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WO2016015675A1 - 抗ctla4的单克隆抗体或其抗原结合片段、药物组合物及用途 - Google Patents

抗ctla4的单克隆抗体或其抗原结合片段、药物组合物及用途 Download PDF

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Publication number
WO2016015675A1
WO2016015675A1 PCT/CN2015/085721 CN2015085721W WO2016015675A1 WO 2016015675 A1 WO2016015675 A1 WO 2016015675A1 CN 2015085721 W CN2015085721 W CN 2015085721W WO 2016015675 A1 WO2016015675 A1 WO 2016015675A1
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Prior art keywords
seq
antibody
ctla4
antigen
monoclonal antibody
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PCT/CN2015/085721
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English (en)
French (fr)
Inventor
李百勇
夏瑜
王忠民
张鹏
庞醒华
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中山康方生物医药有限公司
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Priority to CN202211206595.7A priority Critical patent/CN115960232A/zh
Priority to MX2017001446A priority patent/MX2017001446A/es
Priority to US15/500,744 priority patent/US10449251B2/en
Priority to NZ729158A priority patent/NZ729158A/en
Priority to AP2017009762A priority patent/AP2017009762A0/en
Priority to EP20216429.9A priority patent/EP3858861A1/en
Priority to PL15827441T priority patent/PL3176181T3/pl
Priority to ES15827441T priority patent/ES2857509T3/es
Application filed by 中山康方生物医药有限公司 filed Critical 中山康方生物医药有限公司
Priority to RS20210312A priority patent/RS61590B1/sr
Priority to SI201531555T priority patent/SI3176181T1/sl
Priority to DK15827441.5T priority patent/DK3176181T3/da
Priority to EP15827441.5A priority patent/EP3176181B1/en
Priority to EA201790288A priority patent/EA036396B1/ru
Priority to KR1020177005688A priority patent/KR102017396B1/ko
Priority to TN2017000010A priority patent/TN2017000010A1/en
Priority to MA40474A priority patent/MA40474B1/fr
Priority to MDA20170022A priority patent/MD4795C1/ro
Priority to MYPI2017000162A priority patent/MY192822A/en
Priority to BR112017002080-7A priority patent/BR112017002080B1/pt
Priority to JP2017525666A priority patent/JP6514774B2/ja
Priority to CA2956000A priority patent/CA2956000C/en
Priority to UAA201701943A priority patent/UA119570C2/uk
Priority to LTEP15827441.5T priority patent/LT3176181T/lt
Priority to CN201580040171.XA priority patent/CN106687479B/zh
Priority to AU2015295936A priority patent/AU2015295936C1/en
Priority to SG11201700819QA priority patent/SG11201700819QA/en
Priority to CR20170033A priority patent/CR20170033A/es
Publication of WO2016015675A1 publication Critical patent/WO2016015675A1/zh
Priority to ZA2017/00528A priority patent/ZA201700528B/en
Priority to IL250321A priority patent/IL250321B/en
Priority to CONC2017/0000754A priority patent/CO2017000754A2/es
Priority to PH12017500190A priority patent/PH12017500190A1/en
Priority to AU2018247270A priority patent/AU2018247270A1/en
Priority to US16/562,236 priority patent/US11291720B2/en
Priority to CY20211100197T priority patent/CY1124190T1/el
Priority to HRP20210448TT priority patent/HRP20210448T1/hr
Priority to US17/695,592 priority patent/US20230064544A1/en

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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • G01—MEASURING; TESTING
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    • 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
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    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • 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
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K2317/70—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • G—PHYSICS
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Definitions

  • the present invention relates to the field of tumor therapy and molecular immunology, and relates to monoclonal antibodies against CTLA4 or antigen-binding fragments thereof, pharmaceutical compositions thereof, sequences encoding the same, and methods of using the same for diagnosis, prevention, treatment and/or adjuvant therapy And use.
  • the cytotoxic T lymphocyte sociated antigen 4 (also referred to as CTLA4) has a close relationship with the CD28 molecule in gene structure, chromosomal location, sequence homology and gene expression.
  • the receptor for molecular B7 is mainly expressed on the surface of activated T cells.
  • CTLA4 and CD28 molecules have opposite functions.
  • CTLA4 binds to B7 and inhibits the activation of mouse and human T cells, and plays a negative regulatory role in T cell activation.
  • CTLA4 mAb or CTLA4 ligand can prevent CTLA4 from binding to its natural ligand, thereby blocking CTLA4's negative regulation of T cell signaling and enhancing the reactivity of T cells to various antigens.
  • CTLA4 mAb (10D1, 11.2.2) is currently in clinical trials for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, malignant melanoma, etc. (Grosso JF., Jure-Kunkel MN. , CTLA-4 blockade in tumor models: an overview of preclinical and translational research. Cancer Immun. 2013; 13: 5. Epub 2013 Jan 22; US 6984720 B1 and US 6682736 B1), where 10D1 and 11.2.2 are considered current One of the most effective CTLA4 monoclonal antibodies.
  • Interleukin 2 is produced by T cells, is a growth factor that regulates T cell subsets, is also an important factor regulating immune response, and can promote the proliferation of activated B cells, participate in antibody response, hematopoiesis and tumor surveillance.
  • Recombinant human IL-2 has been approved by the US FDA for the treatment of malignant tumors (including melanoma, kidney tumors, etc.) while ongoing treatment Clinical study of chronic viral infection (Chavez, A.R., et al., Pharmacologic administration of interleukin-2. Ann N Y Acad Sci, 2009.1182: p. 14-27).
  • CTLA4 and CTLA4 mAbs are important factors influencing the function of T cells. By interfering with the immune microenvironment of the body, they can produce specific therapeutic effects on diseases, and exert high therapeutic effects, supplementing the deficiency of traditional drugs, thus opening up new ways of gene therapy. .
  • CTLA4 and CTLA4 mAbs are used in various stages of testing and clinical practice: such as effective inhibition of airway hyperresponsiveness in animal models of asthma, prevention of development of rheumatic diseases, and mediation of immune tolerance in allogeneic transplantation in autoimmune diseases Wait.
  • the present inventors used a mammalian cell expression system to express recombinant CTLA4 as an antigen to immunize mice, and obtained hybridoma cells by fusion of mouse spleen cells with myeloma cells.
  • the inventors obtained a screening of a large number of samples to obtain a specific monoclonal antibody capable of secreting specific binding to CTLA4, and the monoclonal antibody was able to block the binding of CTLA4 and B7 very effectively. Further, humanized antibodies were prepared. The following invention is thus provided:
  • One aspect of the invention relates to a monoclonal antibody or antigen-binding fragment thereof, wherein
  • the monoclonal antibody comprises a complementarity determining region (CDR) selected from the group consisting of:
  • An HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and
  • An HCDR3 comprising the amino acid sequence of SEQ ID NO: 29;
  • An LCDR1 comprising the amino acid sequence of SEQ ID NO: 30,
  • An LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and
  • An LCDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.
  • the amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 and SEQ ID NO: 18.
  • the amino acid sequence of the light chain variable region (VL) of the monoclonal antibody is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22, and SEQ ID NO: 24.
  • the monoclonal antibodies include:
  • VH as shown in SEQ ID NO: 6 and VL as shown in SEQ ID NO: 8;
  • VH as shown in SEQ ID NO: 10 and VL as shown in SEQ ID NO: 12;
  • the above monoclonal antibodies of the (1)-(6) group are the amino acid sequences of the heavy chain variable region and the light chain variable region of 8D2/8D2 (Re), 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17, respectively.
  • the methionine (Met) at position 18 in SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 is independently replaced with an amino acid selected from the group consisting of:
  • Leucine Leu
  • valine Val
  • isoleucine Ile
  • alpha isoleucine
  • Al alanine
  • Antibody therapeutics particularly monoclonal antibodies (MABs) have achieved good results in the treatment of a variety of diseases.
  • Traditional experimental methods for obtaining these therapeutic antibodies are to immunize animals with antigens, to obtain antibodies that target antigens in immunized animals, or to improve antibodies that have lower affinity for antigens by affinity maturation.
  • these methods require a lot of time and effort, and most of the time they do not target specific epitopes on the antigen.
  • variable regions of the light and heavy chains determine the binding of the antigen; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3
  • CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3; which is named by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Vol. 1-3, NIH Publication 91-3242, Bethesda Md ).
  • amino acid sequences of the CDR regions of the monoclonal antibody sequences in the above items (1) to (6) are analyzed by technical means well known to those skilled in the art, for example, by the VBASE2 database, and the results are as follows:
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFTFSDNW (SEQ ID NO: 27)
  • HCDR2 IRNKPYNYET (SEQ ID NO: 28)
  • HCDR3 TAQFAY (SEQ ID NO: 29)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 ENIYGG (SEQ ID NO: 30)
  • LCDR2 GAT (SEQ ID NO: 31)
  • LCDR3 QNVLRSPFT (SEQ ID NO: 32)
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFTFSDNW (SEQ ID NO: 27)
  • HCDR2 IRNKPYNYET (SEQ ID NO: 28)
  • HCDR3 TAQFAY (SEQ ID NO: 29)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 ENIYGG (SEQ ID NO: 30)
  • LCDR2 GAT (SEQ ID NO: 31)
  • LCDR3 QNVLRSPFT (SEQ ID NO: 32)
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFTFSDNW (SEQ ID NO: 27)
  • HCDR2 IRNKPYNYET (SEQ ID NO: 28)
  • HCDR3 TAQFAY (SEQ ID NO: 29)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 ENIYGG (SEQ ID NO: 30)
  • LCDR2 GAT (SEQ ID NO: 31)
  • LCDR3 QNVLRSPFT (SEQ ID NO: 32)
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFTFSDNW (SEQ ID NO: 27)
  • HCDR2 IRNKPYNYET (SEQ ID NO: 28)
  • HCDR3 TAQFAY (SEQ ID NO: 29)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 ENIYGG (SEQ ID NO: 30)
  • LCDR2 GAT (SEQ ID NO: 31)
  • LCDR3 QNVLRSPFT (SEQ ID NO: 32)
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFTFSDNW (SEQ ID NO: 27)
  • HCDR2 IRNKPYNYET (SEQ ID NO: 28)
  • HCDR3 TAQFAY (SEQ ID NO: 29)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 ENIYGG (SEQ ID NO: 30)
  • LCDR2 GAT (SEQ ID NO: 31)
  • LCDR3 QNVLSRHPG (SEQ ID NO: 33)
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFTFSDNW (SEQ ID NO: 27)
  • HCDR2 IRNKPYNYET (SEQ ID NO: 28)
  • HCDR3 TAQFAY (SEQ ID NO: 29)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 ENIYGG (SEQ ID NO: 30)
  • LCDR2 GAT (SEQ ID NO: 31)
  • LCDR3 QNVLSSRPG (SEQ ID NO: 34)
  • the monoclonal antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, dAb, A complementarity determining region fragment, a single chain antibody (eg, scFv), a humanized antibody, a chimeric antibody, or a diabody.
  • the monoclonal antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein said monoclonal antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 K D , 10 -9 M or 10 -10 M or less binds to the CTLA4 protein.
  • the monoclonal antibody comprises a non-CDR region and the non-CDR region is from a species other than a murine, such as from a human antibody.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention is a monoclonal antibody against CTLA4 or an antigen-binding fragment thereof, which is capable of specifically binding to CTLA4.
  • the monoclonal antibody or antigen-binding fragment thereof for use in the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of a tumor; in particular, the tumor is selected from the group consisting of melanoma, renal tumor , prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer and liver cancer.
  • Another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, wherein
  • the antibody heavy chain variable region comprises a CDR having the amino acid sequence of SEQ ID NOs: 27-29;
  • the antibody heavy chain variable region has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 or SEQ ID NO: 18.
  • nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13 or SEQ ID NO: 17.
  • the invention also provides an isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof of the invention.
  • nucleic acid molecules can be isolated from hybridoma cells, or can be obtained by genetic engineering recombinant techniques or chemical synthesis methods.
  • a further aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding a variable region of an antibody light chain, wherein
  • the antibody light chain variable region comprises:
  • amino acid sequence is the CDR of SEQ ID NO: 30-32;
  • amino acid sequences are the CDRs of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 33;
  • amino acid sequences are the CDRs of SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 34;
  • the antibody light chain variable region has the SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: Amino acid sequence;
  • nucleic acid molecule has the nucleotide set forth in SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 or SEQ ID NO: sequence.
  • a further aspect of the invention relates to a vector comprising the isolated nucleic acid molecule of any of the invention.
  • the vector of the present invention may be a cloning vector or an expression vector.
  • the vector of the invention is, for example, a plasmid, a cosmid, a phage, a cosmid, and the like.
  • a further aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of any of the invention, or the vector of the invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the cells of the invention may also be cell lines, such as 293T cells.
  • a further aspect of the invention relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof according to any of the invention, which comprises culturing a host cell of the invention under suitable conditions, and recovering from the cell culture The step of the monoclonal antibody or antigen-binding fragment thereof.
  • a further aspect of the invention relates to a conjugate comprising a monoclonal antibody or antigen-binding fragment thereof, and a conjugated portion, wherein the monoclonal antibody is a monoclonal antibody or antigen-binding thereof according to any one of the invention a fragment, the coupling moiety being a detectable label; in particular, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme (eg horseradish peroxidase).
  • a further aspect of the invention relates to a kit comprising the single of any one of the inventions Cloning an antibody or antigen-binding fragment thereof, or comprising a conjugate of the invention;
  • the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance , a luminescent substance, a colored substance or an enzyme (for example, horseradish peroxidase).
  • a detectable label such as a radioisotope, a fluorescent substance , a luminescent substance, a colored substance or an enzyme (for example, horseradish peroxidase).
  • a further aspect of the invention relates to the use of a monoclonal antibody or antigen-binding fragment thereof according to any of the invention in a kit for detecting the presence or level of CTLA4 in a sample.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of the invention or a conjugate of the invention; optionally further comprising a pharmaceutically acceptable Accepted carriers and/or excipients.
  • a further aspect of the invention relates to a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a medicament for the prophylaxis and/or treatment and/or adjuvant treatment and/or diagnosis of a tumor
  • the tumor is selected from the group consisting of melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, and liver cancer.
  • a further aspect of the invention relates to the use of a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a medicament as follows:
  • a drug that increases the expression of IL-2 in T lymphocytes is a drug that increases the expression of IL-2 in T lymphocytes.
  • a further aspect of the invention relates to an in vivo or in vitro method comprising applying a cell An effective amount of the monoclonal antibody or antigen-binding fragment thereof of any one of the invention or the conjugate of the invention, the method being selected from the group consisting of:
  • a method for increasing IL-2 expression in T lymphocytes is provided.
  • the method can be used for diagnostic or therapeutic purposes, or for non-diagnostic or therapeutic purposes (eg, the sample is a cell sample, not a sample from a patient).
  • a further aspect of the invention relates to a method of preventing and/or treating and/or adjunctively treating and/or diagnosing a tumor comprising administering to the subject an effective amount of the monoclonal antibody or antigen thereof of any one of the invention
  • the step of binding a fragment or a monoclonal antibody conjugate of the present invention; specifically, the tumor is selected from the group consisting of melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, and liver cancer.
  • CTLA4 protein Cytotoxic T-Lymphocyte Antigen 4
  • CTLA4 ECD extracellular fragment of CTLA4
  • Fragment also includes a fusion protein of CTLA4ECD, such as a fragment fused to a Fc protein fragment (mFc) of mouse IgG (SEQ ID NO: 3).
  • mFc Fc protein fragment
  • CTLA4 protein shall include all such sequences, including the sequences set forth in SEQ ID NO: 2, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the CTLA4 protein, it includes not only the sequence fragment of SEQ ID NO: 2 but also the corresponding sequence fragment in its natural or artificial variant.
  • the B7 is B7-1 and/or B7-2; the specific protein sequence is a sequence known in the art, and reference may be made to the existing literature or the sequence disclosed in GenBank. .
  • B7-1 CD80, NCBI Gene ID: 941
  • B7-2 CD86, NCBI Gene ID: 942).
  • EC 50 refers to the term as used herein, half-maximal effective concentration (concentration for 50% of maximal effect ), the concentration refers to cause 50% of maximal effect.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair having one "light” (L) chain and one "heavy” (H) chain. .
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain is comprised of a heavy chain variable region (V H) and a heavy chain constant region (C H) composition.
