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1. (WO2012119989) ANTIBODIES AGAINST CADMI FOR THE DIAGNOSIS AND TREATMENT OF CANCER
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METHODS AND ANTIBODIES FOR THE DIAGNOSIS AND

TREATMENT OF CANCER

FIELD OF THE INVENTION

The invention relates to antibodies/binding molecules that specifically bind to Cell Adhesion Moiecule-1 (CADM 1 ). The use of these antibodies/binding molecules in human and veterinary medicine, for example in the treatment and diagnosis of melanoma, prostate cancer and other cancers is also subject of the present invention. Further, compositions comprising the antibodies are also provided herein.

BACKGROUND OF THE INVENTION

Today prostate-specific antigen (PSA) is the biomarker mostly used in clinical practice for detecting and predicting risk and aggressiveness of prostate cancer (Catalona et al . (1 991 ) N. Engl. J. Med. 324, 1 1 56-1 161 , Cooner et al. (1990) J. Urol. 143, 1 146-1 1 52, Antenor et al. (2005) Urology 66, 1 56-1 60) as well as for monitoring the disease before and after treatment (Ercole et al. (1987) J. Urol. 138, 1 181 -1 184, Pound et al. (1 999) JAMA 281 , 1591 -1597).

However, PSA has shown important limitations in the diagnosis of prostate cancer, especially a low specificity. Several non-cancer conditions frequently lead to serum PSA elevations. Benign prostate hyperplasia (BPH) is the most common cause of false positives due to the increasing prevalence with advancing age. Prostatitis, prostatic manipulation and some medical interventions are other processes which lead to unspecific increased PSA (Nadler et al. (1995) J. Urol. 1 54, 407-413). Moreover, 15% of prostate cancer cases occur in patients with low serum PSA levels (Thompson et al. (2004) N, Engl. J, Med. 350, 2239-2246).

Other PSA related biomarkers have increased the diagnostic potential of total serum PSA. PSA and free PSA are the only two assays approved by the

FDA for the testing of prostate cancer. The ratio of free to total PSA (%fPSA) lowers in men with prostate cancer (Christensson et al. (1 993) J. Urol. 105, 100-105). Despite differences in studies of different populations, %fPSA was able to predict a future diagnosis of aggressive cancer up to 1 0 years prior to diagnosis (Carter et al. (1 997) Urology 49, 379-384, Partin et al. (2003) J. Urol. 1 69, 384). However, the performance of this biomarker is also affected by prostatic manipulation (Lilja et al. (1 999) J. Urol. 1 62, 2029-2034). Recently, additional subforms of PSA have been detected in serum by proteomic approaches. They seem to have a higher sensitivity and specificity than PSA and %fPSA, but more detailed studies are required (Sarrats et al. (201 0) Prostate 70, 1 -9).

The PSA density (PSAD) incorporates the prostate size of each individual and is associated with a greater risk of prostate cancer as compared to BPH (Catalona et al. (2000) Urology 56, 255-260). PSA velocity (PSAV) is defined as the change in PSA per year (ng/m!/year) and has been shown by several studies to be a good predictor of the presence of prostate cancer and aggressiveness (Carter et al. (1992) Cancer Res. 52, 3323-3328, D'Amico et al. (2004) N. Engl. J. Med. 351 , 125-135, Carter et al. (2006) J. Natl. Cancer Inst. 98, 1 521 -1 527). Another modification of PSA determination, namely PSA doubling time (PSADT), is associated with lower survival (Makarov et al. (2008) J. Urol. 1 79, 156-161 ).

Other promising prostate cancer biomarkers are under study for prostate cancer screening, detection and prognostication. This group includes serum markers such as EPCA (Dhir et al. (2004) J Urol. 171 , 1419-1423, Uetsuki et al (2005) J Urol. 1 74, 514-518) and EPCA-2 (Leman et al. (2007) Urology 69, 714-720); tissue markers such as AMACR (Luo et al (2002) Cancer Res. 62, 2220-2226, Rubin et al. (2002) JAMA 287, 1 662-1670), methylated GSTP1 (Lee et al. (1994) PNAS 91 , 1 1733-1 1737, Harden et al. (2003) J Urol. 1 69 , 1 138-1 142), TMPRSS2-ETS gene rearrangement (Tomlms et al. (2005) Science 31 0, 644-648); and urine markers.

Amongst ail of them, current research mainly focuses on serum markers: HAAH (Ghanbari et al. (2006) First AACR International Conference on Molecular Diagnostics in Cancer Therapeutic Development, Sep 1 2-15, Ghanbari (2004) Journal of Biomedicine and Biotechnology 4, 1 75-1 76), AZGP1 (Henshall et al . (2006) J Natl Cancer I nst. 98, 1420, 1424, Bondar et at. (2007) Clin Chem. 53, 673-678), PAP (Taira et al. (2001 ) Oncology

(Williston Park) 21 , 1 003-1 01 0), CXCL1 3 (Singh et al. (2009) Cancer Lett. 283, 29-35), TIMP-2 (Ross et al (2003) Mod Pathol. 16, 198-205), some M MPs (Morgia et al. (2005) Urol Res. 33, 44-50, Brehmer ef al. (2003) prostate Cancer Prostatic Dis. 6, 217-222) including MMP-7 (Hashimoto et al. (1 998) J Urol. 160, 1 872-1876), PSMA (Xiao et al. (2001 ) Cancer Res. 61 , 6029-6033, Chikkaveeraiah et al. (2009) Anal Chem. 81 , 9129-9134) or CGA (Sciarra et al. (2009) Urol Int. 82 , 147-151 ). The up-regulation of these markers is often associated with poor prognosis (see references above).

Recently two different studies found the serum proteins activin A and procollagen type 1 aminoterminal propeptide (P 1 NP) elevated in patients with bone metastases compared to confined prostate cancer (Leto et al.

(2006) Clin. Exp. Metastasis. 23, 1 1 7-22, Klepzig et al. (2009), Anticancer Res. 29, 671 -673). Another study identified serum pro-l collagen peptide (PiCP) as a biomarker useful to detect bone metastases (Zissimopoulos et al. (2009) J BUON 14, 463-472). Other proteins associated with lymph node metastases are FABP5 (detectable in serum), MCCC-2, ezrin , PPA2 and SLP2 (Pang et al . (2010) J Proteome Res. 9, 21 6-26).

The prostate cancer urinary biomarkers identified are mainly DNA-, RNA- or protein-based biomarkers. Methylated GSTP 1 can be detected in urine with methylation specific PCR and used to detect prostate cancer with a high specificity but low sensitivity (Goessl et al. (2000) Cancer Res. 60, 5941 -5945, Jeronimo et al. (2002) Urology 60, 1 131-1 1 35) that can be improved by previous prostate massage (Goessl ef al. (2001 ) Urology 58, 335-338). Several RNA biomarkers can be detected by PCR on urine sediments obtained after prostatic massage with good sensitivity and specificity, mainly PCA3 (DD3) (Fradet et al. (2004) Urology 64, 31 1 -31 6, Deras et al. (2008) J Urol . 1 79, 1 587-1 592) and TMPRSS2: ERG fusion transcripts (Hessels et al.

(2007) Clin Cancer Res. 13, 5103-51 08, Nilsson et al. (2009 ) British Journal of Cancer 100, 1603 - 1607). The group of protein urinary biomarkers includes thymosin β 1 5 (TB-15) (Hutchinson ef al. (2005) prostate 64, 1 16™ 1 27, Hutchinson et al (2005) Clin Biochem 38, 558-571 ), AMACR (Rogers et al. (2004) J Urol 1 72, 1 501 -1 503), mini-chromosome maintenance-5 (MCM-5) (Stoeber et al.(2002) J Natl Cancer Inst. 94, 1071 -1079) and Annexin A3 after DRE combined with PSA (Schostak et al. (2009) The Journal of Urology 181 , 343-353). However, with the exception of Annexin A3, most of these studies are rather small and need confirmation.

Metabolomic profiles have identified sarcosine, an N-methyl derivative of the amino acid glycine, as a differential metabolite that increases during prostate cancer progression to metastasis. Sarcosine can be detected non-invasively in urine and may have a role in prostate cancer invasion (Sreekumar et al. (2009) Nature 457, 910-915).

Today screening for prostate cancer usually includes PSA determination in serum and a DRE (digital rectal examination). Depending on the results, diagnosis of prostate cancer is confirmed or excluded by a prostate biopsy. 6-10 samples of different areas of the prostate are obtained and analyzed by an expert pathologist. Samples undergo microscopic examination and the clinical stage (TIMM system) and Gleason grade are scored.

TNM classification is as follows (European Association of Urology, Aus G , Abbou CC, Bolla M, Heidenreich A, Van Poppei H . , et al . EAU Guidelines on prostate Cancer. 2007):

T: primary tumor extension. Subdivided in different categories from T0 (no evidence of primary tumor) to T4.

N: propagation to regional lymph nodes. N0 (absence) or N1 (presence).

M: propagation to distant lymph nodes or metastatic sites. May be M0 (absence) or M 1 (presence)

The grading system proposed by Gleason (Gleason et al (1974) J Urol. 1 1 1 ,58-64) is based on a pathological examination of prostate tissue obtained by a biopsy. The result is an average index of abnormality, for which values between 2 and 10 can be taken .

GX The degree of differentiation cannot be assessed .

G 1 Well differentiated (weak anaplasia): Gleason 2-4.

G2 Moderately differentiated G2 (moderate anaplasia): Gleason 5-6.

G3-4 Poorly differentiated/undifferentiated (marked anaplasia): Gleason 7-1 0.

There is no model that can reliably predict the ciinical behaviour of prostate cancer. Currently, patients diagnosed with prostate cancer are stratified into three risk groups (low, intermediate and high) based on clinical and pathological variables: PSA at diagnosis, Gleason score and clinical tumor stage (prostate. In; AJCC Cancer Staging Manual. Eth ed. New Your, Springer, 2002, pp 309-316, Montie (1 995) Cancer 75 (7 Suppl), 1814-1 818). (D'Amico et al. (1999) J Clin Oncol. 1 7, 1 68-, Kattan et al. (1998) J Natl Cancer Inst 90, 766-, Partin et al. (1 993) J Urol. 1 50, 1 10-, Ross et at. (2001 ) J Urol 1 65, 1562-).

The D'Amico classification for localised or locally advanced clinical stages helps to predict outcome and define treatment options (D'Amico et al. ( 1998) JAMA. 280(1 1 ):969-74; and D'Amico et al. J Clin Oncol. 2002;20(23):4567-13) Low risk: cT1 -cT2a, Gleason < 7 and PSA ≤10 ng/ml; I ntermediate risk: cT2b, Gleason = 7 or (PSA > 1 0 and ≤20 ng/ml); and H igh risk: cT2c or PSA > 20 ng/ml or Gleason > 7.

Cell adhesion molecule-1 or CADM 1 , also known as tumor suppressor in lung cancer 1 (TSLC 1 ), immunoglobulin superfamily member 4(I GSF4), synaptic cell adhesion molecule (synCAM) and other aliases, is thought to mediate cell-cell adhesion in a Ca÷+ independent manner and in some cancers is thought to act as a tumor suppressor, CADM1 is a member of the immunoglobulin super family of proteins.

It is reported that CADM 1 has tumor suppressor activity with loss of expression associated with poor prognosis in some lung cancers (Goto et al. (2005) Cancer Sci. 96:480-486), colorectal cancer (Chen et al. (201 0) Int J. Cancer 128(2), 266-73), melanoma (You et al, Melanoma Research 2010, 20: 1 79-83) and prostate cancers (Fukuhara et al. ((2002) Jpn J. Cancer Res. 93(6), 605-9)) amongst others. In other cancers CADM 1 is a therapeutic and diagnostic target; in particular, CADM 1 has been proposed as a therapeutic target for some types of lung cancer (Kitamura et al, Biochem. Biophys. Res. Commun. 2009, 383, 480-84). Antibodies to CADM 1 are described in e.g. , US pub. no. 2009/0053243 to Kurosawa et al. and WO 2010/1021 75 to Terret et al. Characteristics of CADM1 include (see e.g., Ito et al. ((2008) J. Smooth Muscle Res 44:83-93); Watabe et al. (2003) Histol. Histopathol. 1 8: 1321 -1 329; and Kawano et al. (2009) J. Biol. Cfrem.284(35)23793-805);

- interaction with CRTAM and promotes natural killer (NK) eel! cytotoxicity and interferon-gamma (I FN-gamma) secretion by CD8+ cells in vitro as wei! as NK cell-mediated rejection of tumors expressing CADM3 in vivo;

- contribution to the less invasive phenotypes of iepidic growth tumor cells;

- mediation of attachment to and promote communication with nerves in mast cells;

- with MITF being essential for development and survival of mast cells in vivo;

- acting as a synaptic cell adhesion molecule that drives synapse assembly;

- the extracellular region of CADM1 directly interacts in cis with that of ErbB3 and reduces the HRG-induced signalling pathways of the ErbB3/Erb82 heterodimer for cell movement and survival;

- involvement in neuronal migration, axon growth, pathfinding, and fasciculation on the axons of differentiating neurons; and

- roles in the spermatogenesis including in the adhesion of spermatocytes and spermatids to Sertoli cells and for their normal differentiation into mature spermatozoa .

Fukuhara et at. {(2002) Jpn J, Cancer Res, 93(6), 605-9) report that CADM1 is absent or markedly reduced in 3 out of 4 prostate cancer cell lines. In PPC-1 cells the CADM 1 promoter was heavily methylated and significant methylation was found in primary prostate cancers also. Lastly restoration of CADM1 in PPC-1 cells in nude mice suppressed tumor formation. Murakami (Cancer Sci 2005 96(9):543-552) reports that CADM1 (TSLC 1 ) is inactivated in numerous cancers including 44% of non-small cell lung cancers and 30-60% of other cancers including liver, pancreatic and prostate cancers, more prominently in those with invasion or metastasis. You et al (Melanoma Research 201 0, 20: 179-83) report that CADM 1 (TSLC1 ) was lost in 84 of 120 cutaneous melanoma samples, and further that CADM1 promoter was methylated in 58 of 1 20 samples. The incidence of the loss of CADM1

expression and methylation of CADM 1 significantly increased as the tumor stage advanced. CADM1 was accordingly speculated as a tumor suppressor in cutaneous melanoma.

What is clear is that current methods and biomarkers for the diagnosis and characterization of cancer, including melanoma, colon and/or colorectal cancer and prostate cancer (e.g., prognosis of prostate cancer, prediction of behavior, selection of therapeutic regiments, differential diagnosis, etc. ) need improvement and new cancer biomarkers such as prostate cancer biomarkers are needed . Furthermore, current methods for treating and/or preventing cancer, such as melanoma, colon and/or colorectal cancer or prostate cancer, are also in need of improvement, and new therapeutic targets are needed with new mechanism of action as compared to previous drugs. The present invention addresses these needs.

BRIEF SUMMARY OF THE INVENTION

The invention relates to the finding that CADM1 is overexpressed in prostate cancer tissue as compared to normal tissue in biological samples at the RNA and protein level. The studies described herein show that CADM1 was overexpressed in prostate cancer tissues as compared to control values obtained from matched non-affected tissue using expression microarray data in two independent sample sets. During the inventor's investigation of CADM1 , it was found that it was also overexpressed at the protein level in prostate tumor tissue in a new set of samples on a tissue microarray. The inventors have shown that CADM1 is upregulated in a statistically significant manner in prostate cancer at the mRNA and upregulation at protein level is also observed. The overexpression of CADM1 was found in prostate cancers having diverse histologies and stages.

The finding that CADM1 is overexpressed in prostate cancer, coupled to investigation of the potential cellular localization and function of CADM1 indicates that it is a new therapeutic target in prostate cancer. Furthermore, CADM1 is a tumor, blood, serum, plasma, and/or urine based biomarker for the detection of prostate cancer. A CADM1 biomarker may be a protein or nucleic acid biomarker which correlates to the levels of CADM1 protein or mRNA.

Furthermore, the invention also provides experimental data showing that CADM1 is expressed in melanoma and colon cancer and that melanoma and colon and/or colorectal cancer can be addressed via targetting CADM1 .

In one embodiment of the invention, CADM1 is a target for a cancer therapeutic target. The invention therefore provides a method for treating cancer comprising administering to an individual in need of treatment a therapeutic agent that modulates CADM1 levels or activity, in a more specific aspect, the invention provides a method for treating melanoma, colon and/or colorectal cancer or prostate cancer comprising administering to an individual in need of treatment a therapeutic agent that modulates CADM1 levels or activity. In one aspect, the therapeutic agent that modulates CADM1 is chosen from an antisense molecule, an interfering RNA molecule, a small organic molecule, and a therapeutic antibody or fragment thereof. In a more specific aspect, the therapeutic agent is an antibody or fragment thereof that binds CADM1 protein.

In one embodiment of the invention, CADM1 is a diagnostic cancer biomarker. In one aspect of this embodiment, CADM1 is a diagnostic marker for prostate cancer. Thus, in one aspect, the invention provides a method for diagnosis of cancer by determining the level of a CADM1 biomarker in a biological sample from an individual wherein an altered level of CADM1 biomarker in the biological sample is diagnostic of cancer or an increased likelihood of cancer. In a more specific aspect, the invention provides a method for diagnosis of prostate cancer by determining the level of a CADM1 biomarker in a biological sample from an individual wherein an altered level of CADM1 biomarker in the biological sample is diagnostic of prostate cancer or an increased likelihood of prostate cancer. In a specific aspect, an increased or elevated level of CADM1 biomarker in the biological sample is diagnostic of prostate cancer or an increased likelihood of prostate cancer. In one aspect, of this embodiment, the biological sample is chosen from tissue, tumor, blood, serum, plasma and urine. According to the invention, the CADM1 biomarker can be a nucleic acid or protein biomarker. Various methods known to the skilled artisan can be used to detect a CADM1 biomarker including, but not limited to, PCR, quantitative PCR, multiplex PCR, nucleic acid microarray, northern blot, RNase protection assays, serial analysis of gene expression (SAGE), immunohistochemistry, western blot, ELISA, and radioimmunoassay. In a more specific aspect of this diagnostic embodiment, protein levels of CADM1 are determined using an antibody or fragment thereof that binds CADM1 protein.

In one embodiment, the invention provides an antibody or fragment thereof to CADM1 protein or a CADM1 fragment or epitope, !n one aspect of this embodiment, the invention provides an antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the CADM1 epitope is chosen from one or more of the peptide sequences of CADM1 as described in more detail below, in one aspect of this embodiment, the CADM1 epitope is an extracellular epitope. In one aspect of this embodiment, the CADM1 epitope is an intracellular epitope. In one aspect of this embodiment, the antibody or fragment thereof is chosen from a monoclonal antibody or a recombinant antibody, or a fragment thereof that binds to CADM1 or a CADM1 epitope, in one aspect of this embodiment, the antibody or fragment thereof is a recombinant antibody chosen from a chimeric antibody, a humanized antibody, a fully human antibody, or a fragment thereof, that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the antibody or fragment thereof, is a monoclonal antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the antibody or fragment thereof is a recombinant antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the antibody or fragment thereof is a chimeric antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the antibody or fragment thereof is a humanized antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the antibody or fragment thereof is a fully human antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the antibody or fragment thereof binds selectively to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the antibody or fragment thereof binds specifically to CADM1 or a CADM1 epitope. In one aspect, the antibody to CADM1 is conjugated to another agent. In one aspect, the antibody or fragment thereof is conjugated to a toxin, a therapeutic agent, or a detectable label. In another aspect, the antibody or fragment thereof blocks an active site or a binding site on CADM1 or inhibits the activity of CADM1 or a CADM1 fragment or epitope, in one aspect of this embodiment, the antibody is a therapeutic antibody or a diagnostic antibody. In one aspect, the antibody is a therapeutic antibody. In one aspect, the antibody is a diagnostic antibody.

Accordingly, the present invention relates to antibodies, antibody fragments or binding molecules specifically binding to CADM1 . The term "binding molecule" in accordance with this invention relates to functional fragments or functional derivatives of the herein disclosed and the herein defined antibodies.

In one embodiment, these antibodies, antibody fragments or binding molecules comprise the variable regions and/or CDRs as provided herein . The invention also provides for antibodies/binding molecules that comprise CDRs and/or variable regions that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the CDRs and variable regions as provided herein. Furthermore, the present invention also provides for antibodies that bind to/recognize the same epitope as any of the antibodies as disclosed herein.

Preferred antibodies of the invention as disclosed below exhibit remarkable functional properties, which make them particularly suitable for use in the therapy or diagnosis of CADM1 -mediated disorders, including cancer, in particular, said antibodies exhibit one or more of the following properties:

- very high affinity for human CADM1 ;

- high specificity for CADM 1 relative to other CADM proteins (CADM2, CADM3 and CADM4);

they bind with high affinity to CADM1 expressed on tumors;

- they show very efficient internalization by CADM 1 -expressing cells, including CADSVS1 -expressing cancer cell lines, resulting in very potent cancer cell-killing effects when administered conjugated with another agent such as a toxin . They are thus very effective carriers for targeted delivery of cancer-cell killing agents into cancer cells. Particularly good results have been obtained against melanoma, colon cancer and prostate cancer cell lines;

- they are not hemolytic.

The invention further provides nucleic acids encoding the antibodies/antibody fragments or binding molecules according to the invention. Also provided are vectors, such as expression vectors, comprising said nucleic acids, and host cells comprising said nucleic acids or said vectors.

The invention further provides a process for the production of an antibody/antibody fragment/binding molecule as disclosed herein , said process comprising culturing a host comprising a nucleic acid or vector encoding said antibody/antibody fragment/binding molecule under conditions allowing the expression of the antibody/antibody fragment/binding molecule

and recovering the produced antibody/antibody fragment/binding molecule from the culture.

In another aspect of the invention, there is provided a method for identifying an antibody or fragment thereof that binds to a CADM1 protein or a CADM1 epitope said method comprising: (a) providing a CADM1 protein or CADM1 epitope; (b) contacting said CADM1 protein or CADM1 epitope with an antibody or fragment thereof; and (c) identifying an antibody or fragment thereof that binds to a CADM1 protein or CADM1 epitope.

in another aspect, there is provided a monoclonal antibody that binds to a CADM1 protein or CADM1 epitope prepared by a process comprising: (a) providing a hybridoma capable of producing the monoclonal antibody; and (b) culturing the hybridoma under conditions that provide for the production of the monoclonal antibody by the hybridoma. in another aspect, the invention further includes a hybridoma that produces the monoclonal antibody described herein.

in another aspect, there is provided a recombinant antibody that binds to CADM1 or a CADM1 epitope prepared by a process comprising: (a) providing a cell line capable of producing a recombinant antibody; and (b) culturing the cell Sine under conditions that provide for the production of the recombinant antibody by the cell Sine. In another aspect, the invention further includes a cell line that produces a recombinant antibody described herein.

In one embodiment, the invention provides nucleic acid based cancer diagnostic reagents for detecting the levels of a CADM1 biomarker.

In one embodiment, the invention provides nucleic acid based cancer therapeutic agents for modulating the level or activity of CADM1.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : FIG. 1 illustrates results of cell viability studies with several antibodies of the invention (squares indicate Antibody-8 and triangles indicate Antibody-10). The upper panel is with LNCaP cells and the bottom panel is with DU145 cells. See Example 8 for description.

Figure 2: FIG. 2 illustrates results of cell adhesion studies with Antibody-10 of the invention using DU145 cells and Fibronectin as indicated. See Example 8 for description.

Figure 3: FIG. 3 illustrates results of cell migration studies with Antibody-10 of the invention using BxPC3 cells. See Example 8 for description.

Figures 4, 5 and 6: Fig 4, 5 and 6 illustrate the results of the specificity study disclosed in Example 19 with AB2 (Fig 4), AB3 (Fig 5) and AB10 (Fig 6), respectively. The graphs show the signal detected on CHO cells, negative for CADM1 expression (grey), no transfected Clon 139 (dotted grey line), Clon 139 transfected with an siRNA control (grey line), and Clon 139 transfected with siRNA s24325 selected to reduce CADM1 expression (black line).

Figures 7, 8 and 9: Fig. 7, 8 and 9 show the signal detected on CHO transfected with each CADM family member (A: CADM1 ; B:CADM2; C: CADM3; D:CADM4) after incubation with AB2 (Fig 7), AB3 (Fig 8) and AB10 (Fig 9) and anti-human fluorescence secondary antibody. See example 20 for description.

Figures 10 and 1 1 : Fig 10 illustrates the results of affinity studies to CADM1 by cytometry with AB10 using A375 cells, and Fig 11 shows the results obtained with AB3 using SKOV3 cells. See example 21 for description. The graphs show a representation of concentration of the antibody versus value of FACS Geometric mean .

Figure 12 and 13: Fig 12 and 13 illustrate the effect on cell viability of the antibody-saporin conjugates AB3-SAP (Fig 12) and AB10-SAP (Fig 13) in the assay disclosed in Example 24 using A375 cells. The graphs show a representation of % cell viability versus concentration of the antibody.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the finding that CADM1 is overexpressed in prostate cancer tissue as compared to normal tissue at the RNA and protein level. The studies described herein show that CADM1 was overexpressed in a first set of samples having 27 primary prostate cancer tumor tissues as compared to matched non-affected tissue. A second study with 7 prostate cancer tumor samples compared to non-affected matched tissue confirmed the first study showing that CADM1 was overexpressed in these samples. During the inventor's investigation of CADM1 , it was found that it was also overexpressed at the protein level in tumors in another set of samples which included high grade PINs, low-grade and high-grade prostate adenocarcinomas on a tissue microarray. The inventors have shown that CADM1 is upregulated in a statistically significant manner in prostate cancer at the mRNA. Furthermore, the overexpression of CADM1 is observed at the protein level. The overexpression of CADM1 was found in prostate cancers having diverse histologies and stages. Without wishing to be bound by any particular theory, the inventors believe that given the above results, and with knowledge of the properties of CADM1 , CADM1 is a biomarker as well as a therapeutic and/or diagnostic target for prostate cancer. In particular, the inventors have found a group of antibodies that bind CADM1 and have surprising therapeutic or diagnostic benefits.

Furthermore, CADM1 is a tissue, tumor, blood, serum, plasma, and/or urine based biomarker for the detection of cancer, including prostate cancer, melanoma and colon and/or colorectal cancer. The CADM1 biomarker can be a protein or nucleic acid biomarker.

Furthermore, the inventors have also found that CADM1 is expressed in melanoma and colon cancer cell lines and that melanoma and colon and/or colorectal cancer can be addressed via targeting CADM1 expressed in said cancers.

in particular, the inventors have identified a group of antibodies or binding agents against CADM1 that exhibit high affinity and specificity for CADM1 and are particularly suitable for use as therapeutic or diagnostic agents for disorders or diseases wherein CADM1 is expressed or involved. These antiCADM1 antibodies are particularly suited for use in the field of cancer, particularly in melanoma, colon and/or colorectal cancer and prostate cancer.

