WO2016077720A1 - Binding agents and uses thereof - Google Patents

Abstract

The technology relates to monoclonal antibodies useful in detecting and / or treating cancer. In some embodiments, binding agents with specificity for at least one tumor antigen and T cell receptors (e.g., human CD3) are provided. In some embodiments, methods for treating cancer using such binding agents are provided.

Description

BINDING AGENTS AND USES THEREOF

[001] RELATED APPLICATIONS

[002] This application claims priority to U.S. Ser. No. 62/079,635 filed November 14, 2014.

[003] FIELD OF THE DISCLOSURE

[004] The technology relates to monoclonal antibodies useful in the identification of cancer cells, as well as the diagnosis and treatment of cancer.

[005] BACKGROUND INFORMATION

[006] Patients with metastatic melanoma have a poor prognosis because the survival rate is only 5%. Although IL-2 can mediate complete regressions in 5-10% of patients, complete regressions are rare when using the anti-CTLA4 antibody ipilimumab or PLX4032, an inhibitor of mutated BRAF gene products. The adoptive transfer of human tumor infiltrating lymphocytes (TIL) selected for specific tumor reactivity led to objective clinical responses in 50-70% of patients with metastatic melanoma, including complete regressions in approximately 10-40% of patients who were pre-treated with lympho- depleting regimens. Therapies that can be more readily applied to a wider patient population, such as the use of non-selected TIL, are also currently being evaluated. However, the clinical efficacy of TIL generated from histologies other than melanoma has not been demonstrated.

[007] Attempts to develop more broadly applicable cancer therapies have focused on genetic modifications that confer autologous peripheral blood mononuclear cells (PBMC) with the ability to recognize antigens specifically expressed on tumor cells. The first clinical cancer trial to evaluate the efficacy of T cells whose target specificity has been re-directed towards tumor cells was carried out using cells that were genetically modified to express a TCR directed against an HLA-A*0201 restricted epitope of the MART-1 antigen. This molecule is a member of the melanocyte differentiation antigen (MDA) family of antigens that are expressed on 80-90% of melanoma but not other cancer types, and are limited in their expression in normal tissues to melanocytes. In this trial, complete regressions were observed in two out of 17 melanoma patients who received autologous PBMC that were retrovirally transduced with a MART-1 -reactive TCR. In a subsequent trial, treatment of patients with autologous PBMC that were transduced either with a second MART-1 reactive TCR or a TCR directed against an HLA-A*0201 - restricted epitope of the MDA gp100 lead to objective clinical response rates of 30% and 19%, respectively. The significant skin, eye and ear toxicities observed in this trial, which presumably resulted from responses to the normal melanocytes resident in these tissues, may have been a consequence of the relatively high avidities of these TCRs.

[008] A recent report detailed the results of a clinical trial carried out with T cells that were transduced with a TCR that recognized NY-ESO-1 , a protein encoded by a member of the cancer/germline family of genes. These genes are expressed in approximately one third of a variety of tumor types that include metastatic melanomas, lung, breast, prostate, bladder, and head and neck cancers, as well as 80% of synovial cell sarcoma, but are limited in their expression in normal adult tissues to the testis. Objective clinical responses were observed in five out of 1 1 melanoma patients and four out of six synovial cell sarcoma patients treated with a high-avidity TCR directed against an HLA-A*0201 -restricted NY-ESO-1 epitope, supporting the efficacy of adoptive immunotherapy for treatment of patients other than those with melanoma.

[009] Binding agents such as antibodies have proven useful in the diagnosis and treatment of diseases such as cancer. Bi-specific antibodies, molecules in which one antibody has been genetically linked to another antibody, have also been used to target T cells to cell surface antigens that are over-expressed on tumors or that are expressed in a highly tissue-specific manner on the tumor cell surface. In contrast to TCR mediated T cell therapy, bi-specific antibodies do not need to be personalized.

[0010] There is a need in the field for improved reagents and methods for treating cancer. Such improved reagents and methods are provided by this disclosure.

[0011] SUMMARY OF THE DISCLOSURE

[0012] The technology relates to monoclonal antibodies useful in the identification of cancer cells, as well as the diagnosis and treatment of cancer. In one embodiment, mAbs with specificity for tumor antigens are provided. In one embodiment, bi-specific antibodies are provided. In some embodiments, methods for using such binding agents to detecting cancerous cells are provided. In some embodiments, methods for treating cancer using such binding agents are provided. In another embodiment, kits for detecting cancerous cells are provided.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Exemplary (BiTE) bi-specific antibody system.

[0015] Figure 2. Gel filtration FPLC profiles of HLA A1 reconstitution from recombinant heavy chain and p2-microglobulin with and without peptides, (a) MAGE A3: EVDPIGHLY, (b) no peptide.

[0016] Figure 3. ELISA with w6/32 showed the right conformation of the refolding proteins.

[0017] Figure 4. Staining of tetramer with TCR transfected T cells and positive staining.

[0018] Figure 5. ELISA of scFv binding to the antigen complexes.

[0019] Figure 6. scFv 8E4 staining 1300A1 cells.

[0020] Figure 7. Western blot hybridization of 8EC3 and CD19X3 generated from yeast.

[0021] Figure 8. Production of IFNy following linking Cancer Cells with 8EC3. [0022] DETAILED DESCRIPTION

[0023] This disclosure relates to binding agents that bind one or more tumor antigens on the surface of cells in vitro and / or in vivo. The binding agents may also bind isolated tumor antigen polypeptides and / or fragments and / or derivatives thereof, typically in vitro. Also provided are methods for using such binding agents to diagnose, treat, prevent and / or ameliorate one or more diseases associated with the existence of cells expressing tumor antigens. For instance, the binding agents may be antibodies (e.g., monoclonal antibodies) that may react with and / or bind to the epitopes of tumor antigens. An exemplary use for the binding agents described herein is the selective targeting and elimination of cells expressing the tumor antigen(s) to which the binding agents bind. In some embodiments, the binding agents may be multi-specific, such as bi-specific, meaning that a single binding agent comprises at least two regions having specificity for different epitopes. The different epitopes may be found on tumor antigens or a tumor antigen and another antigen such as a cell surface receptor. An exemplary cell surface receptor is the T cell receptor (TCR), also known as CD3. Other embodiments, uses and the like are described below. A general illustration of an embodiment of this system is illustrated in Fig. 1. As shown therein, in some embodiments, a bi-specific binding agent having specificity for both TCR (CD3) and tumor antigen (a bi-specific T cell engager or "BiTE") serves to associate a T cell with a tumor cell resulting in killing of the tumor cell by the cell. This and other embodiments are described in greater detail herein.

[0024] In some embodiments, this disclosure provides one or more binding agents comprising at least two binding regions. The first binding region has specificity for CD3 (and / or fragments and / or derivatives thereof). The second binding region has specificity for a tumor antigen (and / or fragments and / or derivatives thereof). Binding "specifically" to a tumor antigen and / or CD3 means that the amount of binding to such a tumor antigen and/or CD3 is more than the amount of binding to non-tumor antigen and / or non-CD3 targets (i.e., there may be some background nonspecific binding in an assay used to measure binding specificity). The specific interaction or binding of a binding agent with its target is thought to be a type of equilibrium reaction that may be quantified using a dissociation constant, or K_d (a type of equilibrium constant that describes the propensity of, in this case, a binding agent to separate from the antigen or epitope to which it has bound). The lower the K_d, which may be measured by any of several widely available assay systems, the higher is the affinity of a binding agent for its target. Also provided are methods, for diagnosing, treating and / or preventing one or more diseases associated with the existence of cells expressing the tumor antigen. In some embodiments, the binding agents may comprise any one or more of the amino acid sequences shown in Table 1 , for example, (and / or one or more fragments and / or derivatives thereof) and may be encoded by any one or more of the nucleotide sequences shown therein (and / or one or more fragments and / or derivatives thereof). This disclosure also provides for the use of these bi-specific antibodies to isolate, identify, and / or target cells expressing the tumor antigen for the prevention and / or treatment of cancer harnessing T cells reactive to the cell.

Table 1

Description Sequence

TATTGTCAACAGGCTGACAGTTTCCCGCTCACTTTCGGCGGAGGGAC CAAGGTGGAGATCAAACGTGGAGGAGCCAGCCTCGTGGAATTCGAGC AGAAGCTGATCTCTGAGGAAGACCTG (SEQ ID NO.: 1)

8E3 light chain GGSSGVGSDIQMTQSPSSVSASVGDRVTITCRASQGISSRLAWYQQK amino acid PGKAPKLLIYAASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATY sequence YCQQADSFPLTFGGGTKVEIKRGGASLVEFEQKLISEEDL ( SEQ ID

NO.: 2)

8E3 light chain QGISSR (SEQ ID NO. : 3 )

CDR1 amino acid

sequence

Exemplary light GGCTTTACCTTTAGCAGCTATGCG (SEQ ID NO.: 4) chain CDR1

nucleotide

sequence

8E3 light chain AAS (SEQ ID NO. : 5)

CDR2 amino acid

sequence

Exemplary light ATTAGCGGCAGCGGCGGCAGCACC ( SEQ ID NO.: 6) chain CDR2

nucleotide

sequence

8E3 light chain QQADSFPLT (SEQ ID NO.: 7)

CDR3 amino acid

sequence

Exemplary light GCGCGCAACCTGGCGAACACCGGCTATTTTGATCCG ( SEQ ID chain CDR3 NO.: 8)

nucleotide

sequence

8E3 heavy chain QVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW amino acid VSAISGSGGSTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY sequence YCARNLANTGYFDPWGRGTLVTVSSGLGGL ( SEQ ID NO.: 9)

Exemplary heavy CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAG chain GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCT nucleotide ATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG sequence GTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATTCTACGCAGACTC (encoding SEQ CGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC ID NO: 9) TGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTAT