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with regions are more conserved, termed framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • 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.
  • an antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full length antibody that retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or compete with the full length antibody.
  • Specific binding to an antigen which is also referred to as an "antigen-binding portion.” See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes.
  • an antigen-binding fragment of an antibody is produced by enzymatic or chemical cleavage of an intact antibody.
  • the antigen-binding fragment includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDRs). Fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Fd fragment means an antibody fragment consisting of V H and C H 1 domains
  • Fv fragment means a single arm of V H and V L domains of an antibody, Antibody fragment
  • dAb fragment means an antibody fragment consisting of a VH domain (Ward et al, Nature 341:544-546 (1989))
  • Fab fragment means by V L , V H , C antibody fragments L and C H 1 domains
  • F (ab ') 2 fragment means antibody fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • the antigen-binding fragment is a single chain antibody (e.g., the scFv), wherein V L and V H domains are paired to form so that it can be produced by a linker to a single polypeptide chain monovalent molecules (see, e.g., Bird Et al, Science 242: 423-426 (1988) and Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).
  • scFv molecules can have the general structure: NH 2 -V L - linker -V H -COOH or NH 2 -V H - linker -V L -COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention 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 antigen-binding fragments are diabodies, i.e., bivalent antibodies in which V H and V L, domains are expressed on a single polypeptide chain, but using a linker that is too short to not allow the same chain in two Pairing between domains forces the domain to pair with the complementary domain of another strand and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 -6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • Antibodies can be obtained from a given antibody (eg, monoclonal antibody 4B3, 13A10, 12B9 or 4H4 provided herein) using conventional techniques known to those skilled in the art (eg, recombinant DNA techniques or enzymatic or chemical cleavage methods).
  • An antigen-binding fragment for example, the above-described antibody fragment
  • an antigen-binding fragment of the antibody is specifically screened in the same manner as used for the intact antibody.
  • antibody As used herein, unless the context clearly dictates otherwise, when referring to the term “antibody”, it includes not only intact antibodies, but also antigen-binding fragments of antibodies.
  • mAb and “monoclonal antibody” refer to a fragment of an antibody or antibody from a population of highly homologous antibody molecules, ie, in addition to a natural mutation that may occur spontaneously, A group of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least two or more different antibodies, which typically recognize different epitopes on the antigen.
  • Monoclonal antibodies are typically obtained using hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see, for example, U.S. Patent 4,816,567).
  • monoclonal antibodies are numbered and are numbered from the same number
  • the monoclonal antibodies obtained by the tumor were the same.
  • monoclonal antibody 4B3 (or 13A10, 12B9 or 4H4) is the same antibody as that obtained from hybridoma cell line 4B3 (or 13A10, 12B9 or 4H4) or its subcloned or progeny cells, respectively.
  • chimeric antibody refers to an antibody whose light chain or/and a portion of a heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or Subclass), and another portion of the light or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belonging to the same or different antibody class or subclass), but in any case, it remains Binding activity to the antigen of interest (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • humanized antibody means that all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by a CDR region of a non-human antibody (donor antibody).
  • An antibody or antibody fragment, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody having the desired specificity, affinity or reactivity.
  • some of the amino acid residues of the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of the corresponding non-human antibody or by amino acid residues of other antibodies to further refine or optimize the performance of the antibody.
  • neutralizing antibody refers to an antibody or antibody fragment that is capable of clearing or significantly reducing the virulence of a target virus (eg, the ability to infect a cell).
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also referred to in the art as an "antigenic determinant.”
  • An epitope or antigenic determinant typically consists of a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and typically has specific three dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes 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, which may be "linear" "or” conformational.
  • the terms “isolated” or “isolated” refer to artificially obtained from a natural state. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or that the substance has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • separation the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • the term “isolated” or “isolated” does not exclude the inclusion of artificial or synthetic materials, nor does it exclude the presence of other impure substances that do not affect the activity of the material.
  • E. coli expression system refers to an expression system consisting of E. coli (strain) and a vector, wherein E. coli (strain) is derived from a commercially available strain such as, but not limited to, GI698 , ER2566, BL21 (DE3), B834 (DE3), BLR (DE3).
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the essential properties of a protein/polypeptide comprising an amino acid sequence. For example, it can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Keep the replacement. Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.). A family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • the term "immunogenicity” refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It means that the antigen can stimulate specific immune cells, activate, proliferate and differentiate immune cells, and finally produce the characteristics of immune effector substances such as antibodies and sensitized lymphocytes. It also means that after the antigen stimulates the body, the body's immune system can form antibodies or A specific immune response to sensitized T lymphocytes. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the mode of immunization.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D refers to a particular antibody - antigen interaction dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • the antibody e.g., monoclonal antibody 4B3, 13A10, 12B9 or 4H4 of the invention
  • the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M
  • a dissociation equilibrium constant (K D ) of 10 -10 M or less binds to an antigen (eg, L1 protein), for example, as determined using surface plasmon resonance (SPR) in a BIACORE instrument.
  • SPR surface plasmon resonance
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • hybridomas and “hybridoma cell lines” are used interchangeably and, when referring to the terms “hybridomas” and “hybridoma cell lines”, they also include subclones of hybridomas. And progeny cells. For example, when referring to hybridoma cell line 4B3, it also refers to subcloning and progeny cells of hybridoma cell line 4B3.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ions. Strength enhancer.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
  • adjuvant refers to a non-specific immunopotentiator that, when brought together with an antigen or pre-delivered into the body, enhances the body's immune response to the antigen or alters the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • an effective amount for preventing a disease for example, a CTLA4 binding to B7 or a CTLA4 activity associated with a disease such as a tumor
  • an amount sufficient to prevent, prevent, or delay a disease for example, a disease associated with CTLA4 binding to B7 or excessive activity of CTLA4 such as a tumor.
  • An amount effective to treat; an amount effective to treat a disease is an amount sufficient to cure or at least partially arrest a disease and a complication thereof in a patient already suffering from the disease. Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • the monoclonal antibody 8D2 of the present invention and the humanized antibody thereof are well specific for binding to CTLA4, wherein the binding efficiency of the antibody 8D2, 8D2(Re) to the murine CTLA4 antigen is stronger than that of the control antibody 10D1 (Alan J. Korman) , Edward L. Halk, et al., HUMAN CTLA-4 ANTIBODIES, United State Patent No. US 6984720 B1) and 11.2.1 (Douglas Charles Hanson, Mark Joseph Neveu, et al., Human monoclonal antibodies to CTLA-4, United State Patent No. US 682736 B1).
  • the binding efficiency of the humanized antibody 8D2H1L1 to the murine CTLA4 antigen was stronger than that of the control antibody 10D1, which was comparable to 11.2.1.
  • the binding efficiency of the humanized antibody 8D2H2L2 to the human CTLA4 antigen was comparable to that of 10D1.
  • the binding efficiency of the humanized antibodies 8D2H2L2 and 8D2H3L3 to the monkey CTLA4 antigen was comparable to that of 10D1.
  • the binding efficiency of the antibodies 8D2H2L15 and 8D2H2L17 to the human CTLA4 antigen was stronger than that of the control antibodies 10D1 and 11.2.1.
  • the antibodies 8D2, 8D2 (Re) and 8D2 humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, 8D2H2L17 competed with B7 for binding to the antigen CTLA4.
  • 8D2, 8D2 (Re), 8D2H1L1, 8D2H2L2 and B7-2 compete with CTLA4 stronger than 10D1; 8D2H1L1, 8D2H2L2, 8D2H3L3; 8D2H2L15, 8D2H2L17 and B7-1, B7-2 competitive binding CTLA4 is stronger than antibody 10D1 And 11.2.1.
  • the monoclonal antibody 8D2 of the present invention and the humanized antibody thereof can block the binding of CLTA4 and B7 very effectively, specifically relieve CTLA4 from immunosuppression of the body, and activate T lymphocytes.
  • 8D2H2L2 and 8D2H2L15 were stronger against T lymphocytes than control antibodies 10D1 and 11.2.1.
  • Figure 1 Results of SDS-PAGE detection of CTLA4 ECD-mFc fusion protein.
  • the samples from the four lanes from left to right and their loadings were: M: marker 10 ⁇ L; CTLA4 ECD-mFc fusion protein 1 ⁇ g; CTLA4 ECD-mFc fusion protein 2 ⁇ g; CTLA4 ECD-mFc fusion protein 3 ⁇ g.
  • Figure 2 Results of SDS-PAGE detection of 8D2 antibody.
  • the samples from the left to the right of the four lanes were loaded with M:marker 10 ⁇ L; reduced protein electrophoresis loading buffer sample antibody 0.3 ⁇ g; non-reduced protein electrophoresis loading buffer 2 ⁇ L; non-reduced type Protein electrophoresis loading buffer sample antibody 0.3 ⁇ g.
  • Figure 3 Results of SDS-PAGE detection of 8D2 recombinant antibody (8D2 (Re)).
  • the samples from the left to the right of the four lanes were loaded with: M:marker 10 ⁇ L; reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g; non-reduced protein electrophoresis loading buffer 2 ⁇ L; non-reduced protein Electrophoresis loading buffer sample antibody 1 ⁇ g.
  • Figure 4 Results of SDS-PAGE detection of humanized antibody 8D2H1L1 of 8D2.
  • the samples from the left to the right of the four lanes were loaded with: M:marker 10 ⁇ L; reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g; non-reduced protein electrophoresis loading buffer 2 ⁇ L; non-reduced protein Electrophoresis loading buffer sample antibody 1 ⁇ g.
  • Figure 5 Results of SDS-PAGE detection of humanized antibody 8D2H2L2 of 8D2.
  • the samples from the left to the right of the four lanes were loaded with: M:marker 10 ⁇ L; reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g; non-reduced protein electrophoresis loading buffer 2 ⁇ L; non-reduced protein Electrophoresis loading buffer sample antibody 1 ⁇ g.
  • Figure 6 Results of SDS-PAGE detection of humanized antibody 8D2H3L3 of 8D2.
  • the samples from the four lanes from left to right and their loadings were: M:marker 10 ⁇ L; Prototype protein electrophoresis loading buffer sample antibody 1 ⁇ g; non-reduced protein electrophoresis loading buffer 2 ⁇ L; non-reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g.
  • Figure 7 Results of SDS-PAGE detection of humanized antibody 8D2H2L15 of 8D2.
  • the sample and the amount of the sample were respectively: M:marker 10 ⁇ L; 1: non-reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g; 2: reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g.
  • Figure 8 Results of SDS-PAGE detection of humanized antibody 8D2H2L17 of 8D2.
  • the sample and the amount of the sample were respectively: M:marker 10 ⁇ L; 1: non-reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g; 2: reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g.
  • Figure 14 Results of the kinetic characteristic parameters of 8D2H2L17.
  • Figure 15 Flow cytometry detection of 293F cell label-free, isotype control, CTLA4 expression histogram of 293F-CTLA4 cells (cell number-fluorescence (FITC)).
  • Figure 16 Flow cytometry to detect the mean fluorescence intensity (MFI) of CTLA4 expression in 293F cells without label, isotype control, 293F-CTLA4 cells.
  • MFI mean fluorescence intensity
  • Figure 17 Binding of monoclonal antibody 8D2 and EC 50 results 293F-CTLA4 labeled cells.
  • Fig 18 EC bound 8D2 (Re) labeled antibody to cells 50 293F-CTLA4 results.
  • FIG 19 EC 50 in combination with the results 8D2H1L1 293F-CTLA4 labeled cells.
  • Figure 20 Binding of the labeled 8D2H2L2 293F-CTLA4 cells EC 50 results.
  • FIG 21 8D2H3L3 293F-CTLA4 binding to cells labeled EC 50 results.
  • Figure 22 ELISA method detects binding of 8D2, 8D2H1L1, 8D2 recombinant antibody to CTLA4.
  • Figure 23 ELISA method detects binding of 8D2H2L2, 8D2H3L3 recombinant antibody to human CTLA4.
  • Figure 24 ELISA method detects binding of 8D2H2L2, 8D2H3L3 recombinant antibody to monkey CTLA4.
  • Figure 25 ELISA method detects binding of 8D2H2L15, 8D2H2L17 recombinant antibody to monkey CTLA4.
  • Figure 26 Results of the 8D2, 8D2H1L1, 8D2 recombinant antibody and B7-1 competition ELISA.
  • Figure 27 Competition ELISA results for 8D2, 8D2H1L1, 8D2 recombinant antibodies and B7-2.
  • Figure 28 Competition ELISA results for 8D2H2L2, 8D2H3L3 antibody and B7-1.
  • Figure 30 Results of competition ELISA between 8D2H2L15 and 8D2H2L17 antibodies and B7-1.
  • Figure 31 Competition ELISA results for 8D2H2L15, 8D2H2L17 antibody and B7-2.
  • Figure 32 Effect of peripheral blood mononuclear cells (PBMC), Raji cells and humanized antibodies 8D2H1L1, 8D2H2L2, and 8D2H3L3 after 72 hours of co-culture for 72 hours.
  • PBMC peripheral blood mononuclear cells
  • the results show that the humanized antibody of monoclonal antibody 8D2 enhances the secretion of IL-2 by T lymphocytes by blocking the receptor of CTLA4.
  • Figure 33 Effect of peripheral blood mononuclear cells (PBMC), Raji cells and humanized antibodies 8D2H2L15, 8D2H2L17 for 72 hours after co-culture for 72 hours.
  • PBMC peripheral blood mononuclear cells
  • the results show that the humanized antibody of monoclonal antibody 8D2 enhances the secretion of IL-2 by T lymphocytes by blocking the receptor of CTLA4.
  • Figure 34 Tumor growth curve of subcutaneously transplanted 8D2H2L2 in the hu-SCID-raji model.
  • the BALB/C mice used were purchased from the Guangdong Medical Laboratory Animal Center.
  • the T cells used were from Zhongshan Kangfang Biomedical Co., Ltd.
  • Control antibody 10D1 was prepared with reference to US Pat. No. 6,984, 420 B1; 11.2.1 was prepared with reference to US 6,682,736 B1.
  • Example 1 Obtainment and monoclonalization of CTLA4-8D2 hybridoma cell line LT001 Preparation of antibody 8D2
  • the mammalian cell expression system is used to express recombinant CTLA4 as an antigen to immunize mice, and the mouse spleen cells are fused with myeloma cells to obtain hybridoma cells.
  • a hybridoma cell line (CTLA4-8D2 hybridoma cell line LT001) was obtained, which was able to secrete monoclonal antibody 8D2 which specifically binds to CTLA4.
  • the specific method is as follows:
  • Amino acid (SEQ ID NO: 2) corresponding to the extracellular fragment CTLA4ECD (Cytotoxic T-Lymphocyte Antigen 4, NCBI Gene ID: 1493, SEQ ID NO: 1) of the gene CTLA4 and an Fc protein fragment (mFc) of mouse IgG A fusion design (SEQ ID NO: 3) was carried out, wherein mFc refers to an Fc protein fragment of murine IgG, the amino acid sequence of which is shown in the underlined portion of SEQ ID NO: 3.
  • CTLA4ECD Cytotoxic T-Lymphocyte Antigen 4, NCBI Gene ID: 1493, SEQ ID NO: 1
  • mFc Fc protein fragment of mouse IgG
  • a fusion design (SEQ ID NO: 3) was carried out, wherein mFc refers to an Fc protein fragment of murine IgG, the amino acid sequence of which is shown in the underlined portion of SEQ ID NO: 3.
  • Jinsi Rui Company was commissioned to optimize the nucleic acid sequence corresponding to the SEQ ID NO: 3 protein sequence, and the optimization mainly considered codon preference, GC content, and mRNA II. Stage structure, repeating sequence and other factors.
  • the sequence of the final CTLA4 ECD-mFc fusion protein gene was optimized as follows (SEQ ID NO: 4) and was commissioned by Kingsray.
  • CTLA4ECD encoded protein sequence (125aa)
  • CTLA4ECD-mFc fusion protein sequence (364aa)
  • the wavy underline is the CTLA4ECD portion
  • the solid underline is the mFc portion.
  • the wavy underline is the CTLA4ECD part
  • the solid underline is mFc part.
  • the synthetic CTLA4ECD-mFc fusion gene (SEQ ID NO: 4) was cloned into the pUC57simple (provided by Kingsray) expression vector by Kingsray, and the pUC57simple-CTLA4ECD-mFc plasmid was obtained.
