WO2019091449A1 - Cd96抗体、其抗原结合片段及医药用途 - Google Patents

Cd96抗体、其抗原结合片段及医药用途 Download PDF

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WO2019091449A1
WO2019091449A1 PCT/CN2018/114797 CN2018114797W WO2019091449A1 WO 2019091449 A1 WO2019091449 A1 WO 2019091449A1 CN 2018114797 W CN2018114797 W CN 2018114797W WO 2019091449 A1 WO2019091449 A1 WO 2019091449A1
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antibody
seq
variable region
chain variable
antigen
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PCT/CN2018/114797
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English (en)
French (fr)
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曹卓晓
付雅媛
于存静
胡齐悦
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN201880047582.5A priority Critical patent/CN110914304B/zh
Priority to US16/763,101 priority patent/US20200347130A1/en
Priority to EP18875520.1A priority patent/EP3712170A4/en
Publication of WO2019091449A1 publication Critical patent/WO2019091449A1/zh

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to CD96 antibodies and antigen-binding fragments thereof. Further, the present disclosure also relates to chimeric antibodies, humanized antibodies comprising the CDR regions of the CD96 antibodies, and to the inclusion of the CD96 antibodies and antigen-binding fragments thereof. Pharmaceutical compositions, and their use as diagnostic and therapeutic agents for CD96-associated diseases.
  • CD96 Human Leukocyte Differentiation Antigen Collaboration Group meeting.
  • CD96 molecules are expressed on normal T cells, T cell clones, and certain transformed T cells.
  • Peripheral blood T cells expressed low levels of CD96 molecules, and their expression was significantly up-regulated after activation, and reached a high peak on the 6th to 9th day after stimulation. Under the condition of allogeneic antigen stimulation, the expression of CD96 on NK cells was also up-regulated.
  • CD96 belongs to the immunoglobulin superfamily, the extracellular domain contains three immunoglobulin-like domains, a total of 15 N-linked glycosylation sites, and a high O-linked glycosylation rich in silk/su
  • the stem domain of the /proline residue has 24 amino acid residues in the transmembrane region, 44 amino acid residues in the cytoplasmic region, and a basic/proline-rich region.
  • CD96 is also expressed in normal human CD4 + T cells, CD8 + T cells, monocytes and NK cells. After PHA stimulation, CD96 expression was significantly up-regulated on CD4 + T cells, CD8 + T cells and NK cells, confirming that CD96 is a molecule with increased expression of activation. However, the up-regulation of PHE on the expression of CD96 in monocytes was not very obvious, suggesting that the expression regulation of CD96 on different cells has different characteristics. Until 2004, Fuchs and colleagues found that NK cells can recognize PVR (CD155) through CD96, promote the adhesion of NK cells to CD155-target cells, promote the cytotoxic activity of NK cells and mediate the internalization of CD155 on the surface of target cells. .
  • PVR CD155
  • CD96 Since PVR (CD155) is highly expressed in certain tumor cells, this receptor system may play an important role in the recognition and killing of tumors by NK cells. However, the understanding of CD96 expression and function to date is still very limited. I believe that with the continuous understanding of the function of the molecule, CD96 will certainly attract more attention.
  • the cytoplasmic domain of CD96 has an immunoreceptor tyrosine inhibition motif (ITIM), which is highly conserved among species. It is generally believed that ITIM is involved in suppressing signal transmission. Recently, it has been found that certain molecules can transmit activation signals through ITIM. The receptors containing ITIM motifs play a key role in the activation signal transmission. Therefore, CD96 may play a key role. Mediates the inhibition of NK cell activity. In addition, CD96 has a certain homology with the CD226 sequence. According to current research, the two inhibit or activate NK cells by binding to CD155, respectively, to achieve the function of regulating NK cell function.
  • ITIM immunoreceptor tyrosine inhibition motif
  • CD96 antibody molecules have been reported to slow immunosuppression in animals, such as CN105636983, CN105636985, etc., or EP2097535 reports that CD96 + AMCSC cells are cleared by CD96 antibody molecules. No specific CD96 antibody molecules have been reported, and no CD96 antibody molecules have been found. Enter the clinical study. Therefore, it is necessary to develop a CD96 antibody with high affinity for a specific epitope to study the effects of CD96 and CD96 antibody drugs.
  • the present disclosure provides monoclonal antibodies or antigen-binding fragments (also referred to as CD96 binding molecules) that specifically bind to the amino acid sequence or three-dimensional structure of CD96.
  • the disclosure provides a monoclonal antibody or antigen-binding fragment thereof, which specifically binds to human CD96, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region ,among them:
  • the heavy chain variable region comprises a HCDR variant selected from the group consisting of a 3, 2, 1 or 0 amino acid mutation, respectively, based on the HCDR1, HCDR2 and HCDR3 regions set forth in SEQ ID NOs: 16, 17 and 18, respectively.
  • the light chain variable region comprises an LCDR variant selected from the group consisting of the LCDR1, LCDR2 and LCDR3 regions shown in SEQ ID NOs: 19, 20 and 21, respectively, having 3, 2, 1 or 0 amino acid mutations;
  • the heavy chain variable region comprises a HCDR variant selected from the group consisting of the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 22, 23 and 24, respectively, having 3, 2, 1 or 0 amino acid mutations, respectively.
  • the light chain variable region comprises an LCDR variant selected from the group consisting of the LCDR1, LCDR2 and LCDR3 regions shown in SEQ ID NOS: 25, 26 and 27, respectively, having 3, 2, 1 or 0 amino acid mutations;
  • the heavy chain variable region comprises a HCDR variant selected from the group consisting of the HCDR1, HCDR2 and HCDR3 regions shown in SEQ ID NOs: 28, 29 and 30, respectively, having 3, 2, 1 or 0 amino acid mutations, respectively.
  • a light chain variable region comprising an LCDR variant selected from the group consisting of 3, 2, 1 or 0 amino acid mutations in the LCDR1, LCDR2 and LCDR3 regions respectively as set forth in SEQ ID NOs: 31, 32 and 33;
  • the heavy chain variable region comprises a HCDR variant selected from the group consisting of the 3, 2, 1 or 0 amino acid mutations in the HCDR1, HCDR2 and HCDR3 regions, respectively, as set forth in SEQ ID NOs: 34, 35 and 36, respectively.
  • the chain variable region comprises an LCDR variant selected from the group consisting of the LCDR1, LCDR2 and LCDR3 regions shown in SEQ ID NOS: 37, 38 and 39, respectively, having 3, 2, 1 or 0 amino acid mutations;
  • the heavy chain variable region comprises a HCDR variant selected from the group consisting of the 3, 2, 1 or 0 amino acid mutations in the HCDR1, HCDR2 and HCDR3 regions, respectively, as set forth in SEQ ID NOs: 40, 41 and 42 respectively.
  • the chain variable region comprises an LCDR variant selected from the group consisting of the LCDR1, LCDR2 and LCDR3 regions shown in SEQ ID NOS: 43, 44 and 45, respectively, having 3, 2, 1 or 0 amino acid mutations.
  • the amino acid mutation is preferably an amino acid substitution, more preferably a conservative substitution of an amino acid.
  • the CDR variants of the CDRs are screened by affinity maturation methods CDR variants with 3, 2 or 1 amino acid mutations obtained.
  • the monoclonal antibody or antigen-binding fragment has an affinity (KD) to CD96 of less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, or less than 10 -11 M.
  • the monoclonal antibody or antigen-binding fragment thereof specifically binds to human CD96, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions set forth in the amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, and the light chain variable region comprises amino acids as SEQ ID NOs: 52, 20 and 21, respectively.
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions set forth in the amino acid sequences of SEQ ID NO: 22, 23 and 64, respectively, and the light chain variable region comprises amino acids as SEQ ID NOs: 25, 26 and 27, respectively.
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions set forth in the amino acid sequences of SEQ ID NOs: 28, 29 and 30, respectively, and the light chain variable region comprises amino acids as set forth in SEQ ID NO: 31, 32 and 33, respectively.
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions set forth in the amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, and the light chain variable region comprises amino acids as SEQ ID NOs: 37, 38 and 39, respectively.
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions set forth in the amino acid sequences of SEQ ID NO: 40, 119 and 42 respectively, and the light chain variable region comprises amino acids as SEQ ID NOs: 43, 44 and 45, respectively.
  • the monoclonal antibody or antigen-binding fragment is a recombinant antibody, preferably selected from the group consisting of a murine antibody, a chimeric antibody, and a humanized antibody.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable region, a heavy chain variable region or a light chain variable region and a heavy chain variable region selected from the group consisting of:
  • the heavy chain variable region of any of SEQ ID NOS: 6, 46, 49, 50, and 51, or variants and/or sequences thereof, is SEQ ID NOs: 7, 47, 48, 53, 54 a light chain variable region of any of 55, 56, 57 and 58 or a variant thereof; or
  • the heavy chain variable region of any of SEQ ID NOS: 8, 59, 62, 63, 65, 66, 67, 68, 69 and 70, or variants and/or sequences thereof, is SEQ ID NO: a light chain variable region of any of 9, 60 and 61 or a variant thereof; or
  • the heavy chain variable region of SEQ ID NO: 10 or a variant and/or sequence thereof is the light chain variable region of SEQ ID NO: 11 or a variant thereof;
  • the heavy chain variable region of any of SEQ ID NOS: 12, 71, 75, 76 and 77, or variants and/or sequences thereof, is any of SEQ ID NOs: 13, 72, 73 and 74 a light chain variable region or variant thereof; or
  • the heavy chain variable region of any of SEQ ID NOS: 14, 78, 82, 83, 84, 122 and 123, or variants and/or sequences thereof, is SEQ ID NOs: 15, 79, 80 and a light chain variable region of any of 81; or a variant thereof;
  • variants in a to e have 1, 2, 3, 4, 5, 6, 7 in the sequence of the framework region of the light chain variable region or the heavy chain variable region , 8, 9, or 10 amino acid mutations.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of:
  • variants in a to e have 1, 2, 3, 4, 5, 6, 7 in the sequence of the framework region of the light chain variable region or the heavy chain variable region , 8, 9, or 10 amino acid mutations.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a variable region selected from the group consisting of:
  • variants in a to e have 1, 2, 3, 4, 5, 6, 7 in the sequence of the framework region of the light chain variable region or the heavy chain variable region A mutation of 8, 8 or 10 amino acids which is preferably a back mutation of the amino acid in the framework region.
  • the antibody or antigen-binding fragment comprises:
  • a heavy chain variable region selected from the group consisting of any one of SEQ ID NOs: 6, 46, 49, 50, and 51 and the sequences are SEQ ID NOs: 7, 47, 48, 53, 54, 55, 56, a light chain variable region as shown in any of 57 and 58; or
  • a heavy chain variable region selected from any of the sequences SEQ ID NO: 8, 59, 62, 63, 65, 66, 67, 68, 69 and 70 and the sequence is SEQ ID NO: 9, 60 and a light chain variable region as shown in any of 61; or
  • a heavy chain variable region selected from any of SEQ ID NOS: 12, 71, 75, 76 and 77 and a light chain of any of SEQ ID NOS: 13, 72, 73 and 74 Variable zone; or
  • a heavy chain variable region selected from any of the sequences SEQ ID NO: 14, 78, 82, 83, 84, 122 and 123 and the sequence is any one of SEQ ID NOs: 15, 79, 80 and 81 The light chain variable region is shown.
  • the antibody is a full length antibody, further comprising a human antibody constant region, preferably comprising a human antibody heavy chain constant region set forth in SEQ ID NO: 117 and/or a human light as set forth in SEQ ID NO: 118 Chain constant region.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F (ab') 2, single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized An antigen-binding fragment of the V region (dsFv) and a peptide comprising a CDR.
  • the antibody or antigen-binding fragment binds to human CD96 with an affinity for a KD value of 1 x 10 -7 M to 1 x 10 -12 M as determined by surface plasmon resonance (BIACORE) technology.
  • the present disclosure also provides an isolated monoclonal antibody or antigen-binding fragment thereof which competes with the monoclonal antibody of (i)-(x) or a-j or an antigen-binding fragment thereof thereof for binding to human CD96.
  • the monoclonal antibody or antigen-binding fragment thereof has at least one of the following characteristics:
  • Iv. increase the activation of NK cells and / or T cells
  • the monoclonal antibody or antigen-binding fragment thereof binds to a region of the human CD96 extracellular region (SEQ ID NO: 3) as indicated by IAVYHPQYGFYCAYGRPCES.
  • the present disclosure also provides a multispecific antibody comprising a light chain variable region and/or a heavy chain variable region of an anti-CD96 antibody or antigen-binding fragment thereof as described above.
  • the present disclosure also provides a single-chain antibody comprising a light chain variable region and/or a heavy chain variable region of an anti-CD96 antibody or antigen-binding fragment thereof as described above.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof according to the above, the multispecific antibody or the single chain antibody, and One or more pharmaceutically acceptable carriers, diluents, buffers or excipients, preferably, the pharmaceutical composition may contain from 0.01 to 99% by weight of the anti-CD96 antibody or antigen-binding fragment thereof or The amount of the monoclonal antibody or antigen-binding fragment thereof contained in the unit dose of the pharmaceutical composition is from 0.1 to 2000 mg, more preferably from 1 to 1000 mg.
  • the present disclosure also provides an isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof, multi-specific antibody or single-chain antibody of any of the foregoing.
  • the present disclosure also provides a recombinant vector comprising the isolated nucleic acid molecule described above.
  • the present disclosure also provides a host cell transformed with the recombinant vector described above, the host cell being selected from the group consisting of a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.
  • the present disclosure provides a method for producing the monoclonal antibody or antigen-binding fragment thereof according to any of the preceding claims, which comprises culturing the aforementioned host cells in a medium to form and accumulate the foregoing The monoclonal antibody or antigen-binding fragment thereof, multi-specific antibody or single-chain antibody of any one, and the monoclonal antibody or antigen-binding fragment thereof recovered from the culture.
  • the present disclosure also provides a method for detecting or determining human CD96 in vitro, the method comprising using the monoclonal antibody or antigen-binding fragment thereof, multi-specific antibody or single-chain antibody of any of the foregoing.
  • the present disclosure provides the use of the monoclonal antibody or antigen-binding fragment thereof, multi-specific antibody or single-chain antibody of any of the foregoing, in the preparation of a reagent for detecting or determining human CD96.
  • the present disclosure also provides a method of reducing or alleviating immunosuppression, the method comprising administering to a subject a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof, multispecific antibody of any of the foregoing Or a single chain antibody, or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above.
  • the present disclosure also provides a method of enhancing NK cell activity, comprising using the monoclonal antibody or antigen-binding fragment thereof, multi-specific antibody or single-chain antibody of any of the foregoing, or comprising the aforementioned
  • the pharmaceutical composition, or the aforementioned isolated nucleic acid molecule reduces the activity of CD96.
  • the present disclosure also provides a monoclonal antibody or antigen-binding fragment thereof, a multispecific antibody or a single chain antibody, or a pharmaceutical composition comprising the same, or the isolated nucleic acid molecule as described above, using any of the foregoing The use of a medicament for enhancing the activity of NK cells.
  • the present disclosure also provides a method of treating a disease associated with human CD96, the method comprising administering to a subject a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof, multispecific An antibody or single chain antibody, or a pharmaceutical composition as described above, or an isolated nucleic acid molecule as described above, for treating a human CD96-associated disease, preferably a tumor, cancer or infectious disease.
  • the present disclosure provides the monoclonal antibody or antigen-binding fragment thereof, multi-specific antibody or single-chain antibody, or the pharmaceutical composition comprising the same, or the isolated nucleic acid molecule of the foregoing or Use in combination with a medicament for the treatment or prevention of a disease or condition, wherein the disease or condition is a human CD96-associated disease, preferably a tumor, cancer or infectious disease.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof according to any of the preceding, a multispecific antibody or a single-chain antibody as described above, or a pharmaceutical composition as described above, Or an isolated nucleic acid molecule as described above.
  • the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof according to any of the preceding, a multispecific antibody or a single-chain antibody as described above, or a pharmaceutical composition as described above, Or an isolated nucleic acid molecule as described above, wherein the medicament is for the treatment of a human CD96-associated disease, preferably a tumor, a cancer or an infectious disease.
  • the cancer can be any cancer that at least partially blocks CD96-mediated immunosuppression, inhibition or peripheral tolerance response.
  • cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy. More specific examples of such cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma.
  • non-Hodgkin's lymphoma diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), T-cell/tissue-rich cells Large B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (canal) cancer, kidney cancer, ovarian cancer, liver cancer, Lymphocytic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancre
  • a disease or condition that is responsive to at least partial blocking of CD96-mediated immunosuppression, suppression or peripheral tolerance is any disease or condition that enhances or restores immune surveillance to benefit a subject having the disease or condition.
  • the diseases and conditions may include persistent diseases or conditions that can be controlled or suppressed by cell-mediated immunity.
  • Non-limiting examples include tumors, cancer, or infectious diseases.
  • examples of cancers contemplated by the present disclosure include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy.
  • cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma.
  • NSCLC non-small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • glioma Hodgkin's lymphoma.