Therapeutic Antibodies to CADM1 protein and CADM1 epitopes

In one embodiment, the invention provides a therapeutic antibody or fragment thereof that binds to a CADM1 protein or a CADM1 epitope. In one aspect of this embodiment, the therapeutic antibody or fragment thereof is a monoclonal antibody or fragment thereof or a recombinant antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the therapeutic antibody or fragment thereof is a monoclonal antibody or fragment thereof that binds to CADM1 or a CADM 1 epitope. In one aspect of this embodiment, the therapeutic antibody or fragment thereof is a recombinant antibody or fragment thereof that binds to CADM1 or a CADM1 epitope, in one aspect of this embodiment, the recombinant antibody or fragment thereof that binds to CADM1 or a CADM1 epitope is chosen from a chimeric, humanized, or fully human antibody or a fragment thereof. In one aspect of this embodiment, the therapeutic antibody or fragment thereof is a chimeric antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the therapeutic antibody or fragment thereof is a humanized antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In one aspect of this embodiment, the therapeutic antibody or fragment thereof is a fully human antibody or fragment thereof that binds to CADM1 or a CADM1 epitope. In an aspect of this embodiment, the therapeutic antibody or fragment thereof to CADM1 or a CADM1 epitope is conjugated to a toxin and/or therapeutic agent. The therapeutic antibody (or fragment thereof) as described above in this paragraph can be provided as a pharmaceutical composition comprising the antibody (or fragment thereof) and a pharmaceutically acceptable carrier. In a preferred aspect, the therapeutic antibody or fragment thereof as described above in this paragraph binds selectively to CADM1 or a CADM1 epitope. In another preferred, the therapeutic antibody or fragment thereof binds specifically to a CADM1 protein or a CADM1 epitope.

The therapeutic antibody of the present invention can be generated against a CADM1 protein or an epitope of a CADM1 protein. The therapeutic antibody of the present invention binds to a CADM1 protein or an epitope of a CADM1 protein, in one embodiment, the therapeutic antibody binds to or can be generated against a polypeptide having the full length sequence of a CADM1 protein as in SEQ ID NO;1-3 (See Example 4). Preferably, the binding site or epitope for therapeutic antibody is located on an extracellular domain of the protein as defined by the amino acid sequence as in SEQ ID NO:4. In another embodiment, the therapeutic antibody binds or can be generated against a epitope having the amino acid sequence of one or more of SEQ ID NOs:5-16, below which represent preferred epitopes of the extracellular domain of CADM1.



A preferred epitope is chosen from the amino acid sequences as in SEQ ID NO:4- 16.

In yet an even more preferred aspect, the epitope is the amino acid sequences as in SEQ ID NO:4.

The inventors have identified a number of antibodies raised against a CADM1 epitope. In particular, the CDR sequences as deduced from their corresponding nucleic acid sequences from the light and heavy chains were determined for these antibodies and are listed below. The nomenclature is as follows: VL = Variabie Light Chain; VH = Variable Heavy Chain; CDR refers to the specified complementary determining region; for a particular antibody. AB1 is Antibody-1 of the invention; AB2 is Antibody-2 of the invention; VL_CDR1 AB3 is the first CDR of the light chain of Antibody-3 of the invention. See Tabie 7 and Table 8 in Example 7.

Given that each of these antibodies can bind to CADM1 , their CDRs can be combined to create other antiCADM1 antibodies/binding molecules of the invention. CADM1 binding of said antibodies generated by combination of the above described CDRs can be tested using the binding assays described in the Examples.

Thus, therapeutic antibodies of the invention comprise one or more of the CDR sequences above or a sequence at least 75% identical, preferably at least 80% identical thereto. Preferably, the therapeutic antibody of the invention comprises one or more of the CDR sequences above or a sequence at least 90% identical thereto (e.g. 90%, 95%,96%,97%,98%,99% or more).

The invention thus provides an isolated antibody that specif ically binds CADM1 wherein said antibody has one or more CDRs, preferably 2 or more CDRs, more preferably 3 or more CDRs, and still more preferably 6 CDRs, each CDR corresponding to a CDR chosen from the CDRs of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 as defined herein, or a CDR sequence having 75% or more (e.g. 80%, 85%, 90%,95% ,96%,97%,98%,99% or more) amino acid identity to said CDR.

The invention also is a nucleic acid encoding one or more of the above listed CDR sequences or a CDR sequence at least 75% identical, preferably at least 80% identical thereto. Preferably, the nucleic acid encodes one or more of the above listed CDR sequences or a CDR sequence at least 90% identical thereto.

The invention also involves one or more of the CDR sequences above or a CDR sequence at least 75% identical, preferably at least 80% identical, more preferably at least 90% identical thereto wherein said CDR sequences is in the context of an antibody framework. Preferably, the antibody framework is a human antibody framework.

The term "CDR" as employed herein relates to "complementary determining region", which is well known in the art. The CDRs are parts of immunoglobulins and T cell receptors that determine the specificity of said molecules and make contact with specific ligand. The CDRs are the most variable part of the molecule and contribute to the diversity of these molecules . There are three CDR regions CDR1 , CDR2 and CDR3 in each V domain. CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain. H means the variable heavy chain and L means the variable light chain. The CDR regions of an Ig-derived region may be determined as described in Kabat ( 1991 ), Sequences of Proteins of Immunological Interest, 5th edit., N I H Publication no. 91 -3242 U .S. Department of Health and Human Services; Chothia (1 987), J . Mo I . Biol . 196, 901 -917; and Chothia (1 989) Nature, 342, 877-883.

Each CDR region of a variable heavy chain is herein interchangeably designated as CDR-H1 or VH-CDR1 , CDR-H2 or VH-CDR2, and CDR-H3 or VH-CDR3, respectively. Likewise, each CDR region of a variable light chain is designated herein CDR-L1 or VL-CDR1 , CDR-L2 or VL-CDR2, and CDR-L3 or VL-CDR3, respectively.

In one embodiment, the invention provides an isolated antibody that specifically binds to CADM1 , wherein the variable region of the heavy chain of said antibody comprises a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 79, SEQ ID NO.: 86, SEQ ID NO. 78, SEQ ID NO.: 77 or SEQ ID NO.: 84. In the context of the CDR3 of the heavy chain (VH_CDR3), the antibodies may also comprise a CDR sequence having at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to one of said CDRs.

In a further embodiment of the present invention, the variable region of the heavy chain of the antibody of this invention comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 55, SEQ ID NO: 62, SEQ ID NO: 54, SEQ ID NO: 53 or SEQ ID NO:60 , or a CDR sequence having 75% or more (e.g. 80%, 85%, 90%,91 %,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid identity to one of said CDRs.

In a further embodiment, the variable region of the heavy chain of the antibody of this invention comprises a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 67, SEQ ID NO: 74, SEQ ID NO: 66, SEQ ID NO: 65 or SEQ ID NO:72, or a CDR sequence having 75% or more (e.g. 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid identity to one of said CDRs.

in a further embodiment, the variable region of the heavy chain of said antibody comprises:

(i) a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 55 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 67 , and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 79;

(ii) a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO; 62 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 74 , and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 86;

(Hi) a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO:

54 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO:

66 , and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 78;

(iv) a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 53 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 65 , and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 77 or

(v) a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 60 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 72 , and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 84. The antibodies may also comprise a CDR sequence having 75% or more (e.g. 80%, 85%, 90% , 91 %, 92%, 93%, 94%, 95%, 96% , 97% , 98%, 99% or more) amino acid identity to one of said CDRs.

In a further embodiment, the variable region of the heavy chain of said antibody comprises:

(i) a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO:

55 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO:

67 , and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 79;

(ii) a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 62 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 74 , and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 86; or

(Hi) a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 54 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 66 , and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 78. The antibodies may also comprise a CDR sequence having 75% or more {e.g. 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%,96%,97%,98%,99% or more) amino acid identity to one of said CDRs.

In one embodiment, the variable region of the light chain of said antibody comprises a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO . : 43, SEQ ID NO.: 50, SEQ ID NO. 42, SEQ ID NO . : 41 or SEQ ID NO. : 48, or a CDR sequence having 75% or more (e.g. 80% , 85%, 90%, 95%, 96%, 97% , 98%, 99% or more) amino acid identity to one of said CDRs. In a further embodiment, the variable region of the light chain of said antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 18, SEQ ID NO: 17 or SEQ ID NO:24, or a CDR sequence having 75% or more {e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) amino acid identity to one of said CDRs. in yet a further embodiment, the variable region of the light chain of said antibody comprises a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 31 , SEQ ID NO: 38, SEQ ID NO: 30, SEQ ID NO: 29 or SEQ ID NO:36, or a CDR sequence having 75% or more (e.g. 80%, 85%, 90%, 91 % , 92%, 93%, 94%, 95% ,96%,97%,98%,99% or more) amino acid identity to one of said CDRs.

In a further embodiment of the present invention, the variable region of the light chain of the antibody of this invention comprises:

(i) a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 19 , a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 31 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 43;

(ii) a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 26 , a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 38 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 50;

(iii) a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO:

18 , a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO:

30 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 42;

(iv) a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 17 , a VL_CDR2 region having an amino acid sequence as depicted in SEQ iD NO: 29 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 41 ; or

(v) a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 24 , a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 36 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO . : 48. The antibodies may also comprise a CDR sequence having 75% or more (e.g. 80%, 85% , 90%, 91 % , 92%, 93% , 94% , 95%,96%,97% ,98%!99% or more) amino acid identity to one of said CDRs.

In a further embodiment of the present invention, the variable region of the light chain of the antibody of this invention comprises:

(i) a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO:

19 , a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO:

31 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 43;

(ii) a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 26 , a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 38 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 50; or

(iii) a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 1 8 , a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 30 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 42. The antibodies may also comprise a CDR sequence having 75% or more (e.g. 80%, 85% , 90%,91 %,92%, 93% , 94%, 95%,96%,97%,98%,99% or more) amino acid identity to one of said CDRs.

The antibodies/binding molecules etc. of the present invention may be characterized by at least one CDR sequence as described above. Preferably, the antibody comprises 2 or more CDRs. More preferably, the antibody comprises 3, 4, 5 or more CDRs. Still even more preferably, said antibody comprises 6 CDRs. Yet, even more preferably the antibody comprises a set of 6 CDRs: 3 CDRs in the variabie region of the Iight chain of the antibody and 3 CDRs in the variable region of the heavy chain of the antibody.

In a further embodiment, the invention provides the antibodies as follows:

(a) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:17, SEQ ID NO:29, SEQ ID NO:41 , SEQ ID NO:53, SEQ ID NO:65, SEQ ID NO:77 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(b) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO: 18, SEQ ID NO:30, SEQ ID NO:42, SEQ ID NO:54, SEQ ID NO:66, SEQ ID NO:78 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences, in a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(c) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO: 19, SEQ ID NO:31 , SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:67, SEQ ID NO:79 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs, Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(d) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:56, SEQ ID NO:68, SEQ ID NO:80 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(e) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:21 , SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, SEQ ID NO: 81 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(f) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:46, SEQ ID NO:58, SEQ ID NO:70, SEQ ID NO:82 or a CDR having an amino acid sequence at least 75%, more preferably at (east 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Stili yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(g) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:23, SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:59, SEQ ID NO:71 , SEQ ID NO:83 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(h) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:84 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer,

(i) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:49, SEQ ID NO:61 , SEQ ID NO:73, SEQ ID NO:85 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(j) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:62, SEQ ID NO:74, SEQ ID NO:86 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(k) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:87 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(I) An isolated antibody comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:28, SEQ ID NO:40, SEQ ID NO:52, SEQ ID NO:64, SEQ ID NO:76, SEQ ID NO:88 or a CDR having an amino acid sequence at least 75%, more preferably at least 90% identical to any of said CDRs. Preferably the one or more CDRs are at least 95% identical to said amino acid sequences. Preferably, said antibody comprises 2 or more CDRs. Even more preferably, said antibody comprises 3 or more CDRs. Still even more preferably, said antibody comprises 4 or more CDRs. Yet even more preferably said antibody comprises 5 or more CDRs. Still yet more preferably said antibody comprises 6 CDR sequences. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

In a further embodiment, the invention provides antibodies designated AB3, AB10, AB2, AB1 and AB8, and preferably antibodies designated AB3, AB10 and AB2, as well as antibodies having CDR sequences having 75% or more (e.g. 80%, 85% , 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid identity to the CDRs in said preferred antibodies.

Accordingly, in a further embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 19, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 31 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 43, or a CDR sequence having 75% or more (e.g . 80%, 85%, 90%,91 %,92%, 93%, 94%, 95%,96%,97%,98%,99% or more) amino acid identity to one of said CDRs; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 55, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 67, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 79, or a CDR sequence having 75% or more (e.g.

80%, 85%, 90%,91 %,92%, 93%, 94%, 95%,96%,97% ,98%,99% or more) amino acid identity to one of said CDRs. In a specific aspect of this embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 19, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 31 , and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 43 ; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 55, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 67, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 79.

In a further embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 26, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 38, and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 50, or a CDR sequence having 75% or more (e.g. 80% , 85%, 90%, 91 %, 92% , 93% , 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid identity to one of said C DRs; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 62, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 74, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO . : 86, or a CDR sequence having 75% or more (e.g. 80% , 85%, 90% , 91 %, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or more) amino acid identity to one of said CDRs. in a specific aspect of this embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 26, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 38, and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 50; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 62, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 74, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 86.

in a further embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in

SEQ ID NO: 18, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 30, and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 42, or a CDR sequence having 75% or more (e.g. 80%, 85% , 90%, 91 %, 92%, 93%, 94%, 95%,96%,97%,98%,99% or more) amino acid identity to one of said CDRs; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO : 54, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 66, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 78, or a CDR sequence having 75% or more (e.g. 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%,96%,97%,98% ,99% or more) amino acid identity to one of said CDRs. Sn a specific aspect of this embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 1 8, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 30, and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 42; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 54, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 66, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 78.

In a further embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 17, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 29, and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 41 , or a CDR sequence having 75% or more (e.g. 80%, 85%, 90%,91 %,92%, 93%, 94%, 95%,96% ,97%,98%,99% or more) amino acid identity to one of said CDRs; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 53, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 65, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 77, or a CDR sequence having 75% or more (e.g. 80% , 85%, 90%, 91 %, 92%, 93%, 94%, 95% , 96% , 97%, 98%, 99% or more)amino acid identity to one of said CDRs. In a specific aspect of this embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 17, a VL_CDR2 region having an amino acid

sequence as depicted in SEQ ID NO: 29, and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 41 ; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 53, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ! D NO: 65, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 77.

In a further embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 24, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 36, and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 48, or a CDR sequence having 75% or more (e.g. 80% , 85%, 90%, 91 %, 92%, 93%, 94%, 95% ,96%,97% ,98% ,99% or more) amino acid identity to one of said CDRs; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 60, a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 72, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO. : 84, or a CDR sequence having 75% or more (e.g . 80%, 85%, 90% , 91 %, 92% , 93%, 94%, 95%, 96%, 97% , 98%, 99% or more) amino acid identity to one of said CDRs. In a specific aspect of this embodiment, the variable region of the light chain of the antibody comprises a VL_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 24, a VL_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 36, and a VL_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 48; and the variable region of the heavy chain of the antibody comprises a VH_CDR1 region having an amino acid sequence as depicted in SEQ ID NO: 60 , a VH_CDR2 region having an amino acid sequence as depicted in SEQ ID NO: 72, and a VH_CDR3 region having an amino acid sequence as depicted in SEQ ID NO.: 84.

Variable Heavy and Light Chain Amino Acid Sequences to corresponding to antibodies of the invention with CDR sequences underlined are shown below (Nucleic sequence for several Heavy and Light Chains are also given). VL refers to variable lights chain and VH refers to variable heavy chain.

Nucleic acid sequence encoding amino acid, sequence of AB1_VL

ABl^VH

Nucleic acid sequence encoding amino acid sequence of AB1_VH

Nucleic acid sequence encoding amino acid sequence of AB2_VL

Nucleic acid sequence encoding amino acid sequence of AB2_VH

Nucleic acid sequence encoding amino acid sequence of AB3_VL

Nucleic acid sequence encoding amino acid sequence of AB3_VH

Nucleic acid sequence encoding amino acid sequence of AB8_VL

Nucleic acid sequence encoding amino acid sequence of AB8_VH

Nucleic acid sequence encoding amino acid sequence of AB10_VL

Nucleic acid sequence encoding amino acid sequence of AB10_VH

Thus, therapeutic antibodies can comprise one or more of the heavy or light chain variable sequences above or a sequence at least 75%, preferably at least 80% identical thereto. More preferably, the therapeutic antibodies can comprise one or more of the heavy or light chain variable sequences above or a sequence at least 90% identical thereto.

The invention thus provides an isolated antibody that specifically binds CADM1 wherein said antibody has one or more light chain variable domain corresponding to Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 as defined herein, or a sequence having 75% or more (e.g. 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95% , 96% , 97%, 98%, 99% or more) amino acid identity to said light chain variable domain.

In another embodiment, the invention provides an isolated antibody that specifically binds CADM1 wherein said antibody has one or more heavy chain variable domain corresponding to Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 as defined herein, or a sequence having 75% or more (e.g. 80% , 85%, 90%, 91 %, 92%, 93%, 94%, 95%,96%,97%,98%,99% or more) amino acid identity to said heavy chain variable domain.

In another embodiment, the invention provides an isolated antibody that specifically binds CADM1 wherein said antibody has:

(i) one or more light chain variable domain corresponding to Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 as defined herein, or a sequence having 75% or more (e.g. 80%, 85%, 90%, 91 %, 92%, 93% , 94% , 95%,96%,97%,98%,99% or more) amino acid identity to said light chain variable domain; and

(ii) one or more heavy chain variable domain corresponding to Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-11 , or Antibody-12 as defined herein, or a sequence having 75% or more (e.g . 80%, 85%, 90%, 91 %, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid identity to said heavy chain variable domain.

The invention also involves nucleic acids encoding one or more of the above listed heavy or light chain variable sequences or a heavy or light chain variable sequences sequence at least 75%, preferably at least 80%, more preferably at least 90% identical thereto. Several Exemplary nucleic acid sequences for variable domains and CDRs are given for Antibody-1 (AB1 ), Antibody-2 (AB2), Antibody-3 (AB3), Antibody-8 (AB8) and Antibody-10 (AB10). Thus, the invention relates to a composition comprising a nucleic acid as in SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:1 10, SEQ ID NO:1 12, SEQ ID 1 17, SEQ ID 1 18; SEQ ID 1 19, SEQ ID 120, SEQ ID 121 , SEQ ID 122 or a sequence 75%, preferably 80%, more preferably 90 % or more identical thereto, or the complement thereof. The invention also relates to a composition comprising a nucleic acid as in SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:1 10, SEQ ID NO:1 12, SEQ ID NO:119 or SEQ ID NO: 120, or a sequence 90 percent or more identical thereto, or the complement thereof.

The invention also involves one or more of the heavy or light chain variable sequences sequences above or a heavy or Sight chain variable sequences sequence at least 75%, preferably at least 80%, more preferably at least 90% identical thereto wherein said heavy or light chain variable sequences are in the context of an antibody framework. Preferably, the antibody framework is a human antibody framework.

Preferred antibody CDR (and/or heavy and light chain variable domains) combinations of the invention correspond to those having the same unique CDR

identifier e.g., AB1 , AB2, AB3, etc. That is to say the 3 light chain CDRs and 3 heavy chain CDRs corresponding to AB1 or 3 light chain CDRs and 3 heavy chain CDRs corresponding to AB2, etc. See Table 7 or Table 8.

In another related aspect, the antibody of the invention is as follows:

(m) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO:89, SEQ ID NO:90, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an isolated amino acid sequence as in SEQ ID NO:89 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO:90 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(n) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO:91 , SEQ ID NO:93, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO:91 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO:93 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(o) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO:95, SEQ ID NO:96, or an amino acid sequence at least 75%,

preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO:95 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO:96 or an amino acid sequence at least 90% identical, in a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(p) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO:97, SEQ ID NO:98, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO:97 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO:98 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(q) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO:99, SEQ ID NO: 1 00, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO:99 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO: 100 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(r) An isolated antibody comprising an amino acid sequence chosen from SEQ ID N O:101 , SEQ ID NO: 102, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO: 101 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO: 102 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(s) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO: 103, SEQ ID NO: 104, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO: 103 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO: 104 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(t) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO: 105, SEQ ID NO: 106, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO: 105 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO: 1 06 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(u) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO: 107, SEQ ID NO: 1 08, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO: 107 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO: 108 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(v) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO: 1 09, SEQ ID NO: 1 1 1 , or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO: 1 09 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO: 1 1 1 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(w)An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO: 1 1 3, SEQ ID NO: 1 14, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO: 1 13 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO: 1 14 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody. In another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

(x) An isolated antibody comprising an amino acid sequence chosen from SEQ ID NO: 1 1 5, SEQ ID NO: 1 16, or an amino acid sequence at least 75%, preferably at least 90% identical to any of said sequences. Preferably the one or more said sequence is at least 95% identical to said amino acid sequence. Preferably, said antibody comprises (1 ) an amino acid sequence as in SEQ ID NO: 1 1 5 or an amino acid sequence at least 90% identical and (2) an amino acid sequence as in SEQ ID NO: 1 1 6 or an amino acid sequence at least 90% identical. In a related aspect, the invention is one or more nucleic acids encoding said antibody, in another related aspect, the invention is the use of said antibody for the treatment or prevention of a disease or disorder in a subject or is a pharmaceutical composition wherein said composition comprises said antibody and a pharmaceutically acceptable carrier. Preferably, the disease or disorder is cancer. Even more preferably, the disease or disorder is melanoma, colon and/or colorectal cancer or prostate cancer.

In a further embodiment, the antibody of the invention comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 120, SEQ ID NO:1 12, SEQ ID NO: 94, SEQ ID NO: 1 18 or SEQ ID

NO: 122, or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to one of said variable VH-regions; or it comprises a variable VH-region having an amino acid sequence as shown in SEQ ID NO:96, SEQ ID NO: 1 1 1 , SEQ ID NO: 93, SEQ ID NO: 90 or SEQ ID NO:106 , or a variable VH-region having an amino acid sequence which has 75% or more identity to one of said variable VH-regions.

In a further embodiment, the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 19, SEQ ID NO:110, SEQ ID NO: 92, SEQ ID NO: 1 17 or SEQ ID NO: 121 , or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to one of said variable VL-regions; or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:95, SEQ ID NO: 109, SEQ ID NO: 91 , SEQ ID NO: 89 or SEQ ID NO: 105 , or a variable VL-region having an amino acid sequence which has 75% or more identity to one of said variable VL-regions.

In a further embodiment, the antibody comprises a variable VH-region and a variable VL-region selected from the group consisting of:

(i) a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 120 or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region , or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:96 or a variable VH-region having an amino acid sequence which has 75% or more identity to said variable VH-region; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 19 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region , or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:95 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region ;

(ii) a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 12 or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region, or a variable VH-region having an amino acid sequence as shown in SEQ ID

NO:111 or a variable VH-region having an amino acid sequence which has 75% or more identity to said variable VH-region ; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 1 0 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region , or a variable VL-region having an amino acid sequence as shown in SEQ ID NO: 1 09 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region;

(iii) a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:94 or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:93 or a variable VH-region having an amino acid sequence which has 75%) or more identity to said variable VH-region; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:92 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:91 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region ;

(iv) a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 18 or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:90 or a variable VH-region having an amino acid sequence which has 75% or more identity to said variable VH-region; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 17 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:89 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region;

(v) a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 22 or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region , or a variable VH-region having an amino acid sequence as shown in SEQ ID NO: 106 or a variable VH-region having an amino acid sequence which has 75% or more identity to said variable VH-region; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 121 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:105 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region.

In a further embodiment, the antibody comprises a variable VH-region and a variable VL-region selected from the group consisting of:

(i) a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 20 or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:96 or a variable VH-region having an amino acid sequence which has 75% or more identity to said variable VH-region; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:1 19 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:95 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region;

(ii) a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 12 or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region , or a variable VH-region having an amino acid sequence as shown in SEQ ID NO: 1 11 or a variable VH-region having an amino acid sequence which has 75% or more identity to said variable VH-region; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 1 0 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region , or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:1 09 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region;

(iii) a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:94 or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:93 or a variable VH-region having an amino acid sequence which has 75% or more identity to said variable VH-region; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:92 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:91 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region.

In a further embodiment, the antibody comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 120, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:96; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 19, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:95.

In a further embodiment, the antibody comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 12, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:1 11 ; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 10, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO: 109.

In a further embodiment the antibody comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:94, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:93; and a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:92, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:91 .

In a further embodiment the antibody comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 1 8, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:90; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO.H 7, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:89.

In a further embodiment the antibody comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 122, or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:106; and

a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO: 1 21 , or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:105.

The herein provided antibodies can comprise one or more of the heavy or light chain variable sequences above or a sequence at least 75%, 80%, 85 %, 90 %, 91 % ,92%, 93%, 94%, 95 %, 96 %, 97 %, 98 %, or 99 % identical thereto.

The invention further relates to an antibody that specifically binds to CADM1 , wherein said antibody binds to an epitope on CADM1 recognized by the antibodies as disclosed above.

In one embodiment, the antibody/binding molecule of the present invention may be a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment, a F(ab)2-fragment or an epitope-binding fragment, as well as a single-chain antibody. The antibodies/binding molecules of the invention may be a monoclonal antibody, a recombinantly produced antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody or a cross-cloned antibody, a diabody, a triabody, a tetrabody, a single chain antibody, a bispecific single chain antibody, etc. The antibody may also be a multispecific antibody, including a bi-specific antibody. The antibodies of the invention may be multifunctional, i.e. they may exert their effects via more than one mode of action, such as for example by promoting internalization of CADM1 and activating ADCC or CDC pathways.

Thus, the antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a CADM1 antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-CADM 1 antibody). In a further preferred embodiment of the invention, the antibodies are humanized or human and/or deimmunized. More preferably, the antibodies are humanized and most preferably the antibodies are fully humanized/human .

Said "fully humanized antibody" are also characterized and described as "completely human" antibodies. All these antibodies can be generated by methods known in the art. For example, by phage display technology, recombinant antibody molecules may be generated due to the use of in vitro maturation which is the usage of a complete human immunoglobulin γ, subclass-1 framework (IgG 1 ) as described by Knappik (2000) J Mol Biol. 296(1 ), 57-86, Rauchenberger (2003) J Biol Chem. 278(40), 38194-205, or Hoet el al (2005) Nature Biotechnol, 23(3), 344-48.

As used herein , the term "CDR-grafted", "humanized" or "humanization" are used interchangeably to refer to a human antibody as defined herein (preferably a igG1 antibody) comprising in its binding domains at least one complementarity determining region ("CDR") from a non-human antibody or fragment thereof. Humanization approaches are described for example in WO 91 /09968 and US 6,407,213. As non-limiting examples, the term encompasses the case in which a variable region of the binding domain comprises a single CDR region , for example the third CDR region (CDR-H3) of the VH , from another non-human animal, for example a rodent, as well as the case in which a or both variable region/s comprise at each of their respective first, second and third CDRs the CDRs from said non-human animal. In the event that all CDRs of a binding domain of the antibody have been replaced by their corresponding equivalents from, for example, a

rodent, one typically speaks of "CDR-grafting", and this term is to be understood as being encompassed by the term "humanized" as used herein. The term "humanized" also encompasses cases in which, in addition to replacement of one or more CDR regions within a VH and/or VL of the binding domain further mutation/s (e.g . substitutions) of at least one single amino acid residue/s within the framework ("FR") regions between the CDRs has/have been effected such that the amino acids at that/those positions correspond/s to the amino acid/s at that/those position/s in the animal from which the CDR regions used for replacement is/are derived. As is known in the art, such individual mutations are often made in the framework regions following CDR-grafting in order to restore the original binding affinity of the non-human antibody used as a CDR-donor for its target molecule. The term "humanized" may further encompass (an) amino acid substitution(s) in the CDR regions from a non-human animal to the amino acid(s) of a corresponding CDR region from a human antibody, in addition to the amino acid substitutions in the framework regions as described above.