  • the plasmid pUC57simple-CTLA4ECD-mFc was digested (Xba I and BamH I), and the fusion gene fragment CTLA4ECD-mFc obtained by electrophoresis was ligated with the pcDNA3.1 expression vector (purchased from Invitrogen) to obtain pcDNA3.1-CTLA4ECD.
  • -mFc transfected competent E. coli cells DH5a (purchased from TIANGEN), transfection and culture were carried out according to the instructions.
  • the positive pcDNA3.1-CTLA4ECD-mFc clone colony was screened, and the Escherichia coli was amplified according to the conventional method, and then extracted by a kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., DP103-03) and according to the instructions of the kit.
  • the pcDNA3.1-CTLA4ECD-mFc recombinant plasmid was obtained.
  • the recombinant plasmid pcDNA3.1-CTLA4ECD-mFc was transfected into 293F cells (purchased from Invitrogen) according to the lipofectamin transfection kit (purchased from Invitrogen).
  • the culture medium was purified by high-speed centrifugation, microfiltration membrane filtration and HiTrap protein A HP column for purification of CTLA4ECD-mFc fusion protein.
  • the sample was added to a reduced protein electrophoresis loading buffer and subjected to SDS-PAGE electrophoresis. As shown in Figure 1, the target protein is approximately 45 kD.
  • CTLA4ECD-mFc fusion protein as antigen, spleen cells from immunized BALB/C mice (purchased from Guangdong Medical Experimental Animal Center) were fused with mouse myeloma cells into hybridoma cells, according to the currently established method (eg, Stewart , SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. GC Howard and DR Bethell, Boca Raton: CRC Press, 2000).
  • CTLA4 as an antigen coating enzyme plate, screening by indirect ELISA Hybridoma cells that secrete new antibodies that specifically bind to CTLA4.
  • Hybridoma cells screened by indirect ELISA were screened by competition ELISA to secrete a single cell that competes with ligand B7-1 (CD80, NCBI Gene ID: 941), B7-2 (CD86, NCBI Gene ID: 942) for binding to CTLA4.
  • the hybridoma cell strain of the antibody was cloned, and a stable hybridoma cell strain was obtained by a limiting dilution method.
  • the hybridoma cell line was named as a CTLA4-8D2 hybridoma cell line, and a CTLA4-8D2 stable cell line (also referred to as LT001 cells in the present invention, and the secreted monoclonal antibody was named 8D2) was obtained by a limiting dilution method.
  • CTLA4-8D2 (LT001) cell line of the present invention was cultured with 10% low IgG fetal bovine serum, and the cell culture supernatant was collected 7 days later to be purified to prepare antibody 8D2.
  • the purified sample is separately added to a reduced protein electrophoresis loading buffer and a non-reduced protein electrophoresis loading buffer, and then boiled for detection.
  • the results showed that the target protein of the reduced protein sample was approximately 50 kD and 25 kD, and the target protein of the non-reduced protein sample was approximately 150 kD (Fig. 2).
  • mRNA was extracted from the CTLA4-8D2 hybridoma cell strain (LT001 cells) prepared in Example 1 according to the method of culturing the cell bacterial total RNA extraction kit (Tiangen, Cat. No. DP430).
  • the cDNA was synthesized by the III First-Strand Synthesis System for RT-PCR kit and subjected to PCR amplification.
  • the PCR amplification product was directly subjected to TA cloning, and the specific procedure was carried out in accordance with the instructions of the pEASY-T1 Cloning Kit (Transgen CT101) kit.
  • the product of the TA clone was directly sequenced, and the sequencing results were as follows:
  • Example 3 Humanized antibody 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 And the design of the light and heavy chain sequences of 8D2H2L17
  • variable region sequences of 8D2H3L3, 8D2H2L15 and 8D2H2L17 are as follows:
  • Example 4 8D2 recombinant antibody 8D2 (Re) and 8D2 humanized antibody 8D2H1L1 Preparation and SDS-PAGE of 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17 Electrophoresis detection
  • the 8D2 heavy chain cDNA sequence (the variable region sequence thereof is shown in SEQ ID NO: 5) and the light chain cDNA sequence (the variable region sequence thereof are shown in SEQ ID NO: 7) were cloned into pUC57simple (Kings) In the vector provided by Rui Company, the pUC57simple-8D2H and pUC57simple-8D2L plasmids were obtained, respectively.
  • the plasmids pUC57simple-8D2H and pUC57simple-8D2L were digested respectively (HindIII & EcoRI), and the heavy chain light chains recovered by electrophoresis were subcloned into pcDNA3.1 vector, and the recombinant plasmid was co-transfected into 293F cells. After 7 days of cell culture, the culture solution was purified by high-speed centrifugation, microfiltration membrane vacuum filtration and HiTrap protein A HP column, and the purified sample was separately added to reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis. The sample buffer was boiled and subjected to SDS-PAGE electrophoresis. As shown in Fig. 3, the reduced protein sample target protein is approximately 50 kD and 25 KD, and the non-reduced protein sample target protein is approximately 150 kD.
  • the heavy chain cDNAs of 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17 (the variable region sequences thereof are SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 13) and the light chain cDNA (the variable region sequences thereof are shown in SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, respectively)
  • the plasmid was cloned into pUC57simple (provided by Kingsray) to obtain pUC57simple-8D2H1L1, pUC57simple-8D2H2L2, pUC57simple-8D2H3L3, pUC57simple-8D2H2L15, pUC57simple-8D2H2L17 plasmid, and subcloned into pcDNA3.1 vector, respectively,
  • the recombinant plasmid was transfected into 293F cells, and the culture solution was purified and detected (the same method as 8D2 (Re) described above). The results are shown in Fig. 4, Fig. 5, Fig. 6, Fig. 7 and Fig. 8, respectively, and reduced proteins.
  • the target protein of the sample is approximately 50kD and 25KD, non-reduced protein-like The target protein is approximately 150 kD.
  • the 8D2 recombinant antibody 8D2 (Re) and the 8D2 humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17 used in the following examples were all prepared according to the method of the present example.
  • Antibody 8D2 and humanized 8D2H1L1, 8D2H2L2, 8D2H3L3 and antigen CTLA4 were determined using a Fortebio molecular interaction instrument (NCBI Gene ID: 1493, encoding the nucleic acid sequence is SEQ ID NO: 25, and the encoded amino acid sequence is SEQ ID NO: 26) Combined kinetic parameters.
  • CTLA4-mFc protein was digested with TEV protease (the synthesis of CTLA4-mFc was the same as that of CTLA4 ECD-mFc described in Example 1), and purified by column to obtain CTLA4 antigen.
  • Antibody 8D2 and its humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, 8D2H2L17 were immobilized on the surface of AR2G sensor by amino coupling, blocked by ethanolamine, equilibrated in PBST, combined with antigen CTLA4, used for CTLA4
  • the PBST was diluted twice, at concentrations of 300, 150, 75, 37.5, 18.75, 9.38, 4.69, 0 nM, and dissociated in PBST.
  • the detection methods of humanized 8D2H1L1, H2L2, H3L3, H2L15, and H2L17 were the same as those of 8D2, and the antigen concentrations were 180, 90, 45, 22.5, 11.25, 5.625, 2.813, and 0 nM.
  • the kinetic parameters of antibody 8D2 and its humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, 8D2H2L17 are shown in Table 1, and the kinetic characteristic parameters are shown in Figure 9-14.
  • Example 6 Flow cytometry method for detecting antibody and hybridoma cell line surface antigen CTLA4 binding activity
  • a host cell 293F expressing the CTLA4 antigen was constructed; then the monoclonal antibody 8D2 prepared in the present invention (see Example 1) and the prepared 8D2 (Re) and 8D2 humanized antibodies 8D2H1L1, 8D2H2L2 and 8D2H3L3 were used (see Example 4). ) Mark the host cell. Flow cytometry analysis was then used to verify that antibodies 8D2, 8D2 (Re) and 8D2 humanized antibodies 8D2H1L1, 8D2H2L2 and 8D2H3L3 have specific binding to antigens with a native conformation on the cell surface.
  • the vector pLenti6.3-CTLA4 (vector pLenti6.3 purchased from Invitrogen) containing CTLA4 was transfected into 293F cells according to the lipofectamin transfection kit (purchased from Invitrogen), and the cloned population 293F-CTLA4 stably expressing CTLA4 was obtained by screening. cell.
  • the host cell 293F expressing the CTLA4 antigen obtained by the above-mentioned steps of the conventional trypsin digestion method was used, and the number of cells in each collection tube was 2 ⁇ 10 5 , and the concentrations were 20 nM, 10 nM, 5 nM, 1 nM in PBS (1% BSA), respectively.
  • the results of CTLA4 expression verification of 293F-CTLA4 cells are shown in Fig. 15 and Fig. 16.
  • the results of binding of 8D2, 8D2 (Re) antibody and 3 humanized antibody to 293F cells are shown in Figures 17-21, respectively.
  • the 8D2 antibody and its humanized antibody can effectively bind to the target CTLA protein on the surface of the host cell 293F, and the binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 2.
  • Table 2 Fluorescence intensity analysis of 8D2, 8D2 (Re) and 8D2 humanized antibodies 8D2H1L1, 8D2H2L2 and 8D2H3L3 binding to CTLA4 host cell 293F surface antigen CTLA4 by flow cytometry
  • Table 3 Flow cytometry detection analysis curve simulation of 8D2, 8D2 (Re) and 8D2 humanized antibody 8D2H1L1, 8D2H2L2, 8D2H3L3 and CTLA4 host cell 293F surface antigen CTLA4 binding efficiency EC 50
  • Example 7 Detection of binding activity of antibody to antigen CTLA4 by ELISA method
  • Table 9 ELISA analysis curve simulating the binding efficiency of 8D2, 8D2 (Re) and 8D2 humanized antibody 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, 8D2H2L17 and CTLA4 antigen EC 50
  • the binding efficiency of the humanized antibody 8D2H2L2 to the human CTLA4 antigen was comparable to that of 10D1.
  • the binding efficiency of the humanized antibodies 8D2H2L2 and 8D2H3L3 to the monkey CTLA4 antigen was comparable to that of 10D1.
  • the binding efficiency of the antibodies 8D2H2L15 and 8D2H2L17 to the human CTLA4 antigen was significantly stronger than the control antibodies 10D1 and 11.2.1.
  • Example 8 Competitive ELISA method detects that an antibody competes with B7-1/2 for binding antigen CTLA4 binding activity
  • the antibody was labeled with CTLA4-mFc at 4 ° C overnight, blocked with 1% BSA at 37 ° C for 2 hours, and anti-CTLA4 antibody monoclonal antibody 8D2, 8D2 (Re) and 8D2 humanized antibody 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17 and the control antibody 10D1 and 11.2.1 were incubated for 10 minutes, and then incubated with B7-2-his at 37 ° C for 40 min, and then added with the enzyme-labeled secondary antibody at 37 ° C for 30 min. The absorbance at 450 nm was measured on a microplate reader.
  • Table 17 Competition ELISA analysis curve simulation of 8D2, 8D2 (Re) and 8D2 humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, 8D2H2L17 compete with B7 for binding antigen CTLA4 binding efficiency EC 50
  • 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15, and 8D2H2L17 all compete with B7 for binding to antigen CTLA4.
  • 8D2, 8D2 (Re), 8D2H1L1, 8D2H2L2 and B7-2 competed with CTLA4 stronger than 10D1; 8D2H2L17 and B7-1, B7-2 competitive binding CTLA4 were significantly stronger than antibodies 10D1 and 11.2.1.
  • Example 9 Monoclonal antibody 8D2 and humanized antibody 8D2H1L1, 8D2H2L2 Analysis of Cellular Biological Activity of 8D2H3L3, 8D2H2L15 and 8D2H2L17
  • peripheral blood mononuclear cells PBMC
  • the peripheral blood of healthy people was collected with a heparin-containing blood collection tube, diluted with PBS and centrifuged (2550 rpm, 20 minutes) to obtain a cell suspension, PBMC, and SEB (1 ⁇ g/mL)/PHA (30 ⁇ l) was added to the cell suspension.
  • the experimental results are statistically analyzed, as shown in Figure 32-33.
  • the humanized antibodies 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17 of monoclonal antibody 8D2 were able to effectively block the binding of CTLA4 to B7 and increase IL-2 in T lymphocytes compared with T cells and with Raji cells. Expression (Figs.
  • Example 10 In vivo antitumor activity of monoclonal antibody 8D2H2L2
  • the in vivo antitumor activity of 8D2H2L2 was evaluated using the hu-SCID-raji animal model.
  • PBMC peripheral blood mononuclear cells
  • PBMCs were activated with SEB 1 ⁇ g/ml for 3 days, and then 1.25 million activated PBMCs were mixed with 5 million Burkitt lymphoma cells raji and 8D2H2L2 (20 mg/kg).