  • non-Hodgkin's lymphoma diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small lymphocytic lymphoma (SLL), T-cell/tissue-rich cells Large B-cell lymphoma, multiple myeloma, myeloid leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), gastrointestinal (canal) cancer, kidney cancer, ovarian cancer, liver cancer, Lymphocytic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancre
  • Infectious diseases are referred to as including all diseases caused by infection with a virus or pathogen and which can be infected by respiratory organs, blood contact or skin contact.
  • infectious diseases include, but are not limited to, hepatitis B, hepatitis C, human papillomavirus (HPV) infection, cytomegalovirus infection, viral respiratory disease, influenza, EBV (EBV) infection.
  • HPV human papillomavirus
  • cytomegalovirus infection viral respiratory disease
  • influenza EBV
  • EBV EBV
  • HSV herpes simplex virus
  • VSV varicella zoster virus
  • CMV cytomegalovirus
  • the CD96 monoclonal antibody or antigen-binding fragment of the present disclosure has high specificity and high affinity with CD96, and the immunogenicity of the humanized antibody is greatly reduced, while completely retaining the specificity, high affinity and activity of the murine antibody.
  • Figure 1 CD96 humanized antibody blocks hCD96-CHOs cell binding assay.
  • Figure 2 CD96 humanized antibody blocks hCD96 antigen and hCD155-CHOs cell binding experiments.
  • Figure 3 Antibody competition experiments.
  • Figure 3A coated antibody (A antibody) is h1718-012;
  • Figure 3B coated antibody is h1719-014;
  • Figure 3C coated antibody is h1721-003;
  • Figure 3D coated antibody is ch1720;
  • Figure 3E coated antibody is h1722- 010.
  • Figure 4 CD96-CHOs cell binding assay (FACS detection) of CD96 chimeric antibody/peptide complex
  • Figure 4A shows the results of ch1718 and 3# peptide detection
  • Figure 4B shows the detection results of ch1719 and 3# peptide
  • Figure 4C For the results of ch1720 and different peptides
  • Figure 4D shows the results of ch1721 and different peptides.
  • an “antibody” as used in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each class Ig of the five classes of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain of the present disclosure may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the present disclosure may further comprise a heavy chain constant region comprising human or murine IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in number and position.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
  • murine antibody in this disclosure is a monoclonal antibody to human CD96 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the CD96 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine CD96 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprising a murine IgG1, IgG2 The heavy chain constant region of IgG3 or a variant thereof.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell, and the variable region gene of the human antibody is cloned as needed, and the murine variable region gene is cloned.
  • the human constant region gene is ligated into a chimeric gene, inserted into an expression vector, and finally expressed in a eukaryotic or prokaryotic system.
  • the antibody light chain of the CD96 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the CD96 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain constant region, or comprising an amino acid mutation
  • An IgGl, IgG2 or IgG4 heavy chain constant region variant (such as a YTE mutation or a back mutation).
  • humanized antibody refers to an antibody produced by engrafting a murine CDR sequence into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. It is possible to overcome the heterologous reaction induced by the chimeric antibody by carrying a large amount of the mouse protein component.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be obtained from the "VBase" human germline sequence database (Internet www.mrccpe.com.ac.uk/vbase), as well as at Kabat, EA et al. , 1991Sequences of Proteins of Immunological Interest, found in the 5th edition.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further affinity matured by phage display.
  • the CDR sequence of the mouse in the CD96 humanized antibody is selected from the group consisting of SEQ ID NOs: 16-21, 22-27, 28-33, 34-39 or 40-45;
  • the antibody variable region framework is designed to be selected, wherein the heavy chain FR region sequence on the antibody heavy chain variable region is derived from a human germline heavy chain sequence, and a human germline light chain sequence.
  • the human antibody variable region can be subjected to minimal reverse mutation (reversion mutation, that is, the amino acid residue of the FR region derived from the human antibody is mutated to the original antibody. Corresponding position amino acid residues) to maintain activity.
  • minimal reverse mutation that is, the amino acid residue of the FR region derived from the human antibody is mutated to the original antibody. Corresponding position amino acid residues
  • the CDR graft can attenuate the affinity of the CD96 antibody or antigen-binding fragment thereof to the antigen due to changes in the framework residues that are contacted with the antigen. Such interactions can be the result of high mutations in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids to the framework of humanized antibodies. Amino acid residues involved in antigen binding from a non-human CD96 antibody or antigen-binding fragment thereof can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be related.
  • the sequence can be compared to a subtype consensus sequence or a consensus sequence of a murine sequence with a high percent similarity.
  • Rare framework residues are thought to be the result of high somatic mutations and thus play an important role in binding.
  • CDR variants like CDR1, CDR2 and CDR3 as shown in SEQ ID NO: X, SEQ ID NO: Y and SEQ ID NO: Z, respectively, "with 3, 2, 1 or 0 amino acid mutations, respectively"
  • An exemplary explanation is that a mutation to a CDR may comprise a mutation of 3, 2, 1 or 0 amino acids, and optionally the same or a different number of amino acid residues may be selected between the CDR1, CDR2 and CDR3 for mutation.
  • CDR1 is mutated by 1 amino acid
  • CDR2 and CDR3 are subjected to 0 amino acids. mutation.
  • a 0 amino acid mutation is made to a certain CDR, then the CDR variant with a 0 amino acid mutation remains the CDR itself.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, CD96). It has been shown that fragments of full length antibodies can be utilized to achieve antigen binding function of antibodies.
  • binding fragment contained in the term "antigen-binding fragment" of an antibody examples include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab') 2 fragment, including a divalent fragment of two Fab fragments joined by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of a VH and CH1 domain; (iv) an Fv fragment consisting of a single arm VH and VL domain of the antibody (v) a single domain or dAb fragment (Ward et al, (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) A combination of two or more separate CDRs, optionally joined by a synthetic linker.
  • CDR complementarity determining region
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined by a synthetic linker using a recombinant method such that they are capable of producing a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chains referred to as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • the antigen binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Antigen-binding fragments of the present disclosure include Fab, F(ab')2, Fab', single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv), inclusion Peptides of CDRs, etc.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity in a fragment obtained by treating an IgG antibody molecule with a protease papain (cleaving an amino acid residue at position 224 of the H chain), wherein the N-terminal side of the H chain About half of the entire L chain is bound by a disulfide bond.
  • the Fab of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure which specifically recognizes human CD96 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with papain. Furthermore, the Fab can be produced by inserting a DNA encoding a Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express a Fab.
  • F(ab')2 is an antibody obtained by digesting the lower portion of two disulfide bonds in the IgG hinge region with an enzyme pepsin, having an molecular weight of about 100,000 and having antigen-binding activity and comprising two Fab regions linked at the hinge position. Fragment.
  • the F(ab')2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure which specifically recognizes human CD96 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof with pepsin. Further, the F(ab') 2 can be produced by linking the Fab' described below with a thioether bond or a disulfide bond.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of the above F(ab')2.
  • the Fab' of the present disclosure can be produced by treating F(ab')2 of the present disclosure which specifically recognizes CD96 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting a DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express Fab'.
  • single-chain antibody single-chain Fv
  • scFv single-chain Fv
  • scFv antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • scFv molecules can have the general structure: NH 2 -VL- linker -VH-COOH or NH 2 -VH- linker -VL-COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variant thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996). , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the scFv of the present disclosure can be produced by obtaining a cDNA encoding VH and VL of a monoclonal antibody that specifically recognizes human CD96 and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof, and constructs a DNA encoding scFv.
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express an scFv.
  • a diabody is an antibody fragment in which an scFv is dimerized, and is an antibody fragment having a bivalent antigen-binding activity.
  • the two antigens may be the same or different.
  • the diabody of the present disclosure can be produced by obtaining the cDNA encoding the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human CD96 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs a cDNA encoding scFv.
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector such that the amino acid sequence of the peptide linker is 8 residues or less in length, and then the expression vector is introduced into a prokaryote or eukaryote In order to express double antibodies.
  • dsFv is obtained by linking a polypeptide in which one of amino acid residues in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues.
  • the amino acid residue substituted with a cysteine residue can be selected based on a three-dimensional structure prediction of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).
  • the dsFv of the present disclosure can be produced by obtaining a cDNA encoding VH and VL of a monoclonal antibody which specifically recognizes human CD96 and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof, and constructs a DNA encoding dsFv
  • the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express dsFv.
  • a peptide comprising a CDR is constructed by one or more regions of a CDR comprising a VH or VL. Peptides comprising a plurality of CDRs can be joined directly or via a suitable peptide linker.
  • the CDR-containing peptide of the present disclosure can be produced by constructing the DNA encoding the CDRs of the VH and VL of the monoclonal antibody of the present disclosure which specifically recognizes human CD96 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and the expression vector is then introduced into a prokaryote or eukaryote to express the peptide.
  • the CDR-containing peptide can also be produced by chemical synthesis methods such as the Fmoc method or the tBoc method.
  • antibody framework refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain that does not have a CDR.
  • amino acid mutation refers to a mutation between a polypeptide and its variant, between a certain amino acid position on a polypeptide fragment, wherein the variant may be substituted, inserted or Deletion of amino acids was obtained.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a CD96 molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refers to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 -8 M, such as less than about 10 -9 M, 10 - 10 M, 10 -11 M or less.
  • KD affinity
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • the antibodies of the present disclosure bind to CD96 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M, or 10 -10 M or less, for example, if a surface is used Plasma resonance (SPR) techniques were measured in a BIACORE instrument.
  • SPR Plasma resonance
  • an antigen binding protein that competes for the same epitope (eg, neutralizing an antigen binding protein or a neutralizing antibody or an antibody that specifically binds)
  • competition between antigen binding proteins is passed through
  • an antigen binding protein eg, an antibody or immunologically functional fragment thereof
  • a reference antigen binding protein eg, a ligand or reference antibody
  • a common antigen eg, a CD96 antigen or Specific binding of the fragment.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, eg, Stahli et al, 1983, Methods in Enzymology 9: 242-253
  • solid phase direct biotin-avidin EIA see, eg, Kirkland et al, 1986, J. Immunol.
  • solid Direct labeling assay solid phase direct label sandwich assay (see, eg, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling with I-125 label RIA (see, eg, Morel et al, 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, eg, Cheung, et al, 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol. 32: 77-82); or by the method of Test Example 7 of the present disclosure.
  • the assay involves the use of a purified antigen (either on a solid surface or on the cell surface) that binds to a reference antigen binding protein with an unlabeled detection antigen binding protein and label.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein to be tested.
  • competitive inhibition is determined by immobilizing antigen-binding protein A, detecting a change in the labeled antigen signal that is pre-bound to the antigen-binding protein.
  • this competitive inhibition assay confirms the location of antigen binding protein A and antigen binding protein B.
  • An antigen binding protein identified by a competitive assay includes: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and a table adjacent to an epitope that is sufficiently close to the binding of the reference antigen binding protein A binding antigen binding protein occurs where the two epitopes interfere with each other spatially.
  • the partially competitive antigen binding protein inhibits (eg, reduces) at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%).
  • Reference antigen binding protein specifically binds to a common antigen.
  • binding of the reference antigen binding protein to the antigen is inhibited by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or more.
  • a suppression of less than 30% is considered to be non-competitive.
  • the competitive antigen-binding protein as antigen-binding protein A or as antigen-binding protein B can inhibit the above-mentioned degree of binding of the reference antigen-binding protein, it is considered to be a competition or a part. competition.
  • amino acid conservative modification or “amino acid conservative substitution” or “conservative substitution of an amino acid” means that the amino acid in the protein is similarly characterized (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) Other amino acid substitutions allow for frequent changes without altering the biological activity of the protein or other desired properties (e.g., antigen affinity and/or specificity).
  • amino acid conservative modification or “amino acid conservative substitution” or “conservative substitution of an amino acid” means that the amino acid in the protein is similarly characterized (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) Other amino acid substitutions allow for frequent changes without altering the biological activity of the protein or other desired properties (e.g., antigen affinity and/or specificity).
  • a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al., (1987)
  • nucleic acid molecule refers to a DNA molecule and an RNA molecule.
  • the nucleic acid molecule may be single stranded or double stranded, but is preferably a double stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to the coding sequence if the promoter or enhancer affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the vector is a "plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (for example, a bacterial vector having an origin of replication of bacteria and an episomal mammalian vector) or can be integrated into the genome of the host cell after introduction into the host cell, thereby The host genome is replicated together (eg, a non-episomal mammalian vector).
  • a mouse can be immunized with human CD96 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include microorganisms (eg, bacteria), plant or animal cells.
  • Bacteria susceptible to transformation include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), HEK293 and NSO cells.
  • the antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human CD96. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • Remission of immunosuppression means at least partially eliminating, removing or overcoming the normal activity or function of CD96 by inhibiting or inhibiting the immune function of one or more cells that normally express CD96.
  • one or more cells that normally express CD96 are T cells, including CD4 + and CD8 + T cells, ⁇ cells, NKT cells, and natural killer (NK) cells.
  • ameliorating immunosuppression may include or involve abolishing a foreign pathogen, a host cell displaying a foreign pathogen (eg, displaying a foreign pathogen-derived polypeptide in autologous MHC), and/or peripheral tolerance of a cancer cell or tissue of the host .
  • blocking refers to the ability of an antibody or antigen-binding fragment thereof of the present disclosure to block, partially block, interfere with, reduce, inhibit, reduce or inactivate a target protein, ie, CD96 and/or its ligand. (eg CD155).
  • a target protein ie, CD96 and/or its ligand. (eg CD155).
  • blocking can encompass the loss of all and/or a portion of the activity of the ligand or receptor.
  • the activity of the ligand or receptor may be inhibited or inhibited by a compound that binds to the active site of the ligand/receptor protein, or by other means, such as inactivating a second protein that activates the inhibited first protein. .
  • total and/or partial inhibition of the interaction between CD96 and CD155 can be indicated by increased NK cell activation.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth.
  • cancer and “cancerous” refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth.
  • cancer and “cancerous” refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth.
  • cancer and “cancerous” refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth.
  • cancer cancer
  • cancer cancer
  • cancer cancer, “cancerous”, “cell proliferative disorder”, “proliferative disorder” and “tumor” are not mutually exclusive when referred to herein.
  • administering when applied to an animal, human, subject, cell, tissue, organ, or biological fluid, refers to an exogenous drug, therapeutic, diagnostic, or combination with an animal, Contact with a human, subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administering “administering,” and “treating” also means treating the cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure or a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, the patient having one or A variety of disease symptoms are known, and the therapeutic agents are known to have a therapeutic effect on these symptoms.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any clinically measured extent.
  • the amount of therapeutic agent (also referred to as "therapeutically effective amount") effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition.
  • any statistical test methods known in the art such as Student's t test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living being, cell or human, depending on the situation.
  • Endogenous refers to a substance produced in a cell, organism or human body, depending on the circumstances.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared x 100.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified.
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • the present disclosure includes an agent for treating a disease associated with CD96, which comprises the monoclonal antibody of the present disclosure or an antibody fragment thereof as an active ingredient.
  • the disease associated with CD96 there is no limitation on the disease associated with CD96 as long as it is a disease associated with CD96, for example, the molecularly induced therapeutic response using the present disclosure can be reduced by binding to human CD96 and then blocking the binding of CD96 to its ligand CD155. Inhibition of cells.
  • the molecules of the present disclosure are very useful for people with tumors, cancer or infectious diseases when in preparations and formulations suitable for therapeutic applications.
  • the present disclosure relates to a method for immunodetection or assay of CD96, a reagent for immunodetection or assay of CD96, a method for immunodetection or assay for cells expressing CD96, and a diagnostic reagent for diagnosing a disease associated with CD96, It comprises as an active ingredient a monoclonal antibody or antibody fragment of the present disclosure which specifically recognizes human CD96 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof.
  • the method for detecting or measuring the amount of CD96 may be any known method.
  • it includes immunodetection or assay methods.
  • the immunodetection or assay method is a method of detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody.
  • immunoassay or assay methods include radioactive substance labeling immunological antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, protein immunoblotting, physicochemical methods Wait.
  • the above CD96-associated diseases can be diagnosed by detecting or measuring cells expressing CD96 with the monoclonal antibodies or antibody fragments of the present disclosure.
  • a known immunodetection method can be used, and immunoprecipitation, fluorescent cell staining, immunohistochemical staining, or the like is preferably used. Further, a fluorescent antibody staining method or the like using the FMAT8100HTS system (Applied Biosystem) can be used.
  • a living sample for detecting or measuring CD96 is not particularly limited as long as it has a possibility of including cells expressing CD96, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, Tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody of the present disclosure or an antibody fragment thereof may further contain an agent for performing an antigen-antibody reaction or an agent for detecting a reaction, depending on a desired diagnostic method.
  • Agents for performing antigen-antibody reactions include buffers, salts, and the like.
  • the reagents for detection include reagents commonly used in immunoassays or assay methods, such as labeled secondary antibodies that recognize the monoclonal antibodies, antibody fragments or conjugates thereof, substrates corresponding to the labels, and the like.
