ANTIBODIES THAT SPECIFICALLY BLOCK THE BIOLOGICAL ACTIVITY OF A TUMOR ANTIGEN

FIELD OF THE INVENTION The present invention relates to monoclonal antibodies and antigen binding fragments thereof that specifically binds to KAAG 1 and their use for treating certain diseases including diagnosing, preventing and treating malignant tumors related to ovarian cancer The present invention also relates to the use of these antibodies for diagnosis, prevention and treatment of various other cancer types

BACKGROUND OF THE INVENTION

Among gynecologic malignancies, ovarian cancer accounts for the highest tumor-related mortality in women in the United States (Jemal et al , 2005) It is the fourth leading cause of cancer-related death in women in the U S (Menon et al , 2005) The American Cancer Society estimated a total of 22,220 new cases in 2005 and attributed 16,210 deaths to the disease (Bonome et al , 2005) For the past 30 years, the statistics have remained largely the same - the majority of women who develop ovarian cancer will die of this disease (Chambers and Vanderhyden, 2006) The disease carries a 1 70 lifetime risk and a mortality rate of >60% (Chambers and Vanderhyden, 2006) The high mortality rate is due to the difficulties with the early detection of ovarian cancer when the malignancy has already spread beyond the ovary Indeed, >80% of patients are diagnosed with advanced staged disease (stage III or IV) (Bonome et al , 2005) These patients have a poor prognosis that is reflected in <45% 5-year survival rate, although 80% to 90% will initially respond to chemotherapy (Berek et al , 2000) This increased success compared to 20% 5-year survival rate years earlier is, at least in part, due to the ability to optimally debulk tumor tissue when it is confined to the ovaries, which is a significant prognostic factor for ovarian cancer (Bπstow R E , 2000, Brown et al , 2004) In patients who are diagnosed with early disease (stage I), the 5-yr survival ranges from >90 (Chambers and Vanderhyden, 2006)

Ovarian cancer comprises a heterogeneous group of tumors that are derived from the surface epithelium of the ovary or from surface inclusions They are classified into serous, mucinous, endometrioid, clear cell, and Brenner (transitional) types corresponding to the different types of epithelia in the organs of the female reproductive tract (Shih and Kurman, 2005) Of these, serous tumors account for -60% of the ovarian cancer cases diagnosed Each histologic subcategory is further divided into three groups benign, intermediate (borderline tumor or low malignancy potential (LMP)), and malignant, reflecting their clinical behavior (Seidman et al , 2002) LMP represents 10% to 15% of tumors diagnosed as serous and is a conundrum as they display atypical nuclear structure and metastatic behavior, yet they are considerably less aggressive than high-grade serous tumors The 5-year survival for patients with LMP tumors is 95% in contrast to a <45% survival for advanced high-grade disease over the same period (Berek et al , 2000)

Presently, the diagnosis of ovarian cancer is accomplished, in part, through routine analysis of the medical history of patients and by performing physical, ultrasound and x- ray examinations, and hematological screening Two alternative strategies have been reported for early hematological detection of serum biomarkers One approach is the analysis of serum samples by mass spectrometry to find proteins or protein fragments of unknown identity that detect the presence or absence of cancer (Mor et al , 2005, Kozak et al , 2003) However, this strategy is expensive and not broadly available Alternatively, the presence or absence of known proteins/peptides in the serum is being detected using antibody microarrays, ELISA, or other similar approaches Serum testing for a protein biomarker called CA-125 (cancer antιgen-125) has long been widely performed as a marker for ovarian cancer However, although ovarian cancer cells may produce an excess of these protein molecules, there are some other cancers, including cancer of the fallopian tube or endometrial cancer (cancer of the lining of the uterus), 60% of people with pancreatic cancer, and 20%-25% of people with other malignancies with elevated levels of CA-125 The CA-125 test only returns a true positive result for about 50% of Stage I ovarian cancer patients and has a 80% chance of returning true positive results from stage II, III, and IV ovarian cancer patients The other 20% of ovarian cancer patients do not show any increase in CA-125 concentrations In addition, an elevated CA-125 test may indicate other benign activity not associated with cancer, such as menstruation, pregnancy, or endometriosis Consequently, this test has very limited clinical application for the detection of early stage disease when it is still treatable, exhibiting a positive predictive value (PPV) of <10% Even with the addition of ultrasound screening to CA-125, the PPV only improves to around 20% (Kozak et al , 2003) Thus, this test is not an effective screening test Despite improved knowledge of the etiology of the disease, aggressive cytoreductive surgery, and modern combination chemotherapy, there has been only little change in mortality. Poor outcomes have been attributed to (1) lack of adequate screening tests for early disease detection in combination with only subtle presentation of symptoms at this stage - diagnosis is frequently being made only after progression to later stages, at which point the peritoneal dissemination of the cancer limits effective treatment and (2) the frequent development of resistance to standard chemotherapeutic strategies limiting improvement in the 5-year survival rate of patients. The initial chemotherapy regimen for ovarian cancer includes the combination of carboplatin (Paraplatin) and paclitaxel (taxol). Years of clinical trials have proved this combination to be most effective after effective surgery - reduces tumor volume in about 80% of the women with newly diagnosed ovarian cancer and 40% to 50% will have complete regression - but studies continue to look for ways to improve it. Recent abdominal infusion of chemotherapeutics to target hard-to-reach cells in combination with intravenous delivery has increased the effectiveness. However, severe side effects often lead to an incomplete course of treatment. Some other chemotherapeutic agents include doxorubicin, cisplatin, cyclophosphamide, bleomycin, etoposide, vinblastine, topotecan hydrochloride, ifosfamide, 5-fluorouracil and melphalan. More recently, clinical trials have demonstrated that intraperitoneal administration of cisplatin confers a survival advantage compared to systemic intravenous chemotherapy (Cannistra and McGuire, 2007). The excellent survival rates for women with early stage disease receiving chemotherapy provide a strong rationale for research efforts to develop strategies to improve the detection of ovarian cancer. Furthermore, the discovery of new ovarian cancer-related biomarkers will lead to the development of more effective therapeutic strategies with minimal side effects for the future treatment of ovarian cancer.

Notwithstanding these recent advances in the understanding and the treatment for ovarian cancer, the use of chemotherapy is invariably associated with severe adverse reactions, which limit their use. Consequently, the need for more specific strategies such as combining antigen tissue specificity with the selectivity of monoclonal antibodies should permit a significant reduction in off-target-associated side effects. The use of monoclonal antibodies for the therapy of ovarian cancer is beginning to emerge with an increasing number of ongoing clinical trials (Oei et al., 2008; Nicodemus and berek, 2005). Most of these trials have examined the use of monoclonal antibodies conjugated to radioisotopes, such as yttπum-90, or antibodies that target tumor antigens already identified in other cancer types An example of this is the use of bevacizumab, which targets vascular endothelial growth factor (Burger, 2007) There are very few ovarian cancer specific antigens that are currently under investigation as therapeutic targets for monoclonal antibodies Some examples include the use of a protein termed B7-H4 (Simon et al , 2006) and more recently folate receptor-alpha (Ebel et al , 2007), the latter of which has recently entered Phase Il clinical trials

Kidney associated antigen 1 (KAAG1) was originally cloned from a cDNA library derived from a histocompatibility leukocyte antιgen-B7 renal carcinoma cell line as an antigenic peptide presented to cytotoxic T lymphocytes (Van den Eynde et al , 1999, Genebank accesssion no Q9UBP8) The locus containing KAAG1 was found to encode two genes transcribed in both directions on opposite strands The sense strand was found to encode a transcript that encodes a protein termed DCDC2 Expression studies by these authors found that the KAAG1 antisense transcript was tumor specific and exhibited very little expression in normal tissues whereas the DCDC2 sense transcript was ubiquitously expressed (Van den Eynde et al , 1999) The expression of the KAAG1 transcript in cancer, and in particular ovarian cancer, renal cancer, lung cancer, colon cancer, breast cancer and melanoma was disclosed in the published patent application No PCT/CA2007/001134 Van den Eynde et al , also observed RNA expression in renal carcinomas, colorectal carcinomas, melanomas, sarcomas, leukemias, brain tumors, thyroid tumors, mammary carcinomas, prostatic carcinomas, oesophageal carcinomas, bladder tumor, lung carcinomas and head and neck tumors Recently, strong genetic evidence obtained through linkage disequilibrium studies found that the VMP/DCDC2/KAAG1 locus was associated with dyslexia (Schumacher et al , 2006, Cope et al , 2005) One of these reports pointed to the DCDC2 marker as the culprit in dyslexic patients since the function of this protein in cortical neuron migration was in accordance with symptoms of these patients who often display abnormal neuronal migration and maturation (Schumacher et al , 2006)

SUMMARY OF THE INVENTION

This invention relates to the expression of KAAG1 in tumor cells The invention also relates to specific antι-KAAG1 antibodies and antigen binding fragments as well as kits useful for the treatment, detection and diagnosis of cancer The antibodies and antigen binding fragments may more particularly be useful for the treatment, detection and diagnosis of cancer where tumor cells expresses KAAG1 , such as ovarian cancer, skin cancer, renal cancer, colorectal cancer, sarcoma, leukemia, brain cancer, cancer of the thyroid, breast cancer, prostate cancer, cancer of the oesophagus, bladder cancer, lung cancer and head and neck cancer.

The present invention provides in one aspect thereof, an isolated or substantially purified antibody or antigen binding fragment which may be capable of specific binding to Kidney associated antigen 1 (KAAG1 defined in SEQ ID NO.:2) or to a KAAG1 variant.

More specifically and in accordance with an embodiment of the invention, the antibody or antigen binding fragment may bind to a domain located between amino acid 30 and amino acid 84 of KAAG1.

In accordance with another embodiment of the invention, the antibody or antigen binding fragment may be capable of binding to an epitope comprised within amino acid 1 to 35 of KAAG 1.

In accordance with a further embodiment of the invention, the antibody or antigen binding fragment may be capable of binding to an epitope comprised within amino acid 36 to 60 Of KAAGl

In accordance with yet a further embodiment of the invention, the antibody or antigen binding fragment may be capable of binding to an epitope comprised within amino acid 61 to 84 of KAAGl

The antibody or antigen binding fragment of the present invention is especially capable of specific binding to a secreted form of KAAG1 , i.e., a form of KAAG1 where the signal peptide has been cleaved.

The antibody or antigen binding fragment of the present invention is especially capable of binding to the extracellular region of KAAGl

As such, the present invention encompasses diagnostic and/or therapeutic antibodies or antigen binding fragments having specificity for a secreted form of KAAG 1 or for an extracellular region of KAAGl Also encompassed by the present invention are antibodies or antigen binding fragments having the same epitope specificity as the antibody of the present invention. A candidate antibody may be identified by determining whether it will bind to the epitope to which the antibodies described herein binds and/or by performing competition assays with antibodies or antigen binding fragments known to bind to the epitope.

Therefore another aspect the present invention provides an isolated antibody or antigen binding fragment capable of competing with the antibody or antigen binding fragment described herein.

Isolated antibodies or antigen binding fragments of the present invention include those which may be capable of inducing killing (elimination, destruction, lysis) of KAAG1- expressing tumor cells or KAAG1 variant-expressing tumor cells (e.g., in an ADCC- dependent manner).

Isolated antibodies or antigen binding fragments of the present invention also include those which are characterized by their ability to reduce spreading of KAAG 1- expressing tumor cells and also those which are characterized by their ability to decrease or impair formation of KAAG 1- expressing tumors.

The antibodies or antigen binding fragments may be particularly effective when KAAG 1 is expressed at the surface of the KAAG 1 -expressing tumor cells and may be particularly useful in targeting KAAG1- expressing tumor cells characterized by anchorage- independent growth.

The invention relates to monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies and human antibodies (isolated) as well as antigen binding fragments having the characteristics described herein. Antibodies or antigen binding fragments encompassing permutations of the light and/or heavy chains between a monoclonal, chimeric, humanized or human antibody are also encompassed herewith.

The antibodies or antigen binding fragments of the present invention may thus comprise amino acids of a human constant region and/or framework amino acids of a human antibody.

The term "antibody" refers to intact antibody, monoclonal or polyclonal antibodies. The term "antibody" also encompasses multispecific antibodies such as bispecific antibodies.

Human antibodies are usually made of two light chains and two heavy chains each comprising variable regions and constant regions The light chain variable region comprises 3 CDRs, identified herein as CDRL1 , CDRL2 and CDRL3 flanked by framework regions The heavy chain variable region comprises 3 CDRs, identified herein as CDRH1 , CDRH2 and CDRH3 flanked by framework regions

The term "antigen-binding fragment", as used herein, refers to one or more fragments of an antibody that retain the ability to bind to an antigen (e g , KAAG1 , secreted form of KAAG 1 or variants thereof) It has been shown that the antigen-binding function of an antibody can be performed by fragments of an intact antibody Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (ι) a Fab fragment, a monovalent fragment consisting of the V _L , V _H , C _L and Cm domains, (ιι) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (ιιι) a Fd fragment consisting of the V _H and Cm domains, (ιv) a Fv fragment consisting of the V _L and V _H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al , (1989) Nature 341 544-546), which consists of a V _H domain, and (vι) an isolated complementarity determining region (CDR), e g , V _H CDR3 Furthermore, although the two domains of the Fv fragment, V _L and V _H , are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single polypeptide chain in which the V _L and V _H regions pair to form monovalent molecules (known as single chain Fv (scFv), see e g , Bird et al (1988) Science 242 423-426, and Huston et al (1988) Proc Natl Acad Sci USA 85 5879-5883) Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody Furthermore, the antigen- binding fragments include binding-domain immunoglobulin fusion proteins comprising (ι) a binding domain polypeptide (such as a heavy chain variable region, a light chain variable region, or a heavy chain variable region fused to a light chain variable region via a linker peptide) that is fused to an immunoglobulin hinge region polypeptide, (n) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (in) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region The hinge region may be modified by replacing one or more cysteine residues with serine residues so as to prevent dimerization Such binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/01 18592 and US 2003/0133939 These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

A typical antigen binding site is comprised of the variable regions formed by the pairing of a light chain immunoglobulin and a heavy chain immunoglobulin. The structure of the antibody variable regions is very consistent and exhibits very similar structures. These variable regions are typically comprised of relatively homologous framework regions (FR) interspaced with three hypervariable regions termed Complementarity Determining

Regions (CDRs). The overall binding activity of the antigen binding fragment is often dictated by the sequence of the CDRs. The FRs often play a role in the proper positioning and alignment in three dimensions of the CDRs for optimal antigen binding.

Antibodies and/or antigen binding fragments of the present invention may originate, for example, from a mouse, a rat or any other mammal or from other sources such as through recombinant DNA technologies.

Further scope, applicability and advantages of the present invention will become apparent from the non-restrictive detailed description given hereinafter. It should be understood, however, that this detailed description, while indicating exemplary embodiments of the invention, is given by way of example only, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows the expression profiling analyses using semi-quantitative RT-PCR reactions carried out to measure the level of KAAG1 mRNA expression in RNA samples derived from greater than 20 ovarian tumors, benign (low malignancy potential) tumors, ovarian cancer cell lines, and 30 normal tissues. The control panels show GAPDH expression, a house-keeping gene used to compare the amount of starting material in each RT-PCR reaction.

Figure 1 B shows semi-quantitative RT-PCR experiments demonstrating that KAAG1 mRNA is expressed in ovarian cancer cell lines, in particular those that are derived from ascites. Figure 1 C shows a diagram illustrating the ability of ovarian cancer cell lines to form 3D structures called spheroids. The left panels show the cells grown in medium lacking serum whereas 5% serum stimulated the formation of the spheroid structures.

Figure 1 D shows semi-quantitative RT-PCR experiments demonstrating that the KAAG1 mRNA is highly induced during the formation of spheroids in ovarian cancer cell lines.

Figure 2A shows a diagram illustrating the wound or scratch assay, a cell-based assay that is a measurement of a cell line's ability to migrate into a denuded area over a predetermined period of time. TOV-21G cells harboring KAAG1 shRNAs display a reduced capacity to fill in the denuded area.

Figure 2B shows an illustration of the clonogenic assay, also known as a colony survival assay. It measured the survival of diluted cells over a period of several days. TOV-21G cells harboring KAAG1 shRNAs display reduced survival.

Figure 3A shows a polyacrylamide gel that was stained with Coomassie Blue and contains a sample (10 μg) of purified Fc-KAAGI fusion protein that was produced in transiently transfected 293E cells.

Figure 3B shows the results of an ELISA of one of the 96-well plates containing individual monoclonal antibodies selected from Omniclonal library #3 containing anti-KAAG1 Fabs. The results showed that 48 (highlighted in grey) of the Fabs interacted very efficiently with KAAG1. The wells indicated by bold numbers contained the exemplary monoclonals 3D3, 3G10, and 3C4.

Figure 4A shows a polyacrylamide gel that was stained with Coomassie Blue and contains a sample (10 μg) of purified Fc-KAAGI fusion protein (lane 1), a truncated mutant of KAAG 1 spanning amino acids 1-60 (lane 2), and another truncated mutant of KAAG 1 spanning amino acids 1-35 (lane 3) that were produced in transiently transfected 293E cells. All proteins were Fc fusion proteins.

Figure 4B is a scheme that illustrates the truncated mutants of KAAG 1 that were generated for the epitope mapping studies.

Figure 4C shows a drawing that describes the results from ELISA analyses to map the epitopes that are bound by the anti-KAAG1 antibodies contained in Omniclonal library #3. The results showed that the majority of monoclonals interact with central region of KAAG 1 and that certain antibodies bound to the amino- or carboxyl-termini of KAAG 1.

Figure 5 presents a scheme that illustrates the steps involved to convert the mouse Fabs into IgGI mouse-human chimeric mAbs.

Figure 6 shows drawings that compare the binding of the mouse anti-KAAG1 Fabs with the binding of the corresponding IgGI chimeric monoclonal antibodies for exemplary antibodies 3D3, 3G10, and 3C4. The results indicate that the relative binding of the Fab variable regions was maintained when transferred to a full human IgGI scaffold.

Figure 7 shows depictions of spheroid formation experiments using TOV-21 G and OV-90 ovarian cancer cell lines in the presence of chimeric IgGI anti-KAAG1 monoclonal antibodies. Loosely packed structures are indicative of less invasive cancer cell lines. The results show spheroids treated with the exemplary anti-KAAG1 antibodies 3D3, 3G10, or 3C4.

Figure 8A shows a scan of a tissue microarray containing approximately 70 biopsy samples obtained from ovarian tumor patients. The samples were blotted with the 3D3 anti-KAAG1 antibody and showed that the vast majority of ovarian tumors expressed very high level of KAAG1 antigen.

Figure 8B a higher magnification picture from the tissue microarray experiment. The arrows show the membrane localization of KAAG1 at the apical surface of the epithelial layer of cells in serous ovarian tumors.

Figure 8C illustrates other immunohistochemical studies that demonstrate that KAAG1 is highly expressed in all ovarian cancer types. The histotypes shown are serous, mucinous and endometroid.

Figure 9A, 9B and 9C is a summary of alignment results obtained for selected CDRL1 , CDRL2 or CDRL3 sequences using the ClustalW2 program; where " * " means that the residues in that column are identical in all sequences in the alignment, " : " means that conserved substitutions have been observed and " . " means that semi-conserved substitutions are observed. Consensus CDRs were generated using the ClustalW program (Larkin M.A., et al., (2007) ClustalW and ClustalX version 2. Bioinformatics 2007 23(21): 2947-2948). Figure 10A, 10B and 10C is a summary of alignment results obtained for selected CDRH1 , CDRH2 or CDRH3 sequences using the ClustalW2 program; where " ^* " means that the residues in that column are identical in all sequences in the alignment, " : " means that conserved substitutions have been observed and " . " means that semi- conserved substitutions are observed. Consensus CDRs were generated using the ClustalW program (Larkin M.A., et al., (2007) ClustalW and ClustalX version 2. Bioinformatics 200723(21): 2947-2948).

Figure 11 represents sequence comparison between each of the light chain variable regions generated and representative light chain variable regions identified in SEQ ID NOs: 16, 20, 24 or 105. Percent sequence identity and percent sequence similarity has been determined unsing Blast2 sequence program as indicated herein.

Figure 12 represents sequence comparison between each of the heavy chain variable regions generated and representative heavy chain variable regions identified in SEQ ID NOs: 18, 22, 26 or 132. Percent sequence identity and percent sequence similarity has been determined unsing Blast2 sequence program as indicated herein.

Figure 13 An IgG ₁ antibody that targets KAAG1 can efficiently mediate ADCC activity in vitro. PBMNCs (AIICeIIs, LLC, Emoryville, CA) were incubated with 3D3 for 30 min and mixed with either OVCAR-3 or WIL2-S cells at a ratio of 1 :25. The cells were incubated for 4 h at 37 C and cell lysis was determined by measuring LDH levels in the medium. Cell cytotoxicity was calculated as follows: % cytotoxicity = (experimental - effector spontaneous - target spontaneous) x 100 / (target maximum - target spontaneous).

Figure 14 Anti-KAAG1 mAbs prevent the spread of TOV-112D ovarian tumors in vivo. 1 x 10 ⁶ cells were implanted in the peritoneal cavity of SCID mice in a volume of 200 μL. Treatment with either PBS or antibodies diluted in PBS was performed 2 days later at a dose of 25 mg/kg qwk. The mice were sacrificed as soon as the tumors were detected by palpation of the abdomen. The number of tumors were scored visually (B) and the data in panel A is expressed as the average number of tumors/mouse ± SE.

Figure 15 shows immunohistochemistry performed with an anti-KAAG1 antibody on human skin tumor tissue microarrays (Pantomics Inc., Richmond, CA) of several sections isolated from squamous cell carcinomas and melanomas. Figure 16 illustrates spheroid formation of melanoma cell lines (A375 and SK-MEL5) and of renal cell carcinoma cell lines (A498 and 786-0) in the presence or absence of the chimeric 3D3 antibody. Figure 17A represents graphs illustrating the binding of increasing concentrations of the 3C4, 3D3 and 3G10 antibodies to cell lines (OV-90, TOV-21G and SKOV-3) fixed under condition that do not permeate the cells.

Figure 17B is a graph illustrating the results of flow cytometry performed on SKOV-3 cell line with the 3D3 antibody. Figure 18A is a schematic illustrating the structure of the 3D3 antibody model. Figure 18B is a schematic illustrating the structure of the 3C4 antibody model.

Figure 19A is a graph illustrating the binding of increasing concentration of the humanized 3D3 antibody in comparison with the chimeric 3D3 antibody to recombinant KAAG1. Figure 19B is a table summarizing the kinetics parameters of the humanized 3D3 antibody, the chimeric 3D3 antibody as well as hybrid antibodies encompassing permutations of the light and heavy chains of the chimeric or humanized antibody.

Figure 19C illustrates spheroid formation of SKOV-3 ovarian cancer cells in the presence of the humanized 3D3 antibody, chimeric 3D3 antibody or in the presence of a buffer or a control IgG.

Figure 2OA represents sequence alignment of the monoclonal 3D3 light chain variable region (SEQ ID NO.: 16) and the humanized 3D3 light chain variable region (SEQ ID NO.: 178). The humanized 3D3 light chain variable region is 86% identical (94% sequence similarity) to the monoclonal 3D3 light chain variable region and their three CDRs are 100% (indicated in bold).

Figure 2OB represents sequence alignment of the monoclonal 3D3 heavy chain variable region (SEQ ID NO.: 18) and the humanized 3D3 heavy chain variable region (SEQ ID NO.: 179). The humanized 3D3 heavy chain variable region is 82% identical (91% sequence similarity) to the monoclonal 3D3 heavy chain variable region and their three CDRs are 100% (indicated in bold).