More specifically, as used herein , "humanized antibodies" or related terms encompass antibodies having the amino acid sequence of a human immunoglobulin with a variable region comprising non-human CDR- and/or framework region- sequences. In contemplating an antibody intended for therapeutic administration to humans, it is highly advantageous that the major part of this antibody is of human origin. Following administration to a human patient, a humanized antibody or a human antibody (or fragment thereof) will most probably not elicit a strong immunogenic response by the patient's immune system, i.e. will not be recognized as being a "foreign", that is non-human protein. This means that no host, i.e. patient antibodies will be generated against the therapeutic antibody which would otherwise block the therapeutic antibody's activity and/or accelerate the therapeutic antibody's elimination from the body of the patient, thus preventing it from exerting its desired therapeutic effect. An antibody as defined herein may also be regarded as humanized if it consists of (a) sequence(s) that deviate(s) from its (their) closest human germline sequence(s) by no more than would be expected due to the imprint of somatic hypermutation . Preferably, the humanized antibodies as defined herein have a human constant region and one or more of the CDR sequences which may be of, but are not limited to, CDRs of non-human, preferably rodent, origin . However, in context of this invention , also antibodies are provided that comprise not only human

constant regions but also CDRs that are of human origin. Accordingly, the present invention also provides for "fully-human" antibodies.

As used herein, the term "chimeric antibody" encompasses antibodies having human constant regions on the light and heavy chains and non-human variable regions on the light and heavy chains. Preferably the non-human regions are from a rodent sequence. For example, the variable regions of the heavy and light chain could be amplified by RT-PCR using RNA extracted from a mouse hybridoma cell which produces the antibody of interest. The amplified sequence could be cloned in frame with the constant heavy-chain or the constant light chain respectively of a human IgG also included in a mammalian expression vector. An expression vector encoding a chimeric IgG could be transfected into the right cell Sine, like for example CHO or HEK293, for chimeric antibody production .

As used herein, the term "deimmunized" or "deimmunization" denotes modification of the binding domain vis-a-vis an original wild type construct by rendering said wild type construct non-immunogenic or less immunogenic in humans. Deimmunization approaches are shown e.g. in WO 00/34317, WO 98/52976, WO 02/079415 or WO 92/1 0755. The term "deimmunized" also relates to constructs, which show reduced propensity to generate T cell epitopes. In accordance with this invention, the term "reduced propensity to generate T cell epitopes" relates to the removal of T-cell epitopes leading to specific T-cell activation. Furthermore, "reduced propensity to generate T cell epitopes" means substitution of amino acids contributing to the formation of T cell epitopes, i.e. substitution of amino acids, which are essential for formation of a T cell epitope. In other words, "reduced propensity to generate T cell epitopes" relates to reduced immunogenicity or reduced capacity to induce antigen independent T cell proliferation . The term "T cell epitope" relates to short peptide sequences which can be released during the degradation of peptides, polypeptides or proteins within cells and subsequently be presented by molecules of the major histocompatibility complex (MHC) in order to trigger the activation of T cells; see inter alia WO 02/066514. For peptides presented by MHC class II such activation of T cells can then give rise to an antibody response by direct stimulation of T cell s to produce said antibodies. "Reduced propensity to generate T-cell epitopes" and/or "deimmunization" may be measured by techniques known in the art. Preferably, de-immunization of proteins may be tested in vitro by T cell

proliferation assay, in this assay PB MCs from donors representing > 80 % of HLA-DR alleles in the world are screened for proliferation in response to either wild type or de-immunized peptides, ideally cell proliferation is only detected upon loading of the antigen-presenting cells with wild type peptides. Alternatively, one may test deimmunization by expressing HLA-DR tetramers representing all haplotypes. These tetramers may be tested for peptide binding or loaded with peptides substitute for antigen-presenting cells in proliferation assays. In order to test whether deimmunized peptides are presented on HLA-DR haplotypes, binding of e.g. fluorescence-labeled peptides on PBMCs can be measured . Furthermore, deimmunization can be proven by determining whether antibodies against the deimmunized molecules have been formed after administration in patients. Preferably, antibody derived molecules are deimmunized in the framework regions and most of the CDR regions are not modified in order to generate reduced propensity to induce T cell epitope so that the binding affinity of the CDR regions is not affected . Even elimination of one T cell epitope results in reduced immunogenicity. In summary, the above approaches help to reduce the immunogenicity of the antibodies provided herein when being administered to patients.

The invention also involves one or more of the disclosed CDR sequences above or a CDR sequence at least 75 % (at least 80%, at least 90%, at least 95%, at least 96 %, at least 97 %, at least 98 % or at least 99 %) identical in their amino acid sequence hereto wherein said CDR sequences is in the context of an antibody framework/framework region . Preferably, the antibody framework is a human antibody framework. Examples for frameworks include an IgG framework, such as fgG 1 , lgG4, lgG2a and lgG2b, preferably a human IgG framework such as lgG 1 , lgG2, lgG3 and lgG4. Accordingly, the antibodies of the invention may also comprise cross-cloned antibodies, i .e. antibodies comprising different antibody regions (e.g. CDR-regions) from one or more parental or affinity-optimized antibody(ies) as described herein . These cross-cloned antibodies may be antibodies in several, different frameworks, e.g. an IgG-framework, e.g. a IgG 1 lgG4, lgG2a or an IgG2b-framework. For example, said antibody framework is a mammalian, e .g . a human framework such as IgG 1 , lgG2, lgG3 or lgG4. It is of note that not only cross-cloned antibodies described herein may be presented in a preferred (human) antibody framework, but also antibody molecules comprising CDRs from antibodies as described herein, may be introduced in

an immunoglobulin framework. Examples for frameworks include IgG frameworks such as lgG1 , lgG4, SgG2a and lgG2b. Most preferred are human frameworks, and particularly human SgG1 or lgG4 frameworks.

As used herein, a "human antibody framework " relates to an antibody framework that is substantially identical (about 85% or more, usually 90 %, more preferably 95%, 96 %, 97 %, 98 %, 99 % or more) to the antibody framework of a naturally occurring human immunoglobulin.

As used herein, a "human framework region" relates to a framework region that is substantially identical (about 85% or more, usually 90 %, more preferably 95%, 96 %, 97 %, 98 %, 99 % or more) to the framework region of a naturally occurring human immunoglobulin.

In accordance with this invention, a framework region relates, accordingly, to a region in the V domain (VH or VL domain) of immunoglobulins and T-cell receptors that provides a protein scaffold for the hypervariable complementarity determining regions (CDRs) that make contact with the antigen. In each V domain, there are four framework regions designated FR1 , FR2, FR3 and FR4. Framework 1 encompasses the region from the N-terminus of the V domain until the beginning of CDR1 , framework 2 relates to the region between CDR1 and CDR2, framework 3 encompasses the region between CDR2 and CDR3 and framework 4 means the region from the end of CDR3 until the C-terminus of the V domain; see, inter alia, Janeway, Immunobiology, Garland Publishing, 2001 , 5th ed. Thus, the framework regions encompass ail the regions outside the CDR regions in VH or VL domains. Furthermore, the term "transition sequence between a framework and a CDR region" relates to a direct junction between the framework and CDR region, in particular, the term "transition sequence between a framework and a CDR region" means the sequence directly located N- and C-terminally of the CDR regions or amino acids surrounding CDR regions. Accordingly, frameworks may also comprise sequences between different CDR regions. The person skilled in the art is readily in a position to deduce from a given sequence the framework regions, the CDRs as well as the corresponding transition sequences; see Kabat (1991 ) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91 -3242 U.S. Department of Health and Human Services, Chothia (1987). J. Mol. Biol. 196, 901 -917 and Chothia (1989) Nature, 342, 877-883.

in a further embodiment of the present invention, the antibody is an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG or SgM antibody, preferably IgG. As used herein, an "antibody" may denote immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to CADM1 . Such antibodies are constructed in the same way. They form paired heavy and light polypeptide chains, and the generic term immunoglobulin is used for all such proteins. Within this general category, however, five different classes of immunoglobulins - IgM, IgD, IgG, IgA, and IgE - can be distinguished by their C regions. IgG antibodies are large molecules, having a molecular weight of approximately 150 kDa, composed of two different kinds of polypeptide chain. One, of approximately 50 kDa, is termed the heavy or H chain, and the other, of 25 kDa, is termed the light or L chain. Each IgG molecule consists of two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. In any given immunoglobulin molecule, the two heavy chains and the two light chains are identical, giving an antibody molecule two identical antigen-binding sites, and thus the ability to bind simultaneously to two identical structures. Two types of light chain, termed lambda and kappa, are found in antibodies. A given immunoglobulin either has lambda chains or kappa chains, never one of each. No functional difference has been found between antibodies having lambda or kappa light chains, and either type of light chain may be found in antibodies of any of the five major classes. The ratio of the two types of light chain varies from species to species. In mice, the average kappa to lambda ratio is 20:1 , whereas in humans it is 2:1 and in cattle it is 1 :20. The reason for this variation is unknown. By contrast, the class, and thus the effector function of an antibody, is defined by the structure of its heavy chain. There are five main heavy-chain classes or isotypes, some of which have several subtypes, and these determine the functional activity of an antibody molecule such as, for example, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The five major classes of immunoglobulin are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). Their heavy chains are denoted by the corresponding lower-case Greek letter (mu, delta, gamma, alpha, and epsilon, respectively). IgG is by far the most abundant immunoglobulin and has several subclasses (lgG1 , 2, 3, and 4 in humans, lgG1 , lgG2a, lgG2b and lgG3 in mice). Their distinctive functional properties are conferred by the carboxy-terminal part of the heavy chain, where it is not associated with the light chain. The general structural features of all the isotypes are similar. The IgG antibody is the most abundant isotype in plasma.

in one embodiment, the antibodies as defined herein are IgG antibodies. As is well known in the art, an IgG comprises not only the variable antibody regions responsible for the highly discriminative antigen recognition and binding, but also the constant regions of the heavy and light antibody polypeptide chains normally present in endogenously produced antibodies and, in some cases, even decoration at one or more sites with carbohydrates. Such glycosylation is generally a hallmark of the IgG format, and portions of these constant regions make up the so called Fc region of a full antibody which is known to elicit various effector functions in vivo, such as e.g. antibody-dependent cellular cytotoxicity (ADCC). In addition, the Fc region mediates binding of the IgG to an Fc receptor, hence prolonging half life in vivo as well as facilitating homing of the IgG to locations with increased Fc receptor presence. Advantageously, the IgG antibody is an lgG1 or lgG4 antibody specifically binding to CADM1 .

In a further embodiment, the invention provides antibodies having a human framework and comprising the amino acid sequence for VL_CDR1 ; VL_CDR2; VL_CDR3; VH_CDR1 ; VH_CDR2; and VH_CDR3 as indicated below :

SEQ ID NOs that correspond to the CDR amino acid sequences for each indicated antibody

In a further embodiment, the invention provides antibodies having a human framework and comprising the amino acid sequence for VL and VH as indicated below .

SEQ ID NOs. that correspond to the amino acid sequence of the variable domain of the light chain and the variable domain of the heavy chain for each indicated antibody

The antibodies of the invention and particularly antibodies AB3, AB10 and AB2 as disclosed above bind with high affinity and specificity to CADM1 , particularly human CADM1 . Affinity and specificity can be determined experimentally by methods known in the art, Such methods comprise, but are not limited to Flow Cytometry (FC), Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. Other methods include the use of siRNA agents against CADM 1 . The appended Examples disclose in more detail some of such methods.

Furthermore, the invention relates to any of the above referenced antibodies that is capable of internalizing. For example, cancer cells or host cells expressing a CADM1 epitope or protein can be contacted with an antibody of the invention and it is expected that the antibody can internalize. Standard assays to evaluate if an antibody internalizes are well known in the art; for example, a humZAP internalization assay such as the one disclosed in the appended Examples can be used.

The therapeutic antibodies of the invention e.g., as disclosed in the preferred embodiments above, and particularly antibodies designated AB3, AB10 and AB2 (as well as antibodies/binding molecules derived therefrom), can be used in methods of treatment to treat cancer, e.g., melanoma, colorectal, prostate, kidney, lung, ovarian, breast, blood (e.g., leukemia), or pancreatic cancer. Accordingly, the antibodies of the invention, including the antibodies AB3, AB10 and AB2 (and antibodies derived therefrom), are particularly useful in the treatment of melanoma, colon and/or colorectal cancer or prostate cancer..

The utility of the antibodies/binding molecules of the invention in the treatment of cancer, such as melanoma, colon and/or colorectal cancer or prostate cancer, can be tested using standard in vitro and in vivo assays well known in the art. In vitro assays that can be used are disclosed for example in the appended Examples. In vivo, the antibodies/binding molecules of the invention can be tested for example in xenograft models, such as melanoma, colon and/or colorectal cancer or prostate cancer xenografts, for example in mice. Briefly, a human cancer cell line, such as a melanoma, colon and/or colorectal cancer or prostate cancer cell line, is s.c. injected (1x 106 to 10x106 cells) into the flank of an immunosuppresed mouse such as Balb/c nude, NOD SCID, Fox Chase SCID, Athymic nude, or Swiss nude, and cells are allowed to grow until the generated tumor reachs a diameter of about 100-200 mm3. At this point, mice are treated with the antibodies/binding molecules to be tested. Routes of antibody administration into mice include intravenous or intraperitoneal administration. Various dosages of potentially therapeutic antibodies or fragments thereof according to the invention (or combinations of a mix of antibodies or combination of the antibodies with chemotherapy) can be tested in the in-vivo model. The treated animals and control animals (treated with a sham antibody or fragment or receiving only the vehicle) are then followed over time and scored for reduction in proliferation; reduction in tumor growth; appearance of cytotoxicity; reduction in cell-adhesion; reduction in metastasis, reduction in cell invasion, or reduction in cell migration.

A detailed in vivo xenograft model that can be used to test the antibodies of the invention is disclosed below.

A375 human malignant melanoma cell line (obtained from ATCC) is grown in DMEM supplemented with 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The tumor cells are routinely subcultured twice weekly. Cells are harvested during the exponential growth period and resuspended in physical PBS with proper cell concentration and kept on ice for tumor inoculation.

Balb/c nude mice, female, 6-8 weeks, weighing approximately 18-22g are used. Each mouse is inoculated subcutaneously at the right flank with A375 tumor cells (5X106) in 0.1 ml of PBS/Matrigel (1 : 1 ) for tumor development. The treatments are started when the mean tumor size reaches approximately 150-200 mm3.

Before commencement of treatment, all animals are weighed and the tumor volumes are measured. Mice are assigned into groups using randomized block design based upon their tumor volumes. This ensures that ail the groups are comparable at the baseline. Treatments to be tested are administered iv to the tumor-bearing mice according to predetermined regimens. At least one week of observation is followed after the final dose

Tumor sizes are measured twice weekly in two dimensions using a caliper, and the volume is expressed in mm3 using the formula: V = 0.5 a x b2 where a and b are the long and short diameters of the tumor, respectively. The tumor sizes are then used for the calculations of T-C and T/C values. T-C is calculated with T as the time (in days) required for the mean tumor size of the treatment group to reach a predetermined size (e.g., 2000 mm3), and C is the time (in days) for the mean tumor size of the control group to reach the same size. The T/C value (in percent) is an indication of antitumor effectiveness, T and C being here the mean volume of the treated and control groups, respectively, on a given day.

The invention further relates to compositions and methods for making the antibodies of the invention.

In one embodiment, the present invention relates to a nucleic acid molecule having a sequence encoding the antibody as defined and provided herein . In a further embodiment, the present invention relates to an isolated nucleic acid molecule having a sequence encoding the antibody as defined and provided herein. The nucleic acid molecules of the invention, for example, those encoding anti-CADM1 antibodies, and its subsequences/alternative transcripts, can be inserted into a vector, which will facilitate expression of the insert. The nucleic acid molecules and the antibodies they encode can be used directly or indirectly as therapeutic (or diagnostic) agents (directly in the case of the antibody or indirectly in the case of a nucleic acid molecule). Accordingly, the present invention relates also to a vector comprising the nucleic acid molecule. The vector may further

comprise a nucleic acid molecule having a regulatory sequence which is operably linked to the nucleic acid molecule. The vector may be an expression vector. Further, the present invention relates to a host, host cell or host cell line transformed or transfected with the vector as defined above. In other words, the host, host cell or host cell line expresses the antibody as provided herein. Said host, host cell or host cell line can be prokaryotic or eukaryotic. The host is preferably a eukaryotic host cell like COS, CHO, HEK293 or a multiple myeloma host cell.

The therapeutic antibody of the invention can be made by any number of methods. For example, an antibody of the invention as described in the embodiments above, including AB3, AB10 and AB2, can be synthesized in a cell line harboring a nucleic acid encoding said antibody as described above, such as AB3, AB10 and AB2, and culturing said cell line under conditions sufficient to allow expression of said antibody.

Accordingly, the present invention relates in one embodiment to a process for the production of the antibody as defined herein, said process comprising culturing a host as defined herein under conditions allowing the expression of the antibody and recovering the produced antibody from the culture. The antibody thus obtained can then be conjugated to a therapeutic agent or to a detectable label for diagnostic purposes, as described above. In the event the antibody is conjugated to a protein (for example a marker or label protein or a therapeutic or a toxic protein) via a fusion protein, a vector encoding the sequence for the fusion protein would be incorporated into the host cell line, which would then be cultured as described above. Techniques for producing and purifying antibodies are well known (see e.g. Liu et al . (2010) MAbs. 2(5):480-99; Shukla et al. (2010) Trends Biotechnol. 28(5):253-61 ; and Backliwal et al . (2008) Nucleic Acids Res. 36(1 5):e96).

The invention also is:

(1 ) A cell or cell line expressing an antibody as defined in the above embodiments, including AB3, AB10 and AB2. Said cell or cell line can be a prokaryotic or eukaryotic. Preferably said cell or cell line is chosen from COS, CHO, HEK293 or a Multiple Myeloma cell or cell line.

(2) A cell or cell line stably transfected with a nucleic acid encoding an antibody as defined in the above embodiments, including AB3, AB10 and AB2. Said cell or cell line can be a prokaryotic or eukaryotic. Preferably said cell or cell line is chosen from COS, CHO, HEK293 or a Multiple Myeloma cell or cell line.

In the following, exemplary methods for the generation of antibodies to CADM1 (like polyclonal, monoclonal, humanized, human antibodies or antibody fragments) are described.

Generation of Antibodies to CADM1

Antibodies and fragments thereof to a CADM1 protein or a CADM1 epitope (also referred to as a target protein) for therapeutic and/or diagnostic uses can be obtained in any number of ways known to those of ordinary skill in the art. These antibodies can be used in the methods of the invention and/or as the basis of engineering new antibodies. Phage display techniques can be used to generate an antibody and/or fragment thereof to a CADM1 protein or a CADM1 epitope. Standard hybridoma technologies can be used to generate antibodies and fragments thereof to a CADM1 protein or a CADM1 epitope. In one aspect, the antibody or fragment thereof to a CADM1 or a CADM1 epitope is a monoclonal antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a CADM1 or a CADM1 epitope is a polyclonal antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a CADM1 or a CADM1 epitope is a recombinant antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a CADM1 or a CADM1 epitope is humanized antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a CADM1 or a CADM1 epitope is fully human antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a CADM1 or a CADM1 epitope is a chimeric antibody or fragment thereof. In one aspect, the antibody or fragment thereof (e.g., CDR(s)) to CADM1 is derived from an animal source (e.g., mouse, rat, or rabbit).

Polyclonal Antibodies

The target protein antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the target protein

polypeptide CADM1 (or fragment or epitope thereof) or a fusion protein thereof, !t may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

Monoclonal Antibodies

The CADM1 protein antibodies may, alternatively, be monoclonal antibodies and/or fragments thereof. Monoclonal antibodies may be prepared using known hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. In a hybridoma method, a mouse, hamster, or other appropriate host animal (e.g., rabbit, goat etc.), is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the target protein polypeptide CADM1 (or fragment thereof) or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp, 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are

sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor (1984) Immunol, 1 33:3001 ; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51 -63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against target protein. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysts of Munson and Pollard (1980) Anal. Biochem. 107:220.

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (e.g., U.S. Pat. No. 4,816,567; Morrison et al., supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies and fragments thereof may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

in vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

Human and Humanized Antibodies

The CADM1 protein antibodies of the invention may further comprise humanized antibodies or human antibodies (and/or fragments thereof). Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies (and/or fragments thereof) include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies (and/or fragments thereof) may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody (and/or fragments thereof) will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially ail of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. (1986) Nature, 321 :522-525; Riechmann ef al.(1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596).

Methods for humanizing non-human antibodies (and/or fragments thereof) are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature, 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239: 1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies (and/or fragments thereof) can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter (1991 ) J. Mol. Biol. 227:381 ; Marks et al. (1991 ) J. Mol. Biol. 222:581 , Hoet el al (2005) Nature Biotechnol, 23(3), 344-48). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole ef al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al. (1991 ) J. Immunol. 147(1 ):86-95). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661 ,016, and in the following scientific

publications: Marks et at. (1992) Bio/Technology 10:779-783; Lonberg et al. (1994) Nature 368:856-859; Morrison (1994) Nature 368:812-13; Fishwild et al. (1996) Nature Biotechnology 14:845-51 ; Neuberger (1996) Nature Biotechnology 14:826; Lonberg and Huszar (1995) Intern. Rev. Immunol. 13 65-93.

The antibodies (and/or fragments thereof) may also be affinity matured using known selection and/or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity which is 5 times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared.

Antibody Fragments

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see Morimoto et al (1992) Journal of Biochemical and Biophysical Methods 24: 107-117; and Brennan et al (1985) Science 229:81 ). Antibody fragments can also be produced directly by recombinant host cells and the antibody phage libraries discussed above. Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al (1992) Bio/Technology 10: 163-167). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571 ,894; and U.S. Pat. No. 5,587,458. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641 ,870, for example. Such linear antibody fragments may be monospecific or bispecific.

Bispecific and multispecific antibodies

Bispecific antibodies with binding specificities for at least two different epitopes (Millstein et al (1983), Nature 305:537-539) may bind to two different epitopes of the CADM1 protein. An anti-CADM1 arm may be combined, for example, with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the CADM1 -expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CADM1 (WO 96/16673; U.S. Pat. No.

5,837,234; WO98/02463; U.S. Pat. No. 5,821 ,337). Purification methods for bispecific antibodies have been disclosed (WO 93/08829; Traunecker et al (1991 ) EMBO J. 10:3655-3659; WO 94/04690; Suresh et al (1986) Methods in Enzymology 121.210; U.S. Pat. No. 5,731 , 168). Bispecific antibodies can be produced using leucine zippers (Kostelny et al (1992) J. Immunol. 148(5):1547-1553), and single-chain Fv (sFv) dimers (Gruber et al (1994) J. Immunol. 152:5368).

Techniques for generating bispecific antibodies from antibody fragments have also been described, such as using chemical linkage wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments (Brennan et al (1985) Science 229:81 ). Fab'-SH fragments can be recovered from E. coli and chemically coupled to form bispecific antibodies (Shalaby et al (1992) J. Exp. Med. 175:217-225. The "diabody" technology provides an alternative method for making bispecific antibody fragments (Hollinger et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448).

Antibodies with more than two valencies are contemplated. Multivalent, Octopus" antibodies with three or more antigen binding sites and two or more variable domains can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody (US 2002/0004586; WO 01/77342). For example, trispecific antibodies can be prepared (Tutt et al (1991 ) J. Immunol. 147:60.

Conjugated antibodies

The antibodies (and fragments thereof) of the invention can be conjugated to molecules for therapeutic purposes. For example, an antibody and/or fragments thereof to CADM1 can be conjugated to a molecule (e.g., a toxin, cytotoxic or therapeutic agent) for therapeutic purposes, for example for treating cancer (e.g., melanoma, colon and/or colorectal cancer or prostate cancer). Antibody conjugates with antibodies to CADM1 can prepared for various types of antibodies (and/or fragments thereof) including chimeric antibodies, humanized antibodies, and fully human antibodies. As used herein, "conjugated" means that the antibody/binding molecule is bound to a molecule such as a toxin or therapeutic agent via any type of bonding, and thus it also includes bonding via fusion proteins (in case the molecule to which the antibody is to be conjugated is of peptidic nature) or any other type of coupling or linkage between the toxin/therapeutic agent and the antibody/binding molecule. "Conjugated to" is thus to be understood as including fused to, linked to or coupled to.

A molecule of antibody may conjugate with more than one molecule of the other therapeutic agent or toxin (as used herein, "conjugation agent"), depending on the number of sites in the antibody available for conjugation and the experimental conditions employed for performing the conjugation. As it will be apparent to those skilled in the art, while each molecule of antibody is conjugated to an integer number of molecules of the conjugation agent, a preparation of the antibody conjugate may analyze for a non-integer ratio of conjugation agent molecules per molecule of antibody, reflecting a statistical average.

Therapeutic agent as used herein refers to any molecule (including small molecules, macromolecules, peptides, polypeptides, proteins (including other therapeutic antibodies), radioactive isotopes, etc) exerting a beneficial effect in the treatment of diseases in humans or other mammals. In a preferred embodiment, such therapeutic agents are suitable for the therapy of cancer. The term "therapeutic agents" also comprises toxins, in particular toxins used in cancer therapy.

Examples of molecules that can be conjugated to the antibodies of the invention targeting CADM1 include, but are not limited to, anticancer agents such as antimetabolites (e.g., methotrexate, azathioprine, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracif decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, ifosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine and tetrazines), anthracyclines (e.g., daunorubicin, doxorubicin, valrubicin, idarubicin, epirubicin, and mitoxantrone), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vinca alkaloids such as vincristine and vinblastine, taxanes such as paclitaxel (also known as taxol) and docetaxel, and other tubulin polimeryzation inhibitors such as monomethyl aurtstatin E (MMAE) and maytansine derivatives like mertansine (also known as DM1 ) and DM4).

Specific examples of cytotoxic and/or therapeutic agents that can be conjugated to the antibodies of the invention to CADM1 include, but are not limited to taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,

tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, glucocorticoids, , monomethyl auristatin E (MMAE), DM1 , DM4, and puromycin and analogs or homologs thereof.

Other therapeutic agents that can be conjugated to the antibodies of the invention include toxins and inhibitory peptides. As used herein, "inhibitory peptide" means any peptide that inhibits cell proliferation or affects cell viability via any mechanism of action.

In one embodiment, the therapeutic agent for conjugation is a toxin. Specific examples of toxins that can be conjugated to the antibodies/binding molecules of the invention to CADM1 include, but are not limited to plant toxins such as saporin, Ricin or Gelonin, and bacterial toxins such as Pseudomona exotoxin or diphteria toxin. Also, ribonucleases can be considered as toxins due to their ability to degrade RNA and cause cell death. Some Rnases which are considered to have cytotoxic effects and can be used also as toxins are Binase (from Bacillus intermedius), α-sarcin (from Aspergillus giganteus), Ranpirnase (from amphinian) and Onconase (from Rana pipiens).