  • the human CD96 protein (Uniprot No.: P40200-2) was used as a template for the CD96 of the present disclosure, and the amino acid sequences of the antigens and the detection proteins involved in the present disclosure were designed. Alternatively, different tags were fused on the CD96 protein and cloned into pHr. On the vector (self-produced) or on the pXC-17.4 vector (LONZA), the 293 cells are transiently expressed or the CHO cells are stably expressed and purified, and the antigen encoding the present invention and the protein for detection are obtained. The following CD96 antigens are not specifically described as human CD96.
  • the horizontal line is the signal peptide and the italic part is the Flag-tag label.
  • CD96-mFc Fusion protein of CD96 extracellular domain and mIgG2a Fc (SEQ ID NO: 2): CD96-mFc, for immunization
  • the cross-hatched portion is the signal peptide and the italicized portion is the mFc.
  • CD96 Full-length CD96 (SEQ ID NO: 3): used to construct CD96 overexpressing cell lines for immunization and detection
  • the underlined part is the signal peptide
  • the normal part is the extracellular domain
  • the italic part is the transmembrane and intracellular regions.
  • the horizontal line is the signal peptide and the italic part is the His tag.
  • Fusion protein of CD96 extracellular domain and hIgG1Fc (SEQ ID NO: 5): CD96-Fc, for detection
  • the cross-hatched portion is the signal peptide and the italicized portion is the Fc.
  • the sample was centrifuged at high speed to remove impurities and concentrated to an appropriate volume.
  • the flag affinity column was equilibrated using 0.5 x PBS and rinsed 2-5 column volumes.
  • the supernatant cells were subjected to supernatant analysis and the supernatant samples were applied to the column.
  • the column was rinsed with 0.5 x PBS until the A280 reading dropped to baseline.
  • the column was washed with PBS containing 0.3 M NaCl, and the protein was washed and collected.
  • the target protein was eluted with 0.1 M acetic acid (pH 3-4) or Flag peptide solution, and the elution peak of the desired product was collected to adjust the pH to neutral.
  • the collected eluted product was concentrated and further purified by, for example, gel chromatography Superdex 200 (GE), the mobile phase was PBS, the polymer and the heteroprotein peak were removed, and the elution peak of the desired product was collected.
  • the obtained protein was identified by electrophoresis, peptide mapping, LC-MS as correct and sub-equipment.
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, and the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM.
  • the nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed 2-5 column volumes. The displaced supernatant sample is combined on the column, and the medium can be selected from nickel columns of different companies.
  • the column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline.
  • the column was washed with PBS + 10 mM imidazole, the non-specifically bound heteroprotein was removed, and the effluent was collected.
  • the protein of interest was eluted with PBS containing 300 mM imidazole, and the eluted peak was collected.
  • the collected eluted product was concentrated and cocoa was further purified by gel chromatography Superdex 200 (GE), the mobile phase was PBS, the polymer and the heteroprotein peak were removed, and the elution peak of the desired product was collected.
  • the obtained protein was identified by electrophoresis, peptide mapping, LC-MS as correct and sub-equipment.
  • Hybridoma supernatant purification/ProteinG affinity chromatography purification of recombinant antibody and Fc fusion protein
  • ProteinG was selected for affinity purification of mouse hybridoma supernatant, and the cultured hybridoma was centrifuged to obtain a supernatant, and 10-15% by volume of 1 M Tris-HCl (pH 8.0-8.5) was added according to the supernatant volume. Clear pH.
  • the ProteinG column is washed with 3-5 times column volume using 6M hydrochloric acid, and then washed with pure water for 3-5 times column volume; using a buffer solution such as 1 ⁇ PBS (pH 7.4) as an equilibration buffer to equilibrate the column 3-5 Column volume; cell supernatant using low flow rate loading, control flow rate to maintain retention time of about 1min or longer; wash the column with 3-5 times column volume using 1 ⁇ PBS (pH 7.4) until UV absorption falls back to Baseline; sample elution with 0.1 M acetic acid/sodium acetate (pH 3.0) buffer, and elution peaks were collected according to UV detection.
  • a buffer solution such as 1 ⁇ PBS (pH 7.4) as an equilibration buffer to equilibrate the column 3-5 Column volume; cell supernatant using low flow rate loading, control flow rate to maintain retention time of about 1min or longer; wash the column with 3-5 times column volume using 1 ⁇ PBS (pH 7.
  • solution displacement can be carried out by methods well known to those skilled in the art, such as ultrafiltration filtration using ultrafiltration tubes and solution displacement to a desired buffer system, or replacement with a molecular exclusion such as G-25 desalting.
  • the cell culture supernatant expressing the Fc fusion protein or antibody is first subjected to high-speed centrifugation to collect the supernatant.
  • the ProteinA affinity column was washed with 3-5 column volumes using 6 M hydrochloric acid, and then washed 3-5 times column volume with pure water.
  • the column was equilibrated with 3-5 column volumes using a buffer solution such as 1 x PBS (pH 7.4) as an equilibration buffer.
  • the cell supernatant was loaded with a low flow rate, and the flow rate was controlled so that the retention time was about 1 min or longer.
  • the column was washed 3-5 times with 1 ⁇ PBS (pH 7.4) until the UV absorption dropped back to the baseline. .
  • the sample was eluted with 0.1 M acetic acid/sodium acetate (pH 3.0-3.5) buffer, and the elution peak was collected according to the ultraviolet detection.
  • the eluted product was quickly adjusted to pH 5-6 by using 1 M Tris-HCl (pH 8.0). Save.
  • solution displacement can be carried out by methods well known to those skilled in the art, such as ultrafiltration filtration using ultrafiltration tubes and solution displacement to a desired buffer system, or replacement with a molecular exclusion such as G-25 desalting.
  • Anti-human CD96 monoclonal antibodies are produced by immunizing mice.
  • Experimental SJL white mice, female, 6-8 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Feeding environment SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. Mice that have adapted to the environment are immunized according to the following protocol.
  • the immunizing antigen is the human CD96 extracellular domain with Flag tag or mFc (SEQ ID NO: 1 or 2), and the CHO cell line overexpressing human CD96.
  • Immunization program A use Gold Adjuvant (Sigma Cat No. T2684) and Thermo Alum (Thermo Cat No. 77161) adjuvant cross-immunization.
  • the antigen was emulsified and inoculated for 0, 14, 28, 42, and 56 days. On day 0, 50 ⁇ g/cell of emulsified antigen was injected intraperitoneally (IP).
  • IP intraperitoneally
  • Immunization Protocol B Mice were immunized with Quick Antibody-Mouse 5W (KX0210041) adjuvant. The ratio of antigen to adjuvant was 1:1, 25 ⁇ g/mouse/time (first exempt/enhanced immunization). The antigen and the adjuvant were quickly and thoroughly mixed and inoculated for 0, 21, and 35 days. On day 0, the mouse calf muscle (IM) was injected with 25 ⁇ g/antigen. On the 21st and 35th day, 25 ⁇ g/mouse was injected in the same manner (depending on the titer, whether or not the third exemption was performed).
  • IM mouse calf muscle
  • Spleen lymphocytes and myeloma cell Sp2/0 cells were optimized using an optimized PEG-mediated fusion step ( CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • the fused hybridoma cells were resuspended in complete medium (DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1 ⁇ 10 ⁇ 6/ml, and 100 ⁇ l/well in 96 wells. After incubating for 3-4 days at 37 ° C, 5% CO 2 in the plate, HAT complete medium was supplemented with 100 ⁇ l/well, and culture was continued for 3-4 days until a needle-like clone was formed.
  • complete medium DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI
  • HT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI was added, and cultured at 37 ° C, 5% CO 2 for 3 days, and then subjected to ELISA.
  • Hybridoma culture supernatants were detected by binding ELISA according to the growth density of hybridoma cells.
  • the positive well cell supernatants detected by ELISA were combined for cell binding assay and cell blocking assay.
  • the well cells that were positive for both binding and blocking were expanded in a timely manner for cryopreservation and two to three subclones until a single cell clone was obtained.
  • the hybridoma clones were screened by the above experiments, and the antibody was further prepared by serum-free cell culture, and the antibody was purified according to the purification example (Example 3, item 3) for use in the test examples.
  • the sequence of cloning from a positive hybridoma is as follows. Hybridoma cells in logarithmic growth phase were collected, using Trizol (Invitrogen, Cat No.15596-018) according to kit instructions an RNA extraction step, with PrimeScript TM Reverse Transcriptase reverse transcription kit (Takara, Cat No.2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer kit (Novagen, TB326 Rev. B 0503), and sent to a sequencing company for sequencing.
  • amino acid sequences corresponding to the heavy and light chain variable region DNA sequences of the murine antibodies m1718, m1719, m1720, m1721, and m1722 were obtained (the amino acid residues of the CDRs in VH/VL were identified and annotated by the Kabat numbering system, and the underlined sequence For CDR):
  • the light heavy chain variable region of the murine antibody is ligated to the light chain constant region (SEQ ID NO: 117) and the heavy chain constant region (SEQ ID NO: 118) of the human antibody to form a chimeric antibody, corresponding to the m1718 antibody.
  • the chimeric antibody was named ch1718, and other antibodies were analogous.
  • the heavy chain and light chain variable region germline genes with high homology to the murine antibody were selected as templates, and the murine antibody was
  • the CDRs are each grafted into the corresponding human template to form a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the backbone sequence are back-mutated to the amino acids corresponding to the murine antibody to ensure the original affinity, that is, the humanized anti-CD96 monoclonal antibody is obtained.
  • the amino acid residues in the back mutation are determined by the natural number and annotated.
  • the humanized light chain template of the murine antibody m1718 was IGKV1-16*01 and hjk4.1, and the humanized heavy chain template was IGHV1-3*01 and hjh6.1. After CDR graft, the humanized antibody h1718- was obtained. 001, the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • the NN of the CDR1 of h1718 series humanized VL was mutated to NQ, NT, NL to increase the stability of the antibody.
  • the h1718VL CDR1 sequence has the general formula KASQDINX 1 YLN (SEQ ID NO: 52), wherein X 1 is selected from the group consisting of N, T, L and Q.
  • Specific h1718VL CDR1 mutants include KASQDINQYLN (SEQ ID NO: 108), KASQDINTYLN (SEQ ID NO: 109), KASQDINLYLN (SEQ ID NO: 110).
  • h1718-VL1 can be changed to the following light chain variable region sequences:
  • h1718-VL2 can be changed to the following light chain variable region sequences:
  • the h1718 series humanized antibody variable region sequence combinations are as follows:
  • the humanized light chain template of the murine antibody m1719 was IGKV1-12*01 and hjk4.1, and the humanized heavy chain template was IGHV2-26*01 and hjh6.1. After CDR grafting, the humanized antibody h1719- was obtained.
  • the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • A43S indicates that the 43-position A is mutated back to S according to the natural sequence of the amino acid sequence. Grafted represents the murine antibody CDRs implanted into the human germline FR region sequence.
  • This table represents the sequence obtained by various combinations of mutations. As indicated by h1719-005, the light chain variable region h1719-VL2 and the heavy chain variable region h1719-VH2 were contained on the humanized antibody h1719-005. Other analogies.
  • the NN in h1719VH CDR3 was mutated to QN, NY, NQ, and M was mutated to L or I to increase the stability of the antibody.
  • h1719VH The CDR3 sequence is of the formula X 2 X 3 YYGSRYGYPX 4 DY (SEQ ID NO: 64), wherein X2 is selected from the group consisting of: N and Q, X3 is selected from the group consisting of N, Y and Q, and X4 is selected from the group consisting of M, L and I.
  • Specific h1719VH CDR3 mutants can include, but are not limited to, QNYYGSRYGYPLDY (SEQ ID NO: 111), NYYYGSRYGYPLDY (SEQ ID NO: 112), NQYYGSRYGYPLDY (SEQ ID NO: 113), QNYYGSRYGYPIDY (SEQ ID NO: 114), NYYYGSRYGYPIDY (SEQ) ID NO: 115), NQYYGSRYGYPIDY (SEQ ID NO: 116).
  • h1719-VH3 can be changed to the following heavy chain variable region sequences:
  • the h1719 series humanized antibody variable region sequence combinations are as follows:
  • the humanized light chain template of the murine antibody m1721 was IGKV1-39*01 and hjk4.1, and the humanized heavy chain template was IGHV2-26*01 and hjh6.1.
  • the humanized antibody h1721- was obtained by CDR graft. 001, the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • the humanized antibody h1721-006 comprises a light chain variable region h1721-VL2 and a heavy chain variable region h1721-VH2. Other analogies.
  • the humanized light chain template of the murine antibody m1722 was IGKV1-39*01 and hjk2.1, and the humanized heavy chain template was IGHV1-69*02 and hjh4.1.
  • the humanized antibody h1722- was obtained by CDR graft. 001, the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • the humanized antibody h1722-006 comprises a light chain variable region h1722-VL2 and a heavy chain variable region h1722-VH2. Other analogies.
  • variable region of the h1722 series humanized antibody is as follows:
  • h1722-VH CDR2 has an acetylated high-risk site NS
  • the sequence of h1722-VH CDR2 is MLHPX 5 SGTTSFNEKFKI (SEQ ID NO: 119), wherein X 5 is selected from N, K or Q.
  • mutants of the h1722-VH CDR2 include, but are not limited to, MLHPKSGTTSFNEKFKI (SEQ ID NO: 120) or MLHPQSGTTSFNEKFKI (SEQ ID NO: 121).
  • h1722-VH2 can be changed to the following heavy chain variable region sequences:
  • Each of the above heavy chain variable regions is recombinantly expressed with the heavy chain constant region sequence as set forth in SEQ ID NO: 117 to give the final intact heavy chain sequence.
  • Each of the above light chain variable regions is recombinantly expressed with a light chain constant region sequence as set forth in SEQ ID NO: 118 to give the final complete light chain sequence.
  • the above light chain variable region, heavy chain variable region can also be recombined and expressed with other alternatively humanized constant regions, and functionally modified humanized constant regions, using conventional techniques in the art.
  • monkey CD96-3 ⁇ Flag (SEQ ID NO: 95)
  • the underlined portion is the signal peptide and the italicized portion is 3 x Flag-tag. See the first item of Example 2 for the preparation method.
  • Test Example 1 ELISA assay of CD96 antibody binding to human CD96 protein
  • the binding capacity of the anti-CD96 antibody was detected by an ELISA assay of the antibody with human CD96 protein.
  • the His-tagged CD96 fusion protein was coated in the ELISA plate, and the signal intensity after the antibody was added was used to judge the binding activity of the antibody and CD96.
  • the specific experimental method is as follows.
  • the CD96-His of the sequence of Example 1 as shown in SEQ ID NO: 4 was diluted to a concentration of 2 ⁇ g/ml with a buffer of PBS (Shanghai Yuanpei Biotechnology Co., Ltd., Cat No. B320) at pH 7.4 to 50 ⁇ l/ The volume of the wells was placed in a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) and placed in an incubator at 37 ° C for 2 hours.
  • PBS Sthai Yuanpei Biotechnology Co., Ltd., Cat No. B320
  • a 5% skim milk (BD, 232100) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 2.5 hours or at 4 ° C overnight (16-18 hours) for blocking. After blocking, discard the blocking solution and wash the plate 4 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), then add 50 ⁇ l/well of different concentrations of the test antibody diluted with the sample dilution (hybridoma purified antibody). Or humanized antibody), incubated in a 37 ° C incubator for 1 hour.
  • PBST buffer pH 7.4 PBS containing 0.05% tween-20
  • Test Example 2 ELISA assay of CD96 antibody binding to cynomolgus CD96 protein
  • the cross-reactive binding of anti-CD96 antibodies to monkey CD96 was detected by ELISA assay of antibodies with cynomolgus CD96 protein.
  • the cynomolgus CD96 fusion protein with the 3 ⁇ FLAG tag was coated in the ELISA plate, and the signal intensity after the antibody was added was used to judge the binding activity of the antibody and the cynomolgus CD96.
  • the specific experimental method is as follows.
  • the amino acid sequence prepared in the above Example 4 was diluted with PBS of pH 7.4 (Shanghai Yuanpei Biotechnology Co., Ltd., Cat No. B320) to the cyno-CD96-3 ⁇ FLAG of SEQ ID NO: 95 to 2 ⁇ g/ The ml concentration was added to a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) in a volume of 50 ⁇ l/well, and placed in an incubator at 37 ° C for 2 hours.
  • a 5% skim milk (BD, 232100) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 2.5 hours or at 4 ° C overnight (16-18 hours) for blocking. After blocking, discard the blocking solution and wash the plate 4 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), then add 50 ⁇ l/well of different concentrations of the test antibody diluted with the sample dilution (hybridoma purified antibody). Or humanized antibody), incubated in a 37 ° C incubator for 1 hour.
  • PBST buffer pH 7.4 PBS containing 0.05% tween-20
  • Test Example 3 Binding experiment of CD96 antibody to human CD96 overexpressing CHO-s cells
  • the binding ability of the anti-CD96 antibody was detected by binding experiments of the antibody to CHO-S cells overexpressing the human CD96 protein.
