 1.78 ~ 0.20 Kidney Adenocareinoma S1'~-839 0.02 ~ 0.01 1.43 t 0.01 Prostate adenocarcinorna PC-3 0.17 ~ 0.01 0.60 ~ 0.01 DU-145 0.1510.01 0.5210.01 ~5tC4geniC Sarcoma sAos-2 0.24 t o.oz 1.2z X0.07 U-2 OS O.I3 X0.04 1.93 ~ 0.05 ay-X37953 iziusisa wru ia: za rw 4iu 'uz usza xmuu~r a mAY~>:;~
>3ladder Ccll Carcinoma T-24 0.13 ~ 0.01 1.Z5 ~ 0.03 Ovarian Adenocarcinoma SK-OV-3 .012 f 0.01 1.14 X0.02 Larynx Carcinoma ~p-Z 0.25 10.01 1.25 ~ 0.01 Normal Human Fibmblast GM-$333 0.13 t 0.01 0.39 t 0.01 P~,~j PLE I 1 lMMiTNOANATOMTG bIfiTRIBUTION ,1ND tMMUNOPAT.~,OCrIC
Hl Itnniunohistochemistry was used to determine expression of H 11 for evaluation of micro-anatomical detail and heterogeneity in tissues and tumors.
Limitations of this technique include possible false negative results due to low levels of expression of the molecule under study, as well as false positive results (crass-reactivity) due to antibody-binding to similar epitopes or epitopes shared by other antigens. To address these limitations, this study was carried out at the highest concentration of antibody that did not show non-specific binding by a control antibody. This allowed for detection of all levels of cross-reactivity in dfffcrent tissues. In addition, fixation analysis to establish the best combination of antigenic staining intensity and morphological preservation, was performed. The present example presents results obtained from IMPAT'I~ Inc., New Yark, which was retained to study the cellular specificity and antigen expression of Eil l, on a selected panel of cryostat-cut frozen sections of normal and tumor tissues. The study used an indirect immunoperoxidase technique.
Histologically normal human tissues were obtained .from surgical and autopsy specimens. These fresh tissues were embedded in OCT compound (Miles Laboratories, Inc., Naperville, IL) in cryamolds and snap-frozen in isopentane, ny-237953 1~0 iziusiua wr~u is:za rvx 4ia aaz usz:~ lcmuu~r ~ mAYS~;~ ~uza cooled by liquid nitrogen. These tissues from 1MPATH's frozen tissue bank ware then cut at 5 microns, placed on poly-L-lysine coated slides, air-dried, and stoned in a -70°C tissue bank until needed.
H11, received on wet ice and stored at 2-8°C, was supplied non-hiotinylated at a concentration of 200 ~glmL, total volur~r~e of 3.0 mL. A human myeloma IgM
(Pierce Cat. #31146), also supplied by Novopharm Biotech, Inc., was used as the negative control antibody. Both the negative control antibody and H11 wore diluted in phosphate buffered saline (p$S) to the same working concentrations dictated by titration analysis of H11. The peroxidase-labeled secondary antibody was a goat anti-human IgM (American Qualex, San Clemente, CA, tot #A112PN) diluted in PBS to 1:500.
Immunopemxidase Techniques: Immunohistochemical studies were performed using an indirect immunopcroxidase method. The cryostat cut sections were removed from the -74°C freezer, air-dried and ~xCd (fixation details prorrided below). Tissue suctions were blocked for 10 minutes with 5% normal goat serum diluted in 15BS, then incubated with the primary antibody overnight at 4°C. Slides were washed in PBS, followed by a wash with 0.5°/a TweenIPBS solution, then another wash in PBS. Endogenous peroxidase activity was blocked with a 30 minute 3% hydrogen peroxide/methanol incubation, followed by 3 washes of 1'BS. The sections were thin incubated with goat anti-human 1gM (peroxidase-labeled) secondary antibody far 15 minutes, at room temperature, and washed ire PBS as described above.
The peroxidase reaction was visualized by incubating tissue sections for 2-5 minutes with 3, 3-diaminobenzidine-tetrahydrochloride (DAB) (Sigma Chemical Co., St. Louis, MO). Tissue sections went thoroughly washed, countcrstairied with a modified Hams hematoxylin (Fisher Scientific, Fairlawn, Nn dehydrated through graded aleohols, cleared in xylene, and eovcrslippecl. Tissues that demonstrated high oy-237953 iziusiaa w~ ia:z;t rv~ 4ia aaz usz~ xmuu~r a mxYt;~;~ ~uz7 levels of background staining with the ncgadvc control antibody were stained again utilizing more extensive washing.
Human breast carcinoma (F95-03b), supplied by IMPATH, was used as the positive control for H11. Negative controls substituted the primary tost antibody with S purified human myeloma Ig.M.
The purpose of the fixation analysis was to establish the conditions which provide the optimal combination of antigenic staining intensity and morphologic preservation. The positive control tissue was tested with five fixatian protocols, including no fixation. The fixation protocols tested were 10% neutral buffertd formalin (23-25°C), acetone (2-8°C), methyllacetone (1:1 VlV, 2-8°C) and 95%
ethanol (23-25°C). For this study, 10% neutral buffered formalin (NHF) gave optimal results far 1-11 I .
Using 10% NBF as the fixative, serial antibody dilutions (20.0 ~g/mL to 0.1 lxg/rnl) were tested on the positive control, human breast carcinoma. A
concentration of 10.0 ugJmL of antibody H11 gave optimal results-~-maximum staining intensity without significant background staining of the negative control.
The results obtained are depicted in Tables 3 and 4. ?able 3 depicts Hl l reactivity on normal tissues and Table 4 shows HI 1 reactivity on human tumors.
ny-z37953 izz iziusiea wry is:za rv~ aia ~~z usz' Hmuu~r ~ mnYt,;~~ ~uz~

