Nothing Special   »   [go: up one dir, main page]

WO2016077720A1 - Agents de liaison et utilisations de ceux-ci - Google Patents

Agents de liaison et utilisations de ceux-ci Download PDF

Info

Publication number
WO2016077720A1
WO2016077720A1 PCT/US2015/060622 US2015060622W WO2016077720A1 WO 2016077720 A1 WO2016077720 A1 WO 2016077720A1 US 2015060622 W US2015060622 W US 2015060622W WO 2016077720 A1 WO2016077720 A1 WO 2016077720A1
Authority
WO
WIPO (PCT)
Prior art keywords
binding agent
seq
antibody
derivative
amino acid
Prior art date
Application number
PCT/US2015/060622
Other languages
English (en)
Inventor
Xiang YANG
Shigui Zhu
Original Assignee
Protein One, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Protein One, Llc filed Critical Protein One, Llc
Publication of WO2016077720A1 publication Critical patent/WO2016077720A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the technology relates to monoclonal antibodies useful in the identification of cancer cells, as well as the diagnosis and treatment of cancer.
  • TIL-2 human tumor infiltrating lymphocytes 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.
  • the clinical efficacy of TIL generated from histologies other than melanoma has not been demonstrated.
  • PBMC peripheral blood mononuclear cells
  • 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.
  • 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.
  • TCR mediated T cell therapy bi-specific antibodies do not need to be personalized.
  • the technology relates to monoclonal antibodies useful in the identification of cancer cells, as well as the diagnosis and treatment of cancer.
  • mAbs with specificity for tumor antigens are provided.
  • bi-specific antibodies are provided.
  • methods for using such binding agents to detecting cancerous cells are provided.
  • methods for treating cancer using such binding agents are provided.
  • kits for detecting cancerous cells are provided.
  • Figure 1 Exemplary (BiTE) bi-specific antibody system.
  • FIG. 1 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.
  • Figure 4 Staining of tetramer with TCR transfected T cells and positive staining.
  • Figure 7 Western blot hybridization of 8EC3 and CD19X3 generated from yeast.
  • 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.
  • 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.
  • 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.
  • 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.
  • TCR T cell receptor
  • 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.
  • 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).
  • 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.
  • K d a dissociation 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 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.
  • Exemplary light GCGCGCAACCTGGCGAACACCGGCTATTTTGATCCG SEQ ID chain CDR3 NO.: 8
  • 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.
  • the MAGE antigen is a peptide of MAGE-A1 or MAGE-A3.
  • the MAGE peptide is MAGE A1 peptide EADPTGHSY (SEQ ID NO:21 ).
  • the MAGE peptide is the MAGE-A3 peptide EVDPIGHLY (SEQ ID NO:22).
  • the binding agent may comprise the amino acid sequences QGISSR (SEQ ID NO: 3) and/or QQADSFPLT (SEQ ID NO: 7).
  • the binding agent may comprise the amino acid sequences QGISSR (SEQ ID NO: 3) and AAS (SEQ ID NO: 5).
  • the binding agent may comprise the amino acid sequences AAS (SEQ ID NO: 5) and QQADSFPLT (SEQ ID NO: 7).
  • 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.
  • the binding agent may be one that competes with such a binding agent for binding to that ligand.
  • 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).
  • 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 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.
  • the binding agent may be one that competes with such a binding agent for binding to that ligand.
  • 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).
  • 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 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).
  • 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).
  • 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.
  • the binding agent comprising any one or more of SEQ ID NOS. 1 , 3, 5, 7, 9, 1 1 , 13 and / or 15.
  • a binding agent may have specificity for a particular ligand.
  • that ligand may have specificity for human MAGE-A3, in particular the peptide EVDPIGHLY (SEQ ID NO:22).
  • 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.
  • 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).
  • 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).
  • TCR T cell receptor
  • 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.
  • 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).
  • CD3 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.
  • 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.
  • 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).
  • 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 .
  • the TcR/CD3 specificity may be provided by the amino acid sequence, or TcR/CD3 binding fragments thereof, shown below:
  • GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRF SGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO.: 30); such as : GGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNY NQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSG GSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKV ASGVPYRFSGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 31) .
  • 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.
  • 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.
  • 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.
  • the binding agents described herein may be glycosylated or not (e.g., "aglycosylated”).
  • 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.
  • 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).