  • the CD96 full-length plasmid was transfected into CHO-S cells by electroporation, and after two weeks of pressure screening, the expression level of CD96 was examined. After the overexpressing cells were fixed in the 96-well plate bottom, the signal intensity after the antibody was added was used to judge the binding activity of the antibody and CD96 overexpressing CHO-S cells, and the specific experimental methods were as follows.
  • the cells were seeded in 96-well plates at a density of 9 ⁇ 10 4 /100 ⁇ L/well overnight. The supernatant was aspirated, washed once with PBS, and then fixed in 100 ⁇ l/well immunostaining fixative (Biyuntian, P0098) for one hour at room temperature, and washed three times with PBS. After discarding the liquid, 250 ⁇ l/well of a 5% skim milk (BD, 232100) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 2.5 hours for blocking.
  • BD 5% skim milk
  • the blocking solution was discarded, and the plate was washed 4 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), and 50 ⁇ l/well of the antibody to be tested diluted with the sample diluent was added (hybridoma purification).
  • PBST buffer pH 7.4 PBS containing 0.05% tween-20
  • Antibody or humanized antibody incubated in a 37 ° C incubator for 2 hours. After the incubation, the plate was washed 4 times with PBST, and 50 ⁇ l/well of HRP-labeled goat anti-mouse secondary antibody diluted with sample dilution (Jackson Immuno Research, Cat No. 115-035-003) or goat anti-human secondary antibody (Jackson) was added.
  • Immuno Research Cat No.
  • Candidate antibody EC50(nM) Candidate antibody EC50(nM) Ch1718 0.026 H1718-012 0.031 Ch1719 0.012 H1719-003 0.028 Ch1720 0.036 H1719-006 0.016 Ch1721 0.036 H1719-014 0.02 Ch1722 0.043 H1721-003 0.074 H1722-005 0.045 H1722-006 0.043 H1722-010 0.047 H1722-017 0.053 H1722-018 0.044
  • the results show that the CD96 chimeric antibody and the humanized antibody are capable of binding to CD96 overexpressing CHO-S cells.
  • Test Example 4 Anti-CD96 antibody blocks CD96 antigen and CD155-CHOs cell binding assay
  • CD155-CHOs cells (Shanghai Hengrui construct) were seeded in 96-well culture plates at 9 ⁇ 10 4 /100 ⁇ L/well overnight, the supernatant was aspirated, washed once with PBS, and 100 ⁇ l/well immunostaining fixative was added. (Biyuntian, P0098) was fixed at room temperature for one hour and washed three times with PBS. After discarding the liquid, 250 ⁇ l/well of a 5% skim milk (BD, 232100) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 2.5 hours for blocking.
  • BD 5% skim milk
  • the NOVOStar microplate reader reads the absorbance at a wavelength of 450 nm and calculates the blocking effect of the CD96 antibody on the binding of the antigen to CD155-CHOs cells (see Figure 2 and Table 16).
  • Candidate antibody IC50(nM) Candidate antibody IC50(nM) Ch1718 0.096 H1718-012 0.133 Ch1719 0.137 H1719-003 0.096 Ch1720 0.149 H1719-006 0.064 Ch1721 0.214 H1719-014 0.046 Ch1722 0.159 H1721-003 0.155 H1722-006 0.398 H1722-010 0.207 H1722-017 0.135 H1722-018 0.134
  • the results show that the CD96 chimeric antibody and the humanized antibody are capable of blocking the binding of CD96 and CD155-CHOs cells.
  • Test Example 5 BIAcore detection of anti-CD96 antibody affinity test
  • the antibody to be tested was affinity-trapped with a Protein A biosensor chip, and then the CD96 antigen was passed through the surface of the chip, and the reaction signal was detected in real time using a Biacore instrument to obtain a binding and dissociation curve. After completion of the dissociation of each experimental cycle, the biosensor chip was washed and regenerated with a glycine-hydrochloric acid regeneration solution (pH 1.5).
  • the antibody to be tested was affinity-captured with a CM5 biosensor chip conjugated with an anti-murine Fc antibody, and then passed through the CD96 antigen on the surface of the chip, and the reaction signal was detected in real time using a Biacore instrument to obtain a binding and dissociation curve. After completion of the dissociation of each experimental cycle, the biochip was washed and regenerated using the regeneration solution in the mouse antibody capture kit.
  • the mobile phase is both CD96-ECD-His.
  • the anti-CD96 chimeric antibodies ch1718, ch1720, ch1721, ch1719, ch1722 have strong affinity with human CD96, and the exemplary humanized antibody also has a strong affinity with human CD96.
  • Antibody A was diluted to a concentration of 2 ⁇ g/mL with PBS pH 7.4 (Cat No. B320) and added to a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) at a volume of 50 ⁇ L/well. In the package, overnight at 4 ° C. After discarding the liquid, 250 ⁇ L/well of a 5% skim milk (BD skim milk, Cat No. 232100) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 3 hours in an incubator.
  • PBS pH 7.4 Cat No. B320
  • the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), and was used.
  • PBST buffer pH 7.4 PBS containing 0.05% tween-20
  • Test Example 7 ELISA experiment of anti-CD96 antibody binding to different polypeptide fragments of CD96 protein
  • the epitope recognized by the anti-CD96 antibody is detected by binding experiments of the antibody to different polypeptide fragments of the CD96 protein.
  • the peptides of different peptides of the CD96 antigen are coated in the ELISA plate, and the signal intensity after the antibody is added is used to determine the binding activity of the antibody and the polypeptide.
  • the specific experimental methods are as follows:
  • the polypeptide was diluted to a concentration of 20 ⁇ g/mL with PBS (Shanghai No. B320) buffer of pH 7.4, and added to a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) in a volume of 50 ⁇ L/well. , coated at 4 ° C overnight. After discarding the liquid, 250 ⁇ L/well of a 5% skim milk (BD skim milk, Cat No. 232100) blocking solution diluted with PBS was added, and the mixture was incubated at 37 ° C for 3 hours in an incubator.
  • PBS Sthai No. B320 buffer of pH 7.4
  • a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) in a volume of 50 ⁇ L/well. , coated at 4 ° C overnight.
  • the blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH 7.4 PBS containing 0.05% tween-20), and 50 ⁇ L/well of the antibody to be tested diluted with the sample diluent was added (hybridoma purified antibody). Or chimeric antibody), incubated in a 37 ° C incubator for 2 hours. After the incubation, the plate was washed 5 times with PBST, and 50 ⁇ L/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) or goat anti-human secondary antibody (Jackson) diluted with the sample dilution was added. Immuno Research, Cat No.
  • the antibodies ch1718, ch1720, ch1721, and ch1719 bind strongly to the 3# antigen fragment (SEQ ID NO: 87), and the 3# antigen fragment contains the epitopes of the antibodies ch1718, ch1720, ch1721, and ch1719.
  • Test Example 8 FACS experiment of CD96 antibody binding to different peptides of CD96
  • the results of the assay showed that the chimeric antibodies ch1721, ch1719, and ch1718 bind to the epitope segment shown by SEQ ID NO: 87 in the extracellular region of human CD96.
  • Test Example 9 NK cell-mediated cell killing function assay
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • WiDr human colorectal cancer cell line WiDr
  • the human colorectal cancer cell line WiDr was cultured in MEM medium, and 10% (v/v) fetal bovine serum (FBS) was added to the medium, and cultured at 37 ° C under 5% CO 2 .
  • Fresh blood was obtained by Ficoll-Hypaque density gradient centrifugation (Stem Cell Technologies) to obtain PBMC, and human primary NK cells were extracted from freshly isolated PBMC (Miltenyi, CAT#130-092-657) and cultured in RPMI 1640 medium. 10% (v/v) FBS was added to the medium, and cultured at 37 ° C under 5% CO 2 .
  • Human primary NK cells were seeded into a 6-well cell culture plate at a cell density of approximately 2 ⁇ 10 6 /ml.
  • the target cell WiDr was co-cultured with human primary NK cells at a ratio of 1:1, and cultured at 37 ° C, 5% CO 2 incubator for 4 h, and the cell culture supernatant was collected.
  • CytoTox The Non-Radioactive Cytotoxicity Assay (Promega, CAT# G1780) method detects the level of LDH secretion in the cell culture supernatant. Refer to the reagent instructions for specific operations.

Abstract

本公开提供CD96抗体、其抗原结合片段及医药用途。进一步地，本公开提供包含所述CD96抗体CDR区的鼠源抗体、嵌合抗体、人源化抗体，以及包含CD96抗体及其抗原结合片段的药物组合物，以及其作为药物的用途。特别地，本公开提供一种人源化的CD96抗体在制备用于治疗CD96相关的疾病或病症的药物中的用途。

Description

CD96抗体、其抗原结合片段及医药用途
本申请要求申请日为2017年11月10日的中国专利申请CN201711107331.5的优先权。本申请引用上述中国专利申请的全文。
技术领域
本公开涉及CD96抗体以及其抗原结合片段，进一步地，本公开还涉及包含所述CD96抗体CDR区的嵌合抗体、人源化抗体，本公开还涉及包含所述CD96抗体及其抗原结合片段的药物组合物，以及其作为CD96相关疾病诊断剂和治疗药物的用途。
背景技术
这里的陈述仅提供与本发明有关的背景信息，而不必然地构成现有技术。
1992年，Wang及其同事发现并且克隆了一种新的细胞膜型分子，当时将其命名为Tactile(T cell activation increased late expression)。随后，在人类白细胞分化抗原协作组会议上将该分子命名为CD96。CD96分子表达于正常T细胞、T细胞克隆以及某些转化的T细胞上。外周血T细胞表达低水平的CD96分子，活化后其表达明显上调，并在刺激后第6～9天表达达到高峰值。在同种异体抗原刺激的条件下，NK细胞上CD96的表达也发生上调。CD96属于免疫球蛋白超家族，胞膜外区包含3个免疫球蛋白样结构域，共有15个N-连接糖基化位点，还有1个高度O-连接糖基化富含丝/苏/脯氨酸残基的茎状结构域，跨膜区有24个氨基酸残基，胞质区含44个氨基酸残基，并有一个富含碱性/脯氨酸的区域。自克隆CD96分子后的十多年间，由于不清楚其配体，该分子的研究无明显进展。CD96在造血和非造血细胞系中表达十分广泛。CD96也表达于正常人CD4 +T细胞、CD8 +T细胞、单核细胞和NK细胞。在PHA刺激后，CD4 +T细胞、CD8 +T细胞和NK细胞上CD96表达明显上调，证实CD96是一种活化表达增加的分子。但PHA对单核细胞CD96的表达上调作用不十分明显，提示不同细胞上CD96分子的表达调节具有不同特点。直到2004年，Fuchs及其同事发现NK细胞可通过CD96识别PVR(CD155)，促进NK细胞对表达CD155靶细胞的黏附，促进NK细胞的细胞杀伤活性并可介导靶细胞表面上CD155的内化。由于PVR(CD155)高表达于某些肿瘤细胞，这一受体***可能在NK细胞对肿瘤的识别和杀伤中起重要作用。然而，迄今为止对CD96表达和功能的认识还十分有限。相信随着对该分子功能的不断认识，CD96必将会引起人们更多的关注。
CD96分子胞质区带有一个免疫受体酪氨酸抑制模体(ITIM)，其在种属间具有很高的保守性。一般认为ITIM参与抑制信号传递，近来研究发现某些分子可通过ITIM传递活化信号，包含ITIM模体的受体在活化信号传递中，SHP-2的募集起着关键性的作用，因此CD96可能会介导抑制NK细胞活性的作用。此外，CD96与CD226序列上有一定的同源性，根据目前的研究认为两者分别通过与CD155的结合来抑制或激活NK细胞，以达到调节NK细胞功能的作用。
截止目前，仅有如CN105636983，CN105636985等报道CD96抗体分子可在动物体内减缓免疫抑制，或EP2097535报道用CD96抗体分子清除CD96 +的AMLSC细胞，尚未发现有具体的CD96抗体分子报道，也没有CD96抗体分子进入临床研究。因此，有必要开发具有高亲和力，针对特定表位的CD96抗体，以研究CD96及CD96抗体药物的作用。
发明内容
本公开提供与CD96的氨基酸序列或三维结构特异性结合的单克隆抗体或抗原结合片段(也可称CD96结合分子)。
一方面，本公开提供一种单克隆抗体或其抗原结合片段，所述单克隆抗体或其抗原结合片段特异性结合人CD96，所述单克隆抗体包含重链可变区和轻链可变区，其中：
(i)重链可变区包含选自在分别如SEQ ID NO：16、17和18所示的HCDR1、HCDR2和HCDR3区基础上分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：19、20和21所示的LCDR1、LCDR2和LCDR3区基础上分别具有3，2，1或0个氨基酸突变的LCDR变体；或
(ii)重链可变区包含选自在分别如SEQ ID NO：22、23和24所示的HCDR1、HCDR2和HCDR3区基础上分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：25、26和27所示的LCDR1、LCDR2和LCDR3区基础上分别具有3，2，1或0个氨基酸突变的LCDR变体；或
(iii)重链可变区包含选自在分别如SEQ ID NO：28、29和30所示的HCDR1、HCDR2和HCDR3区基础上分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：31、32和33所示的LCDR1、LCDR2和LCDR3区分别具有3，2，1或0个氨基酸突变的LCDR变体；或
(iv)重链可变区包含选自在分别如SEQ ID NO：34、35和36所示的HCDR1、HCDR2和HCDR3区分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包 含选自在分别如SEQ ID NO：37、38和39所示的LCDR1、LCDR2和LCDR3区分别具有3，2，1或0个氨基酸突变的LCDR变体；或