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Abstract

本发明属于肿瘤治疗和分子免疫学领域，提供了抗CTLA4的单克隆抗体或其抗原结合片段、其药物组合物及其用途。本发明的单克隆抗体能阻断CTLA4与B7的结合，解除CTLA4对机体的免疫抑制，激活T淋巴细胞。

Description

抗CTLA4的单克隆抗体或其抗原结合片段、药物组合物及用途 技术领域
本发明属于肿瘤治疗和分子免疫学领域，涉及抗CTLA4的单克隆抗体或其抗原结合片段、其药物组合物、编码它们的序列，以及应用它们进行诊断、预防、治疗和/或辅助治疗的方法和用途。
背景技术
细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte sociated antigen 4，亦简称为CTLA4)与CD28分子在基因结构、染色体定位、序列的同源性及基因表达具有十分相近的关系，都是共刺激分子B7的受体，主要表达于被激活T细胞表面。但是作为淋巴细胞激活的共刺激信号，CTLA4与CD28分子的功能是相反的，CTLA4与B7结合后能抑制小鼠和人T细胞的激活，在T细胞活化中起负调节作用。
CTLA4 mAb或CTLA4配体可以阻止CTLA4与其天然配体结合，从而封闭CTLA4对T细胞负性调节信号的传导，增强T细胞对各种抗原的反应性，在这方面体内与体外研究结果基本一致。目前已有CTLA4 mAb(10D1，11.2.2)处于临床试验阶段用于治疗前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、恶性黑色素瘤等(Grosso JF.，Jure-Kunkel MN.，CTLA-4 blockade in tumor models：an overview of preclinical and translational research.Cancer Immun.2013；13：5.Epub 2013 Jan 22；US 6984720 B1以及US 6682736 B1)，其中10D1和11.2.2被视为目前效果最好的CTLA4单克隆抗体之一。
白细胞介素2(IL-2)由T细胞产生，是调节T细胞亚群的生长因子，也是调控免疫应答的重要因子，并可促进活化B细胞增殖，参与抗体反应、造血和肿瘤监视。重组的人IL-2已经被美国FDA批准用于治疗恶性肿瘤(包括黑色素瘤、肾肿瘤等)，同时正在进行治疗 慢性病毒感染的临床研究(Chavez，A.R.，et al.，Pharmacologic administration of interleukin-2.Ann N Y Acad Sci，2009.1182：p.14-27)。
CTLA4及CTLA4 mAb作为T细胞功能状况的重要影响因素，通过干预机体免疫微环境，可对疾病产生特异性治疗作用，并发挥较高疗效，补充传统用药的不足，由此开辟基因治疗的新途径。CTLA4及CTLA4 mAb应用于试验及临床各阶段：如在自身免疫性疾病中有效抑制哮喘动物模型的气道高反应性、阻止风湿性疾病的发展以及在同种异体移植中介导机体免疫耐受等。但同时，尽管生物基因治疗在短期临床试验研究中未发现不良反应，我们亦应注意到其长期应用所存在的潜在影响，如CTLA4 mAb过度阻断CTLA4-B7信号则可导致自身免疫性疾病的发生。由于抗体可以特异结合其配体并导致靶细胞溶解或阻断病理进程，所以抗体尤其人源性抗体药物的开发利用对人类恶性肿瘤及其他免疫性疾病临床治疗有重要意义。
目前，尚需要开发新的阻断CTLA4与B7的结合的抗体及其人源化抗体。
发明内容
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达系统表达出重组的CTLA4作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过进行对大量样本的筛选，得到了一种杂交瘤细胞株能够分泌产生与CTLA4特异性结合的特异性单克隆抗体，并且该单克隆抗体能够十分有效地阻断CTLA4与B7的结合。并进一步制得了人源化抗体。由此提供了下述发明：
本发明的一个方面涉及单克隆抗体或其抗原结合片段，其中，
所述的单克隆抗体包括选自下列的互补决定区(CDR)：
包含氨基酸序列为SEQ ID NO：27的HCDR1，
包含氨基酸序列为SEQ ID NO：28的HCDR2，以及
包含氨基酸序列为SEQ ID NO：29的HCDR3；
和/或
包含氨基酸序列为SEQ ID NO：30的LCDR1，
包含氨基酸序列为SEQ ID NO：31的LCDR2，以及
包含氨基酸序列选自SEQ ID NO：32、SEQ ID NO：33和SEQ ID NO：34的LCDR3。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区(VH)的氨基酸序列选自SEQ ID NO：6、SEQ ID NO：10、SEQ ID NO：14和SEQ ID NO：18；
和/或
所述单克隆抗体的轻链可变区(VL)的氨基酸序列选自SEQ ID NO：8、SEQ ID NO：12、SEQ ID NO：16、SEQ ID NO：20、SEQ ID NO：22和SEQ ID NO：24。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体包括：
(1)如SEQ ID NO：6所示的VH和如SEQ ID NO：8所示的VL；
(2)如SEQ ID NO：10所示的VH和如SEQ ID NO：12所示的VL；
(3)如SEQ ID NO：14所示的VH和如SEQ ID NO：16所示的VL；
(4)如SEQ ID NO：18所示的VH和如SEQ ID NO：20所示的VL；
(5)如SEQ ID NO：14所示的VH和如SEQ ID NO：22所示的VL；或
(6)如SEQ ID NO：14所示的VH和如SEQ ID NO：24所示的VL。
本发明中，上述(1)-(6)组单抗分别为8D2/8D2(Re)、8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17的重链可变区和轻链可变区的氨基酸序列。
具体地，SEQ ID NO：6、SEQ ID NO：10、SEQ ID NO：14中的第18位的蛋氨酸(Met)独立地被选自如下的氨基酸所替代：
亮氨酸(Leu)、缬氨酸(Val)、异亮氨酸(Ile)或丙氨酸(Ala)。
抗体治疗药物，特别是单克隆抗体(MAB)已在多种疾病的治疗中取得了良好的疗效。获取这些治疗性抗体的传统实验方法是采用抗原免疫动物，在免疫动物体内获取靶向抗原的抗体，或通过亲和力成熟的方法来改进那些与抗原的亲和力较低的抗体。然而，这些方法需要大量时间和精力，大多数时候也并不能针对抗原上的特定表位。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Sequences of Proteins of Immunological Interest，Fifth Edition(1991)，第1-3卷，NIH Publication 91-3242，Bethesda Md)。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析上面的(1)-(6)项中的单克隆抗体序列的CDR区的氨基酸序列，结果如下：
第(1)项：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GFTFSDNW        (SEQ ID NO：27)
HCDR2：IRNKPYNYET      (SEQ ID NO：28)
HCDR3：TAQFAY          (SEQ ID NO：29)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：ENIYGG           (SEQ ID NO：30)
LCDR2：GAT              (SEQ ID NO：31)
LCDR3：QNVLRSPFT        (SEQ ID NO：32)
第(2)项：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GFTFSDNW        (SEQ ID NO：27)
HCDR2：IRNKPYNYET      (SEQ ID NO：28)
HCDR3：TAQFAY          (SEQ ID NO：29)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：ENIYGG           (SEQ ID NO：30)
LCDR2：GAT              (SEQ ID NO：31)
LCDR3：QNVLRSPFT        (SEQ ID NO：32)
第(3)项：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GFTFSDNW        (SEQ ID NO：27)
HCDR2：IRNKPYNYET      (SEQ ID NO：28)
HCDR3：TAQFAY          (SEQ ID NO：29)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：ENIYGG         (SEQ ID NO：30)
LCDR2：GAT            (SEQ ID NO：31)
LCDR3：QNVLRSPFT      (SEQ ID NO：32)
第(4)项：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GFTFSDNW        (SEQ ID NO：27)
HCDR2：IRNKPYNYET      (SEQ ID NO：28)
HCDR3：TAQFAY          (SEQ ID NO：29)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：ENIYGG         (SEQ ID NO：30)
LCDR2：GAT            (SEQ ID NO：31)
LCDR3：QNVLRSPFT      (SEQ ID NO：32)
第(5)项：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GFTFSDNW        (SEQ ID NO：27)
HCDR2：IRNKPYNYET      (SEQ ID NO：28)
HCDR3：TAQFAY          (SEQ ID NO：29)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：ENIYGG         (SEQ ID NO：30)
LCDR2：GAT            (SEQ ID NO：31)
LCDR3：QNVLSRHPG      (SEQ ID NO：33)
第(6)项：
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GFTFSDNW       (SEQ ID NO：27)
HCDR2：IRNKPYNYET     (SEQ ID NO：28)
HCDR3：TAQFAY         (SEQ ID NO：29)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：ENIYGG        (SEQ ID NO：30)
LCDR2：GAT           (SEQ ID NO：31)
LCDR3：QNVLSSRPG     (SEQ ID NO：34)
根据本发明任一项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选自Fab、Fab′、F(ab′)2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合CTLA4蛋白。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，
所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
本发明的单克隆抗体或其抗原结合片段为抗CTLA4的单克隆抗体或其抗原结合片段，其能够与CTLA4特异性结合。
根据本发明中任一项所述的单克隆抗体或其抗原结合片段，其用于预防和/治疗和/或辅助治疗和/或诊断肿瘤；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
根据本发明中任一项所述的单克隆抗体，其用于：
阻断CTLA4与B7结合，
调节(例如下调)CTLA4活性或CTLA4水平，
解除CTLA4对机体免疫抑制的，或者
激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2表达。
本发明的另一方面涉及分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，
所述抗体重链可变区包含氨基酸序列为SEQ ID NO：27-29的CDR；
具体地，所述抗体重链可变区具有SEQ ID NO：6，SEQ ID NO：10，SEQ ID NO：14或SEQ ID NO：18所示的氨基酸序列；
更具体地，所述核酸分子具有SEQ ID NO：5，SEQ ID NO：9，SEQ ID NO：13或SEQ ID NO：17所示的核苷酸序列。
本发明还提供了分离的核酸分子，其编码本发明的单克隆抗体或其抗原结合片段。此类核酸分子可以从杂交瘤细胞中分离得到，也可以利用基因工程重组技术或化学合成方法获得。
本发明的再一方面涉及分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中，
所述抗体轻链可变区包含：
1)氨基酸序列为SEQ ID NO：30-32的CDR；
2)氨基酸序列为SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：33的CDR；或
3)氨基酸序列为SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：34的CDR；
具体地，所述抗体轻链可变区具有SEQ ID NO：8，SEQ ID NO：12，SEQ ID NO：16、SEQ ID NO：20、SEQ ID NO：22或SEQ ID NO：24所示的氨基酸序列；
更具体地，所述核酸分子具有SEQ ID NO：7，SEQ ID NO：11，SEQ ID NO：15、SEQ ID NO：19、SEQ ID NO：21或SEQ ID NO：23所示的核苷酸序列。
本发明的再一方面涉及一种载体，其包含本发明任一项所述的分离的核酸分子。本发明的载体可以是克隆载体，也可以是表达载体。在一个优选实施方案中，本发明的载体是例如质粒，粘粒，噬菌体，柯斯质粒等等。
本发明的再一方面涉及一种宿主细胞，其包含本发明任一项所述的分离的核酸分子，或者本发明的载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如293T细胞。
本发明的再一方面涉及制备本发明中任一项所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
本发明的再一方面涉及偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为本发明中任一项所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记；具体地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶(例如辣根过氧化物酶)。
本发明的再一方面涉及试剂盒，其包括本发明中任一项所述的单 克隆抗体或其抗原结合片段，或者包括本发明的偶联物；
具体地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶(例如辣根过氧化物酶)。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测CTLA4在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备预防和/或治疗和/或辅助治疗和/或诊断肿瘤的药物中的用途；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备如下药物中的用途：
检测样品中的CTLA4存在或其水平的药物，
阻断CTLA4与B7结合的药物，
调节(例如下调)CTLA4活性或CTLA4水平的药物，
解除CTLA4对机体免疫抑制的药物，
激活T淋巴细胞的药物，或者
提高T淋巴细胞中IL-2表达的药物。
本发明的再一方面涉及一种在体内或体外方法，包括施加细胞 以有效量的本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物的步骤，所述方法选自如下：
检测样品中的CTLA4存在或其水平的方法，
阻断CTLA4与B7结合的方法，
调节(下调)CTLA4活性或CTLA4水平的方法物，
解除CTLA4对机体免疫抑制的方法，
激活T淋巴细胞的方法，或者
提高T淋巴细胞中IL-2表达的方法。
所述方法可以用于诊断或治疗目的，或者非诊断或治疗目的(例如，所述样品是细胞样品，而非来自患者的样品)。
本发明的再一方面涉及一种预防和/或治疗和/或辅助治疗和/或诊断肿瘤的方法，包括给予受试者有效量的本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的单克隆抗体偶联物的步骤；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，当提及CTLA4蛋白(Cytotoxic T-Lymphocyte Antigen 4)的氨基酸序列时，其包括CTLA4蛋白的全长，或者CTLA4的胞外片段CTLA4ECD(SEQ ID NO：2)或者包含CTLA4ECD的片段；还包括CTLA4ECD的融合蛋白，例如与小鼠IgG的Fc蛋白片段(mFc)进行融合的片段(SEQ ID NO：3)。然而，本领域技术人员理解，在CTLA4蛋白的氨基酸序列中，可天 然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“CTLA4蛋白”应包括所有此类序列，包括SEQ ID NO：2所示的序列以及其天然或人工的变体。并且，当描述CTLA4蛋白的序列片段时，其不仅包括SEQ ID NO：2的序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，如果没有特别说明，所述B7为B7-1和/或B7-2；其具体蛋白序列为现有技术中已知序列，可以参考现有文献或者GenBank中公开的序列。例如，B7-1(CD80，NCBI Gene ID：941)；B7-2(CD86，NCBI Gene ID：942)。
如本文中所使用的，术语EC50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat  Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。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。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology，Ch.7(Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab′、F(ab′)2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人，Nature 341：544-546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab′)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如，Bird等人，Science 242：423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85：5879-5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-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，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90：6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immunol.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。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等人，Proc.Natl.Acad.Sci.USA 90：6444-6448(1993)，和Poljak R.J.等人，Structure 2：1121-1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体4B3、13A10、12B9或4H4)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片断，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature，256：495，1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
如本文中所使用的，以编号提及的单克隆抗体与从相同编号的杂 交瘤获得的单克隆抗体相同。例如，单克隆抗体4B3(或13A10、12B9或4H4)分别是与从杂交瘤细胞株4B3(或13A10、12B9或4H4)或其亚克隆或后代细胞获得的抗体相同的抗体。
如本文中所使用的，术语“嵌合抗体”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567to Cabilly et al.；Morrison et al.，Proc.Natl.Acad.Sci.USA，81：6851 6855(1984))。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.，Nature，321：522 525(1986)；Reichmann et al.，Nature，332：323 329(1988)；Presta，Curr.Op.Struct.Biol.，2：593 596(1992)；和Clark，Immunol.Today 21：397 402(2000)。
如本文中所使用的，“中和抗体”是指，能清除或显著降低目标病毒的毒力(例如，感染细胞的能力)的抗体或抗体片段。
如本文中所使用的，术语“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，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，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping  Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与载体组成的表达系统，其中大肠杆菌(菌株)来源于市场上可得到的菌株，例如但不限于：GI698，ER2566，BL21(DE3)，B834(DE3)，BLR(DE3)。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar，Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4：11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48：444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保 守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32：1180-1187(1993)；Kobayashi等人Protein Eng.12(10)：879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94：412-417(1997)，其通过引用并入本文)。