Figure 21A represents sequence alignment of the monoclonal 3C4 light chain variable region (SEQ ID NO.:24) and the humanized 3C4 light chain variable region (SEQ ID NO 182) The humanized 3C4 light chain variable region is 85% identical (93% sequence similarity) to the monoclonal 3C4 light chain variable region and their three CDRs are 100% (indicated in bold)

Figure 21 B represents sequence alignment of the monoclonal 3C4 heavy chain variable region (SEQ ID NO 26) and the humanized 3C4 heavy chain variable region (SEQ ID NO 183) The humanized 3C4 heavy chain variable region is 86% identical (93% sequence similarity) to the monoclonal 3C4 heavy chain variable region and their three CDRs are 100% (indicated in bold)

DETAILED DESCRIPTION OF THE INVENTION

The expression and biological activity of KAAG 1 in cancer cells

The present invention relates to the use of antibodies to target tumors found in various cancer types, in particular ovarian cancer In order to direct the antibodies to the tumors, the identification of tumor-specific antigens that are expressed at the cell surface of the cancer cells must be carried out There are several technologies that are available to identify tumor-specific antigens and the method that was used to identify KAAG 1 in ovarian tumors, an innovative discovery platform called Subtractive Transcription-based Amplification of mRNA (STAR), is described in the published patent application No PCT/CA2007/001134

Analysis of the ovarian cancer STAR libraries yielded many genes that encode secreted and cell surface proteins One of these, termed AB-0447, contained an open reading frame that encoded a polypeptide of 84 ammo acids, corresponding to SEQ ID NO 2 that was encoded by a cDNA of 885 base pairs with the nucleotide sequence shown in SEQ ID NO 1 A search of publicly available databases revealed that the AB-0447 nucleotide sequence was identical to that of a gene called KAAG 1 Bioinformatic analysis predicted a membrane-anchored protein that presents its functional domain to the extracellular compartment KAAG 1 was originally cloned from a kidney cancer library as a cell surface antigen, a result that confirms its membrane localization Additionally, our studies showed that the protein was processed at its ammo-terminus, a result that was consistent with cleavage of a functional signal peptide at or between amino acids 30 and 34 Furthermore, transient expression of the full-length cDNA resulted in detection of cleaved KAAG 1 in the culture medium This last finding indicated that this membrane- anchored protein could be shed from the cells when expressed at high levels In contrast, expression of an ammo-truncated mutant of KAAG 1 resulted in intra-cellular retention of the protein There are currently no published reports that shed any light on its function and the over-expression of KAAG 1 in ovarian cancer, as disclosed by this invention, has never been previously documented

We have thus investigated whether KAAG1 could be used for antibody-based diagnostics and therapeutics

Several ovarian cancer cell-based models have been established, such as TOV-21G, TOV-112D, OV-90, and others, and are familiar to those skilled in the art These cells are part of a collection of human ovarian cancer cell lines derived from patients with ovarian tumors or ascites fluid These cell lines have undergone an in-depth analysis, including global gene expression patterns on microarrays that make them excellent cell-based models for human ovarian cancer The growth properties, gene expression patterns, and response to chemotherapeutic drugs indicated that these cell lines are very representative of ovarian tumor behavior in vivo (BenoTt et al , 2007) RT-PCR analysis of total RNA isolated from these ovarian cancer cell lines showed that the KAAG 1 transcript was weakly expressed in the cell lines derived from primary tumors In contrast, cell lines derived from ascitic fluid contained high levels of KAAG1 expression The increased expression of KAAG1 in cells from the ascitic fluid suggested that the environment of the cells influences the regulation of the KAAG 1 gene Ascitic cells are associated with advanced disease and this pattern of expression implies that increased KAAG 1 levels are associated with anchorage-independent growth In concordance with this latter suggestion, KAAG1 expression was found to significantly increase in cell lines derived from primary tumors when these cells were cultured as spheroids in 3D cultures These spheroids have been extensively characterized and were found to display many properties associated with tumors in vivo (Cody et al , 2008) Thus, expression of KAAG 1 was found to be significantly increased in models that mimic tumor progression, in particular during the evolution of ovarian cancer

With the demonstration that KAAG 1 expression is regulated in ovarian cancer cells, the function of this gene in ovarian cancer cell behavior was examined in cell-based assays To that effect, RNA interference (RNAi) was used to knock down the expression of the endogenous KAAG1 gene in the ovarian cancer cell lines and it was found that decreased expression of KAAG1 resulted in a significant reduction in the migration of the cells as determined in a standard cell motility assay, as exemplified by a wound healing (or scratch) assay. This type of assay measures the speed at which cells fill a denuded area in a confluent monolayer. Decreased expression of KAAG 1 resulted in a reduction in the survival of ovarian cancer cell lines as measured by a clonogenic assay, such as a colony survival assay. Those skilled in the art may use other methods to evaluate the requirement of KAAG1 in the behavior of cancer cells, in particular ovarian cancer cells.

Based on the expression of KAAG 1 in a large proportion of ovarian tumors, its limited expression in normal tissues, and a concordance between expression levels and increased malignancy, and a putative biological role for KAAG1 in the behavior of ovarian cancer cell lines, KAAG1 was chosen as a therapeutic target for the development of antibodies for the detection, prevention, and treatment of ovarian cancer. Expression of

KAAG 1 in cancer, other than ovarian cancer also lead the Applicant to the evaluation of therapeutic or diagnostic antibodies for other cancer indications.

Therefore, a variety of anti-KAAG1 antibodies and antigen binding fragments thereof, such as monoclonal antibodies, polyclonal antibodies, chimeric and humanized antibodies (including humanized monoclonal antibodies), antibody fragments, single chain antibodies, domain antibodies, and polypeptides with an antigen binding region, useful for targeting KAAG1 are provided.

KAAG 1 as antigen and epitopes derived from KAAG 1

The Applicant has come to the unexpected discovery that KAAG 1 is expressed in several tumor types and is also found in blood and in ascitic fluid of patients. This antigen may thus be useful for targeting tumor cells expressing the antigen in vivo and in the development of detection assays for measuring the tumor associated antigen in vitro or in vivo. The KAAG1 antigen circulating in blood lacks the signal peptide.

The present invention therefore provides a KAAG 1 antigen useful for generating antibodies specific for the circulating form of KAAG1 and/or specific for tumor-expressed KAAG1. The KAAG1 antigen (i.e., epitope) may comprise a fragment of at least 10 amino acids (and up to 84 amino acids) of KAAG1 and may especially bind to the extracellular region of KAAG 1. An exemplary antigen is the whole KAAG 1 protein or a variant form having at least 80% sequence identity with SEQ ID NO 2 or a fragment thereof

Another exemplary antigen derived from KAAG1 is the secreted or circulating form of KAAG1 which lacks the signal peptide or the extracellular region of KAAG1 This antigen may more particularly lack amino acids 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, 1 to 30, 1 to 31 , 1 to 32, 1 to 33, 1 to 34, 1 to 35 or 1 to 36 of KAAG1

The antigen or the epitope described herein may be fused with a carrier such as keyhole limpet (KHL), bovine serum albumin (BSA), ovalbumin (OVA) or else in order to generate antibodies and antigen binding fragments

The present invention also provides an epitope comprised within amino acid 1 to 35 of SEQ ID NO 2, within ammo acid 36 to 60 of SEQ ID NO 2 or within amino acid 61 to 84 of SEQ ID NO 2 to generate antibodies and antigen binding fragments described herein The present invention further provides a composition for generating antibodies to a secreted or circulating form of KAAG1 or to an extracellular region of KAAG1 , the composition may comprise an epitope of KAAG1 comprised within amino acids 30 to 84 of SEQ ID NO 2 and a carrier The epitope may especially comprise at least 10 amino acids of KAAG 1

Exemplary embodiments of compositions are pharmaceutical composition for generating antibodies to a secreted or circulating form of KAAG 1 or to the extracellular region of KAAG1 The pharmaceutical composition may comprise an epitope of KAAG1 comprised within amino acids 30 to 84 of SEQ ID NO 2 and a pharmaceutically acceptable carrier

In yet a further aspect the invention provides a method for generating antibodies to a secreted or circulating form of KAAG 1 The method may comprise administering a polypeptide comprising an epitope of KAAG1 comprised within amino acids 30 to 84 of SEQ ID NO 2 wherein the epitope lacks a KAAG1 signal peptide

Alternatively, the method may comprise administering an epitope which comprises the signal peptide and selecting antibodies which only binds to the secreted form or the extracellular region of the protein In an additional aspect, the present invention provides the use of an epitope of KAAG1 comprised within amino acids 30 to 84 of SEQ ID NO.:2 for generating antibodies to a secreted or circulating form of KAAG 1.

Antibodies and antigen binding frapments that binds to KAAG 1

Antibodies were initially isolated from Fab librairies for their specificity towards the antigen of interest. Comparison of the amino acid sequences of the light chain variable domains or the heavy chain variable domains of antibodies showing the greatest characteristics allowed us to derive consensus sequences within the CDRs and within the variable regions. The consensus for CDRs are provided in SEQ ID Nos: 74 to 90.

The variable regions described herein may be fused with constant regions of a desired species thereby allowing recognition of the antibody by effector cells of the desired species. The constant region may originate, for example, from an IgGI , lgG2, lgG3, or lgG4 subtype. Cloning or synthesizing a constant region in frame with a variable region is well within the scope of a person of skill in the art and may be performed, for example, by recombinant DNA technology.

In certain embodiments of the present invention, antibodies that bind to KAAG1 may be of the IgGI , lgG2, lgG3, or lgG4 subtype. More specific embodiments of the invention relates to an antibody of the IgGI subtype. The antibody may be a humanized antibody of the IgGI subtype that is biologically active in mediating antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CMC), or associated with immune complexes. The typical ADCC involves activation of natural killer (NK) cells and is reliant on the recognition of antibody-coated cells by Fc receptors on the surface of the NK cells. The Fc receptors recognize the Fc domain of antibodies such as is present on IgGI , which bind to the surface of a target cell, in particular a cancerous cell that expresses an antigen, such as KAAG1. Once bound to the Fc receptor of IgG the NK cell releases cytokines and cytotoxic granules that enter the target cell and promote cell death by triggering apoptosis.

In some instances, anti-KAAG1 antibodies with substantially identical light and heavy chain variable regions to antibody 3D3, will interact with an epitope spanned by amino acids 36 - 60, inclusively, of KAAG1. In other instances, anti-KAAG1 antibodies with substantially identical light and heavy chain variable regions to antibody 3G10, will interact with an epitope spanned by amino acids 61 - 84, inclusively, of KAAG1. In yet another instance, anti-KAAG1 antibodies with substantially identical light and heavy chain variable regions to antibody 3C4 will interact with an epitope spanned by amino acids 1 - 35, inclusively, of KAAG1.

The present invention described a collection of antibodies that bind to KAAG1. In certain embodiments, the antibodies may be selected from the group consisting of polyclonal antibodies, monoclonal antibodies such as chimeric or humanized antibodies, antibody fragments such as antigen binding fragments, single chain antibodies, domain antibodies, and polypeptides with an antigen binding region.

In an aspect of the invention, the isolated antibody or antigen binding fragment of the present invention may be capable of inducing killing (elimination, destruction, lysis) of KAAG1 -expressing tumor cells or KAAG1 variant-expressing tumor cells (e.g., in an ADCC-dependent manner).

In a further aspect of the invention, the isolated antibody or antigen binding fragment of the present invention may especially be characterized by its capacity of reducing spreading of KAAG1- expressing tumor cells.

In an additional aspect of the invention, the isolated antibody or antigen binding fragment of the present invention may be characterized by its capacity of decreasing or impairing formation of KAAG 1- expressing tumors.

In accordance with an embodiment of the invention, the antibody or antigen binding fragment may be more particularly effective when KAAG1 is expressed at the surface of the KAAG 1 -expressing tumor cells.

Also in accordance with the present invention, the antibody or antigen binding fragment may be especially useful in targeting KAAG1- expressing tumor cells which are characterized by anchorage-independent growth.

In a further aspect, the present invention relates to an isolated antibody or antigen binding fragment for use in the treatment of cancer comprising tumor cells expressing KAAG 1. In yet a further aspect, the present invention relates to an isolated antibody or antigen binding fragment for use in the detection of cancer comprising tumor cells expressing KAAG 1

In an exemplary embodiment of the invention, the isolated antibody or antigen binding fragment may comprise amino acids of a constant region, which may originate, for example, from a human antibody

In another exemplary embodiment of the invention, the isolated antibody or antigen binding fragment may comprise framework amino acids of a human antibody

Without being limited to the exemplary embodiments presented herein, the Applicant as generated specific antibodies and antigen binding fragments which may be useful for the purposes described herein

The present invention therefore provides in an exemplary embodiment, an isolated antibody or antigen binding fragment comprising a light chain variable domain having, a a CDRL1 sequence selected from the group consisting of SEQ ID NO 74 and SEQ ID NO 75, b a CDRL2 sequence selected from the group consisting of SEQ ID NO 76,

SEQ ID NO 77 and SEQ ID NO 78, or, c a CDRL3 sequence selected from the group consisting of SEQ ID NO 79, SEQ ID NO 80 and SEQ ID NO 81

The isolated antibody or antigen binding fragment may also comprise a heavy chain variable domain having, a a CDRH1 sequence comprising SEQ ID NO 82, b a CDRH2 sequence selected from the group consisting of SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86 and SEQ ID NO 87, or, c a CDRH3 sequence selected from the group consisting of SEQ ID NO 88, SEQ ID NO 89 and SEQ ID NO 90

In an exemplary embodiment, the antibody or antigen binding fragment may comprise any individual CDR or a combination of CDR1 , CDR2 and/or CDR3 of the light chain variable region The CDR3 may more particularly be selected Combination may include for example, CDRL1 and CDRL3; CDRL1 and CDRL2; CDRL2 and CDRL3 and; CDRL1 , CDRL2 and CDRL3.

In another exemplary embodiment, the antibody or antigen binding fragment may comprise any individual CDR or a combination of CDR1 , CDR2 and/or CDR3 of the heavy chain variable region. The CDR3 may more particularly be selected. Combination may include for example, CDRH1 and CDRH3; CDRH1 and CDRH2; CDRH2 and CDRH3 and; CDRH1 , CDRH2 and CDRH3.

In accordance with the present invention, the antibody or antigen binding fragment may comprise at least two CDRs of a CDRL1 , a CDRL2 or a CDRL3.

Also in accordance with the present invention, the antibody or antigen binding fragment may comprise one CDRL1 , one CDRL2 and one CDRL3.

Further in accordance with the present invention, the antibody or antigen binding fragment may comprise: a. At least two CDRs of a CDRL1 , CDRL2 or CDRL3 and; b. At least two CDRs of a CDRH1 , one CDRH2 or one CDRH3.

The antibody or antigen binding fragment may more preferably comprise one CDRL1 , one CDRL2 and one CDRL3.

The antibody or antigen binding fragment may also more preferably comprise one CDRH1 , one CDRH2 and one CDRH3.

Other exemplary embodiments of the invention relates to an isolated antibody or antigen binding fragment comprising a heavy chain variable domain having; a. a CDRH1 sequence comprising SEQ ID NO..82; b. a CDRH2 sequence selected from the group consisting of SEQ ID NO.:83, SEQ ID NO.:84, SEQ ID NO.:85, SEQ ID NO.:86 and SEQ ID NO.:87, or; c. a CDRH3 sequence selected from the group consisting of SEQ ID NO.:88, SEQ ID NO..89 and SEQ ID NO.:90.

In accordance witht the present invention, the antibody or antigen binding fragment may comprise one CDRH1 , one CDRH2 or one CDRH3. In accordance witht the present invention, the antibody or antigen binding fragment may also comprise one CDRH1 , one CDRH2 and one CDRH3.

When only one of the light chain variable domain or the heavy chain variable domain is available, an antibody or antigen-binding fragment may be reconstituted by screening a library of complementary variable domains using methods known in the art (Portolano et al. The Journal of Immunology (1993) 150:880-887, Clarkson et al., Nature (1991) 352:624-628).

Also encompassed by the present invention are polypeptides or antibodies comprising variable chains having at least one conservative amino acid substitution in at least one of the CDRs described herein (in comparison with the original CDR).

The present invention also encompasses polypeptides or antibodies comprising variable chains having at least one conservative amino acid substitution in at least two of the CDRs (in comparison with the original CDRs).

The present invention also encompasses polypeptides or antibodies comprising variable chains having at least one conservative amino acid substitution in the 3 CDRs (in comparison with the original CDRs).

The present invention also encompasses polypeptides or antibodies comprising variable chains having at least two conservative amino acid substitutions in at least one of the CDRs (in comparison with the original CDRs).

The present invention also encompasses polypeptides or antibodies comprising variable chains having at least two conservative amino acid substitutions in at least two of the CDRs (in comparison with the original CDRs).

The present invention also encompasses polypeptides or antibodies comprising variable chains having at least two conservative amino acid substitutions in the 3 CDRs (in comparison with the original CDRs).

In another aspect, the present invention relates to a polypeptide, antibody or antigen binding fragment comprising (on a single polypeptide chain or on separate polypeptide chains) at least one complementarity-determining region of a light chain variable domain and at least one complementarity-determining region of a heavy chain variable domain of one of the antibodies or antigen binding fragment described herein.

The present invention relates in another aspect thereof to anti-KAAG1 antibodies that may comprise (on a single polypeptide chain or on separate polypeptide chains) all six complementarity-determining regions (CDRs) of the antibody or antigen binding fragment described herein.

The antibodies or antigen binding fragment of the present invention may further comprise additional amino acids flanking the amino and/or carboxy region of the CDR(s). Those additional amino acids may be as illustrated in Table A or Table B or may include, for example, conservative amino acid substitution.

In accordance with the present invention, the antibody may comprise a CDRL1 sequence comprising or consisting of formula:

X _1a SSX _2a SLLX ₃ aX4aX5aX6aX7aX8aX9aXi0aLXiia (SEQ ID NO.74) wherein X _1a may be a basic amino acid; wherein X _2a may be a basic amino acid; wherein X ₃₃ may be H, Y or N; wherein X _4a may be S, T, N or R; wherein X _5a may be absent, S or N; wherein X _6a may be D, F or N; wherein X _7a may be G or Q; wherein X _8a may be K, L or N; wherein X _9a may be T or N; wherein X _1Oa may be an aromatic amino acid, and; wherein X ₁₁₃ may be A, N, E or Y.

In an exemplary embodiment of the invention X _1a may be K or R.

In a further embodiment of the invention X _2a may be Q or K.

In yet a further embodiment of the invention X _3a may be N or H.

In an additional embodiment of the invention X _1Oa may be Y or F. More specific embodiments of the invention include CDRL1 of SEQ ID NO.:74 where: X _1a is K; X ₂₃ is Q; X _3a is N; X _3a is H; X _4a is S; X ₄₃ is T; X _5a is S; X _5a is absent; X _6a is N; X _7a is Q; X _7a is G; X _8a is K; X _9a is N; X _9a is T; X ₁₀₃ is Y; or X _11a is A.

In accordance with the present invention, the antibody may comprise a CDRL1 sequence comprising or consisting of formula: KASQDX _1b X _2b X _3b X _4b X _5b X _6b (SEQ ID NO.:75) wherein Xi _b may be an hydrophobic amino acid; wherein X _2b may be G or H; wherein X _3b may be T, N or R; wherein X _4b may be F, Y or A; wherein X _5b may be an hydrophobic amino acid, and; wherein X _6b may be N or A.

In an exemplary embodiment of the invention X _1b may be V or I.

In another exemplary embodiment of the invention X _5b may be V or L.

More specific embodiments of the invention include CDRL1 of SEQ ID NO.:75 where X _1b is I; X _2b is H; X _3b is T; X _3b is N; X _4b is Y; X _4b is F; X _5b is L or X _6b is N.

In accordance with the present invention, the antibody may comprise a CDRL2 sequence comprising or consisting of formula: FX _1c STX _2c X _3c S (SEQ ID NO.:76) Wherein X _1c is A or G; Wherein X _2c is R or T, and; Wherein X _3c is E, K or A.

In an exemplary embodiment of the invention X _1c may be A and X _2c may be T.

In another exemplary embodiment of the invention X _1c may be A and X _2c may be R.

Other specific embodiments of the invention include CDRL2 of SEQ ID NO.:76 where

X _1c is A; X _2c is R or X _3c is E.

In accordance with the present invention, the antibody may comprise a CDRL2 sequence comprising or consisting of formula: X _1d VSX _2d X _3d X _4d S (SEQ ID NO.:77) Wherein X _id may be L or K; Wherein X _2d may be a basic amino acid; Wherein X _3d may be L or R and; Wherein X _4d may be D or F.

In an exemplary embodiment of the invention X _2d may be K or N.

Other specific embodiments of the invention include CDRL2 of SEQ ID NO.:77 where X _1d is L; X _2d is K; X _3d is L or X _4d is D.

In accordance with the present invention, the antibody may comprise a CDRL2 sequence comprising or consisting of formula: X ₁₆ ANRLVX ₂₆ (SEQ ID NO.:78) Wherein Xi _e may be a basic amino acid, and; Wherein X _2e may be D or A.

In an exemplary embodiment of the invention X _1e may be R or H.

Other specific embodiments of the invention include CDRL2 of SEQ ID NO.:78 where X _1e is R or X _2e is D.

In accordance with the present invention, the antibody may comprise a CDRL3 sequence comprising or consisting of formula: X _1f QX _2f X _3f X _4f X _5f PLT (SEQ ID NO.:79)

Wherein X _1f may be Q or L;

Wherein X _2f may be an aromatic amino acid;

Wherein X _3f may be D, F or Y;

Wherein X _4f may be E, A, N or S, and; Wherein X _5f may be I, F or T.

In an exemplary embodiment of the invention X _2f may be Y or H.

In another exemplary embodiment of the invention X _3f may be Y or D.

In yet another exemplary embodiment of the invention X _5f may be I or T. Other specific embodiments of the invention include CDRL3 of SEQ ID NO.:79 where X _lf is Q; X _2f is H; X _3f is D; X _3f is Y; X _4f is S; X _4f is E; X _4f is A; X _5f is T, or X _5f is I.

In accordance with the present invention, the antibody may comprise a CDRL3 sequence comprising or consisting of formula: QQHX ₁₉ X ₂₃ X ₃₉ PLT (SEQ ID NO.:80) Wherein X _1g may be an aromatic amino acid; Wherein X ₂₉ may be N or S, and; Wherein X ₃₉ may be I or T.

In an exemplary embodiment of the invention X ₁₉ may be F or Y

Other specific embodiments of the invention include CDRL3 of SEQ ID NO.:80 where X ₂₉ is S or X _3g is T.

In accordance with the present invention, the antibody may comprise a CDRL3 sequence comprising or consisting of formula: X _1h QGX _2h HX _3h PX _4h T (SEQ ID NO.:81)

Wherein X _1h may be an aromatic amino acid; Wherein X _2h may be a neutral hydrophilic amino acid; Wherein X _3h may be F or V, and; Wherein X _4h may be R or L.

In an exemplary embodiment of the invention X _1h may be W or F.

In another exemplary embodiment of the invention X _2h may be S or T.

Other specific embodiments of the invention include CDRL3 of SEQ ID NO.:81 where X _1h is W; X _2h is T; X _3h is F, or X _4h is R.

In accordance with the present invention, the antibody may comprise a CDRH1 sequence comprising or consisting of formula:

GYX ₁₁ FX ₂₁ X ₃₁ YX ₄₁ X ₅₁ H (SEQ ID NO.:82)

Wherein X ₁₁ may be T, I or K;

Wherein X ₂ , may be a neutral hydrophilic amino acid;

Wherein X ₃₁ may be an acidic amino acid; Wherein X ₄₁ may be E, N or D, and; Wherein X ₅₁ may be hydrophobic amino acid.

In an exemplary embodiment of the invention X ₂ , may be T or S.

In another exemplary embodiment of the invention X ₃ , may be D or E.

In yet another exemplary embodiment of the invention X ₄₁ may be N or E.

In a further exemplary embodiment of the invention X ₅₁ may be M, I or v.

