WO2024088424A1 - 新型抗ctla4抗体 - Google Patents

新型抗ctla4抗体 Download PDF

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Publication number
WO2024088424A1
WO2024088424A1 PCT/CN2023/127601 CN2023127601W WO2024088424A1 WO 2024088424 A1 WO2024088424 A1 WO 2024088424A1 CN 2023127601 W CN2023127601 W CN 2023127601W WO 2024088424 A1 WO2024088424 A1 WO 2024088424A1
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seq
antibody
antigen
binding fragment
cancer
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PCT/CN2023/127601
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English (en)
French (fr)
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杜庆林
吕雅璇
周添添
彭菲
李雪琴
曹欢欢
杨雪艳
丁茜
谭永聪
韩淑华
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嘉和生物药业有限公司
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Publication of WO2024088424A1 publication Critical patent/WO2024088424A1/zh

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present disclosure relates to antibodies and their use. Specifically, the present disclosure relates to antibodies that specifically bind to cytotoxic T lymphocyte-associated antigen-4 (CTLA4), in particular heavy chain antibodies, more particularly single domain antibodies; and therapeutic uses of the antibodies.
  • CTL4 cytotoxic T lymphocyte-associated antigen-4
  • CTLA4 cytotoxic T lymphocyte-associated antigen-4
  • CD152 cytotoxic T lymphocyte-associated antigen-4
  • CTLA4 is constitutively expressed in regulatory T cells (Tregs) and is upregulated in other T cells after activation ( et al., 2015, Jago et al., 2004).
  • CTLA4 shares the same ligands as CD28, namely CD80/B7-1 and CD86/B7-2. Chikuma proposed that the mechanism of CTLA4 is to inhibit T cell activation by outcompeting CD28 for binding to CD80/B7-1 and CD86/B7-2 (Chikuma, 2017).
  • CTLA4 is highly expressed in the tumor microenvironment (TME), especially on Tregs, which are considered to be key molecules for regulating Treg function and modulating anti-tumor immunity (Montler et al., 2016, Sutmuller et al., 2001).
  • TAE tumor microenvironment
  • CTLA4 is constitutively expressed in Foxp3+ Treg cells, inhibiting anti-tumor activity (Montler et al., 2016, Sutmuller et al., 2001).
  • Ipilimumab It is the most well-known anti-CTLA4 antibody and was approved by the US FDA in 2011 for the treatment of advanced melanoma. Ipilimumab is clinically used as a monotherapy (Hodi et al., 2010) and as part of a combination therapy with nivolumab. (Larki et al., 2015) have shown effective cancer immunotherapy effects (CITE).
  • CTLA4 therapies show serious immunotherapy-related adverse reactions (irAEs) (Calabrese et al., 2018), especially when combined with Nivolumab treatment, because systemic activation of T cells by blocking the B7-CTLA4 pathway leads to reduced patient tolerance to antibodies (Hodi, 2010, Bertrand et al., 2015).
  • CTLA4 remains an important immunotherapy target because of its ability to induce durable immune responses in cancer patients (Maio et al., 2015, Whitneydorf et al., 2015).
  • the main challenge in generating CTLA4 antibodies is to improve their safety and efficacy.
  • Zhang et al. reported a novel CTLA4 antibody, HL32 (ONC-392), with a modified Fc, that uniquely targets CTLA4 to selectively eliminate tumor-infiltrating Tregs without affecting T cell activation in peripheral T cells (Zhang et al., 2019).
  • HL32 Compared with other commercial or clinical-stage anti-CTLA4 antibodies, HL32 has more robust CITEs but significantly reduced irAEs in preclinical models (Zhang et al., 2019, Du et al., 2018) and phase 1A/1B clinical trials (PRESERVE-001, NCT04140526). They proposed that clinically effective anti-CTLA4 mAbs cause tumor rejection through a mechanism that is independent of checkpoint blockade but dependent on host Fc receptors (Du et al., 2018).
  • the inventors of the present application screened out improved anti-CTLA4 antibodies with different blocking abilities for CD80/86 and retained the Fc function of the antibody, thereby providing improved response rates and reduced irAEs.
  • the antibodies inhibited the binding of CD80/CD86 to CTLA4 to varying degrees, while exhibiting a strong ADCC response.
  • the present disclosure provides an antibody that specifically binds to CTLA4, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%
  • SEQ ID NO: 1 SEQ ID NO: 2 and SEQ ID NO: 3;
  • the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:1, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:2, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:3.
  • the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:31.
  • the heavy chain variable region comprises SEQ ID NO:4 or consists of SEQ ID NO:4.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5.
  • the heavy chain variable region comprises SEQ ID NO:7 or consists of SEQ ID NO:7.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8.
  • the heavy chain variable region comprises SEQ ID NO:10 or consists of SEQ ID NO:10.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11.
  • the heavy chain variable region comprises SEQ ID NO: 31 or Consists of SEQ ID NO:31.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:32.
  • the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:13, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:14, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:15.
  • the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16 or SEQ ID NO:19.
  • the heavy chain variable region comprises SEQ ID NO:16 or consists of SEQ ID NO:16.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:17.
  • the heavy chain variable region comprises SEQ ID NO:19 or consists of SEQ ID NO:19.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:20.
  • the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:22, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:23, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:24.
  • the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25 or SEQ ID NO:28.
  • the heavy chain variable region comprises SEQ ID NO:25 or consists of SEQ ID NO:25.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26.
  • the heavy chain variable region comprises SEQ ID NO:28 or consists of SEQ ID NO:28.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:29.
  • the antibody or antigen-binding fragment thereof is selected from a whole antibody, a single-chain antibody (scFv), a heavy chain antibody, a single domain antibody, a Fab fragment, a Fab' fragment, F(ab')2, Fv, a Fd fragment, and a bispecific antibody (BsAb).
  • scFv single-chain antibody
  • BsAb bispecific antibody
  • the antibody or antigen-binding fragment thereof is a heavy chain-only antibody.
  • the antibody or antigen-binding fragment thereof comprises or consists of two heavy chains.
  • the antibody or antigen-binding fragment thereof is a single domain antibody.
  • the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
  • the antibody or antigen-binding fragment thereof exhibits significant antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
  • the present disclosure provides an isolated nucleic acid molecule encoding the antibody.
  • the nucleic acid molecule comprises the sequence of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:21, SEQ SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:33 have a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical.
  • the present disclosure provides an expression vector comprising the nucleic acid molecule.
  • the present disclosure provides a host cell, which expresses the antibody, and/or comprises the nucleic acid molecule or the expression vector.
  • the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering the antibody or pharmaceutical composition in a therapeutically effective amount, wherein the disease or disorder is cancer or an autoimmune disease.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.
  • the antibody or pharmaceutical composition is administered in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.
  • the present disclosure provides use of the antibody or the pharmaceutical composition in the preparation of a medicament for treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.
  • the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.
  • the disclosure provides the antibody or the pharmaceutical composition for use in treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.
  • the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.
  • the present disclosure provides a kit comprising
  • Figures 1A and 1B show FACS analysis of the binding specificity of anti-CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, GBD008-hS004-5 and GBD008-hS005-3-2) to cell surface human CTLA4 protein, where Ipi represents Ipilimumab.
  • Figures 2A-2F show FACS analysis of anti-CTLA4 antibodies blocking CD80 and CD86 binding to CTLA4, where Ipi represents Ipilimumab.
  • Figure 2A shows that anti-CTLA4 antibodies (GBD008-hS005-3 and GBD008-hS005-5) block CD80 binding to CTLA4;
  • Figure 2B shows that anti-CTLA4 antibodies (GBD008-S019 and GBD008-hS019-4) block CD80 binding to CTLA4;
  • Figure 2C shows that anti-CTLA4 antibodies (GBD008-S004 and GBD008-hS004-5) block CD80 binding to CTLA4;
  • Figure 2D shows that anti-CTLA4 antibodies (GBD008-hS
  • Figure 2E shows that anti-CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS0
  • FIGS 3A and 3B show the analysis of the CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008- GBD008-hS005-3-2).
  • Figures 4A-4F show the binding ability of anti-CTLA4 antibodies (GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, GBD008-hS004-5 and GBD008-hS005-3-2) to human CTLA4 ( Figures 4A and 4B), cynomolgus monkey CTLA4 ( Figures 4C and 4D) and mouse CTLA4 ( Figures 4E and 4F) (by ELISA).
  • anti-CTLA4 antibodies GBD008-hS005, GBD008-hS005-3, GBD008-hS005-5, GBD008-S019, GBD008-hS019-4, GBD008-S004, GBD008-hS004-5 and GBD008-hS005-3-2
  • human CTLA4 Figures 4A and 4
  • Figure 6 shows the in vivo anti-tumor efficacy of anti-CTLA4 antibodies in the MC38 colorectal tumor model.
  • Figure 6A Average values of tumor volume over time.
  • Figure 6B Average values of tumor volume and TGI (Tumor Growth Inhibition) at the end of the experiment.
  • Figures 7A-7C show the toxicity (arthritis) of anti-CTLA4 antibodies in hCTLA4/hPD1 Balb/C mice.
  • Figure 7A Trend of weight changes in mice.
  • Figure 7B Arthritis (paw and joint inflammation) scores in mice.
  • Figure 7C Incidence of arthritis in mice.
  • the present disclosure provides antibodies that specifically bind to CTLA4.
  • Administering a therapeutically effective amount of the anti-CTLA4 antibodies of the present disclosure to a subject in need thereof can be used to treat a disease or condition, including cancer and autoimmune diseases.