TACTGTGCGAGAAACCTTGCTAATACGGGCTACTTCGACCCCTGGGG CCGGGGAACCCTGGTCACCGTCTCCTCAGGCCTCGGGGGCCTCGGAG GAGGAGGTAGTGGCGGAGGAGGCTCC (SEQ ID NO: 10)

8E3 heavy chain GFTFSSYA (SEQ ID NO.: 11)

CDR1 amino acid

sequence

Exemplary heavy CAGGGCATTAGCAGCCGC ( SEQ ID NO.: 12)

chain CDR1

nucleotide

sequence

8E3 heavy chain ISGSGGST (SEQ ID NO.: 13)

CDR2 amino acid

sequence

Exemplary heavy GCGGCGAGC (SEQ ID NO.: 14)

[0025] In some embodiments, the binding agent may comprise the amino acid sequence of SEQ ID NO: 2 or a portion thereof that provides binding specificity to a ligand. In some embodiments, the binding agent may comprise the amino acid sequence of SEQ ID NO: 9 or a portion thereof that provides specificity to a ligand. In some embodiments, the binding agent may comprise the amino acid sequence of SEQ ID NOS: 1 and 9, and/ or a portion of either that provides binding specificity to a ligand. In some embodiments, the ligand is a tumor antigen expressed on tumor cells. In some embodiments, the tumor antigen is a melanoma antigen such as MAGE. In some embodiments, the MAGE antigen is a peptide of MAGE-A1 or MAGE-A3. In some embodiments, the MAGE peptide is MAGE A1 peptide EADPTGHSY (SEQ ID NO:21 ). In some embodiments, the MAGE peptide is the MAGE-A3 peptide EVDPIGHLY (SEQ ID NO:22).

[0026] In some embodiments, the binding agent may comprise the amino acid sequences QGISSR (SEQ ID NO: 3) and/or QQADSFPLT (SEQ ID NO: 7). In some embodiments, the binding agent may comprise the amino acid sequences QGISSR (SEQ ID NO: 3) and AAS (SEQ ID NO: 5). In some embodiments, the binding agent may comprise the amino acid sequences AAS (SEQ ID NO: 5) and QQADSFPLT (SEQ ID NO: 7). In some embodiments, the binding agent may comprise the amino acid sequences QGISSR (SEQ ID NO: 3), AAS (SEQ ID NO: 5), and QQADSFPLT (SEQ ID NO: 7). Such a binding agent may have specificity for a particular ligand. In some embodiments, the binding agent may be one that competes with such a binding agent for binding to that ligand. In some embodiments, the binding agent comprising any one or more of SEQ ID NOS. 4, 5 and/or 6 may have specificity for human MAGE-A3, in particular the peptide EVDPIGHLY (SEQ ID NO:22). In some embodiments, the binding agent may be one that competes for binding by such binding agents to MAGE-A3, in particular the peptide EVDPIGHLY (SEQ ID NO:22).

[0027] In some embodiments, the binding agent may comprise the amino acid sequence ARNLANTGYFDP (SEQ ID NO: 15). In some embodiments, the binding agent may comprise the amino acid sequences GFTFSSYA (SEQ ID NO: 1 1 ) and ARNLANTGYFDP (SEQ ID NO: 15). In some embodiments, the binding agent may comprise the amino acid sequences ISGSGGST (SEQ ID NO: 13) and ARNLANTGYFDP (SEQ ID NO: 15). In some embodiments, the binding agent may comprise the amino acid sequences GFTFSSYA (SEQ ID NO: 1 1 ), ISGSGGST (SEQ ID NO: 13), and ARNLANTGYFDP (SEQ ID NO: 15). Such a binding agent may have specificity for a particular ligand. In some embodiments, the binding agent may be one that competes with such a binding agent for binding to that ligand. In some embodiments, the binding agent comprising any one or more of SEQ ID NOS. 1 , 2 and/or 3 may have specificity for human MAGE-A3, in particular the peptide EVDPIGHLY (SEQ ID NO:22). In some embodiments, the binding agent may be one that competes for binding by such binding agents to MAGE-A3, in particular the peptide EVDPIGHLY (SEQ ID NO:22).

[0028] In some embodiments, the binding agent may comprise one or more of the amino acid sequences one or more of the amino acid sequences QGISSR (SEQ ID NO: 3), AAS (SEQ ID NO: 5), and/or QQADSFPLT (SEQ ID NO: 7); and one or more of the amino acid sequences GFTFSSYA (SEQ ID NO: 1 1 ), ISGSGGST (SEQ ID NO: 13), and/or ARNLANTGYFDP (SEQ ID NO: 15). In some embodiments, the binding agent may comprise one or more of the amino acid sequences QGISSR (SEQ ID NO: 3), and/or QQADSFPLT (SEQ ID NO: 5), ARNLANTGYFDP (SEQ ID NO: 15). In some embodiments, the binding agent may comprise one or more of the amino acid sequences QGISSR (SEQ ID NO: 3) and ARNLANTGYFDP (SEQ ID NO: 15). In some embodiments, the binding agent may comprise one or more of the amino acid sequences QQADSFPLT (SEQ ID NO: 7) and ARNLANTGYFDP (SEQ ID NO: 15). In some embodiments, the binding agent may comprise one or more of the amino acid sequences QGISSR (SEQ ID NO: 3), QQADSFPLT (SEQ ID NO: 7), and / or ARNLANTGYFDP (SEQ ID NO: 15). In some embodiments, the binding agent may be one that competes with such a binding agent for binding to that ligand. In some embodiments, the binding agent comprising any one or more of SEQ ID NOS. 1 , 3, 5, 7, 9, 1 1 , 13 and / or 15. Such a binding agent may have specificity for a particular ligand. In some embodiments, that ligand may have specificity for human MAGE-A3, in particular the peptide EVDPIGHLY (SEQ ID NO:22). In some embodiments, the binding agent may be one that competes for binding by such binding agents to MAGE-A3, in particular the peptide EVDPIGHLY (SEQ ID NO:22). The specificity for the ligand such as MAGE-A3 may be determined by any combination of amino acid sequences SEQ ID NOS. 2, 3, 5, 7, 9, 1 1 , 13, 15, and / or 19. Such amino acid sequences are found within or compose the complementarity determining regions (CDRs) of antibodies and/or derivatives thereof, for instance.

[0029] The binding agents may also comprise amino acid sequences providing to the binding agent additional binding specificities as may be known in the art (see, e.g., U.S. Pat. No. 4,474,893 and AU 9341957). In certain embodiments, the binding agents may comprise amino acid sequences providing specificity for a tumor antigen and others providing specificity for an immune cell surface protein. An exemplary immune cell surface protein may be the T cell receptor ("TCR", e.g., human CD3). Amino acid sequences providing binding specificity for the tumor antigen may be adjoined to amino acid sequences providing binding specificity for a TCR to provide a bi-specific binding agent. The amino acid sequences may be adjoined to one another by an amino acid sequence providing a linking function. In some embodiments, the binding agent may be arranged as an amino sequence providing binding specificity for a tumor antigen adjoined to a linking amino acid sequence adjoined to an amino acid sequence providing binding specificity for a TCR (i.e., the amino acid sequences are adjoined in series).

[0030] Antibodies binding to TCR (CD3) are known in the art and the amino acid sequences suitable for use as described herein and available to those of ordinary skill in the art. For instance, the binding specificity may be derived from the monoclonal antibody produced by hybridoma strain OKT3 as described in U.S. Pat. No. 4,631 ,549A. CD3 binding specificity may also be provided by using CDRs such as those described in U.S. Pat. Nos. 5,585,097 and 5,698,509 (i.e., SFPMA (SEQ ID NO:23), TISTSGGRTYYRDSVLG (SEQ ID NO:24), FRQYSGGFDY (SEQ ID NO:25), TLSSGNIQNYVH (SEQ ID NO:26), DDDKRPD (SEQ ID NO:27), and HSYVSSFNV (SEQ ID NO:28)). In some embodiments, binding agents having specificity for TCR/CD3 may comprise any of the heavy and / or light chain variable amino acid sequences described in Tables 1 and 2 of U.S. Pat. Pub. No. 2014/0088295A1 (i.e., any of SEQ ID NOS. 1-1240 described therein). In some embodiments, a binding agent having specificity for TCR/CD3 may comprise amino acid sequences having specificity for CD3 and being encoded by nucleotides 847-1203 (heavy chain variable region) and/or 1258- 1575 (light chain variable region) of SEQ ID NO:9 of U.S. Pat. No. 7, 1 12,324B1 . In some embodiments, the TcR/CD3 specificity may be provided by the amino acid sequence, or TcR/CD3 binding fragments thereof, shown below:

GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE

(SEQ ID NO.:29); and / or

GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRF SGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO.: 30); such as : GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSG GSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKV ASGVPYRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 31) .

[0031] The binding agents may be adjoined to one another using a linking amino acid sequence. The linking sequence may be used to adjoin various amino acid sequences described herein, in particular the amino acid sequences providing different specificities thereto. For instance, any of the amino acid sequences of Table 1 may be adjoined to any one or more amino acid sequences that provide specificity for TCR/CD3. As shown above, an exemplary linking amino acid sequence may be GGGGSGGGGS (SEQ ID NO.: 17). Other linking amino acid sequences may also be suitable as may be determined by those of ordinary skill in the art.

[0032] The binding agents described herein may be glycosylated or not (e.g., "aglycosylated"). In some instances, an aglycosylated binding agent such as an antibody may be preferred to avoid problems associated with known first dose responses and / or binding to Fc receptors. Aglycosylated antibodies against CD3, CDRs found within such antibodies, and methods for making the same are described in U.S. Pat. No. 5,585,097. In some situations, the glycosylated binding agent may be preferred. Whether a binding agent is glycosylated or not may depend on the particular amino acid sequences found within the binding agent and / or the cells in which the binding agent is expressed (e.g., from a nucleic acid encoding the same).