Other specific embodiments of the invention include CDRH1 of SEQ ID NO.:82 where X ₂ , is T, X ₃ , is D; X ₄ , is E; X ₅ , is I or X ₅ , is M.

In accordance with the present invention, the antibody may comprise a CDRH2 sequence comprising or consisting of formula:

X ₁₁ X _2J DPX _3J TGX ₄₁ TX _5J (SEQ ID NO.:83)

Wherein X ₁₁ may be V or G

Wherein X _2j may be a hydrophobic amino acid;

Wherein X ₃₁ may be A, G or E; Wherein X ₄ , may be R, G, D, A, S, N or V, and;

Wherein X ₅₁ may be a hydrophobic amino acid.

In an exemplary embodiment of the invention X ₂₁ may be I or L.

In another exemplary embodiment of the invention X ₅₁ may be A or V.

Other specific embodiments of the invention include CDRH2 of SEQ ID NO.:83 where X ₁₁ is V; X ₂₁ is I; X _3j is E; X ₄₁ is D or X _5j is A.

In accordance with the present invention, the antibody may comprise a CDRH2 sequence comprising or consisting of formula: VX _1k DPX _2k TGX _3k TA (SEQ ID NO.:84) Wherein X _1k may be an hydrophobic amino acid; Wherein X _2k may be A, E or G;

Wherein X _3k may be R, G, A, S, N V or D.

In an exemplary embodiment of the invention X _1k may be L or I. Other specific embodiments of the invention include CDRH2 of SEQ ID NO 84 where X _1k is I, X _2K is E, or X _3k is D

In accordance with the present invention, the antibody may comprise a CDRH2 sequence comprising or consisting of formula YIX ₁₁ X _2I X ₃₁ GX _4I X _5I X _6I (SEQ ID NO 85) Wherein X1, may be S or N, Wherein X ₂ | may be an aromatic amino acid Wherein X ₃ , may be D, E or N, Wherein X4| may be a D or H, Wherein X ₅ , may be Y, S or N, Wherein X ₆ ι may be D, E or N

In an exemplary embodiment of the invention X ₃ | may be D or N

In another exemplary embodiment of the invention X ₆ ι may be D or N

Other specific embodiments of the invention include CDRH2 of SEQ ID NO 85 where X ₂ ι is F or Y, X ₃ ι is N, X ₄ , is D or X ₆ | is N

In accordance with the present invention, the antibody may comprise a CDRH2 sequence comprising or consisting of formula X _1m INPYNX _2m VTE (SEQ ID NO 86) wherein X _1m may be N or Y, and, wherein X _2m may be E, D or N

In an exemplary embodiment of the invention X _2m may be D or N

Other specific embodiments of the invention include CDRH2 of SEQ ID NO 86 where X _1m ιs N or X _2m ιs D

In accordance with the present invention, the antibody may comprise a CDRH2 sequence comprising or consisting of formula

DINPX _1n YGX _2n X _3n T (SEQ ID NO 87)

Wherein X _1n may be N or Y,

Wherein X _2n may be G or T and, wherein X _3n may be I or T In accordance with the present invention, the antibody may comprise a CDRH3 sequence comprising or consisting of formula: MX ₁₀ X ₂₀ X _3O DY (SEQ ID NO.:88) Wherein X ₁₀ may be G or S; Wherein X ₂₀ may be Y or H, and; wherein X ₃₀ may be A or S.

Other specific embodiments of the invention include CDRH3 of SEQ ID NO.:88 where X ₁₀ is G; X ₂₀ is Y or X ₃₀ is S.

In accordance with the present invention, the antibody may comprise a CDRH3 sequence comprising or consisting of formula: IX _1p YAX _2p DY (SEQ ID NO.:89) Wherein X _1p may be G or S and; Wherein X _2p may be absent or M.

Other specific embodiments of the invention include CDRH3 of SEQ ID NO.:89 where X _1p is S or X _2p is M.

In accordance with the present invention, the antibody may comprise a CDRH3 sequence comprising or consisting of formula: AX _1q X _2q GLRX _3q (SEQ ID NO.:90) Wherein X _1q may be R or W;

Wherein X _2q may be an aromatic amino acid and; wherein X _3q may be a basic amino acid.

In an exemplary embodiment of the invention X _2q may be W or F.

In another exemplary embodiment of the invention X _3q may be Q or N.

Other specific embodiments of the invention include CDRH3 of SEQ ID NO.:90 where X _1q is R; X _2q is W or X _3q is N.

The framework region of the heavy and/or light chains described herein may be derived from one or more of the framework regions illustrated in Tables A and B. The antibody or antigen binding fragments may thus comprise one or more of the CDRs described herein (e.g., selected from the specific CDRs or consensus CDRs of SEQ ID NO.:74 to 90) and framework regions originating from those illustrated in Tables A and B. In Tables A and B, the expected CDRs are shown in bold, while the framework regions are not.

Table 2 describes the sequences of the nucleotides and the amino acids corresponding to the complete light and heavy chain immunoglobulins of specific examples of anti- KAAG1 antibodies.

TABLE 2 - complete sequences of light and heavy chain immunoglobulins that bind to KAAG 1

An antibody or antigen binding fragment that can bind KAAG1 may comprise any one L chain with any one H chain immunoglobulin that is listed in Table 2. In certain embodiments, the light chain of antibody 3D3 may be combined with the heavy chain of 3D3 or the heavy chain of 3G10 to form a complete antibody with KAAG1 -binding activity. In an exemplary embodiment of the present invention, the 3D3 L chain may be combined with the 3D3 H chain, the 3G10 L chain may be combined with the 3G10 H chain, or the 3C4 L chain may be combined with the 3C4 H chain. Additionally, some examples of antibodies or antigen binding fragment may consist of any combination of two L chains and any two H chains from the list of antibodies listed in Table 2.

The complete nucleotide sequences of the light and heavy immunoglobulin chains of antibody 3D3 are shown in SEQ ID NOS:3 and 5, respectively, and the corresponding amino acid sequences of the light and heavy immunoglobulin chains of antibody 3D3 are shown in SEQ ID NOS:4 and 6, respectively. Thus, in an exemplary embodiment, an antibody that binds to KAAG1 may comprise the light chain amino acid shown in SEQ ID NO.:4 combined with the heavy chain amino acid sequence shown in SEQ ID N0.:6. In another embodiment, the antibody may comprise two identical 3D3 light chains comprising of SEQ ID NO.:4 and two identical 3D3 heavy chains comprising SEQ ID NO.:6.

The complete nucleotide sequences of the light and heavy immunoglobulin chains of antibody 3G10 are shown in SEQ ID NOS:7 and 9, respectively, and the corresponding amino acid sequences of the light and heavy immunoglobulin chains of antibody 3G10 are shown in SEQ ID NOS:8 and 10, respectively. Thus, in an exemplary embodiment, an antibody that binds to KAAG1 may comprise the light chain amino acid shown in SEQ ID NO.:8 combined with the heavy chain amino acid sequence shown in SEQ ID NO.:10. In another embodiment, the antibody may comprise two identical 3G10 light chains comprising SEQ ID NO.:8 and two identical 3G10 heavy chains comprising SEQ ID NO.:10.

The complete nucleotide sequences of the light and heavy immunoglobulin chains of antibody 3C4 are shown in SEQ ID NOS:11 and 13, respectively and the corresponding amino acid sequences of the light and heavy immunoglobulin chains of antibody 3C4 are shown in SEQ ID NOS:12 and 14, respectively. Thus, in an exemplary embodiment, an antibody that binds to KAAG1 may comprise the light chain amino acid shown in SEQ ID NO.:12 combined with the heavy chain amino acid sequence shown in SEQ ID NO.:14. In another embodiment, the antibody may comprise two identical 3C4 light chains comprising SEQ ID NO.:12 and two identical 3C4 heavy chains comprising SEQ ID NO.:14.

Variants of other anti-KAAG1 antibodies or antigen binding fragments formed by the combination of light and/or heavy immunoglobulin chains may each independently have at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to the amino acid sequences listed in Table 2 are also provided. In certain embodiments, the antibody variants may comprise at least one light chain and one heavy chain. In other instances, the antibody variants may comprise two identical light chains and two identical heavy chains. In accordance with the present invention, the region of variation may be located in the constant region or in the variable region. Also in accordance with the present invention, the region of variation may be located in the framework region. Also encompassed by the present invention are antibodies comprising a light chain comprising one of the variable region illustrated in Table A and a heavy chain comprising one of the variable region illustrated in Table B The light chain and heavy chain may comprise a constant domain Combinations of light chains and heavy chains of Table 2, Table A and Table B are also encompassed by the present invention

Antibodies or antigen binding fragments that contain the light chain and heavy chain variable regions are also provided in the present invention Additionally, certain embodiments include antigen binding fragments, variants, and derivatives of these light and heavy chain variable regions

Yet other exemplary embodiments of the invention includes an isolated antibody or antigen binding fragment capable of specific binding to SEQ ID NO 2, to an extracellular portion of SEQ ID NO 2, or to a secreted form of SEQ ID NO 2 or to a variant thereof, the antibody comprising a the light chain variable domain defined in SEQ ID NO 16 and the heavy chain variable domain defined in SEQ ID NO 18, b the light chain variable domain defined in SEQ ID NO 20 and the heavy chain variable domain defined in SEQ ID NO 22, c the light chain variable domain defined in SEQ ID NO 24 and the heavy chain variable domain defined in SEQ ID NO 26, d the light chain variable domain defined in SEQ ID NO 105 and the heavy chain variable domain defined in SEQ ID NO 132, e the light chain variable domain defined in SEQ ID NO 106 and the heavy chain variable domain defined in SEQ ID NO 133, f the light chain variable domain defined in SEQ ID NO 107 and the heavy chain variable domain defined in SEQ ID NO 134, g the light chain variable domain defined in SEQ ID NO 108 and the heavy chain variable domain defined in SEQ ID NO 154, h the light chain variable domain defined in SEQ ID NO 109 and the heavy chain variable domain defined in SEQ ID NO 153, i the light chain variable domain defined in SEQ ID NO 110 and the heavy chain variable domain defined in SEQ ID NO 135, j the light chain variable domain defined in SEQ ID NO 111 and the heavy chain variable domain defined in SEQ ID NO 136, k the light chain variable domain defined in SEQ ID NO 112 and the heavy chain variable domain defined in SEQ ID NO 149,

I the light chain variable domain defined in SEQ ID NO 113 and the heavy chain variable domain defined in SEQ ID NO 137, m the light chain variable domain defined in SEQ ID NO 114 and the heavy chain variable domain defined in SEQ ID NO 140, n the light chain variable domain defined in SEQ ID NO 115 and the heavy chain variable domain defined in SEQ ID NO 141 , o the light chain variable domain defined in SEQ ID NO 116 and the heavy chain variable domain defined in SEQ ID NO 142, p the light chain variable domain defined in SEQ ID NO 1 17 and the heavy chain variable domain defined in SEQ ID NO 139, q the light chain variable domain defined in SEQ ID NO 119 and the heavy chain variable domain defined in SEQ ID NO 143, r the light chain variable domain defined in SEQ ID NO 120 and the heavy chain variable domain defined in SEQ ID NO 152, s the light chain variable domain defined in SEQ ID NO 121 and the heavy chain variable domain defined in SEQ ID NO 146, t the light chain variable domain defined in SEQ ID NO 122 and the heavy chain variable domain defined in SEQ ID NO 138, u the light chain variable domain defined in SEQ ID NO 123 and the heavy chain variable domain defined in SEQ ID NO 150, v the light chain variable domain defined in SEQ ID NO 124 and the heavy chain variable domain defined in SEQ ID NO 144, w the light chain variable domain defined in SEQ ID NO 126 and the heavy chain variable domain defined in SEQ ID NO 145, x the light chain variable domain defined in SEQ ID NO 127 and the heavy chain variable domain defined in SEQ ID NO 157, y the light chain variable domain defined in SEQ ID NO 128 and the heavy chain variable domain defined in SEQ ID NO 155, z the light chain variable domain defined in SEQ ID NO 129 and the heavy chain variable domain defined in SEQ ID NO 156, or, aa the light chain variable domain defined in SEQ ID NO 130 and the heavy chain variable domain defined in SEQ ID NO 151 It is to be understood herein, that the light chain variable region of the specific combination provided above may be changed for any other light chain variable region. Similarly, the heavy chain variable region of the specific combination provided above may be changed for any other heavy chain variable region.

Specific examples of sequences present in these light and heavy chain variable regions are disclosed in Table 3.

Table 3 - Sequences of light and heavy chain variable regions that bind to KAAG1

Therefore, antibodies and antigen binding fragments that bind to KAAG1 may comprise one light variable region and one heavy variable region of the same designated antibody or in any combinations. For example, in an exemplary embodiment, an anti-KAAG1 antibody or fragment may comprise the 3D3 light chain variable region (SEQ ID NO.: 16) and the 3D3 heavy chain variable region (SEQ ID NO.: 18). In an alternate embodiment, an anti-KAAG1 antibody or fragment may comprise the 3D3 light chain variable region (SEQ ID NO.: 16) and the 3G10 heavy chain variable region (SEQ ID NO.:22). In another embodiment, the anti-KAAG1 antibodies may comprise two identical light chain variable regions and two identical heavy chain regions. In yet another embodiment, the anti- KAAG1 antibodies may comprise two different light chain variable regions and two different heavy chain regions.

Variants of other anti-KAAG1 antibodies formed by the combination of light and/or heavy chain variable regions that each have at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identity to the amino acid sequences listed in Table 3 are also provided. Those skilled in the art will also recognize that the anti-KAAG1 antibody variants may include conservative amino acid changes, amino acid substitutions, deletions, or additions in the amino acid sequences of the light and/or heavy chain variable regions listed in Table 3.

In accordance with the present invention, the region of variation may be located in the framework region of the variable region. Table 4 - Sequences of the light and heavy chain CDRs

In certain embodiments of the present invention, the anti-KAAG1 antibodies or antigen binding fragments may comprise the CDR sequences shown in Table 4 or have substantial sequence identity to the CDR sequences of Table 4. In an exemplary embodiment, the 3D3 anti-KAAG1 antibody may comprise a light chain variable region containing CDR1 , 2, and 3 that are encoded by SEQ ID NOS:27, 28, and 29, respectively, and/or a heavy chain variable region containing CDR1 , 2, and 3 that are encoded by SEQ ID NOS:30, 31 , and 32, respectively. In other embodiments the CDR3 region may be sufficient to provide antigen binding. As such polypeptides comprising the CDR3L or the CDR3H or both the CDR3L and the CDR3H are encompassed by the present invention. Additionally, the anti-KAAG1 antibodies or antigen binding fragments may include any combination of the CDRs listed in Table 4. For example, the antibodies or antigen binding fragments may include the light chain CDR3 and the heavy chain CDR3. It is understood that the CDRs that are contained in the anti-KAAG1 antibodies or antigen binding fragments may be variant CDRs with 80%, 85%, 90%, or 95% sequence identity to the CDR sequences presented in Table 4. Those skilled in the art will also recognize that the variants may include conservative amino acid changes, amino acid substitutions, deletions, or additions in the CDR sequences listed in Table 4. Other exemplary embodiments of the invention includes an isolated antibody or antigen binding fragment capable of specific binding to SEQ ID NO.:2, to an extracellular portion of SEQ ID NO.:2 or to a secreted form of SEQ ID NO.:2 or to a variant thereof, the antibody comprising: a. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:16 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:18, b. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:20 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.:22; c. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:24 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.:26; d. the 3CDRs of a light chain variable domain defined in SEQ ID NO.: 105 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.:132, e. the 3CDRs of a light chain variable domain defined in SEQ ID NO.: 106 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.:133, f. the 3CDRs of a light chain variable domain defined in SEQ ID NO.: 107 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.: 134, g. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:108 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.:154, h. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:109 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:153, i. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:110 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:135, j. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:111 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.:136, k. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:1 12 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:149,

I. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:113 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:137, m. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:114 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:140, n. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:115 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:141 , o. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:116 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.:142, p. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:117 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:139, q. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:119 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:143, r. the 3CDRs of a light chain variable domain defined in SEQ ID NO.: 120 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:152, s. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:121 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:146, t. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:122 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:138, u. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:123 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.: 150, v. the 3CDRs of a light chain variable domain defined in SEQ ID NO.: 124 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:144, w. the 3CDRs of a light chain variable domain defined in SEQ ID NO.: 126 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:145, x. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:127 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:157, y. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:128 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:155, z. the 3CDRs of a light chain variable domain defined in SEQ ID NO.: 129 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID NO.:156, or; aa. the 3CDRs of a light chain variable domain defined in SEQ ID NO.:130 and/or the 3CDRs of a heavy chain variable domain defined in SEQ ID

NO.:151.

Again, the light chain variable region of the specific combination provided above may be changed for any other light chain variable region described herein. Similarly, the heavy chain variable region of the specific combination provided above may be changed for any other heavy chain variable region described herein. Variant antibody and antigen binding fragments

The present invention also encompasses variants of the antibodies or antigen binding fragments described herein Variant antibodies or antigen binding fragments included are those having a variation in the amino acid sequence For example, variant antibodies or antigen binding fragments included are those having at least one variant

CDR (two, three, four, five or six variant CDRs or even twelve variant CDRs), a variant light chain variable domain, a variant heavy chain variable domain, a variant light chain and/or a variant heavy chain Variant antibodies or antigen binding fragments included in the present invention are those having, for example, similar or improved binding affinity in comparison with the original antibody or antigen binding fragment

As used herein the term "variant" applies to any of the sequence described herein and includes for example, a variant CDR (either CDRL1 , CDRL2, CDRL3, CDRH1 , CDRH2 and/or CDRH3), a variant light chain variable domain, a variant heavy chain variable domain, a variant light chain, a variant heavy chain, a variant antibody, a variant antigen binding fragment and a KAAG1 variant

Variant antibodies or antigen binding fragments encompassed by the present invention are those which may comprise an insertion, a deletion or an ammo acid substitution (conservative or non-conservative) These variants may have at least one amino acid residue in its amino acid sequence removed and a different residue inserted in its place

The sites of greatest interest for substitutional mutagenesis include the hypervariable regions (CDRs), but modifications in the framework region or even in the constant region are also contemplated Conservative substitutions may be made by exchanging an amino acid (of a CDR, variable chain, antibody, etc ) from one of the groups listed below (group 1 to 6) for another amino acid of the same group

Other exemplary embodiments of conservative substitutions are shown in Table 1A under the heading of "preferred substitutions" If such substitutions result in a undesired property, then more substantial changes, denominated "exemplary substitutions" in Table 1A, or as further described below in reference to amino acid classes, may be introduced and the products screened

It is known in the art that variants may be generated by substitutional mutagenesis and retain the biological activity of the polypeptides of the present invention These variants have at least one amino acid residue in the amino acid sequence removed and a different residue inserted in its place. For example, one site of interest for substitutional mutagenesis may include a site in which particular residues obtained from various species are identical. Examples of substitutions identified as "conservative substitutions" are shown in Table 1A. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in Table 1A, or as further described herein in reference to amino acid classes, are introduced and the products screened.

Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties:

(group 1 ) hydrophobic: norleucine, methionine (Met), Alanine (Ala), Valine (VaI), Leucine (Leu), lsoleucine (He)

(group 2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr) (group 3) acidic: Aspartic acid (Asp), Glutamic acid (GIu) (group 4) basic: Asparagine (Asn), Glutamine (GIn), Histidine (His), Lysine

(Lys), Arginine (Arg)

(group 5) residues that influence chain orientation: Glycine (GIy), Proline (Pro); and (group 6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)

Non-conservative substitutions will entail exchanging a member of one of these classes for another.

Table 1A. Amino acid substitution

Variation in the amino acid sequence of the variant antibody or antigen binding fragment may include an amino acid addition, deletion, insertion, substitution etc., one or more modification in the backbone or side-chain of one or more amino acid, or an addition of a group or another molecule to one or more amino acids (side-chains or backbone).

Variant antibody or antigen binding fragment may have substantial sequence similarity and/or sequence identity in its amino acid sequence in comparison with that the original antibody or antigen binding fragment amino acid sequence. The degree of similarity between two sequences is based upon the percentage of identities (identical amino acids) and of conservative substitution.

Generally, the degree of similarity and identity between variable chains has been determined herein using the Blast2 sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) using default settings, i.e., blastp program, BLOSUM62 matrix (open gap 1 1 and extension gap penalty 1 ; gapx dropoff 50, expect 10.0, word size 3) and activated filters.

Percent identity will therefore be indicative of amino acids which are identical in comparison with the original peptide and which may occupy the same or similar position.

Percent similarity will be indicative of amino acids which are identical and those which are replaced with conservative amino acid substitution in comparison with the original peptide at the same or similar position.

Variants of the present invention therefore comprise those which may have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an original sequence or a portion of an original sequence.

Exemplary embodiments of variants are those having at least 81% sequence identity to a sequence described herein and 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Other exemplary embodiments of variants are those having at least 82% sequence identity to a sequence described herein and 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Further exemplary embodiments of variants are those having at least 85% sequence identity to a sequence described herein and 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Other exemplary embodiments of variants are those having at least 90% sequence identity to a sequence described herein and 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence. Additional exemplary embodiments of variants are those having at least 95% sequence identity to a sequence described herein and 95%, 96%, 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

Yet additional exemplary embodiments of variants are those having at least 97% sequence identity to a sequence described herein and 97%, 98%, 99% or 100% sequence similarity with an original sequence or a portion of an original sequence.

For a purpose of concision the applicant provides herein a Table 1 B illustrating exemplary embodiments of individual variants encompassed by the present invention and comprising the specified % sequence identity and % sequence similarity. Each "X" is to be construed as defining a given variant.

The present invention encompasses CDRs, light chain variable domains, heavy chain variable domains, light chains, heavy chains, antibodies and/or antigen binding fragments which comprise at least 80% identity with the sequence described herein.

Exemplary embodiments of the antibody or antigen binding fragment of the present invention are those comprising a light chain variable domain comprising a sequence selected from the group consisting of a sequence at least 70%, 75%, 80% identical to SEQ ID NO.:16, a sequence at least 70%. 75%, 80% identical to SEQ ID NO.:20, a sequence at least 70%. 75%, 80% identical to SEQ ID NO.:24, a sequence at least 70%. 75%, 80% identical to SEQ ID NO.: 105, a sequence at least 70%. 75%, 80% identical to SEQ ID NO.:109 and a sequence at least 70%. 75%, 80% identical to SEQ ID NO.:126.

These light chain variable domain may comprise a CDRL1 sequence at least 80 % identical to SEQ ID NO.:27, a CDRL2 sequence at least 80 % identical to SEQ ID NO.:28 and a CDRL3 sequence at least 80 % identical to SEQ ID NO.:29.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be at least 90 % identical to SEQ ID NO.:27.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be 100% identical to SEQ ID NO.:27.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence at least 90 % identical to SEQ ID NO.:28.

In yet another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be 100% identical to SEQ ID NO.:28.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be at least 90 % identical to SEQ ID NO.:29.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be 100% identical to SEQ ID NO.:29.

The light chain variable domain listed above may comprise a CDRL1 sequence at least 80 % identical to SEQ ID NO.:33, a CDRL2 sequence at least 80 % identical to SEQ ID NO.:34 and a CDRL3 sequence at least 80 % identical to SEQ ID NO.:35. In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be at least 90 % identical to SEQ ID NO.:33.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be 100% identical to SEQ ID NO.:33.

In yet a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be at least 90 % identical to SEQ ID NO.:34.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be 100% identical to SEQ ID NO.-.34.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be at least 90 % identical to SEQ ID NO.:35.

In yet another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be 100% identical to SEQ ID NO.:35.

The light chain variable domain listed above may comprise a CDRL1 sequence at least 80 % identical to SEQ ID NO.:39, a CDRL2 sequence at least 80 % identical to SEQ ID NO.:40 and a CDRL3 sequence at least 80 % identical to SEQ ID NO.:41.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be at least 90 % identical to SEQ ID NO.:39.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be 100% identical to SEQ ID NO.:39. In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be at least 90 % identical to SEQ ID NO..40.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be 100% identical to SEQ ID NO.:40.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be at least 90 % identical to SEQ ID NO.:41.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be 100% identical to SEQ ID NO.:41.