如本文中使用的，术语“免疫原性(immunogenicity)”是指，能够刺激机体形成特异抗体或致敏淋巴细胞的能力。其既指，抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质如抗体和致敏淋巴细胞的特性，也指抗原刺激机体后，机体免疫系统能形成抗体或致敏T淋巴细胞的特异性免疫应答。免疫原性是抗原最重要的性质，一种抗原能否成功地诱导宿主产生免疫应答取决于三方面的因素：抗原的性质、宿主的反应性和免疫方式。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的 解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体(例如，本发明的单克隆抗体4B3、13A10、12B9或4H4)以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原(例如，L1蛋白)，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株4B3时，其还指杂交瘤细胞株4B3的亚克隆和后代细胞。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如CTLA4与B7结合或者CTLA4活性过高相关的疾病如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如CTLA4与B7结合或者CTLA4活性过高相关的疾病如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明的单克隆抗体8D2及其人源化抗体能够很好地特异性与CTLA4结合，其中，抗体8D2，8D2(Re)与鼠源CTLA4抗原的结合效率强于对照抗体10D1(Alan J.Korman，Edward L.Halk，et al.，HUMAN CTLA-4 ANTIBODIES，United State Patent No.US 6984720 B1)和11.2.1(Douglas Charles Hanson，Mark Joseph Neveu，et al.，Human monoclonal antibodies to CTLA-4，United State Patent No.US 682736 B1)。人源化抗体8D2H1L1与鼠源CTLA4抗原的结合效率强于对照抗体10D1，与11.2.1相当。人源化抗体8D2H2L2与人CTLA4抗原的结合效率与10D1相当。人源化抗体8D2H2L2和8D2H3L3与猴CTLA4抗原的结合效率与10D1相当。抗体8D2H2L15和8D2H2L17与人CTLA4抗原的结合效率强于对照抗体10D1和11.2.1。
抗体8D2，8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15、8D2H2L17能与B7竞争结合抗原CTLA4。其中，8D2，8D2(Re)，8D2H1L1、8D2H2L2与B7-2的竞争结合CTLA4强于10D1；8D2H1L1、8D2H2L2、8D2H3L3；8D2H2L15、8D2H2L17与B7-1，B7-2的竞争结合CTLA4均强于抗体10D1和11.2.1。
本发明的单克隆抗体8D2及其人源化抗体能够十分有效地阻断CLTA4与B7的结合，特异地解除CTLA4对机体免疫抑制，激活T淋巴细胞。其中，8D2H2L2、8D2H2L15对T淋巴细胞强于对照抗体10D1和11.2.1。
附图说明
图1：CTLA4ECD-mFc融合蛋白的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M：marker 10μL；CTLA4ECD-mFc融合蛋白1μg；CTLA4ECD-mFc融合蛋白2μg；CTLA4ECD-mFc融合蛋白3μg。
图2：8D2抗体的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M：marker 10μL；还原型蛋白电泳上样缓冲液样品抗体0.3μg；非还原型蛋白电泳上样缓冲液2μL；非还原型蛋白电泳上样缓冲液样品抗体0.3μg。
图3：8D2重组抗体(8D2(Re))的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M：marker 10μL；还原型蛋白电泳上样缓冲液样品抗体1μg；非还原型蛋白电泳上样缓冲液2μL；非还原型蛋白电泳上样缓冲液样品抗体1μg。
图4：8D2的人源化抗体8D2H1L1的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M：marker 10μL；还原型蛋白电泳上样缓冲液样品抗体1μg；非还原型蛋白电泳上样缓冲液2μL；非还原型蛋白电泳上样缓冲液样品抗体1μg。
图5：8D2的人源化抗体8D2H2L2的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M：marker 10μL；还原型蛋白电泳上样缓冲液样品抗体1μg；非还原型蛋白电泳上样缓冲液2μL；非还原型蛋白电泳上样缓冲液样品抗体1μg。
图6：8D2的人源化抗体8D2H3L3的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M：marker 10μL；还 原型蛋白电泳上样缓冲液样品抗体1μg；非还原型蛋白电泳上样缓冲液2μL；非还原型蛋白电泳上样缓冲液样品抗体1μg。
图7：8D2的人源化抗体8D2H2L15的SDS-PAGE检测结果。样品及其上样量分别为：M：marker 10μL；1：非还原型蛋白电泳上样缓冲液样品抗体1μg；2：还原型蛋白电泳上样缓冲液样品抗体1μg。
图8：8D2的人源化抗体8D2H2L17的SDS-PAGE检测结果。样品及其上样量分别为：M：marker 10μL；1：非还原型蛋白电泳上样缓冲液样品抗体1μg；2：还原型蛋白电泳上样缓冲液样品抗体1μg。
图：9：单抗8D2的动力学特征参数检测结果。
图10：8D2H1L1的动力学特征参数检测结果。
图11：8D2H2L2的动力学特征参数检测结果。
图12：8D2H3L3的动力学特征参数检测结果。
图13：8D2H2L15的动力学特征参数检测结果。
图14：8D2H2L17的动力学特征参数检测结果。
图15：流式细胞仪检测293F细胞无标记，同型对照，293F-CTLA4细胞的CTLA4表达直方图(细胞数-荧光(FITC))。
图16：流式细胞仪检测293F细胞无标记，同型对照，293F-CTLA4细胞的CTLA4表达的平均荧光强度(MFI)。
图17：单抗8D2与标记的293F-CTLA4细胞的结合的EC50结果。
图18：8D2(Re)抗体与标记的293F-CTLA4细胞的结合的EC50结果。
图19：8D2H1L1与标记的293F-CTLA4细胞的结合的EC50结果。
图20：8D2H2L2与标记的293F-CTLA4细胞的结合的EC50结果。
图21：8D2H3L3与标记的293F-CTLA4细胞的结合的EC50结果。
图22：ELISA方法检测8D2、8D2H1L1、8D2重组抗体与CTLA4的结合。
图23：ELISA方法检测8D2H2L2、8D2H3L3重组抗体与人CTLA4结合。
图24：ELISA方法检测8D2H2L2、8D2H3L3重组抗体与猴CTLA4结合。
图25：ELISA方法检测8D2H2L15、8D2H2L17重组抗体与猴CTLA4结合。
图26：8D2、8D2H1L1、8D2重组抗体与B7-1竞争ELISA结果。
图27：8D2、8D2H1L1、8D2重组抗体与B7-2竞争ELISA结果。
图28：8D2H2L2、8D2H3L3抗体与B7-1竞争ELISA结果。
图29：8D2H2L2、8D2H3L3抗体与B7-2竞争ELISA结果。
图30：8D2H2L15、8D2H2L17抗体与B7-1竞争ELISA结果。
图31：8D2H2L15、8D2H2L17抗体与B7-2竞争ELISA结果。
图32：分别为外周血单核细胞(PBMC)，Raji细胞和人源化抗体8D2H1L1、8D2H2L2、8D2H3L3共培养72小时后采用ELISA方法检测T淋巴细胞IL-2分泌水平的影响。结果显示单抗8D2的人源化抗体通过阻止CTLA4的受体提高T淋巴细胞的IL-2的分泌。
图33：分别为外周血单核细胞(PBMC)，Raji细胞和人源化抗体8D2H2L15、8D2H2L17共培养72小时后采用ELISA方法检测T淋巴细胞IL-2分泌水平的影响。结果显示单抗8D2的人源化抗体通过阻止CTLA4的受体提高T淋巴细胞的IL-2的分泌。
图34：8D2H2L2在hu-SCID-raji模型中皮下移植肿瘤生长曲线。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买 获得的常规产品。
在本发明的下述实施例中，使用的BALB/C小鼠购自广东省医学实验动物中心。
在本发明的下述实施例中，使用的T细胞来自中山康方生物医药有限公司。
对照抗体10D1参照美国专利US 6984720 B1制备；11.2.1参照US 6682736 B1制备。
实施例1：CTLA4-8D2杂交瘤细胞株LT001的获得以及单克隆 抗体8D2的制备
利用哺乳动物细胞表达系统表达出重组的CTLA4作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。通过大量的样本筛选，得到了一种杂交瘤细胞株(CTLA4-8D2杂交瘤细胞株LT001)，该细胞株能够分泌产生与CTLA4特异性结合的单克隆抗体8D2。具体方法如下：
1.基因CTLA4ECD-mFc的合成：
对基因CTLA4的胞外片段CTLA4ECD(Cytotoxic T-Lymphocyte Antigen 4，NCBI Gene ID：1493，SEQ ID NO：1)所对应的氨基酸(SEQ ID NO：2)与小鼠IgG的Fc蛋白片段(mFc)进行融合设计(SEQ ID NO：3)，其中，mFc是指鼠IgG的Fc蛋白片段，其氨基酸序列如SEQ ID NO：3的下划线部分所示。
为提高目的基因在293f细胞表达系统中的表达效率，委托金斯瑞公司对SEQ ID NO：3蛋白序列相对应的核酸序列进行优化，优化主要考虑密码子的偏好性、GC含量、mRNA的二级结构、重复序列等因素。最终CTLA4ECD-mFc融合蛋白基因优化后序列如下(SEQ ID NO：4)，并委托金斯瑞公司合成。
基因CTLA4ECD的序列：(375bp)
Figure PCTCN2015085721-appb-000001
Figure PCTCN2015085721-appb-000002
CTLA4ECD编码的蛋白序列：(125aa)
Figure PCTCN2015085721-appb-000003
CTLA4ECD-mFc融合蛋白序列：(364aa)
其中，带波浪下划线的为CTLA4ECD部分，带实线下划线的为mFc部分。
Figure PCTCN2015085721-appb-000004
CTLA4ECD-mFc融合蛋白相对应的基因编码序列：(1092bp)
其中，带波浪下划线的为CTLA4ECD部分，带实线下划线的为 mFc部分。
Figure PCTCN2015085721-appb-000005
2.pUC57simple-CTLA4ECD-mFc质粒的获得：
由金斯瑞公司将合成的CTLA4ECD-mFc融合基因(SEQ ID NO：4)克隆到pUC57simple(金斯瑞公司提供)表达载体中，获得pUC57simple-CTLA4ECD-mFc质粒。
3.pcDNA3.1-CTLA4ECD-mFc重组质粒的构建：
将质粒pUC57simple-CTLA4ECD-mFc进行酶切(Xba I和BamH I)，电泳回收得到的融合基因片段CTLA4ECD-mFc与pcDNA3.1表达载体(购自Invitrogen公司)进行连接反应，获得pcDNA3.1-CTLA4ECD-mFc，转染感受态大肠杆菌细胞DH5a(购自TIANGEN公司)，转染和培养按照说明书进行。筛选得到阳性的pcDNA3.1-CTLA4ECD-mFc克隆菌落，按照常规方法扩增大肠杆菌，然后采用试剂盒(购自天根生化科技(北京)有限公司，DP103-03)并按照试剂盒的说明书提取得到pcDNA3.1-CTLA4ECD-mFc重组质粒。
4.按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将重组质粒pcDNA3.1-CTLA4ECD-mFc转染293F细胞(购自Invitrogen公司)。
5.将重组质粒pcDNA3.1-CTLA4ECD-mFc转染293F细胞7天后，将培养液通过高速离心、微孔滤膜抽真空过滤以及HiTrap protein A HP柱进行纯化CTLA4ECD-mFc融合蛋白，并取纯化后样品加入还原型蛋白电泳上样缓冲液，进行SDS-PAGE电泳检测。如图1所示，目标蛋白大约在45kD处。
6.CTLA4-8D2杂交瘤细胞株LT001的建立
用CTLA4ECD-mFc融合蛋白作为抗原，取免疫BALB/C小鼠(购自广东医学实验动物中心)的脾细胞与小鼠骨髓瘤细胞融合成杂交瘤细胞，参照目前已确立的方法(e.g.，Stewart，S.J.，“Monoclonal Antibody Production”，in Basic Methods in antibody Production and Characterization，Eds.G.C.Howard and D.R.Bethell，Boca Raton：CRC Press，2000)。
用CTLA4作为抗原包被酶标板，进行间接ELISA法筛选，得 到分泌与CTLA4特异性结合的新的抗体的杂交瘤细胞。对间接ELISA筛选得到的杂交瘤细胞，通过竞争ELISA筛选出能够分泌与配体B7-1(CD80，NCBI Gene ID：941)、B7-2(CD86，NCBI Gene ID：942)竞争结合CTLA4的单克隆抗体的杂交瘤细胞株，并经过有限稀释法得到稳定的杂交瘤细胞株。将该杂交瘤细胞株命名为CTLA4-8D2杂交瘤细胞株，并经过有限稀释法得到CTLA4-8D2稳定细胞株(本发明中亦简称为LT001细胞，其分泌的单克隆抗体命名为8D2)。
7.抗体8D2的制备
用含10％的低IgG胎牛血清对本发明CTLA4-8D2(LT001)细胞株进行培养，7天后收集细胞培养上清进行纯化制备抗体8D2。
8.抗体8D2的SDS-PAGE电泳检测：
将纯化后的样品分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行检测。检测结果显示，还原型蛋白样品目标蛋白大约在50kD和25kD处，非还原型蛋白样品目标蛋白大约在150kD处(图2)。
实施例2：单克隆抗体8D2的轻链和重链序列的获得
按照培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1制得的CTLA4-8D2杂交瘤细胞株(LT001细胞)中提取mRNA。
按照Invitrogen
Figure PCTCN2015085721-appb-000006
III First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA，并进行PCR扩增。PCR扩增产物直接进行TA克隆，具体操作参照pEASY-T1Cloning Kit(Transgen CT101)试剂盒说明书进行。将TA克隆的产物直接进行测序，测序结果如下：
重链可变区的DNA测序结果：(345bp)
Figure PCTCN2015085721-appb-000007
Figure PCTCN2015085721-appb-000008
其编码的蛋白序列：(115aa)
Figure PCTCN2015085721-appb-000009
轻链可变区的DNA测序结果：(318bp)
Figure PCTCN2015085721-appb-000010
其编码的蛋白序列：(106aa)
Figure PCTCN2015085721-appb-000011
实施例3：人源化抗体8D2H1L1、8D2H2L2，8D2H3L3，8D2H2L15 和8D2H2L17的轻链和重链序列的设计
根据CTLA4蛋白的三维晶体结构(Nat.Struct.Biol.(1997)4p.527)以及实施例2获得的抗体8D2的序列，通过计算机模拟抗体模型，根据抗体序列和结构模型设计得到抗体8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17的可变区序列(抗体恒定区序列，来自NCBI的数据库)，可变区序列如下：
1.单克隆抗体8D2H1L1的轻链和重链序列
重链可变区的DNA序列：(345bp)
Figure PCTCN2015085721-appb-000012
其编码的蛋白序列：(115aa)
Figure PCTCN2015085721-appb-000013
轻链可变区的DNA序列：(321bp)
Figure PCTCN2015085721-appb-000014
Figure PCTCN2015085721-appb-000015
其编码的蛋白序列：(107aa)
Figure PCTCN2015085721-appb-000016
2.8D2人源化单克隆抗体8D2H2L2的轻链和重链序列
重链可变区的DNA序列：(345bp)
Figure PCTCN2015085721-appb-000017
其编码的蛋白序列：(115aa)
Figure PCTCN2015085721-appb-000018
轻链可变区的DNA序列：(321bp)
Figure PCTCN2015085721-appb-000019
Figure PCTCN2015085721-appb-000020
其编码的蛋白序列：(107aa)
Figure PCTCN2015085721-appb-000021
3.8D2人源化单克隆抗体8D2H3L3的轻链和重链序列
重链可变区的DNA序列：(345bp)
Figure PCTCN2015085721-appb-000022
其编码的蛋白序列：(115aa)
Figure PCTCN2015085721-appb-000023
轻链可变区的DNA序列：(321bp)
Figure PCTCN2015085721-appb-000024
其编码的蛋白序列：(107aa)
Figure PCTCN2015085721-appb-000025
4.8D2人源化单克隆抗体8D2H2L15的轻链和重链序列
重链可变区的DNA序列：(345bp)
Figure PCTCN2015085721-appb-000026
其编码的蛋白序列：(115aa)
Figure PCTCN2015085721-appb-000027
轻链可变区的DNA序列：(321bp)
Figure PCTCN2015085721-appb-000028
其编码的蛋白序列：(107aa)
Figure PCTCN2015085721-appb-000029
5.8D2人源化单克隆抗体8D2H2L17的轻链和重链序列
重链可变区的DNA序列：(345bp)
Figure PCTCN2015085721-appb-000030
Figure PCTCN2015085721-appb-000031
其编码的蛋白序列：(115aa)
Figure PCTCN2015085721-appb-000032
轻链可变区的DNA序列：(321bp)
Figure PCTCN2015085721-appb-000033
其编码的蛋白序列：(107aa)
Figure PCTCN2015085721-appb-000034
实施例4：8D2重组抗体8D2(Re)以及8D2人源化抗体8D2H1L1、 8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17的制备和SDS-PAGE 电泳检测
1.8D2重组抗体即8D2(Re)的制备和SDS-PAGE电泳检测
将8D2的重链cDNA序列(其可变区序列如SEQ ID NO：5所示)和轻链的cDNA序列(其可变区序列如SEQ ID NO：7所示)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-8D2H和pUC57simple-8D2L质粒。
分别将质粒pUC57simple-8D2H和pUC57simple-8D2L进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤以及HiTrap protein A HP柱进行纯化，并将纯化后的样品分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行SDS-PAGE电泳检测。如图3所示，还原型蛋白样品目标蛋白大约在50kD和25KD处，非还原型蛋白样品目标蛋白大约在150kD处。
2.8D2人源化抗体8D2H1L1、8D2H2L2和8D2H3L3的制备和SDS-PAGE电泳检测
将8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17的重链cDNA(其可变区序列分别如SEQ ID NO：9、SEQ ID NO：13、SEQ ID NO：17、SEQ ID NO：13、SEQ ID NO：13所示)和轻链的cDNA(其可变区序列分别如SEQ ID NO：11、SEQ ID NO：15、SEQ ID NO：19、SEQ ID NO：21、SEQ ID NO：23所示)分别克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-8D2H1L1、pUC57simple-8D2H2L2、pUC57simple-8D2H3L3、pUC57simple-8D2H2L15、pUC57simple-8D2H2L17质粒，并分别亚克隆到pcDNA3.1载体中，方法同前述8D2(Re)。
将重组质粒转染293F细胞，将培养液纯化后进行检测(同上所述8D2(Re)的方法)，结果分别如图4、图5、图6、图7和图8所示，还原型蛋白样品目标蛋白大约在50kD和25KD处，非还原型蛋白样 品目标蛋白大约在150kD处。
下面的实施例中所用到的8D2重组抗体8D2(Re)以及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17均按照本实施例的方法制得。
实施例5：抗体的动力学参数测定
使用Fortebio分子相互作用仪测定抗体8D2及人源化8D2H1L1、8D2H2L2、8D2H3L3与抗原CTLA4(NCBI Gene ID：1493，编码核酸序列为SEQ ID NO：25，所编码的氨基酸序列为SEQ ID NO：26)结合的动力学参数。
1.用TEV蛋白酶酶切CTLA4-mFc蛋白(CTLA4-mFc合成方法同实施例1中所述CTLA4ECD-mFc的合成)，并过柱纯化获得CTLA4抗原。
基因CTLA4的序列：(636bp)
Figure PCTCN2015085721-appb-000035
Figure PCTCN2015085721-appb-000036
编码对应的氨基酸序列：(212aa)
Figure PCTCN2015085721-appb-000037
2.分别将抗体8D2及其人源化抗体8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15、8D2H2L17采用氨基偶联的方式固定于AR2G传感器表面，经乙醇胺封闭，于PBST中平衡后，与抗原CTLA4结合，CTLA4用PBST两倍稀释，浓度为300、150、75、37.5、18.75、9.38、4.69、0nM，于PBST中解离。人源化8D2H1L1、H2L2、H3L3、H2L15、H2L17的检测方法与8D2相同，抗原浓度为180、90、45、22.5、11.25、5.625、2.813、0nM。
抗体8D2及其人源化抗体8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15、8D2H2L17的动力学参数见表1，动力学特征参数检测结果分别如图9-14所示。
表1：抗体8D2、8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15、8D2H2L17的动力学参数
Figure PCTCN2015085721-appb-000038
Figure PCTCN2015085721-appb-000039
KD为亲和力常数；kon为抗原抗体结合速率；kdis为抗原抗体解离速率；KD＝kdis/kon。
结果表明，六种抗体均与抗原有较好的亲和力，与对照抗体10D1和11.2.1相当甚至更优。
实施例6：流式细胞仪方法检测抗体与杂交瘤细胞株表面抗原 CTLA4的结合活性
首先构建表达CTLA4抗原的宿主细胞293F；然后用本发明中制备的单克隆抗体8D2(见实施例1)以及制备的8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2和8D2H3L3(见实施例4)对改宿主细胞进行标记。然后采用流式细胞术分析验证抗体8D2，8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2和8D2H3L3对细胞表面具有天然构象的抗原具有特异性的结合。
具体步骤如下：
1.表达CTLA4抗原的宿主细胞293F的构建
按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将包含CTLA4的载体pLenti6.3-CTLA4(载体pLenti6.3购自Invitrogen公司)转染293F细胞，经筛选获得稳定表达CTLA4的克隆群体293F-CTLA4细胞。
2.抗体标记和流式细胞仪检测
采用常规胰酶消化方法上述步骤获得的表达CTLA4抗原的宿主细胞293F，并使每个收集管细胞数为2×105，用PBS(1％BSA)配制浓度分别为20nM，10nM，5nM，1nM，0.1nM，0.01nM，0nM的8D2抗体稀释液，冰上与表达CTLA4的293F细胞孵育2小时， 每管加入100μL FITC-Goat-Anti-Mouse IgG(1∶500)冰上孵育1小时并加入300μLPBS后，在流式细胞仪上用FITC通道检测荧光信号。其它抗体的检测参照8D2抗体。
3.实验结果
293F-CTLA4细胞的CTLA4表达验证结果如图15和图16所示。检测8D2，8D2(Re)抗体及3个人源化抗体与293F细胞的结合结果分别如图17-21所示。由图可见，8D2抗体及其人源化抗体能有效地结合宿主细胞293F表面的靶标CTLA蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强度见表2。
通过对结合的8D2及其人源化抗体进行荧光定量分析，曲线模拟8D2及其人源化抗体的结合效率EC50，如表3。
表2：流式细胞仪检测8D2，8D2(Re)及8D2人源化抗体8D2H1L1，8D2H2L2和8D2H3L3结合CTLA4宿主细胞293F表面抗原CTLA4后荧光强度分析