[0033] The binding agents may be antibodies such as monoclonal antibodies. This disclosure also provides for the use of such monoclonal antibodies to isolate, identify, and / or target cells expressing one or more tumor antigens. In certain embodiments, these monoclonal antibodies may be reactive against a tumor antigen expressed on the surface of cells. The term "antibody" or "antibodies" may refer to whole or fragmented antibodies in unpurified or partially purified form (e.g., hybridoma supernatant, ascites, polyclonal antisera) or in purified form. The antibodies may be of any suitable origin or form including, for example, murine (e.g., produced by murine hybridoma cells), or expressed as humanized antibodies, chimeric antibodies, human antibodies, and the like. For instance, and for example only, antibodies may be wholly or partially derived from human (e.g., IgG (lgG1 , lgG2, lgG2a, Ig2b, lgG3, lgG4), IgM, IgA (lgA1 and lgA2), IgD, and IgE), canine (e.g., IgGA, IgGB, IgGC, IgGD), chicken (e.g., IgA, IgD, IgE, IgG, IgM, IgY), goat (e.g., IgG), mouse (e.g., IgG, IgD, IgE, IgG, IgM), and / or pig (e.g., IgG, IgD, IgE, IgG, IgM), rat (e.g., IgG, IgD, IgE, IgG, IgM) antibodies. Methods of preparing, utilizing and storing various types of antibodies are well-known to those of skill in the art and would be suitable in practicing the present invention (see, for example, Harlow, et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Harlow, et al. Using Antibodies: A Laboratory Manual, Portable Protocol No. 1, 1998; Kohler and Milstein, Nature, 256:495 (1975)); Jones et al. Nature, 321 :522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol., 2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536 (1988); Hoogenboom et al., J. Mol. Biol., 227:381 (1991 ); Marks et al., J. Mol. Biol., 222:581 (1991 ); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1 ):86-95 (1991 ); Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos. 4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and, 5,661 ,016). In certain applications, the antibodies may be contained within hybridoma supernatant or ascites and utilized either directly as such or following concentration using standard techniques. In other applications, the antibodies may be further purified using, for example, salt fractionation and ion exchange chromatography, or affinity chromatography using Protein A, Protein G, Protein A/G, and / or Protein L ligands covalently coupled to a solid support such as agarose beads, or combinations of these techniques. The antibodies may be stored in any suitable format, including as a frozen preparation (e.g., -20°C or -70°C), in lyophilized form, or under normal refrigeration conditions (e.g., 4°C). When stored in liquid form, for instance, it is preferred that a suitable buffer such as Tris-buffered saline (TBS) or phosphate buffered saline (PBS) is utilized. In some embodiments, the binding agent may be prepared as an injectable preparation, such as in suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable vehicles and solvents that may be utilized include water, Ringer's solution, and isotonic sodium chloride solution, TBS and / or PBS, among others. Such preparations may be suitable for use in vitro or in vivo may be prepared as is known in the art and the exact preparation may depend on the particular application. [0034] Where the binding agent is an antibody, it may be identified with reference to the nucleotide and / or amino acid sequence corresponding to the variability and / or complementarity determining regions ("CDRs") thereof. The variable region / CDR sequences may be used in combination with one or more other variable region / CDR amino acid sequences. The variable region / CDR amino acid sequences may alternatively and / or also be adjoined to one or more types of constant region polypeptides of an antibody molecule. For instance, the CDR amino acid sequences shown in Table 1 may be adjoined to or associated with the constant regions of any antibody molecule of the same or a different species (e.g., human, goat, rat, sheep, chicken) and / or antibody subtype of that from which the CDR amino acid sequence was derived. Such CDRs may also be adjoined to CDRs having different specificities such as those providing specificity for TCR/CD3 as described above. CDRs having different specificities may be combined to provide binding agents providing multiple specificities (e.g., bi-specific antibodies). Binding agents having more than two specificities may also be produced by combining CDRs, light chains, and heavy chains (e.g., antibodies) as may be desired by those of ordinary skill in the art.

[0035] It is to be understood that any one or more fragments and / or derivatives of the amino acid sequences of this disclosure, such as those of Table 1 , are also contemplated herein. For instance, any one or more of the amino acids within the amino acid sequences described herein, such as those of Table 1 , may be substituted by any other amino acid as desired by one of ordinary skill in the art. Such substitutions may be considered by those of ordinary skill in the art to be non- conservative or conservative. A non-conservative substitution is typically understood to be the substitution of one amino acid with another that alters the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid originally found at that position. In contrast, a conservative amino acid substitution is typically understood to involve the substitution of one amino acid with another that has little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid originally found at that position. Any of such substitutions typically provide an improved binding agent, the improvement being measured by any technique desired by one of ordinary skill in the art (e.g., measurement of K_d). It is preferred that suitable substitutions do not result in decreased function and / or specificity of the binding agent but, rather, improve the function and / or specificity of the binding agent as such characteristics would be understood by those of ordinary skill in the art. The specific one or more amino acid substitutions selected may depend on the location of the site on the binding agent selected for substitution. One of ordinary skill in the art may, for instance, make conservative substitutions by replacing one or more amino acids with a different amino acid using the exemplary substitutions shown in Table 2 below.

Table 2

In some embodiments, one or more amino acids in any of the amino acid sequences of Table 1 , such as any of SEQ ID NOS. 3, 5, 7, 9, 1 1 , 13, 15, 17, and/or 19, may be substituted with another amino acid, such as but not limited to the conservative substitutions exemplified in Table 2. In some embodiments, the number of amino acid sequences substituted in any amino acid sequence may, in some embodiments, represent up to and / or including about any of 1 %, 5%, 10%, 15%, 20%, and/or 25% of the amino acids present in such an amino acid sequence. In some embodiments, the number of amino acid sequences substituted in any amino acid sequence may, in some embodiments, may up to and / or including about any of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the amino acids present in such an amino acid sequence. In some embodiments, the arginine (R) of QGISSR (SEQ ID NO:3) is not substituted by tryptophan (W). In some embodiments, the aspartic acid (D) of QQADSFPLT (SEQ ID NO:7) is not substituted by asparagine (N). In some embodiments, ARNLANTGYFDP (SEQ ID NO:15) is not substituted to become AKELAVAGYFDY (SEQ ID NO:32) or ARTLANSEVWGQ (SEQ ID NO:33).

[0036] In certain embodiments, a nucleic acid molecule encoding one or more binding agents described herein, or a portion thereof, are also provided. Such nucleic acid sequences may be inserted into one or more expression vectors, as discussed below in greater detail. In such embodiments, the binding agent may be encoded by nucleotides corresponding to the amino acid sequence as the same would be understood by those of ordinary skill in the art. The particular combinations of nucleotides that encode the various amino acids are well known in the art, as described in various references used by those skilled in the art (e.g., Lewin, B. Genes V, Oxford University Press, 1994). The nucleotide sequences encoding the amino acids of said binding agents may be ascertained with reference to Table 3, for example. Nucleic acid variants may use any combination of nucleotides that encode the binding agent.

Table 3

Where the binding agents are antibodies, nucleotide sequences encoding the variable regions thereof may also be isolated from the phage and / or hybridoma cells expressing the same cloned into expression vectors to produce certain preparations (e.g., humanized antibodies). Methods for producing such preparations are well-known in the art.

[0037] Nucleic acid molecules encoding one or more binding agents may be contained within a viral and / or a non-viral vector. In one embodiment, a DNA vector is utilized to deliver nucleic acids encoding one or more binding agents to the patient. In doing so, various strategies may be utilized to improve the efficiency of such mechanisms including, for example, the use of self-replicating viral replicons (Caley, et al. 1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al. 2000. Cancer Res. 60: 51 -55), codon optimization (Liu, et al. 2000. Mol. Then, 1 : 497-500; Dubensky, supra; Huang, et al. 2001. J. Virol. 75: 4947-4951 ), in vivo electroporation (Widera, et al. 2000. J. Immunol. 164: 4635-3640), incorporation of nucleic acids encoding co-stimulatory molecules, cytokines and / or chemokines (Xiang, et al. 1995. Immunity, 2: 129-135; Kim, et al. 1998. Eur. J. Immunol., 28: 1089-1 103; Iwasaki, et al. 1997. J. Immunol. 158: 4591-3601 ; Sheerlinck, et al. 2001. Vaccine, 19: 2647-2656), incorporation of stimulatory motifs such as CpG (Gurunathan, supra; Leitner, supra), sequences for targeting of the endocytic or ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol. 72: 2246-2252; Velders, et al. 2001 . J. Immunol. 166: 5366-5373), prime-boost regimens (Gurunathan, supra; Sullivan, et al.