The light chain variable domain listed above may comprise a CDRL1 sequence at least 80 % identical to SEQ ID NO.: 158, a CDRL2 sequence at least 80 % identical to SEQ ID NO..159 and a CDRL3 sequence at least 80 % identical to SEQ ID NO.:160.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be at least 90 % identical to SEQ ID NO.:158.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be 100% identical to SEQ ID NO.:158.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be at least 90 % identical to SEQ ID NO.:159.

In yet a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be 100% identical to SEQ ID NO.: 159. In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be at least 90 % identical to SEQ ID NO 160

In yet another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be 100% identical to SEQ ID NO 160

The light chain variable domain listed above may comprise a CDRL1 sequence at least 80 % identical to SEQ ID NO 164, a CDRL2 sequence at least 80 % identical to SEQ ID NO 165 and a CDRL3 sequence at least 80 % identical to SEQ ID NO 166

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be at least 90 % identical to SEQ ID NO 164

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be 100% identical to SEQ ID NO 164

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be at least 90 % identical to SEQ ID NO 165

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be 100% identical to SEQ ID NO 165

In yet another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be at least 90 % identical to SEQ ID NO 166

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be 100% identical to SEQ ID NO 166 The light chain variable domain listed above may comprise a CDRL1 sequence at least 80 % identical to SEQ ID NO 170, a CDRL2 sequence at least 80 % identical to SEQ ID NO 171 and a CDRL3 sequence at least 80 % identical to SEQ ID NO 172

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be at least 90 % identical to SEQ ID NO 170

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL1 sequence which may be 100% identical to SEQ ID NO 170

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be at least 90 % identical to SEQ ID NO 171

In yet another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL2 sequence which may be 100% identical to SEQ ID NO 171

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be at least 90 % identical to SEQ ID NO 172

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRL3 sequence which may be 100% identical to SEQ ID NO 172

An exemplary embodiment of a variant antibody light chain variable region encompasses a light chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO 16 and having up to 22 amino acid modifications (e g , conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO 16 A SEQ ID NO 16 variant is provided in SEQ ID NO 178

An exemplary embodiment of a variant antibody light chain variable region encompasses a light chain variable region having CDR ammo acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.:20 and having up to 22 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:20.

An exemplary embodiment of a variant antibody light chain variable region encompasses a light chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.:24 and having up to 21 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:24. A SEQ ID NO.:24 variant is provided in SEQ ID NO.:182.

An exemplary embodiment of a variant antibody light chain variable region encompasses a light chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.: 105 and having up to 22 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.: 105.

An exemplary embodiment of a variant antibody light chain variable region encompasses a light chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.: 109 and having up to 22 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:109.

An exemplary embodiment of a variant antibody light chain variable region encompasses a light chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.:126 and having up to 21 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.: 126.

In some instances, the variant antibody light chain variable region may comprise amino acid deletions or additions (in combination or not with amino acid substitutions). Often 1 , 2, 3, 4 or 5 amino acid deletions or additions may be tolerated.

In an exemplary embodiment, the antibody or antigen binding fragment may comprise a heavy chain variable domain comprising a sequence selected from the group consisting of a sequence at least 80% identical to SEQ ID NO.: 18, a sequence at least 70%. 75%, 80% identical to SEQ ID NO.:22, a sequence at least 70%. 75%, 80% identical to SEQ ID NO.:26, a sequence at least 70%. 75%, 80% identical to SEQ ID NO.:132, a sequence at least 70%. 75%, 80% identical to SEQ ID NO.: 145 and a sequence at least 70%. 75%, 80% identical to SEQ ID NO.:153.

These heavy chain variable domains may comprise a CDRH1 sequence at least 80 % identical to SEQ ID NO.:30, a CDRH2 sequence at least 80 % identical to SEQ ID NO.:31 and a CDRH3 sequence at least 80 % identical to SEQ ID NO.:32.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH1 sequence which may be at least 90 % identical to SEQ ID NO.. -30.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH1 sequence which may be 100% identical to SEQ ID NO..-30.

In yet another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be at least 90 % identical to SEQ ID NO.:31.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be 100% identical to SEQ ID NO.:31.

In yet a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be at least 90 % identical to SEQ ID NO.:32.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be 100% identical to SEQ ID NO.:32.

The heavy chain variable domain listed above may comprise a CDRH 1 sequence at least 80 % identical to SEQ ID NO.:36, a CDRH2 sequence at least 80 % identical to SEQ ID NO.:37 and a CDRH3 sequence at least 80 % identical to SEQ ID NO.:38. In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH1 sequence which may be at least 90 % identical to SEQ ID NO.:36.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH 1 sequence which may be 100% identical to SEQ ID NO.:36.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be at least 90 % identical to SEQ ID NO.:37.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be 100% identical to SEQ ID NO.:37.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be at least 90 % identical to SEQ ID NO.:38.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be 100% identical to SEQ ID NO.:38.

The heavy chain variable domain listed above may comprise a CDRH1 sequence at least 80 % identical to SEQ ID NO.:42, a CDRH2 sequence at least 80 % identical to SEQ ID NO.:43 and a CDRH3 sequence at least 80 % identical to SEQ ID NO.:44.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH1 sequence which may be at least 90 % identical to SEQ ID NO.:42.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH 1 sequence which may be 100% identical to SEQ ID NO.:42. In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be at least 90 % identical to SEQ ID NO .43.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be 100% identical to SEQ ID NO.:43.

In yet a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be at least 90 % identical to SEQ ID NO.:44.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be 100% identical to SEQ ID NO.:44.

The heavy chain variable domain listed above may comprise a CDRH1 sequence at least 80 % identical to SEQ ID NO..161 , a CDRH2 sequence at least 80 % identical to SEQ ID NO..162 and a CDRH3 sequence at least 80 % identical to SEQ ID NO..163.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH 1 sequence which may be at least 90 % identical to SEQ ID NO.:161.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH 1 sequence which may be 100% identical to SEQ ID NO.:161.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be at least 90 % identical to SEQ ID NO.:162.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be 100% identical to SEQ ID NO.: 162. In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be at least 90 % identical to SEQ ID NO.:163.

In yet another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be 100% identical to SEQ ID NO.:163.

The heavy chain variable domain listed above may comprise a CDRH1 sequence at least 80 % identical to SEQ ID NO.:167, a CDRH2 sequence at least 80 % identical to SEQ ID NO.:168 and a CDRH3 sequence at least 80 % identical to SEQ ID NO.:169.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH1 sequence which may be at least 90 % identical to SEQ ID NO.:166.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH 1 sequence which may be 100% identical to SEQ ID NO.: 166.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be at least 90 % identical to SEQ ID NO.:168.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be 100% identical to SEQ ID NO.:168.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be at least 90 % identical to SEQ ID NO.:169.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be 100% identical to SEQ ID NO.:169. The heavy chain variable domain listed above may comprise a CDRH1 sequence at least 80 % identical to SEQ ID NO.: 173, a CDRH2 sequence at least 80 % identical to SEQ ID NO.: 174 and a CDRH3 sequence at least 80 % identical to SEQ ID NO.: 175.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH1 sequence which may be at least 90 % identical to SEQ ID NO.:173.

In an exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH1 sequence which may be 100% identical to SEQ ID NO.: 173.

In an additional exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be at least 90 % identical to SEQ ID NO.: 174.

In a further exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH2 sequence which may be 100% identical to SEQ ID NO.: 174.

In another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be at least 90 % identical to SEQ ID NO.: 175.

In yet another exemplary embodiment of the present invention, any of the antibodies provided herein may comprise a CDRH3 sequence which may be 100% identical to SEQ ID NO.: 175.

An exemplary embodiment of a variant antibody heavy chain variable region encompasses a heavy chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.: 18 and having up to 22 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:18. A SEQ ID NO.:18 variant is provided in SEQ ID NO.:179.

An exemplary embodiment of a variant antibody heavy chain variable region encompasses a heavy chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.:22 and having up to 23 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:22.

An exemplary embodiment of a variant antibody heavy chain variable region encompasses a heavy chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.:26 and having up to 23 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:26. A SEQ ID NO.:26 variant is provided in SEQ ID NO.:183.

An exemplary embodiment of a variant antibody heavy chain variable region encompasses a heavy chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.:132 and having up to 23 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:132.

An exemplary embodiment of a variant antibody heavy chain variable region encompasses a heavy chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.:153 and having up to 23 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:153.

An exemplary embodiment of a variant antibody heavy chain variable region encompasses a heavy chain variable region having CDR amino acid sequences that are 100% identical to the CDR amino acid sequence of SEQ ID NO.: 145 and having up to 22 amino acid modifications (e.g., conservative or non-conservative amino acid substitutions) in its framework region in comparison with the framework region of SEQ ID NO.:145.

In some instances, the variant antibody heavy chain variable region may comprise amino acid deletions or additions (in combination or not with amino acid substitutions). Often 1 , 2, 3, 4 or 5 amino acid deletions or additions may be tolerated. Production of the antibodies in cells

The anti-KAAG1 antibodies that are disclosed herein can be made by a variety of methods familiar to those skilled in the art, such as hybridoma methodology or by recombinant DNA methods.

In an exemplary embodiment of the invention, the anti-KAAG1 antibodies may be produced by the conventional hybridoma technology, where a mouse is immunized with an antigen, spleen cells isolated and fused with myeloma cells lacking HGPRT expression and hybrid cells selected by hypoxanthine, aminoptehn and thymine (HAT) containing media.

In an additional exemplary embodiment of the invention, the anti-KAAG1 antibodies may be produced by recombinant DNA methods.

In order to express the anti-KAAG1 antibodies, nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein or any other may be inserted into an expression vector, i.e , a vector that contains the elements for transcriptional and translational control of the inserted coding sequence in a particular host. These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' un-translated regions. Methods that are well known to those skilled in the art may be used to construct such expression vectors. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

A variety of expression vector/host cell systems known to those of skill in the art may be utilized to express a polypeptide or RNA derived from nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with baculovirus vectors; plant cell systems transformed with viral or bacterial expression vectors; or animal cell systems. For long-term production of recombinant proteins in mammalian systems, stable expression in cell lines may be effected. For example, nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may be transformed into cell lines using expression vectors that may contain viral origins of replication and/or endogenous expression elements and a selectable or visible marker gene on the same or on a separate vector The invention is not to be limited by the vector or host cell employed In certain embodiments of the present invention, the nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may each be hgated into a separate expression vector and each chain expressed separately In another embodiment, both the light and heavy chains able to encode any one of a light and heavy immunoglobulin chains described herein may be hgated into a single expression vector and expressed simultaneously

Alternatively, RNA and/or polypeptide may be expressed from a vector comprising nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein using an in vitro transcription system or a coupled in vitro transcription/translation system respectively

In general, host cells that contain nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein and/or that express a polypeptide encoded by the nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein, or a portion thereof, may be identified by a variety of procedures known to those of skill in the art These procedures include, but are not limited to, DNA/DNA or DNA/RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or amino acid sequences Immunological methods for detecting and measuring the expression of polypeptides using either specific polyclonal or monoclonal antibodies are known in the art Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS) Those of skill in the art may readily adapt these methodologies to the present invention

Host cells comprising nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may thus be cultured under conditions for the transcription of the corresponding RNA (mRNA, siRNA, shRNA etc ) and/or the expression of the polypeptide from cell culture The polypeptide produced by a cell may be secreted or may be retained intracellular^ depending on the sequence and/or the vector used In an exemplary embodiment, expression vectors containing nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein may be designed to contain signal sequences that direct secretion of the polypeptide through a prokaryotic or eukaryotic cell membrane.

Due to the inherent degeneracy of the genetic code, other DNA sequences that encode the same, substantially the same or a functionally equivalent amino acid sequence may be produced and used, for example, to express a polypeptide encoded by nucleotide sequences able to encode any one of a light and heavy immunoglobulin chains described herein. The nucleotide sequences of the present invention may be engineered using methods generally known in the art in order to alter the nucleotide sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. In an exemplary embodiment, anti-KAAG1 antibodies that contain particular glycosylation structures or patterns may be desired. Post-translational processing, which cleaves a "prepro" form of the polypeptide, may also be used to specify protein targeting, folding, and/or activity. Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and W138) are available commercially and from the American Type Culture Collection (ATCC) and may be chosen to ensure the correct modification and processing of the expressed polypeptide.

Those of skill in the art will readily appreciate that natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence resulting in translation of a fusion polypeptide containing heterologous polypeptide moieties in any of the aforementioned host systems. Such heterologous polypeptide moieties may facilitate purification of fusion polypeptides using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein, thioredoxin, calmodulin binding peptide, 6-His (His), FLAG, c-myc, hemaglutinin (HA), and antibody epitopes such as monoclonal antibody epitopes.

In yet a further aspect, the present invention relates to a polynucleotide which may comprise a nucleotide sequence encoding a fusion protein. The fusion protein may comprise a fusion partner (e.g., HA, Fc, etc.) fused to the polypeptide (e.g., complete light chain, complete heavy chain, variable regions, CDRs etc.) described herein.

Those of skill in the art will also readily recognize that the nucleic acid and polypeptide sequences may be synthesized, in whole or in part, using chemical or enzymatic methods well known in the art. For example, peptide synthesis may be performed using various solid-phase techniques and machines such as the ABI 431A Peptide synthesizer (PE Biosystems) may be used to automate synthesis. If desired, the amino acid sequence may be altered during synthesis and/or combined with sequences from other proteins to produce a variant protein.

Antibody conjugates

The antibody or antigen binding fragment of the present invention may be conjugated with a detectable moiety (i.e., for detection or diagnostic purposes) or with a therapeutic moiety (for therapeutic purposes)

A "detectable moiety" is a moiety detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical and/or other physical means. A detectable moiety may be coupled either directly and/or indirectly (for example via a linkage, such as, without limitation, a DOTA or NHS linkage) to antibodies and antigen binding fragments thereof of the present invention using methods well known in the art. A wide variety of detectable moieties may be used, with the choice depending on the sensitivity required, ease of conjugation, stability requirements and available instrumentation. A suitable detectable moiety include, but is not limited to, a fluorescent label, a radioactive label (for example, without limitation, ¹²⁵ I, In ¹¹¹ , Tc", I ¹³¹ and including positron emitting isotopes for PET scanner etc), a nuclear magnetic resonance active label, a luminiscent label, a chemiluminescent label, a chromophore label, an enzyme label (for example and without limitation horseradish peroxidase, alkaline phosphatase, etc.), quantum dots and/or a nanoparticle. Detectable moiety may cause and/or produce a detectable signal thereby allowing for a signal from the detectable moiety to be detected. In another exemplary embodiment of the invention, the antibody or antigen binding fragment thereof may be coupled (modified) with a therapeutic moiety (e.g., drug, cytotoxic moiety). In an exemplary embodiment, the anti-KAAG1 antibodies and antigen binding fragments may comprise a chemotherapeutic or cytotoxic agent. For example, the antibody and antigen binding fragments may be conjugated to the chemotherapeutic or cytotoxic agent. Such chemotherapeutic or cytotoxic agents include, but are not limited to, Yttrium- 90, Scandium-47, Rhenium-186, lodine-131 , lodine-125, and many others recognized by those skilled in the art (e.g., lutetium (e.g., Lu ¹⁷⁷ ), bismuth (e.g., Bi ²¹³ ), copper (e.g., Cu ⁶⁷ )). In other instances, the chemotherapeutic or cytotoxic agent may be comprised of, among others known to those skilled in the art, 5-fluorouracil, adriamycin, irinotecan, taxanes, pseudomonas endotoxin, ricin and other toxins.

Alternatively, in order to carry out the methods of the present invention and as known in the art, the antibody or antigen binding fragment of the present invention (conjugated or not) may be used in combination with a second molecule (e.g., a secondary antibody, etc.) which is able to specifically bind to the antibody or antigen binding fragment of the present invention and which may carry a desirable detectable, diagnostic or therapeutic moiety.

Pharmaceutical compositions of the antibodies and their use

Pharmaceutical compositions of the anti-KAAG1 antibodies (conjugated or not) are also encompassed by the present invention. The pharmaceutical composition may comprise an anti-KAAG1 antibody or an antigen binding fragment and may also contain a pharmaceutically acceptable carrier.

Other aspects of the invention relate to a composition which may comprise the antibody or antigen binding fragment described herein and a carrier.

The present invention also relates to a pharmaceutical composition which may comprise the antibody or antigen binding fragment described herein and a pharmaceutically acceptable carrier.

Yet other aspects of the invention relate to the use of the isolated antibody or antigen binding fragment described herein in the treatment or diagnosis of ovarian cancer. In addition to the active ingredients, a pharmaceutical composition may contain pharmaceutically acceptable carriers comprising water, PBS, salt solutions, gelatins, oils, alcohols, and other excipients and auxiliaries that facilitate processing of the active compounds into preparations that may be used pharmaceutically. In other instances, such preparations may be sterilized.

As used herein, "pharmaceutical composition" means therapeutically effective amounts of the agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers. A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris- HCI., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts). Solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, rectal routes. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermal^, intramuscularly, intravenously, intradermal^, subcutaneously, intraperitonealy, intraventricular^, intracranial^ and intratumorally.

Further, as used herein "pharmaceutically acceptable carrier" or "pharmaceutical carrier" are known in the art and include, but are not limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8 % saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

For any compound, the therapeutically effective dose may be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans. These techniques are well known to one skilled in the art and a therapeutically effective dose refers to that amount of active ingredient that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating and contrasting the ED ₅₀ (the dose therapeutically effective in 50% of the population) and LD ₅₀ (the dose lethal to 50% of the population) statistics. Any of the therapeutic compositions described above may be applied to any subject in need of such therapy, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and humans.

The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

The term "treatment" for purposes of this disclosure refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. The anti-KAAG1 antibodies and antigen binding fragments therein may have therapeutic uses in the treatment of various cancer types, such as ovarian cancer, renal cancer, colon cancer, lung cancer, melanoma, etc. In an exemplary embodiment, the antibodies and fragments have therapeutic uses in ovarian cancer. In certain instances, the anti- KAAG 1 antibodies and fragments may interact with cancer cells that express KAAG 1 and induce an immunological reaction by mediating ADCC. In other instances, the anti- KAAG1 antibodies and fragments may block the interaction of KAAG1 with its protein partners.

The anti-KAAG1 antibodies and antigen binding fragments therein may have therapeutic uses in the treatment of various types of ovarian cancer. Several different cell types may give rise to different ovarian cancer histotypes. The most common form of ovarian cancer is comprised of tumors that originate in the epithelial cell layer of the ovary or the fallopian tube. Such epithelial ovarian cancers include serous tumors, endometroid tumors, mucinous tumors, clear cell tumors, and borderline tumors. In other embodiments, the anti-KAAG1 antibodies and antigen binding fragments therein have uses in the treatment of other types of ovarian cancer such as germ line and sex cord ovarian cancer.

In certain instances, the anti-KAAG1 antibodies and antigen binding fragments therein may be administered concurrently in combination with other treatments given for the same condition. As such, the antibodies may be administered with anti-mitotics (eg., taxanes), platinum-based agents (eg., cisplatin), DNA damaging agents (eg.

Doxorubicin) and other anti-cancer therapies that are known to those skilled in the art. In other instances, the anti-KAAG1 antibodies and antigen binding fragments therein may be administered with other therapeutic antibodies. These include, but are not limited to, antibodies that target EGFR, CD-20, and Her2.

The present invention relates in a further aspect thereof to a method for inhibiting the growth of a KAAG 1 -expressing cell, the method which may comprise contacting the cell with an effective amount of the antibody or antigen binding fragment described herein.

The present invention also encompasses method of treating cancer or inhibiting the growth of a KAAG1 expressing cells in a mammal, the method may comprise administering the antibody or antigen binding fragment described herein to a mammal in need.

In further aspects, the present invention provides method of treatment, diagnostic methods and method of detection using the antibody or antigen binding fragment of the present invention and the use of these antibodies or antigen binding fragment in the manufacture of a pharmaceutical composition or drug for such purposes.

Method of treatment encompassed by the present invention includes administering an antibody or antigen binding fragment described herein to a mammal in need, and especially to a patient having or susceptible of having a cancer.

The invention also provides in further aspects, methods for reducing tumor spread, tumor invasion, tumor formation or for inducing tumor lysis, which may comprise administering an isolated antibody or antigen binding fragment to a mammal in need.

The invention therefore relates to the use of the isolated antibody described herein in the (manufacture of a pharmaceutical composition for) treatment of cancer, reduction of tumor spread, tumor invasion, tumor formation or for inducing tumor lysis of KAAG1- expressing tumor cells.

The antibody or antigen binding fragment may more particularly be applicable for malignant tumor including, for example, a malignant tumor having the ability to metastasize and/or tumor cells characterized by anchorage-independent growth. The antibody or antigen binding fragment of the present invention may also be used in the diagnosis of cancer. The diagnosis of cancer may be performed in vivo by administering the antibody or antigen binding fragment of the present invention to a mammal having or suspected of having a cancer. The diagnosis may also be performed ex vivo by contacting a sample obtained from the mammal with the antibody or antigen binding fragment and determining the presence or absence of cells (tumor cells) expressing KAAG 1.

The present invention also encompasses method of detecting cancer or detecting a KAAG1 expressing cells in a mammal, the method may comprise administering the antibody or antigen binding fragment described herein to a mammal in need. The present invention relates in another aspect thereof to a method for detecting a KAAG 1 -expressing cell, the method may comprise contacting the cell with an antibody or antigen binding fragment described herein and detecting a complex formed by the antibody and the KAAG 1 -expressing cell. Exemplary embodiments of antibodies or antigen binding fragments used in detection methods are those which are capable of binding to the extracellular region of KAAG1.

Other exemplary embodiments of antibodies or antigen binding fragments used in detection methods are those which bind to KAAG 1 expressed at the surface of a tumor cells.

Patients which would benefit from treatment, detection or diagnostic methods described herein are those which have or are suspected of having ovarian cancer (e.g., serous, endometroid, clear cell or mucinous), skin cancer (e.g., melanomas, squamous cell carcinomas), renal cancer (e.g., papillary cell carcinomas, clear cell carcinomas), colorectal cancer (e.g., colorectal carcinomas), sarcoma, leukemia, brain tumor, thyroid tumor, breast cancer (e.g., mammary carcinomas), prostate cancer (e.g., prostatic carcinomas), oesophageal tumor, bladder tumor, lung tumor (e.g., lung carcinomas) or head and neck tumor and especially when the cancer is characterized as being malignant and/or when the KAAG1 -expressing cells are characterized by anchorage- independent growth.

Especially encompassed by the present invention are patients having or susceptible of having ovarian cancer (e.g., serous, endometroid, clear cell or mucinous), skin cancer (e.g., melanomas, squamous cell carcinomas) or renal cancer (e.g., papillary cell carcinomas) and especially when the cancer is characterized as being malignant and/or when the KAAG 1 -expressing cells are characterized by anchorage-independent growth.