  • 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.
  • these monoclonal antibodies may be reactive against a tumor antigen expressed on the surface of cells.
  • 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.
  • 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
  • the antibodies may be contained within hybridoma supernatant or ascites and utilized either directly as such or following concentration using standard techniques.
  • 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).
  • the binding agent 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.
  • TBS Tris-buffered saline
  • PBS phosphate buffered saline
  • 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.
  • 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.
  • 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.
  • any one or more fragments and / or derivatives of the amino acid sequences of this disclosure are also contemplated herein.
  • 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.
  • 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.
  • 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.
  • one or more amino acids in any of the amino acid sequences of Table 1 may be substituted with another amino acid, such as but not limited to the conservative substitutions exemplified in Table 2.
  • 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.
  • 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.
  • the arginine (R) of QGISSR (SEQ ID NO:3) is not substituted by tryptophan (W).
  • the aspartic acid (D) of QQADSFPLT (SEQ ID NO:7) is not substituted by asparagine (N).
  • ARNLANTGYFDP SEQ ID NO:15
  • ARTLANSEVWGQ SEQ ID NO:33.
  • 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.
  • 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.
  • 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.
  • Nucleic acid molecules encoding one or more binding agents may be contained within a viral and / or a non-viral vector.
  • a DNA vector is utilized to deliver nucleic acids encoding one or more binding agents to the patient.
  • 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.
  • 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
  • pFASTBACdual Gibco-BRL, Grand island, NY
  • 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., TOPOTM TA cloning kit, PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, CA.
  • Bacterial vectors may also be used.
  • 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).
  • BCG Bacille Calmette Guerin
  • Streptococcus see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376).
  • Other delivery techniques may also suffice including, for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaP0 4 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.
  • 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.
  • 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).
  • ATCC American Type Culture Collection
  • 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.
  • the binding agents of this disclosure are not in any way limited to antibodies.
  • 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).
  • 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.
  • FACS fluorescence activated cell sorting
  • 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.
  • 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, can
  • Combinations of binding agents may also be identified.
  • 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.
  • combinations exhibiting synergistic ability to restore immune cell function may be identified.
  • 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.
  • 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.
  • 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)).
  • CT cancer-testis
  • MAGE the peptide EVDPIGHLY (SEQ ID NO:22) or EADPTGHSY (SEQ ID NO:21 ) as shown in the Examples herein
  • NY-ESO-1
  • binding agents e.g., monoclonal antibodies
  • 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.
  • multiple single specificity- binding agents may also be combined and used (e.g., administered) to provide an effective multiple specificity reagent.
  • 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).
  • cancer cell populations expressing the tumor antigen(s) may be targeted and eliminated using binding agent / drug conjugates (e.g., antibody-drug conjugates (ADC)).
  • ADC antibody-drug conjugates
  • 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.
  • 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.
  • the binding agents may be adjoined to and / or conjugated to one or more detectable labels.
  • 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'
  • fluorosceins
  • 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.
  • 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).
  • 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
  • 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.
  • CDRs complementarity determining regions
  • 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.
  • 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
  • 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.
  • binding agents e.g., antibodies
  • 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
  • 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
  • the binding agents may also be utilized to detect tumor antigen(s) within a biological sample.
  • 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.
  • 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.
  • base e.g., control
  • the binding agents described herein 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.
  • the binding agents are in purified form.
  • a "purified" binding agent e.g., antibody
  • a purified binding agent 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
  • a purified binding agent 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.
  • 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.