Tested Range of Pogi iyelTo s~ Reactivity (0-,~+,~

Adrenal 0/3 0 Bladder 013 0 Bone Marrow 113 1+

grain 0/3 0 $reast 013 0 Ccrvix 0/3 0 Esophagus 013 0 Eye 013 0 Heart 013 0 Kidney 013 0 Large Intestine 013 4 Liver 013 0 Lung 013 0 Lymph Node 0/3 0 Muscle 013 0 Ovary 012 0 Pancreas 013 0 Parotid 0/3 0 Pituitary 0/1 0 Prostate Ol3 0 Skin 0/3 0 Small intestine 013 0 Spinal cord 0/3 0 Spleen 013 0 Stomach 013 0 Testis 0/3 0 Thymus 013 0 Thyroid 013 p Tonsil 1+

TJtcrus 0/3 0 White Bload Cell0/3 0 ny-237953 i23 iziusiaa w~ is:za rvu am auz uszu xmuu~r ~ mnYt;~~ ~uza .TyE 4 Tested % of Tumor Range of Tumor Poyyc/Tot Celj~, StainipgReactivity [31 Breast carcinoma 30-90 1-3+

Colon carcinoma 3/3 40-70 1-2+

Ctlioma 4/6 30-90 1-2+

Gastric carcinoma 3I3 30-50 1-2+

Lung adenocarcinoma'/, 10-70 1-2+

Lung squamous carcinoma313 10-95 1-3+

Lung small cell '/z 30 1+
carcinoma Lymphoma 8/8 10-95 1-3+

Melanoma 3/3 20-95 1-2+

Ovarian carcinoma 3/3 2b-30 1-3+

Pro$tatc carcinoma 3/3 20-95 1-2-~

Sarcoma 013 0 4 The results obtained indicate that weak (1+) to strong (3+) reactivity was observed in over 70% of tht positive control sample. The antigen recognized by I~11 has a restricted pattern of distribution. H1 I was largely unreactive with normal human tissues tested in the IMPATH system. A11 simple epithelial cells, as wall as the stratified epithelia and squamous cpitb.elia of different organs, were found to be unreactive. No reactivity was observed in neumectodermal cells, including those in the brain, spinal cord and peripheral nerves. Mesenchymal elements such as skeletal and smooth muscle cells, fibroblasts, and endothelial cells were negative.
Tissues of lymphoid origin hlcluding bone marrow, lymph node, spleen, and thymus were largely unreactive witli antibody H11. Weak (1+) reactivity was observed in rare cells in one specimen of bone marrow and in the gernninal centers of one of three 1 ~ specimens of tonsil tasted.
Positive immunoreactivity was observed in almost all specirncns of tumor tested including breast, colon, glioma, gastric, lung (adeno, squamous, and small cell), lymphoma, melanoma, ovarian, and prostate. Reactivity was seen in 10%
to greater than 95% of the tumor cells present in these sgtcimens; staining intensity oy-z~~9s3 t24 1C/U$/88 Wr:U 18:L4 fA~ 4lti 't1G USL3 lilDUli'1' !k mAYB)Jk~ ~øJU:IU
ranged from weak (1+) to strong (3-~). Antibody H11 Was, horwever, uureactive with all three specimens of sarcoma tosted. Some, but not all, normal counterparts of the tumor cells, when present in the specimens, were reactive with H11. A few normal cells present in breast, gastric and prostate carcinoma were reactive with antibody H1 i. The large granular cells that were reactive with antibody H11 are believed to be inflammatory cchs of the eosinophil-mast cell lineage.
In summary, antibody H11 is largely unreactive with normal human tissues with the exception of some normal cells such as infiltrating leukocytes, tissue present in tumors. The H11 antibody detects an antigen that is expressed in almost all of the tumors tested in the present study.
1~.XAMPLE 1?.
PUR1F_ICATI[yN OF SPPCS
(a) Pyrificationyf mixb~~s.
Purification of SPPC mixtures using ADP-affinity ctuomatography is described in Peng et al. (1997) J. Lnmunol. Met, 204 13-2,1; and W~9$/12208.
In particular, a semi-purified cell extract is addal to a column containing an ADP matrix and a buffer containing ADP is then added to the column to elute the SpPCs.
Generally, a tumor cell extract cart be prepared by standard techniques in the art, with.
specific attention paid to inhibiting protease activity, preferably by freeze thaw extract methodology as generally described in Chen et al (1994) J. Irr~munol.
152:3-11. preferably the protease activity i.s inhibited using PIvISF and aprotinin.
(b) Etlrif Lion a i PPC cqntainiryg hs~7b.
C-antigen was isolated in the following manner. A-375 cells (human melanoma cell line) were grown in tissue culture to a cell density of 50-80%
confluent, disrupted, and an extract made by freeze-thaw. In detail, after cell harvest, cells were centrifuged at 1500 rpnrt for 10 min. The cells were washed twice in a PBSh mM PMSFIIOpg/mL apmtinin solution. After washing, the pellet was resuspcnded in the wasli solution and the cell co~xcentration was adjusted to 10-20 x ny-Z~7953 lzs lY/U8/88 Wl::~ 1S:Y4 b'AA 41U ;fl3L USL3 H1DUU'1' ~C ldAYBlrH ~øJU;l1 106 cclls/mL. This suspension was then subjected to five f=eezc-thaw scquex~ces consisting of freezing in a dry-ice-acetone solution, followed immediately by thawing in a 37°C water bath. After the freeze-thaw treatments the extract mixture was centrifuged at 4°C, 2500rprxl for 30 minutes.