  • the binding of the antibody to T cells expressing CTLA4 induces strong antibody-dependent cell-mediated cytotoxicity against cells expressing tumor-associated antigens.
  • the antibodies disclosed herein provide various advantages, such as effective binding to CTLA4, effective induction of strong antibody-dependent cell-mediated cytotoxicity, and/or significantly reduced risk of adverse events (eg, toxicity).
  • anti-CTLA4 antibodies in certain forms bind to CTLA4 more effectively, leading to higher efficacy and improved immunotherapy.
  • range is used as a shorthand form for describing each and all numerical values within the range. Any numerical value within the range, such as an integer value, a value that increases by one tenth (when the end value of the range is one decimal place), or a value that increases by one hundredth (when the end value of the range is two decimal places), can be selected as the end point of the range.
  • range 1-10 is used to describe all numerical values within the range, such as 1, 2, 3, 4, 5, 6, 7, 8 ...
  • an “antibody” may include whole antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding portion") or single chains.
  • an “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof.
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH ) and a heavy chain constant region.
  • VH heavy chain variable region
  • the heavy chain constant region comprises Contains three domains, CH1, CH2 and CH3.
  • each light chain comprises a light chain variable region (abbreviated herein as VL ) and a light chain constant region.
  • the light chain constant region comprises one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which alternate with more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL comprises three CDRs and four framework regions (FRs), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains comprise binding domains that interact with antigens.
  • immunoglobulins can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • immunoglobulins may be from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG and IgM.
  • IgG isotypes are divided into subclasses in certain species: IgGl, IgG2, IgG3, and IgG4 in humans, and IgGl, IgG2a, IgG2b, and IgG3 in mice.
  • Immunoglobulins, e.g., human IgGl exist in several allotypes that differ from each other in at most a few amino acids.
  • antibody may include, for example, whole antibodies, single-chain antibodies (scFv), heavy chain antibodies, single domain antibodies, Fab fragments, Fab' fragments, F(ab')2, Fv, Fd fragments, bispecific antibodies (BsAb), etc.; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; fully synthetic antibodies, etc.
  • scFv single-chain antibodies
  • BsAb bispecific antibodies
  • an "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen bound by a complete antibody.
  • An “antigen-binding fragment” is any proteinaceous structure that can exhibit binding affinity for a specific antigen.
  • Antigen-binding fragments include those provided by any known techniques such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments consist of portions of a complete antibody that retain the antigen-binding specificity of a parent antibody molecule.
  • an antigen-binding fragment may contain at least one variable region (heavy chain variable region or light chain variable region) or one or more CDRs of an antibody known to bind to a specific antigen.
  • suitable antigen-binding fragments include, but are not limited to, diabodies and single-chain molecules, as well as Fab, F(ab')2, Fc, Fabc, and Fv molecules; single-chain (Sc) antibodies; single antibody light chains; single antibody heavy chains; antibody chains or CDRs with other proteins Chimeric fusion between; protein scaffold; heavy chain monomer or dimer; light chain monomer or dimer; dimer consisting of one heavy chain and one light chain; monovalent fragment consisting of VL, VH, CL and CH1 domains; or monovalent antibody as described in WO2007059782; bivalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region; Fd fragment consisting essentially of VH and CH1 domains; Fv fragment consisting essentially of the VL domain and V
  • antigen binding fragments can include non-antibody protein frameworks that can be successfully incorporated into polypeptide segments in an orientation that confers affinity for a given antigen of interest (such as a protein scaffold).
  • Antigen binding fragments can be recombinantly produced or produced by enzymatic or chemical cleavage of intact antibodies.
  • the term “heavy chain antibody” or “heavy chain-only antibody” refers to a single-chain antibody comprising only a heavy chain.
  • a “heavy chain antibody” lacks not only a light chain, but also a constant region CH1 region in a conventional antibody heavy chain. Compared to a heavy chain antibody, a “single domain antibody” as used herein lacks an Fc region.
  • sequence “identity” refers to the relationship between two or more polynucleotide sequences or between two or more polypeptide sequences. When a position in one sequence is occupied by the same nucleic acid base or amino acid residue in the corresponding position of the comparison sequence, the sequence is called “identical” at that position.
  • the percentage sequence “identity” is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of "identical” positions. The number of “identical” positions is then divided by the total number of positions in the comparison window and multiplied by 100 to produce the percentage of sequence "identity".
  • the percentage of "identity” is determined by comparing two optimally aligned sequences in the comparison window.
  • portions of the polynucleotide or polypeptide sequences in the comparison window may contain additions or deletions called gaps, while the reference sequence remains constant.
  • the optimal alignment is the alignment that produces the maximum possible number of "identical” positions between the reference sequence and the comparison sequence, even with gaps.
  • BLAST 2 Sequences obtained from the National Center for Biotechnology Information (NCBI), which includes the programs BLASTN (for nucleotide sequence comparisons) and BLASTP (for polypeptide sequence comparisons), which are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873-5877, 1993).
  • Humanized antibodies refer to antibodies in which some, most or all of the amino acids outside the CDR domains of non-human antibodies, such as mouse antibodies, are replaced by corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains are replaced by amino acids from human immunoglobulins, while some, most or all of the amino acids in one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are allowed as long as they do not abolish the ability of the antibody to bind to a specific antigen. "Humanized” antibodies retain antigenic specificity similar to that of the original antibody.
  • a “chimeric antibody” refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, e.g., an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.
  • a “hybrid” antibody refers to an antibody with heavy and light chains of different types, e.g., a mouse (parent) heavy chain and a humanized light chain, or vice versa.
  • the term "monoclonal antibody” refers to an antibody that displays a single binding specificity and affinity for a particular epitope, or an antibody composition in which all antibodies display a single binding specificity and affinity for a particular epitope.
  • isotype refers to the antibody class encoded by the heavy chain constant region genes (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).
  • heavy chain constant region genes eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • nonspecific cytotoxic cells expressing Fc ⁇ Rs e.g., monocytes, such as natural killer (NK) cells and macrophages
  • NK natural killer
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • any effector cell with an activating Fc ⁇ R can be triggered to mediate ADCC.
  • the primary cell mediating ADCC is the NK cell, which expresses only Fc ⁇ Rs. RIII, while monocytes can express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII depending on their activation, localization or differentiation state.
  • the present disclosure provides an antibody that specifically binds to CTLA4, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 respectively comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to:
  • SEQ ID NO: 1 SEQ ID NO: 2 and SEQ ID NO: 3;
  • the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:1, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:2, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:3.
  • the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:31.
  • the heavy chain variable region comprises SEQ ID NO:4 or consists of SEQ ID NO:4.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5.
  • the heavy chain variable region comprises SEQ ID NO:7 or consists of SEQ ID NO:7.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:8.
  • the heavy chain variable region comprises SEQ ID NO:10 or consists of SEQ ID NO:10.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11.
  • the heavy chain variable region comprises SEQ ID NO:31 or consists of SEQ ID NO:31.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:32.
  • the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:13, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:14, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:15.
  • the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16 or SEQ ID NO:19.
  • the heavy chain variable region comprises SEQ ID NO:16 or consists of SEQ ID NO:16.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:17.
  • the heavy chain variable region comprises SEQ ID NO:19 or consists of SEQ ID NO:19.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:20.
  • the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises or consists of the sequence shown in SEQ ID NO:22, the CDR2 comprises or consists of the sequence shown in SEQ ID NO:23, and the CDR3 comprises or consists of the sequence shown in SEQ ID NO:24.
  • the heavy chain variable region comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:25 or SEQ ID NO:28.
  • the heavy chain variable region comprises SEQ ID NO:25 or consists of SEQ ID NO:25.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:26.
  • the heavy chain variable region comprises SEQ ID NO:28 or consists of SEQ ID NO:28.
  • the heavy chain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:29.
  • the antibody or antigen-binding fragment thereof is selected from a whole antibody, a single-chain antibody (scFv), a heavy chain antibody, a single domain antibody, a Fab fragment, a Fab' fragment, F(ab')2, Fv, a Fd fragment, and a bispecific antibody (BsAb).
  • scFv single-chain antibody
  • BsAb bispecific antibody
  • the antibodies of the present disclosure consist of only heavy chains. In some embodiments, the antibodies of the present disclosure comprise or consist of two heavy chains. In some embodiments, the antibodies of the present disclosure are single domain antibodies.
  • the antibody is a human antibody, a humanized antibody, or a chimeric antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is isolated.
  • the antibody is an isolated monoclonal antibody.
  • nucleotide and amino acid sequence of the antibody of the combination with antigen that is encoded by nucleotide sequence or comprises amino acid sequence are modified.
  • modification can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Consservative sequence modification includes conservative amino acid replacement, wherein amino acid residue is replaced with amino acid residue with similar side chain.
  • the family of amino acid residue with similar side chain has been defined in the art. These families include amino acid with basic side chain (for example, lysine, arginine, histidine), acidic side chain (for example, aspartic acid, glutamic acid), uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chain (for example, alanine, valine
  • the antibodies exhibit potent antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutically acceptable carrier which may be an inert or physiologically active carrier.
  • pharmaceutically acceptable carrier includes any solvent, dispersion medium, coating, antibacterial agent, antifungal agent, etc. that is physiologically compatible. Examples of suitable carriers include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and any combination thereof.
  • the present disclosure provides an isolated nucleic acid molecule encoding the antibody.
  • the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:30 or SEQ ID NO:33.
  • the nucleic acid molecule comprises a nucleic acid having at least In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO:9. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO:9.
  • the nucleic acid molecule comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity to SEQ ID NO:9.
  • the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:9.
  • the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12.
  • the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 12.