Another aspect of the invention relates a method for detecting KAAG1 (SEQ ID NO.:2), a KAAG 1 variant having at least 80% sequence identity with SEQ ID NO.:2 or a secreted form of circulating form of KAAG1 or KAAG1 variant, the method may comprise contacting a cell expressing KAAG1 or the KAAG1 variant or a sample (biopsy, serum, plasma, urine etc.) comprising or suspected of comprising KAAG1 or the KAAG1 variant with the antibody or antigen binding fragments described herein and measuring binding. The sample may originate from a mammal (e.g., a human) which may have cancer (e.g., ovarian cancer) or may be suspected of having cancer (e g , ovarian cancer) The sample may be a tissue sample obtained from the mammal or a cell culture supernatant

In accordance with the invention the sample may be a serum sample, a plasma sample, a blood sample or ascitic fluid obtained from the mammal The antibody or antigen binding fragment described herein may advantageously detect a secreted or circulating form (circulating in blood) of KAAG 1

The method may comprise quantifying the complex formed by the antibody or antigen binding fragment bound to KAAG1 or to the KAAG1 variant

The binding of an antibody to an antigen will cause an increase in the expected molecular weight of the antigen A physical change therefore occurs upon specific binding of the antibody or antigen binding fragment and the antigen

Such changes may be detected using, for example, electrophoresis followed by Western blot and coloration of the gel or blot, mass spectrometry, HPLC coupled with a computer or else Apparatus capable of computing a shift in molecular weight are known in the art and include for example, Phosphoπmager™

When the antibody comprises for example a detectable label, the antigen-antibody complex may be detected by the fluorescence emitted by the label, radiation emission of the label, enzymatic activity of a label provided with its substrate or else

Detection and/or measurement of binding between an antibody or antigen binding fragment and an antigen may be performed by various methods known in the art Binding between an antibody or antigen binding fragment and an antigen may be monitored with an apparatus capable of detecting the signal emitted by the detectable label (radiation emission, fluorescence, color change etc ) Such apparatus provides data which indicates that binding as occurred and may also provide indication as to the amount of antibody bound to the antigen The apparatus (usually coupled with a computer) may also be capable of calculating the difference between a background signal (e g , signal obtained in the absence of antigen-antibody binding) or background noise and the signal obtained upon specific antibody-antigen binding Such apparatuses may thus provide the user with indications and conclusions as to whether the antigen has been detected or not Additional aspects of the invention relates to kits which may include one or more container containing one or more antibodies or antigen binding fragments described herein.

Nucleic acids, vectors and cells

Antibodies are usually made in cells allowing expression of the light chain and heavy chain expressed from a vector(s) comprising a nucleic acid sequence encoding the light chain and heavy chain.

The present therefore encompasses nucleic acids capable of encoding any of the CDRs, light chain variable domains, heavy chain variable domains, light chains, heavy chains described herein.

The present invention therefore relates in a further aspect to a nucleic acid encoding a light chain variable domain and/or a heavy chain variable domain of an antibody which is capable of specific binding to KAAG 1.

In accordance with an embodiment of the invention, the nucleic acid may especially encode a light chain variable domain and/or heavy chain variable domain of an antibody which may be capable of inducing killing (elimination, destruction, lysis) of KAAG1- expressing tumor cells.

In accordance with another embodiment of the invention, the nucleic acid may especially encode a light chain variable domain and/or heavy chain variable domain of an antibody which may be capable of reducing spreading of KAAG1- expressing tumor cells.

In accordance with yet another embodiment of the invention, the nucleic acid may particularly encode a light chain variable domain and/or heavy chain variable domain of an antibody which may be capable of decreasing or impairing formation of KAAG1- expressing tumors.

Exemplary embodiments of nucleic acids of the present invention include nucleic acids encoding a light chain variable domain comprising: a. a CDRL1 sequence selected from the group consisting of SEQ ID NO.74 and SEQ ID NO.:75; b. a CDRL2 sequence selected from the group consisting of SEQ ID NO.:76,

SEQ ID NO.: 77 and SEQ ID NO.:78, or; c a CDRL3 sequence selected from the group consisting of SEQ ID NO 79, SEQ ID NO 80 and SEQ ID NO 81

In accordance with the present invention, the nucleic acid may encode a light chain variable domain which may comprise at least two CDRs of a CDRL1 , a CDRL2 or a CDRL3

Also in accordance with the present invention, the nucleic acid may encode a light chain variable domain which may comprise one CDRL1 , one CDRL2 and one CDRL3

The present invention also relates to a nucleic acid encoding a heavy chain variable domain comprising a a CDRH1 sequence comprising SEQ ID NO 82, b a CDRH2 sequence selected from the group consisting of SEQ ID NO 83,

SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86 and SEQ ID NO 87, or, c a CDRH3 sequence selected from the group consisting of SEQ ID NO 88, SEQ ID NO 89 and SEQ ID NO 90

In accordance with the present invention, the nucleic acid may encode a heavy chain variable domain which may comprise at least two CDRs of a CDRH1 , a CDRH2 or a CDRH3

In accordance with the present invention, the nucleic acid may encode a heavy chain variable domain which may comprise one CDRH1 , one CDRH2 and one CDRH3

Also encompassed by the present invention are nucleic acids encoding antibody variants having at least one conservative ammo acid substitution

In accordance with the present invention, the nucleic acid may encode a CDR comprising at least one conservative amino acid substitution

In accordance with the present invention, the nucleic acid may encode a CDR comprising at least one conservative amino acid substitution in at least two of the CDRs

In accordance with the present invention, the nucleic acid may encode a CDR comprising at least one conservative amino acid substitution in the 3 CDRs In accordance with the present invention, the nucleic acid may encode a CDR comprising at least two conservative amino acid substitutions in at least one of the CDRs

In accordance with the present invention, the nucleic acid may encode a CDR comprising at least two conservative ammo acid substitutions in at least two of the CDRs

In accordance with the present invention, the nucleic acid may encode a CDR comprising at least two conservative amino acid substitutions in the 3 CDRs

Other aspects of the invention relate to a nucleic acid encoding a light chain variable domain having at least 70% 75%, 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO 16, SEQ ID NO 20, SEQ ID NO 24, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ IDNO 111, SEQ IDNO 112, SEQ IDNO 113, SEQ IDNO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127 SEQ ID NO 128, SEQ ID NO 129, SEQ ID NO 130 and SEQ ID NO 131

Yet other aspects of the invention relate to a nucleic acid encoding a heavy chain variable domain having at least 70% 75%, 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO 18, SEQ ID NO 22, SEQ ID NO 26, SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139, SEQ ID NO 140, SEQ ID NO 141, SEQ IDNO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150, SEQ ID NO 151, SEQ ID NO 152, SEQ ID NO 153, SEQ ID NO 154, SEQ ID NO 155, SEQ ID NO 156, SEQ ID NO 157 Other aspects of the invention relates to the use of a nucleic acid selected from the group consisting of SEQ ID NO 1, a fragment of 10 to 884 nucleotides of SEQ ID NO 1 and a complement of any of the preceding for impairing migration or survival of tumor cells expressing KAAG1 Exemplary embodiments of such nucleic acid comprise siRNAs, antisense, ribozymes and the like

In yet another aspect, the present invention relates to a vector comprising the nucleic acids described herein In accordance with the present invention, the vector may be an expression vector

Vector that contains the elements for transcriptional and translational control of the inserted coding sequence in a particular host are known in the art These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' un-translated regions Methods that are well known to those skilled in the art may be used to construct such expression vectors These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination

In another aspect the present invention relates to an isolated cell which may comprise the nucleic acid described herein

The isolated cell may comprise a nucleic acid encoding a light chain variable domain and a nucleic acid encoding a heavy chain variable domain either on separate vectors or on the same vector The isolated cell may also comprise a nucleic acid encoding a light chain and a nucleic acid encoding a heavy chain either on separate vectors or on the same vector

In accordance with the present invention, the cell may be capable of expressing, assembling and/or secreting an antibody or antigen binding fragment thereof

In another aspect, the present invention provides a cell which may comprise and/or may express the antibody described herein

In accordance with the invention, the cell may comprise a nucleic acid encoding a light chain variable domain and a nucleic acid encoding a heavy chain variable domain

The cell may be capable of expressing, assembling and/or secreting an antibody or antigen binding fragment thereof

The examples below are presented to further outline details of the present invention

EXAMPLES Example 1

This example describes the pattern of expression of the KAAG1 gene in ovarian tumors and ovarian cancer cell line PCR analysis was performed to verify the percentage of ovarian tumors that express the mRNA encoding KAAG1 (indicated as AB-0447 in the Figure) The results showed that the KAAG1 gene is expressed in greater than 85% of ovarian tumors from all stages of the disease and 100% of late stage tumors The expression of KAAG1 is lower or undetectable in LMP samples (see Fig 1A) For each sample, 1 μg of amplified RNA was reverse transcribed with random hexamers using Thermoscript RT (Invitrogen) The cDNA was diluted and 1 /200th of the reaction was used as template for each PCR reaction with gene-specific primers as indicated The primers used to amplify the KAAG1 mRNA contained the sequences shown in SEQ ID NOS 45 and 46 PCR reactions were carried out in 96-well plates and half of the 25 μl reaction was electrophoresed on a 1 % agarose gel The gels were visualized and photographed with a gel documentation system (BioRad) The upper panel of Fig 1A shows the results from 6 LMP samples (LMP) and 22 ovarian tumor and 6 ovarian cell line (last 6 lanes on the right, OVCa) samples The lower panel of Fig 1 shows the RNA samples from 30 normal tissues that were tested as indicated

KAAG1 expression was weakly detected in a few normal tissues whereas the mRNA was evident in the fallopian tube and the pancreas (see Fig 1A) The amount of total RNA used in these reactions was controlled with parallel PCR amplifications of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, and the results showed that equivalent starting material was present in each sample (see Fig 1A) The primers used to amplify the GAPDH gene contained the sequences shown in SEQ ID NOs 47 and 48 Thus, the expression of the KAAG1 gene fulfills an important selection criteria it is over-expressed in a large proportion of ovarian tumors and its expression is low or absent in most normal tissues These data suggest that ovarian tumors may be specifically targeted with high affinity monoclonal antibodies against KAAG 1

Early stage cancer or tumors tend to be made up of cells that are in a high state of differentiation but as the tumor progresses to a more aggressive and invasive state, the cancer cells become increasingly undifferentiated There are needs to identify factors that contribute to this transition and exploit these proteins as targets for the development of therapeutics Several ovarian cancer cell lines are available that were derived from primary tumors and serve as excellent models for the functional studies The expression of KAAG1 was examined in these cell lines Four lines termed TOV-21G, TOV-112D, TOV-1946, and TOV-2223G were established from primary tumors whereas OV-90 and OV-1946 are cell lines derived from cells contained in ascites fluid of patients with advanced ovarian cancer. Total RNA from cells established from primary tumors (see in Fig. 1 B, lanes 1 , TOV-21G; 2, TOV-1 12D; 5, TOV-1946; 6, TOV-2223G) and cells established from ascitic cells (lanes 3, OV-90; 4, OV-1946) was converted to cDNA with reverse transcriptase and used as template in PCR reactions with KAAG 1 -specific primers( SEQ ID NOS:45 and 46). As a negative control, the reaction was carried out with total RNA from normal ovary. Equal amounts of starting material were utilized as evidenced by parallel PCR reactions with GAPDH (SEQ ID NOS:47 and 48). A sample of the PCR reaction was electrophoresed on an agarose gel and visualized with ethidium bromide. As shown in Fig. 1 B, KAAG1 was detectable but weakly expressed in the cell lines from the primary tumors and PCR reactions performed at a higher number of cycles revealed the KAAG 1 transcript in all four of these cell lines. Conversely, both cell lines established from the ascitic fluid cells exhibited high level of the KAAG1 transcript. The increased expression in cells from the ascitic fluid suggests that the environment of the cells influences the regulation of the KAAG1 gene.

Ascitic cells are associated with advanced disease and the pattern of expression disclosed in Fig. 1 B implies that increased KAAG1 levels are associated with anchorage- independent growth. This question was addressed by culturing the cells in hanging droplets, a condition that prevents the cells from adhering to the petri dish, as is the case when they are grown as monolayers. These so called three-dimensional cultures allow the cells to associate and the formation of spheroids is observed (see Fig. 1C). Spheroids were cultures as follows: TOV-1 12D, OV-90, or TOV-21G cells (4 000 in 15 μl) were incubated for 4 days in medium in the absence (left panels, Fig. 1C) or presence of 5% FBS {right panels, Fig. 1 C, +5% serum). The magnification of the image was set to 100x. These spheroids have been extensively characterized and exhibit many of the properties found in primary tumors including morphological and functional properties as well as the molecular signature as measured by microarray-based expression profiling.

Total RNA was isolated from spheroid preparations and RT-PCR was performed as described for Fig. 1A. TOV-21G, TOV-1 12D, OV-90 cells were seeded as described in the legend for Fig. 1C under conditions to produce spheroids. After 4 days, total RNA was isolated and used to perform RT-PCR reactions with KAAG 1 -specific primers (SEQ ID NOS:45 and 46). PCR reactions were electrophoresed on agarose gels. Conducting parallel reactions to amplify GAPDH (SEQ ID NOS:47 and 48) demonstrated that equal amounts of starting material were present in each sample. The following acronyms are used in Fig. 1 D: Ce., cells grown as monolayers; Sph., cells grown as spheroids. Strikingly, KAAG1 expression was up-regulated when TOV-21G and TOV-112D were grown as spheroids (see Fig. 1 D). In the case of the OV-90 cells, the level of expression of the KAAG1 gene was unchanged and remained very high. Presumably, the level of expression attained in the cell lines derived from the ascitic fluid, as exemplified by the OV-90 cells and the OV-1946 cells (see Fig. 1A) has reached a maximum.

These results correlated with the previous data showing high expression in cell lines derived from ascitic fluid and confirm that expression of KAAG 1 is influenced by the microenvironment of the cancer cells. Additionally, the up-regulation of KAAG1 transcription that was observed in spheroids implies that high levels of KAAG1 are present in malignant ovarian cancer.

Example 2

This example describes in vitro results that suggest a critical role for KAAG1 in the survival of ovarian cancer cells.

With the demonstration that KAAG 1 expression is regulated in ovarian cancer cells, the function of this gene in these cells was examined. To address this question, in vitro assays were conducted to determine if this protein plays a role in cancer cell proliferation, migration, and/or survival. RNAi was used to knock down the expression of the endogenous KAAG1 gene in the TOV-21G ovarian cancer cell line. The design of two separate short-hairpin RNA (shRNA) sequences was performed using web-based software that is freely available to those skilled in the art (Qiagen for example). These chosen sequences, usually 19-mers, were included in two complementary oligonucleotides that form the template for the shRNAs, i.e. the 19-nt sense sequence, a 9-nt linker region (loop), the 19-nt antisense sequence followed by a 5-6 poly-T tract for termination of the RNA polymerase III. The sequences of the 19-mers that were used to knock down the expression of KAAG1 are shown in SEQ ID NOS:49 and 50. Appropriate restriction sites were inserted at the ends of these oligonucleotides to facilitate proper positioning of the inserts so that the transcriptional start point is at a precise location downstream of the hU6 promoter. The plasmid utilized in all RNA interference studies, pSilencer 2.0 (SEQ ID NO.:51), was purchase from a commercial supplier (Ambion, Austin, TX). Two different shRNA expression vectors were constructed to increase the chance of observing RNAi effects and the specificity of phenotypic observations. TOV- 21 G cells were seeded in 6-well plates and transfected 24h later with 1 μg of pSil-shRNA vector. Sh.1 and sh.2 were used to designate 2 different shRNA sequences targeting the KAAG 1 gene. Stable transfectants were selected for 5 - 7 days, expanded, and grown to confluence. All of the following in vitro cell-based assays were performed using these stably transfected cell lines that contain shRNAs specific for KAAG1.

The migration or mobility of the cells was measured in a standard cell motility assay. This scratch assay, as it is called, measures the speed at which cells fill a denuded area in a confluent monolayer. As illustrated in Fig. 2A, TOV-21G cells containing the scrambled shRNA filled up the wound almost completely after 24h compared to the control untreated cells (compare middle-left panel with left panel). By contrast, the ability of TOV-

21 G cells expressing KAAG1 shRNAs to fill the denuded area was greatly reduced. In fact, the number of cells that filled the denuded area in the presence of the KAAG 1 shRNA cells more closely resembled the number of cells at time Oh (compare the left panel with the right panels).

To examine the longer-term effects of reduced expression of KAAG1 in ovarian cancer cells, the cells were extensively diluted and cultured for 10 days in a colony survival assay. TOV-21G cells were seeded in 12-well plates at a density of 50 000 cells/well and transfected 24h later with 1 μg of pSil-shRNA vector. Sh-1 and sh-2 are used to designate 2 different shRNA sequences targeting the same gene. The next day, fresh medium was applied containing 2 μg/ml puromycin and the selection of the cells was carried out for 3 days. The cells were washed and fresh medium without puromycin was added and growth continued for another 5 days. To visualize the remaining colonies, the cells were washed in PBS and fixed and stained simultaneously in 1 % crystal violet/10% ethanol in PBS for 15 minutes at room temperature. Following extensive washing in PBS, the dried plates were scanned for photographic analysis. A significant decrease in the survival of the cancer cell line was observed and a representative experiment is displayed in Fig. 2B. Identical results were obtained when the shRNAs were transfected into another ovarian cancer cell line, TOV-112D.

Thus, taken together, the regulated expression of KAAG 1 in detached cells coupled with the requirement of this gene in the migration and the survival of ovarian cancer cells supports an important role for KAAG 1 in ovarian cancer cells. Furthermore, these experiments suggest that an antagonist of KAAG1 protein, such as a monoclonal antibody, would result in reduced invasiveness and decreased tumor survival.

Example 3

This example provides details pertaining to the family of monoclonal antibodies that bind to KAAG 1.

The antibodies that bind KAAG1 were generated using the Biosite phage display technology. A detailed description of the technology and the methods for generating these antibodies can be found in the U.S. Patent No. 6,057,098. Briefly, the technology utilizes stringent panning of phage libraries that display the antigen binding fragments (Fabs). After a several rounds of panning, a library, termed the Omniclonal, was obtained that was enriched for recombinant Fabs containing light and heavy chain variable regions that bound to KAAG1 with very high affinity and specificity. From this library, more precisely designated Omniclonal AL0003Z1 , 96 individual recombinant monoclonal Fabs were prepared from E. coli and tested for KAAG1 binding.

To measure the relative binding of each individual monoclonal antibody, recombinant human KAAG1 was produced in 293E cells using the large-scale transient transfection technology (Durocher et al., 2002; Durocher, 2004). The entire coding region of the KAAG1 cDNA was amplified by PCR using a forward primer that incorporated a BamHI restriction site (SEQ ID NO.:52) and a reverse primer that incorporated a Hindlll restriction site (SEQ ID NO.:53). The resulting PCR product measured 276 base pairs and following digestion with BamHI and Hindlll, the fragment was ligated into the expression vector pYD5 (SEQ ID NO.:54) that was similarly digested with the same restriction enzymes. The pYD5 expression plasmid contains the coding sequence for the human Fc domain that allows fusion proteins to be generated as well as the sequence encoding the IgGI signal peptide to allow the secretion of the fusion protein into the culture medium. For each milliliter of cells, one microgram of the expression vector, called pYD5-0447, was transfected in 293E cells grown in suspension to a density of 1.5 - 2.0 million cells/ml. The transfection reagent used was polyethylenimine (PEI), (linear, MW 25,000, Cat# 23966 Polysciences, Inc., Warrington, PA) which was included at a DNA:PEI ratio of 1 :3. Growth of the cells was continued for 5 days after which the culture medium was harvested for purification of the recombinant Fc-KAAGI fusion protein. The protein was purified using Protein-A agarose as instructed by the manufacturer (Sigma- Aldrich Canada Ltd., Oakville, ON). A representative polyacrylamide gel showing a sample of the purified Fc-KAAGI (indicated as Fc-0447) is shown in Fig. 3A.

The 96-well master plate of monoclonal preparations contained different concentrations of purified anti-KAAG1 Fabs in each well. A second stock master plate was prepared by diluting the Fabs to a final concentration of 10 μg/ml from which all subsequent dilutions were performed for ELISA measurements. To carry out the binding of Fc-KAAGI to the monoclonal preparations, the Fc-KAAGI was biotinylated with NHS-biotin (Pierce, Rockford, IL) and 10 ng/well was coated in a streptavidin 96-well plate. One nanogram of each Fab monoclonal preparation was added to each well and incubated at room temperature for 30 minutes. Bound antibody was detected with HRP-conjugated mouse anti-kappa light chain antibody in the presence of TMB liquid substrate (Sigma-Aldrich Canada Ltd., Oakville, ON) and readings were conducted at 450 nm in microtiter plate reader. As shown in Fig. 3B, a total of 48 (highlighted in grey) monoclonal antibodies displayed significant binding in this assay (>0.1 arbitrary OD ₄₅₀ units). The antibodies were purposely diluted to 1 ng/well to accentuate the binding of those antibodies with the most affinity for KAAG1. As a control, the antibodies did not bind to biotinylated Fc domain. These data also revealed that the binding of the antibodies varied from well to well indicating that they exhibited different affinities for KAAG 1.

Example 4

This example describes the epitope mapping studies to determine which region of KAAG 1 the antibodies bind to.

To further delineate the regions of KAAG1 that are bound by the monoclonal antibodies, truncated mutants of KAAG1 were expressed and used in the ELISA. As for the full length KAAG1 , the truncated versions were amplified by PCR and ligated into

BamHI/Hindlll digested pYD5. The primers that were used combined the forward oligonucleotide with the sequence shown in SEQ ID NO.:52 with primers of SEQ ID

NOS:55 and 56, to produce Fc-fused fragments that ended at amino acid number 60 and 35 of KAAG1 , respectively. The expression of these mutants was conducted as was described above for the full length Fc-KAAGI and purified with Protein-A agarose. A representative gel of the protein preparations that were used in the ELISA is shown in Fig. 4A and a schematic of the mutant proteins used for epitope mapping is depicted in Fig. 4B.

The results showed that the library was comprised of antibodies that could bind to each of the delineated KAAG1 regions. In particular, of the 48 mAbs that bound to KAAG1 in the first ELISA, nine (wells A2, A12, C2, C4, D1 , E10, F1 , H3, and H8) were found to interact with the first 35 amino acids of KAAG1 whereas five (D12, E8, F5, G10, and H5) were found to interact with the last 25 amino acids of KAAG1. Thus, the remaining 34 antibodies interacted with a region of KAAG 1 spanned by amino acids 36 - 59. These results were in agreement with the sequence analysis of 24 representative light and heavy chain variable regions. Indeed, alignment of these sequences revealed that the antibodies clustered into three groups based on the percentage identity in their respective CDRs. Antibodies contained in each cluster all interacted with the same region of KAAG 1.

Therefore, based on the relative binding affinity of the mAb, differential epitope interaction characteristics, and the differences in variable domain sequences, three antibodies from the plate described in Example 3 were selected for further analysis as exemplary anti-KAAG1 monoclonal antibodies.

Example 5 This example discloses the methods used to convert the Fabs into full IgGI chimeric monoclonal antibodies. A scheme of the methodology is presented in Fig. 5.

Aside from the possibility of conducting interaction studies between the Fab monoclonals and the KAAG1 protein, the use of Fabs is limited with respect to conducting meaningful in vitro and in vivo studies to validate the biological function of the antigen. Thus, it was necessary to transfer the light and heavy chain variable regions contained in the Fabs to full antibody scaffolds, to generate mouse-human chimeric IgGIs. The expression vectors for both the light and heavy immunoglobulin chains were constructed such that i) the original bacterial signal peptide sequences upstream of the Fab expression vectors were replaced by mammalian signal peptides and ii) the light and heavy chain constant regions in the mouse antibodies were replaced with human constant regions. The methods to accomplish this transfer utilized standard molecular biology techniques that are familiar to those skilled in the art. A brief overview of the methodology is described here (see Fig. 5).