The resulting supernatant was combined with 3M ammonium sulfate buB'er in a 2:1 ratio. This sample was then loaded onto a general purpose hydrophobic chromatographic media (preferably Phenyl Sepharose) at a rate of 0.5 mL/min using a pump. The column was connected to an FPLC system. Once loaded, the column was washed with 15 column volumes (CV) of Buffer A (50 mM sodium phosphate/1 M ammonium sulfate pbI 7.0). The bound proteins were eluted step-wise with buffer H (50 mM sodium phosphate pH 7.0). Active fractions were determined by immunological methods. During elution, the bulk of the bound proteins were eluted with 30% Buffer A 170% buffer H, The 70% bufftr B elution was followed by 100% buffer B. $PPC was eluted in this latter fraction. This positive fraction was concentrated on a membrane concentrator with a MW cut-off of 10 kD (preferably a Centriprep IO). The concentrated sample was passed through a buffer exchange media (preferably G-25) to the ADP-agarose chromatographic Huffer A (20 mM
Tris-acetate, 20 mM NaCI, 3 mM MgClz, pH 7.5).
Six mL of the buffer exchanged material was incubated overnight with an additional 4 mL Huffer A and 5 mL ADF-agarose at 4°C on a platform shaker.
following incubation, the mixture was poured into a XK15 x 40 column. The column was washed with the At~P-agarose chromatographic Buffer A until the QIa at 280 r~achcd base-line. The column was further washed with 0.5M NaCI in chromatographic-Buffer A and re-equilibrated with Huffer A. The bound protein was then eluted with 3 mM ADP in the ADP Suffer A and fractions collected. Tlic active fraction was concentrated on a membrane concentrator with a MW cut-off of 10 kD
(preferably Arnicon).
ny-237953 1LlUS/88 W~ 18:L5 r'AIL 41B ;iUG USLJ ltlDUU'1' !k mAYB~I~JU;IG
The concentrated, eluted, sample was diluted with anionic chromatographic Buffer A (2U mM Tris pH 7.8) at a 1.:10 dilution. dne mL of diluted sample was loaded onto a strong anionic column (preferably a Mono CZ Sepharose) attached t0 an F)?LC. The flow rats was set at 1 mllmin. Fractions were collected and the antigenic Fraction identified as outlined above. This three-step procedure gives a reasonable homogeneous active C-antigen (?95%).
Subsequently, after final concentration from the anionic column, l5pl of 95%
purified SPpC was mixed 50154 with 2X Lacrnmli's buffer. The sample was xun under native, non-denaturing, conditions (no SDS, mercaptoethanol or boiling).
After completion of electrophoresis, the separated protein were blotted onto a membrane (PVDF or nitrocellulose) again under non-denaturing conditions. identification of the antigen location on the blotted membrane was confirmed by incubation with H11-IgG
followed by an appropriately labeled secondary antibody. The C-antigen can then lx "cut" and eluted from the membrane and subjected to fluttter analyses.
An alternate method for the purification of SpPCs was developed from the creation of affinity chromatographic media of C-antigen-specific IgG
antibodies or fragments thereof, in the present cast using Hl l IgG described in W09714446I.
A
5 mL sample from a hydrophobic column (preferably Phenyl Sepharose) was incubated with 2 m>_. of H 11 Sepharose. The IgG-Scpharosclsample was incubated over-night at 4°C on a rotary shaker. After incubation the mixture was poured into a small chromatographic column (preferably BioRad 10 ml l;cono-Column). The column was washed with ten CV of PBS (pH 7.4) followed by three CV of 4.5 M
N'aCl in PBS. The afFnity column was then re-equilibrated with PBS. Following equilibration, C-antigen was eluted using a glycinc buffar p~I 2.$, The eluted material is concentrated on a micro-pore concentrator (preferably Centriprep 3). The acid elution results in the dissociation of SP from its peptide. The small molecular weight fraction (peptide) was concentrated with a peptide concentrator (preferably Microcon SCX). The purified SP was retained on the micro-pore concentrator.
ny-237953 iziusiea wry m:z6 rv~ aia aaz usza xr~uu~r s< mnYSt;x 'puss EX~ P~ LE 13 ~F.RAPRlITiC FFl~FCT OF H11 ,~,GFV CI_N HUMgN~;OR N~FTS
Tumor growth inhibition by H11 scFv in a xcnograft mouse model.
The potential of H 11 scFv as a cancer therapeutic agent was explored using a human tumor xenogrsff mouse model (~alblc athymic nude mice). In these studies, outlined below, an anti-tumor effect was found to be associated with H11 scFv treatment in mice implanted with one of the following human tumors: non-Hodgkin's 13-cull lymphoma, prostate adenocarcinoma, breast adenocarcinoma, and melanoma.
The anti-tumor effects observed, at the doses given, include reduced tumor size, tumor regression, reduced metastatie index and increased survival.
A, lean-Hedgk~'s Lym hpJ~
Mice that were implanted with human non-Hodgkin's lymphoma {>aaudi) tissue exhibited a smaller ,scan tumor volume than their control counterparts (n-3) afttr being treated with 1~111 seFv (n~9) and H 11 scFv-restrictocin (n=9).