  • the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 12. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 18.
  • the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:21. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:21. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:27.
  • the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:27. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: In some embodiments, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO:30. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:30. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO:33 that is at least 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO:33. In some embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:33.
  • the present disclosure provides an expression vector comprising the nucleic acid molecule.
  • the present disclosure provides a host cell, which expresses the antibody, and/or comprises the nucleic acid molecule or the expression vector.
  • the disclosure provides the antibody or the pharmaceutical composition for use in treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.
  • the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.
  • the present disclosure provides use of the antibody or the pharmaceutical composition in the preparation of a medicament for treating a disease or disorder, wherein the disease or disorder is cancer or an autoimmune disease.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer. Cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia and lymphoma.
  • the antibodies or pharmaceutical compositions are for administration in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.
  • the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering the antibody or pharmaceutical composition in a therapeutically effective amount, wherein the disease or disorder is cancer or an autoimmune disease.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, renal cell carcinoma, breast cancer, epithelial squamous cell carcinoma, melanoma, myeloma, gastric cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, head and neck cancer, leukemia, and lymphoma.
  • the antibody or pharmaceutical composition is administered in combination with one or more additional chemotherapeutic agents, radiotherapeutic agents, cytokines, or other antibodies.
  • administering refers to the use of any of the various methods and delivery systems known to those skilled in the art to introduce a composition comprising a therapeutic agent into a subject.
  • the route of administration of the pharmaceutical composition of the present disclosure includes intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, such as by injection or infusion.
  • parenteral administration refers to the mode of administration generally by injection except enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation.
  • the composition is administered by non-parenteral routes, in some embodiments, by oral administration.
  • Non-parenteral routes include topical, epidermal or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual or local. Administration can also be performed, for example, once, multiple times and/or in one or more extended time periods.
  • the term "effective amount” refers to the amount of an active agent required to provide a therapeutic and/or prophylactic benefit to a subject.
  • the "subject in need” refers to any mammal, such as but not limited to humans, horses, cows, cats, mice, rabbits, rats, goats, etc.
  • the mammal is a human.
  • the present disclosure provides a kit comprising
  • VHH antibodies such as camels or alpacas
  • VHH antibodies naturally lack light chains but retain structural stability and effective antigen binding properties.
  • Methods for obtaining antigen or epitope specific VHH domains have been described previously, for example WO2006/040153 and WO2006/122786; R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 Jun. 2009 and WO94/04678.
  • the anti-CTLA4 VHH antibody sequence was obtained by immunizing alpacas with human CTLA4/CD153 protein (Acro Biosystem, Catalog # CT4-H5229).
  • the VHH sequence obtained from alpacas was sequence optimized by changing the amino acids in the original VHH sequence and replacing them with the common amino acids occurring in the VH domain of human antibodies.
  • CHOK1-CTLA4 cells (1x10 5 /well) were washed twice in FACS buffer (PBS + 2% BSA) and resuspended in 100 ⁇ l FACS buffer containing serial dilutions (1:5) of anti-CTLA4 mAb and incubated for 1 hour at 4° C. The cells were then washed twice in FACS buffer and bound antibodies were detected by incubation with APC anti-human IgG Fc (Biolegend, Catalog #410712) for 1 hour at 4° C. Afterwards, the cells were washed twice in FACS buffer and then collected and analyzed using BD LSRFortessa (BD Biosciences).
  • MFI mean fluorescence intensity
  • CHOK1-CTLA4 cells (1x10 5 /well) were washed twice in FACS buffer (PBS + 2% BSA), resuspended in 100 ⁇ l FACS buffer containing serial dilutions (1:5) of anti-CTLA4 mAb and CD80 Biotinylated tag (SinoBiological, Catlog #10698-H49H-B) or CD86 Biotinylated tag (SinoBiological, Catlog #10699-H08H-B), and incubated at 4°C for 0.5 hours.
  • the cells were then washed twice in FACS buffer and bound antibodies were detected by incubation with Streptavidi-Alexa Flour 488 (Invitrogen, Catlog #S32354) for 0.5 hours at 4°C. Afterwards, the cells were washed twice in FACS buffer and then collected and analyzed using BD LSRFortessa (BD Biosciences).
  • the results showed that the anti-CTLA4 antibodies GBD008-S004, GBD008-hS005, GBD008-S019, and the humanized GBD008-hS005-3, GBD008-hS005-5, GBD008-hS005-3-2, GBD008-hS019-4, and GBD008-hS004-5 antibodies partially inhibited the binding of CD80 or CD86 to CTLA4.
  • MFI mean fluorescence intensity
  • MFI mean fluorescence intensity
  • the binding kinetics of CTLA-4 antibody Fc fusion protein and recombinant human CTLA-4 were analyzed by The Octet method was used to qualitatively and quantitatively analyze protein interactions (Octect RH-16, Sartorius).
  • the association rate (k on ) and dissociation rate (k off ) were calculated using a simple one-to-one binding model (Octet evaluation software version 12.2).
  • the equilibrium dissociation constant (k D ) was calculated as the ratio k off /k on .
  • Table 3 The results in Table 3 show that all CTLA4 antibody Fc fusion proteins bind to the CTLA4 antigen with a strong affinity.
  • Example 5 SEB assay for analyzing IL2 production by using CTLA4 antibody
  • Cytotoxic activity was assessed using FACS analysis. Effector cells Human peripheral blood mononuclear cells were obtained from individual human donors ( Biotechnologies) and cultured overnight with 10 ng/ml hIL-2 (PeproTech, 200-02). Target Raji cells expressing full-length human CTLA4 were labeled with 3 ml of detection buffer in the EuTDA kit mixed with 5 ⁇ l of DELFIA BATDA reagent (PerkinElmer, Catlog #AD16) at 37°C for 20 minutes and then labeled with RPMI 1640 medium (Gibco, Catlog #A10491-01) containing 10% FBS (Gibco, Catlog #10099-141) was washed twice and seeded in a 96-well round-bottom plate at a ratio of 20:1 effector cells to target cells.
  • DELFIA BATDA reagent PerkinElmer, Catlog #AD16
  • Example 8 In vivo antitumor efficacy of anti-CTLA4 antibodies in the MC38 colorectal tumor model
  • tumors were formed (about 80 mm 3 )
  • two doses of Ipilimumab 0.3 mg/kg and 3 mg/kg
  • two doses of the anti-CTLA4 antibody of the present disclosure (0.15 mg/kg and 1.5 mg/kg) were given by intraperitoneal injection.
  • Treatment was twice a week for three weeks. Tumor growth was monitored every three days and reported as the average tumor volume. The results are shown in Figure 6.
  • the average tumor volume of mice treated with the anti-CTLA4 antibody of the present disclosure was significantly smaller than that of the IgG1 control.
  • the average tumor volume of mice was reduced by 62% for the 0.15 mpk dose, and by up to 99% for the 1.5 mpk dose.
  • the arthritis of mice treated with the anti-CTLA4 antibody (GBD008-hS005-3-2) of the present disclosure and Nivolumab co-administered was significantly less than that of the IgG1 control and the Ipilimumab and Nivolumab co-administered group.
  • the incidence of arthritis in mice treated with GBD008-hS005-3-2 and Nivolumab co-administered was also significantly less than that of the IgG1 control and the Ipilimumab and Nivolumab co-administered group.
  • the above examples demonstrate that the anti-CTLA4 antibodies disclosed herein inhibit the binding of CD80/CD86 to CTLA4 to varying degrees, while exhibiting a strong ADCC response.
  • the anti-CTLA4 antibodies have varying degrees of IL2 response rates by blocking CD80/86 to varying degrees and retaining the Fc function of the antibody.
  • the anti-CTLA4 antibodies disclosed herein exhibit excellent in vivo anti-tumor efficacy and insignificant toxicity in a mouse model.