The antibodies (and fragments thereof) of the invention can be conjugated to or have a detectable label to molecules for diagnostic purposes. For example, an antibody to CADM1 can be conjugated to a detectable label (e.g. , for imaging purposes) for diagnosing or detecting cancer such as prostate cancer, melanoma or colon and/or colorectal cancer. Suitable detectable markers include, but are not limited to, a radioisotope, a nanoparticle, a fluorescent compound , a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Techniques for conjugating diagnostic agents to antibodies are well known (Holmes et al. (2001 ) Curr Protoc Cytom. May; Chapter 4: Unit 4.2; Kumar et al (2008) ACS Nano. Mar;2(3):449-56; Rosenthal et al. (2006) Laryngoscope Sep; 1 16(9): 1 636-41 ). Additionally kits for conjugating agents (such as detectable labels) to diagnostic antibodies are commercially available.

Such antibody conjugates with antibodies/binding molecules to CADM1 can readily be prepared for various types of antibodies (and/or fragments thereof) as described above, including chimeric antibodies, deimmunized antibodies, humanized

antibodies, fully humanized/human antibodies, single chain antibodies, diabodies and the like.

Techniques for conjugating agents to antibodies are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R, Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, " Immunol. Rev., 62:1 19-58 (1982)). A variety of amino acid residues on the antibody can be used for conjugation, including among others amino groups in lysines, thiol groups in cysteines, carboxyl groups in aspartic and glutamic acids, or hydroxyl groups in tyrosines. Conjugates can be prepared using a variety of cleavable linkers such as for example disulfide-based linkers, hydrazone linkers or peptide linkers (Alley et al. (2010) Curr Opin Chem Biol 14(4):529-37; Webb (201 1 ) Nat.Biotech, 29(4):297-8) or the TAP Sinker technology from ImmunoGen.

The antibodies of the invention may also be conjugated to nanoparticles comprising human serum albumin (HSA) to optimize preparation and uptake of antibodies in cancer cells, as described, for example, by Steinhauser et al., Biomaterials 2006 Oct;27{28):4975-83.

The antibodies of the invention may also be a fusion wherein the antibody portion (comprising one or more CDRs) is fused to another protein or polypeptide. For example, an antibody according to the invention can be fused to another protein or polypeptide wherein said protein or polypeptide is an agent which improve the properties of said antibody e.g. , enhances therapeutic effect. Such proteins or polypeptides which e.g. , can enhance therapeutic effect through a number of mechanisms like attracting or enhancing an immune response or delivering a therapeutic agent such a cytotoxic peptide or inhibitory peptide as defined above. Examples of such proteins or polypeptides are cytokines like IL2 or a IL2 homolog or GM-CSF. Examples of inhibitory peptides include but are not limited to the following peptide sequences:

• YARAAARGARAGRGYVSTT (wherein Y represents a phosphotyrosine), which is a peptide inhibitor of the transcription factor STAT6 which binds only to the phosphorylated form of STAT6 to prevent its dimerization and activity

• PYLKTK (wherein Y represents a phosphotyrosine), which is a phosphopeptide which inhibits the activity of the transcription factor STAT3 in vitro and in vivo

• MVRRFLVTLR!RRACGPPRVRV, which is part of the n-terminal sequence of p14ARF and it is able to induce apoptosis.

A nucleic acid encoding the antibody of the invention operably linked to the desired protein or polypeptide can be prepared and introduced into an expression vector, which is then inserted into a host cell for production of the fusion protein.

Antibody Dependent and Complement Dependent Cytotoxicity.

In one embodiment, the invention relates to a CADM1 antibody that induces, enhances, or mediates antibody-dependent cellular cytotoxicity (ADCC). ADCC is a type of immune reaction in which a target cell is coated with antibodies and killed by certain types of white blood cells. The white blood cells bind to the antibodies and release substances that kill the target cells or microbes. Not all antibodies produce ADCC. Thus, in one embodiment, the invention relates to an antibody to CADM1 that can induce, enhance or mediate ADCC. Furthermore, antibodies of the invention to CADM1 can be engineered to have improved, increased or enhanced ADCC. For example an antibody of the invention that does not induce, enhance, or mediate ADCC can be engineered, e.g., by making certain amino acid modifications to the antibody or by producing the antibody in certain strains of cells, to induce, enhance or mediate ADCC or have improved/enhanced ADCC properties.

In one aspect of this embodiment, an antibody to CADM1 has antibody-dependent cellular cytotoxicity when used in a human subject. One example of an antibody with increased or improved ADCC activity is a antibody to CADM1 that is defucosylated. In one aspect of this embodiment, an antibody to CADM1 having ADCC or increased ADCC is generated by producing the antibody in a cell-line that lacks or has decreased alpha-1 ,6-fucosyitransferase activity. In another aspect of this embodiment, an antibody to CADM1 having ADCC or increased ADCC is generated by producing the antibody in a cell-line that has reduced or lacks GDP-fucose transporter activity, in another aspect of this embodiment, an antibody to CADM1 having ADCC or increased ADCC is generated by producing the antibody in a cell-line that has reduced or lacks GDP-mannose 4,6-dehydratase activity. In another aspect of this embodiment, an antibody to CADM1 having ADCC or increased ADCC is generated by producing the antibody in a cell-line that has reduced or lacks both alpha-1 ,6-fucosyltransferase activity and GDP-mannose 4,6-dehydratase activity. See e.g., Yamane-Ohnuki et al. (2004) Biotechnol Bioeng, 87(5):614-22; Imai-Nishiya et al. (2007) BMC Biotechnology 7:84.

!n another aspect of this embodiment, ADCC can be enhanced or improved by increasing the levels of interieukin-21 (IL-21 ) in a patient or by treating the patient with IL-21 in combination with the antibody of the invention. See e.g., Watanabe et al. Br J Cancer. 2009 Dec 22. [Epub ahead of print].

In one embodiment, the invention relates to a CADM1 antibody that enhances, induces or mediates complement dependent cytotoxicty (CDC). CDC is a cytolytic cascade mediated by complement proteins in the serum. CDC is initiated by the binding of C1 q to the constant region of cell bound antibody molecule. Thus, in one embodiment, the invention relates to an antibody to CADM1 that induces, enhances or mediates CDC. Furthermore, antibodies of the invention to CADM1 can be engineered to have improved, increased or enhanced ADCC. For example an antibody of the invention that does not induce or mediate ADCC can be engineered, e.g., by making certain modifications to the antibody like amino acid muations in Fc or the hinge region thereby improving or enhancing CDC. Another method of producing CDC or enhancing antibodies an antibody's CDC is by shuffling lgG1 and lgG3 sequences within the heavy chain constant region. See e.g., Natsume et al. (2008) Cancer Res. 68:3863-3872.

Therapy

In one embodiment of the invention, CADM1 is a therapeutic target. In a more specific embodiment, CADM1 is a cancer therapeutic target. The invention therefore provides a method for treating cancer comprising administering to an individual in need of treatment a therapeutic agent that modulates CADM1 . in a more specific aspect, said cancer is selected from melanoma, colon and/or colorectal cancer and prostate cancer, in a more specific aspect, the invention provides a method for treating prostate cancer comprising administering to an individual in need of treatment a therapeutic agent that modulates CADM1 . In a more specific aspect, the invention provides a method for treating melanoma comprising administering to an individual in need of treatment a therapeutic agent that modulates CADM1 . In a more specific aspect, the invention provides a method for treating colon and/or colorectal cancer comprising administering to an individual in need of treatment a therapeutic agent that modulates CADM1. In one aspect, the therapeutic agent that modulates CADM1 is chosen from an antisense molecule, an interfering RNA molecule, a small organic molecule, or a therapeutic antibody or fragment thereof, in a more specific aspect, the therapeutic agent is a therapeutic antibody or fragment thereof that binds CADM1 protein. In a more specific aspect, the therapeutic agent is an antibody, antibody fragment or binding molecule specifically binding to CADM1 as provided herein. Preferably, the therapeutic agent has one or more of the follow effects on cancer, cancer cells expressing CADM1 , melanoma, melanoma cells, colon and/or colorectal cancer, colon and/or colorectal cancer cells, prostate cancer, or prostate cancer cells: reduces or inhibits proliferation; reduces or inhibits cellular growth; causes cytotoxicity; reduces or inhibits metastasis; modulates, reduces or inhibits cell-adhesion; modulates, reduces or inhibits cell migration; or modulates, reduces or inhibits cell invasion. Preferably, the therapeutic antibody is chosen from the antibodies defined in the embodiments described herein above, including the antibodies defined in (a)-(x) above and antibodies designated AB3, AB10, AB2, AB1 and AB8. A group of particularly preferred antibodies for use in therapy are the antibodies designated AB3, AB10 and AB8, as well as antibodies/binding molecules derived therefrom.

Antibody-Based Therapy

In one embodiment, the invention provides a method of treating an individual having cancer comprising administering to said individual a therapeutically effective amount of a therapeutic antibody or fragment thereof to CADM1 . In a more specific embodiment, the invention provides a method of treating an individual having melanoma, colon and/or colorectal cancer or prostate cancer comprising administering to said individual a therapeutically effective amount of a therapeutic antibody or fragment thereof to CADM1 . In one aspect of this embodiment, the therapeutic antibody reduces levels of activity of CADM1. in one aspect of this embodiment, the therapeutic antibody or fragment thereof to CADM1 inhibits or reduces proliferation; causes cytotoxicity; inhibits or reduces metastasis; modulates, inhibits or reduces eel! adhesion; modulates, inhibits or reduces migration; or modulates, inhibits or reduces invasion of cancer cells, cancer, melanoma, colon and/or colorectal cancer or prostate cancer, or melanoma, colon and/or colorectal or prostate cancer cells expressing CADM1 . In one aspect of this embodiment, the therapeutic antibody or fragment thereof to CADM1 inhibits or reduces proliferation of melanoma, colon and/or colorectal cancer or prostate cancer, melanoma cancer

cells, colon and/or colorectal cancer cells, prostate cancer cells, cancer, or cancer cells expressing CADM1 . In one aspect of this embodiment, the therapeutic antibody or fragment thereof to CADM1 causes cytotoxicity to melanoma, colon and/or colorectal cancer or prostate cancer, melanoma cancer cells, colon and/or colorectal cancer cells or prostate cancer cells, cancer, or cancer cells expressing CADM1 . in one aspect of this embodiment, the therapeutic antibody or fragment thereof to CADM1 reduces or inhibits metastasis of melanoma, colon and/or colorectal cancer or prostate cancer, melanoma cancer cells, colon and/or colorectal cancer cells or prostate cancer cells, cancer, or cancer cells expressing CADM1 . In one aspect of this embodiment, the therapeutic antibody or fragment thereof to CADM1 reduces or inhibits cell adhesion of melanoma, colon and/or colorectal cancer or prostate cancer, melanoma cancer cells, colon and/or colorectal cancer cells or prostate cancer cells, cancer or cancer cells expressing CADM1 . In one aspect of this embodiment, the therapeutic antibody or fragment thereof to CADM1 reduces or inhibits invasion of melanoma, colon and/or colorectal cancer or prostate cancer, melanoma cancer cells, colon and/or colorectal cancer cells or prostate cancer cells, cancer or cancer cells expressing CADM1. In one aspect of this embodiment, the therapeutic antibody or fragment thereof to CADM1 reduces or inhibits migration of melanoma, colon and/or colorectal cancer or prostate cancer, melanoma cancer cells, colon and/or colorectal cancer cells or prostate cancer cells, cancer or cancer cells expressing CADM1 . In one aspect of this embodiment, the therapeutic antibody to CADM1 induces, enhances, or mediates ADCC (antibody dependent cellular cytotoxicity) against cells to which it binds. In one aspect of this embodiment, the therapeutic antibody to CADM1 induces, enhances, or mediates CDC (complement dependent cytotoxicity) against cells to which it binds. In one aspect of this embodiment, the therapeutic antibody to CADM1 is conjugated to another molecule. In a more specific aspect, the therapeutic antibody is conjugated to a cytotoxin, a radioactive agent, enzyme, toxin, an anti-tumor drug or a therapeutic agent. The therapeutic antibody (or fragment thereof) of this embodiment can be provided as a pharmaceutical composition comprising the antibody (or fragment thereof) and a pharmaceutically acceptable carrier.

in one embodiment, the invention provides a method for treating or preventing prostate cancer. In one aspect of this embodiment, the method comprises identifying a patient having a risk factor for prostate cancer, obtaining a sample from said patient having a risk factor for prostate cancer, and determining the level of CADM1 biomarker in said sample wherein a patient having an increased level of CADM1 biomarker is treated with a therapeutic antibody that binds to or modulates CADM1. In one aspect of this embodiment, the risk factor for prostate cancer is chosen from age, ethnicity, family history of prostate cancer, or genetic predisposing gene or variant thereof, in a preferred aspect of this embodiment, the therapeutic agent is a therapeutic antibody or fragment thereof.

Risk factors for prostate cancer are known to the skilled artisan, in one specific aspect the risk factor for prostate cancer is one or more SNPs that indicated a higher risk of having prostate cancer see e.g., Gudmundsson J, et al. Nat Genet. 2009 Oct;41 (10):1122-6. Epub 2009 Sep 20.

in one embodiment, the invention provides a method for treating prostate cancer in a patient wherein said patient has androgen dependent prostate cancer. In one aspect of this embodiment, the method comprises identifying a patient having androgen dependent prostate cancer and administering to said patient a CADM1 therapeutic antibody. In a further aspect, the method comprises administering to said patient having androgen dependent prostate cancer a CADM1 therapeutic antibody and another therapeutic agent which is hormone therapy. Hormone therapy for prostate cancer are as follows: Luteinizing hormone-releasing hormone agonists, antiandrogens, or modulators of adrenal gland hormone synthesis. Luteinizing hormone-releasing hormone agonists include, but are not limited to, leuprolide, goserelin, or buserelin. Antiandrogens include, but are not limited to, bicalutamide, flutamide, or nilutamide. Modulators of adrenal gland hormone synthesis include, but are not limited to, ketoconazole or aminoglutethimide. Preferably, the therapeutic antibody is chosen from the antibodies defined in the embodiments disclosed herein, , including antibodies AB3, AB10 and AB2, or an antibody/binding molecule derived therefrom.

In one embodiment, the invention provides a method for treating prostate cancer in a patient wherein said patient has hormone-refractory or resistant prostate cancer. In one aspect of this embodiment, the method comprises identifying a patient having hormone-refractory or resistant prostate cancer and administering to said patient a CADM1 therapeutic antibody. (n a further aspect, the method comprises administering to said patient having hormone-refractory or resistant prostate cancer a CADM1 therapeutic antibody and another therapeutic agent which is hormone therapy Preferably, the therapeutic antibody is chosen from the antibodies defined in the embodiments disclosed above, including antibodies AB3, AB10 and AB2, or an antibody/binding molecule derived therefrom.

in one embodiment, the invention provides a method for treating cancer in a patient wherein said patient was previously treated or is currently being treated with radiation therapy, !n a more specific embodiment, the invention provides a method for treating prostate cancer in a patient wherein said patient was previously treated or is currently being treated with radiation therapy. In one aspect of this embodiment, the method comprises identifying a patient previously treated or is currently being treated with radiation therapy and administering to said patient a CADM1 therapeutic antibody. Radiation therapy for prostate cancer is generally classified as external or internal. External radiation therapy usually involves the focusing of high energy beams of energy (e.g., x-rays) on the affected area, internal radiation therapy involves implanting a radioactive substance or composition comprising a radioactive substance near or inside the cancer {also referred to as brachytherapy, internal radiation therapy, and/or radiation brachytherapy). Preferably, the therapeutic antibody is chosen from the antibodies defined as in the embodiments disclosed herein, including antibodies AB3, AB10 and AB2, or an antibody/binding molecule derived therefrom.

In one embodiment, the invention provides a method for treating cancer in a patient wherein said patient was previously treated or is currently being treated with chemotherapy. In a more specific embodiment, the invention provides a method for treating prostate cancer in a patient wherein said patient was previously treated or is currently being treated with chemotherapy. In one aspect of this embodiment, the method comprises identifying a patient previously treated or is currently being treated with chemotherapy and administering to said patient a CADM1 therapeutic antibody. Preferably, the therapeutic antibody is chosen from the antibodies defined in the embodiments described above, including antibodies AB3, AB10 and AB2, or an antibody/binding molecule derived therefrom.

Specially antibodies of the invention designated AB3, AB10 and AB2 (and antibodies/binding molecules derived therefrom) are useful in the diagnosis and therapy of melanoma, colon and/or colorectal cancer and prostate cancer.

In one embodiment, the invention provides an antibody to CADM1 as described herein or as produced by the processes disclosed herein , and particularly as described in the embodiments described above, including an antibody selected from AB3,AB10,AB2,AB 1 or AB8, or an antibody selected from AB3.AB 10 or AB2, or an antibody derived therefrom, for use in medicine.

In one embodiment, the invention provides an antibody to CADM 1 as described herein or as produced by the processes disclosed herein, and particularly as described in the embodiments described above, including an antibody selected from AB3,AB1 0,AB2,AB1 or AB8, or an antibody selected from AB3,AB 1 0 or AB2 , or an antibody derived therefrom, for use in the treatment of diseases or disorders wherein CADM1 is expressed or involved.

The invention further provides an antibody to CADM1 as described herein or as produced by the processes disclosed herein, and particularly as described in the embodiments described above, including an antibody selected from AB3,AB10,AB2,AB1 or AB8, or an antibody selected from AB3,AB 1 0 or AB2, or an antibody derived therefrom, for use in the treatment of cancer.

The invention further provides the use of an antibody to CADM1 as described herein or as produced by the processes disclosed herein , and particularly as described in the embodiments described above, including an antibody selected from AB3,AB 10,AB2,AB 1 or AB8, or an antibody selected from AB3,AB 1 0 or AB2, or an antibody derived therefrom, for the preparation of a pharmaceutical composition for the treatment of cancer.

The invention further provides a method of treating cancer in an individual in need thereof comprising administering to said individual an antibody to CADM 1 as described herein or as produced by the processes disclosed herein, and particularly as described in the embodiments described above, including an antibody selected from AB3.AB 1 0,AB2,AB 1 or AB8, or an antibody selected from AB3,AB10 or AB2, or an antibody derived therefrom , In a preferred embodiment, the individual is a human .

The antibody of the invention for use in the above embodiments can be a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment or a F(ab)2-fragment, a single-chain antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a bispecific single-chain antibody, a synthetic antibody and a cross-cloned antibody. In preferred embodiments, the antibody is a fully human antibody, or a fragment thereof.

The antibodies of the invention have shown high affinity for CADM1 in CADM1 -expressing melanoma, colon cancer and prostate cancer cell lines, as shown in the appended Examples. Moreover, antibodies of the invention have shown very efficient capacity to target CADM1 -expressing cancer cell lines and deliver a therapeutic agent like a toxin to said CADM1-expressing cancer cell lines. Remarkable results have been obtained with antibodies designated AB3, AB10 and AB2 in melanoma, colon cancer and prostate cancer cell lines, as shown in Examples 23 and 24.

Accordingly, in preferred aspects of the above embodiments, said cancer is melanoma, colon and/or colorectal cancer or prostate cancer. In one embodiment, said cancer is melanoma. In another embodiment, said cancer is colon and/or colorectal cancer. In another embodiment, said cancer is prostate cancer.

in further specific aspects of the above embodiments, the antibody of the invention is conjugated to a therapeutic agent or toxin, as disclosed in more detail in the section Conjugated antibodies, above.

In further embodiments, the antibody of the invention (which can be optionally conjugated) is administered in combination with one or more therapeutic agent(s). Administration of the antibody against CADM1 and the therapeutic agent(s) can be in a successive, sequential or simultaneous treatment regimen, and they can be administered via the same or different routes of administration. The antibody of the invention and the one or more other therapeutic agents may also be combined into a single dosage unit. In a specific embodiment, the therapeutic agent is an anticancer agent. Examples of anticancer agents that can be administered in combination with the antibodies of the invention include the drugs fisted below in the section Combination therapy.

While conducting research with the antibodies of the invention in skin cancers, the inventors have come to the surprising finding that the antibodies of the invention are particularly effective in melanoma cancers containing a BRAF mutation, as shown in the table below. See data obtained for melanoma cell Sines A375 or SKMEL28, both reported to have the BRAF mutation V600E, or melanoma cell line MDA-MB-435S, reported to have the BRAF mutation G463V, where a very potent effect on cell viability is observed, as compared to melanoma Hs895T, for which no BRAF mutation has been reported, or the skin cancer A431 cell line, which corresponds to wild-type BRAF. This finding is highly relevant since BRAF gene mutations are highly prevalent in melanomas.

Data in columns "Flow cytometry" and "h-ZAP" correspond to the results obtained for AB2,AB3 and AB10 tested at a concentration of 100 nM following the assays described in examples 22 and 23.

Accordingly, in one embodiment, the invention provides an antibody to CADM1 as described herein or as produced by the processes disclosed herein, and particularly as described in the embodiments described above, including an antibody selected from AB3,AB10,AB2,AB1 or AB8, or an antibody selected from AB3,AB10 or AB2, or an antibody derived therefrom , for use in the treatment of melanoma, wherein said melanoma contains a BRAF mutation . In one embodiment, said BRAF mutation is V600E. In another embodiment, said BRAF mutation is G463V.

The invention further provides the use of an antibody to CADM1 as described herein or as produced by the processes disclosed herein, and particularly as described in the embodiments described above, including an antibody

selected from AB3.AB1 0,AB2,AB 1 or AB8, or an antibody selected from AB3,AB1 0 or AB2, or an antibody derived therefrom, for the preparation of a pharmaceutical composition for the treatment of melanoma, wherein said melanoma contains a BRAF mutation. In one embodiment, said BRAF mutation is V600E. In another embodiment, said BRAF mutation is G463V.

The invention further provides a method of treating melanoma , wherein said melanoma contains a BRAF mutation, in an individual in need thereof comprising administering to said individual an antibody to CADM 1 as described herein or as produced by the processes disclosed herein , and particularly as described in the embodiments described above, including an antibody selected from AB3,AB 10,AB2,AB1 or AB8, or an antibody selected from AB3,AB10 or AB2, or an antibody derived therefrom. In a preferred embodiment, the individual is a human . In one embodiment, said BRAF mutation is V600E. in another embodiment, said B RAF mutation is G463V.

The antibodies of the invention may be administered in combination with one or more therapeutic agents for the treatment of melanoma, particularly melanoma containing a BRAF mutation. One such class of therapeutic agents are B-raf enzyme inhibitors such as vemurafenib (also known as PLX4032); vemurafenib has been recently approved by the FDA for the treatment of late-stage melanoma.

However, acquired drug resistance has been reported to frequently develop in patients receiving vemurafenib (Flaherty et al, N Engl J Med 2010; 363:809-819). Treatment with the antibodies of the invention may be particularly useful to treat melanoma in vemurafenib-resistant patients.

Use of bispecific and multispecific antibodies comprising CDRs of the present invention

As previously described, the invention also contemplates the use of bispecific or multispecific antibodies targeting CADM1 in therapy, in particular in the treatment of cancer, including melanoma, colon and/or colorectal cancer or prostate cancer. Accordingly, also bi- or multispecific antibodies having at least one specificity for/to CADM1 (including full immunoglobulins and/or fragments thereof, including single chain (bispecific or multispecific) antibodies) based on the CDR sequences and/or VH or VL sequences described herein, including the sequences for AB3, AB10 and AB2 (or sequences derived therefrom), can be prepared exhibiting binding specificities for at least one further target in addition to CADM1. Such an anti-

CADM1 arm may be combined, for example, with an arm which binds to/interacts with a triggering molecule and/or surface marker on a leukocyte such as a T-ceN receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus and/or targets cellular defense mechanisms to the CADM1-expressing cell. Alternatively, bispecific or multispecific IgG molecules, full immunoglobulins etc. for the therapy of cancer, including melanoma, colon and/or colorectal cancer or prostate cancer, can be prepared targetting CADM1 and another target involved in cancer, such as prostate-specific membrane antigen (PSMA), Epithelial cell surface antigen (EpCAM), cell adhesion molecules like mucin 1 (MUC1 ), carcinoembryonic antigen (CEA1 ), Tyrosine kinase-type cell surface receptor HER2 (ErbB2), Tyrosine kinase-type cell surface receptor HER3 (ERBB3), cytotoxic T-lymphocyte activator-4 (CTLA4), vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR) or integrin, alpha L (CD1 1 a).

Combination therapy

As previously described, when used in therapy, the antibodies of the invention can be co-administered (i.e. administered in combination) with one or more other therapeutic agents. The antibody of the invention may be, as described above, a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment, a F(ab)2-fragment or an epitope-binding fragment, as well as a single-chain antibody and may be a monoclonal antibody, a recombinantly produced antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody or a cross-cloned antibody, a diabody, a triabody, a tetrabody, a single chain antibody, a bispecific single chain antibody, etc. The antibody of the invention may itself be linked to another agent, i.e. be a conjugated antibody as described above.

The administration of the antibody of the invention and the one or more other therapeutic agent(s) can be in a successive, sequential or simultaneous treatment regimen, and they can be administered via the same or different routes of administration. The antibody of the invention and the one or more other therapeutic agents may also be combined into a single dosage unit.

Such therapeutic agents include, among others, anticancer agents such as antimetabolites (e.g., methotrexate, azathioprine, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracii, dacarbazine, capecitabine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, ifosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine and tetrazines), anthracyclines (e.g., daunorubicin, doxorubicin, valrubicin, idarubicin, epirubicin, and mitoxantrone), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and anthramycin (AMC)), topoisomerase inhibitors (e.g. irinotecan, topotecan and camptothecin), anti-mitotic agents (e.g., vinca alkaloids such as vincristine and vinblastine, taxanes such as paclitaxel (also known as taxol), cabazitaxel and docetaxel, and other tubulin polimeryzation inhibitors such as monomethyl auristatin E (MMAE) and maytansine derivatives like mertansine (also known as DM1 ) and DM4). Also included are protein kinase inhibitors such as imatinib (gleevec), nilotinib and dasatinib, and B-raf inhibitors such as vemurafenib.

Preferred therapeutic agents for combination therapy with the antibodies of the invention in the treatment of melanoma include, among others, dacarbazine, aldesleukin , ipilimumab, and vemurafenib, or combinations thereof.

Preferred therapeutic agents for combination therapy with the antibodies of the invention in the treatment of colorectal cancer include, among others, fluorouracil, bevacizumab, irinotecan. capecitabine, cetuximab, oxaliplatin, leucovorin, and panitumumab, or combinations thereof.

Preferred therapeutic agents for combination therapy with the antibodies of the invention in the treatment of prostate cancer include, among others, abiraterone, cabazitaxel, docetaxel, degarelix, leuprolide acetate, and prednisone, or combinations thereof.

Preferred antibodies of the invention for use in combination therapy include antibodies AB3 , AB 10 and AB2, as well as antibodies/binding molecules derived therefrom .