The total dose ofHl l scFv given was 0.5 mglkg in a regimen that consisted of 0.1 mglinjection given intravenously (i.v.), 5 times, once, every 4 days. In the FIl I scFv-rtstrictocin-treated group, 2 of 8 animals remaining on Day 3$ of the study exhibited partial tumor regression. Despite the reduction in mean tumor volume, the dit~ferences found on the last day of the study (pay 38) were not significantly different from that determined for control animals. (p=0.405, Student's t-test, H11 scFv; ps0.423, H11 scFv-restrictocin).
In a follow-up to the first lymphoma study, mice implanted with Daudi were treated with a total dose of 1 mglkg of H11 scFv (n=13). These H11 scFv-treated mice demonstrated a statistically signif'~cant suppression of tumor growth compared to controls at Day 42 (n=8), (P=0.004, Stud~cnt's t-test). Moreover, 31%
(4/18) of the H11 seFv-treated animals exhibited tumor regression, with 3 being partial and being complete. No spontaneous regression was observed in tht control animals.
uy-237953 1z8 iziusiaa wru ls:za rv~t am auz usz~ Ht~uu~r ~ mAY~r;~ ~uaa The dose used was administered at 0.1 mgllcg i.v., given once a day for 5 days, rcstcd for 9 days, and thcn retreated for 5 days as befort.
A third study implanted Halb/C ath~ymic mice with varying sizes (4-10, 30-60 and 100-200 mm3} of human non-Hodgkin's H-cell lymphoma tumor tissut and given a total dose of 20 mglkg H11 scFv. 'The dose regimen included 4 cycles of H 11 scFv treatment (1 cycle of treatment constituted 5 daily i.v. injections of 1 mg/kg with each cycle being separated by 2 days of rest). There were 22 animals in each treatment group. At the end of treatment all mice treated with H 11 scFv showed a reduction in tumor volume compared to controls. The difference was statically significant at Day 51, compared to controls, for the 4-10 and 30-60 mm3 size tumors (p=0.02 and p=0.006, respectively).
Tumor regression was seen in 22% (4118), 10.5% (?J19) and 12% (3125) of the mice having tumor sizes of 4-10, 30-60 and 100-200 mm3, respectively, at the onsat of trcatrrrtnt. Most notably, for 75% (3/4) of the mice with a tumor size of 4-I O
mm3 at the beginning of the HI I scf'v treatment, tumor regression was complete.
Control animals did not show tumor regression.
H. Mel~a o~mx Mice were implanted with a human melanoma tumor (C31-105) and treated with a total of 1 mgJkg of H11 scFv (0.1 mg/kg) once a day for 5 days, rested for 9 days, anal then retreated for 5 days. Although the mean tumor volumes bathe scFv-treated and control groups at the conclusion of the study (Day 42) were not statistically different, the survival rate was higher in the H11 scFv-treated group. The animals treated with drug had a mortality rate of 21% (3/14). In contrast, 50%
(4 of 8) of the control mice died.
C. Breast Adenoc~~inoma A trial involving mice implanted with a highly metastatic human breast adenoearcinoma (G1-1.01) was also conducted. These animals were given 5 daily i.v.
ny-237953 iziusiaa wru is: ea rw 4ia ~uz uaz' Hmuu~r ~ mnYar;h treatments (0.1 mg/kg) oFHl1 seFv, rested for 9 days, retreated for 5 days and then given twice weekly injections at the same dosage far approximately 7 weeks.
The total dose given was 2..4 mg/kg. All animals were sacrificed on day 77 and the lungs removed for histologic examination and quantificarion of metastatic foci. The number of metastatic foci was expressed as a metastatic index using the following procedure. Briefly, on each slide, two different lung sections were measured with calipers and the number of metastatic foci in each section was counted. Each focus was counted as containing 1-10 cells, 11-50 cells or greater than 50 cells.
When the mctastatic index was calculated, foci with 1-10 cells were assigned a value of 1, foci containing 11-50 cells were assigned a value of 5 and foci containing greater than 50 cells were assigned a value of 10_ The number of foci of each type was multiplied by it's assigned value and these numbers were added together to obtain the total metastatic index (Ml). The MI was oxen divided by the number of mmZ of lung screened_ A. final score of MIluun2 was then reported.
Although the mean tumor volume of the H11 scFv-treated group was approximately half that calculated for the control group at the end of the study, 237 mm'versus 429 m~m3, respectively, the difference was not, statistically different (p~.358 at Day 42). However, the Hl 1 scFv-treated mice exhibited a significantly reduced number of metastatic foci in the lungs than control mitt, 14 versus 21, respectively (Chi-square analysis, p~0.05).
b. Prostate ~allCer Mice were implanted with a human prostate cancer tumor and then given 4 cycles of H11 scFv treatment (1 cycle of treatment constituted 5 daily i.v.
injections of 1 mg/kg with each cycle being separated by 2 days of rest). Upon completion of the i-v. protocol, the same treatment schedule and dose regirr~cn were repeated except that H11 scFv was a~iminister~d intrapcritoneally. The total dose was 20 mg/kg. The treated animals showed marked suppression of tumor size compared to controls.
Treatment also had an effect on survival. Thirty-eight percent (6/lb) of the mice with ny-237953 W