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Abstract

本公开提供了特异性结合细胞毒性T淋巴细胞相关抗原-4(CTLA4)的抗体，特别是重链抗体，更特别是单域抗体。还公开了所述抗体的治疗用途。

Description

新型抗CTLA4抗体 技术领域
本公开涉及抗体及其应用。具体而言，本公开涉及特异性结合细胞毒性T淋巴细胞相关抗原-4(CTLA4)的抗体，特别是重链抗体，更特别是单域抗体；以及所述抗体的治疗用途。
背景技术
作为第一个临床靶向的免疫检查点受体，细胞毒性T淋巴细胞相关抗原-4(Cytotoxic T lymphocyte-associated antigen-4，CTLA4，又称为CD152)在T细胞中表达。CTLA4在调节性T细胞(Tregs)中组成型表达，并在活化后在其他T细胞中上调(et al.,2015,Jago et al.,2004)。CTLA4与CD28共享相同的配体，即CD80/B7-1和CD86/B7-2。Chikuma提出，CTLA4的机制是通过在与CD80/B7-1和CD86/B7-2结合的竞争中胜过CD28来抑制T细胞活化(Chikuma,2017)。CTLA4在肿瘤微环境(TME)中高度表达，尤其是在Tregs上，Tregs被认为是调控Treg功能和调节抗肿瘤免疫的关键分子(Montler et al.,2016,Sutmuller et al.,2001)。此外，CTLA4在Foxp3+Treg细胞中组成型表达，抑制抗肿瘤活性(Montler et al.,2016,Sutmuller et al.,2001)。
到目前为止，已经提出了治疗性CTLA4抗体的几种作用机制，包括通过阻断B7-CTLA4途径活化效应T细胞和通过抗体依赖性细胞介导的细胞毒性(ADCC)消耗Tregs，以及抗体依赖性细胞吞噬作用(ADCP)(Arce Vargas et al.,2018,Du et al.,2018)。然而，这些机制的临床重要性仍然存在争议(Du et al.,2018)。
Ipilimumab是最广为人知的抗CTLA4抗体，于2011年于美国FDA获批用于治疗晚期黑色素瘤。Ipilimumab在临床上作为单一疗法(Hodi et al.,2010)和作为与Nivolumab联合疗法的一部分 (Larki et al.,2015)均表现出有效的癌症免疫治疗效果(CITE)。然而，上述CTLA4疗法表现出严重的免疫疗法相关不良反应(irAE)(Calabrese et al.,2018)，尤其是与Nivolumab治疗相结合时，因为通过阻断B7-CTLA4途径全身活化T细胞，导致降低患者对抗体的耐受性(Hodi,2010,Bertrand et al.,2015)。
即便如此，CTLA4仍然是一个重要的免疫治疗靶点，因为它能够在癌症患者中诱导持久免疫应答(Maio et al.,2015,Schadendorf et al.,2015)。产生CTLA4抗体的主要挑战是提高其安全性和有效性。最近，Zhang等人报道了一种新型CTLA4抗体HL32(ONC-392)，具有修饰的Fc，独特地靶向CTLA4，以选择性消除肿瘤浸润性Treg，而不影响外周T细胞中的T细胞活化(Zhang et al.,2019)。与其他商业或临床阶段的抗CTLA4抗体相比，HL32在临床前模型(Zhang et al.,2019,Du et al.,2018)和1A/1B期临床试验(PRESERVE-001，NCT04140526)中具有更稳健的CITE但显著降低的irAE。他们提出，临床有效的抗CTLA4 mAb通过独立于检查点阻断但依赖于宿主Fc受体的机制引起肿瘤排斥(Du et al.,2018)。
然而，尽管有上述进展，本领域仍需要具有提高的反应率(response rate，RR)和降低的irAE的改进抗CTLA-4抗体以及更有效的免疫疗法。
发明内容
本申请发明人筛选出对CD80/86具有不同阻断能力和保留抗体的Fc功能而提供了具有提高的反应率和降低的irAE的改进抗CTLA4抗体。如本申请实施例证实的，所述抗体不同程度抑制了CD80/CD86与CTLA4的结合，同时表现出强烈的ADCC反应。
因此，在一个方面，本公开提供了一种特异性结合CTLA4的抗体，其中所述抗体包含重链可变区，所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1、CDR2和CDR3分别包含与以下具有至少70％、75％、80％、85％、88％、90％、92％、95％、 97％、98％、99％或100％同一性的氨基酸序列：
(1)SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3；
(2)SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15；或
(3)SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24。
在一些实施方案中，所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1包含SEQ ID NO:1所示的序列或由其组成，所述CDR2包含SEQ ID NO:2所示的序列或由其组成，所述CDR3包含SEQ ID NO:3所示的序列或由其组成。
在一些实施方案中，所述重链可变区包含与SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10或SEQ ID NO:31具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:4或由SEQ ID NO:4组成。
在一些实施方案中，所述重链包含与SEQ ID NO:5具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:7或由SEQ ID NO:7组成。
在一些实施方案中，所述重链包含与SEQ ID NO:8具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:10或由SEQ ID NO:10组成。
在一些实施方案中，所述重链包含与SEQ ID NO:11具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:31或由 SEQ ID NO:31组成。
在一些实施方案中，所述重链包含与SEQ ID NO:32具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1包含SEQ ID NO:13所示的序列或由其组成，所述CDR2包含SEQ ID NO:14所示的序列或由其组成，所述CDR3包含SEQ ID NO:15所示的序列或由其组成。
在一些实施方案中，所述重链可变区包含与SEQ ID NO:16或SEQ ID NO:19具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:16或由SEQ ID NO:16组成。
在一些实施方案中，所述重链包含与SEQ ID NO:17具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:19或由SEQ ID NO:19组成。
在一些实施方案中，所述重链包含与SEQ ID NO:20具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1包含SEQ ID NO:22所示的序列或由其组成，所述CDR2包含SEQ ID NO:23所示的序列或由其组成，所述CDR3包含SEQ ID NO:24所示的序列或由其组成。
在一些实施方案中，所述重链可变区包含与SEQ ID NO:25或SEQ ID NO:28具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:25或由SEQ ID NO:25组成。
在一些实施方案中，所述重链包含与SEQ ID NO:26具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:28或由SEQ ID NO:28组成。
在一些实施方案中，所述重链包含与SEQ ID NO:29具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述抗体或其抗原结合片段选自完整抗体、单链抗体(scFv)、重链抗体、单域抗体、Fab片段、Fab’片段、F(ab’)2、Fv、Fd片段和双特异性抗体(BsAb)。
在一些实施方案中，所述抗体或其抗原结合片段是仅重链的抗体。
在一些实施方案中，所述抗体或其抗原结合片段包含两条重链或由两条重链组成。
在一些实施方案中，所述抗体或其抗原结合片段是单域抗体。
在一些实施方案中，所述抗体是人抗体、人源化抗体或嵌合抗体。
在一些实施方案中，所述抗体或其抗原结合片段表现出显著的抗体依赖性细胞介导的细胞毒性(ADCC)。
在一个方面，本公开提供了一种药物组合物，所述组合物包含治疗有效量的所述抗体或其抗原结合片段和药学上可接受的载体。
在一个方面，本公开提供了一种分离的核酸分子，其编码所述抗体。
在一些实施方案中，所述核酸分子包含与SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、SEQ ID NO:18、SEQ ID NO:21、SEQ  ID NO:27、SEQ ID NO:30或SEQ ID NO:33具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。
在一个方面，本公开提供了一种表达载体，其包含所述核酸分子。
在一个方面，本公开提供了一种宿主细胞，其表达所述抗体，和/或包含所述核酸分子或所述表达载体。
在一个方面，本公开提供了一种治疗有需要的受试者的疾病或病症的方法，包括以治疗有效量施用所述抗体或药物组合物，其中所述疾病或病症是癌症或自身免疫性疾病。
在一些实施方案中，所述癌症选自下组：结直肠癌、结肠癌、肾细胞癌、乳腺癌、上皮鳞状细胞癌、黑色素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、头颈癌、白血病和淋巴瘤。
在一些实施方案中，所述抗体或药物组合物与一种或多种另外的化学治疗剂、放射治疗剂、细胞因子或其它抗体联合施用。
在一个方面，本公开提供了所述抗体或所述药物组合物在制备用于治疗疾病或病症的药物中的用途，其中所述疾病或病症是癌症或自身免疫性疾病。
在一些实施方案中，所述癌症选自下组：结直肠癌、结肠癌、肾细胞癌、乳腺癌、上皮鳞状细胞癌、黑色素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、头颈癌、白血病和淋巴瘤。
在一些实施方案中，所述抗体或药物组合物用于与一种或多种另外的化学治疗剂、放射治疗剂、细胞因子或其它抗体联合施用。
在一个方面，本公开提供了所述抗体或所述药物组合物，其用于治疗疾病或病症，其中所述疾病或病症是癌症或自身免疫性疾病。
在一些实施方案中，所述癌症选自下组：结直肠癌、结肠癌、肾细胞癌、乳腺癌、上皮鳞状细胞癌、黑色素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、头颈癌、白血病和淋巴瘤。
在一些实施方案中，所述抗体或药物组合物用于与一种或多种另外的化学治疗剂、放射治疗剂、细胞因子或其它抗体联合施用。