Figure PCTCN2015085721-appb-000040
表3：流式细胞仪检测分析曲线模拟8D2，8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3与CTLA4宿主细胞293F表面抗原CTLA4的结合效率EC50
  8D2 8D2(Re) 8D2H1L1 8D2H2L2 8D2H3L3
EC50(nM) 3.84 1.38 5.06 4.37 4.54
结果表明，抗体8D2，8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3均与CTLA4宿主细胞293F表面抗原CTLA4有很强的结合能力。
实施例7：ELISA方法检测抗体与抗原CTLA4的结合活性
用CTLA4包被酶标板4℃包被过夜，1％BSA 37℃封闭2h后，分别加入CTLA4抗体8D2，8D2(Re)及8D2人源化抗体8D2H1L1，8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17及对照抗体10D1(Alan J.Korman，Edward L.Halk，et al.，HUMAN CTLA-4ANTIBODIES，United State Patent No.US 6984720 B1)和11.2.1(Douglas Charles Hanson，Mark Joseph Neveu，et al.，Human monoclonal antibodies to CTLA-4，United State Patent No.US 682736 B1)反应30分钟，加入酶标二抗孵育30分钟后，在酶标仪上检测450nm的吸光值。
检测8D2抗体及其人源化抗体与抗原CTLA4结合结果分别如图22-25所示。由图可见，8D2，8D2(Re)抗体及8D2人源化抗体能有效地结合CTLA4蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强度见表4-表8。通过对结合的8D2，8D2(Re)及人源化抗体进行荧光定量分析，曲线模拟8D2，8D2(Re)及人源化抗体的结合效率EC50(表9)。
表4：8D2、8D2重组抗体与鼠CTLA4的结合(ELISA)
Figure PCTCN2015085721-appb-000041
Figure PCTCN2015085721-appb-000042
表5：8D2、8D2H1L1、8D2重组抗体与人CTLA4的结合(ELISA)
Figure PCTCN2015085721-appb-000043
表6：8D2H2L2、8D2H3L3抗体与人CTLA4结合(ELISA)
Figure PCTCN2015085721-appb-000044
Figure PCTCN2015085721-appb-000045
表7：8D2H2L2、8D2H3L3抗体与猴CTLA4结合(ELISA)
Figure PCTCN2015085721-appb-000046
表8：抗体8D2H2L15、8D2H2L17与人CTLA4结合(ELISA)
Figure PCTCN2015085721-appb-000047
Figure PCTCN2015085721-appb-000048
表9：ELISA分析曲线模拟8D2，8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15、8D2H2L17与CTLA4抗原的结合效率EC50
Figure PCTCN2015085721-appb-000049
Figure PCTCN2015085721-appb-000050
注：8D2H2L2平行进行了3次实验。
以上结果表明，抗体8D2，8D2(Re)与鼠源CTLA4抗原的结合效率强于对照抗体10D1和11.2.1。人源化抗体8D2H1L1与鼠源CTLA4抗原的结合效率强于对照抗体10D1，与11.2.1相当。
人源化抗体8D2H2L2与人CTLA4抗原的结合效率与10D1相当。人源化抗体8D2H2L2和8D2H3L3与猴CTLA4抗原的结合效率与10D1相当。抗体8D2H2L15和8D2H2L17与人CTLA4抗原的结合效率显著强于对照抗体10D1和11.2.1。
实施例8：竞争ELISA方法检测抗体与B7-1/2竞争结合抗原 CTLA4的结合活性
1.ELISA检测抗体与B7-1竞争结合抗原CTLA4的结合活性
采用B7-1包被酶标板4℃过夜，1％BSA 37℃封闭2小时，加入抗CTLA4的抗体单克隆抗体8D2，8D2(Re)及8D2人源化抗体8D2H1L1，8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17及对照抗体10D1和11.2.1孵育10分钟后，加入CTLA4-mFc37℃孵育40min后，加入酶标二抗37℃孵育30min。在酶标仪上检测450nm的吸光值。
2.ELISA检测抗体与B7-2竞争结合抗原CTLA4的结合活性
采用CTLA4-mFc包被酶标板4℃过夜，1％BSA 37℃封闭2小时，加入抗CTLA4的抗体单克隆抗体8D2，8D2(Re)及8D2人源化抗体8D2H1L1，8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17 及对照抗体10D1和11.2.1孵育10分钟后，加入B7-2-his37℃孵育40min后，加入酶标二抗37℃孵育30min。在酶标仪上检测450nm的吸光值。检测8D2，8D2(Re)抗体及其人源化抗体与抗原CTLA4结合结果分别如图26-31所示。由图可见，8D2，8D2(Re)抗体及8D2人源化抗体能有效地结合CTLA蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强度见表10-表16。通过对结合的8D2，8D2(Re)及人源化抗体进行荧光定量分析，曲线模拟8D2，8D2(Re)及人源化抗体的结合效率EC50(表17)。
表10：8D2、8D2(Re)与B7-1竞争ELISA
Figure PCTCN2015085721-appb-000051
表11：8D2、8D2H1L1、8D2(Re)与B7-1竞争ELISA
Figure PCTCN2015085721-appb-000052
Figure PCTCN2015085721-appb-000053
表12：8D2、8D2H1L1、8D2重组抗体与B7-2竞争ELISA
Figure PCTCN2015085721-appb-000054
表13：8D2H2L2、8D2H3L3抗体与B7-1竞争ELISA
Figure PCTCN2015085721-appb-000055
Figure PCTCN2015085721-appb-000056
表14：8D2H2L2、8D2H3L3抗体与B7-2竞争结合CTLA4 ELISA
Figure PCTCN2015085721-appb-000057
表15：8D2H2L15、8D2H2L17抗体与B7-1竞争结合CTLA4 ELISA
Figure PCTCN2015085721-appb-000058
表16：8D2H2L15、8D2H2L17抗体与B7-2竞争结合CTLA4 ELISA
Figure PCTCN2015085721-appb-000059
Figure PCTCN2015085721-appb-000060
表17：竞争ELISA分析曲线模拟8D2，8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15、8D2H2L17与B7竞争结合抗原CTLA4的结合效率EC50
Figure PCTCN2015085721-appb-000061
注：8D2H2L2平行进行了3次实验。
以上结果表明，抗体8D2，8D2(Re)及8D2人源化抗体
8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15、8D2H2L17均能与B7竞争结合抗原CTLA4。其中，8D2，8D2(Re)，8D2H1L1、8D2H2L2与B7-2的竞争结合CTLA4强于10D1；8D2H2L17与B7-1，B7-2的竞争结合CTLA4均显著强于抗体10D1和11.2.1。
实施例9：单克隆抗体8D2及人源化抗体8D2H1L1、8D2H2L2、 8D2H3L3、8D2H2L15和8D2H2L17的细胞生物学活性分析
为检测单克隆抗体8D2及人源化抗体8D2H1L1，8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17和对照抗体10D1、11.2.1对外周血单核细胞(Peripheral blood mononuclear cell：PBMC)的IL-2表达的影响，用含肝素钠的血液采集管采集健康者外周血，经PBS稀释和分离液离心(2550rpm，20分钟)后得到细胞悬液即PBMC，向细胞悬液加入SEB(1μg/mL)/PHA(30μl/ml)并置于饱和湿度37℃、5％CO2的培养箱中继续培养，并加入淋巴细胞Raji细胞和抗体进行共孵育培养48小时。将PBMC用PBS洗两次后，加入10000/孔到96孔板，然后分别加入相应浓度梯度的抗体，孵育20min后加入经MMC处理1小时的Raji细胞10000/孔共培养72小时。共孵育培养72小时后收集细胞培养上清，采用ELISA方法(参照试剂盒说明书：DAKEW达科为，DKW12-1020-096)检测细胞共孵育培养上清IL-2表达情况。
实验结果经统计分析，如图32-33所示。与T cells和with Raji cells组相比，单克隆抗体8D2的人源化抗体8D2H1L1、8D2H2L2、8D2H3L3、8D2H2L15和8D2H2L17均能有效地阻断CTLA4与B7的结合并提高T淋巴细胞中IL-2的表达(图32-图33)，其中，本发明人惊奇地发现，8D2H2L2、8D2H2L15、8D2H2L17显著地优于对照抗体10D1和11.2.1，其在浓度为10nM时所实现的IL-2水平与10D1或11.2.1在100nM浓度时相当甚至更优，可见，本发明的抗体能够在较低浓度例如大约为10nM时提高IL-2水平。
实施例10：单克隆抗体8D2H2L2的体内抗肿瘤活性
采用hu-SCID-raji动物模型来评价8D2H2L2的体内抗肿瘤活性。Ficoll试剂分离人人外周血单个核细胞(PBMC)后，以SEB 1μg/ml活化PBMC 3天，然后将125万活化的PBMC与500万人Burkitt淋巴瘤细胞raji与8D2H2L2(20mg/kg)混合后，皮下接种与 SCID-beige小鼠背侧，同时设同型对照组，每组5只动物。之后每周静脉注射给药一次，给药剂量为20mg/kg，连续给药3次。每周测量2次肿瘤体积至实验结束或肿瘤体积达到1000mm3。
如图34所示，8D2H2L2在hu-SCID-raji模型中可以明显抑制肿瘤生长。这个结果显示本抗体可用于临床上治疗淋巴瘤。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (21)

  1. 单克隆抗体或其抗原结合片段，其中，
    所述的单克隆抗体包括选自下列的互补决定区(CDR)：
    包含氨基酸序列为SEQ ID NO：27的HCDR1，
    包含氨基酸序列为SEQ ID NO：28的HCDR2，以及
    包含氨基酸序列为SEQ ID NO：29的HCDR3；
    和/或
    包含氨基酸序列为SEQ ID NO：30的LCDR1，
    包含氨基酸序列为SEQ ID NO：31的LCDR2，以及
    包含氨基酸序列选自SEQ ID NO：32、SEQ ID NO：33和SEQ ID NO：34的LCDR3。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区(VH)的氨基酸序列选自SEQ ID NO：6、SEQ ID NO：10、SEQ ID NO：14和SEQ ID NO：18；
    和/或
    所述单克隆抗体的轻链可变区(VL)的氨基酸序列选自SEQ ID NO：8、SEQ ID NO：12、SEQ ID NO：16、SEQ ID NO：20、SEQ ID NO：22和SEQ ID NO：24。
  3. 根据权利要求3所述的单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体包括：
    (1)如SEQ ID NO：6所示的VH和如SEQ ID NO：8所示的VL；
    (2)如SEQ ID NO：10所示的VH和如SEQ ID NO：12所示的VL；
    (3)如SEQ ID NO：14所示的VH和如SEQ ID NO：16所示的VL；
    (4)如SEQ ID NO：18所示的VH和如SEQ ID NO：20所示 的VL；
    (5)如SEQ ID NO：14所示的VH和如SEQ ID NO：22所示的VL；或
    (6)如SEQ ID NO：14所示的VH和如SEQ ID NO：24所示的VL；
    具体地，SEQ ID NO：6、SEQ ID NO：10、SEQ ID NO：14中的第18位的蛋氨酸独立地被选自如下的氨基酸所替代：
    亮氨酸、缬氨酸、异亮氨酸或丙氨酸。
  4. 根据权利要求1至3中任一项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选自Fab、Fab′、F(ab′)2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
  5. 根据权利要求1至4中任一项所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合CTLA4蛋白。
  6. 根据权利要求1至5中任一项所述的单克隆抗体或其抗原结合片段，其中，
    所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  7. 分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，
    所述抗体重链可变区包含氨基酸序列为SEQ ID NO：27-29的CDR；
    具体地，所述抗体重链可变区具有SEQ ID NO：6，SEQ ID NO： 10，SEQ ID NO：14或SEQ ID NO：18所示的氨基酸序列；
    更具体地，所述核酸分子具有SEQ ID NO：5，SEQ ID NO：9，SEQ ID NO：13或SEQ ID NO：17所示的核苷酸序列。
  8. 分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中，
    所述抗体轻链可变区包含：
    1)氨基酸序列为SEQ ID NO：30-32的CDR；
    2)氨基酸序列为SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：33的CDR；或
    3)氨基酸序列为SEQ ID NO：30、SEQ ID NO：31和SEQ ID NO：34的CDR；
    具体地，所述抗体轻链可变区具有SEQ ID NO：8，SEQ ID NO：12，SEQ ID NO：16、SEQ ID NO：20、SEQ ID NO：22或SEQ ID NO：24所示的氨基酸序列；
    更具体地，所述核酸分子具有SEQ ID NO：7，SEQ ID NO：11，SEQ ID NO：15、SEQ ID NO：19、SEQ ID NO：21或SEQ ID NO：23所示的核苷酸序列。
  9. 一种载体，其包含权利要求7和/或8所述的分离的核酸分子。
  10. 一种宿主细胞，其包含权利要求7和/或8所述的分离的核酸分子，或者权利要求9所述的载体。
  11. 制备权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养权利要求10的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
  12. 偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分， 其中，所述单克隆抗体为权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记；具体地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
  13. 试剂盒，其包括权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段，或者包括权利要求12所述的偶联物；
    具体地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
  14. 权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测CTLA4在样品中的存在或其水平。
  15. 一种药物组合物，其包含权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段或者权利要求12所述的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
  16. 权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段或者权利要求12所述的偶联物在制备预防和/或治疗和/或辅助治疗和/或诊断肿瘤的药物中的用途；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
  17. 权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段或者权利要求12所述的偶联物在制备如下药物中的用途：
    检测样品中的CTLA4水平的药物，
    阻断CTLA4与B7结合的药物，
    调节(例如下调)CTLA4活性或CTLA4水平的药物，
    解除CTLA4对机体免疫抑制的药物，
    激活T淋巴细胞的药物，或者
    提高T淋巴细胞中IL-2表达的药物。
  18. 一种在体内或体外方法，包括施加细胞以有效量的权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段或者权利要求12所述的偶联物的步骤，所述方法选自如下：
    检测样品中的CTLA4水平的方法，
    阻断CTLA4与B7结合的方法，
    调节(下调)CTLA4活性或CTLA4水平的方法物，
    解除CTLA4对机体免疫抑制的方法，
    激活T淋巴细胞的方法，或者
    提高T淋巴细胞中IL-2表达的方法。
  19. 一种预防和/或治疗和/或辅助治疗和/或诊断肿瘤的方法，包括给予受试者有效量的权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段或者权利要求12所述的单克隆抗体偶联物的步骤；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
  20. 根据权利要求1至6中任一项所述的单克隆抗体或其抗原结合片段，其用于预防和/治疗和/或辅助治疗和/或诊断肿瘤；具体地，所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
  21. 根据权利要求1至6中任一项所述的单克隆抗体，其用于：
    阻断CTLA4与B7结合，
    调节(例如下调)CTLA4活性或CTLA4水平，
    解除CTLA4对机体免疫抑制的，或者
    激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2表达。
PCT/CN2015/085721 2014-08-01 2015-07-31 抗ctla4的单克隆抗体或其抗原结合片段、药物组合物及用途 WO2016015675A1 (zh)

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BR112017002080-7A BR112017002080B1 (pt) 2014-08-01 2015-07-31 Anticorpo ou fragmento de ligação ao antígeno do mesmo, molécula de ácido nucleico isolada, vetor, método para preparar o anticorpo ou fragmento de ligação ao antígeno do mesmo, composição farmacêutica, e, uso de um anticorpo ou fragmento de ligação de antígeno do mesmo
US15/500,744 US10449251B2 (en) 2014-08-01 2015-07-31 Anti-CTLA4 monoclonal antibody or its antigen binding fragments, pharmaceutical compositions and uses
NZ729158A NZ729158A (en) 2014-08-01 2015-07-31 Anti-ctla4 monoclonal antibody or antigen binding fragment thereof, medicinal composition and use
AP2017009762A AP2017009762A0 (en) 2014-08-01 2015-07-31 Anti-ctla4 monoclonal antibody or antigen binding fragment thereof, medicinal composition and use
EP20216429.9A EP3858861A1 (en) 2014-08-01 2015-07-31 An anti-ctla4 monoclonal antibody or antigen binding fragment thereof, a pharmaceutical composition and use
PL15827441T PL3176181T3 (pl) 2014-08-01 2015-07-31 Przeciwciało monoklonalne anty-CTLA4 lub jego fragment wiążący antygen, kompozycja lecznicza i zastosowanie
ES15827441T ES2857509T3 (es) 2014-08-01 2015-07-31 Anticuerpo monoclonal anti-CTLA4 o fragmento de unión al antígeno del mismo, composición medicinal y uso
MYPI2017000162A MY192822A (en) 2014-08-01 2015-07-31 An anti-ctla4 monoclonal antibody or antigen binding fragment thereof, a pharmaceutical composition and use
RS20210312A RS61590B1 (sr) 2014-08-01 2015-07-31 Monoklonsko antitelo protiv ctla4 ili njegov antigen-vezujući fragment, medicinska kompozicija i upotreba
SI201531555T SI3176181T1 (sl) 2014-08-01 2015-07-31 Monoklonsko protitelo proti CTLA4 ali njegov antigen-vezavni fragment, zdravilni sestavek in uporaba
DK15827441.5T DK3176181T3 (da) 2014-08-01 2015-07-31 Anti-ctla4 monoklonal antistof eller antigenbindende fragment deraf, medicinal sammensætning og anvendelse
EP15827441.5A EP3176181B1 (en) 2014-08-01 2015-07-31 Anti-ctla4 monoclonal antibody or antigen binding fragment thereof, medicinal composition and use
EA201790288A EA036396B1 (ru) 2014-08-01 2015-07-31 Анти-ctla4 моноклональное антитело или его антигенсвязывающий фрагмент, фармацевтическая композиция и применение
KR1020177005688A KR102017396B1 (ko) 2014-08-01 2015-07-31 항-ctla4 모노클로날 항체 또는 그의 항원 결합 단편, 제약 조성물 및 용도
MX2017001446A MX2017001446A (es) 2014-08-01 2015-07-31 Anticuerpo monoclonal anti-antigeno 4 asociado a linfocitos t citotoxicos (ctla4) o su fragmento de union a antigeno, una composicion farmaceutica y uso.