mu~ma w~ is:z~ rvx am ;~uz usza xmuu~r ~ mnY~t;~; ~uaa 4-20 mm3 size tumors at the start of treatment with. H11 scFv demonstrated long term survival (>140 days). This was significantly different from controls where all animals (10110) were dead before day 100. Together, these results demonstrate that H11 scFv, when administered to athymic mica bearing human tumor tissue implants, possesses potent ir1 vlvo anti-cancer, activity against human ttunors of various origins.
~X_AMPLE 14 pREPARATTOI~OF cDNA ENCODrIVG ANTIBODY FRAGM1~TS
RNA (mRNA) is prepared from PBL (peripheral blood lymphocytes) df normal or immunized humans using standard nnethods. cDNA is prepared from the tnRNA and used as template for PCR amplification of VH and Vlr genes. The VH
and VL genes are spliced together with a linker region using PCR to create scl~v repertoires. Restriction endonuclease sites are added to the gene repertoires to permit forced cloning into a phage display vector.
Gel purified scFv repertoires are digested with the speoific restriction enzymes and ligated directly into phagc display vector. The resulting recombinant vector is used to transform clectrocompetent ,E. colt resulting in the production of a phage antibody library. 1?hage are processed and incubated with the immobilized antigen, non-binding phage then arc removed by washing. Bound phage arc eluted with the addition of alkali. $aund phage are used to infect E. codi to produce more phagc for the next round of selection.
After 3-4 rounds of selection, clones are analyzed for antigen-binding by EL1SA. DNA sequencing can be used to characterize unique, high afFnity binders.
These binders include scFv and a wilt variety of otber antibody .fragments.

