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WO2018036473A1 - 抗ctla4-抗pd-1双功能抗体、其药物组合物及其用途 - Google Patents

抗ctla4-抗pd-1双功能抗体、其药物组合物及其用途 Download PDF

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Publication number
WO2018036473A1
WO2018036473A1 PCT/CN2017/098466 CN2017098466W WO2018036473A1 WO 2018036473 A1 WO2018036473 A1 WO 2018036473A1 CN 2017098466 W CN2017098466 W CN 2017098466W WO 2018036473 A1 WO2018036473 A1 WO 2018036473A1
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seq
antibody
amino acid
acid sequence
ctla4
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English (en)
French (fr)
Inventor
李百勇
夏瑜
王忠民
张鹏
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Akeso Pharmaceuticals Inc
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Akeso Biopharma Inc
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Priority to MX2019002254A priority Critical patent/MX2019002254A/es
Priority to EA201990571A priority patent/EA201990571A1/ru
Priority to NZ751904A priority patent/NZ751904A/en
Priority to JP2019531513A priority patent/JP7425604B2/ja
Priority to AU2017317124A priority patent/AU2017317124B2/en
Priority to CA3034850A priority patent/CA3034850A1/en
Priority to IL264964A priority patent/IL264964B2/en
Priority to EP17842896.7A priority patent/EP3511346A4/en
Priority to KR1020197008463A priority patent/KR102503084B1/ko
Application filed by Akeso Biopharma Inc filed Critical Akeso Biopharma Inc
Priority to BR112019003695-4A priority patent/BR112019003695A2/pt
Priority to US16/327,076 priority patent/US11578128B2/en
Priority to SG11201901583WA priority patent/SG11201901583WA/en
Publication of WO2018036473A1 publication Critical patent/WO2018036473A1/zh
Anticipated expiration legal-status Critical
Priority to JP2022089567A priority patent/JP2022130393A/ja
Priority to US18/067,669 priority patent/US12479919B2/en
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    • 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
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • G01N33/575—
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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    • G01N2800/7019—Ischaemia

Definitions

  • the invention belongs to the field of tumor treatment and molecular immunology, and relates to an anti-CTLA4-anti-PD-1 bifunctional antibody, a pharmaceutical composition thereof and use thereof.
  • the invention relates to a monoclonal antibody against CTLA4-anti-PD-1.
  • 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. When CTLA4 binds to B7, it inhibits the activation of mouse and human T cells and plays a negative regulatory role in T cell activation.
  • CTLA4mAb or CTLA4 ligand can block the binding of CTLA4 to its natural ligand, thereby blocking the transmission of CTLA4 to the negative regulatory signal of T cells and enhancing the reactivity of T cells to various antigens.
  • CTLA4 mAb 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 2013Jan 22).
  • 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.) and is undergoing clinical research for the treatment of chronic viral infections (Chavez, AR, et al., Pharmacologic administration of interleukin- 2. Ann N Y Acad Sci, 2009.1182: p. 14-27).
  • CTLA4 mAb specifically abolishes CTLA4 immunosuppression, activates T cells, induces IL-2 production, and has broad application prospects in gene therapy for diseases such as anti-tumor and parasitic diseases.
  • 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. But at the same time, although biogene therapy has not been published in short-term clinical trials For adverse reactions, we should also notice the potential effects of long-term application.
  • CTLA4 mAb over-blocking CTLA4-B7 signaling can lead to autoimmune diseases. Since antibodies can specifically bind to their ligands and cause target cells to lyse or block pathological processes, the development and utilization of antibodies, especially human-derived antibody drugs, is of great significance for the clinical treatment of human malignant tumors and other immune diseases.
  • the transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 gene family and is expressed in activated T cells, B cells, and bone marrow cells.
  • PD-1 receptors PDL1 and PDL2 belong to the B7 superfamily, in which PDL1 cells are expressed, including T cells, B cells, and endothelial cells and epithelial cells.
  • PDL2 is expressed only in antigen presenting cells such as dendritic cells. And macrophages.
  • T cells play a very important role in clearing viral infections, but T cell antiviral responses are often associated with immunopathology.
  • PD-1 plays a very important role in the activation of negative regulatory T cells.
  • PD-1 mediated negative regulation of T cells can reduce tissue damage caused by the infection process, but block or inhibit PD-1. Negative regulation can lead to autoimmune diseases.
  • PD-1 knockout mice are more effective at clearing pancreatic virus infection, but they cause more severe liver damage (Isai et al., 2003, J. Exp. Med. 198: 39-50).
  • tumors that highly express PD-1 are accompanied by cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104: 3360-5).
  • An effective method for the implementation is to regulate the expression of PD-1 by injecting antibodies in vivo.
  • interferon gamma is mainly produced by natural killer cells (NK), natural killer T cells (NKT), and by specific antigens from CD4 Th1 cells and CD8 cytotoxic T lymphocytes (CTL). Produced after stimulation.
  • NK natural killer cells
  • NKT natural killer T cells
  • CTL CD8 cytotoxic T lymphocytes
  • IFN ⁇ plays an important role in combating or inhibiting the infection of tumors, viruses, certain bacteria and protozoal diseases.
  • IFN ⁇ activates macrophages, induces the expression of class II major histocompatibility complex (MHC), activates immune response, and controls tumor development (SchoenbornJR, Wilson CB. Regulation of Interferon- ⁇ During Innate and Adaptive) Immune Responses. Advances in Immunology 2007; 96: 41-101).
  • MHC major histocompatibility complex
  • Monoclonal antibodies have been used to treat cancer, inflammation and infectious diseases and other diseases, most of which are monospecific. However, some diseases are usually multifaceted in terms of causes and in vivo, including in different aspects. In the signaling pathway, up- or down-regulation of different proteins, cytokines, and receptors inhibits or promotes in vivo functions. Therefore, a variety of different factors can improve the treatment effect. This can be achieved by combining different drugs or by using multiple targeting strategies that replace different drugs, such as multispecific antibodies.
  • a bifunctional antibody also known as a Bispecific Antibody
  • a Bispecific Antibody is a specific drug that simultaneously targets two different antigens, which can be produced by immunostaining.
  • it can also be obtained through genetic engineering. Genetic engineering has certain flexibility in terms of binding site optimization, synthetic form considerations and yield, so it has certain advantages. At present, its form of existence has been proven to have more than 45 species (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24: 89-98).
  • a variety of bispecific antibodies that have been developed so far are in the IgG-ScFv form, the Morrison mode (1997 Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol.
  • the present inventors used a mammalian cell expression system to express recombinant CTLA4 and PD-1 as antigens, and obtained hybridoma cells by fusion of mouse spleen cells with myeloma cells.
  • the inventors obtained the following hybridoma cell lines by screening a large number of samples:
  • Hybridoma cell line LT002 (CTLA4-4G10), which was deposited with the China Center for Type Culture Collection (CCTCC) on June 16, 2015 under the accession number CCTCC NO: C201587;
  • Hybridoma cell line LT003 (PD-1-14C12), which was deposited with the China Center for Type Culture Collection (CCTCC) on June 16, 2015 under the accession number CCTCC NO: C2015105.
  • the hybridoma cell line LT002 is capable of secreting a specific monoclonal antibody (designated 4G10) that specifically binds to CTLA4, and the monoclonal antibody is capable of blocking the binding of CTLA4 to B7 very efficiently;
  • the hybridoma cell line LT003 is capable of secreting a specific monoclonal antibody (designated 14C12) which specifically binds to PD-1, and this monoclonal antibody is capable of blocking the binding of PD-1 to PDL1 very efficiently.
  • the inventors have creatively produced humanized antibodies against CTLA4 (designated 4G10H1L1, 4G10H3L3, 4G10H4L3, and 8D2H14L2, respectively) and humanized antibodies against PD-1 (designated 14C12H1L1).
  • the present inventors creatively fused two types of humanized antibodies into a novel antibody, and obtained a human source capable of binding CTLA4 and PD-1, blocking the binding of CTLA4 and B7, PD-1 and PDL1.
  • Bifunctional antibodies (named BiAb001, BiAb002, BiAb003, BiAb004, BiAb007, and BiAb010, respectively), can effectively bind human T cells, and activate T cells to induce human lymphocytes to secrete IFN- ⁇ and IL-2;
  • the potential to prepare drugs for the prevention and treatment of cancers such as lung cancer, melanoma, kidney tumor, ovarian cancer, and leukemia.
  • One aspect of the invention relates to a bispecific antibody comprising:
  • the bispecific antibody wherein the first protein functional region and the second protein functional region are directly linked or linked by a linker; preferably, the linker is (GGGGS) n, n is a positive integer, such as 1, 2, 3, 4, 5 or 6.
  • the bispecific antibody wherein
  • the first protein functional region and the second protein functional region are independently immunoglobulin or an antigen binding fragment thereof, such as a half antibody, Fab, F(ab') 2 or a single chain antibody;
  • the first protein functional region is an immunoglobulin
  • the second protein functional region is a single chain antibody
  • the first protein functional region is a single chain antibody and the second protein functional region is an immunoglobulin.
  • the bispecific antibody wherein the first protein functional region and The second protein functional regions are independently one, two or two or more.
  • the bispecific antibody wherein the immunoglobulin is IgG, IgA, IgD, IgE or IgM; preferably IgG, such as IgG1, IgG2, IgG3 or IgG4.
  • the bispecific antibody wherein the single chain antibody is linked to the C-terminus of the heavy chain of an immunoglobulin. Since the immunoglobulin is composed of two heavy chains, one immunoglobulin molecule is linked to two single-chain antibody molecules. Preferably, the two single chain antibody molecules are identical.
  • the bispecific antibody wherein:
  • the immunoglobulin having a heavy chain variable region comprising the CDRs of amino acid sequence SEQ ID NOs: 29-31, the light chain variable region comprising the CDRs having the amino acid sequence of SEQ ID NOs: 32-34; and/or
  • the single chain antibody wherein the heavy chain variable region comprises a CDR having the amino acid sequence of SEQ ID NO: 35-37 or a CDR comprising the amino acid sequences of SEQ ID NO: 35, SEQ ID NO: 41 and SEQ ID NO: 37 Or comprising a CDR having the amino acid sequence of SEQ ID NO: 42-44, the light chain variable region comprising the CDRs having the amino acid sequence of SEQ ID NO: 38-40 or comprising the CDRs having the amino acid sequence of SEQ ID NOs: 45-47;
  • the bispecific antibody wherein:
  • the immunoglobulin having a heavy chain variable region comprising the CDRs of amino acid sequence SEQ ID NOs: 35-37 or comprising the CDRs of amino acid sequences SEQ ID NO: 35, SEQ ID NO: 41 and SEQ ID NO: 37 Or comprising a CDR of amino acid sequence SEQ ID NO: 42-44, the light chain variable region comprising the CDRs of amino acid sequence SEQ ID NO: 38-40 or comprising the CDRs having the amino acid sequence of SEQ ID NOs: 45-47; /or
  • the single chain antibody has a heavy chain variable region comprising the CDRs of amino acid sequence SEQ ID NO: 29-31, and the light chain variable region comprising the CDRs having the amino acid sequence of SEQ ID NOS: 32-34.