Light chain expression vector - an existing mammalian expression plasmid, called pTTVHδG (Durocher et al., 2002), designed to be used in the 293E transient transfection system was modified to accommodate the mouse light chain variable region. The resulting mouse-human chimeric light chain contained a mouse variable region followed by the human kappa constant domain. The cDNA sequence encoding the human kappa constant domain was amplified by PCR with primers OGS1773 and OGS1774 (SEQ ID NOS:57 and 58, respectively). The nucleotide sequence and the corresponding amino acid sequence for the human kappa constant region are shown in SEQ ID NOS:59 and 60, respectively. The resulting 321 base pair PCR product was ligated into pTTVHδG immediately downstream of the signal peptide sequence of human VEGF A (NMJD03376). This cloning step also positioned unique restriction endonuclease sites that permitted the precise positioning of the cDNAs encoding the mouse light chain variable regions. The sequence of the final expression plasmid, called pTTVKI , is shown in SEQ ID NO.:61. Based on the sequences disclosed in Table 3, PCR primers specific for the light chain variable regions of antibodies 3D3, 3G10, and 3C4 (SEQ ID NOS:15, 19, and 23, respectively) were designed that incorporated, at their 5'-end, a sequence identical to the last 20 base pairs of the VEGF A signal peptide. The sequences of these primers are shown in SEQ ID NOS:62, 63, and 64. The same reverse primer was used to amplify all three light chain variable regions since the extreme 3'-ends were identical. This primer (SEQ ID NO.:65) incorporated, at its 3'-end, a sequence identical to the first 20 base pairs of the human kappa constant domain. Both the PCR fragments and the digested pTTVKI were treated with the 3' - 5' exonuclease activity of T4 DNA polymerase resulting in complimentary ends that were joined by annealing. The annealing reactions were transformed into competent E. coli and the expression plasmids were verified by sequencing to ensure that the mouse light chain variable regions were properly inserted into the pTTVKI expression vector. Those skilled in the art will readily recognize that the method used for construction of the light chain expression plasmids applies to all anti-KAAG1 antibodies contained in the original Fab library.

Heavy chain expression vector - the expression vector that produced the heavy chain immunoglobulins was designed in a similar manner to the pTTVKI described above for production of the light chain immunoglobulins Plasmid pYD11 (Durocher et al , 2002), which contains the human IgGK signal peptide sequence as well as the CH2 and CH3 regions of the human Fc domain of IgGI , was modified by ligating the cDNA sequence encoding the human constant CH1 region PCR primers OGS1769 and OGS1770 (SEQ ID NOS 66 and 67), designed to contain unique restriction endonuclease sites, were used to amplify the human IgGI CH1 region containing the nucleotide sequence and corresponding amino acid sequence shown in SEQ ID NOS 68 and 69 Following ligation of the 309 base pair fragment of human CH1 immediately downstream of the IgGK signal peptide sequence, the modified plasmid (SEQ ID NO 70) was designated pYD15 When a selected heavy chain variable region is hgated into this vector, the resulting plasmid encodes a full IgGI heavy chain immunoglobulin with human constant regions Based on the sequences disclosed in Table 3, PCR primers specific for the heavy chain variable regions of antibodies 3D3, 3G10, and 3C4 (SEQ ID NOS 17, 21 , and 25, respectively) were designed that incorporated, at their 5'-end, a sequence identical to the last 20 base pairs of the IgGK signal peptide The sequences of these primers are shown in SEQ ID NOS 71 (3D3 and 3G10 have the same 5'-end sequence) and 72 The same reverse primer was used to amplify all three heavy chain variable regions since the extreme 3'- ends were identical This primer (SEQ ID NO 73) incorporated, at its 3'-end, a sequence identical to the first 20 base pairs of the human CH1 constant domain Both the PCR fragments and the digested pYD15 were treated with the 3' - 5' exonuclease activity of T4 DNA polymerase resulting in complimentary ends that were joined by annealing The annealing reactions were transformed into competent E coli and the expression plasmids were verified by sequencing to ensure that the mouse heavy chain variable regions were properly inserted into the pYD15 expression vector Those skilled in the art will readily recognize that the method used for construction of the heavy chain expression plasmids applies to all antι-KAAG1 antibodies contained in the original Fab library

Expression of human IgGIs in 293E cells - The expression vectors prepared above that encoded the light and heavy chain immunoglobulins were expressed in 293E cells using the transient transfection system (Durocher et al , 2002) The methods used for co- transfecting the light and heavy chain expression vectors were described in Example 3 The ratio of light to heavy chain was optimized in order to achieve the most yield of antibody in the tissue culture medium and it was found to be 9 1 (L H) The ability of the chimeric antι-KAAG1 monoclonal antibodies to bind to recombinant Fc-KAAGI was measured in the ELISA and compared with the original mouse Fabs The method was described in Example 3 As depicted in Fig 6, the binding of the 3D3, and 3G10 chimeric IgGI monoclonal antibodies was very similar to the Fabs In the case of the 3C4, the binding activity of the chimeric was slightly less than the Fab Despite this, this result shows that the transposition of the variable domains from the mouse Fabs into a human IgGI backbone did not significantly affect the capacity of the light and heavy chain variable regions to confer KAAG1 binding

Example 6

This example describes the use of antι-KAAG1 antibodies to block the activity of KAAG 1 in ovarian cancer cell models

Example 2 disclosed RNAi studies showing that KAAG1 played an important role in the behavior of ovarian cancer cells The monoclonal antibodies described above were used to determine whether it was possible to reproduce these results by targeting KAAG 1 at the cell surface TOV-21G and OV-90 cells were cultured under conditions to produce spheroids and treated with 10 μg/ml of 3D3, 3G10, or 3C4 antι-KAAG1 chimeric monoclonal antibody As illustrated in Fig 7, both cell lines efficiently formed spheroids when left untreated (parental) or when treated with antibody dilution buffer (control) In contrast, the presence of antι-KAAG1 antibodies resulted in loosely packed structures and in certain cases, the cells were unable to assemble into spheroids These results confirm the earlier observations and suggest that the antι-KAAG1 monoclonal antibodies can modulate the activity of KAAG 1 during the formation of spheroids Since spheroid formation by cancer cell lines is an in vitro model for tumor formation, the results also suggest that blocking KAAG 1 could lead to decreased tumor formation in vivo

Example 7

This example describes the use of antι-KAAG1 antibodies for detecting the expression of KAAG1 in ovarian tumors

As a means of confirming the expression of KAAG1 protein in ovarian cancer tumors and in order determine if expression of the gene correlated with the presence of the protein, immunohistochemistry was conducted Tissue microarrays were obtained that contained dozens of ovarian tumor samples generated from patient biopsies Paraffin-embedded epithelial ovarian tumor samples were placed on glass slides and fixed for 15 mm at 50 ⁰ C Deparaffinization was conducted by treating 2x with xylene followed by dehydration in successive 5 mm washes in 100%, 80%, and 70% ethanol The slides were washed 2x in PBS for 5 mm and treated with antigen retrieval solution (citrate-EDTA) to unmask the antigen Endogenous peroxide reactive species were removed by incubating slides with H ₂ O ₂ in methanol and blocking was performed by incubating the slides with serum-free blocking solution (Dakocytomation) for 20 mm at room temperature The primary mAb (antι-KAAG1 3D3) was added for 1 h at room temperature KAAG 1 -reactive antigen was detected by incubating with biotin-conjugated mouse anti-kappa followed by streptavidin- HRP tertiary antibody Positive staining was revealed by treating the slides with DAB- hydrogen peroxide substrate for less than 5 mm and subsequently counterstained with hematoxylin The KAAG 1 protein was found to be expressed at very high levels in the vast majority of ovarian tumor samples A representative array containing 70 tumors is depicted in Fig 8A As demonstrated by the expression profiling studies that were performed using RT-PCR, KAAG 1 transcripts were present in greater than 85% of ovarian tumor samples analyzed Clearly, there is an excellent correlation between the transcription of the KAAG 1 gene and the presence of the protein in ovarian cancer Some of the samples were inspected at a higher magnification to determine which cells were expressing the KAAG 1 protein As depicted in Fig 8B, KAAG 1 is predominantly expressed in the surface epithelium of ovarian tumors In addition, strong intensity was observed on the apical side of these epithelial cells (see arrows in Fig 8B, magnification 2Ox) Finally, immunohistochemistry was repeated on ovarian tumor samples that originated from different histotypes As explained earlier, epithelial ovarian cancer can be classified into 4 major histotypes serous, endometroid, clear cell, and mucinous The expression of KAAG1 was detected in all types of epithelial ovarian cancer, in particular serous and endometroid histotypes (see Fig 8C)

Taken together, these immunohistochemical studies illustrate the utility of detecting KAAG 1 in ovarian cancer with the monoclonal antibodies

Example 8

IgG ₁ antibodies against KAAG1 can mediate ADCC

Antibody-Dependent Cell Cytotoxicity (ADCC) is a mechanism of cell-mediated immunity whereby effector cells, typically natural killer (NK) cells, of the immune system actively lyse target cells that have been bound by specific antibodies. The interaction between the NK cells and the antibody occurs via the constant Fc domain of the antibody and high-affinity Fcγ receptors on the surface of the NK cells. IgGiS have the highest affinity for the Fc receptors while IgG ₂ mAbs exhibit very poor affinity. For this reason the chimeric antibodies targeting KAAG1 were designed as IgG ₁ S. This type of effector function that is mediated in this manner can often lead to the selective killing of cancer cells that express high level of antigen on their cell surfaces.

An in vitro assay to measure ADCC activity of the anti-KAAG1 IgG ₁ chimeric antibodies was adapted from a previously published method, which measured the ADCC activity of the anti-CD20 rituxan in the presence of a lymphoma cell line called WIL2-S (Idusogie et al., (2000) J. Immunol. 164, 4178-4184). Human peripheral blood mononuclear cells (PBMNCs) were used as a source of NK cells which were activated in the presence of increasing concentration of the 3D3 chimeric IgG ₁ antibody (see Fig. 12). The target cells were incubated with the activated PBMNCs at a ratio of 1 to 25. As shown, cell death increased in a dose-dependent manner both in the presence of OVCAR-3 and the lymphoma cell line, the latter of which was shown to express KAAG1 by RT-PCR (not shown). As a positive control, the results from the published method were reproduced where high level of ADCC was obtained for rituxan in the presence of WIL2-S cells.

ADCC was also observed with other ovarian cancer cell lines that express relatively high levels of KAAG1. These results demonstrate that IgG ₁ antibodies that are specific for KAAG1 , as exemplified by 3D3, can enhance the lysis of cancer cells which express the antigen on their cell surface.

Example 9 Antibodies against KAAG1 can reduce the invasion of ovarian tumors

Patients that develop ovarian cancer have lesions that typically initiate by an uncontrolled growth of the cells in the epithelial layer of the ovary or, in some instances, the fallopian tube. If detected early, these primary tumors are surgically removed and first-line chemotherapy can result in very good response rates and improved overall survival. Unfortunately, 70% of the patients will suffer recurrent disease resulting in the spread of hundreds of micro-metastatic tumors throughout the abdominal cavity. Second-line therapies can be efficacious, but often patients either respond poorly or the tumors develop chemoresistance. Treatment options are limited and there are urgent needs for new therapies to circumvent resistance to cytotoxic drugs.

In order to test the efficacy of anti- KAAG1 antibodies in vivo, an animal model of ovarian cancer was used that is the closest representation of the clinical manifestation of the disease in humans. The TOV-112D cell line is of endometrioid origin and expresses the KAAG1 antigen as measured by RT-PCR. Previous IHC studies showed that ovarian tumors of the endometrioid histotype contain strong expression of KAAG1 thus rendering the 112D cell line an appropriate selection for testing anti- KAAG 1 antibodies.

The intra-peritoneal inoculation of the TOV-112D cell line in SCID mice resulted in the implantation of dozens of micro-metastatic tumors that closely resemble those that are observed in humans. Mice treated with PBS, the diluent for the antibodies, contained upon examination, an average of 25 - 30 tumors per animal (Fig. 13A and B). In some cases, the number of tumors was so high in the abdominal cavity of these mice that the number of tumors could not be easily determine; these mice were excluded from the statistical analysis. When the mice were treated with the 3C4 and 3D3 antibodies, the number of micro-metastatic tumors was drastically reduced. In addition, there was at least one animal per group treated with anti- KAAG1 where no tumors were seen. A second experiment was conducted in mice containing a larger number of TOV-112D tumors (> 50/animal) and very similar results were obtained. Moreover, there was very little difference between the groups treated with the 3C4 compared to the 3D3 antibody. However, the tendency in these in vivo experiments as well as the results obtained in the cell-based assays show that the 3D3 antibody displayed slightly more efficacy. Whether, this is due to a more accessible epitope or a higher affinity of 3D3 compared to 3C4 for the antigen still remains to be established. The results from these two experiments demonstrated that targeting KAAG 1 on the surface of ovarian cancer cells could lead to a significant reduction in the spread of the tumors in vivo.

Furthermore, these findings are in complete agreement with the observations that were made in the cell-based assays. For example, the increased expression of the KAAG1 mRNA in the spheroids compared to cell lines grown as monolayers; the reduction in cell migration in the presence of KAAG1 shRNAs, the reduction in the ability of cell lines to form spheroids when treated with KAAG1 antibodies; and finally, enhancement of ADCC activity by anti- KAAG1 IgG ₁ S. Taken together, the results strongly suggest that targeting KAAG 1 with an antibody has great therapeutic potential in recurrent ovarian cancer.

Example 10 KAAG1 is expressed in skin tumors and renal cell carcinomas and is a therapeutic target in these indications.

The mRNA profiling studies that were conducted showed that the transcript encoding the KAAG1 antigen was highly expressed in cell lines derived from melanoma samples and renal carcinomas. These results were disclosed in Sooknanan et al., 2007. To confirm the transcriptional regulation of the KAAG1 gene in these cancer types, immunohistochemistry was performed with an anti-KAAG1 antibody on human skin tumor tissue microarrays (Pantomics Inc., Richmond, CA) containing several sections isolated from squamous cell carcinomas and melanomas. The analysis of this array showed that there was very strong staining in biopsies isolated from squamous cell carcinomas and melanomas (Figure 14, top panel). Both of these types are among the most common forms of skin cancers and interestingly, the squamous cell carcinomas are the most metastatic, a fact that again links the expression of KAAG 1 to an invasive phenotype. As previously observed, the presence of KAAG1 was very weak or absent on the three normal skin samples that were contained on the array. Similarly, KAAG1 was detected in many of the samples contained in an array of renal cancer. Most of the positive samples were predominantly of the papillary cell carcinoma type and a few clear cell carcinomas expressed KAAG1 protein. Papillary carcinomas represent approximately 20% of renal cancer cases.

In order to test if the function of KAAG1 is the same in these types of cancer compared to its role in ovarian cancer, cell lines derived from melanoma and renal cell carcinomas were obtained and tested in the spheroid culture assay (see Example 1 and 6). For the melanoma model, A375 and SK-MEL5 cells, two malignant melanoma cell lines, were cultured under conditions that allowed them to form spheroids in the presence of 5% FBS. The cultures were incubated with or without the anti-KAAG1 chimeric 3D3 antibody at a concentration of 5 μg/ml. As shown in Figure 15, inclusion of 3D3 antibody in the cultures prevented the proper assembly of spheroid structures in melanoma cell lines. This result suggested that KAAG1 plays a similar role in melanoma as it does in ovarian cancer. Cell lines derived from renal cell carcinoma were also tested. The A-498 cell line is a renal papillary cell carcinoma cell line whereas the 786-0 is a renal clear cell carcinoma. As depicted in Figure 15, only the A-498 spheroids were affected by the presence of the 3D3 anti-KAAG1 antibody while the 786-0 cell line was unaffected in this assay. These results parallel the immunohistochemistry results described above and indicate that the inhibition of spheroids formation is dependent on the presence of KAAG1 on the surface of renal cancer cells derived predominantly from papillary kidney cancers. It is possible however, that the anti-KAAG1 antibody may work in other types of assays for renal clear cell carcinoma.

Taken together, these data are strongly supportive of a critical function in role of KAAG 1 in melanoma and kidney cancer and indicate that blocking KAAG1 with antibodies in these indications has therapeutic potential.

Example 11

KAAG1 is expressed on the surface of ovarian cancer cells. The combined results from the bioinformatics analysis of the primary structure of the cDNA encoding KAAG1 , biochemical studies, and immunohistochemical detection of the protein in epithelial cells suggested that the KAAG1 antigen was located on the cell surface. However, more direct evidence was required to demonstrate that KAAG 1 is indeed a membrane-bound protein. In one approach, ovarian cancer cell lines known to express KAAG1 were plated in micro-titer plates, fixed under conditions that do not permeate the cells, and incubated with increasing concentration of anti- KAAG 1 chimeric antibodies. Following extensive washing of the cells, bound antibody was detected with HRP-conjugated anti-human IgG as a secondary antibody in a modified cell-based ELISA (see Figure 16A). The first observation that can be made from these experiments is that the antibodies could be specifically captured by the cells suggesting that the KAAG 1 was present at the cell surface. Secondly, the amount of binding was strongest on SKOV-3 cells and the TOV- 21 G cells exhibited the weakest binding. This was in complete agreement with RT-PCR data which demonstrated that the KAAG 1 mRNA was expressed in similar proportions in these cell lines (not shown). Additionally, the 3D3 antibody produced the strongest signal implying that the epitope targeted by this antibody was the most accessible in this assay. The 3G10 could only detect KAAG1 in the cell line that expressed the highest level of AB-0447 (SKOV-3 cells, see right panel of Figure 16A). A second approach used was flow cytometry. In this case, a mouse 3D3 anti-KAAG1 antibody was incubated with SKOV-3 ovarian cancer cells at saturating conditions and following extensive washing, the bound 3D3 anti-KAAG1 antibody was detected with anti-mouse IgG conjugated to FITC in a flow cytometer As shown in Fig 16B, the signal at the surface of SKOV-3 cells was much higher compared to same cells labeled with the negative control, an anti- KLH (Keyhole limpet hemocyanin) antibody, specific for a non-mammalian unrelated protein, which was at a fluorescence level the same as the background readings Taken together, these results demonstrate that KAAG 1 is located on the surface of cells

Example 12

Methods for the use of humanized anti-KAAG1 antibodies.

On the basis of both the in vitro and preliminary in vivo results, two mouse antι-KAAG1 antibody candidates, designated 3D3 and 3C4, were selected for humanization using in silico modeling using methods familiar to those in the art In brief, the variable regions of the murine antibodies were modeled in 3D based on available crystal structures of mouse, humanized, and fully human variable regions that displayed high sequence homology and similar CDR loop lengths The CDRs are the amino acid sequences that contribute to antigen binding, there are 3 CDRs on each antibody chain Additionally, the framework regions, the amino acid sequences that intervene between the CDRs, were modified by standard homology comparison between mouse and human antibody sequences resulting in the 'best-fit' human sequence These modifications ensured that the proper positioning of the CDR loops was maintained to ensure maximum antigen binding in the humanized structure as well as preserving the potential N- and O-linked glycosylation sites The sequence of both the heavy and light chain variable regions in the humanized (h) 3D3 and 3G4 resulted in 96% and 94% humanization, respectively The structure of the 3D3 and 3C4 models for each antibody is shown in Figure 17A and 17B, respectively As illustrated in these structures, the 3D3 required the maintenance of 3 unusual amino acids (Figure 17A, Met93 and Gly94 on the heavy chain and Ser57 on the light chain) because of their proximity to the CDRs Modeling predicted that replacement of these mouse amino acids with human equivalents might compromise binding of the antibody with the KAAG1 antigen In the case of 3C4, 6 amino acids were considered unusual (Figure 17B, GIuI , Gln72 and Ser98 on the heavy chain and Thr46, Phe49 and Ser87 on the light chain) In both figures, the light chain CDRs are indicated by L1 , L2, and L3 for CDR1 , CDR2, and CDR3, respectively, whereas the heavy chain CDRs are indicated by H1 , H2, and H3 for CDR1 , CDR2, and CDR3, respectively The sequences that encode the complete antι-KAAG1 3D3 immunoglobulin light and heavy chains are shown in SEQ ID NO 176 and 177, respectively The variable region of the humanized 3D3 light chain is contained between amino acids 21-133 of SEQ ID NO 176 and is shown in SEQ ID NO 178 The variable region of the humanized 3D3 heavy chain is contained between amino acids 20-132 of SEQ ID NO 177 and is shown in SEQ ID NO 179 The sequences that encode the complete antι-KAAG1 3C4 immunoglobulin light and heavy chains are shown in SEQ ID NO 180 and 181 , respectively The variable region of the humanized 3C4 light chain is contained between amino acids 21-127 of SEQ ID NO 180 and is shown in SEQ ID NO 182 The variable region of the humanized 3C4 heavy chain is contained between amino acids 19-136 of SEQ ID NO 181 and is shown in SEQ ID NO 183

Following assembly of expression vectors and production of the h3D3 in transfected mammalian cells (see Example 5), several assays were performed to demonstrate the bio-equivalence of the humanization process Since an antibody harboring effector functions was required, the h3D3 was assembled as a human IgG ₁ ELISA-based assays were performed to directly compare the ability of the h3D3 to recombinant KAAG1 The methods used to perform these tests were as described in Example 3 using recombinant Fc-KAAGI As shown in Figure 18A, the binding activity of the h3D3 was identical to that of the chimeric 3D3

More precise measurements were conducted using Surface Plasmon Resonance (SPR) in a Biacore instrument Kinetic analysis was used to compare the affinity of the chimeric 3D3 with the h3D3 as well as with hybrid antibodies encompassing different permutations of the light and heavy chains (see Figure 18B) Briefly, anti-human Fc was immobilized on the Biacore sensor chip and chimeric or h3D3 was captured on the chip Different concentrations of monomeric recombinant KAAG1 were injected and the data were globally fitted to a simple 1 1 model to determine the kinetic parameters of the interaction The kinetic parameters of the chimeric 3D3 were tabulated in Figure 18B (m3D3) The average K ₀ of the chimeric 3D3 was 2 35 x 10 ¹⁰ M In comparison, all permutations of the chιmerιc(C)/humanιzed(H) displayed very similar kinetic parameters The average K ₀ of the chimeric light chain expressed with the chimeric heavy chain (indicated as 'CC in Figure 18B) was 2 71 x 10 ¹⁰ M, the average K _D of the humanized light chain expressed with the chimeric heavy chain (indicated as 'HC in Figure 18B) was 3 09 x 10 ¹⁰ M, the average K ₀ of the chimeric light chain expressed with the humanized heavy chain (indicated as 'CH' in Figure 18B) was 5.05 x 10 ^"10 M, and the average K _D of the humanized light chain expressed with the humanized heavy chain (indicated as 'HH' in Figure 18B) was 4.39 x 10 ^"10 M. The analyses indicated that the humanization of 3D3 conserved the binding activity of the original mouse antibody.

The biological function of the h3D3 was evaluated in the spheroid culture assay (see Example 6). SKOV-3 ovarian cancer cells were cultured in the presence of 5% FBS in the presence of h3D3 or a non-KAAG1 binding isotype control antibody. The results (shown in Figure 18C), indicated that treatment with either the buffer or the non-related IgG did not inhibit the formation of the compact 3-D structures. In contrast, both the chimeric 3D3 and the humanized 3D3 prevented the spheroids from forming. The results are shown in duplicate (left and right panels). These results indicate that the biological activity of the chimeric 3D3 was conserved in the humanized 3D3 and suggests that the h3D3 will behave in an identical manner.