ii~TU'.tA'hr 1'~AF3S' T,IRECTED AGAI''Tc'~" TLTI~~pR-A$SOC1ATED SPPC
Human-human hybridomas secreting monaelonal antibodies (Mobs) specific for tumor-associated SPPCs are generated by fusing peripheral blood lymph,ocytcs (PBS) from a patient presenting with a malignancy. The fusion protocol has been previously described by GalFre and Milstein (1981) Met. Enzymol. 73:1-46_ ny-Z37953 1C/U8/88 WL'U 1S:C7 b'A~ 41d BUG U81J HlUlJll'1' !k mAYB~H ~JU;17 Briefly, PHL isolated by Fieoll gradient density centrifugation are mixed at a PBLabsion partner ratio of 3:1 in serum-free medium. The fusion partner is an Epstsin-Barn nuclear antigen-negative, Human myeloma-like cell line, TM-fit-(Sullivan et al. Hybridoma Technology, pp. b3-68, L. Russ, D. Carltorl, eds.
Ortho Pharmaceuticals Canada Ltd., Toronto, 1982). The cell mixture is centrifuged (400 xg, 5 rnin), the supernatant removed and membrane ftlsion facilitated by the addition of 1 mL of pre-warmed (37°C) 50% polyethylene glycol (>?EG) in serum-fret medium directly into the pellet over a period of 1 min. One nnL of serum-free medium is added again directly into the pellet over 1 min and this step repeated twice 1.0 more.
An additional 7 mL scrum free medium is added slowly (over the course of 2 min) to the pellet with stirring and a final 12 to 13 mL added dropwise to the mixture.
After which, the cell nvixture is ccntrii~ged (400 xg, 5 min), the supernatant discarded and the cell pellet resuspended to a final cell concentration of 1.0 x 106 eellslmL in 1 S complete medium containing hypoxanthine (H, 100 ,'~ivl) and thymidine (T, 1, 6 ,~M}. A 200 ~L volume of tb~e cell mixture is aliquoted into each will of a sterile 96 well flat-bottom tissue culture plate. The following day l04 I,eL is removed from each well and replaced with 100 pL of complete medium containing aminopterin (A, 2X. 0.8 ~l.Vi). Several wells containing the fusion partner alone are added to one of 20 the plates to onsure the selectivity of the medium. Every 3 to 4 days, half of the medium is removed and replactd with medium containing HAT and monitored for the growth of hybridomas. Wells containing hybrids are screened for anti-SPPC
reactivity using the dot-blot procedurt detailed in Example 3. Wells exhibiting reactivity towards the SPPC fraction are expanded into 24 well plates.
Supernatants 25 from these cultures arc tested against the cell membrane fraction containing the SPPC
in the presence and absence of ATP. Clones demonstrating antibody reactivity in the absence of ATP, but negative in the presence of ATP, are cloned by limiting dilution oy-Z37953 iziusiaa w~u is:z7 rvx am aaz u~ia Hmuu~r a mAYS>ar; ~u;~s into 96 well plates at 1 cell per Well. All subclones arc re-tested and the cloning procedure repeated twice more with the best positive subclones.
Example 16 Identification of peptides containing the consensus peptide motif X",(HyX)3Xm Synthesis and screening of an oligonucl.eotide array is carried out using commercially availablt DNA GeneChip technology, produced by AfPymetrix~. (Sec US 5,510,270; US 5,744,305; 5,571,639; and US 5,527,681). An oligdnucleotide probe array of all DNA sequences corresponding to the consensus sequence HyXHyXHyX, where Hy is valine, leucine or isoleucine, and where X can be any amino acid, with the exception of glycine and proline, is generated.
Alternative sets of probes can use less hydrophobic amino acid residues for Hy.
Photolithographie masks designed by computational algorithms are used in manufacturing the probe arrays. (See U5 5,571,639). A total of 327,082,769 oligonucleotides are calculated, where Hy can be encoded by any of GUU, GUC, GUA, csUQ, UUA, UUG, CUU, CUC, CUA. CUG, AUU, AUC, or AUA and X can be encoded by any of UUU, UUC, UUA, UUG, CUU, CUC, CUA, CUG, AUU, AUC, AUA, AUG, GUU, GUC, GUA, GUG, UCU, UCC, UCA, UCG, ACU, ACC, ACA, ACG, GCU, GCC, GGA. GCG, UAU, UAC, CAU, CAC. CAA, CAG, AAU>
AAC, AAA, AAG, GAU, GAC, GAA, GAG, UOU, UGC, UGG, CCiU, CGC, CGA, CGG, AGU, AGC, AGA or AGG. 'thus, [133x533] results in 327,0$2,759 possible permutations factored into synthesis of tha oligonucleotidc array.
Approximately 4 GeneChips, each holding up to 1X10a oligonucleotides (Sec US 5,510,270, column 15, lint 32), are hybridized to a cDNA library generated from a tumor cell, for instance the A-375 melanoma cell lime. ~iybridi.zation data arc collected to identify each probe sequence that hybridizes to individual cDNA consensus sequences.
The sequence of each probe giving rise to a positive match is recorded and grouped according to the peptide sequence encoded. This subset of probes is referred to as the optimized oligonucleotide array. There can be a maximum number of [3x183] or ay-237953 mamas wru is:z7 rvx 4m asz usz' Hmuu~r rx mnYti~ra ~uaa 157,464 possible peptide permutations. Some number of the possible peptide permutations do not exist in the A-375 melanoma cell line. Screening over GeneChips reduces this number to the actual number of peptide consensus sequences that exist in the A-375 melanoma cell line.
Secondary screening over GtneChips is used to identify a consensus sequence motif Xm(HyX)3Xm, where (I~yX) consists of the optimized oligonucleotide array identifed above and where X is any amino acid, with the exception of glycine and praline, and II xm ~ 5 amino acids.
Secondary screening over gene chips can also be carried out where Xm in the 1 Q consensus sequence motif Xm(HyX)aX," is ascertained by isolating and sequencing cDNAs that hybridize to the optimized oligonucleotide array identified about.
Secondary screening over gene chips can also be carried out where X," in the consensus sequence motif X",(HyXj~X", is ascertained by isolating and sequencing cDN.As that encode antigenic peptides recognized by Hl 1.
All references cited herein, both supra and infra, are hereby incorporated herein. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications can be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.
ny-z3~9~3 i34