  • the bispecific antibody wherein:
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 20; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO: 22; and/or
  • the amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 and SEQ ID NO: 25;
  • the amino acid sequence of the light chain variable region of the antibody is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO:
  • the bispecific antibody wherein:
  • amino acid sequence of the heavy chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14 and SEQ ID NO: 25; the amino acid sequence of the light chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12 and SEQ ID NO: 27; and/or
  • amino acid sequence of the heavy chain variable region of the single chain antibody is selected from the group consisting of SEQ ID NO: 16 and SEQ ID NO: 20; the amino acid sequence of the light chain variable region of the immunoglobulin is selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO:22.
  • the bispecific antibody wherein
  • the immunoglobulin 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 constant region of the immunoglobulin is humanized, for example, the heavy chain constant region adopts Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region adopts Ig Kappa chain C region, ACCESSION: P01834.
  • the bispecific antibody wherein the bispecific 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 CTLA4 protein and/or PD-1 protein.
  • the invention also relates to a bispecific antibody, the heavy chain variable region comprising:
  • amino acid sequence is the CDR of SEQ ID Nos: 29-31,
  • amino acid sequence is the CDR of SEQ ID Nos: 32-34,
  • amino acid sequence is the CDR of SEQ ID Nos: 38-40, or
  • amino acid sequence is the CDR of SEQ ID Nos: 45-47;
  • the CDRs of the light chain variable region are not identical to the CDRs comprised by the heavy chain variable region.
  • 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 heavy chain variable region of the antibody comprises:
  • amino acid sequence is the CDR of SEQ ID Nos: 29-31,
  • the invention also 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 Nos: 32-34,
  • amino acid sequence is the CDR of SEQ ID Nos: 38-40, or
  • amino acid sequence is the CDRs of SEQ ID Nos: 45-47.
  • a further aspect of the invention relates to a vector comprising the isolated nucleic acid molecule of the invention.
  • a further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a vector of the invention.
  • a further aspect of the invention relates to a method of preparing a bispecific antibody of the invention comprising culturing a host cell of the invention under suitable conditions, and recovering said bispecific antibody from cell culture.
  • a further aspect of the invention relates to a conjugate comprising a bispecific antibody and a coupling moiety, wherein the bispecific antibody is a bispecific antibody of the invention, the coupled moiety being a detectable label;
  • the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  • a further aspect of the invention relates to a kit comprising a bispecific antibody of the invention or comprising a conjugate of the invention;
  • the kit further includes a second antibody that specifically recognizes the bispecific antibody; optionally, the second antibody further includes a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, Colored substances or enzymes.
  • a detectable label such as a radioisotope, a fluorescent substance, a luminescent substance, Colored substances or enzymes.
  • a further aspect of the invention relates to the use of a bispecific antibody of the invention in a kit for detecting the presence or level of CTLA4 and /PD-1 in a sample.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a bispecific antibody of the invention or a conjugate of the invention; optionally, further comprising a pharmaceutically acceptable carrier and/or excipient.
  • a further aspect of the invention relates to the use of a bispecific antibody of the invention or a conjugate of the invention in the manufacture of a medicament for the prevention and/or treatment of a tumor or anemia, or in the preparation of a medicament for diagnosing a tumor or anemia Use; specifically, the tumor is selected from the group consisting of melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer, and leukemia.
  • the present inventors also found through animal experiments that the bispecific antibody BiAb004 of the present invention can effectively reduce the growth of PD-1 HuGEMM mice (human PD-1 transgenic mice) by subcutaneously inoculated MC38 tumor cells on the right side, which is markedly significant. Inhibition of the increase in tumor volume of PD-1 HuGEMM MC38 tumor-bearing mice.
  • a further aspect of the invention relates to the use of a bispecific antibody of the invention or a conjugate of the invention in the manufacture of a medicament as follows:
  • a drug that increases the expression of IFN- ⁇ in T lymphocytes A drug that increases the expression of IFN- ⁇ in T lymphocytes.
  • a further aspect of the invention relates to a method, in vivo or in vitro, comprising administering a cell or administering to a subject in need thereof an effective amount of a bispecific antibody of the invention or a conjugate of the invention, said method Selected from the following:
  • a method for increasing the expression of IFN- ⁇ in T lymphocytes A method for increasing the expression of IFN- ⁇ in T lymphocytes.
  • anti-CTLA4 antibody, anti-PD-1 antibody and anti-CTLA4-anti-PD-1 bifunctional antibody can induce the secretion of IFN ⁇ to activate the immune response.
  • a further aspect of the invention relates to a method of preventing and/or treating a tumor or anemia, or a method of diagnosing a tumor or an anemia comprising administering to a subject in need thereof an effective amount of any one of the inventions
  • the step of the bispecific antibody or the 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, liver cancer, non-small Cellular lung cancer, ovarian cancer, and leukemia.
  • a bispecific antibody or conjugate according to any one of the present invention for use in the prevention and/or treatment of a tumor or an anemia, or for diagnosing a tumor or an anemia; in particular, the tumor is selected from the group consisting of melanoma , kidney tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer and leukemia.
  • 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 following items (1) to (13) 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 sequence of the heavy chain variable region is set forth in SEQ ID NO: 16
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 18.
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFAFSSYD (SEQ ID NO: 29)
  • HCDR2 ISGGGRYT (SEQ ID NO: 30)
  • HCDR3 ANRYGEAWFAY (SEQ ID NO: 31)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 QDINTY (SEQ ID NO: 32)
  • LCDR2 RAN (SEQ ID NO: 33)
  • LCDR3 LQYDEFPLT (SEQ ID NO: 34)
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 20
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 22.
  • amino acid sequences of the three CDR regions of the heavy chain variable region are identical to those of 14C12.
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of 14C12.
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region is SEQ ID NO: 4;
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GYSFTGYT (SEQ ID NO: 35)
  • HCDR2 INPYNNIT (SEQ ID NO: 36)
  • HCDR3 ARLDYRSY (SEQ ID NO: 37)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 TGAVTTSNF (SEQ ID NO: 38)
  • LCDR2 GTN (SEQ ID NO: 39)
  • LCDR3 ALWYSNHWV (SEQ ID NO: 40)
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • amino acid sequences of the three CDR regions of the heavy chain variable region are identical to those of 4G10.
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of 4G10.
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequences of the three CDR regions of the heavy chain variable region are identical to those of 4G10.
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of 4G10.
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12.
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GYSFTGYT (SEQ ID NO: 35)
  • HCDR2 INPYNDIT (SEQ ID NO: 41)
  • HCDR3 ARLDYRSY (SEQ ID NO: 37)
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of 4G10.
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region is SEQ. ID NO: 27 is shown.
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFTFSDNW (SEQ ID NO: 42)
  • HCDR2 IRNKPYNYET (SEQ ID NO: 43)
  • HCDR3 TAQFAY (SEQ ID NO: 44)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 ENIYGG (SEQ ID NO: 45)
  • LCDR3 QNVLRSPFTF (SEQ ID NO: 47)
  • amino acid sequences of the nine CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFAFSSYD (SEQ ID NO: 29)
  • HCDR2 ISGGGRYT (SEQ ID NO: 30)
  • HCDR3 ANRYGEAWFAY (SEQ ID NO: 31)
  • HCDR4 GYSFTGYT (SEQ ID NO: 35)
  • HCDR5 INPYNNIT (SEQ ID NO: 36)
  • HCDR6 ARLDYRSY (SEQ ID NO: 37)
  • HCDR7 TGAVTTSNF (SEQ ID NO: 38)
  • HCDR8 GTN (SEQ ID NO: 39)
  • HCDR9 ALWYSNHWV (SEQ ID NO: 40)
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCDR1 QDINTY (SEQ ID NO: 32)
  • LCDR2 RAN (SEQ ID NO: 33)
  • LCDR3 LQYDEFPLT (SEQ ID NO: 34)
  • amino acid sequences of the nine CDR regions of the heavy chain variable region are identical to those of BiAb001.
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of BiAb001.
  • amino acid sequences of the nine CDR regions of the heavy chain variable region are identical to those of BiAb001.
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of BiAb001.
  • amino acid sequences of the nine CDR regions of the heavy chain variable region are identical to those of BiAb001.
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of BiAb001.
  • amino acid sequences of the nine CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFAFSSYD (SEQ ID NO: 29)
  • HCDR2 ISGGGRYT (SEQ ID NO: 30)
  • HCDR3 ANRYGEAWFAY (SEQ ID NO: 31)
  • HCDR4 GYSFTGYT (SEQ ID NO: 35)
  • HCDR5 INPYNDIT (SEQ ID NO: 41)
  • HCDR6 ARLDYRSY (SEQ ID NO: 37)
  • HCDR7 TGAVTTSNF (SEQ ID NO: 38)
  • HCDR8 GTN (SEQ ID NO: 39)
  • HCDR9 ALWYSNHWV (SEQ ID NO: 40)
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of BiAb001.
  • amino acid sequences of the nine CDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFAFSSYD (SEQ ID NO: 29)
  • HCDR2 ISGGGRYT (SEQ ID NO: 30)
  • HCDR3 ANRYGEAWFAY (SEQ ID NO: 31)
  • HCDR4 GFTFSDNW (SEQ ID NO: 42)
  • HCDR5 IRNKPYNYET (SEQ ID NO: 43)
  • HCDR6 TAQFAY (SEQ ID NO: 44)
  • HCDR7 ENIYGG (SEQ ID NO: 45)
  • HCDR8 GAT (SEQ ID NO: 46)
  • HCDR9 QNVLRSPFTF (SEQ ID NO: 47)
  • amino acid sequences of the three CDR regions of the light chain variable region are identical to those of BiAb001.
  • CTLA4 protein when referring to the amino acid sequence of the CTLA4 protein (Cytotoxic T-Lymphocyte Antigen 4), it includes the full length of the CTLA4 protein, or the extracellular fragment CTLA4 ECD of CTLA4 or a fragment comprising CTLA4 ECD; also includes CTLA4 ECD.
  • a fusion protein such as a fragment fused to an Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • Fc protein fragment mFc or hFc
  • mutations or variations including but not limited to substitutions, deletions and/or additions
  • the term "CTLA4 protein” shall include all such sequences, including natural or artificial variants thereof.
  • a sequence fragment of a CTLA4 protein it also includes corresponding sequence fragments in its native or artificial variant.