MA40474A MA40474B1 (fr) 2014-08-01 2015-07-31 Anticorps monoclonal anti-ctla4 ou fragment de celui-ci se liant à l'antigène, composition médicinale et son utilisation
MDA20170022A MD4795C1 (ro) 2014-08-01 2015-07-31 Anticorp monoclonal anti-CTLA4 sau fragment de legare a acestuia la antigen, compoziţie farmaceutică şi utilizarea lor
CN202211206595.7A CN115960232A (zh) 2014-08-01 2015-07-31 抗ctla4的单克隆抗体或其抗原结合片段、药物组合物及用途
TN2017000010A TN2017000010A1 (en) 2014-08-01 2015-07-31 Anti-ctla4 monoclonal antibody or antigen binding fragment thereof, medicinal composition and use.
AU2015295936A AU2015295936C1 (en) 2014-08-01 2015-07-31 Anti-CTLA4 monoclonal antibody or antigen binding fragment thereof, medicinal composition and use
CA2956000A CA2956000C (en) 2014-08-01 2015-07-31 An anti-ctla4 monoclonal antibody or antigen binding fragment thereof, a pharmaceutical composition and use
UAA201701943A UA119570C2 (uk) 2014-08-01 2015-07-31 Анти-ctla4 моноклональне антитіло або його антигензв'язувальний фрагмент, фармацевтична композиція і їх застосування
LTEP15827441.5T LT3176181T (lt) 2014-08-01 2015-07-31 Monokloninis antikūnas prieš ctla4 ar antigeną surišantis jo fragmentas, gydomoji kompozicija ir panaudojimas
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ZA2017/00528A ZA201700528B (en) 2014-08-01 2017-01-23 Anti-ctla4 monoclonal antibody or antigen binding fragment thereof, medicinal composition and use
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Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017040790A1 (en) 2015-09-01 2017-03-09 Agenus Inc. Anti-pd-1 antibodies and methods of use thereof
WO2017165742A1 (en) 2016-03-24 2017-09-28 Millennium Pharmaceuticals, Inc. Methods of treating gastrointestinal immune-related adverse events in anti-ctla4 anti-pd-1 combination treatments
WO2017165778A1 (en) 2016-03-24 2017-09-28 Millennium Pharmaceuticals, Inc. Methods of treating gastrointestinal immune-related adverse events in immune oncology treatments
WO2017198212A1 (zh) * 2016-05-19 2017-11-23 苏州康宁杰瑞生物科技有限公司 针对ctla4的单域抗体及其衍生蛋白
WO2017220990A1 (en) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 antibodies
WO2018071500A1 (en) 2016-10-11 2018-04-19 Agenus Inc. Anti-lag-3 antibodies and methods of use thereof
JP2019533475A (ja) * 2016-08-23 2019-11-21 康方▲藥▼▲業▼有限公司Ａｋｅｓｏ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 抗ｃｔｌａ４−抗ｐｄ−１二機能性抗体、その医薬組成物および使用
CN110505882A (zh) * 2017-03-31 2019-11-26 默沙东公司 用pd-1的拮抗剂和抗ctla4抗体的组合治疗癌症的组合物和方法
WO2019241730A2 (en) 2018-06-15 2019-12-19 Flagship Pioneering Innovations V, Inc. Increasing immune activity through modulation of postcellular signaling factors
CN110678199A (zh) * 2017-05-02 2020-01-10 默沙东公司 单独的和与程序性死亡受体1(pd-1)抗体组合的抗ctla4抗体的稳定制剂及其使用方法
WO2020127377A1 (en) 2018-12-21 2020-06-25 Ose Immunotherapeutics Bifunctional anti-pd-1/il-7 molecule
JP2020520655A (ja) * 2017-05-19 2020-07-16 ウーシー バイオロジクス（シャンハイ）カンパニー リミテッド 細胞傷害性ｔリンパ球関連タンパク質４（ｃｔｌａ−４）に対する新規モノクローナル抗体
WO2020165374A1 (en) 2019-02-14 2020-08-20 Ose Immunotherapeutics Bifunctional molecule comprising il-15ra
CN111727056A (zh) * 2018-02-13 2020-09-29 默沙东公司 用抗pd-1抗体和抗ctla-4抗体治疗癌症的方法
WO2020227159A2 (en) 2019-05-03 2020-11-12 Flagship Pioneering Innovations V, Inc. Methods of modulating immune activity
WO2021127217A1 (en) 2019-12-17 2021-06-24 Flagship Pioneering Innovations V, Inc. Combination anti-cancer therapies with inducers of iron-dependent cellular disassembly
WO2021122866A1 (en) 2019-12-17 2021-06-24 Ose Immunotherapeutics Bifunctional molecules comprising an il-7 variant
WO2021178611A1 (en) * 2020-03-05 2021-09-10 Merck Sharp & Dohme Corp. Methods for treating cancer or infection using a combination of an anti-pd-1 antibody, an anti-ctla4 antibody, and an anti-tigit antibody
WO2022006179A1 (en) 2020-06-29 2022-01-06 Flagship Pioneering Innovations V, Inc. Viruses engineered to promote thanotransmission and their use in treating cancer
WO2022026330A1 (en) * 2020-07-27 2022-02-03 Promab Biotechnologies, Inc. Humanized cd37 and bi-specific cd19-humanized cd37 car-t cells
WO2022112198A1 (en) 2020-11-24 2022-06-02 Worldwide Innovative Network Method to select the optimal immune checkpoint therapies
WO2022212784A1 (en) 2021-03-31 2022-10-06 Flagship Pioneering Innovations V, Inc. Thanotransmission polypeptides and their use in treating cancer
WO2022214652A1 (en) 2021-04-09 2022-10-13 Ose Immunotherapeutics Scaffold for bifunctioanl molecules comprising pd-1 or cd28 and sirp binding domains
WO2022214653A1 (en) 2021-04-09 2022-10-13 Ose Immunotherapeutics New scaffold for bifunctional molecules with improved properties
US11479608B2 (en) 2016-08-23 2022-10-25 Akeso Biopharma, Inc. Anti-CTLA4 antibodies
WO2023278641A1 (en) 2021-06-29 2023-01-05 Flagship Pioneering Innovations V, Inc. Immune cells engineered to promote thanotransmission and uses thereof
US11845798B2 (en) 2017-05-02 2023-12-19 Merck Sharp & Dohme Llc Formulations of anti-LAG3 antibodies and co-formulations of anti-LAG3 antibodies and anti-PD-1 antibodies
WO2024003360A1 (en) 2022-07-01 2024-01-04 Institut Curie Biomarkers and uses thereof for the treatment of neuroblastoma
WO2024028386A1 (en) 2022-08-02 2024-02-08 Ose Immunotherapeutics Multifunctional molecule directed against cd28
WO2024056049A1 (zh) * 2022-09-16 2024-03-21 同润生物医药(上海)有限公司 具有pH依赖性的抗CTLA4抗体或抗原结合片段
WO2024077191A1 (en) 2022-10-05 2024-04-11 Flagship Pioneering Innovations V, Inc. Nucleic acid molecules encoding trif and additionalpolypeptides and their use in treating cancer
WO2024151687A1 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations V, Inc. Genetic switches and their use in treating cancer
US12076398B2 (en) 2016-08-23 2024-09-03 CTTQ-Akeso (ShangHai) Biomed. Tech. Co., Ltd. Anti-PD1 monoclonal antibody, pharmaceutical composition thereof and use thereof
WO2024200826A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Lipid-based nanoparticle targeted at activated immune cells for the expression of immune cell inhibiting molecule and use thereof
WO2024200823A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Lipid-based nanoparticle targeted at activated immune cells for the expression of immune cell enhancing molecule and use thereof

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105296433B (zh) * 2014-08-01 2018-02-09 中山康方生物医药有限公司 一种ctla4抗体、其药物组合物及其用途
SI3303394T1 (sl) 2015-05-29 2020-10-30 Agenus Inc. Protitelesa proti-CTLA-4 in postopki njihove uporabe
JP6976322B2 (ja) * 2016-10-10 2021-12-08 クラウン バイオサイエンス，インコーポレイテッド（タイツァン） 新規抗ｃｔｌａ４抗体
MX2019006340A (es) 2016-12-07 2019-11-07 Agenus Inc Anticuerpos anti antígeno 4 del linfocito t citotóxico (ctla-4) y métodos de uso de los mismos.
EP3597735A4 (en) * 2017-03-15 2020-11-25 Suzhou Galaxy Biopharma, Co., Ltd. CTLA4 ANTIBODIES, PHARMACEUTICAL COMPOSITION AND USES THEREOF
JOP20190260A1 (ar) 2017-05-02 2019-10-31 Merck Sharp & Dohme صيغ ثابتة لأجسام مضادة لمستقبل الموت المبرمج 1 (pd-1) وطرق استخدامها
AR111760A1 (es) 2017-05-19 2019-08-14 Novartis Ag Compuestos y composiciones para el tratamiento de tumores sólidos mediante administración intratumoral
CN108948194B (zh) * 2017-05-19 2023-02-17 上海药明生物技术有限公司 一种新的ctla-4单克隆抗体
CN107325181A (zh) * 2017-07-05 2017-11-07 无锡傲锐东源生物科技有限公司 抗ctla4蛋白单克隆抗体及其用途
WO2019036855A1 (en) 2017-08-21 2019-02-28 Adagene Inc. ANTI-CD137 MOLECULES AND THEIR USE
WO2019056281A1 (en) * 2017-09-21 2019-03-28 Eucure (Beijing) Biopharma Co., Ltd ANTI-CTLA4 ANTIBODIES AND USES THEREOF
WO2019148445A1 (en) 2018-02-02 2019-08-08 Adagene Inc. Precision/context-dependent activatable antibodies, and methods of making and using the same
WO2019148444A1 (en) 2018-02-02 2019-08-08 Adagene Inc. Anti-ctla4 antibodies and methods of making and using the same
WO2019179388A1 (en) * 2018-03-19 2019-09-26 Wuxi Biologics (Shanghai) Co., Ltd. Novel anti-ctla-4 antibody polypeptide
CN109336975B (zh) * 2018-04-18 2022-05-13 中国科学院微生物研究所 一种靶向pd-1的肿瘤抑制性抗体及其应用
CN112399973B (zh) 2018-06-05 2022-12-16 江苏康宁杰瑞生物制药有限公司 二聚体及其用途
CN110760002A (zh) * 2018-07-25 2020-02-07 南京金斯瑞生物科技有限公司 人源化抗人ctla4单克隆抗体及其制备方法和用途
CN110256561B (zh) * 2019-06-13 2021-04-16 东大生物技术(苏州)有限公司 一组ctla-4单克隆抗体及其医药用途
CN110256563A (zh) * 2019-07-05 2019-09-20 石河子大学 Ctla-4纳米抗体、制备方法及其应用
WO2021208838A1 (en) * 2020-04-13 2021-10-21 Biosion Inc. Antibodies binding ctla4 and uses thereof
CN112079926A (zh) * 2020-09-17 2020-12-15 北京华大蛋白质研发中心有限公司 抗人ctla4单克隆抗体及其应用
CN114316053B (zh) * 2021-12-20 2023-09-29 南京诺唯赞检测技术有限公司 一种vce的单克隆抗体及其制备方法
CN118541390A (zh) * 2022-01-30 2024-08-23 百奥赛图(北京)医药科技股份有限公司 抗ctla4/ox40双特异性抗体及其用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1328571A (zh) * 1998-12-23 2001-12-26 辉瑞大药厂 抗ctla－4的人单克隆抗体
CN101074264A (zh) * 2006-05-17 2007-11-21 上海中信国健药业有限公司 一种重组抗ctla4单克隆抗体及其制备方法和用途
CN103221544A (zh) * 2010-09-24 2013-07-24 昂克斯治疗有限公司 编码单克隆抗-ctla-4抗体的溶瘤腺病毒载体
CN103547595A (zh) * 2011-03-09 2014-01-29 安迪拓普有限公司 人源化ctla-4抗体

Family Cites Families (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US682736A (en) 1900-08-02 1901-09-17 James C Owen Oil-can.