(v)重链可变区包含选自在分别如SEQ ID NO：40、41和42所示的HCDR1、HCDR2和HCDR3区分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：43、44和45所示的LCDR1、LCDR2和LCDR3区分别具有3，2，1或0个氨基酸突变的LCDR变体。所述氨基酸突变优选氨基酸替代，更优选氨基酸的保守替代。
在一些实施方式中，所述单克隆抗体或抗原结合片段的CDR(包括3个重链CDR和3个轻链CDR)具有3，2或1个氨基酸突变的CDR变体是经亲和力成熟方法筛选获得的具有3，2或1个氨基酸突变的CDR变体。
在一些实施方式中，所述单克隆抗体或抗原结合片段与CD96的亲和力(KD)小于10 -7M、小于10 -8M、小于10 -9M、小于10 -10M或小于10 -11M。
在一些实施方式中，所述单克隆抗体或其抗原结合片段特异性结合人CD96，所述单克隆抗体包含重链可变区和轻链可变区，其中：
(vi)重链可变区包含分别如SEQ ID NO：16、17和18氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：52、20和21氨基酸序列所示的LCDR1、LCDR2和LCDR3区，其中所述LCDR1优选如SEQ ID NO：19、108、109或110任一所示LCDR1；或
(vii)重链可变区包含分别如SEQ ID NO：22、23和64氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：25、26和27氨基酸序列所示的LCDR1、LCDR2和LCDR3区，其中所述HCDR3优选如SEQ ID NO：24、111、112、113、114、115和116任一所示HCDR3；或
(viii)重链可变区包含分别如SEQ ID NO：28、29和30氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：31、32和33氨基酸序列所示的LCDR1、LCDR2和LCDR3区；或
(ix)重链可变区包含分别如SEQ ID NO：34、35和36氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：37、38和39氨基酸序列所示的LCDR1、LCDR2和LCDR3区；或
(x)重链可变区包含分别如SEQ ID NO：40、119和42氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：43、44和45氨基酸序列所示的LCDR1、LCDR2和LCDR3区，其中所述的HCDR2优选如SEQ ID NO：41、120 和121所示。
在一些实施方式中，所述单克隆抗体或抗原结合片段是重组抗体，优选选自鼠源抗体、嵌合抗体、人源化抗体。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，其包含选自如下轻链可变区、重链可变区或轻链可变区和重链可变区：
a.序列为SEQ ID NO:6、46、49、50和51任一所示的重链可变区或其变体和/或序列为SEQ ID NO：7、47、48、53、54、55、56、57和58任一所示的轻链可变区或其变体；或
b.序列为SEQ ID NO:8、59、62、63、65、66、67、68、69和70任一所示的重链可变区或其变体和/或序列为SEQ ID NO：9、60和61任一所示的轻链可变区或其变体；或
c.序列为SEQ ID NO:10所示的重链可变区或其变体和/或序列为SEQ ID NO:11所示的轻链可变区或其变体；
d.序列为SEQ ID NO:12、71、75、76和77任一所示的重链可变区或其变体和/或序列为SEQ ID NO：13、72、73和74任一所示的轻链可变区或其变体；或
e.序列为SEQ ID NO:14、78、82、83、84、122和123任一所示的重链可变区或其变体和/或序列为SEQ ID NO:15、79、80和81任一所示的轻链可变区或其变体；
其中a至e中所述变体是在所述轻链可变区或重链可变区的框架区序列上具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸突变。
在一些实施方式中，其中所述的单克隆抗体或其抗原结合片段，其包含选自如下重链链可变区：
(a)氨基酸序列如SEQ ID NO:6、46、49、50和51任一所示的重链可变区或其变体；
(b)氨基酸序列如SEQ ID NO:8、59、62、63、65、66、67、68、69和70任一所示的重链可变区或其变体；
(c)氨基酸序列如SEQ ID NO:10所示的重链可变区或其变体；
(d)氨基酸序列如SEQ ID NO:12、71、75、76和77任一所示的重链可变区或其变体；
(e)氨基酸序列如SEQ ID NO:14、78、82、83、84、122和123任一所示的重链可变区或其变体；
其中，a至e中所述变体是在所述轻链可变区或重链可变区的框架区序列上具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸突变。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段包含选自如下轻链链可变区：
(a)氨基酸序列如SEQ ID NO：7、47、48、53、54、55、56、57和58任一所示的轻链可变区或其变体；
(b)氨基酸序列如SEQ ID NO：9、60和61任一所示的轻链可变区或其变体；
(c)氨基酸序列如SEQ ID NO:11所示的轻链可变区或其变体；
(d)氨基酸序列如SEQ ID NO：13、72、73和74任一所示的轻链可变区或其变体；(e)氨基酸序列如SEQ ID NO:15、79、80和81任一所示的轻链可变区或其变体；
其中，a至e中所述变体是在所述轻链可变区或重链可变区的框架区序列上具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸突变，所述氨基酸突变优选框架区氨基酸的回复突变。
在一些实施方式中，其中所述抗体或抗原结合片段包含：
f.选自序列为SEQ ID NO:6、46、49、50和51任一所示的重链可变区和序列为SEQ ID NO：7、47、48、53、54、55、56、57和58任一所示的轻链可变区；或
g.选自序列为SEQ ID NO:8、59、62、63、65、66、67、68、69和70任一所示的重链可变区和序列为SEQ ID NO：9、60和61任一所示的轻链可变区；或
h.序列为SEQ ID NO:10所示的重链可变区和序列为SEQ ID NO:11所示的轻链可变区；
i.选自序列为SEQ ID NO:12、71、75、76和77任一所示的重链可变区和序列为SEQ ID NO：13、72、73和74任一所示的轻链可变区；或
j.选自序列为SEQ ID NO:14、78、82、83、84、122和123任一所示的重链可变区和序列为SEQ ID NO:15、79、80和81任一所示的轻链可变区。
在一些实施方式中，所述抗体为全长抗体，进一步包括人抗体恒定区，优选包含SEQ ID NO:117所示的人抗体重链恒定区和/或SEQ ID NO:118所示的人轻链恒定区。
在一些实施方式中，所述抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
在一些实施方式中，所述抗体或抗原结合片段以如通过表面等离子共振(BIACORE)技术所测定的1×10 -7M至1×10 -12M的KD值的亲和力结合人CD96。
另一方面，本公开还提供一种分离的单克隆抗体或其抗原结合片段，其与前述(i)-(x)或a-j的单克隆抗体或其抗原结合片段竞争结合人CD96。
在一些实施方式中，所述的单克隆抗体或其抗原结合片段，具有以下特征中的至少一种：
i.以如通过表面等离子共振(BIACORE)技术所测定的1×10 -7M至1×10 -12M的KD值的亲和力结合人CD96；
ii.与食蟹猴或恒河猴CD96交叉反应；
iii.阻断人CD96与人CD155的结合；
iv.增加NK细胞和/或T细胞的活化；
v.阻断由CD96与CD155结合所诱导的NK细胞活化抑制。
在一些实施方式中，所述单克隆抗体或其抗原结合片段结合人CD96胞外区(SEQ ID NO：3)中如IAVYHPQYGFYCAYGRPCES所示的区域。
在另一方面，本公开还提供一种多特异性抗体，其含有如上所述的抗CD96抗体或其抗原结合片段的轻链可变区和/或重链可变区。
在另一方面，本公开还提供一种单链抗体，其含有如上所述的抗CD96抗体或其抗原结合片段的轻链可变区和/或重链可变区。
在另一方面，本公开还提供一种药物组合物，其含有治疗有效量的根据如上所述的单克隆抗体或其抗原结合片段、所述的多特异性抗体或所述的单链抗体以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂，优选地，所述药物组合物单位计量中可含有0.01至99重量％的抗CD96抗体或其抗原结合片段或药物组合物单位剂量中含单克隆抗体或其抗原结合片段的量为0.1-2000mg，更优选为1-1000mg。
在另一方面，本公开还提供一种分离的核酸分子，其编码前述任一项的单克隆抗体或其抗原结合片段、多特异性抗体或单链抗体。
在另一方面，本公开还提供一种重组载体，其包含上述的分离的核酸分子。
在另一方面，本公开还提供一种用上述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
在另一方面，本公开还提供用于生产前述任一项所述的单克隆抗体或其抗原结合片段的方法，所述方法包括将前述的宿主细胞在培养基中进行培养以形成并积累前述任一项的单克隆抗体或其抗原结合片段、多特异性抗体或单链抗体，以及从培养物回收所述单克隆抗体或其抗原结合片段。
在另一方面，本公开还提供用于体外检测或测定人CD96的方法，所述方法包括使用前述任一项的单克隆抗体或其抗原结合片段、多特异性抗体或单链抗体。
在另一方面，本公开还提供前述任一项的单克隆抗体或其抗原结合片段、多特异性 抗体或单链抗体在制备用于检测或测定人CD96的试剂中的用途。
在另一方面，本公开还提供一种减少或缓解免疫抑制的方法，所述方法包括向受试者施用治疗有效量的前述任一项的单克隆抗体或其抗原结合片段、多特异性抗体或单链抗体，或包含前述的药物组合物，或前述的分离的核酸分子。
在另一方面，本公开还提供一种增强NK细胞活性的方法，所述方法包括使用前述任一项的单克隆抗体或其抗原结合片段、多特异性抗体或单链抗体，或包含前述的药物组合物，或前述的分离的核酸分子降低CD96活性的步骤。
在另一方面，本公开还提供一种利用前述任一项的单克隆抗体或其抗原结合片段、多特异性抗体或单链抗体，或包含前述的药物组合物，或前述的分离的核酸分子制备增强NK细胞活性的药物的用途。
在另一方面，本公开还提供一种治疗与人CD96相关的疾病的方法，所述方法包括向受试者施用治疗有效量的前述任一项的单克隆抗体或其抗原结合片段、多特异性抗体或单链抗体，或包含前述的药物组合物，或前述的分离的核酸分子，以治疗人CD96相关的疾病，所述疾病优选肿瘤、癌症或感染性疾病。
在另一方面，本公开还提供如前述任一项的单克隆抗体或其抗原结合片段、多特异性抗体或单链抗体，或包含前述的药物组合物，或前述的分离的核酸分子或其组合在制备治疗或预防疾病或病症的药物中的用途，其中所述疾病或病症是人CD96相关疾病，所述疾病或病症优选肿瘤、癌症或感染性疾病。
在另一方面，本公开还提供作为药物的如前述任一项的单克隆抗体或其抗原结合片段、前述的多特异性抗体或前述的单链抗体，或如前所述的药物组合物，或如前所述的分离的核酸分子。
在另一方面，本公开还提供作为药物的如前述任一项的单克隆抗体或其抗原结合片段、前述的多特异性抗体或前述的单链抗体，或如前所述的药物组合物，或如前所述的分离的核酸分子，其中所述的药物用于人CD96相关的疾病的治疗，所述疾病优选肿瘤、癌症或感染性疾病。
适当地，癌症可以是任何对至少部分封闭CD96介导的免疫抑制、抑制或外周耐受应答的癌症。癌症癌症的例子包括但不限于癌瘤，淋巴瘤，胚细胞瘤(blastoma)，肉瘤，和白血病或淋巴样恶性。这种癌症的更具体的例子包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病 (CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、***癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子***、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、肝胆癌(hepatobiliary cancer)、中枢神经***癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。在一些实施方案中，癌症是转移性癌症，其有能力迁移到机体内的另一位点、组织或器官，并且在该位点形成肿瘤。
典型地，对至少部分封闭CD96介导的免疫抑制、压制或外周耐受是应答的疾病或病症是提高或恢复免疫监视能够使患有疾病或病症的受试者受益的任何疾病或病症。该疾病和病症可能包括那些能够被细胞介导的免疫所控制或压制的持久性疾病或病症。非限制性的实施例包括肿瘤、癌症或感染性疾病。具体地，本公开所涉及癌症的例子包括但不限于癌瘤，淋巴瘤，胚细胞瘤(blastoma)，肉瘤，和白血病或淋巴样恶性。这种癌症的更具体的例子包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌(NSCLC)、头和颈鳞状细胞癌(HNSCC)、神经胶质瘤、何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤(DLBCL)、滤泡性淋巴瘤、急性成淋巴细胞性白血病(ALL)、急性髓细胞样白血病(AML)、慢性淋巴细胞性白血病(CLL)、慢性髓细胞样白血病(CML)、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤(MCL)、小淋巴细胞性淋巴瘤(SLL)、富含T-细胞/组织细胞的大B-细胞淋巴瘤、多发性骨髓瘤、髓样细胞白血病-1蛋白(Mcl-1)、骨髓异常增生综合征(MDS)、胃肠(道)癌、肾癌、卵巢癌、肝癌、成淋巴细胞性白血病、淋巴细胞白血病、结肠直肠癌、子宫内膜癌、肾癌、***癌、甲状腺癌、黑素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性成胶质细胞瘤、胃癌、骨癌、尤因氏肉瘤、子***、脑癌、胃癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、肝细胞癌(HCC)、透明细胞肾细胞癌(RCC)、头和颈癌、肝胆癌(hepatobiliary cancer)、中枢神经***癌、食管癌、恶性胸膜间皮瘤、全身性轻链淀粉样变性、淋巴浆细胞性淋巴瘤(lymphoplasmacytic lymphoma)、骨髓异常增生综合征、骨髓增生性肿 瘤、神经内分泌肿瘤、梅克尔细胞癌、睾丸癌和皮肤癌。在一些实施方案中，癌症是转移性癌症，其有能力迁移到机体内的另一位点、组织或器官，并且在该位点形成肿瘤。
所涉及的感染性疾病指包括由病毒或病原菌感染引起的并且可通过呼吸器官、血液接触或皮肤接触感染的所有疾病。这些感染性疾病的非限制性的实例包括但不限于乙型肝炎、丙型肝炎、人***状瘤病毒(HPV)感染、巨细胞病毒感染、病毒性呼吸道疾病、流感、EB病毒(EBV)感染、单纯疱疹病毒(HSV包括HSV1和HSV2)感染、水痘带状疱疹病毒(VSV)感染以及巨细胞病毒(CMV)感染等。
本公开CD96单克隆抗体或抗原结合片段具有很高的特异性、与CD96的高亲和力，人源化抗体的免疫原性大大降低，同时完全保留了鼠源抗体的特异性，高亲和力和活性。
附图说明
图1：CD96人源化抗体阻断hCD96-CHOs细胞结合实验。
图2：CD96人源化抗体阻断hCD96抗原和hCD155-CHOs细胞结合实验。
图3：抗体竞争实验。图3A包被抗体(A抗体)是h1718-012；图3B包被抗体是h1719-014；图3C包被抗体是h1721-003；图3D包被抗体是ch1720；图3E包被抗体是h1722-010。
图4：CD96嵌合抗体/肽段复合物的CD96-CHOs细胞结合实验(FACS检测)，图4A为ch1718与3#肽段检测结果，图4B为ch1719与3#肽段检测结果，图4C为ch1720与不同肽段检测结果，图4D为ch1721与不同肽段检测结果。
图5：ch1718、ch1719、ch1720促进NK细胞对肿瘤细胞的杀伤作用，E:T＝1:1。
发明详述
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本公开所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同 一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
在本公开中，本公开所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本公开中，本公开所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人CD96的单克隆抗体。制备时用CD96抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个优选的实施方案中，所述的鼠源CD96抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后***表达载体中，最后在真核***或原核***中表达嵌合抗体分子。在本公开一个具体的实施方案中，所述的CD96嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的CD96嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者包含氨基酸突变(如YTE突变或回复突变)的IgG1、IgG2或IgG4重链恒定区变体。
术语“人源化抗体(humanized antibody)”，是指将鼠的CDR序列移植到人的抗体可 变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(因特网www.mrccpe.com.ac.uk/vbase)中获得，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。在本公开一个优选的实施方案中，所述的CD96人源化抗体中鼠的CDR序列选自SEQ ID NO:16-21、22-27、28-33、34-39或40-45；人的抗体可变区框架经过设计选择，其中所述抗体重链可变区上的重链FR区序列，来源于人种系重链序列，和人种系轻链序列。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区可进行最少反向突变(回复突变，即将人抗体来源的FR区氨基酸残基突变成原始来源抗体对应位置的氨基酸残基)，以保持活性。
CDR的移植可由于与抗原接触的构架残基的变化而导致产生的CD96抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人CD96抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查鼠单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
在类似“分别具有3、2、1或0个氨基酸突变的，如SEQ ID NO:X、SEQ ID NO:Y和SEQ ID NO:Z所示的CDR1、CDR2和CDR3的CDR变体”的描述中，示例性的解释是对CDR的突变可以包含3个、2个、1个或0个氨基酸的突变，CDR1、CDR2和CDR3之间可以任选地选择相同或不同数目的氨基酸残基进行突变，例如在如SEQ ID NO:X、SEQ ID NO:Y和SEQ ID NO:Z所示的CDR1、CDR2和CDR3的基础上，对CDR1进行1个氨基酸的突变，对CDR2和CDR3进行0个氨基酸突变。当对某个CDR进行0个氨基酸突变时，则该具有0个氨基酸突变的CDR变体仍是该CDR本身。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，CD96)的能力的一个或多个片段。已显示可利用全长抗体的片段来实现抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的 两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science 242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
本公开的抗原结合片段包括Fab、F(ab')2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)、包含CDR的肽等。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
本公开的Fab可以通过用木瓜蛋白酶处理本公开的特异性识别人CD96并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA***到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
本公开的F(ab')2可以通过用胃蛋白酶处理本公开的特异性识别人CD96并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab')2。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的特异性识别CD96并与胞外区的氨基酸序列或其三维结构结合的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA***到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本公开的scFv可以通过以下步骤来生产：获得本公开的特异性识别人CD96并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA，将所述DNA***到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
双抗体是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
本公开的双抗体可以通过以下步骤来生产：获得本公开的特异性识别人CD96并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA***到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering,7,697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的dsFv可以通过以下步骤来生产：获得本公开的特异性识别人CD96并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA***到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
包含CDR的肽是通过包含VH或VL的CDR中的一个或多个区域而构成的。包含多个CDR的肽可以被直接相连或经由适合的肽接头相连。
本公开的包含CDR的肽可以通过以下步骤来生产：构建本公开的特异性识别人CD96并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的CDR的编码DNA， 将所述DNA***到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达所述肽。也可以通过化学合成方法例如Fmoc方法或tBoc方法来生产所述包含CDR的肽。
本文中使用的术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“氨基酸突变”是指多肽与其变体之间，在多肽片段上某个或某些氨基酸位点之间的突变，其中变体可以由多肽上某个或某些位点经替代、***或缺失氨基酸获得。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，CD96分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 - 10M、10 -11M或更小的亲和力(KD)结合。