Claims (69)

1. A composition comprising at least one SPPC which is specifically immunogenioally cross-reactive with one or more cell surface-associated SFPCs specific to a target cancer.

2. The composition according to claim 1, wherein at least oue SPPC contains a non-covalently bound peptide which confers the specific immunogenicity.

3. A composition comprising a plurality of SPPCs, which are specifically immunogenically cross-reactive with one or more cell surface-associated SPPCs specific to a target cancer.

4. The composition according to claim 3, wherein the SPPCs contain different non-covalently bound peptides, which confer the specific immunogenicity.

5. The composition according to claim 3, where the plurality is selected from the group consisting of, 2-10 SPPCs.

6. The composition according to claim I, wherein the cancer is selected from the group consisting of astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinonna, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bileduct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas and leiomyosarcomas.

7. The composition according to claim 1, where the SPPC is specifically immunogenically cross-reactive with more than one type of cancer cell population.

8. The composition according to claim 1, 2, 3 or 4, wherein the SP is a member of a heat shock protein family selected from the group consisting of hsp20-30, hsp60.
hsp70, hsp90 and combinations thereof.

9. The composition according to claim 8, wherein the heat shock protein is hsp72.

10. The composition according to claim 8, wherein the heat shock protein is hsp85.

11. The composition according to claim 8, wherein the heat shock protein is hsp96 (gp96).

12. The composition according to claim 8, wherein the peptide is derived from a tumor.

13, The composition according to claim 8, wherein the tumor is obtained from a human or a non-human mammal.

14. The composition according to claim 8, wherein the tumor is human and is selected from the group consisting of astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell. lung adenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bileduct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia. multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas and leiomyosarcomas.

15. A composition of matter comprising an isolated antigen-binding fragment of an antibody which binds specifically to at least one SPPC which is specifically immunogenically cross-reactive with one or more cell surface-associated SPPCs specific to a target cancer.

16. The composition of matter according to claim 15, wherein the at least one SPPC
contains a non-covalently bound peptide which confers the specific immunogenicity.

17. A composition comprising an antigen-binding fragment of an antibody which binds specifically to a plurality of SPPCs which is specifically immunogenically cross-reactive with one or more cell surface-associated SPPCs specific to a target cancer.

18. The composition according to claim 17, wherein the SPPCs contain different non-covalently bound peptides, which confer the specific immunogenicity.

19. The composition according to claim 15, 16, 17, or 18, wherein the antigen-binding fragment is selected from the group consisting of whole antibodies, bispecific antibodies, chimeric antibodies, Fab, F(ab')2, single chain V
region fragments (soFv) and fusion polypeptides.

20. The composition according to claim 19, wherein the antigen-binding fragment is of human origin.

21. The composition according to claim 20, wherein the antigen-binding-fragment is encoded by a phage display library.

22. The composition according to claim 19, wherein the antigen-binding fragment consists essentially of a scFv.

23. The composition according to claim 19, wherein the whole antibody is a human immunoglobulin of any isotype.

24. The composition according to claim 19, wherein the antigen-binding fragment comprises a variable region derived from an IgM and a constant region derived from an IgG.

25. The composition according to claim 19, wherein the fusion peptide comprises the antigen-binding fragment fused to a chemically functional moiety.

26. The composition according to claim 25, wherein the moiety is selected from the group consisting of signal peptides, agents that enhance immunologic reactivity, agents that facilitate coupling to a solid support, bioresponse modifiers, immunotoxins, toxins, detectable labels, paramagnetic labels and drugs.

27. The composition according to claim 26, wherein the agent that facilitates coupling to a solid support is selected from the group consisting of biotin and avidin.

28. The composition according to claim 26, wherein the immunogen carrier is selected from the group consisting of any physiologically acceptable buffer.

29. The composition according to claim 26, wherein the bioresponse modifier is a cytokine.

30. The composition according to claim 26, wherein the cytokine is selected from the group consisting of tumor necrosis factor, interleukin-2, interleukin-4, interleukin-12, granulocyte macrophage colony stimulating factor and .gamma.-interferons.

31. The composition according to claim 26, wherein the drug is an antineoplastic agent selected from the group consisting of radioisotopes, vinca alkaloids, adriamycin, bleomycin sulfate, carboplatin, cisplatin, cyclophosphamidc, cytarabint, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, lomustine, mechloroethamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mototane, pentostatin, pipobroman, procarbaze hydrochloride, streptozotocin, taxol, thioguanine and uracil mustard.

32. The composition according to claim 31, wherein the vinca alkaloid is selected from the group consisting of vinblastine sulfate, vincristine sulfate and vindesine sulfate.

33. The composition according to claim 26, wherein the toxin is selected from the group consisting of ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal ribosome inactivating proteins and phospholipase enzymes.

34. The composition according to claim 33, wherein the detectable label is selected from the group consisting of radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, bioluminescent compounds, enzymes, substrates, cofactors and inhibitors.

35. A composition of matter according to claim 1, 2, 3, 4, 16, 17, 18 or 19 wherein the SPPC is specifically immunogenic to the target cancer in a plurality of genetically different individuals having spontaneously arising tumors of the same type.

36.A composition of matter according to claim 1, 2, 3, 4, 16, 17, 18 or 19 wherein the SPPCs arc specifically immunogenic with respect to a plurality of different target cancers.