  • a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • buffers e.g., TBS, PBS
  • blocking agents solutions including nonfat dry milk, normal sera, Tween-20 Detergent, BSA, or casein
  • detection reagents e.g., goat anti-mouse IgG biotin, streptavidin-HRP conjugates, allophycocyan
  • 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.
  • the kit provides a binding agent in purified form.
  • the binding agent may be provided in biotinylated form either alone or along with an avidin-conjugated detection reagent (e.g., antibody).
  • the kit includes a binding agents comprising one or more detectable labels that may be used to directly detect the binding agent ligands.
  • kits 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.
  • 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.
  • this disclosure provides a binding agent having specificity for a T cell receptor and HLA A1 -tumor antigen peptide complex.
  • the binding agent of claim 1 wherein the tumor antigen is a melanoma tumor antigen.
  • the melanoma tumor antigen is MAGE such as MAGE-A3 (e.g., or i.e., EVDPIGHLY (SEQ ID NO:22)).
  • 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.
  • 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.
  • the binding agent is one that competes for binding to MAGE-A3 with any such binding agents.
  • 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).
  • 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).
  • the binding specificity for the T cell receptor is provided by one or more of the amino acid sequences:
  • GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRF SGSGSGTSYSLTI SSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO.:30); and/or,
  • 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)).
  • a binding agent having specificity for the T cell receptor e.g., or i.e., GGGGSGGGGS (SEQ ID NO.: 17
  • such a binding agent may be a bi-specific antibody.
  • the binding agent may have the amino acid sequence:
  • 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.
  • the binding agent may comprise a detectable label.
  • 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.
  • the kit may include one or more such binding agents and/or derivatives in lyophilized form.
  • 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.
  • 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.
  • such methods may further include the administration of one or more chemotherapeutic agents.
  • methods for preparing such binding agents 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.
  • the first binding agent is selected from a human antibody phage display library.
  • 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.
  • the binding agent is a polypeptide and / or a bi-specific antibody.
  • the binding agent may be expressed in a host cell such as Pichia.
  • nucleic acid molecules encoding the binding agents and/or derivatives described herein.
  • the nucleic acid molecule may be part of an expression vector.
  • 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.
  • 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.
  • livestock e.g., cattle, horses, pigs, sheep, and goats
  • laboratory animals e.g., mice, rabbits, rats, guinea pigs
  • 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.
  • 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.
  • 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 are meant to include the range per se as well as each independent value within the range as if each value was individually listed.
  • 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.
  • 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.
  • HLA A1 -peptide complex MAGE-A3:168- 176/HLA A1 : HLA A1 heavy chain with Bir (biotinylation) site at 5' end and ⁇ 2 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).
  • HLA A1 heavy chain and ⁇ 2 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.
  • 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).
  • scFv antibody 8E3 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).
  • 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.
  • Cell lines 1300A1 cells are described above.
  • Cole renal cancer cells (RCC) are HLA A1 positive and MAGE A3 negative.
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 8E3 was shown to bound to 1300A1 cells and shifted contrasted to 1300 cells (green line in Fig. 6).
  • 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.
  • 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.
  • G4S GGGGSGGGGS ( SEQ ID NO . : 1 7 )
  • 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).
  • 8EC3 was expressed in Pichia and purified using Ni-affinity column (Fig. 7).
  • the amino acid sequence of 8EC3 is:
  • 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.
  • 1300A1 cells pulsed with peptides stimulated T cells and generated at least 4 folds of IFN- ⁇ compared to 1300 cells, suggesting 8EC3 could bridge T cells with MAGE A3 antigen on cancer cells and 8EC3 activated T cells.
  • Besser MJ Shapira-Frommer R, Treves AJ, et al. Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clin Cancer Res.2010;16:2646-2655.
  • AAS (SEQ ID NO. : 5)
  • GCGCGCAACCTGGCGAACACCGGCTATTTTGATCCG SEQ ID NO.: 8
  • CAGGGCAT AGCAGCCGC SEQ ID NO.: 12
  • CAGCAGGCGGATAGCTTTCCGCTGACC SEQ ID NO.: 16
  • GGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRF SGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK SEQ ID NO.: 30
  • ARTLANSEVWGQ (SEQ ID NO: 33) QVQLVQSGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI SGSGGSTFYADSVK GRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARNLANTGYFDPWGRGTLVTVSSGLGGLGGGGSG GGGSGGSSGVGSDIQMTQSPSSVSASVGDRVTITCRASQGI SSRLAWYQQKPGKAPKLLIYAASG LQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQADSFPLTFGGGTKVEIKRGGASLVEFEQ KLI SEEDLGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPG QGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLD YWGQGTTL