  • the amino acid sequence of the PD-1 protein when referring to the amino acid sequence of the PD-1 protein (Programmed cell death protein 1, NCBI GenBank: NM_005018), it includes the full length of the PD-1 protein, or the extracellular fragment PD of PD-1.
  • -1 ECD or a fragment comprising PD-1 ECD also a fusion protein of PD-1 ECD, such as a fragment fused to a Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • Fc protein fragment mFc or hFc
  • 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).
  • EC50 refers to a concentration for 50% of maximal effect and refers to a concentration that causes a 50% maximal effect.
  • the term "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. .
  • a heavy chain can be understood as a polypeptide chain having a relatively large molecular weight in an antibody
  • a light chain refers to a polypeptide chain having a relatively small molecular weight in an antibody.
  • Light chains can be classified into kappa and lambda light chains.
  • Heavy chains can generally be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • 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.
  • V H and V L 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 heavy chain may also comprise more than 3 CDRs, for example 6, 9, or 12.
  • the heavy chain may be the ScFv of the heavy chain of the IgG antibody linked to the other antibody, in which case the heavy chain contains 9 CDRs.
  • antibody is not limited by any particular method of producing 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.
  • IgG eg, IgGl, IgG2, IgG3 or IgG4 subtype
  • IgA1, IgA2, IgD, IgE or IgM antibodies 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)).
  • An antigen-binding fragment of an antibody (eg, an antibody fragment described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (eg, recombinant DNA techniques or enzymatic or chemical cleavage methods), and Antibodies are specifically screened for antigen-binding fragments of antibodies in the same manner.
  • 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).
  • 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 light chain or / and heavy Another part of the 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 retains binding activity to the antigen of interest (USP 4,816,567 to Cabilly) Et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (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.
  • 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.).
  • the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been integrated into the ALIGN program (version 2.0), can also be used, using the PAMl20 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 PAM250 matrix and gap weight of 16, 14, 12, 10, 8, 6 or 4 The length weights of 1, 2, 3, 4, 5 or 6 are used to determine the percent identity between the two amino acid sequences.
  • the term “specifically binds” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and the antigen to which it is directed.
  • 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.
  • the term “targeting” refers to specific binding.
  • 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 has a dissociation equilibrium constant (K D ) of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • the antigen is bound, for example, as measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • 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.
  • hybridoma cell line LT002 or LT003 it also refers to the subcloned and progeny cells of the hybridoma cell line LT002 or LT003.
  • 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 include but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant). Agent), Corynebacterium parvum, lipopolysaccharide, cytokines, and the like. 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 antibodies 4G10H1L1 and 4G10H3L3 according to the present invention can bind specifically to CTLA4, and can effectively block the binding of CLTA4 and B7, specifically relieve CTLA4 from immunosuppression and activate T lymphocytes.
  • the monoclonal antibody 14C12H1L1 of the present invention is capable of specifically binding to CTLA4, and is capable of blocking the binding of CLTA4 and B7 very effectively, specifically releasing CTLA4 to immunosuppress the body and activating T lymphocytes.
  • the bifunctional antibody of the present invention has a medicament for preparing a medicament for antitumor such as anti-melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer or leukemia. potential.
  • Figure 1 Results of SDS-PAGE detection of monoclonal murine antibody 4G10.
  • the samples from the left to the right of the four lanes were loaded with: non-reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; Marker, 5 ⁇ l; BSA , 1 ⁇ g.
  • Figure 2 Results of SDS-PAGE detection of monoclonal humanized antibody 4G10H1L1.
  • the samples from the left to the right of the three lanes and their loadings were: non-reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; Marker, 5 ⁇ l.
  • Figure 3 Results of SDS-PAGE detection of monoclonal humanized antibody 4G10H3L3.
  • the samples from the left to the right of the two lanes and their loadings were: reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; Marker, 5 ⁇ l.
  • Figure 4 Results of SDS-PAGE detection of monoclonal humanized antibody 14C12H1L1.
  • the samples from the left to the right of the four lanes were loaded with: non-reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; reduced protein electrophoresis loading buffer sample antibody, 1 ⁇ g; Marker, 5 ⁇ l; BSA , 1 ⁇ g.
  • Figure 5 Results of SDS-PAGE detection of the bifunctional antibody BiAb001. Lane samples and their loading were: Marker, 5 ⁇ l; 1, Non-reduced: non-reduced protein electrophoresis loading buffer sample, 1 ⁇ g; 2, Reduced: reduced protein electrophoresis loading buffer sample, 1 ⁇ g; BSA, 1 ⁇ g.
  • Figure 6 Results of SDS-PAGE detection of the bifunctional antibody BiAb002. Lane samples and their loading were: Marker, 5 ⁇ l; 1, Non-reduced: non-reduced protein electrophoresis loading buffer sample, 1 ⁇ g; 2, Reduced: reduced protein electrophoresis loading buffer sample, 1 ⁇ g; BSA, 1 ⁇ g.
  • Figure 7 Results of SDS-PAGE detection of the bifunctional antibody BiAb003. Lane samples and their loading were: Marker, 5 ⁇ l; 1, Non-reduced: non-reduced protein electrophoresis loading buffer sample, 1 ⁇ g; 2, Reduced: reduced protein electrophoresis loading buffer sample, 1 ⁇ g; BSA, 1 ⁇ g.
  • Figure 8 Results of SDS-PAGE detection of the bifunctional antibody BiAb004. Lane samples and their loading were: Marker, 5 ⁇ l; 1, Non-reduced: non-reduced protein electrophoresis loading buffer sample, 1 ⁇ g; 2, Reduced: reduced protein electrophoresis loading buffer sample, 1 ⁇ g; BSA, 1 ⁇ g.
  • Figure 9 Results of SDS-PAGE detection of the bifunctional antibody BiAb007. Lane samples and their loading were: Marker, 5 ⁇ l; 1, Non-reduced: non-reduced protein electrophoresis loading buffer sample, 1 ⁇ g; 2, Reduced: reduced protein electrophoresis loading buffer sample, 1 ⁇ g; BSA, 1 ⁇ g.
  • Figure 10 Results of SDS-PAGE detection of the bifunctional antibody BiAb0010. Lane samples and their loading were: Marker, 5 ⁇ l; 1, Non-reduced: non-reduced protein electrophoresis loading buffer sample, 1 ⁇ g; 2, Reduced: reduced protein electrophoresis loading buffer sample, 1 ⁇ g; BSA, 1 ⁇ g
  • Figure 12 Results of kinetic characteristic parameter detection of antibody 4G10H1L1.
  • Figure 14 Results of kinetic characteristic parameter detection of antibody 4G10H4L3.
  • Figure 16 Results of kinetic characteristic parameter detection of antibody 14C12H1L1.
  • Figure 17 Results of kinetic characteristic parameter detection of CTLA4 and antibody BIAb001.
  • Figure 18 Results of kinetic characteristic parameter detection of CTLA4 and antibody BIAb002.
  • Figure 19 Results of kinetic characteristic parameter detection of CTLA4 and antibody BIAb003.
  • Figure 20 Results of kinetic characteristic parameter detection of CTLA4 and antibody BIAb004.
  • Figure 21 Results of kinetic characteristic parameter detection of CTLA4 and antibody BIAb007.
  • Figure 22 Results of kinetic characteristic parameter detection of PD-1 and antibody BIAb001.
  • Figure 23 Results of kinetic characteristic parameter detection of PD-1 and antibody BIAb002.
  • Figure 24 Results of kinetic characteristic parameter detection of PD-1 and antibody BIAb003.
  • Figure 25 Results of kinetic characteristic parameter detection of PD-1 and antibody BIAb004.
  • Figure 26 Results of kinetic characteristic parameter detection of PD-1 and antibody BIAb007.
  • Figure 27 Results of kinetic characteristic parameter detection of PD-1 and antibody BIAb010.
  • Figure 28 Detection of binding of antibodies 4G10H1L1 and 4G10H3L3 to antigen CTLA4 by indirect ELISA.
  • Figure 29 Detection of the binding activity of the antibodies 4G10H1L1 and 4G10H3L3 to B7 for binding to the antigen CTLA4 by a competitive ELISA method.
  • Figure 30 Detection of binding of antibody 14C12, 14C12H1L1 to PD-1 by indirect ELISA.
  • Figure 31 Competition for binding of antibody 14C12, 14C12H1L1 to PDL1 to PD-1 by competitive ELISA.
  • Figure 32 Binding of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 to CTLA4 was detected by indirect ELISA.
  • Figure 33 Detection of binding of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 to PD-1 by indirect ELISA.
  • Figure 34 Detection of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 competed with B7 for binding to CTLA4 using a competitive ELISA method.
  • Figure 35 Detection of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 competed with PDL1 for binding to PD-1 using a competitive ELISA method.
  • Figure 36 Antibody 4G10H1L1 and 293T-CTLA4 EC CTLA4 binding cell surface protein of 50.
  • Figure 37 293T-CTLA4 antibody 4G10H3L3 with a cell surface protein binding of CTLA4 EC 50.
  • Figure 38 Antibody 14C12H1L1 and 293T-PD-1 cell surface PD-1 binding protein EC 50.
  • Figure 39 293T-CTLA4 antibody BiAb001 with a cell surface protein binding CTLA4 EC 50.
  • Figure 40 293T-CTLA4 antibody BiAb002 with a cell surface protein binding CTLA4 EC 50.
  • Figure 41 Antibody BiAb003 293T-CTLA4 protein to the surface of cells binding CTLA4 EC 50.
  • Figure 42 Antibody BiAb004 293T-CTLA4 protein to the surface of cells binding CTLA4 EC 50.
  • Figure 43 antibody BiAb001 surface 293T-PD-1 with the PD-1 binding cells EC 50.
  • Figure 44 antibody BiAb002 surface 293T-PD-1 with the PD-1 binding cells EC 50.
  • Figure 45 antibody BiAb003 surface 293T-PD-1 with the PD-1 binding cells EC 50.
  • Figure 46 antibody BiAb004 surface 293T-PD-1 with the PD-1 binding cells EC 50.
  • Figure 47 Binding activity of antibody 4G10H3L3 to T cell surface antigen CTLA4.
  • Figure 48 Binding activity of antibody 14C12H1L1 to T cell surface antigen PD-1.
  • Figure 49 Binding activity of antibodies BiAb003 and BiAb004 to T cells, and comparison with antibodies 14C12H1L1 and 4G10H3L3.
  • Figure 50 Effect of antibodies 4G10H1L1, 4G10H3L3 on secretion of cytokine IFN- ⁇ from mixed lymphocytes.
  • Figure 51 Effect of antibody 14C12H1L1 on secretion of cytokine IFN- ⁇ from mixed lymphocytes.