Therapeutic Reagents

The invention provides therapeutic reagents targeting the CADM1 biomarker of the invention. The invention provides a therapeutic reagent for treating cancer

expressing and/or overexpressing the CADM1 biomarker of the invention. The invention provides a therapeutic reagent for treating prostate cancer. In one aspect, the therapeutic agent of the invention for treating prostate cancer is chosen from a an antisense molecule to a CADM1 nucleic acid, an interfering RNA molecule to a CADM1 nucleic acid, a small organic molecule that binds covalently or non-covalently to CADM1 protein, or a therapeutic antibody or fragment thereof that binds to a CADM1 protein or epitope. The invention includes a pharmaceutical composition which comprises a pharmaceutically acceptable carrier and a therapeutic agent (e.g., therapeutic antibody or fragment thereof to CADM1 ).

Nucleic Acid Based Therapeutic Agents

The invention relates to molecules that modulate the expression of CADM1 protein, CADM1 gene, or CADM1 .

In one embodiment, an antisense compound specific to a nucleic acid encoding a CADM1 protein invention is administered an individual in need of such treatment.

The antisense compound for use in the invention specifically inhibits the expression of CADM1 protein. As is known to the skilled artisan, antisense drugs generally act by hybridizing to a particular target nucleic acid thus blocking gene expression. Methods for designing antisense compounds and using such compounds in treating diseases are well known and well developed in the art. For example, the antisense drug, fomivirsen, a 21-base long oligonucleotide, has been successfully developed and marketed by Isis Pharmaceuticals, Inc, for treating cytomegalovirus (CMV)-induced retinitis.

Methods for designing and making antisense compounds are known to the skilled artisan. See generally, Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993. Antisense compounds are oligonucleotides designed based on the nucleotide sequence of the mRNA or gene of the CADM1 protein. In one aspect, the antisense compound can be designed to specifically hybridize to a particular region of the gene sequence or mRNA of CADM1 to modulate (increase or decrease) replication, transcription, or translation. As used is this context, "specifically hybridize" means a sufficient degree of complementarity or pairing between an antisense compound and a target DNA or mRNA such that stable and specific binding occurs therebetween. 100% complementary or pairing is not required. Specific hybridization takes place when sufficient hybridization occurs between the antisense compound and its intended target nucleic acids in the

substantial absence of non-specific binding of the antisense compound to non-target sequences under predetermined conditions, e.g., for purposes of in vivo treatment, preferably under physiological conditions. Preferably, specific hybridization results in the interference with normal expression of the target DNA or mRNA.

For example, antisense oligonucleotides can be designed to specifically hybridize to target genes, in regions critical for regulation of transcription; to pre-mRNAs, in regions critical for correct splicing of nascent transcripts; and to mature mRNAs, in regions critical for translation initiation or mRNA stability and localization.

As is generally known in the art, commonly used oligonucleotides are oligomers or polymers of ribonucleotides or deoxyribonucleotides, that are composed of a naturally-occurring nitrogenous base, a sugar (ribose or deoxyribose) and a phosphate group. In nature, the nucleotides are linked together by phosphodiester bonds between the 3' and 5' positions of neighboring sugar moieties. However, it is noted that the term "oligonucleotides" also encompasses various non-naturally occurring mimetics and derivatives, i.e., modified forms, of naturally occurring oligonucleotides as described below. Typically an antisense compound of the present invention is an oligonucleotide having from about 6 to about 200, and preferably from about 8 to about 30 nucleoside bases.

The antisense compounds preferably contain modified backbones or non-natural internucleoside linkages, including but not limited to, modified phosphorous-containing backbones and non-phosphorous backbones such as morpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones; formacetyl and thioformacetyl backbones; alkene-containing backbones; methyleneimino and methylenehydrazino backbones; amide backbones, and the like.

Diagnostics

In one embodiment of the invention, CADM1 is a diagnostic cancer biomarker. In one aspect of this embodiment, CADM1 is a diagnostic marker for melanoma, colon and/or colorectal cancer or prostate cancer. Thus, in one aspect, the invention provides a method for diagnosis of cancer by determining the level or activity of a CADM1 biomarker in a biological sample from an individual wherein an altered level of CADM1 biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer or an increased likelihood of cancer. In a more specific aspect, the invention provides a method for diagnosis of melanoma, colon and/or colorectal cancer or prostate cancer by determining the level of a CADM1

biomarker in a biological sample from an individual wherein an altered level of CADM1 biomarker in the biological sample as compared to a control or normal value is diagnostic of melanoma, colon and/or colorectal cancer or prostate cancer, respectively, or an increased likelihood of melanoma, colon and/or colorectal cancer or prostate cancer. The CADM1 biomarker can be CADM1 nucleic acid biomarker or a CADM1 protein biomarker. In one aspect of this embodiment, the agent capable of detecting CADM1 is a diagnostic antibody or fragment thereof to a CADM1 protein or CADM1 epitope, particularly the antibodies against CADM1 having the CDRs and/or variable domains as described herein, including antibodies AB3, AB10 or AB2, or antibodies/binding molecules derived therefrom.

The invention therefore relates to a method for the diagnosis of prostate cancer by contacting an agent capable of detecting a CADM1 biomarker with a biological sample from an individual and determining the level of the CADM1 biomarker wherein an altered level of CADM1 biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer or an increased likelihood of cancer. In a specific aspect, the invention provides a method for the diagnosis of prostate cancer by contacting an agent capable of detecting a CADM1 biomarker with a tissue, tumor, blood, serum, plasma or urine sample from an individual and determining the level of the CADM1 biomarker wherein an altered Ievel of CADM1 biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer or an increased likelihood of cancer. In an even more specific aspect, the biological sample is a blood, serum, plasma or urine. In a preferred aspect, the biological sample is a blood, serum, or plasma sample. In a more preferred aspect, the biological sample is a plasma sample. In a preferred aspect, an increased or elevated level of CADM1 biomarker as compared to a control or normal value is indicative of prostate cancer or an increased likelihood of prostate cancer. In one aspect of this embodiment, the agent capable of detecting CADM1 is a diagnostic antibody or fragment thereof to a CADM1 protein or CADM1 epitope, in one aspect of this embodiment, the agent capable of detecting CADM1 is a diagnostic antibody or fragment thereof to a CADM1 protein as in SEQ ID NOs:1 -3, a CADM1 epitope which is chosen from one or more of the peptide sequences of SEQ ID NOs: 4-16 as described above which are extracellular domain epitopes of CADM1 or a transmembrane and/or intracellular domain epitopes. In one aspect of this embodiment, the agent capable of detecting CADM1 is a diagnostic antibody or fragment thereof having the CDRs and/or variable domain sequences as described herein. In one aspect of this embodiment, the agent capable of detecting CADM1 is a monoclonal antibody or fragment thereof that binds to CADML In one aspect of this embodiment, the agent capable of detecting CADM1 is a recombinant antibody that binds to CADM1 protein or epitope. In one aspect of this embodiment, the agent capable of detecting CADM1 is a polyclonal antibody that binds to CADM1 or epitope. In one aspect, the diagnostic antibody or fragment thereof binds selectively to CADM1 or epitope, in one aspect, the diagnostic antibody or fragment thereof binds specifically to CADM1 or epitope. In one aspect, the diagnostic antibody or fragment thereof is conjugated to a detectable label. In one aspect, the agent capable of detecting CADM1 is a nucleic probe capable of hybridizing to a CADM1 nucleic acid. In one aspect, the agent capable of detecting CADM1 is a set of primers capable of hybridizing to a CADM1 nucleic acid and are useful for amplifying said CADM1 nucleic acid. In another aspect of the invention, CADM1 activity may be measured so as to determine relative leveIs of CADM1 when compared to a control level of activity. CADM1 activity may be measured using any method available to one of ordinary skill in the art including, but not limited to, measurement of product levels.

In one aspect of this embodiment, the invention includes a method of diagnosing prostate cancer comprising:

(a) obtaining or providing a biological sample from an individual and

(b) determining the level of CADM 1 biomarker in the biological sample wherein an increased level of CADM1 biomarker compared to control or a normal value indicates prostate cancer or an increased likelihood of prostate cancer, !n a preferred aspect of this method, the sample is a serum , blood, plasma, or urine sample. In a more preferred aspect of this method, the sample is a serum, blood, plasma or sample. In an even more preferred aspect of this method, the sample is a plasma sample. In one aspect, the CADM 1 biomarker is a nucleic acid biomarker. In one aspect, the CADM1 biomarker is a protein biomarker. Preferably said CADM 1 biomarker is detected with an antibody that binds to CADML Preferably, said antibody to CADM1 is an antibody having the CDRs and/or variable domain sequences as described herein. In one embodiment, said antibody to CADM 1 is an antibody as described in (a)-(x). In another embodiment, said antibody to CADM1 is AB3, AB10 or AB2, or antibodies/binding molecules derived therefrom.

In a preferred aspect, the invention provides a method for diagnosing prostate cancer comprising obtaining a biological sample from an individual and determining the expression level or activity level of CADM 1 in the

biological sample wherein if CADM 1 levels are altered compared to a control value, then the individual is diagnosed with prostate cancer or an increased likelihood of prostate cancer. Preferably the sample is a serum , blood , plasma, or urine sample. In a more preferred aspect, the sample is a plasma sample. According to a preferred aspect of this embodiment, increased levels or increasing levels of CADM1 over time indicates prostate cancer or an increased likelihood of prostate. Preferably said CADM1 biomarker is detected with an antibody that binds to CADM 1 . Preferably, said antibody to CADM1 is an antibody having the CDRs and/or variable domain sequences as described herein. In one embodiment, said antibody to CADM1 is an antibody as described in (a)-(x). in another embodiment, said antibody to CADM1 is AB3, AB10 or AB2, or antibodies/binding molecules derived therefrom.

In one embodiment, the present invention provides a method for characterizing a sample obtained from a patient for prognostic, diagnostic and/or pharmacogenomic uses. Characterization of a sample obtained from a patient by determining the level of a CADM1 biomarker can be used to provide information regarding diagnosis of prostate cancer, prognosis of prostate, disease progression of prostate, diagnosis of prostate cancer type (and/or subtype), and selection of an appropriate therapeutic treatment for prostate. According to the method of the invention, a biological sample is obtained from an individual. The individual can be a healthy person, an individual diagnosed with cancer, an individual suspected of having cancer, an individual displaying one or more symptoms of cancer and/or an individual desiring screening for cancer. The method comprises the step of determining the level of a CADM 1 biomarker in a sample obtained for a patient wherein the CADM1 biomarker is a protein or nucleic acid biomarker. Preferably the CADM 1 biomarker is a CADM1 protein biomarker. In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of CADM1 can be used to provide information regarding prognosis of prostate. In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of CADM1 can be used to provide information regarding disease progression of prostate. In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of CADM 1 can be used to provide information diagnosis of prostate cancer cell type (and/or subtype). In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of CADM1 can be used to provide information regarding selection of an appropriate therapeutic treatment for prostate.

In one aspect of the diagnostic embodiments, one or more additional auxiliary prostate biomarkers are detected .

In one aspect of this embodiment, the one or more auxiliary prostate cancer diagnostic biomarkers are chosen from differential diagnosis biomarkers, prognostic biomarkers, biomarkers useful for detecting prostate cancer, biomarkers for classifying prostate cancer and additional biomarkers for detecting prostate cancer.

Auxiliary biomarkers for differential diagnosis biomarkers, prognostic biomarkers, biomarkers useful for detecting prostate cancer, biomarkers for classifying prostate cancer and additional biomarkers for detecting prostate cancer are know in the art including patients with LNM PCa who had significantly higher levels of serum e-FABP5 (Pang et al (2010) J Proteome Res. Jan;9(1 ):216-26); BSP, DSPP, and OPN were elevated in serum from prostate cancer subjects, with serum DSPP exhibiting the greatest difference, yielding an area under the receiver operator characteristic curve value of 0.98. Clin Cancer Res. 2009 Aug 1 5; 1 5(1 6):5199-207. Epub 2009 Aug 1 1 ; HSP90alpha as a serum protein biomarker Burgess et al. Volume 2 Issue 9, Pages 1223 - 1 233. Kuefer et al. Br J Cancer. 2005 June 6; 92(1 1 ): 2018-2023 showed a significant difference in the expression level of the 80kDa fragment in the serum of healthy individuals vs patients with BPH and between BPH vs Loc PCA and Met PCA (Ρ <0.001 ) with highest expression levels are observed in advanced metastatic disease.

Brown et al. Clin Cancer Res. 2009 Nov 1 ; 15(21 ):6658-64. Epub 2009 Oct 20. MIC-1 is a serum PCa prognostic marker. Albanes et al. J Natl Cancer Inst, 2009 Sep 1 6; 101 (18): 1 272-9. Epub 2009 Aug 21 showed that elevated fasting levels of serum insulin within the normal range appear to be associated with a higher risk of prostate cancer. Meyer et al. Cancer Epidemiol Biomarkers Prev. 2009 Sep;18(9):2386-90. Epub 2009 Aug 1 8. showed that decreased SEPP concentration in serum might represent an additional valuable marker for prostate cancer diagnostics.

The diagnostic methods of the invention may be used in combination with other well-known markers used in the clinical for characterizing prostate cancer, prostate cancer risk, prostate cancer prognosis, prostate cancer diagnosis, and prostate cancer therapy selection . For example, standard staging procedures, PSA levels, PSA velocity, PCA-3 levels. Other new markers for characterizing prostate cancer include TMPRSS2 fusions as detected in primary tumor and/or blood . See e.g., Barwick et al. Br. J, Cancer. 2010 Jan 12. [Epub ahead of print] and Rostad et al . APMI S. 2009 Aug; 1 17(8):575-82.

Additional auxiliary biomarkers which can be used in the methods of the invention are known to the skilled artisan and are also discussed in the background of the invention.

Diagnostic Antibody CADM1 Binding Sites

The diagnostic antibodies of the present invention can be generated against a CADM1 protein or an epitope of a CADM1 protein. The diagnostic antibodies of the present invention bind to a CADM1 protein or an epitope of a CADM1 protein. In one embodiment, the diagnostic antibody binds to or can be generated against a polypeptide having the full length sequence of a CADM1 protein as in SEQ ID NO:1 -3 as described herein. In one aspect, the binding site or epitope for the diagnostic antibody is located on an extracellular domain of the protein as defined by the amino acid sequence as in SEQ ID NO:4. In another embodiment, the diagnostic antibody binds or can be generated against a epitope having the amino acid sequence of one or more of SEQ ID NOs: 5-16.

In an even more preferred aspect the epitope is chosen from the amino acid sequences as in SEQ ID NOs:1-3.

In yet an even more preferred aspect, the epitope as in SEQ ID NO:4

In a preferred embodiment, said diagnostic antibody to CADM1 is an antibody having the CDRs and/or variable domain sequences as described above.

Nucleic Acid Based Diagnostic Reagents

In one aspect of this embodiment, the invention provides a nucleic acid chosen from CADM1 mRNA, cDNA, or complement thereof; for use for diagnosing prostate cancer or an increased likelihood of having prostate cancer.

In one aspect of this embodiment, the invention provides a primers for a CADM1 nucleic acid for use for diagnosing prostate cancer and/or an increased likelihood of having prostate cancer.

In one aspect of this embodiment, the invention provides a probe for CADM1 for diagnosing prostate cancer and/or an increased likelihood of having prostate cancer.

in one aspect, the invention provides primers that can hybridize to a nucleic acid corresponding to a biomarker described herein and be used to amplify a nucleic acid or fragment thereof corresponding to said biomarker for diagnosing prostate cancer according to the methods of the invention. In a more specific aspect, the primers are designed to amplify one or more exons of the biomarker. In another aspect, the primers are designed to amplify a fragment of one or more exons of the biomarker. In one aspect, the primers are suitable for RT-PCR analysis. In one aspect, the method of the invention involves the use of primers to amplify a nucleic acid corresponding to a CADM1 , and detecting the amplification product with a probe to the amplification product. In another aspect, the method of the invention involves the use of primers to amplify a nucleic acid corresponding to CADM1 , and detecting the amplification product with a dye that allows for quantification of the amplification product.

In one aspect, the invention provides probes to the CADM1 biomarker for detecting a nucleic acid or fragment thereof corresponding to the biomarker. The probes can be used in the methods of the invention e.g., for diagnosing prostate cancer. In a specific aspect, the probe is for the biomarker mRNA or a nucleic acid, is obtained from the mRNA corresponding to the biomarker. In a specific aspect, the probe corresponds to two contiguous exons of the biomarker, (or fragments of two or more contiguous exons). In a specific aspect, the probe corresponds to an exon of the biomarker or a fragment thereof. In a specific aspect, the probe corresponds to at least a portion of the promoter region of the biomarker and at least a portion of exon 1 of the biomarker.

In some embodiments, the invention relies on quantitative PCR to determine the level of one or more biomarkers corresponding to CADM1 . In a specific aspect the quantitative PCR method is quantitative RT-PCR. The methods can be semi-quantitative or fully quantitative.

The methods of the invention for detecting the biomarkers of the invention can comprise competitive quantitative PCR or real-time quantitative PCR which both estimate target gene concentration in a sample by comparison with standard curves constructed from amplifications of serial dilutions of standard DNA. Quantitative PCR or real-time quantitative PCR differ substantially in how the standard curves are generated. In competitive QPCR, an internal competitor DNA is added at a known concentration to both serially diluted standard samples and unknown (e.g., obtained from a patient) samples. After co-amplification, ratios of the internal competitor and target PCR products are calculated for both standard dilutions and unknown samples, and a standard curve is constructed that plots competitor-target PCR product ratios against the initial target DNA concentration of the standard dilutions. Given equal amplification efficiency of competitor and target DNA, the concentration of the latter in patient samples can be extrapolated from this standard curve.

In real-time QPCR, the accumulation of amplification product is measured continuously in both standard dilutions of target DNA and samples containing unknown amounts of target DNA. A standard curve is constructed by correlating initial template concentration in the standard samples with the number of PCR cycles (Ct) necessary to produce a specific threshold concentration of product. In the test samples, target PCR product accumulation is measured after the same Ct, which allows interpolation of target DNA concentration from the standard curve. Although real-time QPCR permits more rapid and facile measurement of target DNA during routine analyses, competitive QPCR remains an important alternative for target quantification in environmental samples. The co- amplification of a known amount of competitor DNA with target DNA is an intuitive way to correct for sample-to-sample variation of amplification efficiency due to the presence of inhibitory substrates and large amounts of background DNA that are obviously absent from the standard dilutions.

Another type of QPCR is applied quantitatively PCR. Often termed "relative quantitative PCR," this method determines the relative concentrations of specific nucleic acids. In the context of the present invention, RT-PCR is performed on mRNA species isolated from patients. By determining that the concentration of a specific mRNA species, it can be determined if the gene encoding the specific mRNA species is differentially expressed.

Identification of Small Molecule Therapeutics

In one embodiment, the invention relates to the use of the identification of CADM1 being overexpressed in prostate cancer for screening for small molecule therapeutics. The invention therefore provides therapeutic methods for diseases which overexpress CADM1 , and in particular prostate cancer using small molecule therapeutics. CADM1 can be used as a target for screening assays. For example, cells overexpressing CADM1 can be treated with a test compound to see the effect of the test compound on biomarker expression. Compounds that effect expression of the biomarker are candidates for modulating expression and therefore modulating the disease (e.g., treating and/or preventing). Any number of typical screening formats can be used to identify compounds that effect biomarkers levels. In some formats, the screen for modulators of CADM1 expression is performed in a medium to high throughput format.

As described herein, the modulators contemplated by the present invention can be small organic compounds. Such modulators can be identified by assays (e.g., in microtiter formats on microtiter plates in robotic assays) used to screen iarge numbers of compounds. There are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.

in particular, modulators displaying a desired activity can be identified from combinatorial libraries (i.e., collections of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of "building, blocks"). Preparation and screening of combinatorial libraries is well known to those of skill in the art. Such combinatorial libraries include, but are not limited to, peptide libraries (see e.g., U.S. Pat. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991 ) and Houghton et al., Nature 354:84-88 (1991 )). Other chemistries for generating chemical diversity libraries also can be used. Such chemistries include, but are not limited to, peptoids (see, for example, PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091 ), benzodiazepines (see, for example, U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al. (1993) Proc. Nat. Acad. Sci. USA 90:6909-6913), vinylogous polypeptides (Hagihara ef al. (1992) J. Amer. Chem. Soc. 1 14:6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al. (1992) J. Amer. Chem. Soc. 114:9217-9218), analogous organic syntheses of small compound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 1 16:2661 ), oligocarbamates (Cho et al. (1993) Science 261 :1303), and/or peptidyl phosphonates (Campbell et al. (1994) J. Org. Chem. 59:658), nucleic acid libraries (see e.g., Ausubel; Berger and Sambrook, all supra), peptide nucleic acid libraries (see e.g., U.S. Pat. No. 5,539,083), antibody libraries (see e.g., Vaughn et al. (1996) Nature Biotechnology 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see e.g., Liang et al. Science, 274:1520-1522 and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, for example, benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519, 134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are commercially available (see e.g. , ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo.; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md., etc.).

High-throughput assays also can be used to identify the modulators. Using the high-throughput assays, it is possible to screen thousands of potential modulators in a single day. For example, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (for example, 96) modulators. If 1536 well plates are used, then a single plate easily can assay from about 100 to about 1500 different compounds.

Additionally, when the biomarker has a known assayable activity, this activity can be the target of the screen.

Methods for Identifying and Evaluating Cancer Treatment Using CADM1 Modulators in one embodiment, the present invention provides methods for treating or controlling a cancer or tumor and the symptoms associated therewith. Any compounds, for example, those identified in the aforementioned assay systems, can be tested for the ability to prevent and/or ameliorate symptoms of tumors and cancers. As used herein, inhibit, control, ameliorate, prevent, treat, and suppress collectively and interchangeably mean stopping or slowing cancer formation, development, or growth and/or eliminating or reducing cancer symptoms. Cell-

based and animal model-based trial systems for evaluating the ability of the tested compounds to prevent and/of ameliorate tumors and cancer symptoms are used according to the present invention, in a specific aspect, the modulator of CADM1 is useful for treating prostate cancer. In another specific aspect, the modulator of CADM1 is an antibody to CADM1.

For example, cell based systems can be exposed to a compound suspected of ameliorating cancer symptoms, at a sufficient concentration and for a time sufficient to elicit such an amelioration in the exposed population of cells. After exposure, the populations of cells are examined to determine whether one or more tumor/cancer phenotypes represented in the population has been altered to resemble a more normal or more wild-type, non-cancerous phenotype. Further, the levels of target gene mRNA expression and DNA amplification within these cells may be determined, according to the methods provided herein. A decrease in the observed level of expression and amplification would indicate the successful intervention of tumors and cancers (e.g. , prostate cancer).

In addition, animal models can be used to identify compounds for use as drugs and pharmaceuticals (including the antibodies of the inventions) that are capable of treating or suppressing symptoms of tumors and cancers. For example, animal models can be exposed to a test compound at a sufficient concentration and for a time sufficient to elicit such amelioration in the exposed animals. The response of the animals to the exposure can be monitored by assessing the reversal of symptoms associated with the tumor or cancer, or by evaluating the changes in DNA copy number in cell populations and levels of mRNA expression of the target gene. Any treatments which reverse any symptom of tumors and cancers, and/or which reduce overexpression and amplification of the target gene may be considered as candidates for therapy in humans. Dosages of test agents can be determined-by deriving dose-response curves.

In another aspect, the present invention also provides assays for compounds that interfere with gene and cellular protein interactions involving CADM1 . The target gene product protein may interact in vivo with one or more cellular or extracellular macromolecules, for example, proteins and nucleic acid molecules. Such cellular and extracellular macromolecules are referred to as "binding partners." Compounds that disrupt such interactions can be used to regulate the activity of the target gene product protein, especially mutant target gene product. Such compounds can

include, but are not limited to, molecules, for example, antibodies, peptides and other chemical compounds.

Formulations and Routes and Administrations

In another aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, containing an antibody of the invention to CADM1 , formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination of (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of this disclosure. For example, a pharmaceutical composition of this disclosure can comprise a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or that have complementary activities.

Pharmaceutical compositions of this disclosure also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include an anti-CADM1 antibody of the present disclosure combined with at least one other anti-cancer agent. Examples of therapeutic agents that can be used in combination therapy are described in greater detail above in the section Combination therapy.

As used herein, "pharmaceutically acceptable carrier" relates to any component of a pharmaceutical composition other than the active ingredient and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The pharmaceutical compounds of this disclosure may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et ai. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of this disclosure also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA)5 butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any

conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of this disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, in many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of this disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. For administration of the antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1 -10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Exemplary dosage regimens for an anti-CADM1 antibody of this disclosure include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.

In some methods, two or more therapeutic antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals between single dosages can be, for example, weekly, monthly, every three months or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to the target antigen in the patient, in some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1 -1000 μg/ml and in some methods about 25-300 μg/ml.

Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human

antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime. For use in the prophylaxis and/or treatment of diseases related to abnormal cellular proliferation, a circulating concentration of administered compound of about 0.001 μΜ to 20 μΜ is preferred, with about 0.01 μΜ to 5 μΜ being preferred.

Patient doses for oral administration of the compounds described herein typically range from about 1 mg/day to about 10,000 mg/day, more typically from about 10 mg/day to about 1 ,000 mg/day, and most typically from about 50 mg/day to about 500 mg/day. Stated in terms of patient body weight, typical dosages range from about 0.01 to about 150 mg/kg/day, more typically from about 0.1 to about 15 mg/kg/day, and most typically from about 1 to about 10 mg/kg/day, for example 5 mg/kg/day or 3 mg/kg/day. In at least some embodiments, patient doses that retard or inhibit tumor growth can be 1 μmol/kg/day or less. For example, the patient doses can be 0.9, 0.8, 0.7, 0.6, 0.5, 0.45, 0.3, 0.2, 0.15, 0.1 , 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01 , or 0.005 μmol/kg or less {referring to moles of the drug). Preferably, the antibody-drug conjugate retards growth of the tumor when administered in the daily dosage amount over a period of at least five days. In at least some embodiments, the tumor is a human-type tumor in a SCID mouse. As an example, the SCID mouse can be a CB 17. SCID mouse (available from Taconic, Germantown, NY).

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dosage" of an anti-CADM1 antibody of this disclosure preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of CADM1 + tumors, a "therapeutically effective dosage" preferably inhibits cell growth or tumor growth by at least about 5%, more preferably by at least about 10%, even more preferably by at least about 20%, and still more preferably by at least about 60% relative to untreated subjects (or cells in cell based studies). The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth. Such inhibition can be measured in vitro by assays known to the skilled practitioner (cell proliferation, metastasis, cytotoxicty, invasion, migration, ,etc). A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

A composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for antibodies of this disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Alternatively, an antibody of this disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, eds, Marcel Dekker, Inc., New York, 1978. Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of this disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in U .S. Patent Nos. 5,399, 163; 5,383,851 ; 5,312,335; 5,064,413; 4,941 ,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486, 194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art. In certain embodiments, therapeutic antibodies of this disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of this disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,81 1 ; 5,374,548; and 5,399,331 . The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U .S. Patent 5,416,016 to Low et al.); marmosides (Umezawa et al. (1988) Biochem, Biophys.

Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); pi 20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett 346: 123; JJ. Killion; IJ. Fidier (1994) Immunomethods 4:273.