37. A composition comprising a population of one or more different SPPC's predominantly consisting of at least one immunogenic cancer cell surface-associated SPPC of a target cancer wherein the SPPC comprises a SP associated peptide that renders the SPPC immunogenic with respect to the target cancer.

38. The composition of matter according to claim 16, 17, 18 or 19 further comprising a physiologically acceptable excipient.

39. The composition according to claim 38, wherein the antigen-binding fragment is present in an amount effective to elicit a cancer-associated immune response in a subject upon administration to the subject.

40. The composition according to claim 38, wherein the antigen-binding fragment is selected from the group consisting of whole antibodies, bispecific antibodies, chimeric antibodies, Fab, F(ab')2, single chain V region fragments (scFv) and fusion polypeptides.

41. The composition according to claim 40, wherein the antigen-binding fragment is of human origin.

42. The composition according to claim 41, wherein the antigen-binding fragment is encoded by a phage display library.

43. The composition according to claim 40, wherein the antigen-binding fragment consists essentially of a scFv.

44. The composition according to claim 40, wherein the whole antibody is a human immunoglobulin of any isotype.

45. The composition according to claim 40, wherein antigen-binding fragment comprises a variable region derived from an IgM and a constant region derived from an IgG.

46. The composition according to claim 40, wherein the fusion peptide comprises the antigen-binding fragment fused to a chemically functional moiety.

47. The composition according to claim 44, wherein the moiety is selected from the group consisting of signal peptides, agents that enhance immunologic reactivity, agents that facilitate coupling to a solid support, bioresponse modifiers, immunotoxins, toxins, detectable labels, paramagnetic labels and drugs.

48. The method according to claim 47, wherein the agent that facilitates coupling to a solid support is selected from the group consisting of biotin and avidin.

49. The composition according to claim 47, wherein the bioresponse modifier is a cytokine.

50. The composition according to claim 49, wherein the cytokine is selected from the group consisting of tumor necrosis factor, interleukin-2, interleukin-4, interleukin-12, granulocyte macrophage colony stimulating factor and .gamma.-interferons.

51. The composition according to claim 47, wherein the drug is an antineoplastic agent selected from the group consisting of radioisotopes, vinca alkaloids, adriamycin, bleomycin sulfate, carboplatin, cisplatin, cyclophosphamide cytarabine, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorauracil, lomustine, mechloroethaminc hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mototane, pentostatin, pipobroman., procarbaze hydrochloride, streptozotocin, taxol, thioguanine and uracil mustard.

52. The composition according to claim 51, wherein the vinca alkaloid is selected from the group consisting of vinblastine sulfate, vincristine sulfate and vindesine sulfate.

53. The composition according to claim 47, wherein the toxin is selected from the group consisting of ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, as fungal ribosome inactivating proteins and phospholipase enzymes.

54. The composition according to claim 47, wherein the detectable label is selected from the group consisding of radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, bioluminescent compounds, enzymes, substrates, cofactors and inhibitors.

55. The composition according to claim 38, wherein the stress protein is selected from a heat shock protein family selected from the group consisting of hsp26, hsp60, hsp70, hsp90 and combinations thereof.

56. The composition according to claim 55, wherein the heat shock protein is hsp72.

57. The composition according to claim 55, wherein the heat shock protein is hsp85.

58. The composition according to claim 55, wherein the heat shock protein is hsp96.

59. The composition according to claim 38, wherein the peptide is derived from a tumor.

60. The composition according to claim 59, wherein the tumor is obtained from a human or a non-human mammal.

61. The composition according to claim 60, wherein the tumor is human and is selected from the group consisting of astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinonoa, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenie carcinoma, renal cell carcinoma, bileduct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas and leiomyosarcomas.

62. A method of treating a cancer subject comprising administering to the subject an amount of a composition of matter according to claim 38.

63. The method according to claim 62, wherein the peptide is pan-carcinomic.

64. A method of identifying antigen-binding fragments of as antibody specific for a tumor-associated SPPC comprising the steps of:
(a) generating a suitable phage display library;
(b) generating stress protein-peptide complex from a tumor;
(c) screening the product of step (a) with the product of step (b) to obtain phage which display an antigen-binding-fragment that binds specifically to SPPC; and, (d) screening tha phage obtained in step (c) for cell surface tumor-associated reactivity.

65. A method of isolating an antigenic tumor-associated SPPC, comprising the steps of:
(a) fractionating a tumor-cell extract on an antigen-binding fragment affinity medium to bind the complex;
(b) applying tht eluate from the affinity medium to molecular sieve which is capable of separating the stress protein from the peptide;
(c) isolating the peptide; and (d) re-associating the stress protein with the isolated peptide.

66. A method as claimed in claim 65, wherein said stress protein is a member of the HSP70 family.

67. A method as claimed in claim 66, wherein said stress protein is HSP72.

68. A method as claimed in claim 64, wherein said SPPC is C-antigen.

69. A method as claimed in claim 64 wherein said SPPC is first fractionated on a hydrophobic column to isolate a hydrophobic fraction.