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Selon l'invention, cette technologie concerne des anticorps monoclonaux utiles dans la détection et/ou le traitement du cancer. Dans certains modes de réalisation, il est prévu des agents de liaison ayant une spécificité pour au moins un antigène tumoral et des récepteurs de lymphocytes T (par exemple, CD3 humain ). Certains modes de réalisation portent sur des méthodes de traitement du cancer faisant appel auxdits agents de liaison.
PCT/US2015/060622 2014-11-14 2015-11-13 Agents de liaison et utilisations de ceux-ci WO2016077720A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462079635P 2014-11-14 2014-11-14
US62/079,635 2014-11-14

Publications (1)

Publication Number Publication Date
WO2016077720A1 true WO2016077720A1 (fr) 2016-05-19

Family

ID=55955122

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/060622 WO2016077720A1 (fr) 2014-11-14 2015-11-13 Agents de liaison et utilisations de ceux-ci

Country Status (1)

Country Link
WO (1) WO2016077720A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019210144A1 (fr) * 2018-04-27 2019-10-31 Vanderbilt University Anticorps largement neutralisants dirigés contre le virus de l'hépatite c
WO2019235915A1 (fr) * 2018-06-04 2019-12-12 Apo-T B.V. Procédés et moyens pour attirer des cellules immunitaires effectrices vers des cellules tumorales
WO2020023679A1 (fr) * 2018-07-25 2020-01-30 Accurus Biosciences, Inc. Nouveaux anticorps monoclonaux spécifiques à cldn 18.2 et leurs méthodes d'utilisation
WO2021112676A3 (fr) * 2019-12-04 2021-09-30 Apo-T B.V. Procédés et moyens pour attirer des cellules immunitaires effectrices vers des cellules tumorales

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066803A2 (fr) * 2008-12-09 2010-06-17 Genmab A/S Anticorps humains du facteur tissulaire
US20120114654A1 (en) * 2010-11-08 2012-05-10 Regeneron Pharmaceuticals, Inc. Human antibodies to human tnf-like ligand 1a (tl1a)
WO2013041865A1 (fr) * 2011-09-22 2013-03-28 Immunocore Limited Récepteurs de lymphocytes t
WO2013048243A1 (fr) * 2011-09-29 2013-04-04 Apo-T B.V. Molécules de liaison multispécifiques ciblant des cellules aberrantes
WO2014047231A1 (fr) * 2012-09-21 2014-03-27 Regeneron Pharmaceuticals, Inc. Anticorps anti-cd3, molécules de liaison à un antigène bispécifiques qui se lient à cd3 et cd20, et leurs utilisations
US20140205599A1 (en) * 2010-12-27 2014-07-24 Maria Johanna J.E. Van Driel Polypeptide that binds aberrant cells and induces apoptosis

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066803A2 (fr) * 2008-12-09 2010-06-17 Genmab A/S Anticorps humains du facteur tissulaire
US20120114654A1 (en) * 2010-11-08 2012-05-10 Regeneron Pharmaceuticals, Inc. Human antibodies to human tnf-like ligand 1a (tl1a)
US20140205599A1 (en) * 2010-12-27 2014-07-24 Maria Johanna J.E. Van Driel Polypeptide that binds aberrant cells and induces apoptosis
WO2013041865A1 (fr) * 2011-09-22 2013-03-28 Immunocore Limited Récepteurs de lymphocytes t
WO2013048243A1 (fr) * 2011-09-29 2013-04-04 Apo-T B.V. Molécules de liaison multispécifiques ciblant des cellules aberrantes
WO2014047231A1 (fr) * 2012-09-21 2014-03-27 Regeneron Pharmaceuticals, Inc. Anticorps anti-cd3, molécules de liaison à un antigène bispécifiques qui se lient à cd3 et cd20, et leurs utilisations

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019210144A1 (fr) * 2018-04-27 2019-10-31 Vanderbilt University Anticorps largement neutralisants dirigés contre le virus de l'hépatite c
WO2019235915A1 (fr) * 2018-06-04 2019-12-12 Apo-T B.V. Procédés et moyens pour attirer des cellules immunitaires effectrices vers des cellules tumorales
JP2021528382A (ja) * 2018-06-04 2021-10-21 エーピーオー‐ティー ビー．ヴイ． 免疫エフェクター細胞を腫瘍細胞へ誘引する方法及び手段
WO2020023679A1 (fr) * 2018-07-25 2020-01-30 Accurus Biosciences, Inc. Nouveaux anticorps monoclonaux spécifiques à cldn 18.2 et leurs méthodes d'utilisation
US12054543B2 (en) 2018-07-25 2024-08-06 Accurus Biosciences, Inc. CLDN 18.2-specific monoclonal antibodies and methods of use thereof
WO2021112676A3 (fr) * 2019-12-04 2021-09-30 Apo-T B.V. Procédés et moyens pour attirer des cellules immunitaires effectrices vers des cellules tumorales

Similar Documents

Publication Publication Date Title
US20220169733A1 (en) Immunological Reagents
JP7089474B2 (ja) 免疫学的試薬
US20200317806A1 (en) Bispecific antibody molecules with antigen-transfected t-cells and their use in medicine
US9982052B2 (en) Immunological reagents
US20220298212A1 (en) Recombinant 4-1bb binding proteins and their use
WO2016077720A1 (fr) Agents de liaison et utilisations de ceux-ci
JP2016531583A (ja) 免疫系調節薬
CA3154450A1 (fr) Anticorps a domaine unique diriges contre lilrb2
US20220259315A1 (en) Immunological Reagents
CA3108192A1 (fr) Nouveaux antigenes associes a une tumeur independante du cmh
US20190375819A1 (en) Glycosylated transferrin receptor 1 tumor antigen
TW202404646A (zh) 抗cdh6抗體-藥物結合物之給藥方案

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15858943

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15858943

Country of ref document: EP

Kind code of ref document: A1