Data and Information

In one aspect of the invention, the present invention relates to methods for comparing and compiling data wherein the data is stored in electronic or paper format. Electronic format can be selected from the group consisting of electronic mail, disk, compact disk (CD), digital versatile disk (DVD), memory card, memory chip, ROM or RAM, magnetic optical disk, tape, video, video clip, microfilm, internet, shared network, shared server and the like; wherein data is displayed, transmitted or analyzed via electronic transmission, video display, telecommunication, or by using any of the above stored formats; wherein data is compared and compiled at the site of sampling specimens or at a location where the data is transported following a process as described above. The data of this embodiment is information regarding the results of the analysis of CADM1.

The compounds, targets, assays, tests, inquiries and methodologies described herein can be employed in a variety of contexts, including diagnostic and therapeutic discovery, diagnostic and therapeutic development, safety and efficacy monitoring, compound and treatment regimen potency determination and validation, treatment assessment, comparative studies, marketing and the like. The information provided by the invention can be communicated to regulators, physicians and other healthcare providers, manufacturers, owners, investors, patients, and/or the general public. This information and the like can be used in exploratory research, pre-clinical and clinical settings, labeling, production, advertising, and sales, for example.

Definitions

As used herein a "CADM1 biomarker" refers to a "CADM1 nucleic acid" or a "CADM1 protein" that can be specifically detected for diagnostic purposes or targeted for therapeutic purposes. A CADM1 nucleic acid can be a RNA molecule, DNA molecule, or other nucleic acid that corresponds to the CADM1 gene or a fragment thereof. For example, a CADM1 gene may correspond to a human CADM1 gene. For example, a CADM1 nucleic acid can be a cDNA, or fragment thereof, corresponding to a CADM1 mRNA molecule. A CADM1 protein refers to a

protein (or fragment thereof) encoded or expressed by the CADM1 gene. Examples of CADM1 biomarkers are given in the examples as well as some reagents useful for detecting CADM1 biomarkers, nucleic acids, and proteins.

As used herein, the term "antibody to CADM1 ," "antiCADM1 antibody", "anti-CADM1 antibody", "antibody or fragment thereof to CADM1" and "antibodies or fragments thereof that bind to CADM1 " as used herein refers to antibodies or fragments thereof that specifically bind to a CADM1 polypeptide or a fragment or epitope of a CADM1 polypeptide and do not specifically bind to other non-CADM1 polypeptides. Preferably, antibodies or fragments bind immunospecifically to a CADM1 polypeptide or fragment thereof do not non-specifically cross-react with other antigens (e.g. , binding cannot be competed away with a non-CADM1 protein, e.g., BSA in an appropriate immunoassay). Antibodies or fragments that immunospecifically bind to a CADM1 polypeptide can be identified, for example, by immunoassays or other techniques known to those of skill in the art. Antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a CADM1 antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-CADM1 antibody).

As used herein, a CADM1 protein refers to a protein (or fragment or epitope thereof) encoded or expressed by the CADM1 gene.

A "cancer" in an animal refers to the presence of cells possessing one or more characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. Preferably the cancer is melanoma, colon and/or colorectal cancer or prostate cancer. Colorectal cancer, as used herein, comprises colon cancer.

The phrase "detecting a cancer" or "diagnosing a cancer" refers to determining the presence or absence of cancer or a precancerous condition in an animal. "Detecting a cancer" also can refer to obtaining indirect evidence regarding the likelihood of the presence of precancerous or cancerous cells in the animal or assessing the predisposition of a patient to the development of a cancer. Detecting a cancer can be accomplished using the methods of this invention alone, in combination with other methods, or in light of other information regarding the state of health of the animal.

A "tumor," as used herein, refers to ail neoplastic cell growth and proliferation, whether malignant or benign, and ail precancerous and cancerous cells and tissues.

The term "precancerous" refers to cells or tissues having characteristics relating to changes that may lead to malignancy or cancer.

"Treating" or "treatment" does not require a complete cure. It means that the symptoms of the underlying disease are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated . It is understood that reduced , as used in this context, means relative to the state of the disease, including the molecular state of the disease, not just the physiological state of the disease.

A "target gene," as used herein, refers to a differentially expressed gene in which modulation of the level of gene expression or of gene product activity prevents and/or ameliorates tumor and cancer symptoms. Thus, compounds that modulate the expression of a target gene, the target gene, or the activity of a target gene product can be used in the diagnosis, treatment or prevention of tumors and cancers.

In general, a "CADM1 gene" is a region on the genome that is capable of being transcribed to an RNA that encodes a CADM1 protein as well as the regulatory sequences associated with or operably linked to the coding region. The skilled artisan will appreciate that the present invention encompasses all encoding transcripts that may be found, including splice variants, allelic variants and transcripts that occur because of alternative promoter sites or alternative polyadenylation sites of CADM1. A "full-length" gene or RNA therefore encompasses any naturally occurring splice variants, allelic variants, other

alternative transcripts, splice variants generated by recombinant technologies which bear the same function as the naturally occurring variants, and the resulting RNA molecules. A "fragment" of a gene, can be any portion from the gene, which may or may not represent a functional domain, for example, a catalytic domain, a DNA binding domain, etc. A fragment may preferably include nucleotide sequences that encode for at least 25 contiguous amino acids, and preferably at least about 30, 40, 50, 60, 65, 70, 75 or more contiguous amino acids or any integer thereabout or therebetween.

As used herein, the term "transformed cell" means a cell into which {or into predecessor or an ancestor of which) a nucleic acid molecule encoding a polypeptide of the invention has been introduced, by means of, for example, recombinant DNA techniques or viruses.

The nucleic acid molecules of the invention, for example, those corresponding to CADM1 , and its subsequences/alternative transcripts, can be inserted into a vector, as described below, which will facilitate expression of the insert. The nucleic acid molecules and the polypeptides they encode can be used directly as therapeutic agents, or can be used (directly in the case of the polypeptide or indirectly in the case of a nucleic acid molecule) to generate antibodies that, in turn, are clinically useful as a therapeutic agent. Accordingly, vectors containing the nucleic acids of the invention, cells transfected with these vectors, the polypeptides expressed, and antibodies generated against either the entire polypeptide or an antigenic fragment or epitope thereof, are among the aspects of the invention.

An "isolated DNA molecule" is a fragment of DNA that has been separated from the chromosomal or genomic DNA of an organism. Isolation also is defined to connote a degree of separation from original source or surroundings.

"Complementary DNA" (cDNA), often referred to as "copy DNA," is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA molecule that comprises such a single-stranded DNA molecule and its complement DNA strand.

The term "expression" refers to the biosynthesis of a gene product, such as a protein or an mRNA molecule.

An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.

A "recombinant host" may be any prokaryotic or eukaryotic cell that contains a cloning vector, expression vector, or other heterologous nuclide acid seqeunces. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.

The term "operably linked" is used to describe the connection between regulatory elements and a gene or its coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene or coding region is said to be "operably linked to" or "operatively linked to" or "operably associated with" the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.

The term "identity", "sequence identity", "homology" or "Sequence homology" {the terms are used interchangeably herein) is used to describe the sequence relationships between two or more nucleic acids, polynucleotides, proteins, or polypeptides, and is understood in the context of and in conjunction with the terms including: (a) reference sequence, (b) comparison window, (c) sequence identity, (d) percentage of sequence identity, and (e) substantial identity or "homologous."

A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence, for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence can be chosen from at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence can be chosen from at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

A "comparison window" includes reference to a contiguous and specified segment of a polynucleotide or polypeptide sequence, wherein the polynucleotide or polypeptide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a misleadingly high similarity to a reference sequence due to inclusion of gaps in the polynucleotide or polypeptide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482, 1981 ; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 7 Science Dr., Madison, Wise, USA; the CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73: 237-244; Corpet et al. (1988) Nucleic Acids Research 16:881 -90; Huang, et al. (1992) Computer Applications in the Biosciences, 8:1 -6; and Pearson, et al. (1994) Methods in Molecular Biology, 24:7-331 . The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995. New versions of the above programs or new programs altogether will undoubtedly become available in the future, and can be used with the present invention.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs, or their successors, using default parameters. Altschul et al. (1997) Nucleic Acids Res, 2:3389-3402. It is to be understood that default settings of these parameters can be readily changed as needed in the future.

As those ordinary skilled in the art will understand, BLAST searches assume that proteins or nucleic acids can be modeled as random sequences. However, many real proteins and nucleic acids comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids or nucleic acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein or nucleic acid are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten et al. (1993) Comput. Chem. 17: 149-163) and XNU (Claverie et al. (1993) Comput. Chem. 17:191 -1 ) low-complexity filters can be employed alone or in combination.

"Sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window, and can take into consideration additions, deletions and substitutions. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (for example, charge or hydrophobicity) and therefore do not deleteriously change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have sequence similarity. Approaches for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1 . The scoring of conservative substitutions is

calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Set., 4: 11-17, 1988, for example, as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

"Percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or nucleic acid sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" or "homologous" in their various grammatical forms in the context of polynucleotides means that a polynucleotide comprises a sequence that has a desired identity, for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%, 96%, 97%, 98% or 99%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, still more preferably at least 75%, 80%, 90%, and even more preferably at least 95%.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Thus, the detection of only specifically hybridizing sequences wilt usually require stringent hybridization and washing conditions such as, for example, the highly stringent hybridization conditions of 0.1 x SSC, 0.1 % SDS at 65°C or 2 x SSC , 60°C, 0.1 % SDS. Low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1 % SDS at 55°C or 60°C. However, nucleic acids

which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical.

The term "substantial identity" or "homologous" in their various grammatical forms in the context of peptides indicates that a peptide comprises a sequence that has a desired identity, for example, at least 60% identity, preferably at least 70% sequence identity to a reference sequence, more preferably at least 75%, more preferably 80%, still more preferably 85%, even more preferably at least 90% or 95% or even 96%, 97%, 98% or 99% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol., 48:443. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative substitutions are known to those skilled in the art and typically include, but are not limited to, substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine, and others as known to the skilled person. Examples of conservative substitutions include, but are not limited to, substitutions listed in the following table:

"Subject" as used herein refers to a biological subject that contains or is suspected of containing nucleic acids or polypeptides corresponding to CADM1 . In embodiments, the subject may be a mammalian subject such as a human.

"Biological sample" as used herein refers to a sample obtained from a subject, including sample of biological tissue or fluid origin, obtained, reached, or collected in vivo, ex-vivo, or in situ, that contains or is suspected of containing nucleic acids or polypeptides corresponding to CADM1 . A biological sample also includes samples from a region of a biological subject containing or suspected of containing precancerous or cancer cells or tissues. Such samples can be, but are not limited to, organs, tissues, fractions and cells isolated from mammals including, humans such as a patient. Biological samples also may include sections of the biological sample including tissues, for example, frozen sections taken for histologic purposes. A biological sample, as described herein, can be: a "control" or a "control sample" or a "test sample". A biological sample can be obtained from the prostate using commonly employed clinical practices (e.g., fine needle biopsy, blood from a blood draw, serum or plasma derived from blood, tumor sections, circulating tumor cells, sample obtained from prostate massage techniques).

A "control" refers to a representative of healthy, cancer-free biological subject or information obtained from a different individual or a normalized value, which can be based on baseline data obtained from a population or other acceptable sources. A control also can refer to a given level of CADM1 , representative of the cancer-free population, that has been previously established based on measurements from normal, cancer-free animals. A control also can be a reference data point in a database based on data obtained from control samples representative of a cancer-free population. Further, a control can be established by a specific age, sex, ethnicity or other demographic parameters, in some situations, the control is implicit in the particular measurement.

A "control sample" refers to a sample of biological material representative of healthy, cancer-free animals or a normal biological subject obtained from a cancer-free population. The level of CADM1 , in a control sample is desirably typical of the general population of normal, cancer-free animals of the same species. This sample either can be collected from an animal for the purpose of being used in the methods described in the present invention or it can be any biological material representative of normal, cancer-free animals suitable for use in the methods of this invention. A control sample also can be obtained from normal tissue from the animal that has cancer or is suspected of having cancer.

A "test sample" as used herein refers to a biological sample, including sample of biological tissue or fluid origin, obtained, reached, or collected in vivo, ex-vivo, or in situ, that contains or is suspected of containing nucleic acids or polypeptides corresponding to CADM1 , A test sample also includes biological samples containing or suspected of containing precancerous or cancer cells or tissues. A test sample also may include sections of the biological sample including tissues, for example, frozen sections taken for histologic purposes.

"Providing a sample, a biological sample, or a test sample" means to obtain from a subject a sample, in vivo, ex-vivo, or in situ, including tissue or cell sample for use in the methods described in the present invention. Most often, this will be done by removing a sample of cells from an animal, but also can be accomplished in vivo, ex-vivo, or in situ, or by using previously isolated cells (for example, isolated from another subject, at another time, and/or for another purpose).

"Data" includes, but is not limited to, information obtained that relates to "biological sample," "test sample." "control sample," and/or "control," as described above, wherein the information is applied in generating a test level for diagnostics, prevention, monitoring or therapeutic use. The present invention relates to methods for comparing and compiling data wherein the data is stored in electronic or paper formats. Electronic format can be selected from the group consisting of electronic mail, disk, compact disk (CD), digital versatile disk (DVD), memory card, memory chip, ROM or RAM, magnetic optical disk; tape, video, video clip, microfilm, internet, shared network, shared server and the like; wherein data is displayed, transmitted or analyzed via electronic transmission, video display, telecommunication, or by using any of the above stored formats; wherein data is compared and compiled at the site of sampling specimens or at a location where the data is transported following a process as described above.

"Overexpression" of a gene or an "increased," or "elevated," level of a ribonucleotide or protein refers to a level of the gene, ribonucleotide or polypeptide that, in comparison with a control level of gene, ribonucleotides or polypeptide, is detectably higher. Comparison may be carried out by statistical analyses on numeric measurements of the expression; or, it may be done through visual examination of experimental results by qualified researchers.

A level of ribonucleotide or polypeptide, that is "expected" in a control sample refers to a level that represents a typical, cancer-free sample, and from which an elevated, or diagnostic, presence of the polypeptide or polynucleotide, can be distinguished. Preferably, an "expected" level will be controlled for such factors as the age, sex, medical history, etc., of the mammal, as well as for the particular biological subject being tested.

The phrase "functional effects" in the context of an assay or assays for testing compounds that modulate a particular gene's activity includes the determination of any parameter that is indirectly or directly under the influence of the gene, for example, a functional, physical, or chemical effect, for example, of the genes activity, activity of a polypeptide encoded by the gene, the ability to induce gene amplification or overexpression in cancer cells, and to aggravate cancer cell proliferation. "Functional effects" include in vitro, in vivo, and ex vivo activities.

"Determining the functional effect" refers to assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of the gene or the polypeptide encoded by the gene, for example, functional, physical, and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, for example, changes in spectroscopic characteristics (for example, fluorescence, absorbance, refractive index), hydrodynamic {for example, shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of the gene, measuring binding activity or binding assays (for example, substrate binding, and measuring cellular proliferation), measuring signal transduction, or measuring cellular transformation.

"Inhibitors," "activators," "modulators," and "regulators" refer to molecules that activate, inhibit, modulate, regulate and/or block an identified function. Any molecule having potential to activate, inhibit, modulate, regulate and/or block an identified function can be a "test molecule," as described herein. For example, referring to oncogenic function or anti-apoptotic activity of the gene, such molecules may be identified using in vitro and in vivo assays of gene. Inhibitors are compounds that partially or totally block the genes activity, respectively, decrease, prevent, or delay their activation, or desensitize their cellular response. This may be accomplished by binding to protein expressed by the gene directly or via other intermediate molecules. An antagonist or an antibody that blocks the activity of the genes expression product, including inhibition of oncogenic function or anti-apoptotic activity of gene's expression product, respectively, is considered to be such an inhibitor. Activators are compounds that bind to a gene's protein expression product directly or via other intermediate molecules, thereby increasing or

enhancing their activity, stimulating or accelerating their activation, or sensitizing their cellular response. An agonist of a genes expression product is considered to be such an activator. A modulator can be an inhibitor or activator, A modulator may or may not bind the gene or its protein expression product directly; it affects or changes the activity or activation of gene's expression product or the cellular sensitivity to the gene or its expression product, respectively. A modulator also may be a compound, for example, a small molecule, that inhibits transcription of the gene's mRNA. A regulator of a gene includes any element, for example, nucleic acid, peptide, polypeptide, protein, peptide nucleic acid or the like, that influences and/or controls the transcription/expression of the gene or its coding region.

The group of inhibitors, activators, modulators and regulators of this invention also includes genetically modified versions of CADM1 , for example, versions with altered activity. Thus, unless otherwise indicated, the group is inclusive of the naturally occurring protein as well as synthetic ligands, antagonists, agonists, antibodies, small chemical molecules and the like.

"Assays for inhibitors, activators, modulators, or regulators" refer to experimental procedures including, for example, expressing CADM1 , in vitro, in cells, applying putative inhibitor, activator, modulator, or regulator compounds, and then determining the functional effects on the gene's (or its expressed protein product) activity or transcription, as described above. Samples that contain or are suspected of containing the gene (or its expressed protein product) are treated with a potential activator, inhibitor, or modulator. The extent of activation, inhibition, or change is examined by comparing the activity measurement from the samples of interest to control samples. A threshold level is established to assess activation or inhibition. For example, inhibition of a polypeptide is considered achieved when the activity value relative to the control is 80% or lower. Similarly, activation of a polypeptide is considered achieved when the activity value relative to the control is two or more fold higher.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this

invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. Various levels of purity may be applied as needed according to this invention in the different methodologies set forth herein. The customary purity standards known in the art may be used if no standard is otherwise specified.

An "isolated nucleic acid molecule" can refer to a nucleic acid molecule, depending upon the circumstance, that is separated from the 5' and 3' coding sequences of genes or gene fragments contiguous in the naturally occurring genome of an organism. The term "isolated nucleic acid molecule" also includes nucleic acid molecules which are not naturally occurring, for example, nucleic acid molecules created-by recombinant DNA techniques.

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

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively, modified variants thereof (for example, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with suitable mixed base and/or deoxyinosine residues (Batzer et al. (1991 ) Nucleic Acid Res, 19:081 ; Ohtsuka et al., J. Biol. Chem. (1985) 260:2600-2608 Rossolini et al. (1994) Mol. Cell Probes, 8:91 -98). The term nucleic

acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

A "host cell" is a naturally occurring cell or a transformed cell or a transfected cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be cultured cells, explants, cells in vivo, and the like. Host cells may be prokaryotic cells, for example, E. coli, or eukaryotic cells, for example, yeast, insect, amphibian, or mammalian cells, for example, Vero, CHO, HeLa, and others.

A "label" or a "detectable moiety" is a composition that when linked with the nucleic acid or protein molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes {for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens. A "labeled nucleic acid or oligonucleotide probe" is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic bonds, van der Waals forces, electrostatic attractions, hydrophobic interactions, or hydrogen bonds, to a label such that the presence of the nucleic acid or probe may be detected by detecting the presence of the label bound to the nucleic acid or probe.

The term "CADM1 gene" or "CADM1 biomarker" or "CADM1 nucleic acid" or "CADM1 protein" can refer to a target nucleic acid (DNA and RNA) or protein (or polypeptide), (e.g., corresponding to CADM1 ) and can include their polymorphic variants, alleles, mutants, and interspecies homologs that have (i) substantial nucleotide sequence homology (for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%) with the nucleotide sequence indicated in EnsembI database for the indicated ID number; or (ii) at least 65% sequence homology with the amino acid sequence as indicated in the EnsembI record; or (iii) substantial nucleotide sequence homology (for example, at least 60% identity, preferably at least 70% sequence identity, more preferably at least 80%, still more preferably at least 90% and even more preferably at least 95%) with the nucleotide sequence as set forth in the EnsembI record with substantial sequence homology with the encoded amino acid sequence. As used in herein, and unless otherwise specified, these terms refer to the entire gene sequence, mRNA sequence, and/or protein sequence as well as fragments of these sequences. In a

more specific definition, these terms refer to the minimal amount of nucleic acid or amino acid sequence that can be used to identify biomarker in a specific manner. The skilled artisan recognizes that the target genes/biomarker can have numerous splice forms and variants. When referring to a specific target gene or locus by a reference number (e.g., Entrez gene ID or ENSEMBL), all splices forms and variant which are included in the various embodiments of the invention. The target gene/biomarker can also comprise a regulatory element. These sequences are representative of one particular individual in the population of humans. Humans vary from one to another in their gene sequences. These variations are very minimal, sometimes occurring at a frequency of about 1 to 10 nucleotides per gene. Different forms of any particular gene exist within the human population. These different forms are called allelic variants. Allelic variants often do not change the amino acid sequence of the encoded protein; such variants are termed synonymous. Even if they do change the encoded amino acid (non-synonymous), the function of the protein is not typically affected. Such changes are evolutionary or functionally neutral. When a gene ID (e.g., Genbank or ENSEMBL) is referred to in the present application all allelic variants are intended to be encompassed by the term. The gene ID sequences given for a biomarker are provided merely as representative examples of a wild-type human sequence. The invention is not limited to a single allelic form of the amplified genes or regions (and proteins they encode).

As used herein, "binds selectively to" or "binds specifically to" means that a compound binds (immunologically in the context) to CADM1 and not to any other materials in the medium containing CADM1. Selective binding refers to compound (e.g., antibody) binding to CADM1 preferentially as compared to non-CADM1 molecules. Specific binding refers to compound (e.g., antibody) binding only to CADM1 or a portion of CADM1 and not binding other material in the sample.

Additional embodiments

1 . An isolated antibody that specifically binds CADM1 wherein said antibody has one or more CDRs each CDR corresponding to a CDR chosen from the CDRs of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 or a CDR sequence having 75% or more amino acid identity to said CDR.

2. The isolated antibody of embodiment 1 having 2 or more CDRs each CDR corresponding to a CDR chosen from the CDRs of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 or a CDR sequence having 75% or more amino acid identity to said CDR.

3. The isolated antibody of embodiment 1 having 3 or more CDRs each CDR corresponding to a CDR chosen from the CDRs of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or antibody-12 or a CDR sequence having 75% or more amino acid identity to said CDR,

4. The isolated antibody of embodiment 1 having 6 CDRs each CDR corresponding to a CDR chosen from the CDRs of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 or a CDR sequence having 75% or more amino acid identity to said CDR.

5. An isolated antibody having one or more Sight chain variable domain corresponding to Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 or a sequence having 90% or more amino acid identity to said light chain variable domain.

6. An isolated antibody having one or more heavy chain variable domains corresponding to Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 or a sequence having 90% or more amino acid identity to said light chain variable domain.

7. An isolated antibody having (a) one or more light chain variable domains corresponding to Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 or a sequence having 90% or more identity thereto and (b) one or more heavy chain variable domains corresponding to Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-11 , or Antibody-12 or a sequence having 90% or more amino acid identity thereto.

8. The isolated antibody of embodiment 1 comprising one or more consensus light chain CDR amino acid sequences derived from the alignment of 2 or more light chain CDRs of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 wherein said consensus sequence has 75% or more identical amino acid residues.

9. The isolated antibody of embodiment 1 comprising one or more consensus heavy chain CDR sequences derived from the alignment of 2 or more heavy chain sequence CDRs of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 wherein said consensus sequence has 75% or more identical amino acid residues.

10. The isolated antibody of embodiment 1 comprising a consensus variable heavy chain sequence derived from the alignment of 2 or more variable heavy chain sequences of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-11 , or Antibody-12 wherein said consensus sequence has 75% or more identical amino acid residues.

11. The isolated antibody of embodiment 1 comprising a consensus variable light chain sequence derived from the alignment of 2 or more variable light chain sequences of Antibody-1 , Antibody-2, Antibody-3, Antibody-4, Antibody-5, Antibody-6, Antibody-7, Antibody-8, Antibody-9, Antibody-10, Antibody-1 1 , or Antibody-12 wherein said consensus sequence has 75% or more identical amino acid residues.

12. The isolated antibody of embodiment 8 comprising 3 or more consensus light chain CDR sequences.

13. The isolated antibody of embodiment 8 comprising 6 consensus light chain CDR sequences.

14. The isolated antibody of embodiment 9 comprising 3 or more consensus heavy chain CDR sequences.

15. The isolated antibody of embodiment 9 comprising 6 consensus heavy chain CDR sequences.

16. The isolated antibody of embodiment 10 comprising 2 consensus variable heavy chain sequences.

17. The isolated antibody of embodiment 11 comprising 2 consensus variable light chain sequences.

18. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO: 1 7, SEQ ID NO:29, SEQ ID NO:41 , SEQ ID NO:53, SEQ ID NO;65 , SEQ ID NO: 77 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

19. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID 1x1O: 18, SEQ ID NO:30, SEQ ID NO:42, SEQ ID NO:54, SEQ ID NO:66, SEQ ID NO:78 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs,

20. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO: 19, SEQ ID NO:31 , SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:67, SEQ ID NO: 79 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

21. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:56, SEQ ID NO:68, SEQ ID NO:80 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

22. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:21 , SEQ ID NO: 33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, SEQ ID NO:81 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

23. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:46, SEQ ID NO:58, SEQ ID NO:70, SEQ ID NO :82 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

24. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:23, SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:59, SEQ ID NO:71 , SEQ ID NO:83 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

25. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:84 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

26. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:49, SEQ ID NO:61 , SEQ ID

NO:73, SEQ ID NO:85 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

27. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:62, SEQ ID NO:74, SEQ ID NO: 86 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

28. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:51 , SEQ ID NO:63 , SEQ ID NO:75, SEQ ID NO:87 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

29. The isolated antibody of embodiment 1 comprising one or more CDRs each CDR having an independent amino acid sequence chosen from SEQ ID NO :28, SEQ ID NO;40, SEQ ID NO:52, SEQ ID NO:64, SEQ ID NO:76, SEQ ID NO:88 or a CDR having an amino acid sequence at least 90% identical to any of said CDRs.

30. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO:89, SEQ ID NO:90, or an amino acid sequence at least 90% identical to any of said sequences.

31. The isolated antibody of embodiment 30 which comprises (1 ) an amino acid sequence as in SEQ ID NO:89 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO:90 or an amino acid sequence at least 90%.

32. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO:91 , SEQ ID NO:93, or an amino acid sequence at least 90% identical to any of said sequences.

33. The isolated antibody of embodiment 32 which antibody comprises (1 ) an amino acid sequence as in SEQ ID NO:91 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO:93 or an amino acid sequence at least 90%.

34. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO:95, SEQ ID NO:96, or an amino acid sequence at least 90% identical to any of said sequences.

35. The isolated antibody of embodiment 34 which comprises (1 ) an amino acid sequence as in SEQ ID NO: 95 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO:96 or an amino acid sequence at least 90%

36. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO:97, SEQ ID NO:98, or an amino acid sequence at least 90% identical to any of said sequences.

37. The isolated antibody of embodiment 36 which comprises (1 ) an amino acid sequence as in SEQ ID NO:97 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO:98 or an amino acid sequence at least 90%.

38. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO:99, SEQ ID NO: 100, or an amino acid sequence at least 90% identical to any of said sequences.

39. The isolated antibody of embodiment 38 which comprises (1 ) an amino acid sequence as in SEQ ID NO:99 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO: 100 or an amino acid sequence at least 90%.

40. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO: 101 , SEQ ID NO: 102, or an amino acid sequence at least 90% identical to any of said sequences.

41. The isolated antibody of embodiment 40 which comprises (1 ) an amino acid sequence as in SEQ ID NO: 101 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO: 102 or an amino acid sequence at least 90%.

42. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO: 103, SEQ ID NO: 104, or an amino acid sequence at least 90% identical to any of said sequences.

43. The isolated antibody of embodiment 42 which comprises (1 ) an amino acid sequence as in SEQ ID NO: 103 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO: 104 or an amino acid sequence at least 90%.

44. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO: 1 05, SEQ ID NO: 106, or an amino acid sequence at least 90% identical to any of said sequences.

45. The isolated antibody of embodiment 44 which comprises (1 ) an amino acid sequence as in SEQ ID NO: 105 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO: 106 or an amino acid sequence at least 90.

46. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO: 1 07, SEQ ID NO: 1 08, or an amino acid sequence at least 90% identical to any of said sequences.

47. The isolated antibody of embodiment 46 which comprises (1 ) an amino acid sequence as in SEQ ! D NO: 107 or an amino acid sequence at !east 90% and (2) an amino acid sequence as in SEQ ID NO: 1 08 or an amino acid sequence at least 90%.

48. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO: 1 09, SEQ ID NO: 1 1 1 , or an amino acid sequence at least 90% identical to any of said sequences.

49. The isolated antibody of embodiment 48 which comprises (1 ) an amino acid sequence as in SEQ ID NO: 1 09 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO: 1 1 1 or an amino acid sequence at least 90%.

50. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO: 1 1 3, SEQ ID NO: 1 14, or an amino acid sequence at least 90% identical to any of said sequences.

51. The antibody of embodiment 50 which comprises (1 ) an amino acid sequence as in SEQ ID NO.1 1 3 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO: 1 14 or an amino acid sequence at least 90%.

52. The isolated antibody of embodiment 5 comprising an amino acid sequence chosen from SEQ ID NO: 1 15, SEQ ID NO: 1 16, or an amino acid sequence at least 90% identical to any of said sequences.

53. The isolated antibody of embodiment 52 which comprises (1 ) an amino acid sequence as in SEQ ID NO: 1 15 or an amino acid sequence at least 90% and (2) an amino acid sequence as in SEQ ID NO: 1 16 or an amino acid sequence at least 90%.

54. The isolated antibody of one of embodiments 1-53, wherein said antibody is a monoclonal antibody.

55. The isolated antibody of one of embodiments 1-53, wherein said antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

56. The isolated antibody of one of embodiments 1 -53, wherein said antibody is a chimeric antibody.

57. The isolated antibody of any one of embodiments 1 -53, wherein said antibody is a humanized antibody.

58. The isolated antibody of any one of embodiments 1-53, wherein said antibody is a fully human antibody.

59. The isolated antibody of any one of embodiments 1-53, wherein said antibody is an antibody conjugated to a toxin and/or therapeutic agent.

60. The isolated antibody of any one of embodiments 1-53, wherein said antibody binds to a protein having SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.

61. The isolated antibody of any one of embodiments 1-53, wherein said antibody binds to an epitope having an amino acid sequence as in one of SEQ ID NOs: 4-16.

62. An isolated antibody that blocks or inhibits the binding of an antibody of anyone of embodiments 1-53 to CADM1 or a protein having SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3.

63. The isolated antibody of one of embodiments 1 -53 wherein said antibody is a diagnostic antibody.

64. The isolated antibody of embodiments 63 having a detectable label. 65. The isolated antibody of any one of embodiments 1 -53 wherein said antibody modulates cell adhesion.

66. The isolated antibody of any one of embodiments 1 -53 wherein said antibody modulates cell migration.

67. The isolated antibody of any one of embodiments 1 -53 wherein said antibody is effective for treating or preventing a cancer when administered to a mammal or human.

68. An isolated nucleic acid, or the complement thereof, encoding an isolated antibody as in any of embodiments 1 -53.

69. The isolated nucleic acid of embodiment 68 in a vector.

71. A host cell comprising an isolated nucleic acid of embodiment 68 or the vector of embodiment 69.

72. The host cell of embodiment 71 wherein said host cell is capable of producing an antibody of one of embodiments 1 -53.

73. The host cell of embodiment 72 which is a prokaryotic or eukaryotic host cell. 74. The host cell of embodiment 73 which is a eukaryotic host cell chosen from COS, CHO, HEK293 or a multiple myeloma host cell.

75. A pharmaceutical composition comprising the isolated antibody of any one of embodiments 1-53 and a pharmaceutically acceptable carrier.

76. The pharmaceutical composition comprising an isolated antibody as in any of embodiments 1 -53 wherein said pharmaceutical acceptable carrier is a carrier suitable for IV infusion.

77. A method for identifying an antibody that binds to CADM1 said method comprising providing a CADM1 antigen;contacting said CADM1 antigen with an antibody or fragment thereof; and identifying an antibody or fragment thereof that binds to a CADM1 antigen.

78. An antibody as in one of embodiments 1 -53, prepared by a process comprising:

(a) providing a hybridoma capable of producing said antibody; and

(b) culturing the hybridoma under conditions that provide for the production of said antibody by the hybridoma.

79. A hybridoma that produces an antibody of any one of embodiments 1-53.

80. A method of treating an individual having cancer comprising administering to said individual a therapeutically effective amount of a therapeutic antibody to CADM1 wherein said antibody is as in any one of embodiments 1 -53.

81 . The method of embodiment 80, wherein said therapeutic antibody is administered in a successive, sequential or simultaneous treatment regimen, and is administered intravenously or intraperitoneally to said individual.

82. A method of diagnosing prostate cancer comprising:

obtaining or providing a sample from an individual and determining the protein level of CADM1 in the sample wherein increased levels of CADM 1 in the sample indicate prostate cancer or an increased likelihood of prostate cancer.

83. The method of embodiment 82 wherein said sample is a serum, blood , or plasma sample.

84. The method of embodiment 82 wherein the protein level of CADM1 is determined with an antibody as in any one of embodiments 1 -53,

85. A method of diagnosing prostate cancer comprising:

a . obtaining or providing a sample from an individual

b. determining the protein level of CADM1 in the sample

wherein increased levels of CADM1 indicate prostate cancer and/or an increased likelihood of prostate cancer and wherein the protein level of CADM1 is determined using an antibody of any one of embodiments 1 -53.

85. An isolated antibody of any one of embodiments 1 -53 in a human antibody framework.

86. A method of treating an individual having cancer comprising administering to said individual a therapeutically effective amount the isolated antibody of embodiment 85.

87. The method of embodiment 86 wherein said cancer is prostate, kidney, lung, ovarian, breast, blood, colorectal, melanoma, or pancreatic cancer..

88. The method of embodiment 86 wherein said cancer is prostate cancer.

89. The method of any one of embodiments 86-88 wherein said antibody reduces levels of activity of CADM1 .

90. The method of any one of embodiments 86-88 wherein said antibody inhibits or reduces proliferation; causes cytotoxicity; inhibits or reduces metastasis; modulates, inhibits or reduces cell adhesion; modulates, inhibits or reduces migration; or modulates, inhibits or reduces invasion of cancer cells, cancer, prostate cancer or prostate cancer cells expressing CADM1.

91. The method of any one of embodiments 86-88 wherein said antibody inhibits or reduces proliferation of prostate cancer, prostate cancer cells, cancer, or cancer cells expressing CADM1 .

92. The method of any one of embodiments 86-88 wherein said antibody causes cytotoxicity to prostate cancer, prostate cancer cells, cancer, or cancer cells expressing CADM1 .

93. The method of any one of embodiments 86-88 wherein said antibody reduces or inhibits metastasis of prostate cancer, prostate cancer cells, cancer, or cancer cells expressing CADM1 .

94. The method of any one of embodiments 86-88 wherein said antibody reduces or inhibits cell adhesion of prostate cancer, prostate cancer cells, cancer or cancer cells expressing CADM1 .

95. The method of any one of embodiments 86-88 wherein said reduces or inhibits invasion of prostate cancer, prostate cancer cells, cancer or cancer cells expressing CADM1 .

96. The method of any one of embodiments 86-88 wherein said antibody reduces or inhibits migration of prostate cancer, prostate cancer cells, cancer or cancer cells expressing CADM1 .

97. The method of any one of embodiments 86-88 wherein said antibody induces, enhances, or mediates ADCC (antibody dependent cellular cytotoxicity) against cells to which it binds.

98. The method of any one of embodiments 86-88 wherein said antibody to CADM1 induces, enhances, or mediates CDC (complement dependent cytotoxicity) against cells to which it binds.

99. The method of any one of embodiments 86-88 wherein said antibody is conjugated to another molecule.

100. The method of any one of embodiments 86-88 wherein said antibody is conjugated to a cytotoxin, a radioactive agent, enzyme, toxin, an anti-tumor drug or a therapeutic agent.

101. An antibody as in one of embodiments 1 -53 wherein said antibody is capable of internalizing when bound to a CADM1 .

Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques used by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 : identification of Prostate Cancer Biomarkers

In order to identify biomarkers for predicting and/or diagnosing prostate cancer, gene expression levels from prostate primary tumors in several differentiation stages were compared with normal matched prostate tissues by RNA expression microarray techniques. This technique allowed us to check the expression of the whole genome in a particular type of cell, tissue, organ, or in this case, check the differential gene expression between prostate cancer and healthy prostate tissue. A microarray chip contains small oligonucleotide sequences arranged in a regular pattern with specific addresses for probes for typically thousands of genes.

The amount of specific mRNAs in a sample can be estimated by its hybridization signal on the array.

Sample Description

Tumor samples were obtained from prostate cancer patients who underwent surgery and had not received any treatment previous to surgery. Normal samples were also obtained from non-affected regions of the prostate of the same patients (paired samples). During preparation of the specimens, care was taken to microdissect the epithelial tissue away from any adjacent stroma. 27 paired tissue samples were included in set 1 (Table 1 ) and 7 in set 2 (Table 2). Both tables summarize some tumor sample characteristics and the stage (Gleason grade) of prostate cancer biopsy assigned by the pathologists. In the first set, three samples of RNA isolated from prostate stroma were co-hybridized with a pool of normal RNA.

In addition, 3 samples from prostate stroma were analysed to rule out that stroma contamination could account for the differential expression of candidate genes in normal versus tumoral tissue.

The basic characteristics of the test samples for sample set 1 are summarized in the Table 1 below. The basic characteristics of the test samples in sample set 2 are summarized in Table 2.

Total RNA was extracted with the RNeasy mini kit (Qiagen, Hilden, Germany), following the instructions provided by the manufacturer. Quantity and quality of the obtained RNA was measured with a Nandrop ND-1000, Agilent 2100 Bioanalyzer and low quality RNA was discarded from the array hybridization process.

Microarray Design

Microarrays for gene expression were designed by the Tethys algorithm using the ENSEMBL database. For sequences where we did not find high quality probes, we complemented the design with Oryzon optimized Agilent probes. DNA microarray synthesis was outsourced to Agilent.

The Whole Genome Gene Expression Array contains:

• 20148 Oryzon high quality probes from ENSEMBL Database.

• 5698 Oryzon Tm optimized Agilent probes.

The total number of probes was 25846.

aRNA Labeling

Cy3- and Cy5-labeled aRNA was produced using the MessageAmplification Kit by Ambion {Ref: 1819 for 96x kit or Ref: 1751 for 20x kit). These kits are used with some modifications introduced by Oryzon genomics. RNA labeling was performed essentially using the Eberwine protocol (Van Gelder, 1992) commercialized by Ambion with the MessageAmplification Kit (Ambion/Applied Biosystems) with minor modifications. 500 ng of total RNA was reverse transcribed in the presence of oligo{dT)24 , second-strand synthesis was generated and transcription of this dsDNA was prepared using CTP_Cy3 or CTP_Cy5 (PerkinElmer). Amplified cRNA was quantified by Nanodrop ND-1000 and cRNA quality was controlled with the Agilent RNA Bionalyzer 2100.

Microarray Hybridization

Microarray hybridization was performed at 60°C and 17 hours hybridization time according to Agilent indications, using Agilent gaskets (G2534-60002), Agilent hybridization chambers (G2534A) and in an Agilent DNA Hybridization Oven (G2545A). Oryzon hybridization controls were also used in the hybridization process. Controls for the hybridization process corresponding to 3 cDNA clones of maize (Xet, Zm42,Exp) were included in all analysis. Exp is used as the negative spike control and was not amplified or labeled. For Xet and Zm42 PCR fragments were generated by PCR amplification from the vector with universal primers and cRNA was generated using in vitro transcription systems (T7 or T3 Megascript kit; Ambion) with CTP_Cy3 or CTP_Cy5 (PerkinElmer). Both of the positive spike controls Xet and Zm42 were with both the Cy5 and Cy3 fluorophore.

Data Acquisition

initial raw data were obtained using an Agilent DNA Microarray Scanner (G2505B) and Agilent acquisition software (Feature Extraction Software). The extraction protocol performed does not use background subtraction, computation of dye biases and ratio correction.

Data Analysis

A large number of controls were included in the microarray designs to monitor scanner and array performance and to control spatial homogeneity and correct deviations. This way, the overall error on the microarray data measurements can be estimated by the spreading analysis of the data from the controls.

The mean fold change or M values can be ranked based on their probability of being different from 0, according to the absolute value of the regularized t-statistic (Baldi and Long, 2001 ) which uses a Bayesian framework to derive a modified and improved t-student statistics. To make Fold Change based selection, the mean M distribution was used. This distribution is adjusted to a normal distribution and an iterative process is used to define the mean M numbers that are outside the distribution. The cut-off is chosen as n times the Standard deviations (σ) from the mean. This method generates a robust mean and standard deviation and allows to dynamically adjusting the cut-off value to the noise distribution of the data. Typically, values with mean FC>3σ or mean FC<-3σ of the sample data distribution were selected.

Results of microarray analysis

mRNA expression of CADM1 was analysed with 2 probes.

Table 3 below shows the fold change in CADM1 mRNA of the 27 tumoral and 3 stroma samples (set 1 of microarray analysis). For the probe ENSG00000182985 the mean fold change of tumoral versus normal samples was +1 .43 with a p-value of 2Ί 0-7. The mean fold change of the stroma versus normal tissue was -1 .14. Accordingly, in case of the probe A_24_P227230, the mean fold change of tumoral versus normal samples was +1 .29 with a p-value of 9.6· 10-5 . The mean fold change of the stroma versus normal tissue was -1 .25.

As shown in Table 4, CADM1 expression levels were confirmed using a different set of tumor samples (set 2, see Table 2). For the probe ENSG00000182985 the mean fold change of tumoral versus normal samples was +1 .41 with a p-value of 0.143. For the probe A_24_P227230 the fold change was +1.18 and the p-value 0.235.

Example 2: Confirmation of Overexpression of CADM1 by RT-PCR

CADM1 candidate was selected for its up-regulated fold change. Increased expression levels can be validated by an independent technique (RT-PCR using Microfluidic Cards from Applied Biosystems) using RNA isolated from tumor and normal prostate tissue (set 3).

Sample Description

Samples can be used as described in Example 1 .

RNA can be extracted as described in the gene expression microarray analysis. RT-PCR can be performed following Applied Biosystem standard protocol for the 7900HT system. The protocol consisted in a two-step method where the first step is the generation of cDNA from the RNA samples using a High Capacity cDNA Kit and the second step is the amplification of cDNA. cDNA is loaded in the wells of Microfluidic Card, which contain Applied Biosystems fluorogenic 5' nuclease assays that detect the real-time amplification of the array selected target by the ABI PRISM® 7900 HT system.

Data analysis

Relative levels of gene expression can be determined from the fluorescence data generated during PCR using the ABI PRISM® 7900HT Sequence Detection System (7900HT SDS) Relative Quantification software. Data analysis for the set of samples can be made using the comparative ΔΔCt method of relative quantification. Firstly, Ct of normal and tumoral samples can be normalized respect to the Ct of the endogenous gene (18S) to get the delta Ct (ΔCt CADM1 = Ct CADM1 - Ct 18S).

Next the mean of all the normal samples is calculated (mnΔCt: mean normal ΔCt). Finally, individual normal and tumoral ΔΔCt values are calculated (ΔΔCt = ΔCt CADM1 - mnΔCt). RQ is calculated for each normal and tumoral sample using the formula RQ = 2-(ΔΔCt).

RQ: relative quantity, it is the relative amount of RNA for a specific gene present on the tumour samples referred to the amount present on the control samples for the same gene.

Results of TaqMan analysis

The RQ values corresponding to the normal and tumoral samples are expected to show increased levels of CADM1 is tumor samples.

Example 3: Study of CADM1 at the protein level

A TMA of prostate cancer, other types of cancer and a panel of normal tissues can be analysed for CADM1 expression by using antibodies specific to CADM1 which have been developed by phage display as described herein

Tissue microarray (TMA) method enables to extract small cylinders of tissue from normal or pathology specimens and arrange them on a matrix. A large number of tissues can be included in one slide and be analysed at the same time, for instance, analyze the patter of expression of a protein in normal and tumor biopsies by immunohistochemistry staining with the same experimental conditions protocol and antibodies. Antibody-2 gave positive results in TMA studies.

Example 4: Characteristics of CADM1

CADM1 was found to be overexpressed in prostate cancer primary tissue as compared to normal prostate tissue by the microarray experiment described in Example 1.

The gene identification number corresponding to CADM1 is given in ENSEM BL accession number below. This accession numbers has information related to the gene, mRNA, and associated protein sequences for CADM1 .


The corresponding amino acid sequence for one CADM1 isoform is given in the ENSEMBL protein accession numbers below and have a sequences as in SEQ ID NO: 1 (ENSP00000329797), 2{ENSP00000385329) and 3 (EP000000395359),


Example 5: Preparation of Monoclonal Antibodies

Monoclonal antibodies to CADM1 can be prepared using known hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. In a hybridoma a host animal (e.g., mammal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent (or the lymphocytes may be immunized in vitro).

The immunizing agent will typically include the target protein polypeptide CADM1 (or fragment thereof) or a fusion protein thereof or epitope thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against CDAM1 target protein or epitope. The binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard (1980) Anal, Biochem. 107:220.

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using

conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (e.g., U.S. Pat. No. 4,816,567; Morrison et al., supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

Example 6: Humanization of Murine Antibodies

This example sets out a procedure for humanization of a murine anti-CADM1 antibody.

Design of Genes for Humanized CADM1 Antibody Light and Heavy Chains

The VL and VH amino acid sequences for murine antibodies are known or can be determined using standard molecular biology techniques (e.g., from the monoclonal antibody as prepared according to Example 5 or commercially available hybridomas encoding such an antibody). The sequence of a human antibody identified using the National Biomedical Foundation Protein Identification Resource or similar database can be used to provide the framework of the humanized antibody. To select the sequence of the humanized heavy chain, the murine heavy chain sequence is aligned with the sequence of the human antibody heavy chain. At each position, the human antibody amino acid is selected for the humanized sequence, unless that position falls in any one of four categories defined below, in which case the murine amino acid is selected:

(1 ) The position falls within a complementarity determining region (CDR), as defined by Kabat (1980) J. Immunol. 125:961 -969;

(2) The human antibody amino acid is rare for human heavy chains at that position, whereas the murine amino acid is common for human heavy chains at that position;

(3) The position is immediately adjacent to a CDR in the amino acid sequence of the murine heavy chain; or

(4) 3-dimensional modeling of the murine antibody suggests that the amino acid is physically close to the antigen binding region.

To select the sequence of the humanized light chain, the murine light chain sequence is aligned with the sequence of the human antibody light chain. The human antibody amino acid is selected at each position for the humanized sequence, unless the position again falls into one of the categories described above and repeated below:

(1 ) CDR's;

(2) murine amino acid more typical than human antibody;

(3) Adjacent to CDR's; or

(4) Possible 3-dimensional proximity to binding region.

The actual nucleotide sequence of the heavy and light chain genes is selected as follows:

(1 ) The nucleotide sequences code for the amino acid sequences chosen as described above;

(2) 5' of these coding sequences, the nucleotide sequences code for a leader (signal) sequence. These leader sequences are chosen as typical of antibodies;

(3) 3' of the coding sequences, the nucleotide sequences are the sequences that follow the mouse light chain J5 segment and the mouse heavy chain J2 segment, which are part of the murine sequence. These sequences are included because they contain splice donor signals; and

(4) At each end of the sequence is a specific restriction site (e.g., Xba I site) to allow cutting at the restriction site (e.g., Xba I sites and cloning into the restriction site (e.g., Xba I site) of a vector.

Construction of Humanized Light and Heavy Chain Genes

The genes encoding the humanized light and heavy chain genes can be prepared by any method. One method involves annealing fragments of the gene together to synthesize the full length genes.

To synthesize the heavy chain, four oligonucleotides are synthesized using a DNA synthesizer (e.g., Applied Biosystems 380B DNA synthesizer). Two of the oligonucleotides are part of each strand of the heavy chain, and each oligonucleotide overlaps the next one by about 20 nucleotides to allow annealing. Together, the oligonucleotides cover the entire humanized heavy chain variable region with a few extra nucleotides at each end to allow cutting at the restriction site (e.g., Xba I sites). The oligonucleotides are purified from polyacrylamide gels.

One method for annealing oligonucleotides is as follows: Each oligonucleotide is phosphorylated using ATP and T4 polynucleotide kinase by standard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). To anneal the phosphorylated oligonucleotides, they are suspended together in 40 μl of TA (e.g., 33 mM Tris acetate, pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate) at a concentration of about 3.75 μΜ each, heated to 95° C for about 4 min. and cooled slowly to 4° C. The complete gene can be synthesized from the oligonucleotides by synthesizing the opposite strand of each oligonucleotide, for example the following components are added in a final volume of 100 μl:

• 10 ul annealed oligonucleotides

• 0.16 mM each deoxyribonucleotide

• 0.5 mM ATP

• 0.5 mM DTT

• 100 ug/ml BSA 3.5 ug/ml

• T4 g43 protein (DNA polymerase)

25 ug/ml T4 g44/62 protein (polymerase accessory protein)

• 25 ug/ml 45 protein (polymerase accessory protein)

The mixture is incubated at 37° C for about 30 min. Then 10 ug of T4 DNA ligase is added and incubation at 37° C is resumed for about 30 min. The polymerase and ligase are inactivated by incubation of the reaction at 70° C for about 15 min. To digest the gene with Xba I, 50 ul of 2xTA containing BSA at 200 ug/ml and DTT at 1 mM, 43 ul of water, and 50 ug of Xba I in 5 ul of buffer is added to the reaction. The reaction is incubated for 3 hr at 37° C and then purified on a gel. The Xba I fragment is purified from a gel and cloned into the Xba I site of the plasmid pUC19 by standard methods. Plasmids are purified using standard techniques and sequenced using the dideoxy method.

Construction of plasmids to express humanized light and heavy chains can be accomplished, -e.g., by isolating the light and heavy chain Xba f fragments from the pUC19 plasmid in which it had been inserted and then inserting it into the Xba I site of an appropriate expression vector which will express high levels of a complete heavy chain when transfected into an appropriate host cell.

Synthesis and Affinity of Humanized Antibody

The expression vectors are transfected into mouse Sp2/0 cells, and cells that integrate the plasmids are selected on the basis of the selectable marker(s) conferred by the expression vectors by standard methods. To verify that these cells secreted antibody that binds to CADM1 , supernatant from the cells are incubated with cells that are known to express CADM1. After washing, the cells they can be, e.g., incubated with fluorescein-conjugated goat anti-human antibody, washed, and analyzed for fluorescence on a FACSCAN cytofluorometer.

The cells producing the humanized antibody are cultured in vitro. Humanized antibody is purified to substantial homogeneity from the cell supernatants by passage through an affinity column of Protein A (Pro-Chem. Inc., Littleton, Mass. or equivalent) according to standard techniques. The affinity of the humanized antibody relative to the original murine antibody is determined according to techniques known in the art.

Example 7: Selection and Screening of Anti-CADM1 antibody fragments via phage display

A number of strategies can be used to generate recombinant antibodies against CADM1 protein or a CADM1 epitope.

A Target CADM1 protein or epitope can be immobilised to immunotube and contacted with a phage display library. The library can be used screening on cell, fragment of cells or tissue overexpressing the target.

A phage display library is a library of genetically engineered phage (virus that can infect bacteria). The phage library is engineered in such a way that it encodes a diversity of antibodies, single chain antibodies or fragments thereof which are expressed in a bacteriophage library as the form of fusions with the bacteriophage coat protein where the antibodies, single chain antibodies or fragments thereof protein are displayed on the surface of the phage. Each individual phage corresponds to and displays an antibody, single chain antibody or fragment thereof that corresponds to the genetic sequence within the phage. Phage with specific binding properties can be isolated and the gene for the antibody can be sequenced, cloned or otherwise isolated.

Thus, the phage-display library is added to the immunotube containing the CADM1 target protein or CADM1 epitope and incubated for a length of time. After incubation for a time sufficient to allow phage to bind to the target protein or epitope, the dish is washed. Phage-displayed antibodies that bind with the target protein or epitope remain attached to the dish, while the other phage are washed away.

Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant (i.e. non-binding phage) than were present in the initial mixture.

The DNA within the interacting phage contains the sequences of interacting proteins, and following further bacterial-based amplification, can be sequenced to identify the relevant, interacting proteins or protein fragments.

A fully human antibody library was used to identify and isolate fully human antibodies which have amino acid sequences of CDRs and variable sequences corresponding to the SEQ ID NOs in the following tables.

Example 8: Testing Anti-CADM1 Antibodies for Anti-Cancer Properties

Antibodies or fragments thereof to CADM1 , such as those identified or created by any of the foregoing Examples are tested for anti-cancer properties

A cell line of prostate cancer is grown and propagated in culture according to methods well known to one of ordinary skill in the art. Various dosages of potentially therapeutic antibodies or fragments thereof according to the invention are applied to various cultures of the prostate cell line. The treated cultures and control cultures (treated with a sham antibody or fragment) are then followed over time and scored for reduction in proliferation; reduction in cellular growth; appearance of cytotoxicity; reduction in cell-adhesion; or reduction in cell migration.

Comparison of control and treated cultures reveals that at least some of the potentially therapeutic antibodies or fragments thereof significantly modulate at least one of proliferation; cellular growth; cytotoxicity; cell-adhesion; or cell migration.

Antibody-10 as described herein was tested in cell viability assays and shown to have an effect. See figure 1 . More specifically, Antibody-10 was shown to have an effect in hexosaminaidase assays of cell viability in both LNCaP and DU145 prostate cancer cells.

Cell adhesion studies were performed with several of the antibodies of the invention in cell-adhesion assays with fibronectin. Antibody-10 had an effect -50% at 0.05uM with DU145 cells. See figure 2.

In these cell migration studies Antibody-10 were found to have some effects in the pancreatic cancer line model BxPC3. See figure 3.