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5851795A (en) * 1991-06-27 1998-12-22 Bristol-Myers Squibb Company Soluble CTLA4 molecules and uses thereof
US6719972B1 (en) * 1994-06-03 2004-04-13 Repligen Corporation Methods of inhibiting T cell proliferation or IL-2 accumulation with CTLA4- specific antibodies
NZ306304A (en) 1995-04-27 1999-03-29 Advanced Tissue Sciences Inc Apparatus and method for sterilizing, seeding, culturing, storing, shipping and testing tissue, synthetic or native vascular grafts
US6051227A (en) 1995-07-25 2000-04-18 The Regents Of The University Of California, Office Of Technology Transfer Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US5855887A (en) 1995-07-25 1999-01-05 The Regents Of The University Of California Blockade of lymphocyte down-regulation associated with CTLA-4 signaling
US5811097A (en) 1995-07-25 1998-09-22 The Regents Of The University Of California Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
JP2001523958A (ja) 1997-03-21 2001-11-27 ブライハム アンド ウィミンズ ホスピタル，インコーポレイテッド 免疫療法のｃｔｌａ−４結合ペプチド
US7122615B1 (en) 1998-09-10 2006-10-17 Rutgers, The State University Of New Jersey Polyanhydrides with therapeutically useful degradation products
US6682736B1 (en) 1998-12-23 2004-01-27 Abgenix, Inc. Human monoclonal antibodies to CTLA-4
US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
EP1212422B1 (en) 1999-08-24 2007-02-21 Medarex, Inc. Human ctla-4 antibodies and their uses
US7605238B2 (en) 1999-08-24 2009-10-20 Medarex, Inc. Human CTLA-4 antibodies and their uses
JP2003520828A (ja) 2000-01-27 2003-07-08 ジェネティクス インスティテュート，エルエルシー Ｃｔｌａ４（ｃｄ１５２）に対する抗体、これを含む結合体、およびその使用
IL149701A0 (en) 2001-05-23 2002-11-10 Pfizer Prod Inc Use of anti-ctla-4 antibodies
CA2481207C (en) 2002-04-12 2015-06-30 Medarex, Inc. Methods of treatment using ctla-4 antibodies
EP2484660A3 (en) 2002-05-24 2013-03-13 Castrol Limited Preparation of monomers for grafting to polyolefins
DK2206517T3 (da) 2002-07-03 2023-11-06 Ono Pharmaceutical Co Immunpotentierende sammensætninger omfattende af anti-PD-L1-antistoffer
AU2003260871A1 (en) 2002-09-30 2004-04-19 Pfizer Products Inc. Hybridomas producing high levels of human sequence antibody
US8168170B2 (en) 2002-10-03 2012-05-01 The Procter And Gamble Company Compositions having an inner core and at least three surrounding layers
US7465446B2 (en) 2003-05-30 2008-12-16 Medarex, Inc. Surrogate therapeutic endpoint for anti-CTLA4-based immunotherapy of disease
CN1964739A (zh) 2004-03-26 2007-05-16 辉瑞产品公司 抗ctla－4抗体的用途
DE102004063494A1 (de) * 2004-12-23 2006-07-13 Tegenero Ag Antikörper
CA2600608A1 (en) * 2005-03-08 2006-09-14 Pharmacia & Upjohn Company Llc Composition comprising human igg2 antibody and chelating agent
WO2006110277A1 (en) 2005-04-11 2006-10-19 Medarex, Inc. Protein purification using hcic amd ion exchange chromatography
EP2161336B2 (en) 2005-05-09 2017-03-29 ONO Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
EP2007423A2 (en) 2006-04-05 2008-12-31 Pfizer Products Incorporated Ctla4 antibody combination therapy
WO2009100140A1 (en) 2008-02-04 2009-08-13 Medarex, Inc. Anti-clta-4 antibodies with reduced blocking of binding of ctla-4 to b7 and uses thereof
US9540426B2 (en) 2009-10-06 2017-01-10 Bristol-Myers Squibb Company Mammalian cell culture processes for protein production
CA2777226A1 (en) 2009-10-12 2011-04-21 Pfizer Inc. Cancer treatment
NO2501822T3 (zh) 2009-11-17 2018-01-13
CN102134276A (zh) * 2010-01-22 2011-07-27 上海抗体药物国家工程研究中心有限公司 一种抗ctla-4嵌合抗体
RU2657440C2 (ru) 2011-06-30 2018-06-13 Джензим Корпорейшн Ингибиторы т-клеточной активации
WO2013142796A2 (en) 2012-03-23 2013-09-26 Bristol-Myers Squibb Company Methods of treatments using ctla4 antibodies
US9856320B2 (en) 2012-05-15 2018-01-02 Bristol-Myers Squibb Company Cancer immunotherapy by disrupting PD-1/PD-L1 signaling
US8844207B2 (en) 2012-06-04 2014-09-30 Mity-Lite, Inc. Portable dance floor panel with floating magnet retention system
CN102801616B (zh) 2012-08-02 2015-04-15 华为技术有限公司 报文发送和接收的方法、装置和系统
EP2759305A1 (en) 2013-01-24 2014-07-30 Thrombotargets Europe, S.L. Hemostatic compositions
US10570204B2 (en) 2013-09-26 2020-02-25 The Medical College Of Wisconsin, Inc. Methods for treating hematologic cancers
KR20160119867A (ko) 2014-03-05 2016-10-14 브리스톨-마이어스 스큅 컴퍼니 항-pd-1 항체 및 또 다른 항암제의 조합물을 이용한 신장암의 치료
MX2016014753A (es) 2014-05-15 2017-03-06 Bristol Myers Squibb Co Tratamiento de cancer de pulmon con el uso de una combinacion de un anticuerpo de muerte anti-programada 1 (anti-pd-1) y otro agente anti-cancerigeno.
CN105296433B (zh) * 2014-08-01 2018-02-09 中山康方生物医药有限公司 一种ctla4抗体、其药物组合物及其用途
MX2017007390A (es) 2014-12-16 2017-11-06 Bristol Myers Squibb Co Uso de inhibidores de punto de control inmunitario en neoplasmas de sistemas nerviosos centrales.
CN104479019B (zh) 2014-12-26 2020-07-21 上海复宏汉霖生物技术股份有限公司 一种抗ctla-4人源抗体
SG10202006863YA (en) 2015-02-16 2020-08-28 Lonza Ag Cl and/or ch1 mutated antibodies for drug conjugation
US20180256715A1 (en) 2015-05-13 2018-09-13 The Board Of Regents Of The University Of Texas System Anti-ctla-4 blockade
JP2019503387A (ja) 2016-01-27 2019-02-07 ブリストル−マイヤーズ スクイブ カンパニーＢｒｉｓｔｏｌ−Ｍｙｅｒｓ Ｓｑｕｉｂｂ Ｃｏｍｐａｎｙ 抗ｐｄ−１抗体および別の抗癌剤の組合せを用いる肺癌の処置法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1328571A (zh) * 1998-12-23 2001-12-26 辉瑞大药厂 抗ctla－4的人单克隆抗体
CN101074264A (zh) * 2006-05-17 2007-11-21 上海中信国健药业有限公司 一种重组抗ctla4单克隆抗体及其制备方法和用途
CN103221544A (zh) * 2010-09-24 2013-07-24 昂克斯治疗有限公司 编码单克隆抗-ctla-4抗体的溶瘤腺病毒载体
CN103547595A (zh) * 2011-03-09 2014-01-29 安迪拓普有限公司 人源化ctla-4抗体

Cited By (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017040790A1 (en) 2015-09-01 2017-03-09 Agenus Inc. Anti-pd-1 antibodies and methods of use thereof
WO2017165742A1 (en) 2016-03-24 2017-09-28 Millennium Pharmaceuticals, Inc. Methods of treating gastrointestinal immune-related adverse events in anti-ctla4 anti-pd-1 combination treatments
WO2017165778A1 (en) 2016-03-24 2017-09-28 Millennium Pharmaceuticals, Inc. Methods of treating gastrointestinal immune-related adverse events in immune oncology treatments
CN109195665A (zh) * 2016-05-19 2019-01-11 苏州康宁杰瑞生物科技有限公司 针对ctla4的单域抗体及其衍生蛋白
WO2017198212A1 (zh) * 2016-05-19 2017-11-23 苏州康宁杰瑞生物科技有限公司 针对ctla4的单域抗体及其衍生蛋白
US11091549B2 (en) 2016-05-19 2021-08-17 Suzhou Alphamab Co., Ltd. Single domain antibody and derivative proteins thereof against CTLA4
CN109195665B (zh) * 2016-05-19 2022-09-09 苏州康宁杰瑞生物科技有限公司 针对ctla4的单域抗体及其衍生蛋白
US11912768B2 (en) 2016-05-19 2024-02-27 Suzhou Alphamab Co., Ltd. Single domain antibody and derivative proteins thereof against CTLA4
WO2017220988A1 (en) 2016-06-20 2017-12-28 Kymab Limited Multispecific antibodies for immuno-oncology
WO2017220990A1 (en) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 antibodies
WO2017220989A1 (en) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 and il-2 cytokines
US11578128B2 (en) * 2016-08-23 2023-02-14 Akeso Pharmaceuticals, Inc. Anti-CTLA4 and anti-PD-1 bifunctional antibody, pharmaceutical composition thereof and use thereof
US12076398B2 (en) 2016-08-23 2024-09-03 CTTQ-Akeso (ShangHai) Biomed. Tech. Co., Ltd. Anti-PD1 monoclonal antibody, pharmaceutical composition thereof and use thereof
JP2019533475A (ja) * 2016-08-23 2019-11-21 康方▲藥▼▲業▼有限公司Ａｋｅｓｏ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 抗ｃｔｌａ４−抗ｐｄ−１二機能性抗体、その医薬組成物および使用
EP3511346A4 (en) * 2016-08-23 2020-04-29 Akeso Pharmaceuticals, Inc. ANTI-CTLA4 AND ANTI-PD -1 BIFUNCTIONAL ANTIBODIES, PHARMACEUTICAL COMPOSITION AND USE THEREOF
US11479608B2 (en) 2016-08-23 2022-10-25 Akeso Biopharma, Inc. Anti-CTLA4 antibodies
JP7425604B2 (ja) 2016-08-23 2024-01-31 康方▲藥▼▲業▼有限公司 抗ｃｔｌａ４－抗ｐｄ－１二機能性抗体、その医薬組成物および使用
US11993651B2 (en) 2016-10-11 2024-05-28 Agenus Inc. Anti-lag-3 antibodies and methods of use thereof
WO2018071500A1 (en) 2016-10-11 2018-04-19 Agenus Inc. Anti-lag-3 antibodies and methods of use thereof
US10844119B2 (en) 2016-10-11 2020-11-24 Agenus Inc. Anti-LAG-3 antibodies and methods of use thereof
US10882908B2 (en) 2016-10-11 2021-01-05 Agenus Inc. Anti-LAG-3 antibodies and methods of use thereof
JP2020512354A (ja) * 2017-03-31 2020-04-23 メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． Ｐｄ−１のアンタゴニストと抗ｃｔｌａ４抗体との組み合わせでがんを処置するための組成物および方法
CN110505882A (zh) * 2017-03-31 2019-11-26 默沙东公司 用pd-1的拮抗剂和抗ctla4抗体的组合治疗癌症的组合物和方法
EP3618866A4 (en) * 2017-05-02 2021-07-14 Merck Sharp & Dohme Corp. STABLE FORMULATIONS OF ANTI-CTLA4 ANTIBODIES ALONE AND IN COMBINATION WITH PROGRAMMED DEATH RECEPTOR 1 (PD-1) ANTIBODIES AND METHOD OF USING THEREOF
US11845798B2 (en) 2017-05-02 2023-12-19 Merck Sharp & Dohme Llc Formulations of anti-LAG3 antibodies and co-formulations of anti-LAG3 antibodies and anti-PD-1 antibodies
JP2020518598A (ja) * 2017-05-02 2020-06-25 メルク・シャープ・アンド・ドーム・コーポレーションＭｅｒｃｋ Ｓｈａｒｐ ＆ Ｄｏｈｍｅ Ｃｏｒｐ． 単独およびプログラム死受容体１（ｐｄ−１）抗体と組み合わされた抗ｃｔｌａ４抗体の安定な製剤、ならびにその使用方法
CN110678199A (zh) * 2017-05-02 2020-01-10 默沙东公司 单独的和与程序性死亡受体1(pd-1)抗体组合的抗ctla4抗体的稳定制剂及其使用方法
JP7173993B2 (ja) 2017-05-19 2022-11-17 ウーシー バイオロジクス（シャンハイ）カンパニー リミテッド 細胞傷害性ｔリンパ球関連タンパク質４（ｃｔｌａ－４）に対する新規モノクローナル抗体
JP2020520655A (ja) * 2017-05-19 2020-07-16 ウーシー バイオロジクス（シャンハイ）カンパニー リミテッド 細胞傷害性ｔリンパ球関連タンパク質４（ｃｔｌａ−４）に対する新規モノクローナル抗体
JP2022024054A (ja) * 2017-05-19 2022-02-08 ウーシー バイオロジクス（シャンハイ）カンパニー リミテッド 細胞傷害性ｔリンパ球関連タンパク質４（ｃｔｌａ－４）に対する新規モノクローナル抗体
CN111727056A (zh) * 2018-02-13 2020-09-29 默沙东公司 用抗pd-1抗体和抗ctla-4抗体治疗癌症的方法
WO2019241730A2 (en) 2018-06-15 2019-12-19 Flagship Pioneering Innovations V, Inc. Increasing immune activity through modulation of postcellular signaling factors
WO2020127377A1 (en) 2018-12-21 2020-06-25 Ose Immunotherapeutics Bifunctional anti-pd-1/il-7 molecule
WO2020165374A1 (en) 2019-02-14 2020-08-20 Ose Immunotherapeutics Bifunctional molecule comprising il-15ra
WO2020227159A2 (en) 2019-05-03 2020-11-12 Flagship Pioneering Innovations V, Inc. Methods of modulating immune activity
WO2021127217A1 (en) 2019-12-17 2021-06-24 Flagship Pioneering Innovations V, Inc. Combination anti-cancer therapies with inducers of iron-dependent cellular disassembly
WO2021122866A1 (en) 2019-12-17 2021-06-24 Ose Immunotherapeutics Bifunctional molecules comprising an il-7 variant
WO2021178611A1 (en) * 2020-03-05 2021-09-10 Merck Sharp & Dohme Corp. Methods for treating cancer or infection using a combination of an anti-pd-1 antibody, an anti-ctla4 antibody, and an anti-tigit antibody
WO2022006179A1 (en) 2020-06-29 2022-01-06 Flagship Pioneering Innovations V, Inc. Viruses engineered to promote thanotransmission and their use in treating cancer
WO2022026330A1 (en) * 2020-07-27 2022-02-03 Promab Biotechnologies, Inc. Humanized cd37 and bi-specific cd19-humanized cd37 car-t cells
WO2022112198A1 (en) 2020-11-24 2022-06-02 Worldwide Innovative Network Method to select the optimal immune checkpoint therapies
WO2022212784A1 (en) 2021-03-31 2022-10-06 Flagship Pioneering Innovations V, Inc. Thanotransmission polypeptides and their use in treating cancer
WO2022214653A1 (en) 2021-04-09 2022-10-13 Ose Immunotherapeutics New scaffold for bifunctional molecules with improved properties
WO2022214652A1 (en) 2021-04-09 2022-10-13 Ose Immunotherapeutics Scaffold for bifunctioanl molecules comprising pd-1 or cd28 and sirp binding domains
WO2023278641A1 (en) 2021-06-29 2023-01-05 Flagship Pioneering Innovations V, Inc. Immune cells engineered to promote thanotransmission and uses thereof
WO2024003360A1 (en) 2022-07-01 2024-01-04 Institut Curie Biomarkers and uses thereof for the treatment of neuroblastoma
WO2024028386A1 (en) 2022-08-02 2024-02-08 Ose Immunotherapeutics Multifunctional molecule directed against cd28
WO2024056049A1 (zh) * 2022-09-16 2024-03-21 同润生物医药(上海)有限公司 具有pH依赖性的抗CTLA4抗体或抗原结合片段
WO2024077191A1 (en) 2022-10-05 2024-04-11 Flagship Pioneering Innovations V, Inc. Nucleic acid molecules encoding trif and additionalpolypeptides and their use in treating cancer
WO2024151687A1 (en) 2023-01-09 2024-07-18 Flagship Pioneering Innovations V, Inc. Genetic switches and their use in treating cancer
WO2024200826A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Lipid-based nanoparticle targeted at activated immune cells for the expression of immune cell inhibiting molecule and use thereof
WO2024200823A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Lipid-based nanoparticle targeted at activated immune cells for the expression of immune cell enhancing molecule and use thereof
WO2024200820A1 (en) 2023-03-30 2024-10-03 Ose Immunotherapeutics Method of synthesis of targeted lipid nanoparticle and uses thereof

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