2000. Nature, 408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al.

2001 . Science, 292: 69-74), proteasome-sensitive cleavage sites, and the use of mucosal delivery vectors such as Salmonella (Darji, et al. 1997. Cell, 91 : 765-775; Woo, et al. 2001. Vaccine, 19: 2945-2954). Other methods are known in the art, some of which are described below. Various viral vectors that have been successfully utilized for introducing a nucleic acid to a host include retrovirus, adenovirus, adeno- associated virus (AAV), herpes virus, and poxvirus, among others. The vectors may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991 . Academic Press, San Diego, CA), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, ca). "Non-viral" plasmid vectors may also be suitable in certain embodiments. Preferred plasmid vectors are compatible with bacterial, insect, and / or mammalian host cells. Such vectors include, for example, PCR-ii, PCR3, and pcDNA3.1 (Invitrogen, San Diego, CA), pBSii (Stratagene, La Jolla, CA), pet15 (Novagen, Madison, Wl), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFp-n2 (Clontech, Palo Alto, CA), pETI (Bluebacii, Invitrogen), pDSR-alpha (PCT pub. No. WO

90/14363) and pFASTBACdual (Gibco-BRL, Grand island, NY) as well as Bluescript^® plasmid derivatives (a high copy number COLel-based phagemid, Stratagene Cloning Systems, La Jolla, CA), PCR cloning plasmids designed for cloning TAQ-amplified PCR products (e.g., TOPO™ TA cloning kit, PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, CA). Bacterial vectors may also be used. These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille Calmette Guerin (BCG), and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376). Many other non-viral plasmid expression vectors and systems are known in the art and may be use. Other delivery techniques may also suffice including, for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaP0₄ precipitation, gene gun techniques, electroporation, and colloidal dispersion systems. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et al., 1981 , Trends Biochem. Sci., 6: 77). The composition of the liposome is usually a combination of phospholipids, particularly high-phase- transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0038] A cultured cell comprising the vector is also provided. The cultured cell may be a cultured cell transfected with the vector or a progeny of the cell, wherein the cell expresses the immunogenic polypeptide. Suitable cell lines are known to those of skill in the art and are commercially available, for example, through the American Type Culture Collection (ATCC). The transfected cells can be used in a method of producing an immunogenic polypeptide. The method comprises culturing a cell comprising the vector under conditions that allow expression of the immunogenic polypeptide, optionally under the control of an expression sequence. The immunogenic polypeptide can be isolated from the cell or the culture medium using standard protein purification methods.

[0039] The binding agents of this disclosure are not in any way limited to antibodies. For example, the binding agent may be any compound exhibiting similar binding properties as another (e.g., a mimetic). An exemplary binding agent may be one that binds one or more tumor antigens and / or one or more cell surface receptors and / or can compete with binding agent having specificity therefor (e.g., a monoclonal antibody). In some embodiments, the mimetic may exhibit substantially the same affinity in binding assays as the binding agent to which it is being compared. The affinity a particular binding agent may be measured by any suitable assay including but not limited to fluorescence activated cell sorting (FACS) staining of endogenous cell surface tumor antigen and / or cell surface receptor on cells. One binding agent may be said to have "substantially the same affinity" as another where the measurements (e.g., nm) are within about any of 1 -20, 1 -5 5-10, 10-15, or 15-20 percent of one another. Exemplary mimetics may include, for example, organic compounds that specifically bind the tumor antigen(s) and / or cell surface receptor(s), or an affibody (Nygren, et al. FEBS J. 275 (1 1 ): 2668-76 (2008)), affilin (Ebersbach, et al. J. Mol. Biol. 372 (1 ): 172- 85 (2007)), affitin (Krehenbrink, et al. J. Mol. Biol. 383 (5): 1058-68 (2008)), anticalin (Skerra, A. FEBS J. 275 (1 1 ): 2677-83 (2008)), avimer (Silverman, et al. Nat. Biotechnol. 23 (12): 1556-61 (2005)), DARPin (Stumpp, et al. Drug Discov. Today 13 (15-16): 695-701 (2008)), Fynomer (Grabulovski, et al. J. Biol. Chem. 282 (5): 3196- 3204 (2007)), Kunitz domain peptide (Nixon, et al. Curr. Opin. Drug Discov. Devel. 9 (2): 261-8 (2006)), and / or a monobody (Koide, et al. Methods Mol. Biol. 352: 95-109 (2007)). Other mimetics may include, for example, a derivative of an antibody such as, for example, an F_ab, F_ab2, Fab' single chain antibody, F_v, single domain antibody, single chain variable fragment (scFv) (a fusion protein of the variable regions of the heavy (VH) and light chains (V_L) of immunoglobulins, connected with a short linker peptide of seven to about 25 amino acids (e.g., SEQ ID NO:27, an exemplary linker being SEQ ID NO:17)), mono-specific antibody, bi-specific antibody, tri-specific antibody, multi-valent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized, CDR-grafted antibody, shark antibody, nanobody, canelid antibody, microbody, and / or intrabody, or derivative thereof. Other binding agents are also provided herein as would be understood by one of ordinary skill in the art.

[0040] Combinations of binding agents may also be identified. In some embodiments, the combinations may be identified to provide statistically significant differences from results obtained using only one or more of the binding agents and not others. In some embodiments, combinations exhibiting synergistic ability to restore immune cell function may be identified. In some embodiments, the combination may comprise a first binding agent that binds to a tumor antigen(s) with a second binding agent that binds to another tumor antigen(s) (or a different epitope of the same tumor antigen) and / or a binding agent that binds to a cell surface receptor such as the T cell receptor. These binding agents may be different entities such as two or more different monoclonal antibodies or derivatives thereof, or may be found on the same entity such as a bi-functional antibody (a single antibody or derivative thereof comprising multiple binding specificities). Multiple multi-specific binding agents may also be combined. Also contemplated are combinations that provide multiple types of each binding agent. Such combinations as described herein may also be combined with one or more other agents that may effect immune cell function such as antibodies against CTLA-4 and the like. One of ordinary skill in the art would recognize that many such combinations may be suitable for use as described herein.

[0041] For treating a disease such as cancer, one or more the binding agents may have specificity to one or more tumor antigens (e.g., cancer-testis (CT) antigen (e.g., MAGE (the peptide EVDPIGHLY (SEQ ID NO:22) or EADPTGHSY (SEQ ID NO:21 ) as shown in the Examples herein), NY-ESO-1 )); melanocyte differentiation antigen (e.g., Melan A/MART-1 , tyrosinase, gp100); mutational antigen (i.e., MUM-1 , p53, CDK-4); overexpressed 'self antigen (i.e., HER-2/neu, p53); and / or viral antigens (i.e., HPV, EBV)). The binding agents (e.g., monoclonal antibodies) may be generated as generally described herein. The specificities of such binding agents may be recombined into a single binding agent using techniques that are widely available to those of ordinary skill in the art. In some embodiments, multiple single specificity- binding agents may also be combined and used (e.g., administered) to provide an effective multiple specificity reagent.

[0042] In some embodiments, the binding agents described herein may be conjugated to active agents to target and inhibit the function of and /or eliminate cell populations expressing the tumor antigen(s). For instance, cancer cell populations expressing the tumor antigen(s) may be targeted and eliminated using binding agent / drug conjugates (e.g., antibody-drug conjugates (ADC)). Mono- and/or bi-specific candidate binding agents may be conjugated with one or more types of drugs (e.g., drugs damaging DNA, targeting microtubules). The binding agents described herein and/ or derivatives thereof may also be adjoined to and / or conjugated to functional agents for in vitro and / or in vivo use. For instance, the binding agent may be adjoined to and / or conjugated to functional moieties such as cytotoxic drugs or toxins, and / or active fragments thereof such as diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, among others. Suitable functional moieties may also include radiochemicals. Binding agents, such as antibodies, may be adjoined to and / or conjugated to the one or more functional agents using standard techniques in the art.

[0043] In some embodiments, the binding agents may be adjoined to and / or conjugated to one or more detectable labels. For instance, suitable detectable labels may include, for instance, fluorosceins (e.g., DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5- HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 6-JOE; 6- carboxyfluorescein (6-FAM); FITC; 6-carboxy-1 ,4-dichloro-2',7'-dichlorofluorescein (TET); 6-carboxy-1 ,4-dichloro-2',4', 5', 7'-tetrachlorofluorescein (HEX); 6-carboxy-4',5'- dichloro-2', 7'-dimethoxyfluorescein (JOE); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570, 576/589, 581/591 , 630/650-X, 650/665-X, 665/676, FL, FL ATP, Fl-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE)), rhodamines (e.g., 1 10, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5- TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA (6-carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), WT), Texas Red, and / or Texas Red-X. Other detectable labels known in the art may also be suitable for use. Binding agents, such as antibodies, may be adjoined to and / or conjugated to the one or more detectable labels using standard techniques in the art.

[0044] Any method known to those of ordinary skill in the art may be used to generate binding agents having specificity for the tumor antigen(s) and / or cell surface receptor(s) and combinations thereof. For instance, to generate and isolate monoclonal antibodies an animal such as a mouse may be administered (e.g., immunized) with one or more tumor antigens or fragments thereof (e.g., a tumor antigen polypeptide). Animals exhibiting serum reactivity to the tumor antigen / cell surface receptor antigen may then be selected for generation of anti-tumor antigen / cell surface receptor antigen hybridoma cell lines. This may be repeated for multiple rounds. Hybridomas may then be selected for further processing based on antigen- binding affinity or other similar measurements. Multi-specific binding agents may then be prepared using standard techniques available to those of ordinary skill in the art including both biochemical (e.g., joining of polypeptide regions of two or more binding agents to one another to form a hybrid, or bi-specific binding agent) and / or recombinant techniques (e.g., cloning of complementarity determining regions (CDRs) of two or more binding agents and recombining the CDRs into a single bi-specific binding agent). Other embodiments of the binding agents and methods for making the same are available in the art and may be apparent from this disclosure.

[0045] As mentioned above, the binding agents described herein may be used to treat and / or prevent and / or ameliorate the symptoms of cancer. Exemplary cancers may include, for instance, any of the breast, blood, colon, stomach, rectum, skeletal tissue, skin (e.g., melanoma) brain, lung, bladder, kidney, ovary, and / or liver, among others. One or more of the binding agents may also be combined with and / or administered with or in conjunction with one or more agents used to prevent, treat and / or ameliorate cancer such as for example, an alkylating agent (e.g., any nitrogen mustard, nitrosourea, tetrazine, aziridine, cisplatin and / or derivative thereof), anti-metabolite (e.g., any of the methotrexates, pemetrexeds, fluoropyrimidines and / or derivative thereof), anti-microbtubule agent (e.g., vinca alkyloids, taxanes, podophyllotoxin and / or derivative thereof), topoisomerase I and / or II inhibitors (e.g., a camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, aclarubicin and / or derivative thereof) and / or cytotoxic antibiotic (e.g., any anthracyclines, actinomycin, bleomycin, plicamycin, and mitomycin and / or derivative thereof). The one or more binding agents may also, or alternatively, be combined with one or more other binding agents available to those of ordinary skill in the art for treating, preventing and / or ameliorating cancer such as, for example, Nivolumab, Lambrolizumab, Pidilizumab and / or other similar agents and / or derivatives thereof. Other suitable agents are known to those of skill in the art and may be suitable for use as described herein. Such agents may either be used prior to, during, or after administration of the binding agents and / or use of the methods described herein.