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Sequences referred to in the description

SEQ ID NO.:1

GAGGGGCATCAATCACACCGAGAAGTCACAGCCCCTCAACCACTGAGGTGTGGGGGG GTAGGGAT CTGCATTTCTTCATATCAACCCCACACTATAGGGCACCTAAATGGGTGGGCGGTGGGGGA GACCG ACTCACTTGAGTTTCTTGAAGGCTTCCTGGCCTCCAGCCACGTAATTGCCCCCGCTCTGG ATCTG GTCTAGCTTCCGGATTCGGTGGCCAGTCCGCGGGGTGTAGATGTTCCTGACGGCCCCAAA GGGTG CCTGAACGCCGCCGGTCACCTCCTTCAGGAAGACTTCGAAGCTGGACACCTTCTTCTCAT GGATG ACGACGCGGCGCCCCGCGTAGAAGGGGTCCCCGTTGCGGTACACAAGCACGCTCTTCACG ACGGG CTGAGACAGGTGGCTGGACCTGGCGCTGCTGCCGCTCATCTTCCCCGCTGGCCGCCGCCT CAGCT CGCTGCTTCGCGTCGGGAGGCACCTCCGCTGTCCCAGCGGCCTCACCGCACCCAGGGCGC GGGAT CGCCTCCTGAAACGAACGAGAAACTGACGAATCCACAGGTGAAAGAGAAGTAACGGCCGT GCGCC TAGGCGTCCACCCAGAGGAGACACTAGGAGCTTGCAGGACTCGGAGTAGACGCTCAAGTT TTTCA CCGTGGCGTGCACAGCCAATCAGGACCCGCAGTGCGCGCACCACACCAGGTTCACCTGCT ACGGG CAGAATCAAGGTGGACAGCTTCTGAGCAGGAGCCGGAAACGCGCGGGGCCTTCAAACAGG CACGC CTAGTGAGGGCAGGAGAGAGGAGGACGCACACACACACACACACACAAATATGGTGAAAC CCAAT TTCTTACATCATATCTGTGCTACCCTTTCCAAACAGCCTA

SEQ IDNO.:2 MDDDAAPRVEGVPVAVHKHALHDGLRQVAGPGAAAAHLPRWPPPQLAASRREAPPLSQRP HRTQG AGSPPETNEKLTNPQVKEK

SEQ IDNO.:3

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAATAGGACAGAAGGTC ACTATGAA CTGCAAGTCCAGTCAGAGCCTTTTAAATAGTAACTTTCAAAAGAACTTTTTGGCCTGGTA CCAGC AGAAACCAGGCCAGTCTCCTAAACTTCTGATATACTTTGCATCCACTCGGGAATCTAGTA TCCCT GATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAG GCTGA AGACCTGGCAGATTACTTCTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGG GACCA AGCTGGAGCTGAAAGCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGC AGTTG AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA GTACA GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGA CAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC ACAAA GTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGG AGAGTGT

SEQ IDNO.:4

DIVMTQSPSSLAVSIGQKVTMNCKSSQSLLNSNFQKNFLAWYQQKPGQSPKLLI YFASTRESSI P DRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELKAVAAPSVFI FPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC

SEQIDNO.:5

GAGGTTCAGCTGCAGCAGTCTGTAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACG CTGTCCTG CAAGGCTTCGGGCTACATATTTACTGACTATGAGATACACTGGGTGAAGCAGACTCCTGT GCATG GCCTGGAATGGATTGGGGTTATTGATCCTGAAACTGGTAATACTGCCTTCAATCAGAAGT TCAAG GGCAAGGCCACACTGACTGCAGACATATCCTCCAGCACAGCCTACATGGAACTCAGCAGT TTGAC ATCTGAGGACTCTGCCGTCTATTACTGTATGGGTTATTCTGATTATTGGGGCCAAGGCAC CACTC TCACAGTCTCCTCAGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGGCCCCCTCCTCCA AGAGC ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG ACGGT GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC CTCAG GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCT ACATC TGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCT TGTGA ATTCACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT CCTCT TCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG TGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG CATAA TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCT CACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCA GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCT GCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCT ATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAG AGCAG GTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA CACGC AGAAGAGCCTCTCCCTGTCTCCCGGGAAA SEQIDNO.:6

EVQLQQSVAELVRPGASVTLSCKASGYI FTDYEIHWVKQTPVHGLEWIGVIDPETGNTAFNQKFK GKATLTADI SSSTAYMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYI CNVNHKPSNTKVDKKVEPKSCEFTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALP APIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQIDNO.:7 GATGTTTTGATGACCCAAACTCCACGCTCCCTGTCTGTCAGTCTTGGAGATCAAGCCTCC ATCTC TTGTAGATCGAGTCAGAGCCTTTTACATAGTAATGGAAACACCTATTTAGAATGGTATTT GCAGA AACCAGGCCAGCCTCCAAAGGTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCC CAGAC AGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCGGAGTGGAGGCT GAGGA TCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCTCACGTTCGGTGCTGGGAC CAAGC TGGAGCTGAAAGCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGT TGAAA TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTG GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG CAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AAGTC TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGG GGAGA GTGT

SEQ ID NO.:8

DVLMTQTPRSLSVSLGDQAS I SCRS SQSLLHSNGNTYLEWYLQKPGQPPKVLI YKVSNRFSGVPD RFSGSGSGTDFTLKI SGVEAEDLGVYYCFQGSHVPLTFGAGTKLELKAVAAPSVFI FPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKV YACEVTHQGLSSPVTKSFNRGEC

SEQIDNO..9

GAGATCCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTCAGTGAAG ATATCCTG TAAGGCTTCTGGATACACCTTCACTGACAACTACATGAACTGGGTGAAGCAGAGCCATGG AAAGA

GCCTTGAGTGGATTGGAGATATTAATCCTTACTATGGTACTACTACCTACAACCAGA AGTTCAAG

GGCAAGGCCACATTGACTGTAGACAAGTCCTCCCGCACAGCCTACATGGAGCTCCGC GGCCTGAC

ATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGATGACTGGTTTGATTATTGGGG CCAAGGGA

CTCTGGTCACTGTCTCTGCAGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGGCCC CCTCCTCC AAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGT

GACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT CCTACAGT

CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCA CCCAGACC TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCC AAATC TTGTGAATTCACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC AGTCT TCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT GCGTG GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGT GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG CGTCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG GTGTA CACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAG GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAA GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA CCACT ACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAA SEQIDNO.:10

EIQLQQSGPELVKPGASVKISCKASGYTFTDNYMNWVKQSHGKSLEWIGDINPYYGT TTYNQKFK GKATLTVDKSSRTAYMELRGLTSEDSAVYYCARDDWFDYWGQGTLVTVSAASTKGPSVFP LAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQT YICNVNHKPSNTKVDKKVEPKSCEFTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO.:11 GACATCGTTATGTCTCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACT ATCAC TTGCAAGGCGAGTCAGGACATTCATAACTTTTTAAACTGGTTCCAGCAGAAACCAGGAAA ATCTC CAAAGACCCTGATCTTTCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTG GCAGT GGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTTTGAAGATTTGGGAATT TATTC TTGTCTACAGTATGATGAGATTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAG AGCTG TGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG CCTCT GTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGC CCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTA CAGCC TCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCG AAGTC ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

SEQ IDNO.:12

DIVMSQSPSSMYASLGERVTITCKASQDIHNFLNWFQQKPGKS PKTLI FRANRLVDGVPSRFSGS

GSGQDYSLTI SSLEFEDLGI YSCLQYDE I PLTFGAGTKLELRAVAAPSVFI FPPS DEQLKSGTAS

VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEV THQGLSSPVTKSFNRGEC

SEQIDNO.:13

GAGGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCA CTCACCTG CACTGTCACTGGCTTCTCCATCACCAGTGGTTATGGCTGGCACTGGATCCGGCAGTTTCC AGGAA ACAAACTGGAGTGGATGGGCTACATAAACTACGATGGTCACAATGACTACAACCCATCTC TCAAA AGTCGAATCTCTATCACTCAAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCT GTGAC TACTGAGGACACAGCCACATATTACTGTGCAAGCAGTTACGACGGCTTATTTGCTTACTG GGGCC AAGGGACTCTGGTCACTGTCTCTGCAGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGG CCCCC TCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC CCCGA ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGC TGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG GCACC CAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT GAGCC _C AAATCTTGTGAATTCACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGT _C AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC TGAGGTCACA TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGT GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT GGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCT CCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCΆAAGCCAAAGGGCAGCCCCGAGA ACCACA GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTG CCTGG

TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAAC TACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGT GGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT GCACA ACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAA

SEQIDNO.:14 EVQLQESGPDLVKPSQSLSLTCTVTGFSITSGYGWHWIRQFPGNKLEWMGYINYDGHNDY NPSLK SRISITQDTSKNQFFLQLNSVTTEDTATYYCASSYDGLFAYWGQGTLVTVSAASTKGPSV FPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGT QTYICNVNHKPSNTKVDKKVEPKSCEFTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSN KALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQIDNO.:15

GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCAATAGGACAGAAGGTC ACTATGAA CTGCAAGTCCAGTCAGAGCCTTTTAAATAGTAACTTTCAAAAGAACTTTTTGGCCTGGTA CCAGC AGAAACCAGGCCAGTCTCCTAAACTTCTGATATACTTTGCATCCACTCGGGAATCTAGTA TCCCT GATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAG GCTGA AGACCTGGCAGATTACTTCTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGG GACCA AGCTGGAGCTGAAA

SEQ ID NO.:16

DIVMTQSPSSLAVSIGQKVTMNCKSSQSLLNSNFQKNFLAWYQQKPGQSPKLLIYFA STRESSIP DRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPLTFGAGTKLELK SEQ ID NO.:17

GAGGTTCAGCTGCAGCAGTCTGTAGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGACG CTGTCCTG CAAGGCTTCGGGCTACATATTTACTGACTATGAGATACACTGGGTGAAGCAGACTCCTGT GCATG GCCTGGAATGGATTGGGGTTATTGATCCTGAAACTGGTAATACTGCCTTCAATCAGAAGT TCAAG GGCAAGGCCACACTGACTGCAGACATATCCTCCAGCACAGCCTACATGGAACTCAGCAGT TTGAC ATCTGAGGACTCTGCCGTCTATTACTGTATGGGTTATTCTGATTATTGGGGCCAAGGCAC CACTC TCACAGTCTCCTCA

SEQ ID NO.:18

EVQLQQSVAELVRPGASVTLSCKASGYI FTDYEIHWVKQTPVHGLEWIGVI DPETGNTAFNQKFK GKATLTADISSSTAYMELSSLTSEDSAVYYCMGYSDYWGQGTTLTVSS

SEQIDNO.:19

GATGTTTTGATGACCCAAACTCCACGCTCCCTGTCTGTCAGTCTTGGAGATCAAGCC TCCATCTC

TTGTAGATCGAGTCAGAGCCTTTTACATAGTAATGGAAACACCTATTTAGAATGGTA TTTGCAGA AACCAGGCCAGCCTCCAAAGGTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCC CAGAC

AGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCGGAGTGGAG GCTGAGGA TCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCTCACGTTCGGTGCTGGGAC CAAGC TGGAGCTGAAA SEQ ID NO.:20

DVLMTQTPRSLSVSLGDQASISCRSSQSLLHSNGNTYLEWYLQKPGQPPKVLIYKVS NRFSGVPD RFSGSGSGTDFTLKISGVEAEDLGVYYCFQGSHVPLTFGAGTKLELK

SEQIDNO.:21 GAGATCCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTCAGTGAAGATA TCCTG TAAGGCTTCTGGATACACCTTCACTGACAACTACATGAACTGGGTGAAGCAGAGCCATGG AAAGA GCCTTGAGTGGATTGGAGATATTAATCCTTACTATGGTACTACTACCTACAACCAGAAGT TCAAG GGCAAGGCCACATTGACTGTAGACAAGTCCTCCCGCACAGCCTACATGGAGCTCCGCGGC CTGAC ATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGATGACTGGTTTGATTATTGGGGCCA AGGGA CTCTGGTCACTGTCTCTGCA

SEQIDNO.:22

EIQLQQSGPELVKPGASVKI SCKASGYT FTDNYMNWVKQSHGKSLEWIGDINPYYGTTTYNQKFK GKATLTVDKSSRTAYMELRGLTSEDSAVYYCARDDWFDYWGQGTLVTVSA

SEQ ID NO.:23

GACATCGTTATGTCTCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTC ACTATCAC TTGCAAGGCGAGTCAGGACATTCATAACTTTTTAAACTGGTTCCAGCAGAAACCAGGAAA ATCTC CAAAGACCCTGATCTTTCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTG GCAGT GGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTTTGAAGATTTGGGAATT TATTC TTGTCTACAGTATGATGAGATTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAG A

SEQ ID NO.:24

DIVMSQSPSSMYASLGERVTITCKASQDIHNFLNWFQQKPGKSPKTLI FRANRLVDGVPSRFSGS GSGQDYSLTISSLEFEDLGIYSCLQYDEIPLTFGAGTKLELR

SEQ ID NO.:25

GAGGTGCAGCTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCA CTCACCTG CACTGTCACTGGCTTCTCCATCACCAGTGGTTATGGCTGGCACTGGATCCGGCAGTTTCC AGGAA ACAAACTGGAGTGGATGGGCTACATAAACTACGATGGTCACAATGACTACAACCCATCTC TCAAA AGTCGAATCTCTATCACTCAAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCT GTGAC TACTGAGGACACAGCCACATATTACTGTGCAAGCAGTTACGACGGCTTATTTGCTTACTG GGGCC AAGGGACTCTGGTCACTGTCTCTGCA SEQ ID NO.:26

EVQLQESGPDLVKPSQSLSLTCTVTGFSITSGYGWHWIRQFPGNKLEWMGYINYDGH NDYNPSLK SRISITQDTSKNQFFLQLNSVTTEDTATYYCASSYDGLFAYWGQGTLVTVSA

SEQ ID NO.:27 KSSQSLLNSNFQKNFLA

SEQ IDNO.:28

FASTRES SEQ ID NO..29

QQHYSTPLT SEQ IDNO.:30

GYIFTDYEIH SEQ ID NO.:31

VIDPETGNTA

SEQ IDNO.:32

MGYSDY

SEQ ID NO.:33

RSSQSLLHSNGNTYLE

SEQ IDNO.:34 KVSNRFS

SEQ IDNO.:35

FQGSHVPLT SEQ ID NO.:36

GYTFTDNYMN

SEQIDNO.:37

DINPYYGTTT

SEQ IDNO.:38

ARDDWFDY

SEQ IDNO.:39 KASQDIHNFLN

SEQ IDNO.:40

RANRLVD SEQ IDNO.:41

LQYDEIPLT

SEQ ID NO.:42

GFSITSGYGWH

SEQ ID NO.:43

YINYDGHND

SEQ ID NO.:44 ASSYDGLFAY

SEQ ID NO.:45

GAGGGGCATCAATCACACCGAGAA SEQ ID NO.:46

CCCCACCGCCCACCCATTTAGG SEQ ID NO.:47

TGAAGGTCGGAGTCAACGGATTTGGT SEQ ID NO.:48

CATGTGGGCCATGAGGTCCACCAC

SEQ ID NO.:49

GGCCTCCAGCCACGTAATT

SEQ ID NO.:50

GGCGCTGCTGCCGCTCATC

SEQIDNO.:51 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA CAGCT TGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGC GGGTG TCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG GTGTG AAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGC TGCGC AACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGG GGATG TGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAAC GACGG CCAGTGCCAAGCTTTTCCAAAAAACTACCGTTGTTATAGGTGTCTCTTGAACACCTATAA CAACG GTAGTGGATCCCGCGTCCTTTCCACAAGATATATAAACCCAAGAAATCGAAATACTTTCA AGTTA CGGTAAGCATATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAA ATTAT TACTTTCTACGTCACGTATTTTGTACTAATATCTTTGTGTTTACAGTCAAATTAATTCTA ATTAT CTCTCTAACAGCCTTGTATCGTATATGCAAATATGAAGGAATCATGGGAAATAGGCCCTC TTCCT GCCCGACCTTGGCGCGCGCTCGGCGCGCGGTCACGCTCCGTCACGTGGTGCGTTTTGCCT GCGCG TCTTTCCACTGGGGAATTCATGCTTCTCCTCCCTTTAGTGAGGGTAATTCTCTCTCTCTC CCTAT AGTGAGTCGTATTAATTCCTTCTCTTCTATAGTGTCACCTAAATCGTTGCAATTCGTAAT CATGT CATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCG GAAGC ATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGC TCACT GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC GGGGA GAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGG TCGTT CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA GGGGA TAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGC CGCGT TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAA GTCAG AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTC GTGCG CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG CGTGG CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC TGGGC TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT GAGTC CAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGA GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA GAACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT TGATC CGGCAAAAAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCG CAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACG AAAAC TCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA AATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCA ATGCT TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGAC TCCCC GTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATA CCGCG AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGA GCGCA GAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA GAGTA AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTG TCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATG ATCCC CCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGT TGGCC GCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCC GTAAG ATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGΆGAATAGTGTATGCGGC GACCGA GTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAG TGCTC ATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCC AGTTC GATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTC TGGGT GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT GAATA CTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGC GGATA CATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA AGTGC CACCTATTGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCA TCTCA ATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAA GCATG CATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACT CCGCC CAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGA GGCCG CCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTT GCAAA AAGCTAGCTTGCATGCCTGCAGGTCGGCCGCCACGACCGGTGCCGCCACCATCCCCTGAC CCACG CCCCTGACCCCTCACAAGGAGACGACCTTCCATGACCGAGTACAAGCCCACGGTGCGCCT CGCCA CCCGCGACGACGTCCCCCGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCG CCACG CGCCACACCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTC CTCAC GCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGT CTGGA CCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCG AGTTG AGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCC AAGGA GCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCAAGGGTCTGGG CAGCG CCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGA CCTCC GCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAG GTGCC CGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGACGCCCGCCCCACGA CCCGC AGCGCCCGACCGAAAGGAGCGCACGACCCCATGGCTCCGACCGAAGCCACCCGGGGCGGC CCCGC CGACCCCGCACCCGCCCCCGAGGCCCACCGACTCTAGAGGATCATAATCAGCCATACCAC ATTTG TAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAA TGAAT GCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGC ATCAC AAATTTCACAAATAAAGCATTTTTTTCACTGCAATCTAAGAAACCATTATTATCATGACA TTAAC CTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

SEQ ID NO.:52 GTAAGCGGATCCATGGATGACGACGCGGCGCCC

SEQ ID NO.53

GTAAGCAAGCTTCTTCTCTTTCACCTGTGGATT SEQ ID NO.:54

GTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGAC TAGTTATT AATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACAT AACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATG ACGTA TGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACG GTAAA CTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCA ATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGG CAGTA CATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGG GCGTG GATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTT TGTTT TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAA ATGGG CGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAT CCTCA CTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGCTCGCGGTTGAGGACAAACT CTTCG CGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACGGTACTCCGCCACC GAGGG ACCTGAGCCAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGAAAGGCGTCTAACCAGT CACAG TCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGTGGCGGTCGGGGTTGTTTCTG GCGGA GGTGCTGCTGATGATGTAATTAAAGTAGGCGGTCTTGAGCCGGCGGATGGTCGAGGTGAG GTGTG GCAGGCTTGAGATCCAGCTGTTGGGGTGAGTACTCCCTCTCAAAAGCGGGCATGACTTCT GCGCT AAGATTGTCAGTTTCCAAAAACGAGGAGGATTTGATATTCACCTGGCCCGATCTGGCCAT ACACT TGAGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTCCAAG TTTGC CGCCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTC CACTG GCGCCGGATCAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGT CAGTC TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA TGCGT GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT GGAGG TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTC CTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGC CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA GGTGT ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGG TCAAA GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAAC TACAA GACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGT GGACA AGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA ACCAC TACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAAGCTAGCGGAGCCGGAAGCACAACC GAAAA CCTGTATTTTCAGGGCGGATCCGAATTCAAGCTTGATATCTGATCCCCCGACCTCGACCT CTGGC TAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTC GGAAG GACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGCCCC GCCGC CGGACGAACTAAACCTGACTACGGCATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAA TCCCT TCAGTTGGTTGGTACAACTTGCCAACTGAACCCTAAACGGGTAGCATATGCTTCCCGGGT AGTAG TATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCATATG CTATC GAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATGTAGGTGGGCGGGCCAAGATAG GGGCG CGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGG TTGTT GGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCAT ATACT ACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCC TAATC TATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATT TATAT CTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATAT CTGGG

TAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAΆTAGAGATT AGGGTAGTA TATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATCTGGATAGCATATG CTATC CTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATC CTAAT CTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT TTATA TCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATA TCTGG GTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTCACGATGATAAGC TGTCA

AACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAG GTTAATGT CATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC CCCTA TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT AAATG CTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATT CCCTT TTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGA TGCTG AAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCC TTGAG AGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGC GCGGT ATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAA TGACT TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAAT TATGC AGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGA GGACC GAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTG GGAAC CGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGG CAACA ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATA GACTG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTT TATTG CTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAG ATGGT AAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGA AATAG ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTA CTCAT ATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC TTTTT GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC GTAGA AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC AAAAA AACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGA AGGTA ACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC CACCA CTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC TGCTG CCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG CGCAG CGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACC GAACT GAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA CAGGT ATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACG CCTGG TATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC TCGTC AGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTT TTGCT GGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCC TTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGC GAGGA AGC

SEQ ID NO.:55 GTAAGCAAGCTTAGGCCGCTGGGACAGCGGAGGTGC

SEQ ID NO.:56

GTAAGCAAGCTTGGCAGCAGCGCCAGGTCCAGC SEQ ID NO.:57

GTAAGCAGCGCTGTGGCTGCACCATCTGTCTTC

SEQIDNO.:58

GTAAGCGCTAGCCTAACACTCTCCCCTGTTGAAGC

SEQ ID NO.:59

GCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT GGAACTGC CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGT GGATA ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCA CCTAC AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC TGCGA AGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA G

SEQ ID NO.:60

AVAAPSVFI FPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ IDNO.:61

CTTGAGCCGGCGGATGGTCGAGGTGAGGTGTGGCAGGCTTGAGATCCAGCTGTTGGG GTGAGTAC TCCCTCTCAAAAGCGGGCATTACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAG GATTT GATATTCACCTGGCCCGATCTGGCCATACACTTGAGTGACAATGACATCCACTTTGCCTT TCTCT CCACAGGTGTCCACTCCCAGGTCCAAGTTTAAACGGATCTCTAGCGAATTCATGAACTTT CTGCT GTCTTGGGTGCATTGGAGCCTTGCCTTGCTGCTCTACCTCCACCATGCCAAGTGGTCCCA GGCTT GAGACGGAGCTTACAGCGCTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATG AGCAG TTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGT ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA GGACA GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGA AACAC AAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTC AACAG GGGAGAGTGTTAGGGTACCGCGGCCGCTTCGAATGAGATCCCCCGACCTCGACCTCTGGC TAATA AAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAG GACAT ATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGCCCCGCCGC CGGAC GAACTAAACCTGACTACGGCATCTCTGCCCCTTCTTCGCGGGGCAGTGCATGTAATCCCT TCAGT TGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACATGTGACACGGGGGGGGACCAAAC ACAAA GGGGTTCTCTGACTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGA GTGGC TTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGT AACTC TTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGGGGCCCAGGAAGAC TACGG GAGGCTACACCAACGTCAATCAGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAG AGGGC ATTAGCAATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCC GGGTA GTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGAAGCA TATGC TATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATATCTCCCACCCCATGAGC TGTCA CGGTTTTATTTACATGGGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGG GCAGT GGCTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCT CCTTC GTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAGGTGCTCGAA AACAA GGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGC TATGA CACCAATATAACCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATT CTGAA TATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCT CACAC GAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATACTGGGGTTATTAAGA TGTGT CCCAGGCAGGGACCAAGACAGGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAA AGAGA GTGGACGCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGG GGCTC CACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTGTGGAG TGGGG GCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACTGTAAAATAAGGGTGTAATA ACTTG GCTGATTGTAACCCCGCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGG CACCC CGGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCT GGAGG ACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCCTCATATTCAC GAGGT CGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTAGCATATACTACCCAAATATCTGGA TAGCA TATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCA TATGC TATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGGC TATCC TAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCC TAATC TGTATCCGGGTAGCATATGCTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATT TATAT CTGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGG GTAGC ATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGC ATATG CTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGG CTATC CTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATC CTAAT CTGTATCCGGGTAGCATATGCTATCCTCACGATGATAAGCTGTCAAACATGAGAATTAAT TCTTG AAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGT TTCTT AGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCT AAATA CATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA AAAAG GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG CCTTC CTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTG CACGA GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAA GAACG TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGA CGCCG GGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCAC CAGTC ACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC ATGAG TGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGC TTTTT TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAG CCATA CCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTA TTAAC TGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAA AGTTG CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAG CCGGT GAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC GTAGT TATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGAT AGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG ATTTA AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACC AAAAT CCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATC TTCTT GAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG CGGTG GTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA GCGCA GATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGT AGCAC CGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGT CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGG TTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG TGAGC ATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCA GGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCT GTCGG GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCT ATGGA AAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACA TGTTC TTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT ACCGC TCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCC AATAC GCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTC CCGAC TGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCC CAGGC TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCA CACAG GAAACAGCTATGACCATGATTACGCCAAGCTCTAGCTAGAGGTCGACCAATTCTCATGTT TGACA GCTTATCATCGCAGATCCGGGCAACGTTGTTGCATTGCTGCAGGCGCAGAACTGGTAGGT ATGGC AGATCTATACATTGAATCAATATTGGCAATTAGCCATATTAGTCATTGGTTATATAGCAT AAATC AATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATT GGCTC ATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAAT TACGG GGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCC CGCCT GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTA ACGCC AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGC AGTAC ATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG CCTGG CATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTA GTCAT CGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGA CTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATC AACGG GACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTA CGGTG GGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCCTCACTCTCTTCCGCAT CGCTG TCTGCGAGGGCCAGCTGTTGGGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGT ACTCT TGGATCGGAAACCCGTCGGCCTCCGAACGGTACTCCGCCACCGAGGGACCTGAGCGAGTC CGCAT CGACCGGATCGGAAAACCTCTCGAGAAAGGCGTCTAACCAGTCACAGTCGCAAGGTAGGC TGAGC ACCGTGGCGGGCGGCAGCGGGTGGCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATG ATGTA ATTAAAGTAGGCGGT SEQ Dl NO.:62