  • Figure 52 Effect of antibodies BiAb001 and BiAb002 on the secretion of cytokine IFN- ⁇ from mixed lymphocytes and compared with antibodies 14C12H1L1, 4G10H1L1.
  • Figure 53 Effect of antibodies BiAb003 and BiAb004 on the secretion of cytokine IFN- ⁇ from mixed lymphocytes and compared with antibodies 14C12H1L1, 4G10H3L3.
  • Figure 54 Effect of antibody 4G10H3L3 on secretion of mixed lymphocyte cytokine IL-2.
  • Figure 55 Effect of antibody 14C12H1L1 on IL-2 secretion by mixed lymphocytes.
  • Figure 56 Effect of antibodies BiAb003 and BiAb004 on the secretion of cytokine IL-2 in mixed lymphocytes and compared with antibodies 14C12H1L1, 4G10H3L3.
  • Figure 57 Effect of antibodies 4G10H1L1 and 4G10H3L3 on secretion of cytokine IL-2 induced by co-culture of mixed PBMC, MDA-MB-231 and Raji cells.
  • Figure 58 Effect of antibody 14C12H1L1 on secretion of cytokine IL-2 induced by co-culture of mixed PBMC, MDA-MB-231 and Raji cells.
  • Figure 59 Effect of antibodies BiAb001, BiAb002, BiAb003, BiAb004 on the secretion of cytokine IL-2 induced by co-culture of mixed PBMC, MDA-MB-231 and Raji cells, and compared with antibodies 4G10H1L1, 4G10H3L3 and 14C12H1L1.
  • Figure 60 Effect of antibody BiAb004 on tumor volume of PD-1 HuGEMM MC38 tumor-bearing mice.
  • Hybridoma cell line LT002 (CTLA4-4G10), which was deposited with the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with the accession number CCTCC NO: C201587, deposited at Wuhan University, Wuhan, China. :430072.
  • Hybridoma cell line LT003 (PD-1-14C12), which was deposited with the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with the accession number CCTCC NO: C2015105, and the deposit address is China. Wuhan. Wuhan University , Zip code: 430072.
  • the T cells used were from Zhongshan Kangfang Biomedical Co., Ltd.; the BALB/C mice used were purchased from the Guangdong Medical Laboratory Animal Center; the PD-1 HuGEMM mice used were derived from Nanjing Yinhe Biomedical Co., Ltd. The company used MC38 cells from the Shanghai Fudan IBS Cell Resource Center.
  • the antigen CTLA4-mFc used for the preparation of the anti-CTLA4 antibody is a fusion protein of the extracellular domain of human CTLA4 (Genbank ID: NP_005205.2) and murine IgG1Fc.
  • the spleen cells of the immunized BALB/C mice purchased from the Guangdong Medical Laboratory Animal Center) were fused with the mouse myeloma cells into hybridoma cells, and reference was made to the currently established methods (for example, Stewart, SJ, "Monoclonal Antibody Production", In Basic Methods in antibody Production and Characterization, Eds. GC Howard and DR Bethell, Boca Raton: CRC Press, 2000).
  • the fusion protein CTLA4-mFc was digested with TEV protease and purified by column to obtain CTLA4 protein.
  • CTLA4 protein was used as an antigen-coated ELISA plate and screened by indirect ELISA to obtain specific secretion of CTLA4.
  • New antibodies to hybridoma cells 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 hybridoma cell line LT002 (CTLA4-4G10), and the secreted monoclonal antibody was named 4G10.
  • Hybridoma cell line LT002 (CTLA4-4G10), which was deposited with the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with the accession number CCTCC NO: C201587, deposited at Wuhan University, Wuhan, China. :430072.
  • the LT002 cell line prepared above was cultured in IMDM medium containing 10% low IgG fetal bovine serum (IMDM medium containing 1% streptomycin in a 5% CO 2 , 37 ° C cell incubator The culture supernatant was collected, and the cell culture supernatant was collected 7 days later, and purified by high-speed centrifugation, microfiltration membrane filtration, and HiTrap protein A HP column to obtain antibody 4G10. The purified 4G10 sample was subjected to SDS-PAGE electrophoresis, and the results are shown in Fig. 1.
  • mRNA was extracted from the LT002 cell strain cultured 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 by referring to the pEASY-T1 Cloning Kit (Transgen CT101) kit instructions.
  • Example 3 Design of humanized antibodies against CTLA4 4G10H1L1, 4G10H3L3 and 4G10H4L3 And preparation
  • the antibody 4G10H1L1, 4G10H3L3 and 4G10H4L3 were obtained by computer simulation of the antibody model and design mutation according to the model.
  • Variable region sequence antibody constant region sequence, number from NCBI According to the library, the heavy chain constant region is Ig gamma-1chain C region, ACCESSION: P01857, and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834).
  • variable regions designed is as follows:
  • the nucleic acid sequence of the light chain variable region is identical to the light chain variable region sequence of 4G10H3L3.
  • the heavy chain constant region adopts Ig gamma-1chain C region, ACCESSION: P01857; the light chain constant region adopts Ig kappa chain C region, ACCESSION: P01834.
  • the 4G10H1L1 heavy chain cDNA and light chain cDNA, 4G10H3L3 heavy chain cDNA and light chain cDNA, and 4G10H4L3 heavy chain cDNA and light chain cDNA were cloned into pUC57simple (provided by Kingsray) vector to obtain pUC57simple.
  • pUC57simple provided by Kingsray
  • pUC57simple provided by Kingsray
  • the recombinant plasmid was transfected into 293F cells, and the culture solution was collected and purified to obtain humanized antibodies 4G10H1L1, 4G10H3L3, and 4G10H4L3.
  • the purified 4G10H1L1 sample was subjected to SDS-PAGE electrophoresis, and the results are shown in Fig. 2.
  • the purified 4G10H3L3 sample was subjected to SDS-PAGE electrophoresis, and the results are shown in FIG.
  • spleen cells from immunized BALB/C mice purchased from Guangdong Medical Laboratory Animal Center
  • mouse myeloma cells into hybridoma cells, with reference to currently established methods (eg , Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. G. C. Howard and D. R. Bethell, Boca Raton: CRC Press, 2000).
  • the PD-1-mFc was used as an antigen-coated ELISA plate, and subjected to indirect ELISA screening to obtain a hybridoma cell secreting a novel antibody which specifically binds to PD-1.
  • a hybridoma cell line capable of secreting a monoclonal antibody that competes with the ligand PDL1-hFc (PDL1Genbank ID: NP_054862.1) for binding to PD-1 was screened by competitive ELISA, and a stable hybridoma cell line was obtained by limiting dilution method.
  • the LT003 stable cell line (PD-1-14C12) was obtained by limiting dilution, and the secreted monoclonal antibody was named 14C12.
  • Hybridoma cell line LT003 (PD-1-14C12), which was deposited with the China Center for Type Culture Collection (CCTCC) on June 16, 2015, with the accession number CCTCC NO: C2015105, and the deposit address is China. Wuhan. Wuhan University , Zip code: 430072.
  • the LT003 cell line prepared above was cultured in IMDM medium containing 10% low IgG fetal bovine serum (IMDM medium containing 1% streptomycin in a 5% CO 2 , 37 ° C cell incubator) The culture was carried out), and after 7 days, the cell culture supernatant was collected and purified to obtain Antibody 14C12.
  • the mRNA was extracted from the hybridoma cell line LT003 prepared in Example 4 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 by referring to the pEASY-T1 Cloning Kit (Transgen CT101) kit instructions.
  • Example 6 Design, preparation and detection of humanized antibody 14C12H1L1 against PD-1
  • variable regions designed is as follows:
  • the heavy chain constant region was Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant region was Ig kappa chain C region, ACCESSION: P01834.
  • the heavy chain eDNA of 14C12H1L1 and the eDNA of the light chain were separately cloned into the pcDNA3.1 vector to obtain a recombinant expression plasmid of antibody 14C12H1L1.
  • the recombinant plasmid was transfected into 293F cells.
  • the 293F cell culture medium was purified and tested.
  • the target protein of the reduced protein sample was approximately 24.5 kD and 49 KD
  • the reduced protein sample target protein is approximately 147 kD.
  • the structural mode of the bifunctional antibodies BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 in the present invention belongs to Morrison mode (IgG-scFv), that is, seFv which is linked to another antibody at the C-terminus of both heavy chains of one IgG antibody. Fragments, the major constituents of their heavy and light chains are designed as shown in Table 1 below.
  • Table 1 Composition design of heavy and light chains of BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010
  • V in the lower right corner refers to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. Where no “V” is indicated, the corresponding heavy or light chain is the full length comprising the constant region.
  • the variable region or full-length amino acid sequence and the nucleic acid sequence encoding the same are referred to the corresponding sequences described in the above examples.
  • the amino acid sequence of Linker2 is (GGGGS) 4 (SEQ ID NO: 24)
  • the heavy chain cDNA sequence and the light chain cDNA sequence of BiAb001 were cloned into pUC57simple (provided by Kingsray) vector, respectively, to obtain pUC57simple-BiAb001H and pUC57simple-BiAb001L plasmids, respectively.
  • the plasmids pUC57simple-BiAb001H and pUC57simple-BiAb001L were digested (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 centrifuged by high speed, concentrated, and loaded onto a HiTrap MabSelect SuRe column, and the protein was eluted in one step with Elution Buffer and the target sample was recovered and exchanged to PBS.
  • the purified sample is separately added to the reduced protein electrophoresis loading buffer and the non-reduced protein electrophoresis loading buffer
  • the solution was boiled and subjected to SDS-PAGE electrophoresis.
  • the electropherogram of BiAb001 is shown in Figure 5.
  • the reduced protein sample target protein is at 23.6 kD and 75.8 kD
  • the non-reduced protein sample (single antibody) target protein is at 199 kD.
  • BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 were obtained according to the above expression and purification method of BiAb001.
  • the purified sample was separately added to a reduced protein electrophoresis loading buffer and a non-reduced protein electrophoresis loading buffer, and then boiled and subjected to SDS-PAGE electrophoresis.
  • the electropherograms of BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010 are shown in Figure 6, Figure 7, Figure 8, Figure 9, and Figure 10, respectively.
  • the reduced protein samples are at 23.6kD and 75.8kD, non-reduced protein samples. (Single antibody)
  • the target protein is at 199 kD.
  • the kinetic parameters of antigen-antibody binding were determined using a Fortebio molecular interaction instrument.
  • CTLA4-mFc protein was digested with TEV protease and purified by column to obtain CTLA4 antigen.
  • Antibody 4G10 was immobilized on the surface of AR2G sensor by amino coupling, blocked by ethanolamine, equilibrated in PBST, and bound to antigen CTLA4.