[0046] The skilled artisan has many suitable techniques for using the binding agents (e.g., antibodies) described herein to identify biological samples containing proteins that bind thereto. For instance, antibodies may be utilized to isolate a tumor antigen(s) and / or cell surface receptor and / or cells and / or proteins to which a multi-sp

[0047] cific binding agent binds using, for example, immunoprecipitation or other capture-type assay. This well-known technique may be performed by attaching the antibody to a solid support or chromatographic material (e.g., a bead coated with Protein A, Protein G and / or Protein L). The bound antibody may then introduced into a solution either containing or believed to contain the tumor antigen(s), cell surface receptor, cells comprising both a tumor antigen(s) and a cell surface receptor to which a multi-specific binding agent binds, or different cells that each express one or both of the tumor antigen(s) and the cell surface receptor. The ligand(s) of the binding agent may then bind to the antibody and non-binding materials may be washed away under conditions in which the ligand(s) remains bound to the antibody. The bound ligand(s) may then be separated from the antibody and analyzed as desired. Similar methods for isolating ligands using an antibody are well-known in the art. The binding agents (e.g., antibodies) may also be utilized to detect tumor antigen(s) within a biological sample. For instance, the antibodies may be used in assays such as, for example, flow cytometric analysis, ELISA, immunoblotting (e.g., western blot), in situ detection, immunocytochemistry, and / or immunhistochemistry. Methods of carrying out such assays and others similar thereto are well-known in the art.

[0048] The binding agents described herein may be also be used to determine the presence of a disease state in a patient, to predict prognosis, or to determine the effectiveness of a chemotherapeutic or other treatment regimen. Expression profile assays, performed as described herein or as is otherwise known in the art, may be used to determine the relative level of expression of tumor antigen(s) and / or cell surface receptors, and / or cells expressing both tumor antigen(s) and cell surface receptor(s), and / or different cells expressing at least one or the other of the tumor antigen(s) and cell surface receptor(s). The level of expression may then be correlated with base (e.g., control) levels to determine whether a particular disease is present within the patient, the patient's prognosis, or whether a particular treatment regimen is effective. For example, if the patient is being treated with a particular anti-infective regimen, an increased or decreased level of expression of such ligands in the patient's tissues (e.g., in peripheral blood, breast tissue biopsy) may indicate the regimen is worsening or improving the load of the infectious agent in that host. The increase or decrease in expression may indicate the regimen is having or not having the desired effect and another therapeutic modality may therefore be selected.

[0049] It is also possible to use the binding agents described herein as reagents in drug screening assays to test, for example, new drug candidates. The reagents may be used to ascertain the effect of a drug candidate on the expression of the immunogenic target in a cell line, or a cell or tissue of a patient. The expression profiling technique may be combined with high throughput screening techniques to allow rapid identification of useful compounds and monitor the effectiveness of treatment with a drug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8 (1998)). Drug candidates may be chemical compounds, nucleic acids, proteins, antibodies, or derivatives therefrom, whether naturally occurring or synthetically derived. Drug candidates thus identified may be utilized, among other uses, as pharmaceutical compositions for administration to patients or for use in further screening assays. [0050] In some embodiments, the binding agents are in purified form. A "purified" binding agent (e.g., antibody) may be one that is separated from at least about 50% of the proteins and / or other components with which it is initially found (e.g., as part of a hybridoma supernatant or ascites preparation in the case of a monoclonal antibody). A purified binding agent (e.g., antibody) may be one that is separated from at least about 50%, 60%, 75%, 90%, or 95% of the proteins and / or other components with which it is initially found.

[0051] The binding agents and/or nucleic acids described herein may be combined with one or more pharmaceutically acceptable carriers prior to administration to a host. A pharmaceutically acceptable carrier is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Suitable pharmaceutical carriers and their formulations are described in, for example, Remington's: The Science and Practice of Pharmacy, 21^st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally from about 5 to about 8 or from about 7 to about 7.5. Other carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing polypeptides or fragments thereof. Matrices may be in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of polypeptides and / or fragments thereof to humans or other subjects. Pharmaceutical compositions may also include carriers, thickeners, diluents, buffers, preservatives, surface-active agents, adjuvants, immunostimulants, in addition to the immunogenic polypeptide. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents and anesthetics. The pharmaceutical composition may be administered orally, parentally, by inhalation spray, rectally, intranodally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a nucleic acid, polypeptide, or peptide as a pharmaceutical composition. A "pharmaceutical composition" is a composition comprising a therapeutically effective amount of a nucleic acid or polypeptide. The terms "effective amount" and "therapeutically effective amount" each refer to the amount of a binding agent, nucleic acid or the like used to observe the desired therapeutic effect (e.g., restore T cell function).

[0052] Methods for treating one or more disease conditions (e.g., cancer) in a mammalian host comprising administering to the mammal at least one or more effective doses of one or more binding agents (and / or derivative(s) thereof) described herein are also provided. The one or more binding agents may be administered in a dosage amount of about 1 to about 50 mg / kg, about 1 to about 30 mg / kg, or about 5 to about 30 mg / kg (e.g., about any of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 mg / kg). In certain embodiments, the one or more binding agents may be administered to the mammal (e.g., intradermally, intravenously, orally, rectally) at about 10 mg / kg one or more times. When multiple doses are administered, the doses may comprise about the same or different amount of binding agent in each dose. The doses may also be separated in time from one another by the same or different intervals. For instance, the doses may be separated by about any of 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 1 1 months, 12 months, 1.5 years, 2 years, 3 years, 4 years, 5 years, or any time period before, after, and / or between any of these time periods. In some embodiments, the binding agents may be administered in conjunction with other agents (e.g., anti- infective agents and/or chemotherapeutic agent). Such other agents may be administered about simultaneously with the binding agents, or at a different time and / or frequency. Other embodiments of such methods may also be appropriate as could be readily determined by one of ordinary skill in the art.

[0053] To assist the skilled artisan in using the antibodies described herein, the same may be provided in kit format. A kit including such antibodies and optionally other components necessary for using the antibodies to detect cells expressing the tumor antigen(s) and / or cell surface receptor(s) is provided. The antibodies of the kit may be provided in any suitable form, including frozen, lyophilized, or in a pharmaceutically acceptable buffer such as TBS or PBS. The kit may also include other reagents required for utilization of the antibodies in vitro or in vivo such as buffers (e.g., TBS, PBS), blocking agents (solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein), and / or detection reagents (e.g., goat anti-mouse IgG biotin, streptavidin-HRP conjugates, allophycocyanin, B-phycoerythrin, R-phycoerythrin, peroxidase, detectable labels, and other labels and / or staining kits (e.g., ABC Staining Kit, Pierce)). The kits may also include other reagents and / or instructions for using the antibodies in commonly utilized assays described above such as, for example, flow cytometric analysis, ELISA, immunoblotting (e.g., western blot), in situ detection, immunocytochemistry, immunhistochemistry. In one embodiment, the kit provides a binding agent in purified form. In another embodiment, the binding agent may be provided in biotinylated form either alone or along with an avidin-conjugated detection reagent (e.g., antibody). In another embodiment, the kit includes a binding agents comprising one or more detectable labels that may be used to directly detect the binding agent ligands. Buffers and the like required for using any of these systems are well- known in the art and / or may be prepared by the end-user or provided as a component of the kit. The kit may also include a solid support containing positive- and negative- control protein and / or tissue samples. For example, kits for performing spotting or western blot-type assays may include control cell or tissue lysates for use in SDS-PAGE or nylon or other membranes containing pre-fixed control samples with additional space for experimental samples. Kits for visualization of the binding agent ligands in cells on slides may include pre-formatted slides containing control cell or tissue samples with additional space for experimental samples. Other embodiments of kits are also contemplated herein as would be understood by those of ordinary skill in the art.

[0054] Thus, in some embodiments, this disclosure provides a binding agent having specificity for a T cell receptor and HLA A1 -tumor antigen peptide complex. In some embodiments, the binding agent of claim 1 wherein the tumor antigen is a melanoma tumor antigen. In some embodiments, the melanoma tumor antigen is MAGE such as MAGE-A3 (e.g., or i.e., EVDPIGHLY (SEQ ID NO:22)). In some embodiments, the binding agent comprises any one or more of the amino acid sequences QGISSR (SEQ ID NO: 3), AAS (SEQ ID NO: 5), QQADSFPLT (SEQ ID NO: 7), GFTFSSYA (SEQ ID NO: 1 1 ), ISGSGGST (SEQ ID NO: 13), and/or ARNLANTGYFDP (SEQ ID NO: 15); and / or any of such amino acid sequences comprising one or more substitutions; and / or a fragment and / or derivative thereof. In some embodiments, the binding agent may comprise SEQ ID NO:2 and/or SEQ ID NO:9; and / or either of such amino acid sequences comprising one or more substitutions; and / or a fragment and / or derivative thereof. In some embodiments, the binding agent is one that competes for binding to MAGE-A3 with any such binding agents. In some embodiments, the binding agent may be a monoclonal antibody or a polypeptide fragment thereof, or a derivative thereof such as a one or more single chain variable fragments (scFv). In some embodiments, the binding agent may further comprise binding specificity for a T cell receptor (e.g., or i.e., a human T cell receptor (also known as CD3)) that may be provided by an amino acid sequence derived from a monoclonal antibody or polypeptide fragment thereof (e.g., or i.e., OKT3). In some embodiments, the binding specificity for the T cell receptor is provided by one or more of the amino acid sequences:

GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE

(SEQ ID NO. : 29) ;

GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRF SGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO.:30); and/or,

GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSG GSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKV ASGVPYRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 31) ; and/ or a fragment and/or derivative thereof. Any of such amino acid sequences may also comprise one or more amino acid substitutions. Such binding agents may also comprise a linking amino acid sequence adjoining a binding agent having specificity for a tumor antigen and / or a HLA-A1/tumor antigen peptide complex, and a binding agent having specificity for the T cell receptor (e.g., or i.e., GGGGSGGGGS (SEQ ID NO.: 17)). In some embodiments, such a binding agent may be a bi-specific antibody. In some embodiments, the binding agent may have the amino acid sequence:

QVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI SGSGGSTFYADSVK GRFTI SRD SKNTLYLQM SLRAEDTAVYYCARNLANTGYFDPWGRGTLVTVSSGLGGLGGGGSG GGGSGGSSGVGSDIQMTQSPSSVSASVGDRVTITCRASQGISSRLAWYQQKPGKAPKLLIYAASG LQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQADSFPLTFGGGTKVEIKRGGASLVEFEQ KLI SEEDLGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCRTSGYTFTRYTMHWVKQRPG QGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLD YWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAG TKLELK (SEQ ID NO.:34);

and/or be a conservatively substituted derivative thereof; and/or be a fragment and/or derivative thereof; and/or comprise a linking amino acid sequence other than GGGGSGGGGS (SEQ ID NO.: 17). In some embodiments, the binding agent may be or be derived from any type of antibody, F_ab, F_ab2, Fab' single chain antibody, F_v, single chain, mono-specific antibody, bi-specific antibody, trimeric antibody, multi-specific antibody, multivalent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized antibody, CDR-grafted antibody, shark antibody, nanobody, camelid antibody, and a de-fucosylated antibody. In some embodiments, the binding agent may comprise a detectable label. Also provided in some embodiments are compositions comprising any one or more such binding agents and and a pharmaceutically acceptable carrier. Kits for detecting the expression of an antigen in a biological sample and / or activating cells (e.g., T cells) and / or assaying cells (e.g., T cells) comprising any one or more such binding agents and/or derivatives instructions for use are also provided. In some embodiments, the kit may include one or more such binding agents and/or derivatives in lyophilized form. In some embodiments, methods for detecting cancer cells, or activating immune cells against such cells, in a biological sample by contacting a test biological sample with a binding agent, derivative or composition comprising the same and detecting the binding agent bound to the biological sample or components thereof and / or the activity of the immune cells are provided. Also provided, in some embodiments, are methods for treating cancer in a mammal by administering to the mammal at least one effective dose (e.g., or i.e., 1 to 50 mg / kg) of one or more binding agents, derivatives, and/or compositions comprising the same. In some embodiments, such methods may further include the administration of one or more chemotherapeutic agents.

[0055] In some embodiments, methods for preparing such binding agents are provided, the methods comprising: selecting a first binding agent having specificity for a tumor antigen from a phage display library using a complex of an HLA A1 heavy chain and a peptide of a tumor antigen, the binding agent optionally having specificity for the complex; and, adjoining the first binding agent to a second binding agent having specificity for a T cell receptor. In some embodiments, the first binding agent is selected from a human antibody phage display library. In some embodiments, the methods may comprise isolating a first nucleic acid sequence encoding the binding agent or a fragment thereof and operably linking the first nucleic acid to a second nucleic acid encoding the T cell receptor or a fragment thereof to produce a fusion construct that may direct the expression of a polypeptide having binding specificity for the tumor antigen or complex, and the T cell receptor. In some embodiments, the binding agent is a polypeptide and / or a bi-specific antibody. In some embodiments, the binding agent may be expressed in a host cell such as Pichia.

[0056] Also provided, in some embodiments, are nucleic acid molecules encoding the binding agents and/or derivatives described herein. In some embodiments, the nucleic acid molecule may be part of an expression vector. In some embodiments, host cells comprising any such nucleic acid molecules and / or expression vectors are also provided. Methods for producing such binding agents and/or derivatives by expressing the binding agent or derivative from any such nucleic acid molecule and / or expression vector are also provided.

[0057] It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a fragment may include mixtures of fragments and reference to a pharmaceutical carrier or adjuvant may include mixtures of two or more such carriers or adjuvants. The terms "about", "approximately", and the like, when preceding a list of numerical values or range, refer to each individual value in the list or range independently as if each individual value in the list or range was immediately preceded by that term. The terms mean that the values to which the same refer are exactly, close to, or similar thereto. As used herein, a subject or a host is meant to be an individual. The subject or host may include domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs) birds, and / or human beings, for example. In some embodiments, the subject or host may be a mammal such as a canine animal. "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase optionally the composition can comprise a combination means that the composition may comprise a combination of different molecules or may not include a combination such that the description includes both the combination and the absence of the combination (e.g., individual members of the combination). Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed. When the terms prevent, preventing, and prevention are used herein in connection with a given treatment for a given condition (e.g., preventing infection by Streptococcus sp.), it is meant to convey that the treated patient either does not develop a clinically observable level of the condition at all, or develops it more slowly and/or to a lesser degree than he/she would have absent the treatment. These terms are not limited solely to a situation in which the patient experiences no aspect of the condition whatsoever. For example, a treatment will be said to have prevented the condition if it is given during exposure of a patient to a stimulus that would have been expected to produce a given manifestation of the condition, and results in the patient's experiencing fewer and/or milder symptoms of the condition than otherwise expected.

[0058] All references cited herein are hereby incorporated by reference into this disclosure in their entirety. Certain embodiments are further described in the following examples. A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.

EXAMPLES

[0059] Example 1

[0060] Production of biotinylated HLA A1 -peptide complex: MAGE-A3:168- 176/HLA A1 : HLA A1 heavy chain with Bir (biotinylation) site at 5' end and β2 microglobulin were cloned into the pET28 vector and expressed in BL21 (DE3). Inclusion bodies were washed three times and dissolved in 2M urea at a concentration of 100mg/ml. HLA-A1 complex was reconstituted by dialysis as described (Garboczi, et al). Briefly, refolding of HLA A1 heavy chain and β2 microglobulin was performed in the presence of MAGE-A3:168-176 peptide (EVDPIGHLY (SEQ ID NO:22)) and L-arginine for 72 hrs. The mixture was dialyzed against a refolding buffer for at least 48 hrs at 4°C, concentrated, and finally, biotinylated with BirA enzyme. The biotinylated and concentrated HLA-A1 was subject to gel filtration (FPLC), and the peak at 23 min was collected.

[0061] Selection of phage antibodies on MAGE-A3:168-176/HLA A1 complexes: A large number of human scFv libraries containing 10¹² diverse scFv clones were used for selection. Phage were preincubated with skimmed dried milk and streptavidin Dynabeads. Biotilynated MAGE-A3/HLA A1 complexes were incubated with phage for 60 minutes and the bound phage captured on streptavidin Dynabeads. The captured phage were eluted with triethylamine and re-infected XL-1 blue cells for next selection. At least 4 rounds of selection were performed.

[0062] Phage ELISA: Antigen complexes were bound to streptavidin in a streptavidin- coated plate which was pre-blocked with BSA. Phages were incubated in the plate and anti-Myc antibody was used to detect the bound phage. After stopping the reactions, the OD at 450 nm was measured with SpectroMax M5 (Molecular Devices Corp., Sunnyvale, CA).

[0063] Production of scFv antibody 8E3 and labeling with FITC: A 6-his-tag was added at the C terminal of the plasmid pYT100-8E by site-directed mutation. The new plasmid pYT100-8E3-6 his was expressed in BL21 (DE3) following induction with 0.5mM IPTG at 37°C for 3 hours. The scFv antibody, 8E3, was purified from the periplasm by Ni-sepharose affinity resin. The scFv antibody 8E3 was labeled by FITC with the FluoReporter FITC protein labeling kit according to the manufacturer's protocol (Life Technologies, Frederick, MD) catalog number F6434).

[0064] Flow Cytometric analysis of TCR-like antibody binding to the cells: The tumor cells were trypsinized and stained with 8E3/FITC for 1 hr at room temperature. Cells were washed once and analyzed by a BD FACS Calibur flow cytometer (BD Biosciences, CA). Some cancer cells were also pulsed with peptide MAGE A3:168-176 for 1 hr, and washed once with PBS, subsequent to the staining of the 8E3/FITC.

[0065] Generation of bi-specific antibody 8EC3 in yeast: The overlapping PCR was used to fuse 8E3 gene with OKT3 scFv sequences, designated as 8EC3 and cloned into PJ912. The 8EC3 was linearized by Sacl and transfected into Pichia competent cells using heat shock method. Expression of antibody was induced with methanol and purified with complete his-tag purification resin (Roche).

[0066] Characterization of 8EC3: 10⁵ 1300A1 cells (MAGE A3-positive melanoma cell line 1300 stably transfected to express HLA A1 ) and 10⁵ T cells were co-cultured in the presence of 8EC3 overnight. ELISA was undertaken to examine the secretion of IFN-γ .

[0067] Cell lines: 1300A1 cells are described above. Cole renal cancer cells (RCC) are HLA A1 positive and MAGE A3 negative.