ATGCCAAGTGGTCCCAGGCTGACATTGTGATGACCCAGTCTCC

SEQ ID NO.:63

ATGCCAAGTGGTCCCAGGCTGATGTTTTGATGACCCAAACTCC

SEQ ID NO.:64

ATGCCAAGTGGTCCCAGGCTGACATCGTTATGTCTCAGTCTCC

SEQ ID NO.:65 GGGAAGATGAAGACAGATGGTGCAGCCACAGC SEQ ID NO.:66 GTAAGCGCTAGCGCCTCAACGAAGGGCCCATCTGTCTTTCCCCTGGCCCC

SEQ ID NO.:67 GTAAGCGAATTCACAAGATTTGGGCTCAACTTTCTTG

SEQ ID NO.:68

GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT GGGGGCAC AGCAGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA CTCAG GCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT CCCTC AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG AATCA CAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT

SEQ IDNO.:69 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO.:70

CTTGAGCCGGCGGATGGTCGAGGTGAGGTGTGGCAGGCTTGAGATCCAGCTGTTGGG GTGAGTAC TCCCTCTCAAAAGCGGGCATTACTTCTGCGCTAAGATTGTCAGTTTCCAAAAACGAGGAG GATTT GATATTCACCTGGCCCGATCTGGCCATACACTTGAGTGACAATGACATCCACTTTGCCTT TCTCT CCACAGGTGTCCACTCCCAGGTCCAAGTTTGCCGCCACCATGGAGACAGACACACTCCTG CTATG GGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGCGGAGACGGAGCTTACGGGCCCATCTG TCTTT CCCCTGGCCCCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTC AAGGA CTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCA CACCT TCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT CCAGC AGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTG GACAA GAAAGTTGAGCCCAAATCTTGTGAATTCACTCACACATGCCCACCGTGCCCAGCACCTGA ACTCC TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC GGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAAC TGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG CACGT ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACA AGTGC AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCC CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT CAGCC TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAG CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT GCATG AGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATGATCCC CCGAC CTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTT GTGTC TCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGGTCGAGATCCCTCGGAGATCTC TAGCT AGAGCCCCGCCGCCGGACGAACTAAACCTGACTACGGCATCTCTGCCCCTTCTTCGCGGG GCAGT GCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGAACCCTAAACGGGTAGCAT ATGCT TCCCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAA CGGGA AGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGATGTAGGTGGG CGGGC CAAGATAGGGGCGCGATTGCTGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGC GCCAA GCACAGGGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTG CCATG GGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTA GCATA GGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATA TGCTA TCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTA TCCTA ATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTA ATAGA GATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTACCCAAATATC TGGAT AGCATATGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGT AGCAT AGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTAT ATGCT ATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCT ATCCT AATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCT CACGA TGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCT ATTTT TATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAA ATGTG CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGA CAATA ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCG TGTCG CCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG TGAAA GTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC AGCGG TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGT TCTGC TATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATAC ACTAT TCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATG ACAGT AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT GACAA CGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTC GCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG CCTGC AGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG GCAAC AATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC CGGCT GGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCA GCACT GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAAC TATGG ATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGT CAGAC CAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC TAGGT GAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTG AGCGT CAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCT GCTGC TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCA ACTCT TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTA GCCGT AGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCC TGTTA CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG TTACC GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCG AACGA CCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAG GGAGA AAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTT CCAGG GGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCG ATTTT TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTAC GGTTC CTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTG GATAA CCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG CGAGT CAGTGAGCGAGGAAGCGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGAC ATTGA TTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATG GAGTT CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC ATTGA CGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTG GAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCG CCCCC TATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACG GGACT TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTT GGCAG TACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATT GACGT CAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCC CGCCC CGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTT TAGTG AACCGTCAGATCCTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGTTGGGCTCG CGGTT GAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATCGGAAACCCGTCGGCCTCCGA ACGGT ACTCCGCCACCGAGGGACCTGAGCGAGTCCGCATCGACCGGATCGGAAAACCTCTCGAGA AAGGC GTCTAACCAGTCACAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGTGGCG GTCGG GGTTGTTTCTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGT

SEQ ID NO.:71 GGGTTCCAGGTTCCACTGGCGAGGTTCAGCTGCAGCAGTCTGT

SEQ ID NO 72 GGGTTCCAGGTTCCACTGGCGAGGTGCAGCTTCAGGAGTCAGG

SEQ ID NO 73

GGGGCCAGGGGAAAGACAGATGGGCCCTTCGTTGAGGC SEQ ID NO 91 Exemplary embodiment of CDRL1

K-S-S-Q-S-L-L-N/H-S/T-S/N/D-N/G-Q/N/K-K/L-N-Y-L-A

SEQ ID NO 92 Exemplary embodiment of CDRL1 K-A-S-Q-D-l-H-N/T-Y/F-L-N

SEQ ID NO93 Exemplary embodiment of CDRL2 F-A-S-T-R-E-S

SEQ ID NO 94 Exemplary embodiment of CDRL2 L-V-S-K-L-D-S

SEQ ID NO 95 Exemplary embodiment of CDRL2 R-A-N-R-L-V-D SEQ ID NO 96 Exemplary embodiment of CDRL3 Q-Q-H-Y-S-T-P-L-T

SEQ ID NO 97 Exemplary embodiment of CDRL3 W/L-Q-Y/G-D/T-A/E/H-F-P-R-T

SEQ ID NO 98 Exemplary embodiment of CDRH1 1 G-Y-T/l-F-T-D/E-Y-E/N-M/l/V-H

SEQ ID NO 99 Exemplary embodiment of CDRH1 G-F-T/S-l-T-S-G-Y-G-W-H

SEQ ID NO 100 Exemplary embodiment of CDRH2 V/N/G-l/L-D-P-E/A/G-T/Y-G-X-T-A SEQ ID NO 101 Exemplary embodiment of CDRH2 Y-l-N/S-F/Y-N/D-G

SEQ ID NO 102 Exemplary embodiment of CDRH3 M-G-Y-S/A-D-Y

SEQ ID NO 103 Exemplary embodiment of CDRH3 A-S-S-Y-D-G-F-L-A-Y

SEQ ID NO 104 Exemplary embodiment of CDRH3 3 A-R/W-W/F-G-L-R-Q/N

SEQ ID NO 158 KSSQSLLHSDGKTYLN

SEQ ID NO.:159 LVSKLDS

SEQ ID NO.:160 WQGTHFPRT SEQ IDNO.:161 GYTFTD YNMH

SEQ ID NO.:162 YINPYNDVTE

SEQ ID NO.:163 AWFGL RQ

SEQ ID NO.:164 RSSKSLLHSNGN TYLY

SEQ ID NO.:165 RMSNLAS SEQ ID NO.:166 MQHLEYPYT

SEQ ID NO.:167 GDTFTD YYMN

SEQ ID NO.:168 DINPNYGGIT

SEQ IDNO.:169 QAYYRNS DY

SEQ ID NO.:170 KASQDVGTAVA SEQ ID NO.:171 WTSTRHT

SEQ IDNO.:172 QQHYSIPLT

SEQ IDNO.:173 GYIFTDYEIH

SEQ IDNO.:174 VIDPETGNTA

SEQ IDNO.:175 MGYSDY

SEQ ID NO.:176 MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVSLGERATINCKSSQSLLNSNFQKNFLA WYQQKPGQPPKLLIYFASTRESSVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHY STPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

SEQ ID NO.:177

MDWTWRILFLVAAATGTHAEVQLVQSGAEVKKPGASVKVSCKASGYIFTDYEIHWVR Q APGQGLEWMGVIDPETGNTAFNQKFKGRVTITADTSTSTAYMELSSLTSEDTAVYYCM GYSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID N:178

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSNFQKNFLAWYQQKPGQPPKLLIYFA ST

RESSVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTKLEIK SEQ ID NO.:179

EVQLVQSGAEVKKPGASVKVSCKASGYIFTDYEIHWVRQAPGQGLEWMGVIDPETGN TAFNQKFKGRVTITADTSTSTAYMELSSLTSEDTAVYYCMGYSDYWGQGTLVTVSS

SEQ ID NO.:180 MVLQTQVFISLLLWISGAYGDIVMTQSPSSLSASVGDRVTITCKASQDIHNFLNWFQQK PGKAPKTLIFRANRLVDGVPSRFSGSGSGTDYTLTISSLQPEDFATYSCLQYDEIPLTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO.:181

MDWTWRILFLVAAATGTHAEVQLQESGPGLVKPSQTLSLTCTVSGFSITSGYGWHWI R QHPGKGLEWIGYINYDGHNDYNPSLKSRVTISQDTSKNQFSLKLSSVTAADTAVYYCAS SYDGLFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

SEQ ID No.:182

DIVMTQSPSSLSASVGDRVTITCKASQDIHNFLNWFQQKPGKAPKTLIFRANRLVDG VP

SRFSGSGSGTDYTLTISSLQPEDFATYSCLQYDEIPLTFGQGTKLEIK SEQ ID NO.:183

EVQLQESGPGLVKPSQTLSLTCTVSGFSITSGYGWHWIRQHPGKGLEWIGYINYDGH N DYNPSLKSRVTISQDTSKNQFSLKLSSVTAADTAVYYCASSYDGLFAYWGQGTLVTVS Table A: Light chains variable region of selected antibodies

SEQID NO:

3zlA02L 105 DAVMTQIPLTLSVTIGQPASLSC KSSQSLLHSDGK TYLN WLLQRPGQSPKRLIS LVSKLDS GVPDRFTGSGSGTDFTLKISRVEAEDLGLYYC WQGTHFPRT FAGGTNLEIK

3zlF06L 106 SIVMTQTPLTLSVTIGQPASITC KSSQSLLYSDGK TYLN WLLQRPGQSPKRLIS LVSKLDS GVPDGFTGSGSGTDFTLKI ΞRVEAEDLGVYYC WQGTHFPRT FGGGTKLEIK

3zlE08L 107 DAVMTQIPLTLSVTIGQPASISC KSSQSLLHSDGK TYLN WLLQRPGQSPKRLIY LVSKLDS GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPRT FGGGTKLEIK

3zlG10L 108 DVLMTQTPRSLSVSLGDQASISC RSSQSLLHSNGN TYLE WYLQKPGQPPKVLIY KVSNRFS GVPDRFSGΞGSGTDFTLKI SGVEAEDLGVYYC FQGSHVPLT FGAGTKLELK

3ZlElOL 109 DIVMTQAAPSVPVTPGESVSISC RSSKSLLHSNGN TYLY WFLQRPGQSPQLLIY RMSNLAS GVPDRFSGSGSGTAFTLRIΞRVEAEDVGVYYC MQHLEYPYT FGGGTKLEIK

3zlA09L 110 DIVMTQSPSSLAMΞLGQKVTMSC KSSQSLLNSNNQLNYLA WYQQKPGQSPKLLVY FASTRKS GVPDRFIGSGSGTDFTLTITSVQAEDLADYFC QQHFNTPLT FGAGTKLELK

3zlB01L 111 DIVMTQSPSSLAISVGQKVTMΞC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVF FASTRES GVPDRFIGSGSGTDFTLTI SΞVQAEDLADYFC QQHYSIPLT FGAGTKLELK

3zlG05L 112 DIVMTQΞPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVF FASTRES GVPDRFIGSGSGTDFTLTITSVQAEDLADYFC QQHYSIPLT FGSGTKLELK

3zlB02L 113 DIVMTQSPΞSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVY FASTRES GVPDRFIGSGSGTDFTLTISSVQAEDLADYFC QQHYSIPLT FGAGTKLELK

3zlB08L 114 DIVMTQΞPSSLAMΞVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVY FASTRES GVPDRFIGSGSGTDFTLTISSVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlG08L 115 DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVY FASTRES GVPDRFIGSGSGTDFTLTI ΞSVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlF07L 116 DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLIY FASTRES GVPDRFIGSGSGTDFTLTI ΞSVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlE09L 117 DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVY FASTRES GVPDRFIGSGSGTEFTLTITSVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlC03L 118 DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVY FGSTRES GVPDRFIGSGSGTDFTLTISGVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlE12L 119 DIVMTQSPSSLAMΞVGQKVTMNC KSSQSLLNRSNQKNYLA WYQQKPGQSPKLLVY FASTRES GVPDRFIGSGSGTDFTLTISSVQAEDLADYFC QQHYSIPLT FGAGTKLELK

4zlA02L 120 DIVMTQSPSSLAMSVGQKVTMNC KSSQSLLNNSNQKNYLA WYQQKPGQSPKLLLY FASTRES GVPDRFIGSGSGTYFTLTISSVQAEDLADYFC QQHYSTPLT FGAGTKLDLK

3zlF10L 121 DIVMTQSPSSLTMΞVGQKVTMSC KSSQSLLNTSNQLNYLA WYQQKPGQS PKLLVY FASTTES GVPDRFIGSGSGTDFTLTISSVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlF04L 122 DIVMTQSPSΞLTVTAGEKVTMSC KSSQSLLNTSNQKNYLA WYQQKPGQSPKLLVY FASTRAS GVPDRFIGSGSGTDFTLTISSVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlBllL 123 DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVY FASTRES GVPDRFIGSGSGTDFTLTISSVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlD03L 124 DIVMTQSPSSLAVSIGQKVTMNC KSSQSLLNSNFQKNFLA WYQQKPGQSPKLLIY FASTRES SIPDRFIGSGSGTDFTLTISSVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlC03L 125 DIVMTQSPSSLAMSVGQKVTMSC KSSQSLLNSSNQKNYLA WYQQKPGQSPKLLVY FGSTRES GVPDRFIGSGSGTDFTLTISGVQAEDLADYFC QQHYSTPLT FGAGTKLELK

3zlG12L 126 DIVMTQSPKFMSTSVGDRVΞITC KASQDVG TAVA WYQQKPGQSPELLIY WTSTRHT GVPDRFSGSGSGTDFTLTISSVQAEDLADYFC QQHYSIPLT FGAGTKLELR

3zlC04L 127 DIVMSQSPSSMYAΞLGERVTITC KASQDIH NFLN WFQQKPGKSPKTLIF RANRLVD GVPSRFSGΞGSGQDYSLTI SSLEFEDLGIYSC LQYDEIPLT FGAGTKLELR

3zlD01L 128 DIKMTQSPSSMYASLGERVTITC KASQDIH TYLN WFQQKPGKSPETLIY RANRLVD GVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC LQYDEFPLT FGAGTKLELK

3zlC02L 129 DIQMTQSPSSMYASLGERVTLTC KASQDIH NYLN WFQQKPGKSPKTLIH RANRLVA GVPSRFSGSGSGQDYSLTISSLEYEDLGIYYC LQYDAFPLT FGAGTKLELK

3zlE06L 130 DIQMTQSPSSMYAΞLGERVTLTC KASQDIH NYLN WFQQKPGKSPKTLIH RANRLVA GVPSRFSGSGSGQDYSLTISSLEYEDLGIYYC LQYDAFPLT FGAGTKLELK

3zlH03L 131 DIVMSQSPSSMYASLGERVTITC KASQDIH RFLN WFQQKPGKSPKTLIF HANRLVD GVPSRFSGSGSGLDYSLTISSLEYEDMGIYFC LQYDAFPLT FGAGTKLELK

Table B: Heavy chains variable region of selected antibodies

SEQID NO:

3zlA02H 132 HEIQLQQSGPELVKPGASVKMSCKTS GYTFTD YNMH WVKQKPGQGLEWIG YINPYNDVTE YNEKFKGRATLTSDKSSΞTAYMDLSSLTSDDSAVYFC AWFGL RQ WGQGTLVTVST

3zlF06H 133 HEVQLQQSGPELVKPGASVKMSCKAS GYIFTE YNIH WVKQKPGQGPEWIG NINPYNDVTE YNEKFKGKATLTSDKASSTAYMDLΞSLTSEDSAVYYC ARWGL RN WGQGTLVTVSA

3zlE08H 134 HEVQLQQSVPELVKPGASVKMSCKTS GYTFTE YNMH WVKQKPGQGPEWIG NINPYNNVTE YNEKFKGKATLTSDKSSSTAYLDLSSLTSEDSAVYYC ARWGL RN WGQGTLVTVSA

3zlA09H 135 HQVQVQQPGAELVRPGASVTLSCKAS GYIFTD YEVH WVRQRPVHGLEWIG VIDPETGDTA YNQKFKGKATLTADKSSΞTAYMELSSLTAEDSAVYYC IGYA DY WGQGTTLTVSS

3zlB01H 136 HQVQLQQPGAELVRPGASVTLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VIDPETGGTA YNQKFKGKATLTTDKSSSTAYMELRSLTSEDSAVYYC MGYS DY WGQGTTLTVSS

3zlB02H 137 HEVQLQQSGAELVRPGASVTLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VIDPETGATA YNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYC MGYS DY WGQGTTLTVSS

3zlF04H 138 HEVQLQQSGAELVRPGASVTLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VIDPETGSTA YNQKFKGKATLTADKASSTAYMELSSLTSEDSAVYYC MGYS DY WGQGTTLTVSS

3Z1E09H 139 HEVQLQQSGAELVRPGASATLSCKAΞ GYTFTD YEMH WVKQTPVHGLEWIG VIDPETGSTA YNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYC MGYA DY WGQGTTLTVSS

3zlB08H 140 HEVQLQQSGAELVRPGASVTLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VIDPETGDTA YNQNFTGKATLTADKSSSTAYMELS SLTSEDΞAVYYC MGYA DY WGQGTTLTVSS

3zlG08H 141 HQVQLKQSGAELVRPGASVTLSCKAS GYTFTD YEVH WVKQTPVHGLEWIG VIDPATGDTA YNQKFKGKATLTADKSSSTAYMEVSSLTSEDSAVYYC MGYS DY WGQGTTLTVSS

3zlF07H 142 HQAYLQQSGAELVRPGASVTLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VIDPETGDTA YNQKFKDKATLTADKASSTAYMELSSLTSEDSAVYYC MGYS DY WGQGTTLTVSS

3zlE12H 143 HQVQLQQSEAELVKPGASVKLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VIDPETGDTA YNQKFKGKATLTADKSSSTAYMELSRLTSEDSAVYYC MGHS DY WGQGTTLTVSS

3Z1D03H 144 HEVQLQQSVAELVRPGAΞVTLSCKAΞ GYIFTD YEIH WVKQTPVHGLEWIG VIDPETGNTA FNQKFKGKATLTADISSSTAYMELSSLTSEDSAVYYC MGYS DY WGQGTTLTVSS

3zlG12H 145 HEVQLQQSVAELVRPGASVTVSCKAS GYIFTD YEIH WVKQTPAHGLEWIG VIDPETGNTA FNQKFKGKATLTADI ΞΞΞTAYMELSSLTSEDSAVYYC MGYS DY WGQGTTLTVSS

3zlF10H 146 HEVQLQQSVAELVRPGAPVTLSCKAS GYTFTD YEVH WVKQTPVHGLEWIG VIDPETGATA YNQKFKGKATLTADKSSSAAYMELSRLTSEDΞAVYYC MSYS DY WGQGTTLTVSS

3zlC03H 147 HEVQLQQSVAEVVRPGASVTLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VIDPETGVTA YNQRFRDKATLTTDKSSSTAYMELSSLTSEDSAVYFC MGYS DY WGQGTTLTVSS

3zlC03H 148 HEVQLQQΞVAEVVRPGASVTLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VIDPETGVTA YNQRFRDKATLTTDKSΞSTAYMELSSLTSEDSAVYFC MGYS DY WGQGTTLTVSS

3zlG05H 149 HQVQLQQPGAELVRPGASVTLSCKAS GYTFTD YEIH WVKQTPVHGLEWIG VLDPGTGRTA YNQKFKDKATLSADKΞSSTAYMELSΞLTSEDSAVYYC MSYS DY WGPGTTLTVSS

3ZlBIlH 150 HEVQLQQSVAELVRPGASVTLSCKAΞ GYTFTD YEMH WVKQTPVRGLEWIG VIDPATGDTA YNQKFKGKATLTADKSSSAAFMELSSLTSEDSAVYYC MGYS DY WGQGTTLTVSS

3zlE06H 151 HQVQLQQSGAELVRPGASVTLSCKAS GYTFSD YEMH WVKQTPVHGLEWIG GIDPETGDTV YNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYC ISYAM DY WGQGTSVTVSS

4Z1A02H 152 HQVKLQQSGTELVRPGASVTLSCKAΞ GYKFTD YEMH WVKQTPVHGLEWIG GIDPETGGTA YNQKFKGKAILTADKSSTTAYMELRΞLTSEDSAVYYC ISYAM DY WGQGTSVTVSS

3ZlElOH 153 HEVQLQQSGPELVKPGASVKISCKAΞ GDTFTD YYMN WVKQSHGKSLEWIG DINPNYGGIT YNQKFKGKATLTVDTSΞSTAYMELRGLTSEDSAVYYC QAYYRNS DY WGQGTTLTVSS

3Z1G10H 154 HEIQLQQSGPELVKPGASVKISCKAS GYTFTD NYMN WVKQSHGKSLEWIG DINPYYGTTT YNQKFKGKATLTVDKSSRTAYMELRGLTSEDSAVYYC ARDDWF DY WGQGTLVTVSA

3zlD01H 155 HEVQLQESGPDLVKPSQSLSLTCTVT GFSITSGYGWH WIRQFPGDKLEWMG YIS FNGDYN YNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYC ASSYDGLFAY WGQGTLVTVSA

3zlC02H 156 HDVQLQESGPDLVKPSQSLSLTCTVT GFSITSGYGWH WIRQFPGNKLEWMG YIS FNGDSN YNPSLKSRI S ITRDTSKNQFFLQLNSVTSEDTATYYC ASSYDGLFAY WGQGPLVTVSA

3zlC04H 157 HEVQLQESGPDLVKPSQSLSLTCTVT GFSITSGYGWH WIRQFPGNKLEWMG YIN YDGHND YNPSLKSRI SITQDTSKNQFFLQLNSVTTEDTATYYC ASSYDGLFAY WGQGTLVTVSA