  • CTLA4 was diluted twice with PBST at concentrations of 268.1, 134.1, 67, 33.5, 16.8. 8.38, 4.19, 0 nM, dissociated in PBST.
  • the detection methods of humanized 4G10H1L1, 4G10H3L3, and 4G10H4L3 were similar to those of 4G10, and the antigen concentrations were 180, 90, 45, 22.5, 11.25, 5.625, 2.813, and 0 nM.
  • the PD-1-mFc protein was digested with TEV protease and purified by column to obtain PD-1 antigen.
  • Antigen PD-1 (antigen concentration 1 ⁇ g/ml) was biotinylated and fixed on the surface of the SA sensor. After equilibration in PBST, it was bound to antibody 14C12 and 14C12H1L1, respectively, and the antibody was diluted three times from 200 nM with PBST. Dissociated in PBST.
  • the antigen PD-1 (antigen concentration 1 ⁇ g/ml) was labeled with biotin and immobilized on the surface of the SA sensor. After equilibration in PBST, it was bound to the antibodies BiAb001, BiAb002, BiAb003, BiAb004, BiAb007 and BiAb010, respectively. Dilute three times from 200 nM and dissociate in PBST.
  • Both antibody 4G10 and its humanized antibody have a good affinity for the antigen.
  • 14C12 and 14C12H1L1 have good affinity with antigen PD-1.
  • Both bifunctional antibodies have better affinity to the antigens CTLA4 and PD-1.
  • Example 9 Detection of binding activity of an antibody to an antigen by an ELISA method
  • the test results are shown in Fig. 28.
  • the humanized antibodies 4G10H1L1, 4G10H3L3 can effectively bind to CTLA4 protein, and the binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 3.
  • the binding efficiencies EC 50 of the antibodies 4G10H1L1 and 4G10H3L3 obtained by curve simulation were 0.048 and 0.067 nM, respectively.
  • B7/1-hFc (B7/1 Genbank ID: NP_005182.1) was coated with the plate at 4 ° C overnight, and 1% BSA was blocked for 2 h. Then the antibody and antibody mixture of CTLA4-mFc were added separately (see Table 4 for dilution concentration). Incubate at 37 ° C for 30 min, incubate with enzyme-labeled secondary antibody for 1 h, and incubate at 37 ° C for 30 min. The absorbance at 450 nm was measured on a microplate reader (see Table 4).
  • the results of detecting the antibody binding to B7-1 for binding to the antigen CTLA4 are shown in FIG.
  • the antibodies 4G10H1L1 and 4G10H3L3 can effectively compete with B7-1 for binding to CTLA4 protein, and the binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 4.
  • the curve simulation binding efficiency EC 50 was 1.297 nM and 1.229 nM, respectively.
  • Indirect ELISA was used to determine the binding activity of monoclonal recombinant antibodies 14C12 and 14C12H1L1 to PD-1, respectively. The method is as follows:
  • the plate was incubated with PD-1-mFc at 4 ° C overnight, blocked with 1% BSA at 37 ° C for 2 h, then added to the antibody, incubated at 37 ° C for 30 min, and added HRP-labeled goat anti-human IgG (H + L) secondary antibody. (Jackson, 109-035-088), a color reaction was carried out with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was measured in a microplate reader.
  • the results of binding of the detection antibody 14C12, 14C12H1L1 to the antigen PD-1 are shown in FIG.
  • both antibody 14C12 and 14C12H1L1 were able to efficiently bind to PD-1 protein, and their binding efficiency was dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 5.
  • simulation curve and 14C12 antibody binding efficiency 14C12H1L1 EC 50 respectively 0.175nM and 0.043nM.
  • Different concentrations of antibody 14C12 and 14C12H1L1 were mixed with PDL1-hFc and PDL1-mFc for 10 min (diluted concentration) after plating with PD-1-hFc or PD-1-mFc at 4 °C overnight and blocking with 1% BSA for 2 h. See Table 6), incubate at 37 ° C for 30 min, then add the enzyme-labeled secondary antibody for 30 min at 37 ° C. The absorbance at 450 nm was measured on a microplate reader (see Table 6).
  • Fig. 32 The results of binding of the antibodies BiAb001, BiAb002, BiAb003 and BiAb004 to the antigen PD-1 are shown in Fig. 32.
  • the antibodies BiAb001, BiAb002, BiAb003 and BiAb004 all bind PD-1 efficiently, and the binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 7.
  • the results of binding of the antibodies BiAb001, BiAb002, BiAb003 and BiAb004 to the antigen PD-1 are shown in FIG.
  • the antibodies BiAb001, BiAb002, BiAb003 and BiAb004 all bind PD-1 efficiently, and the binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 7.
  • simulation curve of antibody binding efficiency EC 50 is shown in Table 8.
  • the test results are shown in Figure 34.
  • the antibodies BiAb001, BiAb002, BiAb003 and BiAb004 can effectively bind the antigen CTLA4, inhibit CTLA4 binding to B7/1, and the antibody inhibits the efficiency of CTLA4 binding to B7/1 in a dose-dependent manner.
  • the absorbance of each dose is shown in Table 9. .
  • By binding an antibody BiAb001, BiAb002, BiAb003 and BiAb004 absorbance intensity for the quantitative analysis of antibody binding efficiency curve obtained analog binding EC 50 (Table 9).
  • the test results are shown in Figure 35.
  • the antibodies BiAb001, BiAb002, BiAb003 and BiAb004 can effectively bind to the antigen PD-1, inhibit PD-1 binding to its ligand PDL1, and the antibody inhibits the efficiency of PD-1 binding to its ligand PDL1 in a dose-dependent manner.
  • the absorbance of each dose is shown in Table 10.
  • Table 10 By binding an antibody BiAb001, BiAb002, BiAb003 and BiAb004 absorbance intensity for the quantitative analysis of antibody binding efficiency curve obtained analog binding EC 50 (Table 10).
  • Example 10 Flow cytometry method for detecting binding activity of antibodies to cell surface antigens
  • host cells 293T expressing CTLA4 and PD-1 antigens were separately constructed, and the host cells were labeled with the humanized antibodies prepared in the present invention. Flow cytometry analysis was then used to verify the antigen-specific binding ability of the antibody to the native conformation of the cell surface.
  • CTLA4 or PD-1 vectors pLenti6.3-CTLA4, pLenti6.3-PD-1 (vector pLenti6.3 purchased from Invitrogen) were transfected into 293T cells, and 293T-CTLA4 cells stably expressing CTLA4 were obtained by screening. A clonal population of 293T-PD-1 cells stably expressing PD-1.
  • the antigen-expressing host cells obtained by the above steps of the conventional trypsin digestion method were used, and the number of cells in each collection tube was 2 ⁇ 10 5 , and the antibody concentration gradient dilution solution was prepared with PBS (1% BSA), and the corresponding antigen was expressed on ice.
  • the results of binding of the humanized antibodies 4G10H1L1 and 4G10H3L3 to 293T-CTLA4 cells are shown in Fig. 36 and Fig. 37, respectively.
  • the 4G10H1L1 and 4G10H3L3 antibodies can effectively bind to the target CTLA4 protein on the surface of the host cell 293T-CTLA4, and the binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 11.
  • 4G10H1L1 4G10H3L3 by antibody binding and quantitative analysis of fluorescence curves and analog 4G10H1L1 antibody binding efficiency 4G10H3L3 EC 50 were 7.58nM, 10.54nM.
  • Table 11 Fluorescence intensity analysis of 4G10H1L1 and 4G10H3L3 binding to CTLA4 host cell 293T-CTLA4 surface antigen by flow cytometry
  • Table 12 Fluorescence intensity analysis of 14C12H1L1 binding to PD-1 host cell 293T-PD-1 surface antigen by flow cytometry
  • Table 13 Flow cytometry analysis of the fluorescence intensity of antibodies BiAb001, BiAb002, BiAb003 and BiAb004 293F-CTLA4 binding cell surface antigens and EC 50
  • PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare LOT No.: 171440-02), CD4 + cells were isolated from PBMC, washed with PHA (50 ⁇ l/ml) for three days, washed once with PBS, and added with different concentrations of antibody, ice. Incubate for 1.5 h. After the completion of the incubation, it was washed once with PBS, and FITC-labeled anti-human secondary antibody IgG (Jackson immunoresearch lot. 102155) was added. After incubating for 1 h in the dark on ice, it was washed once with PBS and detected by flow cytometry.
  • control antibody Nivolumab used is an antibody against PD-1, which is commercially available, and the information can also be referred to http://www.drugbank.ca/drugs/DB09035;
  • the control antibody Ipilimumab is an antibody against CTLA4 and is commercially available. For information, see http://www.drugbank.ca/drugs/DB06186.
  • FIG. 3 Binding results of humanized antibody 4G10H3L3 and T cells are shown in FIG. As can be seen from the figure, the 4G10H3L3 antibody can effectively bind to the target CTLA4 protein on the surface of T cells, and its binding efficiency is dose dependent.
  • Table 15 Binding activity of antibodies 14C12H1L1, 4G10H3L3 and BiAb003 and BiAb004 to T cells
  • Example 11 Mixed lymphocyte reaction: secretion of cytokine IFN- ⁇ , IL-2
  • PBMC peripheral blood mononuclear cells
  • Ficoll-Paque Plus GE Healthcare LOT No.: 171440-02
  • IL-4 Peprotech K2513, 1000 U/ml
  • GM-CSF Peprotech H1513, 1000 U/ml
  • DC cells were obtained by inducing TNF- ⁇ (Peprotech G1513, 200 U/ml) for 3 days.
  • T cells were isolated from PBMC, and the obtained DC cells were mixed with T cells in a ratio of 1:10, and different proportions of antibodies (hIgG as a control) were added for 5-6 days, and IFN- ⁇ was detected by ELISA kit.
  • the amount of secretion purchased from Daktronics for the company
  • IL-2 purchased from Daktronics for the company.
  • the secretion detection results of IFN- ⁇ after mixed culture of DC cells and T cells are shown in Fig. 50-53, respectively.
  • the secretion detection results of IL-2 after mixed culture of DC cells and T cells are shown in Fig. 54 to Fig. 56, respectively.
  • 4G10H1L1, 4G10H3L3, 14C12H1L1, and bifunctional antibodies BiAb001, BiAb002, BiAb003 and BiAb004 were all effective in inducing secretion of IFN- ⁇ and IL-2 by mixed lymphocytes.
  • the anti-PD-1 antibody 14C12H1L1 induced the secretion of IFN- ⁇ at a concentration of 1 nM and 10 nM, which was comparable to the control antibody Nivolumab at 100 nM.
  • the CTLA4 antibodies 4G10H1L1 and 4G10H3L3 induced more IFN- ⁇ secretion than the control antibody Ipilimumab at a concentration of 100 nM (Fig. 52).