在一个方面，本公开提供了一种药盒，其包含
a)所述抗体或所述药物组合物；和
b)使用说明。
附图说明
图1A和1B显示抗CTLA4抗体(GBD008-hS005、GBD008-hS005-3、GBD008-hS005-5、GBD008-S019、GBD008-hS019-4、GBD008-S004、GBD008-hS004-5和GBD008-hS005-3-2)与细胞表面人CTLA4蛋白结合特异性的FACS分析，其中Ipi代表Ipilimumab。
图2A-2F显示抗CTLA4抗体阻断CD80和CD86结合CTLA4的FACS分析，其中Ipi代表Ipilimumab。图2A显示抗CTLA4抗体(GBD008-hS005-3和GBD008-hS005-5)阻断CD80结合CTLA4；图2B显示抗CTLA4抗体(GBD008-S019和GBD008-hS019-4)阻断CD80结合CTLA4；图2C显示抗CTLA4抗体(GBD008-S004和GBD008-hS004-5)阻断CD80结合CTLA4；图2D显示抗CTLA4抗体(GBD008-hS005-3-2)阻断CD80结合CTLA4；图2E显示抗CTLA4抗体(GBD008-hS005、GBD008-hS005-3、GBD008-hS005-5、GBD008-S019、GBD008-hS019-4、GBD008-S004和GBD008-hS004-5)阻断CD86结合CTLA4；图2F显示抗CTLA4抗体(GBD008-hS005-3-2)阻断CD86结合CTLA4。
图3A和3B显示通过SEB测定法，分析使用CTLA4抗体(GBD008-hS005、GBD008-hS005-3、GBD008-hS005-5、GBD008- S019、GBD008-hS019-4、GBD008-S004、GBD008-hS004-5和GBD008-hS005-3-2)而产生的IL2。
图4A-4F显示抗CTLA4抗体(GBD008-hS005、GBD008-hS005-3、GBD008-hS005-5、GBD008-S019、GBD008-hS019-4、GBD008-S004、GBD008-hS004-5和GBD008-hS005-3-2)对人CTLA4(图4A和4B)、食蟹猴CTLA4(图4C和4D)和小鼠CTLA4(图4E和4F)(通过ELISA)的结合能力。
图5显示ADCC的结果，其中Ipi代表Ipilimumab，所述测定法使用PBMC作为效应细胞，表达全长人CTLA4的Raji细胞作为靶细胞(E：T＝20:1)，用抗CTLA4抗体诱导。
图6显示抗CTLA4抗体在MC38结直肠肿瘤模型中的体内抗肿瘤功效。图6A)肿瘤体积随时间变化的平均值。图6B)实验结束时的肿瘤体积平均值和TGI(Tumor Growth Inhibition，肿瘤生长抑制)。
图7A-7C显示抗CTLA4抗体在hCTLA4/hPD1 Balb/C小鼠中的毒性(关节炎)。图7A)小鼠体重变化的趋势。图7B)小鼠的关节炎(爪子以及关节炎症)评分。图7C)小鼠关节炎的发病率。
发明详述
本公开提供了与CTLA4特异性结合的抗体。对有需要的受试者施用治疗有效量的本公开的抗CTLA4抗体可用于治疗疾病或病症，包括癌症和自身免疫性疾病。所述抗体与表达CTLA4的T细胞的结合诱导针对表达肿瘤相关抗原的细胞的强烈抗体依赖性细胞介导的细胞毒性。
本公开的所述抗体提供了多种优点，例如，有效结合CTLA4，有效诱导强烈的抗体依赖性细胞介导的细胞毒性，和/或不良事件(例如毒性)风险显著降低。
在某些方面，抗CTLA4抗体以某些形式(例如，与典型的全长抗体相比的仅重链的抗体，即重链抗体或单域抗体)与CTLA4更有效地结合，导致更高的效力和改进的免疫疗法。
除非另有定义，否则本文使用的所有科学和技术术语具有与本公开内容所属领域的普通技术人员通常理解的相同的含义。在冲突的情况下以本说明书中的定义为准。
本文使用的术语仅用于描述特定实施方案的目的，并且不预期限制本发明。虽然本文仅描述了某些示例性材料和方法，但与本文描述的那些类似或等价的许多方法和材料可以用于本公开内容的实践中。
除非上下文另有明确指示，否则本文所用的单数形式“一”、“一个”和“所述”也意图包括复数形式。此外，开放式的表述“包括”和“包含”解释为还可以含有没有述及的结构组成部分或方法步骤，但需要注意的是，该开放式的表述也涵盖仅由所述的组分和方法步骤组成的情形(即涵盖了封闭式表述“由……组成”的情形)。
一般而言，术语“约”在本文中用于指所述值之上和之下的数值修改5％的变化。
如全文所用，范围用作描述该范围内的每个数值和所有数值的简写形式。范围内的任何数值例如整数值、以十分之一递增的值(当范围的端值为小数点后一位时)或以百分之一递增的值(当范围的端值为小数点后二位时)都可选做该范围的终点。例如，范围1-10用作描述该范围内的所有数值，例如1、2、3、4、5、6、7、8……9、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9和10(以十分之一递增的值)，并且包括所有子范围，例如1-1.5、2.0-3.0、4.0-5.0、6.0-7.0、8.0-9.0等。
本文使用的术语“抗体”可包括整个抗体和其任何抗原结合片段(即，“抗原-结合部分”)或单一链。在一个实施方案中，“抗体”是指包含通过二硫键互相连接的至少两条重(H)链和两条轻(L)链的糖蛋白，或其抗原结合片段。各重链包含重链可变区(本文简称为VH)和重链恒定区。在某些天然存在的IgG、IgD和IgA抗体中，重链恒定区包 含三个结构域，CH1、CH2和CH3。在某些天然存在的抗体中，各轻链包含轻链可变区(本文简称为VL)和轻链恒定区。轻链恒定区包含一个结构域，CL。VH和VL区可进一步细分为超变性的区域，称为互补决定区(CDR)，其与称为框架区(FR)的较保守的区域交替。各VH和VL包含三个CDR和四个框架区(FR)，从氨基端至羧基端按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区包含与抗原相互作用的结合结构域。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(Clq)结合。除非另外指明，免疫球蛋白可来自任何通常已知的同种型，包括但不限于IgA、分泌性IgA、IgG和IgM。IgG同种型在某些物种中分为亚类：在人中IgG1、IgG2、IgG3和IgG4，和在小鼠中IgG1、IgG2a、IgG2b和IgG3。免疫球蛋白，例如，人IgG1，以若干同种异型存在，其在至多几个氨基酸上彼此不同。
除非另外指明，"抗体"可包括例如，完整抗体、单链抗体(scFv)、重链抗体、单域抗体、Fab片段、Fab’片段、F(ab’)2、Fv、Fd片段、双特异性抗体(BsAb)等；单克隆和多克隆抗体；嵌合和人源化抗体；人和非-人抗体；全合成抗体等。
如本文所用，抗体的“抗原结合片段”是指抗体的一个或多个片段，其保留特异性结合完整抗体所结合的抗原的能力。“抗原结合片段”是可以对特定抗原表现出结合亲和力的任何蛋白质性结构。抗原结合片段包括通过任何已知技术诸如酶切割、肽合成和重组技术提供的那些。一些抗原结合片段由完整抗体的保留亲本抗体分子的抗原结合特异性的部分组成。例如，抗原结合片段可包含已知结合特定抗原的抗体的至少一个可变区(重链可变区或轻链可变区)或者一个或多个CDR。合适的抗原结合片段的示例包括但不限于双体抗体和单链分子以及Fab、F(ab’)2、Fc、Fabc和Fv分子；单链(Sc)抗体；单个抗体轻链；单个抗体重链；抗体链或CDR与其它蛋白质 之间的嵌合融合体；蛋白质支架；重链单体或二聚体；轻链单体或二聚体；由一条重链和一条轻链组成的二聚体；由VL、VH、CL和CH1结构域组成的单价片段；或如WO2007059782中所述的单价抗体；包含由铰链区的二硫键连接的两个Fab片段的二价片段；基本上由VH和CH1结构域组成的Fd片段；基本上由抗体的单臂的VL结构域和VH结构域组成的Fv片段；基本上由VH结构域组成的dAb片段，并且也被称为结构域抗体；羊驼或纳米抗体；分离的互补决定区(CDR)等。所有抗体同种型均可用于产生抗原结合片段。另外，抗原结合片段可包括非抗体蛋白质性框架，其可成功地以赋予感兴趣的给定抗原(诸如蛋白质支架)亲和力的取向并入多肽区段。抗原结合片段可重组产生或通过对完整抗体的酶切割或化学切割产生。
如本文所用，术语“重链抗体”或“仅重链的抗体”是指仅包含重链的单链抗体。“重链抗体”不仅缺失轻链，而且缺失常规抗体重链中的恒定区CH1区。与重链抗体相比，如本文所用的“单域抗体”缺少Fc区。
如本文所用，术语序列“同一性”是指两个或更多个多核苷酸序列之间或两个或更多个多肽序列之间的关系。当一个序列中的位置被比较序列的相应位置中相同的核酸碱基或氨基酸残基占据时，序列在该位置处称为“相同的”。通过确定两个序列中相同的核酸碱基或氨基酸残基出现的位置的数目以产生“相同的”位置的数目来计算百分比序列“同一性”。然后，将“相同的”位置的数目除以比较窗口中的位置的总数并乘以100以产生序列“同一性”的百分比。通过比较在比较窗口中两个最佳比对的序列来确定“同一性”的百分比。为了最佳地比对序列以进行比较，比较窗口中的多核苷酸或多肽序列的部分可以包含称为空位的添加或缺失，而参考序列保持恒定。最佳的比对是即使具有空位也在参考序列和比较序列之间产生最大可能数目的“相同的”位置的比对。例如，可以使 用自美国国家生物技术信息中心(National Center for Biotechnology Information，NCBI)获得的程序“BLAST 2序列”的版本确定两个序列之间的百分比“同一性”，该程序包含程序BLASTN(用于核苷酸序列比较)和BLASTP(用于多肽序列比较)，这些程序是基于Karlin和Altschul的算法(Proc.Natl.Acad.Sci.USA 90(12):5873-5877,1993)。
"人源化"抗体是指其中非人抗体，例如小鼠抗体的CDR结构域外的一些、大部分或所有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在人源化形式的抗体的一个实施方案中，CDR结构域外的一些、大部分或所有的氨基酸被来自人免疫球蛋白的氨基酸置换，而一个或多个CDR区内的一些、大部分或所有的氨基酸未改变。氨基酸的小的添加、缺失、插入、置换或修饰是允许的，只要它们不废除抗体结合特定抗原的能力。"人源化"抗体保留类似于原始抗体的抗原特异性。
"嵌合抗体"是指其中可变区源自一个物种和恒定区源自另一个物种的抗体，例如其中可变区源自小鼠抗体和恒定区源自人抗体的抗体。“杂合”抗体是指具有不同类型的重链和轻链的抗体，例如小鼠(亲本)重链和人源化轻链，或反之亦然。
本文使用的术语“单克隆抗体”是指显示对特定表位的单一结合特异性和亲和力的抗体，或其中所有抗体显示对特定表位的单一结合特异性和亲和力的抗体组合物。
如本文使用的，"同种型"是指由重链恒定区基因编码的抗体类型(例如，IgG1,IgG2,IgG3,IgG4,IgM,IgA1,IgA2,IgD和IgE抗体)。
如本文所用，术语“抗体依赖性细胞介导的细胞毒性”和“ADCC”是指细胞介导的过程，其中表达FcγR的非特异性细胞毒性细胞(例如单核细胞，如自然杀伤(NK)细胞和巨噬细胞)识别靶细胞上的结合抗体(或其他能够结合FcγR的蛋白质)，并随后引起靶细胞裂解。原则上，任何具有激活FcγR的效应细胞都可以经触发来介导ADCC。介导ADCC的主要细胞是NK细胞，其仅表达Fcγ RIII，而单核细胞根据其激活、定位或分化状态可以表达FcγRI、FcγRII和FcγRIII。