[0068] Production and Use of HLA A1 Complex: In order to generate soluble T cell antigen complexes, HLA A1 heavy chain and light chain were generated by recombinant DNA technology as described above and refolded with MAGE A3 peptides. Three assays were performed to evaluate the refolding correctness. First, in the FPLC gel filtration the success of refolding gives an extra peak between the peaks of heavy chain and light chain, as shown Fig. 2. The refolding of HLA A1 was also assessed by ELISA using conformation-specific anti-HLA antibody W6/32, which only recognizes correct conformation of HLA molecules. As shown in Fig. 3, W6/32 recognized the refolded HLA A1 with peptides from MAGE A1 and MAGE A3, but not HLA A1 heavy chain and p2m refolded mixtures, suggesting that HLA A1 refolded with MAGE A1 and A3 peptides have correct confirmations. The complexes of HLA A1 MAGE A3/A1 were then tetramerized with PE conjugated streptavidin. This tetramer was used to stain T cells transduced with the genes of TCR recognized MAGE A3 with the constitution of HLA A1. Fig. 4 shows that MAGE A3/A1 complexes stained the T cells with the TCRs. All of these results suggest that refolding of HLA A1 with MAGE A3 peptides to be in correct conformation and functional as desired.

[0069] Screening of phage human antibody library

[0070] MAGE A3:168-176/HLA A1 specific single chain antibodies were selected from a large diverse human antibody phage display library as described in Methods. After fourth round of screening 96 clones were selected and sequenced. Three different clones were obtained (8E3, 5F3, and 12G1 ). Specific clones were detected by an ELISA where binding was tested with specific and nonspecific complexes. Antigen complex MAGE A1 :161 -169/HLA A1 was chosen as a nonspecific one since MAGE A1 peptide (EADPTGHSY (SEQ ID NO:21 )) has only 3 amino acids different to that of the MAGE A3 peptide (EVDPIGHLY (SEQ ID NO:22)). In order to demonstrate MAGE A1 :161-169/ HLA A1 complex was truly active to its own target anti-MAGE A1/HLA A1 , scFv was synthesized based on the published sequences. Fig. 5 shows the results of ELISA of phages bearing TCR like scFv antibodies against MAGA A1 :161-169/HLA A1 , MAGE A3:168-176/HLA A1 , and HLA A1 only complexes. Clones 8E3 and 12G1 were shown to be specifically recognized by the MAGE A3:168-176/HLA A1 , not with MAGE A1 :161-169/HLA A1. Clone 5F3 was reactive to MAGE A3:168-176/HLA A1 , but also to MAGE A1 :161-169/HLA A1 , suggesting the specificity to MAGE A3:168-176/HLA A1 was not satisfactory in this phage ELISA level. Clones 8E4 and 12G1 shared the same translated amino acids sequences. However, the gene sequence of clone 8E3 was different. Thus, we selected clone 8E3 for further investigations. [0071] TCR-like antibody 8E3 bound to MAGE A3 HLA A1 positive cancer cells

[0072] To generate the soluble scFv, the insert of 8E3 was cloned with a his-tag into a PelB secretion vector and purified from periplasm using Ni-affinity column. The purified antibodies were then conjugated by FITC and tested on two cancer lines. One of the cancer cell lines 1300 expresses MAGE A3 and HLA A2. The other cell line, 1300A1 while identical to 1300, it expresses HLA A1. Thus, 1300 was negative with HLA A1 and was used as a base line (blue line in Fig. 6) to MAGE A3 antigen in the context of HLA A1 because 1300 was not able to present any HLA A1 specific epitope. On the other hand, 8E3 was shown to bound to 1300A1 cells and shifted contrasted to 1300 cells (green line in Fig. 6). To explore the possibility that 8E3 binding is epitope specific and concentration dependent, 1300A1 was pulsed with 1 mg/ml and 10 mg/ml of MAGE A3: 168-176. The 1300A1 cells pulsed with 1 mg/ml peptides was shifted a little (red line in Fig. 6), while the cells pulsed with 10 mg/ml peptides (brown line) were shifted a dramatically, suggesting 8E3 was binding 1300A1 cells related on HLA A1 and peptide concentration.

[0073] Generation of bi-specific antibody against MAGE A: 3168-176/HLA A1 and CD3

[0074] The OKT3 gene was chemically synthesized according to the published sequence (3). The 8E3 sequence was fused to OKT3 sequences with a linker (G4S) ( GGGGSGGGGS ( SEQ ID NO . : 1 7 ) ) by overlapping PCR and designated 8EC3. Attempts were made to express 8EC3 in PelB signal secretion/expression system. However, it was not secreted successfully as most part of the protein in E. coli was in insoluble. Then, we tried to express it in mammalian HEK 293 expression system. The yield was too low (data not shown). Finally, 8EC3 was expressed in Pichia and purified using Ni-affinity column (Fig. 7). The amino acid sequence of 8EC3 is:

QVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI SGSGGSTFYADSVK GRFTI SRD SKNTLYLQM SLRAEDTAVYYCARNLANTGYFDPWGRGTLVTVSSGLGGLGGGGSG GGGSGGSSGVGSDIQMTQSPSSVSASVGDRVTITCRASQGISSRLAWYQQKPGKAPKLLIYAASG LQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQADSFPLTFGGGTKVEIKRGGASLVEFEQ KLI SEEDLGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPG QGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLD YWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAG TKLELK (SEQ ID NO.:34)

[0075] Evaluation of bi-specific antibody 8EC3 to target cancer cells

[0076] Peripheral blood T cells were stimulated with OKT3 and were incubated at 37°C for 9-14 days. The cancer cells (10e5) were incubated with or without MAGE A3/ A1 peptides (0.25 mM) for 1 hr. T cells (10e5) and 8EC3 (1 ng) was added to the cancer cells and the co-cultures (200 I) were incubated overnight. ELISA was employed to test the IFN- production. As shown in Fig. 8, 1300A1 cells pulsed with peptides stimulated T cells and generated at least 4 folds of IFN-γ compared to 1300 cells, suggesting 8EC3 could bridge T cells with MAGE A3 antigen on cancer cells and 8EC3 activated T cells.

[0077] While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed.

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SEQUENCE LISTING

GGTGGATCCAGCGGTGTGGGTTCCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATC TGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGTAGCCGGTTAGCCTGGT ATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCGGTTTGCAAAGTGGG GTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCA GCCTGAAGATTCTGCAACTTATTATTGTCAACAGGCTGACAGTTTCCCGCTCACTTTCGGCGGAG GGACCAAGGTGGAGATCAAACGTGGAGGAGCCAGCCTCGTGGAATTCGAGCAGAAGCTGATCTCT GAGGAAGACCTG ( SEQ ID NO.: 1)

GGSSGVGSDIQMTQSPSSVSASVGDRVTITCRASQGISSRLAWYQQKPGKAPKLLIYAASGLQSG VPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQADSFPLTFGGGTKVEIKRGGASLVEFEQKLIS EEDL ( SEQ ID NO. : 2)

QGISSR (SEQ ID NO. : 3 )

GGCTTTACCTTTAGCAGCTATGCG (SEQ ID NO.: 4)

AAS (SEQ ID NO. : 5)

ATTAGCGGCAGCGGCGGCAGCACC ( SEQ ID NO.: 6)

QQADSFPLT (SEQ ID NO.: 7)

GCGCGCAACCTGGCGAACACCGGCTATTTTGATCCG ( SEQ ID NO.: 8)

QVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTFYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNLANTGYFDPWGRGTLVTVSSGLGGL (SEQ ID NO. : 9 )

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTG TGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGG GGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATTCTACGCAGACTCCGTGAAG GGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAG AGCCGAGGACACGGCCGTGTATTACTGTGCGAGAAACCTTGCTAATACGGGCTACTTCGACCCCT GGGGCCGGGGAACCCTGGTCACCGTCTCCTCAGGCCTCGGGGGCCTCGGAGGAGGAGGTAGTGGC GGAGGAGGCTCC (SEQ ID NO: 10)

GFTFSSYA (SEQ ID NO.: 11)

CAGGGCAT AGCAGCCGC ( SEQ ID NO.: 12)

ISGSGGST (SEQ ID NO.: 13)

GCGGCGAGC (SEQ ID NO.: 14)

ARNLANTGYFDP (SEQ ID NO.: 15)

CAGCAGGCGGATAGCTTTCCGCTGACC ( SEQ ID NO.: 16)

GGGGSGGGGS (SEQ ID NO.: 17)

GGCGGCGGCGGCAGCGGCGGCGGCGGCAGC ( SEQ ID NO.: 18) QVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTFYADSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNLANTGYFDPWGRGTLVTVSSGLGGLGGGGSG GGGSGGSSGVGSDIQMTQSPSSVSASVGDRVTITCRASQGISSRLAWYQQKPGKAPKLLIYAASG LQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQADSFPLTFGGGTKVEIKRGGASLVEFEQ KLISEEDL (SEQ ID NO.: 19)

CAGGTGCAGCTGGTGCAGAGCGGCGGCGGCCTGGTGCAGCCGGGCCGCAGCCTGCGCCTGAGCTG CGCGGCGAGCGGCTTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAGGCGCCGGGCAAAG GCCTGGAATGGGTGAGCGCGATTAGCGGCAGCGGCGGCAGCACCTTTTATGCGGATAGCGTGAAA GGCCGCTTTACCATTAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCG CGCGGAAGATACCGCGGTGTATTATTGCGCGCGCAACCTGGCGAACACCGGCTATTTTGATCCGT GGGGCCGCGGCACCCTGGTGACCGTGAGCAGCGGCCTGGGCGGCCTGGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGGCGGCAGCAGCGGCGTGGGCAGCGATATTCAGATGACCCAGAGCCCGAGCAG CGTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGGCATTAGCAGCC GCCTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATGCGGCGAGCGGC CTGCAGAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCAT TAGCAGCCTGCAGCCGGAAGATAGCGCGACCTATTATTGCCAGCAGGCGGATAGCTTTCCGCTGA CCTTTGGCGGCGGCACCAAAGTGGAAATTAAACGCGGCGGCGCGAGCCTGGTGGAATTTGAACAG AAACTGATTAGCGAAGAAGATCTG (SEQ ID NO.: 20)

EADPTGHSY (SEQ ID NO : 21 )

EVDPIGHLY (SEQ ID NO: 22)

SFPMA (SEQ ID NO: 23)