  • the isolated PBMC (method as in Example 10) was stimulated with PHA (Shanghai CDC Biotechnology Co., Ltd., 50 ⁇ l/mL) for 3 days, and then stimulated mature PBMC was added to the 96-well plate (from volunteer blood donors, 5 ⁇ 10). 4 cells/well), Raji cells (from ATCC, 5 ⁇ 10 4 cells/well) and MDA-MB-231 cells (from ATCC) (1 ⁇ 10 4 cells/well), plus antibody (100 nM) mixed Evenly co-cultured. After 3 days, the secretion amount of IL-2 (purchased from Daktronics Co., Ltd.) was detected by an ELISA kit, and the specific operation was carried out in accordance with the kit instructions.
  • PHA Sthai CDC Biotechnology Co., Ltd., 50 ⁇ l/mL
  • mice human PD-1 transgenic mice, subcutaneously inoculated MC38 tumor cells 1 ⁇ 10 6 /only
  • the mice were randomly divided into 4 according to the tumor volume.
  • One experimental group was administered intraperitoneally, and 8 mice in each group.
  • the specific grouping and dosage are as follows:
  • BiAb004 low dose group (administered dose 0.267mg/kg),
  • the above three groups were administered twice a week for a total of 5 times. Tumor size was measured twice weekly after group administration.
  • the tumor volume of the BiAb004 high dose group and the BiAb004 low dose group was statistically significantly lower than that of the Isotype control group (P ⁇ 0.001, ⁇ 0.05).
  • the low dose group of BiAb004 showed a statistically significant anti-tumor effect in the PD-1 HuGEMM mouse MC38 tumor model.

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Abstract

一种抗CTLA4-抗PD-1双功能抗体、其药物组合物及其用途。具体地，所述的抗CTLA4和PD-1的双功能抗体，包括：靶向PD-1的第一蛋白功能区，和靶向CTLA4的第二蛋白功能区。该双功能抗体能够很好地特异性地与CTLA4和PD-1结合，特异地解除CTLA4和PD-1对机体免疫抑制，激活T淋巴细胞，具有良好的应用前景。

Description

抗CTLA4-抗PD-1双功能抗体、其药物组合物及其用途 技术领域
本发明属于肿瘤治疗和分子免疫学领域，涉及一种抗CTLA4-抗PD-1双功能抗体、其药物组合物及其用途。具体地，本发明涉及一种抗CTLA4-抗PD-1的单克隆抗体。
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。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。
CTLA4mAb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 2013Jan 22)。
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)。体外实验中，CTLA4 mAb可特异地解除CTLA4对机体免疫抑制，激活T细胞，诱导IL-2产生，在抗肿瘤及寄生虫等疾病的基因治疗中有广泛应用前景。
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。
跨膜受体PD-1(程序性细胞死亡-1)是CD28基因家族成员之一，在活化的T细胞，B细胞以及骨髓系细胞都有表达。PD-1的受体PDL1和PDL2均属于B7超家族，其中PDL1多种细胞都有表达，包括T细胞，B细胞以及内皮细胞和上皮细胞，PDL2则仅表达于抗原呈递细胞如树突状细胞和巨噬细胞。
T细胞对清除病毒感染起着非常重要的作用，但T细胞抗病毒反应通常与免疫病理相关。PD-1在负调节T细胞的活化过程中起着非常重要的作用，PD-1介导的对T细胞负调节作用可减少促感染过程引起的组织损伤，但是阻断或抑制PD-1的负调节作用可导致自身免疫性疾病，例如，PD-1基因敲除小鼠能更有效的清除胰腺病毒感染，但是却导致了更严重的肝脏损伤(Isai et al.，2003，J.Exp.Med.198：39-50)。另外，高表达PD-1的肿瘤伴随着很难被检测到的癌症(Hamanishi et al.，2007，Proc.Natl.Acad.Sci.USA 104：3360-5)。一种实施有效的方法是通过体内注射抗体对PD-1的表达进行调控。
由于PD-1抗体的广谱抗肿瘤前景和惊人的药效，业界普遍认为针对PD-1通路的抗体将带来治疗多种肿瘤治疗的突破性的进展：用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色素瘤(Homet M.B.，Parisi G.，et al.，Anti-PD-1 Therapy in Melanoma.Semin Oncol.2015Jun；42(3)：466-473)，白血病以及贫血病(Held SA，Heine A，et al.，Advances in immunotherapy of chronic myeloid leukemia CML.Curr Cancer Drug Targets.2013 Sep；13(7)：768-74)。在2012年和2013年的美国癌症协会(AACR)年会以及美国临床肿瘤协会(ASCO)年会上揭晓的前所未有的临床药效数据后，既成为全球制药行业最炙手可热的在研新药。
此外，干扰素γ(IFNγ)，主要由自然杀伤细胞(NK)，自然杀伤T细胞(NKT)先天产生，和由CD4 Th1细胞和CD8细胞毒性T淋巴细胞(CTL)这些效应T细胞经由特定抗原刺激后产生。IFNγ作为一种重要的先天性和获得性免疫细胞因子在对抗或抑制肿瘤，病毒，某些细菌和原虫性疾病感染起着重要作用。同时，IFNγ能激活巨噬细胞，诱导II类主要组织相容性复合体(MHC)的表达激活免疫反应控制肿瘤的发展(SchoenbornJR，Wilson CB.Regulation of Interferon-γ During Innate and Adaptive  Immune Responses.Advances in Immunology 2007；96：41-101)。
单克隆抗体目前已被用于治疗癌症，炎症和感染性疾病和其它疾病，大部分是单特异性的，然而，有些疾病在成因上以及体内的影响因素通常是多方面的，包括在不同的信号通路中，不同的蛋白、细胞因子和受体等的上调或者下调，抑制或促进体内机能作用。因此，多种不同的因素阻断可以改善治疗效果。这可以通过组合不同的药物，或者使用替代合不同药物如采用多特异性抗体的多重靶向策略来实现。
双功能抗体亦称为双特异性抗体(Bispecific Antibody)，是同时靶向结合两种不同抗原的特异性药物，其可通过免疫分选纯化生产。另外，也可通过基因工程获得，基因工程在结合位点优化，合成形式的考量以及产量等方面都具有相应的灵活性，所以具有一定的优势。目前，其存在形式已被证明有超过45种(Müller D，Kontermann RE.Bispecific antibodies for cancer immunotherapy：Current perspectives.BioDrugs 2010；24：89-98)。目前已开发的多种双特异性抗体为IgG-ScFv形式即Morrison模式(1997Coloma MJ，Morrison SL.Design and production of novel tetravalent bispecific antibodies.Nat Biotechnol.Nature Biotechnology，1997；15：159-163)，由于这种类似于天然存在的IgG形式，其在抗体工程、表达和纯化上所具有的优势，已被证明是双功能抗体的其中一种理想存在形式(Miller BR，Demarest SJ，et al.，Stability engineering of scFvs for the development of bispecific and multivalent antibodies.Protein Eng Des Sel 2010；23：549-57；Fitzgerald J，Lugovskoy A.Rational engineering of antibody therapeutics targeting multiple oncogene pathways.MAbs 2011；3：299-309)。
然而，目前市场上甚至还没有靶向同一类细胞上两个以上靶点的双功能抗体，目前世界上有的是针对两种不同细胞表面分子的抗体。需要开发同时抗PD-1和CTLA4的双功能抗体药物。
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分别表达出重组的CTLA4和PD-1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，分别得到了如下的杂交瘤细胞株：
杂交瘤细胞株LT002(CTLA4-4G10)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C201587；以及
杂交瘤细胞株LT003(PD-1-14C12)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015105。
本发明人惊奇地发现：
杂交瘤细胞株LT002能够分泌产生与CTLA4特异性结合的特异性单克隆抗体(命名为4G10)，并且该单克隆抗体能够十分有效地阻断CTLA4与B7的结合；
杂交瘤细胞株LT003能够分泌产生与PD-1特异性结合的特异性单克隆抗体(命名为14C12)，并且该单克隆抗体能够十分有效地阻断PD-1与PDL1的结合。
进一步地，本发明人创造性地制得了抗CTLA4的人源化抗体(分别命名为4G10H1L1、4G10H3L3、4G10H4L3和8D2H14L2)和抗PD-1的人源化抗体(命名为14C12H1L1)。
进一步地，本发明人创造性地将两类人源化抗体进行蛋白重组融合成新的抗体，获得了能够结合CTLA4和PD-1，阻断CTLA4与B7、PD-1与PDL1的结合的人源化双功能抗体(分别命名为BiAb001、BiAb002、BiAb003、BiAb004、BiAb007和BiAb010)，能有效地结合人T细胞，并且激活T细胞，诱导人淋巴细胞分泌IFN-γ和IL-2；具有用于制备防治肺癌、黑色素瘤、肾肿瘤、卵巢癌、白血病等癌症的药物的潜力。
由此提供了下述发明：
本发明的一个方面涉及一种双特异性抗体，其包括：
靶向PD-1的第一蛋白功能区，和
靶向CTLA4的第二蛋白功能区。
在本发明的一个实施方案中，所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；优选地，所述连接片段为(GGGGS)n，n为正整数，例如1、2、3、4、5或6。
在本发明的一个实施方案中，所述的双特异性抗体，其中，
所述第一蛋白功能区和第二蛋白功能区独立地为免疫球蛋白或其抗原结合片段，例如半抗体、Fab、F(ab’)2或单链抗体；
优选地，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；或者
优选地，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白。
在本发明的一个实施方案中，所述的双特异性抗体，其中，所述第一蛋白功能区和 第二蛋白功能区独立地为1个、2个或者2个以上。
在本发明的一个实施方案中，所述的双特异性抗体，其中，所述免疫球蛋白为IgG、IgA、IgD、IgE或IgM；优选为IgG，例如IgG1、IgG2、IgG3或IgG4。
在本发明的一个实施方案中，所述的双特异性抗体，其中，所述单链抗体连接在免疫球蛋白的重链的C末端。由于免疫球蛋白由两条重链，因此，一个免疫球蛋白分子连接有两个单链抗体分子。优选地，两个单链抗体分子相同。
在本发明的一个实施方案中，所述的双特异性抗体，其中：
所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：29-31的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：32-34的CDR；和/或