Protocols for the above assays are as follows:

Viability Assay: LnCap cells were plated at 1 0000 cells/well in 100 μl media in tissue culture 96 well plates (Cultek). After 24h, media was supplemented by 100 μl/well of diluted monoclonal {e.g., recombinant) antibody at final concentration of 1 μΜ . Dilutions were done, so the range of compound concentration was 1 μΜ to 0.1 nM . Three replicates were done. Negative control did not contain compounds. Reagent blanks, containing media plus colorimetric reagent without cells were run on each plate. Blank values were subtracted from test values and were routinely 10-15% of uninhibited control values. Plates were incubated 24h and 48h and living cell number was determined by hexosaminidase test. The enzymatic activity was measured according to the following protocol: the media was removed and cells were washed once with PBS . 60 pi of substrate solution (p-nitrophenol-N-acetyl-beta-D-glucosamide 7.5 mM [Sigma N-9376], sodium citrate 0.1 M, pH 5.0, 0.25% Triton X-1 00) was added to each well and incubated at 37°C for 1 -2 hours; after this incubation time, a bright yellow appears; then, plates could be developed by adding 90 μl of developer solution (Glycine 50 mM, pH 10.4; EDTA 5 mM), and absorbance was recorded at 410 nM. Blank values were subtracted from test values and were routinely 5-1 0% of uninhibited control values.

Methods of evaluation and statistics: Data analysis was done by calculating the percentage of cell viability normalized in front of negative control values, that were considered as a 1 00%. The curve was adjusted using a sigmoidal dose-response (variable slope) equation , and EC50 values were obtained from the equation:

Y=Bottom + (Top-Bottom)/(1 +10^((LogEC50-X)*HillSlope)),

where X is the logarithm of concentration, and Y is the response.

Cell adhesion Assay:

Cell lines used in the study were DU145 and LnCap human prostate cancer cells.

Examplary Coating proteins include: Vitronectin (VN); Fibrinogen (FB); or Collagen (CL)

Inhibition of cell adhesion: Non-tissue culture treated ELISA plates were coated ON at 4°C with the appropriate concentration of the ligand. Coating solution was discarded and wells were blocked with blocking solution (BSA 1.5%; 60 minutes at 37°C). Blocking solution was discarded by flicking and serial dilutions of mAbs were plated in duplicates/triplicates. Immediately, harvested cells were plated at a given concentration (40000/100 μl) to the same plate. Plates were incubated for 60-90 min at 37°C to allow cell adhesion and spreading on the ligands. Plates were washed three times with PBS and 100 ul/well of Hexosaminidase Substrate solution was added. Plates were incubated at 37°C during 3-5 hours and then stop solution was added. Adsorption was read at OD 405nm in a Multiskan ASCENT.

Each plate contains positive and negative controls and human IgG was also tested.

Cell migration assay: BxPC3 were thawed in DMEM high glucose (Gibco-BRL 31966-21 ) plus 1 0% heat-inactivated Foetal Calf Serum (FCS) (Gibco-BRL 101 06-169). Cells were passaged at 90% confluence by washing once in HBSS cation-free followed by a 3 min incubation with trypsin ([0.5 μg/ml]/EDTA [0.2 μg/ml]) (Gibco-BRL, 1 5400054) solution in HBSS at 37°C, and transferred to DMEM high glucose. Cells were counted and concentrated to 1 25000 cell s/20p l.

Twenty-four-well cell culture plates with light-opaque PET membrane filter inserts with 8 μm-pores (Transwell HTS FluoroBlokTM Multiwell Insert Systems from Becton Dickinson) were used to test the migratory activity. The upper and lower surfaces of the Transwell membranes were coated for 2 hours at 37°C with 15 μg/mL of Type I Collagen (Upstate). Appropriate mAb concentrations were prepared in 80 μl/well of DMEM high glucose. Then, 20

μl of the cell suspension were added and seeded, after the collagen coating, onto the upper side of each Transwell chamber. Then, 500 μl DMEM high glucose plus 10% FCS (complete media) were added to the lower compartment of the 24-well plates to test their chemotactic capacity. Negative controls (lower compartment with DEMEM high glucose without FCS), positive controls (DMEM high glucose plus 10% FCS, without mAb) and human IgG at 3-0.5 μΜ we included in the assays. All treatments were triplicates. After 24 hours at 37°C, cells that had migrated to the lower side of the transwell were incubated, after removing media from the lower compartment, with 3 μΜ Calcein-AM (Calbiochem) in DMEM high glucose for 25-30 min at 37°C. Migrated cells were photographed under a light microscope at a magnification of X10 and, then, media with calcein was removed from the lower compartment and replaced with media without calcein and fluorescence was measured at 485/538 (excitation/emission) using a Fluoroscan ASCENT. Nine values from the center of the insert were taken for statistical analysis.

For statistical analysis, an unpaired Mann-Whitney non-parametric t test (p<0.05) was applied.

Example 9: Testing of Anti-CADM1 Antibodies Using In-vivo models

Antibodies or fragments thereof identified as modulating a CADM1 expressing cell line in Example 8 can be tested using an in-vivo model.

An in-vivo model of a CADM1 expressing is provided for testing. The in-vivo model may be an animal having prostate and/or an animal that has had a cell line of prostate implanted and allowed to proliferate to form a tumor. Various dosages of potentially therapeutic antibodies or fragments thereof according to the invention are used to treat the in-vivo model. The treated models and control models (treated with a sham antibody or fragment) are then followed over time and scored for reduction in proliferation; reduction in cellular growth; appearance of cytotoxicity; reduction in cell-adhesion; reduction in metastasis, reduction in cell invasion, or reduction in cell migration.

Comparison of control and treated models can be carried out and can reveal the utility of the therapeutic antibodies of the invention or fragments thereof for example via modulation of proliferation; cellular growth; cytotoxicity; cell-adhesion; cell invasions, metastasis, or cell migration.

Example 10: Diagnosis Using Anti-CADM1 Antibodies

Antibodies or fragments thereof to CADM1 , such as those identified or created by any of the foregoing Examples can be tested for usefulness in the diagnosis of prostate and/or increased risk for developing prostate.

A cohort of subject is identified and a sample collected from each subject. The sample is tested for levels of CADM1 using the antibodies or fragments thereof to CADM1 . All subjects are further tested for the presence of prostate using techniques standard in the art. All subjects are followed and periodically tested using the antibodies or fragments thereof to CADM1 and further tested for the presence of prostate using techniques standard in the art. After each round of testing, the levels of CADM1 are correlated with the presence of prostate and/or increased risk for developing prostate.

The foregoing correlation can be carried out and can reveal the use of the antibodies or fragments thereof to CADM1 in the diagnosis of prostate and/or identifying subjects with an increased risk for developing prostate.

Example 1 1 : identification and Development of Nucleic Acids Capable of Binding CADM1 Nucleic Acids

Nucleic acids capable of binding CADM1 nucleic acids are identified.

The nucleotide sequence of a CADM1 gene is identified and potential regulatory and/or splice donor or acceptor sites are identified. Nucleic acids complementary to the potential regulatory and/or splice donor or acceptor sides are identified. The complementary nucleic acids are examined for homology and/or potential binding to known sequence using the available nucleotide sequence data bases. Those complementary nucleic acids that are likely to specifically bind CADM1 are synthesized.

The secondary structure of the CADM1 mRNA and pre-mRNA are also identified. Within the determined secondary structures, areas which remain single stranded and accessible are identified. Nucleic acids complementary to the single stranded portions are identified. The complementary nucleic acids are examined for homology and/or potential binding to known sequence using the available nucleotide sequence data bases. Those complementary nucleic acids that are likely to specifically bind CADM1 are synthesized.

The synthesized nucleic acids are tested for the ability to specifically bind CADM1 using microarray and/or northern or southern blot techniques.

Example 12: Testing Anti-CADM1 Nucleic Acids for Anti-Cancer Properties

Nucleic acids complementary to CADM1 , such as those identified or created by any of the foregoing Examples can be tested for anti-cancer properties

A cell line of prostate cancer is grown and propagated in culture according to methods well known to one of ordinary skill in the art. Various dosages of potentially nucleic acids according to the invention are applied to various cultures of the prostate cell line. The treated cultures and control cultures (treated with a sham nucleic acid) are then followed over time and scored for reduction in proliferation; reduction in cellular growth; appearance of cytotoxicity; reduction in cell-adhesion; or reduction in cell migration.

Comparison of control and treated cultures can be carried out and can reveal the utility of therapeutic nucleic acids to modulate proliferation; cellular growth; cytotoxicity; cell-adhesion; or cell migration.

Example 13: Testing of Anti-CADM1 Nucleic Acids Using In-vivo models

Nucleic acids identified as modulating a cancer cell line expressing CADM1 in

Example 12 can be tested using an in-vivo mode!.

An in-vivo mode! of a cancer expressing CADM1 is provided for testing. The in-vivo model may be an animal having prostate and/or an animal that has had a cell line of prostate implanted and allowed to proliferate to form a tumor. Various dosages of potentially therapeutic nucleic acids according to the invention are used to treat the in-vivo model. The treated models and control models (treated with a sham nucleic acids) are then followed over time and scored for reduction in proliferation; reduction in cellular growth; appearance of cytotoxicity; reduction in cell-adhesion; reduction in metastasis, reduction in cell invasion, or reduction in cell migration.

Comparison of control and treated models can be carried out and can reveal the utility of the therapeutic nucleic acids to modulate proliferation; cellular growth; cytotoxicity; cell-adhesion; eel! invasions, metastisis, or cell migration.

Example 14: Diagnosis Using Anti-CADM1 Nucleic Acids

Antibodies or fragments thereof to CADM1 , such as those identified or created by any of the foregoing Examples can be tested for usefulness in the diagnosis of prostate and/or increased risk for developing prostate.

A cohort of subject is identified and a sample collected from each subject. The sample is tested for levels of CADM1 using the nucleic acids to CADM1. All subjects are further tested for the presence of prostate using techniques standard in the art. All subjects are followed and periodically tested using the nucleic acids to CADM1 and further tested for the presence of prostate using techniques standard in the art. After each round of testing, the levels of CADM1 are correlated with the presence of prostate and/or increased risk for developing prostate. The foregoing correlation can be carried out and can reveal the utility of the nucleic acids to CADM1 in the diagnosis of prostate and/or identifying subjects with an increased risk for developing prostate.

Example 15: Flow Cytometry

Flow cytometry experiments were undertaken with antibodies of the invention.

Flow cytometry is a technique that allows measuring certain physical and chemical characteristics of cells or microscopic particles as they pass in a fluid stream by a beam of laser light. Millions of cells can be analyzed by staining their proteins with specific fluorescent antibodies. Antibody staining of cell membrane proteins and its analysis by flow cytometry is the best way to characterize cell populations. A major application is to separate cells according to subtype or epitope expression for further biological studies. This process is called cell sorting or FACS. Other interesting applications are comparation of membrane protein levels, monitorization of internalization of receptors or even studies of antibody affinities and specificities. See e.g..Handbook of Flow Cytometry Methods by J. Paul Robinson, et al. ISBN 0471596345. The results of studies with various cell lines and various antibodies of the invention are shown in the Tables below. CHO-FIpIn is a CHO cell line. The CHO-FIpIn CADM1 line in a CHO cell line derived from the CHO-FIPIn line and engineered to express the CADM1 epitope described herein (corresponding to CADM1 extracellular domain). The values equal percent of cell that are bound by the indicated CADM1 antibodies.

NF(means no fucosylated to use in ADCC experiments).

Example 16: ELISA

An ELISA assay was constructed for antibodies of the invention.

Enzyme-linked immunosorbent assay (ELISA) is a plate-based technique designed for detecting and quantifying substances such as peptides, proteins, antibodies or hormones using specific labeled antibodies. The antigen must be immobilized to a solid surface, usually a polystyrene microtiter plate, either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). If the antigen is in a live cell, this can be growth in the same place where they are going to be detected with a specific antibody. The detection requires using a specific fluorescent or covalently linked to an enzyme antibody that is applied over the antigen coated surface of the plates. The most commonly used enzyme labels horseradish peroxidase (HRP) and alkaline phosphatase (AP). if the detection antibody is is biotin labeled a secondary antibody linked to a protein such as streptavidin is required. Therefore the assay combines the specificity of antibodies with the sensitivity of simple enzyme assays or fluorescence intensity signal. The ELISA it is very useful as a diagnostic tool in basic research, medicine.

Lequin R (2005). "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA).". Clin. Chem. 51 (12): 2415-8. doi:10.1373/clinchem.2005.051532. PMID 16179424

It was found that in this ELISA assay Antibody-2 was positive for: human prostate cancer cell lines like LnCaP and DU145 and also the HELA cervical cancer line whereas it was negative for human prostate cancer line PC3.

Example 17: Western Blot Assay

Western blotting is an analytical method that allows identifying native or denatured proteins immobilized on a PVDF or nitrocellulose membrane using specific antibodies for their detection. The target protein can be in a tissue homogenate or cell extract and must be separated using a SDS polyacrylamide gel electrophoresis (SDS-PAGE). The separation by size must be using denaturing conditions and by the 3-D structure of the protein with native conditions. W. Neil Burnette (April 1981 ). '"Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate — polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A". Analytical Biochemistry (2): 195-203. PMID 6266278.

Antibody-2 described herein was successfully used in a western blot assay to detect CADM1 protein.

Example 1 8: In vitro internalization assay

CADM 1 antibody internalization has been tested with a Hum-ZAP assay. Cancer cell lines that express CADM 1 on their surface and have been positive by flow cytometry are chosen for this assay as well as a cell line without CADM 1 expression as a negative control . Cells were plated in 96-well microplates at 1000 - 2,500 cells/well in a volume of 100ul of complete tissue culture medium and incubated at 37C in the presence of 5% CO2. After 24h 1 00ul of different concentrations of primary antibody (1 0E-7 to 1 0E-1 1 ) pre-incubated during 30 minutes with 100ng (10ul at 10ng/ul) of either mAb-ZAP or Hum-ZAP secondary antibodies were added per well. Some negative control plates were also treated with 100ul containing 1 00ng of secondary antibody-toxin conjugated but without primary antibody or with

isotype control IgG . Recognition and internalization of the primary antibody results in delivery of the saporin-antibody complex to the cell interior followed by cell killing. Cell viability was assayed after 72h using Cell Proliferation Assay AlamarBlue according to the manufacturer's instructions ( Invitrogen, Ref. DAL1 025), and absorbance was monitored at 570nm and 600nm in a microplate reader. Percent cell viability was calculated by assigning the average of the readings ( n≥6) from mAb-ZAP or Hum-ZAP secondary antibodies without anti-CADM 1 antibody as 1 00% cell viability % viability= (average of sample/average of negative control)x 100

Results

Anti-CADM 1 monoclonal antibodies AB_02 and AB_10 incubated with Hum-ZAP secondary antibodies were shown to be internalized upon binding CADM1 expressing cell lines. Cell death is only detected in cancer cell lines that express CADM 1 and no cytotoxicity was detected at ail in negative control cells or in any cell incubated only with secondary saporin-antibody.

Example 19 : anti-CADM1 antibodies recognize CADM1 specifically

In order to demonstrate that the antibodies of the invention recognize CADM1 and not any other protein expressed as a result of CADM1 overexpression, an experiment was performed using the small interfering RNA technique (siRNAs). siRNAs were used to modulate expression levels of CADM1 and demonstrate specificity of binding of the antibodies of the invention.

More specifically, the level of CADM1 protein detected by flow cytometry in the original CHO cell was compared to the level of protein detected in the same cell line overexpressing human CADM1 protein (CLON 139). Clon 139 is a CHO stable cell line that has integrated in its genome the human CADM1 DNA sequence IOH26601 . Clon 139 cells were transiently transfected with a siRNA control or a siRNA which blocked the expression of CADM1 (Invitrogen siRNA ID; s24327, s24325, s24326) and labelled with the antibodies described herein.

Briefly, cells were plated on 6-well plate dishes and transiently transfected with lipofectamine 2000 (Invitrogen) with the corresponding siRNA. Cell were transfected at 70-80% confluence with 50 pmoles of siRNA per well for two consecutive days. 48 hours after the second transfection, the expression of cell surface CADM1 was evaluated by flow cytometry. Cells were detached with PBS without using enzymatic treatments as trypsin and counted. Around 250.000 cells were transferred to tubes and incubated with the corresponding anti-CADM1 antibody (human IgG1 isotype) at 10ug/ml final concentration or without antibody in the case of the negative control, on ice for an hour. After that, cells were washed with 1x PBS plus 1 % of FBS three times by centrifugation.The centrifugation step (5 min at 1500 g and 4C) pellets cells from the staining suspension, thus permitting removal of the supernatant fluid, and the washing of unbound antibody from the cells. The second incubation is with 1 :250 dilution of a goat anti-human secondary antibody conjugated to Alexa Fluor 488 (Jackson InmunoResearch) for one hour at 4C in the dark. Three final wash steps (5 min at 1500 g and 4C) with 1x PBS plus 1 % of FBS, before the suspension of the cells with 300ul of PBS/1 % FBS and their analysis by flow cytometry.

Fig. 4, 5 and 6 show that the signal detected with the antibody AB2, AB-3 and AB10 of the invention, respectively, is drastically reduced when clone 139 cells were analyzed 48 hours after transfeccion with siRNA s24325 against CADM1 . By contrast in cells transfected with control siRNA, there is no change in CADM1 expression levels if we compare with untrasfected clon 139 cells. These results show that the antibodies of the invention recognize CADM1 in a specific manner.

Similar results were obtained when using siRNA s24327 or s24326.

AB2, AB3 and AB10 exhibited the same specific binding to CADM1 when tested using DU145 prostate cancer cells.

Example 20 : Binding of antibodies of the invention to other members of the CADM1 family

The CADM1 family comprises four genes, encoding proteins with three !g-like domains, a single trans membrane region, and a short cytosolic tail. It has been reported that the four human CADM1 family members (CADM1 , CADM2, CADM3

and CADM4) share a high degree of amino acid identity ranging for the full-length proteins from 50% (CADM2 vs CADM3) to 36% (CADM 1 vs 3 or 4). (Biederer, T. Bioinformatic characterization of the SynCAM family of immunoglobulin-like domain containing adhesion molecules. Genomics 87, 139-150 (2006)) In order to demonstrate that the antibodies of the invention recognize CADM1 and not CADM2, CADM3 or CADM4, full-length cDNAs of each one cloned in pCMV6-XL5 vector (OriGene) were transiently transfected in CHO cells and their expression under CMV promoter tested by cytometry with the antibodies of the invention.

CHO cells were plated on 100mm dishes and transiently transfected at 60% of confluence. Cells were transfected with lipofectamine 2000 (Invitrogen) with 6ug of vector DNAs per plate for two consecutive days. 48 hours after the second transfection, the expression of each cell surface CADM1 , CADM2, CADM3 and CADM4, was evaluated by flow cytometry following the same protocol described in Example 19, using AB2, AB3 and AB10 as primary antibodies.

Results are shown in Figs 7 to 9. AB2, AB3 and AB10 were shown to only recognize CHO cells that express CADM1 but not the CHO cells that express the other three members of the family: CADM2, CADSV13 and CADM4. The secondary antibody doesn't have any fluorescence background.

Example 21 : Measurement of the affinity of the antibodies of the invention to CADM1 by cytometry

The measurement of binding affinity of anti-CADM1 antibodies of the invention to CADM1 was done by flow cytometry.

The antibodies were tested with several human cancer cell lines that express CADM1 as: LnCaP prostate cancer cell line (ATCC), SKOV3 ovarian cancer cell line (ATCC) or A375 melanoma cell line (ATCC). Nine tubes with 250.000 cells are prepared and kept on ice during the whole process. The cells were carefully detach with PBS without using enzymatic treatments, such as trypsin, counted and resuspended in a volume of 100u( of 1xPBS + 1 % FBS with the primary antibody to the following final concentrations: 10ug/ml, 5ug/ml, 1 ug/ml, 0.5ug/ml, 0.25ug/ml, 0.1 ug/mS, 50ng/ml and 10ng/ml. One of the tubes hasn't got primary antibody because it is going to be the negative control (it contains only secondary antibody to quantify background). The mixture was incubated for 1 hour. After that, cells were washed with 1x PBS plus 1 % of FBS three times and incubated for another hour at 4C with the 1 :250 dilution of the secondary antibody: a goat anti-human secondary antibody conjugated to Alexa Fluor 488 (Jackson InmunoResearch) when we are testing lgG1 antibodies or anti-mouse conjugated to Alexa Fluor 488 (Invitrogen) if we are testing mouse lgG2a antibodies.

The results obtained with AB2, AB3 and AB10 are shown in table 9 below. A representative graph obtained for AB10 in A375 and AB3 in SKOV3 is shown in Fig 10 and 1 1.

The antibodies of the invention show very high binding affinities to CADM1 in human cancer cell lines, with EC50 values in the low nM range and in some cell lines under 1 nM (see Table 9) .

The comparison of affinity binding between the isotypes human IgGl and mouse lgG2a are very similar: for instance, 1 .26 and 0.76 nM, respectively for anti-CADM1 AB-10 in A375 cells.

No significant differences in binding between fucosilated and non-fucosilated antibodies of the invention were detected.

The antibodies of the invention exhibit higher affinities as compared to prior art antibodies disclosed in WO2010102175A1 . The EC50 values in SKOV3 ovary cancer cells for AB2, AB3 and AB10 are in the range of about 1 -2 nM; for example the EC50 value for AB3 is 1 nM. By contrast, the anti-CADM1 antibody disclosed in WO2010102175A1 is reported to exhibit an EC50 value of 12.55 nM in SKOV3 (PTA021 -A3, see fig 12 in WO'175), when tested under similar experimental conditions.

Example 22: Testing Anti-CADM1 antibody in different cancer cell lines via flow cytometry

The isolated antibodies were used to identify cell lines which express CADM1 by flow cytometry. The assays were performed as follows: 0.5 millions of cells were incubated with 10ug/ml of different primary antibodies (anti-CADM1 antibodies of the invention) in 200u! of PBS/1 %FBS for an hour at 4C. After the incubation, cells were washed twice with PBS/ 1 %FBS and they were incubated for an hour at 4C with a goat anti-human IgG Alexa Fluor 488 labelled antibody (Jackson InmunoResearch). The experiment was done in non-permeabilized cells so the levels of CADM1 detected correspond to the plasma membrane protein.

Positive staining was also observed in further cancer cell lines.

Example 23 : Antibodies of invention are useful as carriers using a Hum-ZAP assay in cancer cell lines

The isolated antibodies of the invention are able to internalize inside cell lines which express CADM1 on the plasma membrane. The following shows these antibodies can be used as carriers to introduce inside cells conjugates or bound agents such as toxins and are able to work as immunotoxins.

Cells were incubated with serial dilutions of the isolated antibodies of the invention with a secondary goat anti-human IgG secondary antibody conjugated to saporin. Saporin is a ribosome-inactivating protein which comes from the seeds of the plant Saponaria officinalis. Once inside the cells, saporin causes inhibition of protein synthesis which leads to cell death. Human cancer cell lines were plated on 96 well plates (2000 cells per well) in complete growth media. The day after, serial dilutions of the antibodies to be tested were added to the corresponding well, from 10-6 to 10-13 M. The antibodies were added in combination with a human secondary antibody coupled to the toxin Saporin, the Hum-ZAP secondary antibody from Advanced Targeting Systems. The secondary antibody was used at a constant concentration of 100 ng per well. Each antibody concentration is tested in three wells (n=3) and additional wells are used as negative controls: cells without primary and secondary antibodies (n=3) and cells without primary antibodies containing only 100ng of Hum-ZAP secondary antibody (n=3). As positive control of cell death 0.1 % triton-X100 (Sigma) was added to lyse and kill cells in another three wells. 72 hours after the addition of the antibodies, an Alamar Blue assay was performed and the results are summarized in Table 1 1 . AlamarBlue® (invitrogen) is a proven cell viabiiity indicator that uses the natural reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin. The active ingredient of AlamarBlue® (resazurin) is a nontoxic, cell permeable compound that is blue in color and virtually nonfluorescent. Upon entering cells, resazurin is reduced to

resorufin, which produces very bright red fluorescence. Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability—and cytotoxicity. Basically, AlamarBlue® was added to the cells following the manufacturer's instructions and after 4 hours incubation fluorescence signal was read with an spectrophotometer, The amount of fluorescence is proportional to the number of living cells and corresponds to the cells metabolic activity. Damaged and nonviable cells have lower innate metabolic activity and thus generate a proportionally lower signal than healthy cells. The average value of each antibody concentration is divided by the average value of negative control (cells without antibodies). The percentages obtained are adjusted with respect to the negative control for the secondary antibody conjugated to saporine that gives us an estimation of background.

Example 24 : I nternalization of CADM 1 antibody conjugates in cancer cell lines

Conjugation of 1 5 mg of unfucosilated purified antibodies of the invention AB-3 and AB-10 were conjugated to saporin using Advanced Targeting Systems technology for conjugation to saporin . 2-5 milligrams of conjugated material were obtained with a ratio moles saporin/mole antibody = 2.1 , To test internalization potential of conjugated antibodies (AB3-SAP and AB- 10-SAP) and calculate the EC50 of cell viability an Alamar Blue assay was performed. The cell lines to test were plated on 96 well plates (2000 cells per well) in complete growth media . The day after, serial dilutions of the antibodies were added to the corresponding well, from 10-6 to 10-1 3 M. Each concentration is tested in sixtuplicated . As negative control unconjugted antibody is used, as well as, cell s without any antibodies. 72 hours after the addition of the antibodies, an Alamar Blue assay was performed and the results analysed as in Example 23.

Results obtained with AB3 and AB 1 0 conjugated to saporin (AB3-SAP and AB 1 0-SAP) in melanoma cancer A375 cell line are shown in Fig 1 2 and 1 3, respectively. EC50 values of 1 .5 nM and 2 nM were obtained for AB3-SAP and AB10-SAP, respectively.

Example 25: Human hemotoxicity study

Preparation of red blood cell s (RBC):

Erythrocytes and mononuclear cells were isolated from 20 tubes with 10 ml of human whole blood from donor of Blood & Tissue Bank. Histopaque 1077 (Sigma 1 0771 ) was used for a density gradient separation. 3 ml of Histopaque 1 077 and 3 ml of each of the blood were mixed. After 30 minutes of centrifugation at 300g separate pools were obtained from mononuclear cells and erythrocytes. Subsequently, successive washings were performed with PBS. The erythrocytes were resuspended en 4ml of PBS and divided in

4 tubes (concentrated RBC). Each tube was diluted with 19 m!_ of PBS to obtain the RBC working solution.

Hemolysis assay:

Ail antibodies were tested at the same concentrations, 1 50, 75, 37.5, 1 8.75, 9.38, 4.69 and 2.34 ug / mL. Concentrations were prepared by mixing appropriate amount of each of the Ab in a solution containing 200 uL of working RBC and 2 mi of PBS. After leaving the antibodies act for 1 0 minutes under constant stirring, the solution was centrifuged at 3000 rpm for 2 min . The supernatant was recovered and read the absorbance at 541 nm. Optical densities (OD) allowed us to calculate percentage of hemolysis with the following equation:

% Hemolysis = 1 00 * (OD sample-OD negative control) / (OD Total - OD negative control)

Where:

OD Total is the maximum OD of positive control hemolysis.

Positive control was performed with 7 concentrations of Sodium dodecyl sulfate (SDS), SDS (80, 70, 60, 50, 40, 30, 20 ug / mL) and a negative control with PBS

Hemolysis was not observed for any of the antibodies and conditions tested in this assay ( <1 % of hemolysis).

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. Ail publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.