术语"KD"是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合CD96，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体或特异性结合的抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如CD96抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)；或通过如本公开测试例7的方法进行测定。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的 纯化抗原(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。或者如在本文测试例7中提供的关于用于测定竞争性结合的方法，通过固定抗原结合蛋白A，检测与抗原结合蛋白预先结合的标记过的抗原信号变化来测定竞争性抑制。通常，这种竞争性抑制测试会调换抗原结合蛋白A和抗原结合蛋白B的位置进行确认。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与充分接近参考抗原结合蛋白结合的表位所邻近的表位发生结合的抗原结合蛋白，所述两个表位在空间上互相妨碍结合的发生。通常当竞争的抗原结合蛋白过量存在时，部分竞争性抗原结合蛋白抑制(例如降低)至少40％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％)参考抗原结合蛋白与共同抗原的特异性结合。在完全竞争情况下，参考抗原结合蛋白与抗原的结合被抑制至少80％、至少85％、至少90％、至少95％、至少97％、至少98％、至少99％或更多。抑制小于30％被认为是不竞争。在以测试例7提供的测试竞争性结合的方法中，无论竞争性抗原结合蛋白作为抗原结合蛋白A或作为抗原结合蛋白B能够抑制参考抗原结合蛋白上述程度的结合，都被认为是竞争或部分竞争。
“氨基酸保守修饰”或“氨基酸保守取代”或“氨基酸的保守替代”指蛋白质中的氨基酸被具有相似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其他氨基酸取代，从而使得在不改变蛋白质的生物活性或其他所需特性(例如抗原亲和力和/或特异性)的情况下，可以经常进行改变。本领域技术人员认识到，通常，多肽的非必需区域中的单个氨基酸取代基本上不改变生物活性(参见，例如，Watson等人，(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页(第4版))。此外，结构上或功能上相似的氨基酸的取代不太可能破坏生物活性。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。
示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人CD96或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括微生物(例如细菌)、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、HEK293和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人CD96特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“缓解免疫抑制”表示以抑制或抑制一种或更多种通常表达CD96的细胞的免疫功能，至少部分消除、去除或克服CD96的正常活性或功能。典型地，一种或更多种通常表达CD96的细胞是T细胞，包括CD4 +和CD8 +T细胞、γδΤ细胞、NKT细胞、以及自然杀伤(NK)细胞。在一些实施方案中，缓解免疫抑制可能包括或涉及废除对外来病原体、显示外来病原体的宿主细胞(例如在自体MHC中显示外来病原体衍生的多肽)和/或宿主的癌细胞或组织的外周耐受。
术语“阻断”是指本公开的抗体或其抗原结合片段封闭、部分封闭、干扰、降低、抑制、减少靶蛋白或使其失活的能力，所述靶蛋白即CD96和/或其配体(如CD155)。因此，本领域的技术人员理解术语“阻断”可以涵盖所述配体或受体的全部和/或部分活性丧失。所述配体或受体的活性可以通过与配体/受体蛋白的活性位点结合的化合物，或通过其它方式，诸如使激活受抑制的第一蛋白的第二蛋白失活来阻抑或抑制。例如，CD96和CD155之间的相互作用的全部和/或部分抑制可以通过NK细胞活化增加来表示。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理 疾患。术语“癌症”，“癌性”，“细胞增殖性病症”，“增殖性病症”和“肿瘤”在本文中提到时并不互相排斥。
“给予”、“施用”和“处理”当应用于动物、人、受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种抗体或其抗原结合片段的组合物或编码抗体或其抗原结合片段的核酸分子，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床有测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳 比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
此外，本公开包括用于治疗与CD96相关的疾病的药剂，所述药剂包含本公开的单克隆抗体或其抗体片段作为活性成分。
对与CD96相关的疾病没有限制，只要它是与CD96相关的疾病即可，例如利用本公开的分子诱导的治疗反应可通过结合人类CD96然后阻断CD96与其配体CD155的结合，从而降低对NK细胞的抑制。因此，当处于适于治疗应用的制备物和制剂中时，本公开的分子对这样一些人是非常有用的，他们患有肿瘤、癌症或感染性疾病。
此外，本公开涉及用于免疫检测或测定CD96的方法、用于免疫检测或测定CD96的 试剂、用于免疫检测或测定表达CD96的细胞的方法和用于诊断与CD96相关的疾病的诊断剂，其包含本公开的特异性识别人CD96并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定CD96的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与CD96相关的疾病可以通过用本公开的单克隆抗体或抗体片段检测或测定表达CD96的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS***(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定CD96的活体样品没有特别限制，只要它具有包含表达CD96的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
具体实施方式
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1、CD96抗原蛋白设计及表达
以人CD96蛋白(Uniprot号：P40200-2)作为本公开CD96的模板，设计本公开涉及的抗原及检测用蛋白的氨基酸序列，可选的在CD96蛋白基础上融合不同的标签，分别克隆到pHr载体上(自产)或pXC-17.4载体上(LONZA)上，在293细胞瞬转表达或CHO细胞 稳定表达纯化，获得编码本公开抗原及检测用蛋白。以下CD96抗原未特殊说明的均指人CD96。
带Flag标签的CD96胞外区(SEQ ID NO：1)：CD96-Flag，用于免疫小鼠
Figure PCTCN2018114797-appb-000001
注释：划横线部分为信号肽，斜体部分为Flag-tag标签。
CD96胞外区和mIgG2a Fc的融合蛋白(SEQ ID NO：2)：CD96-mFc，用于免疫
Figure PCTCN2018114797-appb-000002
注释：划横线部分为信号肽，斜体部分为mFc。
全长CD96(SEQ ID NO：3)：用于构建CD96过表达细胞株，用于免疫和检测
Figure PCTCN2018114797-appb-000003
注释：下划横线部分为信号肽，正体部分为胞外区，斜体部分为跨膜区和胞内区
带His标签的CD96胞外区(SEQ ID NO：4)：CD96-His，用于检测
Figure PCTCN2018114797-appb-000004
注释：划横线部分为信号肽，斜体部分为His tag。
CD96胞外区和hIgG1Fc的融合蛋白(SEQ ID NO：5)：CD96-Fc，用于检测
Figure PCTCN2018114797-appb-000005
Figure PCTCN2018114797-appb-000006
注释：划横线部分为信号肽，斜体部分为Fc。
实施例2、CD96相关重组蛋白的纯化，以及杂交瘤抗体、重组抗体的纯化
1.带Flag标签的CD96-Flag重组蛋白的纯化步骤：
将样品高速离心去除杂质，并浓缩至适当体积。使用0.5×PBS平衡flag亲和柱，冲洗2-5倍柱体积。将除杂后的细胞表达上清样品上柱。用0.5×PBS冲洗柱子，至A280读数降至基线。用含有0.3M NaCl的PBS冲洗柱子，冲洗杂蛋白，并收集。用0.1M乙酸(pH3-4)或者Flag肽溶液洗脱目的蛋白，并收集目的产物洗脱峰，调节pH至中性。
收集的洗脱产物浓缩后可示例性地使用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS，去除聚体及杂蛋白峰，收集目的产物洗脱峰。所得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。
2.带His标签的CD96-His重组蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，并将缓冲液置换为PBS，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的上清样品上柱结合，介质可以选择不同公司的镍柱。用含有5mM咪唑的PBS溶液冲洗柱子，至A280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。
收集的洗脱产物浓缩后可可示例性地用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS，去除聚体及杂蛋白峰，收集目的产物洗脱峰。所得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。
3.杂交瘤上清分离纯化/ProteinG亲和层析：重组抗体，Fc融合蛋白的纯化
对于小鼠杂交瘤上清纯化首选ProteinG进行亲和层析，将培养所得杂交瘤离心取上清，根据上清体积加入10-15％体积的1M Tris-HCl(pH8.0-8.5)调节上清pH。ProteinG 柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积；利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积；细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间；利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线；利用0.1M醋酸/醋酸钠(pH3.0)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
4.Protein A亲和层析提取带Fc标签的融合蛋白或者抗体
首先将表达Fc融合蛋白或者抗体的细胞培养上清进行高速离心收取上清。ProteinA亲和柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积。利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用0.1M醋酸/醋酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
实施例3、抗人CD96杂交瘤单克隆抗体的制备
1.免疫
抗人CD96单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。免疫抗原为带Flag标签或mFc的人CD96胞外区(SEQ ID NO:1或2)，以及过表达人CD96的CHO细胞株。
免疫方案A：用
Figure PCTCN2018114797-appb-000007
Gold Adjuvant(Sigma Cat No.T2684)与Thermo
Figure PCTCN2018114797-appb-000008
Alum(Thermo Cat No.77161)佐剂交叉免疫。抗原与佐剂(
Figure PCTCN2018114797-appb-000009
Gold Adjuvant) 比例为1:1，抗原与佐剂(Thermo
Figure PCTCN2018114797-appb-000010
Alum)比例为3:1，50μg/只/次(首免)，25μg/只/次(加强免疫)。抗原乳化后进行接种，时间为第0、14、28、42、56天。第0天腹膜内(IP)注射50μg/只的乳化后抗原。第14天皮下(sc)多点(一般背部6-8点)注射25μg/只。第28，42天根据背部结块和腹部肿胀情况，选择背部或腹膜内注射抗原。于第21，35，49，63天取血，用ELISA方法确定小鼠血清中的抗体滴度。在4-5免以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射50μg/只的生理盐水配制的抗原溶液。
免疫方案B：用Quick Antibody-Mouse 5W(KX0210041)佐剂对小鼠进行免疫。抗原与佐剂比例为1:1，25μg/只/次(首免/加强免疫)。抗原与佐剂迅速充分混匀后接种，时间为第0、21、35天。第0天小鼠后小腿肌肉(IM)注射25μg/只的抗原。第21，35天按同样方式注射25μg/只(根据滴度决定第3免是否进行)。于第28，42天取血，用ELISA方法确定小鼠血清中的抗体滴度。选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射50μg/只的生理盐水配制的抗原溶液。
2.脾细胞融合
采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2018114797-appb-000011
CRL-8287 TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞以0.5-1×10^6/ml的密度用完全培养基(含20％FBS、1×HAT、1×OPI的DMEM培养基)重悬，100μl/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全培养基100μl/孔，继续培养3-4天至形成针尖般克隆。去除上清，加入200μl/well的HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
3.杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法进行杂交瘤培养上清检测。并将结合ELISA检测的阳性孔细胞上清进行细胞结合实验和细胞阻断实验。结合和阻断均为阳性的孔细胞及时进行扩增冻存保种和二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞也均需进行CD96结合ELISA、细胞结合实验和细胞阻断实验检测。通过以上实验筛选得到杂交瘤克隆，用无血清细胞培养法进一步制备抗体，按纯化实例(实施例2第3项)纯化抗体，供在检测例中使用。
4.杂交瘤阳性克隆序列测定
从阳性杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScript TMReverse Transcriptase试剂盒反转录(Takara,Cat No.2680A)。将反转录得到的cDNA采用小鼠Ig-Primer试剂盒(Novagen,TB326Rev.B 0503)进行PCR扩增后送测序公司测序。得到鼠源抗体m1718、m1719、m1720、m1721、m1722的重链和轻链可变区DNA序列对应的氨基酸序列(VH/VL中CDR的氨基酸残基由Kabat编号***确定并注释，下划线标注的序列为CDR)：
m1718VH(SEQ ID NO：6)
Figure PCTCN2018114797-appb-000012
m1718VL(SEQ ID NO：7)
Figure PCTCN2018114797-appb-000013
m1719VH(SEQ ID NO：8)
Figure PCTCN2018114797-appb-000014
m1719VL(SEQ ID NO：9)
Figure PCTCN2018114797-appb-000015
m1720VH(SEQ ID NO：10)
Figure PCTCN2018114797-appb-000016
m1720VL(SEQ ID NO：11)
Figure PCTCN2018114797-appb-000017
m1721VH(SEQ ID NO：12)
Figure PCTCN2018114797-appb-000018
m1721VL(SEQ ID NO：13)
Figure PCTCN2018114797-appb-000019
m1722VH(SEQ ID NO：14)
Figure PCTCN2018114797-appb-000020
m1722VL(SEQ ID NO：15)
Figure PCTCN2018114797-appb-000021
其中各抗体轻重链中CDR序列如表1所示。
表1 各抗体重链及轻链CDR区序列
Figure PCTCN2018114797-appb-000022
Figure PCTCN2018114797-appb-000023
上述鼠源抗体的轻重链可变区分别与人源抗体的轻链恒定区(SEQ ID NO：117)和重链恒定区(SEQ ID NO：118)连接后形成嵌合抗体，m1718抗体对应的嵌合抗体命名为ch1718，其他抗体类推。
实施例4、鼠源抗CD96抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，将骨架序列中关键氨基酸回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力，即得到人源化抗CD96单克隆抗体。其中回复突变时氨基酸残基由自然编号确定并注释。
4.1杂交瘤克隆m1718的人源化
(1)m1718人源化构架选择
鼠源抗体m1718的人源化轻链模板为IGKV1-16*01和hjk4.1，人源化重链模板为IGHV1-3*01和hjh6.1，经CDR graft后得到人源化抗体h1718-001，人源化可变区序列如下：
h1718-001 VH-CDR graft
Figure PCTCN2018114797-appb-000024
                                         SEQ ID NO：46
h1718-001 VL-CDR graft
Figure PCTCN2018114797-appb-000025
                                         SEQ ID NO：47
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1718系列回复突变设计如下表：
表2.h1718抗体的回复突变
Figure PCTCN2018114797-appb-000026
注：如S46T表示依照氨基酸序列自然顺序编号，将46位S突变回T。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1718系列人源化抗体可变区序列组合如下表：
表3.h1718系列人源化抗体可变区序列组合
  h1718-VH1 h1718-VH2 h1718-VH3 h1718-VH4
h1718-VL1 h1718-001 h1718-002 h1718-003 h1718-004
h1718-VL2 h1718-005 h1718-006 h1718-007 h1718-008
注：该表表示各种突变组合所得的序列。如h1718-006表示，在人源化的抗体h1718-006包含有轻链可变区h1718-VL2和重链可变区h1718-VH2。其它类推。
(4)h1718系列人源化抗体可变区具体序列如下：
>h1718-VL1(同H1718-001VL-CDR graft)
Figure PCTCN2018114797-appb-000027
                                    SEQ ID NO：47
>h1718-VL2
Figure PCTCN2018114797-appb-000028
                                    SEQ ID NO：48
>h1718-VH1(同H1718-001VH-CDR graft)
Figure PCTCN2018114797-appb-000029
                                    SEQ ID NO：46
>h1718-VH2
Figure PCTCN2018114797-appb-000030
                                  SEQ ID NO：49
>h1718-VH3
Figure PCTCN2018114797-appb-000031
                                     SEQ ID NO：50
>h1718-VH4
Figure PCTCN2018114797-appb-000032
                                    SEQ ID NO：51
(5)h1718-VL热点突变：
将h1718系列人源化VL的CDR1(即SEQ ID NO:19，KASQDINNYLN)中NN突变为NQ、NT、NL，以提高抗体的稳定性。h1718VL CDR1序列通式为KASQDINX 1YLN(SEQ ID NO：52)，其中X 1选自：N、T、L和Q。具体的h1718VL CDR1突变体包括KASQDINQYLN(SEQ ID NO：108)，KASQDINTYLN(SEQ ID NO：109)，KASQDINLYLN(SEQ ID NO：110)。
h1718-VL1可变为以下轻链可变区序列：
>h1718.VL1a(SEQ ID NO：53)
Figure PCTCN2018114797-appb-000033
>h1718.VL1b(SEQ ID NO：54)
Figure PCTCN2018114797-appb-000034
>h1718.VL1c(SEQ ID NO：55)
Figure PCTCN2018114797-appb-000035
h1718-VL2可变为以下轻链可变区序列：
>h1718.VL2a(SEQ ID NO：56)
Figure PCTCN2018114797-appb-000036
>h1718.VL2b(SEQ ID NO：57)
Figure PCTCN2018114797-appb-000037
>h1718.VL2c(SEQ ID NO：58)
Figure PCTCN2018114797-appb-000038
h1718系列人源化抗体可变区序列组合如下表：
表4.h1718系列人源化抗体可变区序列组合
  h1718-VH3 h1718-VH4 h1718-VH1 h1718-VH2
h1718-VL1a h1718-009 h1718-015 h1718-021 h1718-027
h1718-VL1b h1718-010 h1718-016 h1718-022 h1718-028
h1718-VL1c h1718-011 h1718-017 h1718-023 h1718-029
h1718-VL2a h1718-012 h1718-018 h1718-024 h1718-030
h1718-VL2b h1718-013 h1718-019 h1718-025 h1718-031
h1718-VL2c h1718-014 h1718-020 h1718-026 h1718-032
4.2杂交瘤克隆m1719的人源化
(1)m1719人源化构架选择