所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：35-37的CDR或者包含氨基酸序列为SEQ ID NO：35、SEQ ID NO：41和SEQ ID NO：37的CDR或者包含氨基酸序列为SEQ ID NO：42-44的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：38-40的CDR或者包含氨基酸序列为SEQ ID NO：45-47的CDR；
在本发明的另一个实施方案中，所述的双特异性抗体，其中：
所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：35-37的CDR或者包含氨基酸序列为SEQ ID NO：35、SEQ ID NO：41和SEQ ID NO：37的CDR或者包含氨基酸序列为SEQ ID NO：42-44的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：38-40的CDR或者包含氨基酸序列为SEQ ID NO：45-47的CDR；和/或
所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：29-31的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：32-34的CDR。
在本发明的一个实施方案中，所述的双特异性抗体，其中：
所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO：16和SEQ ID NO：20；所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO：18和SEQ ID NO：22；和/或
所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6、SEQ ID NO：10、SEQ ID NO：14和SEQ ID NO：25；所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12和SEQ ID NO：27；
在本发明的另一个实施方案中，所述的双特异性抗体，其中：
所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6、 SEQ ID NO：10、SEQ ID NO：14和SEQ ID NO：25；所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12和SEQ ID NO：27；和/或
所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：16和SEQ ID NO：20；所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO：18和SEQ ID NO：22。
在本发明的一个实施方案中，所述的双特异性抗体，其中，
所述的免疫球蛋白包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
在本发明的一个实施方案中，所述免疫球蛋白的恒定区是人源化的，例如，重链恒定区均采用Ig gamma-1 chain C region，ACCESSION：P01857；轻链恒定区均采用Ig kappa chain C region，ACCESSION：P01834。
在本发明的一个实施方案中，所述的双特异性抗体，其中，所述的双特异性抗体以小于大约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蛋白和/或PD-1蛋白。
本发明还涉及一种双特异性抗体，其重链可变区包含：
氨基酸序列为SEQ ID NO：29-31的CDR、
氨基酸序列为SEQ ID NO：35-37的CDR或者氨基酸序列为SEQ ID NO：35、SEQ ID NO：41和SEQ ID NO：37的CDR或者氨基酸序列为SEQ ID NO：42-44的CDR，和
氨基酸序列为SEQ ID NO：32-34的CDR或者氨基酸序列为SEQ ID NO：38-40的CDR或者氨基酸序列为SEQ ID NO：45-47的CDR；
并且其轻链可变区包含：
氨基酸序列为SEQ ID NO：32-34的CDR，
氨基酸序列为SEQ ID NO：38-40的CDR，或者
氨基酸序列为SEQ ID NO：45-47的CDR；
优选地，轻链可变区的CDR与重链可变区包含的CDR不相同。
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，
所述抗体的重链可变区包含：
氨基酸序列为SEQ ID NO：29-31的CDR、
氨基酸序列为SEQ ID NO：35-37的CDR或者氨基酸序列为SEQ ID NO：35、SEQ ID NO：41和SEQ ID NO：37的CDR或者氨基酸序列为SEQ ID NO：42-44的CDR，和
氨基酸序列为SEQ ID NO：32-34的CDR或者氨基酸序列为SEQ ID NO：38-40的CDR或者氨基酸序列为SEQ ID NO：45-47的CDR。
本发明还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：
氨基酸序列为SEQ ID NO：32-34的CDR，
氨基酸序列为SEQ ID NO：38-40的CDR，或者
氨基酸序列为SEQ ID NO：45-47的CDR。
本发明的再一方面涉及一种载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及制备本发明的双特异性抗体的方法，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双特异性抗体的步骤。
本发明的再一方面涉及偶联物，其包括双特异性抗体以及偶联部分，其中，所述双特异性抗体为本发明的双特异性抗体，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。
本发明的再一方面涉及试剂盒，其包括本发明的双特异性抗体，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，其特异性识别所述双特异性抗体；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。
本发明的再一方面涉及本发明的双特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测CTLA4和/PD-1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，其包含本发明的双特异性抗体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的双特异性抗体或者本发明的偶联物在制备预防和/或治疗肿瘤或者贫血病的药物中的用途，或者在制备诊断肿瘤或者贫血病的药物中的用途；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、肝癌、非小细胞性肺癌、卵巢癌和白血病。
本发明人还通过动物实验发现，本发明的双特异性抗体BiAb004能够有效地降低PD-1 HuGEMM小鼠(人PD-1转基因小鼠)右侧皮下接种MC38肿瘤细胞的生长，表现为显著性地抑制PD-1 HuGEMM MC38荷瘤鼠的肿瘤体积的增加。
本发明的再一方面涉及本发明的双特异性抗体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；
和/或
阻断PD-1与PDL1结合的药物，
调节(例如下调)PD-1活性或水平的药物，
解除PD-1对机体免疫抑制的药物，或者
提高T淋巴细胞中IFN-γ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，包括施加细胞或者给予有需求的受试者以有效量的本发明的双特异性抗体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；
和/或
阻断PD-1与PDL1结合的方法，
调节(例如下调)PD-1活性或水平的方法，
解除PD-1对机体免疫抑制的方法，或者
提高T淋巴细胞中IFN-γ表达的方法。
本发明的体外实验中，抗CTLA4抗体、抗PD-1抗体及抗CTLA4-抗PD-1双功能抗体均能诱导IFNγ的分泌激活免疫反应。
本发明的再一方面涉及一种预防和/或治疗肿瘤或者贫血病的方法，或者一种诊断肿瘤或者贫血病的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的双特异性抗体或者本发明的偶联物的步骤；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、肝癌、非小细胞性肺癌、卵巢癌和白血病。
根据本发明中任一项所述的双特异性抗体或者偶联物，其用于预防和/治疗肿瘤或者贫血病，或者用于诊断肿瘤或者贫血病；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、肝癌、非小细胞性肺癌、卵巢癌和白血病。
根据本发明中任一项所述的双特异性抗体或者偶联物，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；
和/或
阻断PD-1与PDL1结合，
调节(例如下调)PD-1活性或水平，
解除PD-1对机体免疫抑制，或者
提高T淋巴细胞中IFN-γ表达。
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数据库分析下面的(1)-(13)项中的单克隆抗体序列的CDR区的氨基酸序列，结果如下：
(1)14C12
重链可变区的氨基酸序列如SEQ ID NO：16所示，轻链可变区的氨基酸序列如SEQ ID NO：18所示。
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GFAFSSYD(SEQ ID NO：29)
HCDR2：ISGGGRYT(SEQ ID NO：30)
HCDR3：ANRYGEAWFAY(SEQ ID NO：31)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：QDINTY(SEQ ID NO：32)
LCDR2：RAN(SEQ ID NO：33)
LCDR3：LQYDEFPLT(SEQ ID NO：34)
(2)14C12H1L1
重链可变区的氨基酸序列如SEQ ID NO：20所示，轻链可变区的氨基酸序列如SEQ ID NO：22所示。
其重链可变区的3个CDR区的氨基酸序列与14C12相同。
其轻链可变区的3个CDR区的氨基酸序列与14C12相同。
(3)4G10
重链可变区的氨基酸序列如SEQ ID NO：2所示，轻链可变区的氨基酸序列如SEQ ID  NO：4所示；
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GYSFTGYT(SEQ ID NO：35)
HCDR2：INPYNNIT(SEQ ID NO：36)
HCDR3：ARLDYRSY(SEQ ID NO：37)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：TGAVTTSNF(SEQ ID NO：38)
LCDR2：GTN(SEQ ID NO：39)
LCDR3：ALWYSNHWV(SEQ ID NO：40)
(4)4G10H1L1
重链可变区的氨基酸序列如SEQ ID NO：6所示，轻链可变区的氨基酸序列如SEQ ID NO：8所示；
其重链可变区的3个CDR区的氨基酸序列与4G10相同。
其轻链可变区的3个CDR区的氨基酸序列与4G10相同。
(5)4G10H3L3
重链可变区的氨基酸序列如SEQ ID NO：10所示，轻链可变区的氨基酸序列如SEQ ID NO：12所示；
其重链可变区的3个CDR区的氨基酸序列与4G10相同。
其轻链可变区的3个CDR区的氨基酸序列与4G10相同。
(6)4G10H4L3
重链可变区的氨基酸序列如SEQ ID NO：14所示，轻链可变区的氨基酸序列如SEQ ID NO：12所示。
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GYSFTGYT(SEQ ID NO：35)
HCDR2：INPYNDIT(SEQ ID NO：41)
HCDR3：ARLDYRSY(SEQ ID NO：37)
其轻链可变区的3个CDR区的氨基酸序列与4G10相同。
(7)8D2H14L2
重链可变区的氨基酸序列如SEQ ID NO：25所示，轻链可变区的氨基酸序列如SEQ  ID NO：27所示。
其重链可变区的3个CDR区的氨基酸序列如下：
HCDR1：GFTFSDNW(SEQ ID NO：42)
HCDR2：IRNKPYNYET(SEQ ID NO：43)
HCDR3：TAQFAY(SEQ ID NO：44)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：ENIYGG(SEQ ID NO：45)
LCDR2：GAT(SEQ ID NO：46)
LCDR3：QNVLRSPFTF(SEQ ID NO：47)
(8)BiAb001
其重链可变区的9个CDR区的氨基酸序列如下：
HCDR1：GFAFSSYD(SEQ ID NO：29)
HCDR2：ISGGGRYT(SEQ ID NO：30)
HCDR3：ANRYGEAWFAY(SEQ ID NO：31)
HCDR4：GYSFTGYT(SEQ ID NO：35)
HCDR5：INPYNNIT(SEQ ID NO：36)
HCDR6：ARLDYRSY(SEQ ID NO：37)
HCDR7：TGAVTTSNF(SEQ ID NO：38)
HCDR8：GTN(SEQ ID NO：39)
HCDR9：ALWYSNHWV(SEQ ID NO：40)
其轻链可变区的3个CDR区的氨基酸序列如下：
LCDR1：QDINTY(SEQ ID NO：32)