在一个方面，本公开提供了一种特异性结合CTLA4的抗体，其中所述抗体包含重链可变区，所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1、CDR2和CDR3分别包含与以下具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列：
(1)SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3；
(2)SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15；或
(3)SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24。
在一些实施方案中，所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1包含SEQ ID NO:1所示的序列或由其组成，所述CDR2包含SEQ ID NO:2所示的序列或由其组成，所述CDR3包含SEQ ID NO:3所示的序列或由其组成。
在一些实施方案中，所述重链可变区包含与SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10或SEQ ID NO:31具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:4或由SEQ ID NO:4组成。
在一些实施方案中，所述重链包含与SEQ ID NO:5具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:7或由SEQ ID NO:7组成。
在一些实施方案中，所述重链包含与SEQ ID NO:8具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:10或由SEQ ID NO:10组成。
在一些实施方案中，所述重链包含与SEQ ID NO:11具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:31或由SEQ ID NO:31组成。
在一些实施方案中，所述重链包含与SEQ ID NO:32具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1包含SEQ ID NO:13所示的序列或由其组成，所述CDR2包含SEQ ID NO:14所示的序列或由其组成，所述CDR3包含SEQ ID NO:15所示的序列或由其组成。
在一些实施方案中，所述重链可变区包含与SEQ ID NO:16或SEQ ID NO:19具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:16或由SEQ ID NO:16组成。
在一些实施方案中，所述重链包含与SEQ ID NO:17具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:19或由SEQ ID NO:19组成。
在一些实施方案中，所述重链包含与SEQ ID NO:20具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含CDR1、CDR2和 CDR3，其中所述CDR1包含SEQ ID NO:22所示的序列或由其组成，所述CDR2包含SEQ ID NO:23所示的序列或由其组成，所述CDR3包含SEQ ID NO:24所示的序列或由其组成。
在一些实施方案中，所述重链可变区包含与SEQ ID NO:25或SEQ ID NO:28具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:25或由SEQ ID NO:25组成。
在一些实施方案中，所述重链包含与SEQ ID NO:26具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:28或由SEQ ID NO:28组成。
在一些实施方案中，所述重链包含与SEQ ID NO:29具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述抗体或其抗原结合片段选自完整抗体、单链抗体(scFv)、重链抗体、单域抗体、Fab片段、Fab’片段、F(ab’)2、Fv、Fd片段和双特异性抗体(BsAb)。
在一些实施方案中，本公开的抗体仅由重链组成。在一些实施方案中，本公开的抗体包含两条重链或由两条重链组成。在一些实施方案中，本公开的抗体是单域抗体。
在一些实施方案中，所述抗体是人抗体、人源化抗体或嵌合抗体。
在一些实施方案中，所述抗体是单克隆抗体。
在一些实施方案中，所述抗体是分离的。
在一些实施方案中，所述抗体是分离的单克隆抗体。
还提供了对本文提供的抗体序列的“保守序列修饰”，即没有消除 由核苷酸序列编码或包含氨基酸序列的抗体与抗原的结合的核苷酸和氨基酸序列修饰。例如，修饰可通过本领域已知的标准技术引入，例如定点诱变和PCR-介导的诱变。保守序列修饰包括保守氨基酸置换，其中氨基酸残基被替换为具有类似侧链的氨基酸残基。具有类似侧链的氨基酸残基的家族已在本领域中定义。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、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分支的侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。
在一些实施方案中，所述抗体表现出强烈的抗体依赖性细胞介导的细胞毒性(ADCC)。
在一个方面，本公开提供了一种药物组合物，所述组合物包含治疗有效量所述抗体和药学上可接受的载体，其可以是惰性或生理活性载体。如本文所用，术语“药学上可接受的载体”包括生理上相容的任何溶剂、分散介质、包衣、抗菌剂和抗真菌剂等。合适载体的示例包括水、盐水、磷酸盐缓冲盐水、右旋糖、甘油、乙醇等以及它们的任意组合。
在一个方面，本公开提供了一种分离的核酸分子，其编码所述抗体。
在一些实施方案中，所述核酸分子包含与SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、SEQ ID NO:18、SEQ ID NO:21、SEQ ID NO:27、SEQ ID NO:30或SEQ ID NO:33具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。
在一些实施方案中，所述核酸分子包含与SEQ ID NO:6具有至 少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。在一些实施方案中，所述核酸分子包含SEQ ID NO:6的核苷酸序列。在一些实施方案中，所述核酸分子的核苷酸序列如SEQ ID NO:6所示。在一些实施方案中，所述核酸分子包含与SEQ ID NO:9具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。在一些实施方案中，所述核酸分子包含SEQ ID NO:9的核苷酸序列。在一些实施方案中，所述核酸分子的核苷酸序列如SEQ ID NO:9所示。在一些实施方案中，所述核酸分子包含与SEQ ID NO:12具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。在一些实施方案中，所述核酸分子包含SEQ ID NO:12的核苷酸序列。在一些实施方案中，所述核酸分子的核苷酸序列如SEQ ID NO:12所示。在一些实施方案中，所述核酸分子包含与SEQ ID NO:18具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。在一些实施方案中，所述核酸分子包含SEQ ID NO:18的核苷酸序列。在一些实施方案中，所述核酸分子的核苷酸序列如SEQ ID NO:18所示。在一些实施方案中，所述核酸分子包含与SEQ ID NO:21具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。在一些实施方案中，所述核酸分子包含SEQ ID NO:21的核苷酸序列。在一些实施方案中，所述核酸分子的核苷酸序列如SEQ ID NO:21所示。在一些实施方案中，所述核酸分子包含与SEQ ID NO:27具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。在一些实施方案中，所述核酸分子包含SEQ ID NO:27的核苷酸序列。在一些实施方案中，所述核酸分子的核苷酸序列如SEQ ID NO:27所示。在一些实施方案中，所述核酸分子包含与SEQ ID NO: 30具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。在一些实施方案中，所述核酸分子包含SEQ ID NO:30的核苷酸序列。在一些实施方案中，所述核酸分子的核苷酸序列如SEQ ID NO:30所示。在一些实施方案中，所述核酸分子包含与SEQ ID NO:33具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。在一些实施方案中，所述核酸分子包含SEQ ID NO:33的核苷酸序列。在一些实施方案中，所述核酸分子的核苷酸序列如SEQ ID NO:33所示
在一个方面，本公开提供了一种表达载体，其包含所述核酸分子。
在一个方面，本公开提供了一种宿主细胞，其表达所述抗体，和/或包含所述核酸分子或所述表达载体。
在一个方面，本公开提供了所述抗体或所述药物组合物，其用于治疗疾病或病症，其中所述疾病或病症是癌症或自身免疫性疾病。
在一些实施方案中，所述癌症选自下组：结直肠癌、结肠癌、肾细胞癌、乳腺癌、上皮鳞状细胞癌、黑色素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、头颈癌、白血病和淋巴瘤。
在一些实施方案中，所述抗体或药物组合物用于与一种或多种另外的化学治疗剂、放射治疗剂、细胞因子或其它抗体联合施用。
在一个方面，本公开提供了所述抗体或所述药物组合物在制备用于治疗疾病或病症的药物中的用途，其中所述疾病或病症是癌症或自身免疫性疾病。
在一些实施方案中，所述癌症选自下组：结直肠癌、结肠癌、肾细胞癌、乳腺癌、上皮鳞状细胞癌、黑色素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺 癌、睾丸癌、甲状腺癌、头颈癌、白血病和淋巴瘤。
在一些实施方案中，所述抗体或药物组合物用于与一种或多种另外的化学治疗剂、放射治疗剂、细胞因子或其它抗体联合施用。
在一个方面，本公开提供了一种治疗有需要的受试者的疾病或病症的方法，包括以治疗有效量施用所述抗体或药物组合物，其中所述疾病或病症是癌症或自身免疫性疾病。
在一些实施方案中，所述癌症选自下组：结直肠癌、结肠癌、肾细胞癌、乳腺癌、上皮鳞状细胞癌、黑色素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、头颈癌、白血病和淋巴瘤。
在一些实施方案中，所述抗体或药物组合物与一种或多种另外的化学治疗剂、放射治疗剂、细胞因子或其它抗体联合施用。