鼠源抗体m1719的人源化轻链模板为IGKV1-12*01和hjk4.1，人源化重链模板为IGHV2-26*01和hjh6.1，经CDR graft后得到人源化抗体h1719-001，人源化可变区序列如下：
h1719-001 VH-CDR graft
Figure PCTCN2018114797-appb-000039
                                      SEQ ID NO：59
h1719-001 VL-CDR graft
Figure PCTCN2018114797-appb-000040
                                      SEQ ID NO：60
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1719-001回复突变设计如下表：
表5.h1719人源化抗体回复突变
Figure PCTCN2018114797-appb-000041
注：如A43S表示依照氨基酸序列自然顺序编号，将43位A突变回S。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1719系列人源化抗体可变区序列组合如下表：
表6.h1719系列人源化抗体可变区序列组合
  h1719-VH1 h1719-VH2 h1719-VH3
h1719-VL1 h1719-001 h1719-002 h1719-003
h1719-VL2 h1719-004 h1719-005 h1719-006
注：该表表示各种突变组合所得的序列。如h1719-005表示，在人源化的抗体h1719-005上包含轻链可变区h1719-VL2和重链可变区h1719-VH2。其它类推。
(4)h1719系列人源化抗体可变区具体序列如下：
>h1719-VL1(同h1719-001 VL-CDR graft)
Figure PCTCN2018114797-appb-000042
                                        SEQ ID NO：60
>h1719-VL2
Figure PCTCN2018114797-appb-000043
                                        SEQ ID NO：61
>h 1719-VH1(同h1719-001 VH-CDR graft)
Figure PCTCN2018114797-appb-000044
                                        SEQ ID NO：59
>h1719-VH2
Figure PCTCN2018114797-appb-000045
                                     SEQ ID NO：62
>h1719-VH3
Figure PCTCN2018114797-appb-000046
                                         SEQ ID NO：63
(5)h1719-VH热点突变：
将h1719VH CDR3(NNYYGSRYGYPMDY)中NN突变为QN、NY、NQ，M突变 为L或I，以提高抗体的稳定性。h1719VH CDR3序列通式为X 2X 3YYGSRYGYPX 4DY(SEQ ID NO：64)，其中X2选自：N和Q，X3选自：N、Y和Q，X4选自M、L和I。具体的h1719VH CDR3突变体可以包括但不限于QNYYGSRYGYPLDY(SEQ ID NO：111)，NYYYGSRYGYPLDY(SEQ ID NO：112)，NQYYGSRYGYPLDY(SEQ ID NO：113)，QNYYGSRYGYPIDY(SEQ ID NO：114)，NYYYGSRYGYPIDY(SEQ ID NO：115)，NQYYGSRYGYPIDY(SEQ ID NO：116)。
h1719-VH3可变为以下重链可变区序列：
>h1719.VH3a(SEQ ID NO：65)
Figure PCTCN2018114797-appb-000047
>h1719.VH3b(SEQ ID NO：66)
Figure PCTCN2018114797-appb-000048
>h1719.VH3c(SEQ ID NO：67)
Figure PCTCN2018114797-appb-000049
>h1719.VH3d(SEQ ID NO：68)
Figure PCTCN2018114797-appb-000050
>h1719.VH3e(SEQ ID NO：69)
Figure PCTCN2018114797-appb-000051
>h1719.VH3f(SEQ ID NO：70)
Figure PCTCN2018114797-appb-000052
Figure PCTCN2018114797-appb-000053
h1719系列人源化抗体可变区序列组合如下表：
表7.h1719系列人源化抗体可变区序列组合
Figure PCTCN2018114797-appb-000054
4.3杂交瘤克隆m1721的人源化
(1)m1721人源化构架选择
鼠源抗体m1721的人源化轻链模板为IGKV1-39*01和hjk4.1，人源化重链模板为IGHV2-26*01和hjh6.1，经CDR graft后得到人源化抗体h1721-001，人源化可变区序列如下：
h1721-001 VH-CDR graft
Figure PCTCN2018114797-appb-000055
                                        SEQ ID NO：71
h1721-001 VL-CDR graft
Figure PCTCN2018114797-appb-000056
                                        SEQ ID NO：72
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1721-001回复突变设计如下表：
表8.h1721人源化抗体回复突变
Figure PCTCN2018114797-appb-000057
Figure PCTCN2018114797-appb-000058
注：如A48S表示依照氨基酸序列自然顺序编号，将48位A突变回S。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1721系列人源化抗体可变区序列组合如下表：
表9.h1721系列人源化抗体可变区序列组合
  h1721-VH1 h1721-VH2 h1721-VH3 h1721-VH4
h1721-VL1 h1721-001 h1721-002 h1721-003 h1721-004
h1721-VL2 h1721-005 h1721-006 h1721-007 h1721-008
h1721-VL3 h1721-009 h1721-010 h1721-011 h1721-0012
注：该表表示各种突变组合所得的序列。如h1721-006表示，在人源化的抗体h1721-006包含轻链可变区h1721-VL2、重链可变区h1721-VH2。其它类推。
(4)h1721系列人源化抗体可变区具体序列如下：
>h1721V-L1(同h1721-001 VL-CDR graft)
Figure PCTCN2018114797-appb-000059
                                          SEQ ID NO：72
>h1721-VL2
Figure PCTCN2018114797-appb-000060
                                          SEQ ID NO：73
>h1721-VL3
Figure PCTCN2018114797-appb-000061
                                          SEQ ID NO：74
>h1721-VH1(同h1721-001 VH-CDR graft)
Figure PCTCN2018114797-appb-000062
                                       SEQ ID NO：71
>h1721-VH2
Figure PCTCN2018114797-appb-000063
                                      SEQ ID NO：75
>h1721-VH3
Figure PCTCN2018114797-appb-000064
                                         SEQ ID NO：76
>h1721-VH4
Figure PCTCN2018114797-appb-000065
                                        SEQ ID NO：77
4.4杂交瘤克隆m1722的人源化
(1)m1722人源化构架选择
鼠源抗体m1722的人源化轻链模板为IGKV1-39*01和hjk2.1，人源化重链模板为IGHV1-69*02和hjh4.1，经CDR graft后得到人源化抗体h1722-001，人源化可变区序列如下：
h1722-001 VH-CDR graft
Figure PCTCN2018114797-appb-000066
                                          SEQ ID NO：78
h1722-001 VL-CDR graft
Figure PCTCN2018114797-appb-000067
                                       SEQ ID NO：79
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)h1722-001回复突变设计如下表：
表10.h1722人源化回复突变
Figure PCTCN2018114797-appb-000068
注：如L45R表示依照氨基酸序列自然顺序编号，将45位L突变回R。Grafted代表鼠抗体CDR植入人种系FR区序列。
(3)h1722系列人源化抗体可变区序列组合如下表：
表11.h1722系列人源化抗体可变区序列组合
  h1722-VH1 h1722-VH2 h1722-VH3 h1722-VH4
h1722-VL1 h1722-001 h1722-002 h1722-003 h1722-004
h1722-VL2 h1722-005 h1722-006 h1722-007 h1722-008
h1722-VL3 h1722-009 h1722-010 h1722-011 h1722-012
注：该表表示各种突变组合所得的序列。如h1722-006表示，在人源化的抗体h1722-006包含轻链可变区h1722-VL2、重链可变区h1722-VH2。其它类推。
(4)h1722系列人源化抗体可变区具体序列如下：
>h1722-VL1(同h1722-001 VL-CDR graft)
Figure PCTCN2018114797-appb-000069
                                         SEQ ID NO：79
>h1722-VL2
Figure PCTCN2018114797-appb-000070
                                         SEQ ID NO：80
>h1722-VL3
Figure PCTCN2018114797-appb-000071
                                      SEQ ID NO：81
>h1722-VH1(同h1722-001 VH-CDR graft)
Figure PCTCN2018114797-appb-000072
                                      SEQ ID NO：78
>h1722-VH2
Figure PCTCN2018114797-appb-000073
                                      SEQ ID NO：82
>h1722-VH3
Figure PCTCN2018114797-appb-000074
                                      SEQ ID NO：83
>h1722-VH4
Figure PCTCN2018114797-appb-000075
                                       SEQ ID NO：84
(5)h1722-VH热点突变：
由于h1722-VH CDR2有一个乙酰化高风险位点NS，因此我们将NS突变为KS或QS，以提高抗体的化学稳定性。h1722-VH CDR2的序列通式为MLHPX 5SGTTSFNEKFKI(SEQ ID NO：119)，其中X 5选自N、K或Q。具体地，h1722-VH CDR2的突变体包括但不限于；MLHPKSGTTSFNEKFKI(SEQ ID NO：120)或MLHPQSGTTSFNEKFKI(SEQ ID NO：121)。
h1722-VH2可变为以下重链可变区序列：
>h1722-VH2a(N54K)
Figure PCTCN2018114797-appb-000076
                                     SEQ ID NO：122
>h1722-VH2b(N54Q)
Figure PCTCN2018114797-appb-000077
                                        SEQ ID NO：123
h1722系列人源化抗体可变区序列突变后的组合如下表：
表12.h1722系列人源化抗体可变区序列突变后组合
  h1722-VH2a h1722-VH2b
h1722-VL1 h1722-013 h1722-014
h1722-VL2 h1722-015 h1722-016
h1722-VL3 h1722-017 h1722-018
以上各重链可变区与如SEQ ID NO:117所示的重链恒定区序列重组表达得到最终的完整重链序列。以上各轻链可变区与如SEQ ID NO:118所示的轻链恒定区序列重组表达得到最终的完整轻链序列。
Figure PCTCN2018114797-appb-000078
                                       SEQ ID NO：117
轻链恒定区：
Figure PCTCN2018114797-appb-000079
                                       SEQ ID NO：118
采用所属领域的常规技术手段，将以上轻链可变区、重链可变区还可与其他可选的人源化恒定区，及经过功能性修饰的人源化恒定区，重组并表达。
用于检测的猴CD96抗原：猴CD96-3×Flag(SEQ ID NO：95)
Figure PCTCN2018114797-appb-000080
Figure PCTCN2018114797-appb-000081
下划线部分为信号肽，斜体部分为3×Flag-tag。制备方法见实施例2第1项。
以下用生化测试方法验证本公开性能及有益效果。
测试例1：CD96抗体结合人CD96蛋白的ELISA实验
抗CD96抗体的结合力通过抗体与人CD96蛋白的ELISA实验来检测。用带His标签的CD96融合蛋白包被在酶标板中，抗体加入后信号的强弱被用于判断抗体和CD96的结合活性，具体实验方法如下。
用pH7.4的PBS(上海源培生物科技有限公司，Cat No.B320)缓冲液将实施例1中序列如SEQ ID NO:4所示的CD96-His稀释至2μg/ml浓度，以50μl/孔的体积加入96孔酶标板(Corning，Cat No.CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD，232100)封闭液250μl/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％tween-20)洗板4次后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板4次，加入50μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板4次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算CD96抗体对人CD96的结合EC50值(见表13)。
表13.CD96抗体与人CD96的亲和力ELISA测定
Figure PCTCN2018114797-appb-000082
Figure PCTCN2018114797-appb-000083
结果显示抗CD96的嵌合抗体和人源化抗体能够与人CD96特异性结合。
测试例2：CD96抗体结合食蟹猴CD96蛋白的ELISA实验
抗CD96抗体的与猴CD96的交叉反应结合力通过抗体与食蟹猴CD96蛋白的ELISA实验来检测。用带3×FLAG标签的食蟹猴CD96融合蛋白包被在酶标板中，抗体加入后信号的强弱被用于判断抗体和食蟹猴CD96的结合活性，具体实验方法如下。
用pH7.4的PBS(上海源培生物科技有限公司，Cat No.B320)缓冲液将上述实施例4制备得到的氨基酸序列为SEQ ID NO：95的cyno-CD96-3×FLAG稀释至2μg/ml浓度，以50μl/孔的体积加入96孔酶标板(Corning，Cat No.CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD，232100)封闭液250μl/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％tween-20)洗板4次后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板4次，加入50μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板4次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算CD96抗体对猴CD96的结合EC50值(见表14)。
表14 CD96抗体与食蟹猴CD96结合ELISA测定
Figure PCTCN2018114797-appb-000084
结果显示CD96嵌合抗体和人源化抗体能够与猴CD96具有交叉反应。
测试例3：CD96抗体与人CD96过表达CHO-s细胞的结合实验
抗CD96抗体的结合力通过抗体与过表达人CD96蛋白的CHO-S细胞的结合实验来检测。通过电转染的方法将CD96全长质粒转染进CHO-S细胞中后加压筛选两周后，检测CD96的表达量。将过表达细胞固定于96孔板底后，抗体加入后信号的强弱被用于判断抗体和CD96过表达CHO-S细胞的结合活性，具体实验方法如下。
将细胞以9×10 4/100μL/well密度，接种于96孔板中过夜培养。吸掉上清，用PBS洗一遍后，加入100μl/孔免疫染色固定液(碧云天，P0098)室温固定一小时，PBS洗三遍。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD，232100)封闭液250μl/孔，37℃孵育箱孵育2.5小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4 PBS含0.05％tween-20)洗板4次后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育2小时。孵育结束后用PBST洗板4次，加入50μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板4次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育10-25min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算CD96抗体对CD96过表达CHO-S细胞的结合EC50值(见图1和表15)。
表15 CD96抗体与过表达人CD96-CHOs细胞的亲和力ELISA测定
候选抗体 EC50(nM) 候选抗体 EC50(nM)
ch1718 0.026 h1718-012 0.031
ch1719 0.012 h1719-003 0.028
ch1720 0.036 h1719-006 0.016
ch1721 0.036 h1719-014 0.02
ch1722 0.043 h1721-003 0.074
h1722-005 0.045 h1722-006 0.043
h1722-010 0.047 h1722-017 0.053
h1722-018 0.044    
结果显示CD96嵌合抗体和人源化抗体能够结合CD96过表达CHO-S细胞。
测试例4：抗CD96抗体阻断CD96抗原和CD155-CHOs细胞结合实验
CD155-CHOs细胞(上海恒瑞构建)以9×10 4/100μL/well的数量接种于96孔培养板中过夜培养，吸掉上清，用PBS洗一遍后，加入100μl/孔免疫染色固定液(碧云天，P0098)室温固定一小时，PBS洗三遍。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD，232100)封闭液250μl/孔，37℃孵育箱孵育2.5小时进行封闭。封闭结束后，弃去封闭液， 并用PBST缓冲液(pH7.4PBS含0.05％tween-20)洗板4次后，加入50μl/孔抗原-抗体预孵育液(梯度浓度的杂交瘤待测抗体与终浓度0.5μg/mL的CD96-Fc 37℃预混合30分钟，或者梯度浓度的人源化抗体样品与终浓度0.5μg/mL的Bio-CD96-His 37℃预混合30分钟)(生物素标记试剂盒，东仁化学，Cat No.LK03)，置37℃孵育箱孵育2小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板4次后，加入50μl/孔用样品稀释液稀释HRP标记的羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)或者HRP标记的链霉亲和素(Sigma，Cat No.S2438)，37℃孵育1小时。用PBST洗板4次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育10-20min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算CD96抗体对抗原与CD155-CHOs细胞结合的阻断作用(见图2和表16)。
表16 CD96抗体阻断CD96与表达人CD155-CHOs细胞的结合测定
候选抗体 IC50(nM) 候选抗体 IC50(nM)
ch1718 0.096 h1718-012 0.133
ch1719 0.137 h1719-003 0.096
ch1720 0.149 h1719-006 0.064
ch1721 0.214 h1719-014 0.046
ch1722 0.159 h1721-003 0.155
h1722-006 0.398 h1722-010 0.207
h1722-017 0.135 h1722-018 0.134
结果显示CD96嵌合抗体和人源化抗体能够阻断CD96和CD155-CHOs细胞的结合。
测试例5：BIAcore检测抗CD96抗体亲和力实验
1.人Fc抗体亲和力测定方法：
用Protein A生物传感芯片亲和捕获待测抗体，然后于芯片表面流经CD96抗原，用Biacore仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用甘氨酸-盐酸再生溶液(pH 1.5)将生物传感芯片洗净再生。
2.鼠Fc抗体亲和力测定方法：
用偶联有抗鼠Fc抗体的CM5生物传感芯片亲和捕获待测抗体，然后于芯片表面流经CD96抗原，利用Biacore仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用小鼠抗体捕获试剂盒里的再生溶液将生物芯片洗净再生。
流动相均为CD96-ECD-His。
表17候选抗体的亲和力BIACORE测定
抗体 亲和力(M) 抗体 亲和力(M)
ch1718 3.32E-9 h1721-001 2.73E-9
ch1719 9.74E-9 h1721-002 2.20E-9
ch1720 1.35E-9 h1721-003 1.61E-9
ch1721 2.28E-9 h1721-004 2.00E-9