TISTSGGRTYYRDSVLG (SEQ ID NO: 24)

FRQYSGGFDY (SEQ ID NO: 25)

TLSSGNIQNYVH (SEQ ID NO : 26 )

DDDKRPD (SEQ ID NO: 27)

HSYVSSFNV (SEQ ID NO: 28)

GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVE

(SEQ ID NO. :29)

GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRF SGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO.: 30)

GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSG GSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKV ASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK ( SEQ ID NO: 31)

AKELAVAGYFDY (SEQ ID NO: 32)

ARTLANSEVWGQ (SEQ ID NO: 33) QVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI SGSGGSTFYADSVK GRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARNLANTGYFDPWGRGTLVTVSSGLGGLGGGGSG GGGSGGSSGVGSDIQMTQSPSSVSASVGDRVTITCRASQGI SSRLAWYQQKPGKAPKLLIYAASG LQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQADSFPLTFGGGTKVEIKRGGASLVEFEQ KLI SEEDLGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPG QGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLD YWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAG TKLELK ( SEQ ID NO . : 34 )

Claims

CLAIMS What is claimed is:

1. A binding agent having specificity for a T cell receptor and HLA A1-tumor antigen peptide complex.

2. The binding agent of claim 1 wherein the tumor antigen is a melanoma tumor antigen.

3. The binding agent of claim 1 wherein the melanoma tumor antigen is MAGE.

4. The binding agent of claim 3 wherein the melanoma tumor antigen is MAGE-A3.

5. The binding agent of claim 4 wherein melanoma tumor antigen is the MAGE-A3 peptide EVDPIGHLY (SEQ ID NO:22).

6. A binding agent comprising the amino acid sequence QGISSR (SEQ ID NO: 3), SEQ ID NO:3 comprising one or more substitutions, or a fragment or derivative thereof.

7. A binding agent comprising the amino acid sequence QQADSFPLT (SEQ ID NO: 7), SEQ ID NO:7 comprising one or more substitutions, or a fragment or derivative thereof.

8. The binding agent of claim 6 or 7 further comprising the amino acid sequence AAS (SEQ ID NO: 5), SEQ ID NO:5 comprising one or more substitutions, or a fragment or derivative thereof.

9. A binding agent comprising the amino acid sequence ARNLANTGYFDP (SEQ ID NO: 15), SEQ ID NO:15 comprising one or more substitutions, or a fragment or derivative thereof.

10. The binding agent of claim 9 further comprising at least one of GFTFSSYA (SEQ ID NO: 1 1 ), SEQ ID NO:1 1 comprising one or more substitutions, or a fragment or derivative thereof; and/or ISGSGGST (SEQ ID NO: 13), SEQ ID NO:13 comprising one or more substitutions, or a fragment or derivative thereof.

1 1 . The binding agent of any one of claims 1-10 comprising SEQ ID NO:2, SEQ ID NO:2 comprising one or more substitutions, or a fragment or derivative thereof.

12. The binding agent of any one of claims 1-1 1 comprising SEQ ID NO:9, SEQ ID NO:9 comprising one or more substitutions, or a fragment or derivative thereof.

13. The binding agent of any one of claims 1-12 comprising SEQ ID NO:2, SEQ ID NO:2 comprising one or more substitutions, or a fragment or derivative thereof; and SEQ ID NO:9, SEQ ID NO:9 comprising one or more substitutions, or a fragment or derivative thereof.

14. The binding agent of any one of claims 6-13 having specificity for MAGE-A3.

15. The binding agent of claim 15 wherein the specificity is for the MAGE-A3 peptide EVDPIGHLY (SEQ ID NO:21 ).

16. A binding agent that competes for binding to MAGE-A3 with the binding agent of any one of claims 1-16.

17. The binding agent of any one of claims 1 -17 that is an isolated monoclonal antibody or a polypeptide fragment thereof.

18. The binding agent of any one of claims 1 -17 that is a single chain variable fragment (scFv).

19. The binding agent of any one of claims 6-18 further comprising binding specificity for a T cell receptor.

20. The binding agent of claim 19 wherein the T cell receptor is human.

21 . The binding agent of claim 19 or 20 wherein the binding specificity for the T cell receptor is provided by an amino acid sequence.

22. The binding agent of claim 21 wherein the amino acid sequence is derived from a monoclonal antibody or polypeptide fragment thereof.

23. The binding agent of claim 22 wherein the antibody is OKT3.

24. The binding agent of any one of claims 1-5 or 19-23 wherein the binding specificity for the T cell receptor is provided by the amino acid sequence:

GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGY TNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS VE (SEQ ID NO. : 29) ;

GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVP YRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID

NO. : 30) ; and/or

GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGY TNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS VEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPK RWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

(SEQ ID NO: 31) ;

and/ or a fragment thereof.

25. The binding agent of any one of claims 19-24 comprising a linking amino acid sequence adjoining a binding agent having specificity for a tumor antigen and / or a HLA-A1/tumor antigen peptide complex, and a binding agent having specificity for the T cell receptor.

26. The binding agent of claim 25 wherein the linking amino acid sequence comprises GGGGSGGGGS (SEQ ID NO.: 17).

27. The binding agent of any one of claims 19-26, the binding agent being a bi-specific antibody.

28. The binding agent of any one of claims 19-27 having the amino acid sequence:

QVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI SGSGGSTFYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNLANTGYFDPWGRGTLVTVSSGLGGL GGGGSGGGGSGGSSGVGSDIQMTQSPSSVSASVGDRVTITCRASQGISSRLAWYQQKPGKAP KLLIYAASGLQSGVPSRFSGSGSGTDFTLTI SSLQPEDSATYYCQQADSFPLTFGGGTKVEI KRGGASLVEFEQKLI SEEDLGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGY TFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSE DSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSAS PGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISS MEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO.:34);

and/or a conservatively substituted derivative thereof;

and/or a fragment and/or derivative thereof;

and/or comprising a linking amino acid sequence other than GGGGSGGGGS (SEQ ID NO.: 17).

29. The binding agent of any one of claims 1 -28 derived from any human IgG, human lgG1 , human lgG2, human lgG3, human lgG4, human IgM, human IgA, human lgA1 , human lgA2, human IgD, human IgE, canine antibody, canine IgGA, canine IgGB, canine IgGC, canine IgGD, canine IgA, canine IgD, canine IgE, canine IgM, chicken antibody, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goat antibody, goat IgG, mouse antibody, mouse IgG, mouse IgA, mouse IgD, mouse IgE, mouse IgM, pig antibody, pig IgG, rat antibody, rat IgG and feline antibody, feline IgG.

30. A derivative of a binding agent of any one of claims 1 -29.

31 . The derivative of claim 30 selected from the group consisting of F_ab, F_ab2, Fab' single chain antibody, F_v, single chain, mono-specific antibody, bi-specific antibody, trimeric antibody, multi-specific antibody, multivalent antibody, chimeric antibody, canine- human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized antibody, CDR-grafted antibody, shark antibody, nanobody, camelid antibody, and a de-fucosylated antibody.

32. The derivative of claim 30 or 31 further comprising a detectable label fixably attached thereto.

33. A composition comprising the binding agent or derivative of any one of claims 1 -32 and a pharmaceutically acceptable carrier.

34. A kit for detecting the expression of an antigen in a biological sample, the kit comprising a binding agent or derivative of any one of claims 1-32 and instructions for use.

35. A kit of claim 34 wherein the binding agent or derivative is in lyophilized form.

36. A method for detecting cancer cells in a biological sample, the method comprising contacting a test biological sample with a binding agent, derivative or composition of any one of claims 1 -33 and detecting the binding agent bound to the biological sample or components thereof.

37. A method for treating cancer in a mammal, the method comprising administering to the mammal at least one effective dose of the binding agent, derivative or composition of any one of claims 1-33.

38. The method of claim 37 wherein multiple doses of the binding agent, derivative or composition are administered to the mammal.

39. The method of claim 37 or 38 wherein the binding agent or derivative is administered in a dosage amount of about 1 to 50 mg / kg.

40. The method of any one of claims 37-39 wherein the binding agent, derivative or composition is administered in conjunction with one or more chemotherapeutic agents.

41 . A method for preparing a binding agent, the method comprising:

a. selecting a first binding agent having specificity for a tumor antigen from a phage display library using a complex of an HLA A1 heavy chain and a peptide of a tumor antigen, the binding agent optionally having specificity for the complex; and,

b. adjoining the first binding agent to a second binding agent having specificity for a T cell receptor.

42. The method of claim 41 wherein the T cell receptor is human.

43. The method of claim 41 or 42 wherein the first binding agent is a monoclonal antibody or polypeptide fragment thereof.

44. The method of claim 43 wherein the first binding agent is a single chain antibody.

45. The method of any one of claims 40-44 wherein the first binding agent is selected from a human antibody phage display library.

46. The method of any one of claims 40-45 comprising isolating a first nucleic acid sequence encoding the binding agent or a fragment thereof and operably linking the first nucleic acid to a second nucleic acid encoding the T cell receptor or a fragment thereof to produce a fusion construct.

47. The method of claim 41 wherein the fusion construct directs the expression of a polypeptide having binding specificity for the tumor antigen or complex, and the T cell receptor.

48. The method of claim 47 wherein the polypeptide is a bi-specific antibody.

49. The method of claim 47 or 48 wherein the expression is in Pichia.

50. A nucleic acid molecule encoding the binding agent or derivative of any one of claims 1-32.

51 . An expression vector comprising the nucleic acid molecule of claim 50.

52. A host cell comprising the nucleic acid molecule of claim 50 and / or the expression vector of claim 51.

53. A method of producing the binding agent or derivative of any one of claims 1 -32 comprising expressing the binding agent or derivative from a nucleic acid molecule of claim 50 or the expression vector of claim 51 in a cell and isolating the binding agent therefrom.