“施用”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将包含治疗剂的组合物引入到受试者。本公开的药物组合物的施用途径包括静脉内、肌内、皮下、腹膜内、脊髓或其它肠胃外施用途径，例如通过注射或输注。本文所用的短语“肠胃外施用”是指除了肠内和局部施用以外通常通过注射的施用方式，且包括但不限于静脉内、肌内、动脉内、鞘内、淋巴内、损伤内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注以及体内电穿孔。在一些实施方案中，组合物通过非肠胃外途径施用，在一些实施方案中，通过口服施用。其它非肠胃外途径包括局部、表皮或粘膜施用途径，例如鼻内、阴道、直肠、舌下或局部。施用也可以例如进行一次、多次和/或在一个或多个延长的时间段内进行。
如本文所用，术语“有效量”是指用于向受试者提供治疗性和/或预防性益处所需的活性剂的量。
所述“有需要的受试者”意指任何哺乳动物，所述哺乳动物例如但不限于人、马、牛、猫、小鼠、兔、大鼠、山羊等。优选地， 所述哺乳动物是人。
在一个方面，本公开提供了一种药盒，其包含
a)所述抗体或所述药物组合物；和
b)使用说明。
表1A.序列信息






表1B.序列说明

本公开的示例性抗体的序列如下表2所示：
表2：示例性抗体的全长序列
参考以下的非限制性实施例进一步描述本发明。
实施例
以下示例性描述了上述八种抗体：GBD008-hS005、GBD008-hS005-3、GBD008-hS005-5、GBD008-S019、GBD008-hS019-4、GBD008-S004、GBD008-hS004-5和GBD008-hS005-3-2抗体的开 发、表征和体内抗肿瘤功效。
实施例1.抗CTLA4抗体的制备
骆驼科动物，例如骆驼或羊驼，是生产包含仅一个重链可变区(VHH)和两个常规CH2和CH3区的抗体的理想动物模型。VHH抗体天然缺乏轻链，但保持结构稳定性和有效的抗原结合特性。先前已经描述了获得抗原或表位特异性VHH结构域的方法，例如WO2006/040153和WO2006/122786；R.van der Linden et al.,Journal of Immunological Methods,240(2000)185-195；Li et al.,J Biol Chem.,287(2012)13713-13721；Deffar et al.,African Journal of Biotechnology Vol.8(12),pp.2645-2652,17 Jun.2009和WO94/04678。通过用人CTLA4/CD153蛋白免疫羊驼获得抗CTLA4 VHH抗体序列(Acro Biosystem,Catalog#CT4-H5229)。通过改变原始VHH序列中的氨基酸，用人类抗体VH结构域中出现的常规氨基酸将其替换，对从羊驼获得的VHH序列进行序列优化。
实施例2.抗CTLA4抗体与细胞表面人CTLA4蛋白的结合特异性的FACS分析
CHOK1-CTLA4细胞(1x105/孔)在FACS缓冲液(PBS+2％BSA)中洗涤两次，并重悬于100μl含有连续稀释(1:5)抗CTLA4 mAb的FACS缓冲液中，并在4℃下孵育1小时。然后将细胞在FACS缓冲液中洗涤两次，并通过与APC抗人IgG Fc(Biolegend,Catalog#410712)在4℃下孵育1小时来检测结合的抗体。之后，细胞在FACS缓冲液中洗涤两次，然后使用BD LSRFortessa(BD Biosciences)收集和分析。
如图1A和B所示，结果表明GBD008-S004、GBD008-hS005、GBD008-S019，人源化后的GBD008-hS005-3、GBD008-hS005-5、GBD008-hS019-4、GBD008-hS004-5和GBD008-hS005-3-2抗体均显示与CTLA4蛋白特异性结合。特别是，GBD008-S019和GBD008-hS019-4的平均荧光强度(MFI)趋势与Ipilimumab相近，表明其结合 特异性程度与Ipilimumab相当。GBD008-S005、GBD008-hS005-3、GBD008-hS005-3-2和GBD008-hS005-5的平均荧光强度(MFI)趋势次之，GBD008-S004和GBD008-hS004-5与CTLA4平均荧光强度(MFI)趋势较弱。
实施例3.抗CTLA4抗体阻断CD80和CD86结合CTLA4的FACS分析
CHOK1-CTLA4细胞(1x105/孔)在FACS缓冲液(PBS+2％BSA)中洗涤两次，重悬于100μl含有连续稀释(1:5)抗CTLA4 mAb和CD80 Biotinylated tag(SinoBiological,Catlog#10698-H49H-B)或CD86 Biotinylated tag(SinoBiological,Catlog#10699-H08H-B)的FACS缓冲液中，并在4℃下孵育0.5小时。然后将细胞在FACS缓冲液中洗涤两次，并通过与Streptavidi-Alexa Flour 488(Invitrogen,Catlog#S32354)在4℃下孵育0.5小时来检测结合的抗体。之后，细胞在FACS缓冲液中洗涤两次，然后使用BD LSRFortessa(BD Biosciences)收集和分析。
如图2A-F所示，结果表明抗CTLA4抗体GBD008-S004、GBD008-hS005、GBD008-S019，人源化后的GBD008-hS005-3、GBD008-hS005-5、GBD008-hS005-3-2、GBD008-hS019-4和GBD008-hS004-5抗体均部分抑制了CD80或CD86与CTLA4的结合。特别是，GBD008-S019和GBD008-hS019-4的平均荧光强度(MFI)趋势与Ipilimumab几乎等同，表明其阻断能力与Ipilimumab相当。GBD008-S005、GBD008-hS005-3、GBD008-hS005-3-2和GBD008-hS005-5的平均荧光强度(MFI)趋势次之，表明其阻断能力次之，GBD008-S004和GBD008-hS004-5与CTLA4的平均荧光强度(MFI)趋势较弱，表明其阻断能力较弱。
实施例4.CTLA-4抗体Fc融合蛋白对CTLA-4的亲和力(Octet方法)
CTLA-4抗体Fc融合蛋白与重组人CTLA-4的结合动力学通过 Octet方法对蛋白质相互作用进行定性定量分析(Octect RH-16,Sartorius)。结合速率(kon)和解离速率(koff)使用简单的一对一结合模型(Octet评估软件版本12.2)计算。平衡解离常数(kD)计算为比率koff/kon。结果如表3所示。表3中的结果表明所有CTLA4抗体Fc融合蛋白均以较强的亲和力与CTLA4抗原结合。
表3.CTLA4抗体Fc融合蛋白对CTLA4的亲和力结果
实施例5.通过使用CTLA4抗体分析IL2产生的SEB测定
将连续稀释的抗体与葡萄球菌肠毒素B(SEB)(Sigma-Aldrich,Catlog#S4881)添加到效应细胞人外周血单核细胞(1x105/孔)中，这些细胞是从个体供体(Biotechnologies)获得的并在含有10％FBS(Gibco,Catlog#10099-141)的RPMI 1640培养基(Gibco,Catlog#A10491-01)中培养。将细胞在37℃下培养4天，然后收集样品上清液并与预混合的IL2-HTRF抗体(PerkinElmer,Catlog#62HIL02PET)混合。密封样品板并在室温下孵育3小时。之后，移除板密封并在EnVision 2105多模式读板器(PerkinElmer)上读取。
如图3A和B所示，结果显示GBD008-S004、GBD008-hS005、 GBD008-S019，人源化后的GBD008-hS005-3、GBD008-hS005-3-2、GBD008-hS005-5、GBD008-hS019-4和GBD008-hS004-5抗体均以浓度依赖性方式显著促进效应细胞中IL2的产生，其中GBD008-S019和GBD008-hS019-4的IL2的产生与Ipilimumab相当。GBD008-S004、GBD008-hS005以及人源化后的GBD008-hS005-3、GBD008-hS005-3-2和GBD008-hS005-5的IL2的产生次之，GBD008-hS004-5与CTLA4的IL2的产生低。这些结果表明本公开的抗体有治疗癌症或自身免疫性疾病的潜力。
实施例6.抗CTLA4抗体对食蟹猴和小鼠CTLA4的结合能力(通过ELISA)
用1μg/ml人(Acro Biosystem,Catlog#CT4-H52H9)、食蟹猴(Acro Biosystem,Catlog#Ct4-C82E5)或小鼠CTLA4(Acro Biosystem,Catlog#CT4-M52H5)在4℃将板包被过夜。随后用封闭缓冲液在室温进行封闭。之后，加入梯度稀释系列的抗-CTLA4抗体并使其在室温下反应1小时。加入山羊抗人IgG Fc(HRP)(Abcam,Catlog#ab97225)并在室温下反应1小时。加入显色溶液后随后终止，使用EnVision 2105多模式读板器(PerkinElmer)在450nm波长处读取吸光度。结果如图4A-C所示。
图4A-F的吸光度数据表明GBD008-S004、GBD008-hS005、GBD008-S019，人源化后的GBD008-hS005-3、GBD008-hS005-3-2、GBD008-hS005-5、GBD008-hS019-4和GBD008-hS004-5抗体均具有与人类和食蟹猴CTLA4的结合，但不结合小鼠CTLA4。
实施例7.抗CTLA4抗体的ADCC杀伤试验
使用FACS分析评估细胞毒活性。效应细胞人外周血单核细胞获自个体人类供体(Biotechnologies)，并与10ng/ml hIL-2(PeproTech,200-02)一起培养过夜。表达全长人CTLA4的靶Raji细胞用EuTDA试剂盒中的3ml检测缓冲液与5μl DELFIA BATDA试剂混合(PerkinElmer，Catlog#AD16)在37℃下标记20分钟，用 含有10％FBS(Gibco,Catlog#10099-141)的RPMI 1640培养基(Gibco,Catlog#A10491-01)洗涤两次，并以20:1效应细胞与靶细胞比例接种在96孔圆底板中。将系列稀释的抗CTLA4 mAb添加到测定板的指定行。在37℃下孵育2小时后，离心后取20μl上清与200μl铕溶液室温共孵育10分钟并在EnVision 2105多模式读板器(PerkinElmer)上读取。结果如图5所示。
结果表明，本公开的抗CTLA4抗体有效诱导靶细胞裂解和死亡。
实施例8.抗CTLA4抗体在MC38结肠直肠肿瘤模型中的体内抗肿瘤功效
用MC38(NTCC-MC38)C57BL/6人CTLA4敲入小鼠皮下接种结肠癌细胞(每组n＝6，雌性，7-8周龄)。当肿瘤形成时(约80mm3)，通过腹腔注射给予两个剂量的Ipilimumab(0.3mg/kg和3mg/kg)和两剂本公开的抗CTLA4抗体(0.15mg/kg和1.5mg/kg)。治疗每周两次，持续三周。每三天一次监测肿瘤生长并报告为平均肿瘤体积。结果如图6所示。
从图6可见，用本公开的抗CTLA4抗体治疗的小鼠的平均肿瘤体积显著小于IgG1对照。另外，实验结束时，对于0.15mpk剂量，小鼠的平均肿瘤体积减少62％，而对于1.5mpk剂量，小鼠的平均肿瘤体积减少高达99％。
实施例9.抗CTLA4抗体在hCTLA4/hPD1 Balb/C小鼠模型中的毒性(关节炎)