ch1722 1.05E-9 h1721-005 2.25E-9
h1718-007 3.88E-9 h1721-006 2.29E-9
h1718-008 3.37E-9 h1721-007 1.87E-9
h1718-012 3.98E-9 h1721-008 2.22E-9
h1718-013 4.08E-9 h1721-009 2.01E-9
h1718-014 4.31E-9 h1721-010 2.04E-9
h1718-018 3.82E-9 h1721-011 2.12E-9
h1718-019 3.70E-9 h1721-012 2.14E-9
h1718-020 3.41E-9 h1722-005 9.87E-9
h1719-006 1.31E-8 h1722-006 1.19E-9
h1719-007 2.55E-8 h1722-007 1.31E-9
h1719-008 1.76E-8 h1722-008 1.00E-9
h1719-010 4.65E-7 h1722-009 4.55E-9
h1719-013 1.98E-8 h1722-010 8.60E-10
h1719-014 1.49E-8 h1722-011 9.48E-10
h1719-016 9.07E-8 h1722-012 7.81E-10
*亲和力数值3.32E-9M是3.32×10 -9M。
抗CD96嵌合抗体ch1718、ch1720、ch1721、ch1719、ch1722与人CD96均有很强的亲和力，示例性人源化抗体也与人CD96有很强的亲和力。
抗人CD96单克隆抗体的表位测定
测试例6：抗CD96抗体的竞争实验
将不同抗体与CD96蛋白结合的竞争实验用来进行抗体的结合表位分类。具体实验方法如下：
用pH7.4的PBS(上海源培，Cat No.B320)缓冲液将抗体A稀释至2μg/mL浓度，以50μL/孔的体积加入96孔酶标板(Corning，Cat No.CLS3590-100EA)中，于4℃包被过夜。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD skim milk,Cat No.232100)封闭液250μL/孔，37℃孵育箱孵育3小时。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％tween-20)洗板5次后，备用。1％BSA稀释抗体B至40μg/ml和8μg/ml，同时稀释Biotinylated CD96抗原至4μg/mL，将抗体B和抗原按1:1体积比混匀，37℃预孵育45分钟后，加入到已包被抗体A的酶标板中，放于37℃孵育箱孵育1.5小时。孵育结束后用PBST洗板5次，加入50μL/孔用样品稀释液稀释的HRP标记的链霉亲和素(Sigma，Cat No.S2438)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)进行显色，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，根据OD值的大小确定A和B两种 CD96抗体是否竞争。彼此竞争的抗体分为同一类的抗体，不能竞争的抗体则是不同类的抗体。检测结果见图3和表18。
表18不同组抗体间竞争关系检测
Figure PCTCN2018114797-appb-000085
竞争实验结果表明h1721-003、h1719-014、h1718-012和ch1720抗体之间存在竞争关系，它们都与h1722-010抗体不竞争。
测试例7：抗CD96抗体结合CD96蛋白不同多肽片段的ELISA实验
抗CD96抗体所识别的抗原表位通过抗体与CD96蛋白不同多肽片段的结合实验来检测。用CD96抗原的不同肽段的多肽包被在酶标板中，抗体加入后信号的强弱被用于判断抗体和多肽的结合活性，具体实验方法如下：
用pH7.4的PBS(上海源培，Cat No.B320)缓冲液将多肽稀释至20μg/mL浓度，以50μL/孔的体积加入96孔酶标板(Corning，Cat No.CLS3590-100EA)中，于4℃包被过夜。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD skim milk,Cat No.232100)封闭液250μL/孔，37℃孵育箱孵育3小时。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％tween-20)洗板5次后，加入50μL/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或嵌合抗体)，放于37℃孵育箱孵育2小时。孵育结束后用PBST洗板5次，加入50μL/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)或者anti-Rat-IgG HRP(Jackson Immuno Research，Cat No.112-035-003)，37℃孵育1小时。用PBST洗板5次后，加入50μL/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15min，加入50μL/孔1M H 2SO 4终止反应，用酶标仪(Thermo scientific MuLtiskan MK3)在波长450nm处读取吸收值，推测分析CD96抗体可能所识别的抗原表位。
表19抗原表位肽列表
序号(#) 多肽序列(在CD96胞外区中的位置，序列编号) ch1718 ch1720 ch1721 ch1719
1 GSDVNLTCQTQTVGFFVQMQ(17-36，SEQ ID NO：85) +++ - + -
2 FVQMQWSKVTNKIDLIAVYH(32-51，SEQ ID NO：86) +++ - +/- -
3 IAVYHPQYGFYCAYGRPCES(47-66，SEQ ID NO：87) ++++ +++ ++++ +++
4 RPCESLVTFTETPENGSKWT(62-81，SEQ ID NO：88) +++ - - -
5 GSKWTLHLRNMSCSVSGRYE(77-86，SEQ ID NO：89) +++ - + -
6 SGRYECMLVLYPEGIQTKIY(92-111，SEQ ID NO：90) +++ - +/- -
7 QTKIYNLLIQTHVTADEWNS(107-126，SEQ ID NO：91) +++ - +/- -
8 DEWNSNHTIEIEINQTLEIP(122-141，SEQ ID NO：92) +++ - +/- -
9 TLEIPCFQNSSSKISSEFTY(137-156，SEQ ID NO：93) +++ - - -
10 KISSEFTYAWSVEDNGTQETLI(149-170，SEQ ID NO：94) ++ - - -
11 QETLISQNHLISNSTLLKDR(166-185，SEQ ID NO：96) +++ - +/- -
12 LLKDRVKLGTDYRLHLSPVQ(181-200，SEQ ID NO：97) +++ - +/- -
13 LSPVQIFDDGRKFSCHIRVG(196-215，SEQ ID NO：98) +++ - +/- -
14 HIRVGPNKILRSSTTVKVFA(211-230，SEQ ID NO：99) +++ - + -
15 LRSSTTVKVFAKPEIPVIVE(220-239，SEQ ID NO：100) +++ - +/- -
16 PVIVENNSTDVLVERRFTCL(235-254，SEQ ID NO：101) +++ - +/- -
17 RFTCLLKNVFPKANITWFID(250-269，SEQ ID NO：102) ++++ - + -
18 TWFIDGSFLHDEKEGIYITN(265-284，SEQ ID NO：103) +++ - - -
19 IYITNEERKGKDGFLELKSV(280-299，SEQ ID NO：104) +++ - - -
20 ELKSVLTRVHSNKPAQSDNL(295-314，SEQ ID NO：105) ++ - +/- -
21 QSDNLTIWCMALSPVPGNKV(310-329，SEQ ID NO：106) ++ - - -
22 PGNKVWNISSEKITFLLGSE(325-344，SEQ ID NO：107) ++ - +/- -
注：“+”表示结合强度，“+”越多，表示结合力越强；“-”表示无结合。
根据上述结果，抗体ch1718、ch1720、ch1721、ch1719与3#抗原片段(SEQ ID NO：87)结合较强，3#抗原片段中含抗体ch1718、ch1720、ch1721、ch1719的表位。
测试例8：CD96抗体结合CD96不同肽段的FACS实验
为了验证抗体与CD96蛋白多肽片段的结合是特异的，我们设计了以下FACS实验进行验证。用多肽片段与抗体孵育结合后，抗体与CD96蛋白结合的表位即被占据，则抗体再与细胞表面的CD96蛋白结合的信号下降。具体实验方法如下：
1％BSA稀释抗体样品至1.2μg/mL，同时稀释多肽至40μg/mL，将多肽和抗体样品按1:1体积比混匀，37℃预孵育90分钟；收集CD96-CHOs细胞，PBS洗涤一次，按0.5×10 6/test分配；用100μL的多肽-抗体混合液重悬细胞，4℃孵育60分钟，1％BSA洗涤3次；Alexa Fluor 488anti-human IgG Fc(thermofisher，A-11013),或者PE goat anti-mouse IgG(Biolegend,405307)，或者PE goat anti-Rat IgG(Biolegend，405406)稀释液(1:400，1:40，1:100)100μL重悬细胞，4℃孵育40分钟，1％BSA洗涤3次，以200μL的1％BSA重悬细胞，在流式细胞仪BD FACSCantoⅡ上读取各样品MFI，GraphPad Prism 5分析数据做出柱状图，通过未与多肽孵育的和与多肽孵育的两者之间的MFI差值来判断CD96抗体可识别的抗原表位。检测结果见图4。
检测结果显示，嵌合抗体ch1721、ch1719、ch1718结合人CD96胞外区中SEQ ID NO：87所示的表位区段。
测试例9：NK细胞介导的细胞杀伤功能实验
为了研究CD96抗体对NK细胞杀伤功能的影响，收集和纯化人外周血单核细胞(PBMC)，提取自然杀伤细胞(NK)，与人大肠癌细胞WiDr(Cat No.ATCC-CCL-218)共培养4h，检测乳酸脱氢酶(LDH)的分泌水平。
实验过程简单描述如下：
人大肠癌细胞系WiDr于MEM培养基中培养，该培养基中添加10％(v/v)胎牛血清(FBS)，37℃、5％CO 2条件下培养。新鲜血液利用Ficoll-Hypaque密度梯度离心(Stem Cell Technologies)得到PBMC，人原代NK细胞从新鲜分离的PBMC中提取(Miltenyi,CAT#130-092-657)，于RPMI 1640培养基中培养，该培养基中添加10％(v/v)FBS，37℃、5％CO 2条件下培养。人原代NK细胞接种至6孔细胞培养板，细胞密度约为2×10 6/ml，加入100U/mL人IL-2(Peprotech，200-02-100)过夜培养后，利用无酚红RPMI1640培养基洗涤，重悬，并接种至96孔U底板中，细胞密度约为3×10 5/孔，同时加入梯度稀释的抗体样品(用PBS稀释)或等量的同型IgG作为空白对照，以只加入IL-2、NK细胞和癌细胞的孔作为空白对照(control)。37℃，5％CO 2培养箱孵育1h后，靶标细胞WiDr以1:1比例与人原代NK细胞共培养，37℃，5％CO 2培养箱培养4h后，收集细胞培养上清。采用CytoTox
Figure PCTCN2018114797-appb-000086
Non-Radioactive Cytotoxicity Assay(Promega,CAT#G1780)方法检测细胞培养上清内LDH分泌水平。具体操作参考试剂说明书。特异性细胞溶解的百分比以下式决定：％溶解＝100×(ER-SR1-SR2)/(MR-SR1)，其中ER、SR(1&2)及MR分别代表实验、自发(1为靶标细胞，2为人原代NK细胞)及最大LDH释放。自发释放系由单独培养于培养基中的靶标细胞或人原代NK细胞所释放的LDH，最大释放系利用裂解液裂解所有靶标细胞时所测定的LDH。
实验结果见图5，结果表明CD96的抗体ch1718，ch1720，以及ch1719均能不同程度增强NK细胞对肿瘤细胞的杀伤。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (26)

  1. 一种单克隆抗体或其抗原结合片段，所述单克隆抗体或其抗原结合片段特异性结合人CD96，所述单克隆抗体包含重链可变区和轻链可变区，其中：
    (i)重链可变区包含选自在分别如SEQ ID NO：16、17和18所示的HCDR1、HCDR2和HCDR3区基础上分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：19、20和21所示的LCDR1、LCDR2和LCDR3区基础上分别具有3，2，1或0个氨基酸突变的LCDR变体；或
    (ii)重链可变区包含选自在分别如SEQ ID NO：22、23和24所示的HCDR1、HCDR2和HCDR3区基础上分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：25、26和27所示的LCDR1、LCDR2和LCDR3区基础上分别具有3，2，1或0个氨基酸突变的LCDR变体；或
    (iii)重链可变区包含选自在分别如SEQ ID NO：28、29和30所示的HCDR1、HCDR2和HCDR3区基础上分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：31、32和33所示的LCDR1、LCDR2和LCDR3区基础上分别具有3，2，1或0个氨基酸突变的LCDR变体；或
    (iv)重链可变区包含选自在分别如SEQ ID NO：34、35和36所示的HCDR1、HCDR2和HCDR3区基础上分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：37、38和39所示的LCDR1、LCDR2和LCDR3区基础上分别具有3，2，1或0个氨基酸突变的LCDR变体；或
    (v)重链可变区包含选自在分别如SEQ ID NO：40、41和42所示的HCDR1、HCDR2和HCDR3区基础上分别具有3，2，1或0个氨基酸突变的HCDR变体，轻链可变区包含选自在分别如SEQ ID NO：43、44和45所示的LCDR1、LCDR2和LCDR3区基础上分别具有3，2，1或0个氨基酸突变的LCDR变体。
  2. 如权利要求1所述的单克隆抗体或其抗原结合片段，其中：
    (vi)重链可变区包含分别如SEQ ID NO：16、17和18氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：52、20和21氨基酸序列所示的LCDR1、LCDR2和LCDR3区，其中所述LCDR1优选如SEQ ID NO：19、108、109和110任一所示LCDR1；或
    (vii)重链可变区包含分别如SEQ ID NO：22、23和64氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：25、26和27氨基酸序列所 示的LCDR1、LCDR2和LCDR3区，其中所述HCDR3优选如SEQ ID NO：24、111、112、113、114、115和116任一所示HCDR3；或
    (viii)重链可变区包含分别如SEQ ID NO：28、29和30氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：31、32和33氨基酸序列所示的LCDR1、LCDR2和LCDR3区；或
    (ix)重链可变区包含分别如SEQ ID NO：34、35和36氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：37、38和39氨基酸序列所示的LCDR1、LCDR2和LCDR3区；或
    (x)重链可变区包含分别如SEQ ID NO：40、119和42氨基酸序列所示的HCDR1、HCDR2和HCDR3区，轻链可变区包含分别如SEQ ID NO：43、44和45氨基酸序列所示的LCDR1、LCDR2和LCDR3区，其中所述的HCDR2优选如SEQ ID NO：41、120或121所示。
  3. 如权利要求1或2所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体或其抗原结合片段是重组抗体，优选自鼠源抗体、嵌合抗体、人源化抗体。
  4. 如权利要求3所述的单克隆抗体或其抗原结合片段，其包含：
    a.序列为SEQ ID NO:6、46、49、50和51任一所示的重链可变区或其变体和/或序列为SEQ ID NO：7、47、48、53、54、55、56、57和58任一所示的轻链可变区或其变体；或
    b.序列为SEQ ID NO:8、59、62、63、65、66、67、68、69和70任一所示的重链可变区或其变体和/或序列为SEQ ID NO：9、60和61任一所示的轻链可变区或其变体；或
    c.序列为SEQ ID NO:10所示的重链可变区或其变体和/或序列为SEQ ID NO:11所示的轻链可变区或其变体；或
    d.序列为SEQ ID NO:12、71、75、76和77任一所示的重链可变区或其变体和/或序列为SEQ ID NO：13、72、73和74任一所示的轻链可变区或其变体；或
    e.序列为SEQ ID NO:14、78、82、83、84、122和123任一所示的重链可变区或其变体和/或序列为SEQ ID NO:15、79、80和81任一所示的轻链可变区或其变体；
    其中a至e中所述变体是在所述轻链可变区或重链可变区的框架区序列上具有1-10个氨基酸突变，所述的突变较佳地为回复突变。
  5. 如权利要求4所述的单克隆抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含：
    f.选自序列为SEQ ID NO:6、46、49、50和51任一所示的重链可变区和序列为SEQ  ID NO：7、47、48、53、54、55、56、57和58任一所示的轻链可变区；或
    g.选自序列为SEQ ID NO:8、59、62、63、65、66、67、68、69和70任一所示的重链可变区和序列为SEQ ID NO：9、60和61任一所示的轻链可变区；或
    h.序列为SEQ ID NO:10所示的重链可变区和序列为SEQ ID NO:11所示的轻链可变区；或
    i.选自序列为SEQ ID NO:12、71、75、76和77任一所示的重链可变区和序列为SEQ ID NO：13、72、73和74任一所示的轻链可变区；或
    j.选自序列为SEQ ID NO:14、78、82、83、84、122和123任一所示的重链可变区和序列为SEQ ID NO:15、79、80和81任一所示的轻链可变区。
  6. 如权利要求1～5中任一项所述的单克隆抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括人抗体恒定区，优选包含SEQ ID NO:117所示的人抗体重链恒定区和/或SEQ ID NO:118所示的人抗体轻链恒定区。
  7. 如权利要求1～5任一项所述的单克隆抗体或其抗原结合片段，其中所述抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段的形式。
  8. 如权利要求1～7任一项所述的单克隆抗体或其抗原结合片段，其中所述抗体或抗原结合片段以如通过表面等离子共振(BIACORE)技术所测定的1×10 -7M至1×10 -12M的KD值的亲和力结合人CD96。
  9. 一种分离的单克隆抗体或其抗原结合片段，其与权利要求1～8任一项所述的单克隆抗体或其抗原结合片段竞争结合人CD96。
  10. 如权利要求9所述的单克隆抗体或其抗原结合片段，具有以下特征中的至少一种：
    i.以如通过表面等离子共振(BIACORE)技术所测定的1×10 -7M至1×10 -12M的KD值的亲和力结合人CD96；
    ii.与食蟹猴或恒河猴CD96交叉反应；
    iii.阻断人CD96与人CD155的结合；
    iv.增加NK细胞和/或T细胞的活化；
    v.阻断由CD96与CD155结合所诱导的NK细胞的活化抑制。
  11. 如权利要求9或10所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体或其抗原结合片段结合人CD96胞外区中如IAVYHPQYGFYCAYGRPCES所示的区域。
  12. 一种多特异性抗体，含有如权利要求1～11任一项所述的单克隆抗体或其抗原结 合片段的轻链可变区和/或重链可变区。
  13. 一种单链抗体，含有如权利要求1～11任一项所述的单克隆抗体或其抗原结合片段的轻链可变区和/或重链可变区。
  14. 一种药物组合物，其含有治疗有效量的根据权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  15. 一种分离的核酸分子，其编码权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体。
  16. 一种重组载体，其包含权利要求15所述的分离的核酸分子。
  17. 一种用如权利要求16所述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞。
  18. 用于生产如权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体的方法，所述方法包括将权利要求15的宿主细胞在培养基中进行培养以形成并积累权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体，以及从培养物回收所述单克隆抗体或其抗原结合片段、所述多特异性抗体或所述单链抗体。
  19. 用于体外检测或测定人CD96的方法，所述方法包括使用权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体。
  20. 权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体在制备用于检测或测定人CD96的试剂中的用途。
  21. 一种减少或缓解免疫抑制的方法，所述方法包括向受试者施用治疗有效量的权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体，或权利要求14所述的药物组合物，或权利要求15所述的分离的核酸分子。
  22. 一种增强NK细胞活性的方法，所述方法包括使用权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体，或权利要求14所述的药物组合物，或权利要求15所述的分离的核酸分子降低CD96活性的步骤。
  23. 一种治疗与人CD96相关的疾病的方法，所述方法包括向受试者施用治疗有效量的权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体，或权利要求14所述的药物组合物，或权利要求15所述的分离的核酸分子，以治疗人CD96相关的疾病，所述疾病优选肿瘤、癌症或感染性疾病。
  24. 如权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体，或权利要求14所述的药物组合物，或权利要求15所述的分离的核酸分子或其组合在制备治疗或预防疾病或病症的药物中的用途，其中所述疾病或病症是人CD96相关疾病，所述疾病或病症优选肿瘤、癌症或感染性疾病。
  25. 作为药物的权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体，或权利要求14所述的药物组合物，或权利要求15所述的分离的核酸分子。
  26. 如权利要求25所述的作为药物的权利要求1～11任一项所述的单克隆抗体或其抗原结合片段、权利要求12所述的多特异性抗体或权利要求13所述的单链抗体，或权利要求14所述的药物组合物，或权利要求15所述的分离的核酸分子，其中所述的药物用于人CD96相关的疾病的治疗，所述疾病优选肿瘤、癌症或感染性疾病。
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