人CTLA4/hPD1敲入Balb/C鼠(每组n＝8，雌性，4周龄)通过腹腔注射分别给予IgG1(30mg/kg)、Ipilimumab和Nivolumab联合给药(15mg/kg+15mg/kg)、GBD008-hS005-3-2和Nivolumab联合给药(7.5mg/kg+15mg/kg)。治疗每周两次，直至小鼠关节炎(爪子以及关节炎症)消失。每三或四天一次监测小鼠体重以及对小鼠关节炎进行评分。结果如图7A-C所示。
从图7B可见，用本公开的抗CTLA4抗体(GBD008-hS005-3-2)与Nivolumab联合给药治疗的小鼠的关节炎显著小于IgG1对照以及Ipilimumab和Nivolumab联合给药组。另外，从图7C可见，GBD008-hS005-3-2与Nivolumab联合给药治疗的小鼠的关节炎发病率也显著小于IgG1对照以及Ipilimumab和Nivolumab联合给药组。
以上实施例证实，本公开的抗CTLA4抗体不同程度抑制了CD80/CD86与CTLA4的结合，同时表现出强烈的ADCC反应。通过对CD80/86不同程度的阻断能力和保留抗体的Fc功能，所述抗CTLA4抗体具有不同程度的IL2反应率。另外，本公开的抗CTLA4抗体在小鼠模型上显示出优异的体内抗肿瘤功效以及不显著的毒性。
虽然上文已描述了本发明的各种实施方案，但是应理解的是，其仅以示例的方式提供，而并非限制。在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都将落入要求保护的本发明范围内。本发明要求保护的范围由所附的权利要求书及其等效物界定。

Claims (41)

  1. 一种特异性结合CTLA4的抗体或其抗原结合片段，其中所述抗体包含重链可变区，所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1、CDR2和CDR3分别包含与以下序列具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列：
    (1)SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3；
    (2)SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15；或
    (3)SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1包含SEQ ID NO:1所示的序列或由其组成，所述CDR2包含SEQ ID NO:2所示的序列或由其组成，所述CDR3包含SEQ ID NO:3所示的序列或由其组成。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，其中所述重链可变区包含与SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10或SEQ ID NO:31具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  4. 根据权利要求3所述的抗体或其抗原结合片段，其中所述重链可变区包含SEQ ID NO:4或由SEQ ID NO:4组成。
  5. 根据权利要求4所述的抗体或其抗原结合片段，其中所述重链包含与SEQ ID NO:5具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  6. 根据权利要求3所述的抗体或其抗原结合片段，其中所述重链可变区包含SEQ ID NO:7或由SEQ ID NO:7组成。
  7. 根据权利要求6所述的抗体或其抗原结合片段，其中所述重链包含与SEQ ID NO:8具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  8. 根据权利要求3所述的抗体或其抗原结合片段，其中所述重链可变区包含SEQ ID NO:10或由SEQ ID NO:10组成。
  9. 根据权利要求8所述的抗体或其抗原结合片段，其中所述重链包含与SEQ ID NO:11具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  10. 根据权利要求3所述的抗体或其抗原结合片段，其中所述重链可变区包含SEQ ID NO:31或由SEQ ID NO:31组成。
  11. 根据权利要求10所述的抗体或其抗原结合片段，其中所述重链包含与SEQ ID NO:32具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  12. 根据权利要求1所述的抗体或其抗原结合片段，其中所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1包含SEQ ID NO:13所示的序列或由其组成，所述CDR2包含SEQ ID NO:14所示的序列或由其组成，所述CDR3包含SEQ ID NO:15所示的序列或由其组成。
  13. 根据权利要求12所述的抗体或其抗原结合片段，其中所述重链可变区包含与SEQ ID NO:16或SEQ ID NO:19具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  14. 根据权利要求13所述的抗体或其抗原结合片段，其中所述重链可变区包含SEQ ID NO:16或由SEQ ID NO:16组成。
  15. 根据权利要求14所述的抗体或其抗原结合片段，其中所述 重链包含与SEQ ID NO:17具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  16. 根据权利要求13所述的抗体或其抗原结合片段，其中所述重链可变区包含SEQ ID NO:19或由SEQ ID NO:19组成。
  17. 根据权利要求14所述的抗体或其抗原结合片段，其中所述重链包含与SEQ ID NO:20具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  18. 根据权利要求1所述的抗体或其抗原结合片段，其中所述重链可变区包含CDR1、CDR2和CDR3，其中所述CDR1包含SEQ ID NO:22所示的序列或由其组成，所述CDR2包含SEQ ID NO:23所示的序列或由其组成，所述CDR3包含SEQ ID NO:24所示的序列或由其组成。
  19. 根据权利要求18所述的抗体或其抗原结合片段，其中所述重链可变区包含与SEQ ID NO:25或SEQ ID NO:28具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  20. 根据权利要求19所述的抗体或其抗原结合片段，其中所述重链可变区包含SEQ ID NO:25或由SEQ ID NO:25组成。
  21. 根据权利要求20所述的抗体或其抗原结合片段，其中所述重链包含与SEQ ID NO:26具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  22. 根据权利要求19所述的抗体或其抗原结合片段，其中所述重链可变区包含SEQ ID NO:28或由SEQ ID NO:28组成。
  23. 根据权利要求22所述的抗体或其抗原结合片段，其中所述重链包含与SEQ ID NO:29具有至少70％、75％、80％、85％、 88％、90％、92％、95％、97％、98％、99％或100％同一性的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的抗体或其抗原结合片段，所述抗体或其抗原结合片段选自完整抗体、单链抗体(scFv)、重链抗体、单域抗体、Fab片段、Fab’片段、F(ab’)2、Fv、Fd片段和双特异性抗体(BsAb)。
  25. 根据权利要求1-24中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是仅重链的抗体。
  26. 根据权利要求1-25中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含两条重链或由两条重链组成。
  27. 根据权利要求1-24中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段是单域抗体。
  28. 根据权利要求1-27中任一项所述的抗体或其抗原结合片段，其中所述抗体是人抗体、人源化抗体或嵌合抗体。
  29. 根据权利要求1-28中任一项所述的抗体，其中所述抗体或其抗原结合片段表现出显著的抗体依赖性细胞介导的细胞毒性(ADCC)。
  30. 一种药物组合物，所述组合物包含治疗有效量的权利要求1-19中任一项的抗体或其抗原结合片段和药学上可接受的载体。
  31. 一种分离的核酸分子，其编码权利要求1-29中任一项的抗体或其抗原结合片段。
  32. 根据权利要求31所述的核酸分子，其中所述核酸分子包含与SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、SEQ ID NO:18、SEQ ID NO:21、SEQ ID NO:27、SEQ ID NO:30或SEQ ID NO:33具有至少70％、75％、80％、85％、88％、90％、92％、95％、97％、98％、99％或100％同一性的核苷酸序列。
  33. 一种表达载体，其包含权利要求31或32的核酸分子。
  34. 一种宿主细胞，其表达权利要求1-29中任一项的抗体或其抗原结合片段，和/或包含权利要求31或32的核酸分子或权利要求33的表达载体。
  35. 一种治疗有需要的受试者的疾病或病症的方法，所述方法包括以治疗有效量施用权利要求1-29中任一项的抗体或其抗原结合片段或权利要求30的药物组合物，其中所述疾病或病症是癌症或自身免疫性疾病。
  36. 根据权利要求35所述的方法，其中所述癌症选自下组：结直肠癌、结肠癌、肾细胞癌、乳腺癌、上皮鳞状细胞癌、黑色素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、头颈癌、白血病和淋巴瘤。
  37. 根据权利要求35或36所述的方法，其中所述抗体或其抗原结合片段或药物组合物与一种或多种另外的化学治疗剂、放射治疗剂、细胞因子或其它抗体联合施用。
  38. 权利要求1-29中任一项的抗体或其抗原结合片段或权利要求30的药物组合物在制备用于治疗疾病或病症的药物中的用途，其中所述疾病或病症是癌症或自身免疫性疾病。
  39. 根据权利要求38所述的用途，其中所述癌症选自下组：结直肠癌、结肠癌、肾细胞癌、乳腺癌、上皮鳞状细胞癌、黑色素瘤、骨髓瘤、胃癌、脑癌、肺癌、胰腺癌、宫颈癌、卵巢癌、肝癌、膀胱癌、前列腺癌、睾丸癌、甲状腺癌、头颈癌、白血病和淋巴瘤。
  40. 根据权利要求38或39所述的用途，其中所述抗体或其抗原结合片段或药物组合物用于与一种或多种另外的化学治疗剂、放射治疗剂、细胞因子或其它抗体联合施用。
  41. 一种药盒，其包含
    a)权利要求1-29中任一项的抗体或其抗原结合片段或权利要求 30的药物组合物；和
    b)使用说明。
PCT/CN2023/127601 2022-10-28 2023-10-30 新型抗ctla4抗体 WO2024088424A1 (zh)

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