LCDR2：RAN(SEQ ID NO：33)
LCDR3：LQYDEFPLT(SEQ ID NO：34)
(9)BiAb002
其重链可变区的9个CDR区的氨基酸序列与BiAb001相同。
其轻链可变区的3个CDR区的氨基酸序列与BiAb001相同。
(10)BiAb003
其重链可变区的9个CDR区的氨基酸序列与BiAb001相同。
其轻链可变区的3个CDR区的氨基酸序列与BiAb001相同。
(11)BiAb004
其重链可变区的9个CDR区的氨基酸序列与BiAb001相同。
其轻链可变区的3个CDR区的氨基酸序列与BiAb001相同。
(12)BiAb007
其重链可变区的9个CDR区的氨基酸序列如下：
HCDR1：GFAFSSYD(SEQ ID NO：29)
HCDR2：ISGGGRYT(SEQ ID NO：30)
HCDR3：ANRYGEAWFAY(SEQ ID NO：31)
HCDR4：GYSFTGYT(SEQ ID NO：35)
HCDR5：INPYNDIT(SEQ ID NO：41)
HCDR6：ARLDYRSY(SEQ ID NO：37)
HCDR7：TGAVTTSNF(SEQ ID NO：38)
HCDR8：GTN(SEQ ID NO：39)
HCDR9：ALWYSNHWV(SEQ ID NO：40)
其轻链可变区的3个CDR区的氨基酸序列与BiAb001相同。
(13)BiAb010
其重链可变区的9个CDR区的氨基酸序列如下：
HCDR1：GFAFSSYD(SEQ ID NO：29)
HCDR2：ISGGGRYT(SEQ ID NO：30)
HCDR3：ANRYGEAWFAY(SEQ ID NO：31)
HCDR4：GFTFSDNW(SEQ ID NO：42)
HCDR5：IRNKPYNYET(SEQ ID NO：43)
HCDR6：TAQFAY(SEQ ID NO：44)
HCDR7：ENIYGG(SEQ ID NO：45)
HCDR8：GAT(SEQ ID NO：46)
HCDR9：QNVLRSPFTF(SEQ ID NO：47)
其轻链可变区的3个CDR区的氨基酸序列与BiAb001相同。
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或者包含CTLA4ECD的片段；还包括CTLA4ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。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，包括其天然或人工的变体。并且，当描述CTLA4蛋白的序列片段时，其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，当提及PD-1蛋白(Programmed cell death protein 1，NCBI GenBank：NM_005018)的氨基酸序列时，其包括PD-1蛋白的全长，或者PD-1的胞外片段PD-1ECD或者包含PD-1ECD的片段；还包括PD-1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段。然而，本领域技术人员理解，在PD-1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“PD-1蛋白”应包括所有此类序列，包括其天然或人工的变体。并且，当描述PD-1蛋白的序列片段时，其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。从一般意义上，重链可以理解为抗体中分子量较大的多肽链，轻链是指抗体中分子量较小的多肽链。轻链可分类为κ和λ轻链。重链通常可分类为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。特别地，重链还可以包含3个以上CDR，例如6、9、或12个。例如在本发明的双功能抗体中，重链可以是IgG抗体的重链的C端连接另一个抗体的ScFv，这种情况下重链含有9个CDR。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，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，81：68516855(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，321：522525(1986)；Reichmann et al.，Nature，332：323329(1988)；Presta，Curr.Op.Struct.Biol.，2：593596(1992)；和Clark，Immunol.Today 21：397402(2000)。
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PAMl20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“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。在本发明的一些实施方案中，术语“靶向”是指特异性结合。
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，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LT002或LT003时，其还指杂交瘤细胞株LT002或LT003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
本发明涉及的单克隆抗体4G10H1L1、4G10H3L3能够很好地特异性与CTLA4结合，并且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。
本发明涉及的单克隆抗体14C12H1L1能够很好地特异性与CTLA4结合，并且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。
本发明的双功能抗体具有应用于制备抗肿瘤例如抗黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌或白血病的药物的潜力。
附图说明
图1：单克隆鼠源抗体4G10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：非还原型蛋白电泳上样缓冲液样品抗体，1μg；还原型蛋白电泳上样缓冲液样品抗体，1μg；Marker，5μl；BSA，1μg。
图2：单克隆人源化抗体4G10H1L1的SDS-PAGE检测结果。从左至右的3个泳道的样品及其上样量依次为：非还原型蛋白电泳上样缓冲液样品抗体，1μg；还原型蛋白电泳上样缓冲液样品抗体，1μg；Marker，5μl。
图3：单克隆人源化抗体4G10H3L3的SDS-PAGE检测结果。
从左至右的2个泳道的样品及其上样量依次为：还原型蛋白电泳上样缓冲液样品抗体，1μg；Marker，5μl。
图4：单克隆人源化抗体14C12H1L1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：非还原型蛋白电泳上样缓冲液样品抗体，1μg；还原型蛋白电泳上样缓冲液样品抗体，1μg；Marker，5μl；BSA，1μg。
图5：双功能抗体BiAb001的SDS-PAGE检测结果。泳道样品及其上样量为：Marker，5μl；1，Non-reduced：非还原型蛋白电泳上样缓冲液样品，1μg；2，Reduced：还原型蛋白电泳上样缓冲液样品，1μg；3，BSA，1μg。
图6：双功能抗体BiAb002的SDS-PAGE检测结果。泳道样品及其上样量为：Marker，5μl；1，Non-reduced：非还原型蛋白电泳上样缓冲液样品，1μg；2，Reduced：还原型蛋白电泳上样缓冲液样品，1μg；3，BSA，1μg。
图7：双功能抗体BiAb003的SDS-PAGE检测结果。泳道样品及其上样量为：Marker，5μl；1，Non-reduced：非还原型蛋白电泳上样缓冲液样品，1μg；2，Reduced：还原型蛋白电泳上样缓冲液样品，1μg；3，BSA，1μg。
图8：双功能抗体BiAb004的SDS-PAGE检测结果。泳道样品及其上样量为：Marker，5μl；1，Non-reduced：非还原型蛋白电泳上样缓冲液样品，1μg；2，Reduced：还原型蛋白电泳上样缓冲液样品，1μg；3，BSA，1μg。
图9：双功能抗体BiAb007的SDS-PAGE检测结果。泳道样品及其上样量为：Marker，5μl；1，Non-reduced：非还原型蛋白电泳上样缓冲液样品，1μg；2，Reduced：还原型蛋白电泳上样缓冲液样品，1μg；3，BSA，1μg。
图10：双功能抗体BiAb0010的SDS-PAGE检测结果。泳道样品及其上样量为：Marker，5μl；1，Non-reduced：非还原型蛋白电泳上样缓冲液样品，1μg；2，Reduced：还原型蛋白电泳上样缓冲液样品，1μg；3，BSA，1μg
图11：抗体4G10的动力学特征参数检测结果。
图12：抗体4G10H1L1的动力学特征参数检测结果。
图13：抗体4G10H3L3的动力学特征参数检测结果。
图14：抗体4G10H4L3的动力学特征参数检测结果。
图15：抗体14C12的动力学特征参数检测结果。
图16：抗体14C12H1L1的动力学特征参数检测结果。
图17：CTLA4和抗体BIAb001的动力学特征参数检测结果。
图18：CTLA4和抗体BIAb002的动力学特征参数检测结果。
图19：CTLA4和抗体BIAb003的动力学特征参数检测结果。
图20：CTLA4和抗体BIAb004的动力学特征参数检测结果。
图21：CTLA4和抗体BIAb007的动力学特征参数检测结果。
图22：PD-1和抗体BIAb001的动力学特征参数检测结果。
图23：PD-1和抗体BIAb002的动力学特征参数检测结果。
图24：PD-1和抗体BIAb003的动力学特征参数检测结果。
图25：PD-1和抗体BIAb004的动力学特征参数检测结果。
图26：PD-1和抗体BIAb007的动力学特征参数检测结果。
图27：PD-1和抗体BIAb010的动力学特征参数检测结果。
图28：采用间接ELISA方法检测抗体4G10H1L1和4G10H3L3与抗原CTLA4的结合。
图29：采用竞争ELISA方法检测抗体4G10H1L1和4G10H3L3与B7竞争结合抗原CTLA4的结合活性。
图30：采用间接ELISA方法检测抗体14C12、14C12H1L1与PD-1的结合。
图31：采用竞争ELISA方法检测抗体14C12、14C12H1L1与PDL1的竞争结合PD-1。
图32：采用间接ELISA方法检测抗体BiAb001、BiAb002、BiAb003和BiAb004与CTLA4的结合。
图33：采用间接ELISA方法检测抗体BiAb001、BiAb002、BiAb003和BiAb004与PD-1的结合。
图34：采用竞争ELISA方法检测抗体BiAb001、BiAb002、BiAb003和BiAb004与B7竞争结合CTLA4。
图35：采用竞争ELISA方法检测抗体BiAb001、BiAb002、BiAb003和BiAb004与PDL1竞争结合PD-1。
图36：抗体4G10H1L1与293T-CTLA4细胞表面蛋白CTLA4的结合EC50。
图37：抗体4G10H3L3与293T-CTLA4细胞表面蛋白CTLA4的结合EC50。
图38：抗体14C12H1L1与293T-PD-1细胞表面蛋白PD-1结合EC50。
图39：抗体BiAb001与293T-CTLA4细胞表面蛋白CTLA4结合EC50。
图40：抗体BiAb002与293T-CTLA4细胞表面蛋白CTLA4结合EC50。
图41：抗体BiAb003与293T-CTLA4细胞的表面蛋白CTLA4结合EC50。
图42：抗体BiAb004与293T-CTLA4细胞的表面蛋白CTLA4结合EC50。
图43：抗体BiAb001与293T-PD-1细胞的表面蛋白PD-1结合EC50。
图44：抗体BiAb002与293T-PD-1细胞的表面蛋白PD-1结合EC50。
图45：抗体BiAb003与293T-PD-1细胞的表面蛋白PD-1结合EC50。
图46：抗体BiAb004与293T-PD-1细胞的表面蛋白PD-1结合EC50。
图47：抗体4G10H3L3与T细胞表面抗原CTLA4的结合活性。
图48：抗体14C12H1L1与T细胞表面抗原PD-1的结合活性。
图49：抗体BiAb003和BiAb004与T细胞的结合活性，并与抗体14C12H1L1和4G10H3L3进行对比。
图50：抗体4G10H1L1、4G10H3L3对混合淋巴细胞的细胞因子IFN-γ分泌的影响。
图51：抗体14C12H1L1对混合淋巴细胞的细胞因子IFN-γ分泌的影响。
图52：抗体BiAb001、BiAb002对混合淋巴细胞的细胞因子IFN-γ分泌的影响，并与抗体14C12H1L1、4G10H1L1进行对比。
图53：抗体BiAb003、BiAb004对混合淋巴细胞的细胞因子IFN-γ分泌的影响，并与抗体14C12H1L1、4G10H3L3进行对比。
图54：抗体4G10H3L3对混合淋巴细胞细胞因子IL-2分泌的影响。
图55：抗体14C12H1L1对混合淋巴细胞的IL-2分泌的影响。
图56：抗体BiAb003、BiAb004对混合淋巴细胞的细胞因子IL-2分泌的影响，并与抗体14C12H1L1、4G10H3L3进行对比。
图57：抗体4G10H1L1和4G10H3L3对混合PBMC、MDA-MB-231和Raji细胞共培养所诱导的细胞因子IL-2的分泌的影响。
图58：抗体14C12H1L1对混合PBMC、MDA-MB-231和Raji细胞共培养所诱导的细胞因子IL-2的分泌的影响。
图59：抗体BiAb001、BiAb002、BiAb003、BiAb004对混合PBMC、MDA-MB-231和Raji细胞共培养所诱导的细胞因子IL-2的分泌的影响，并与抗体4G10H1L1、4G10H3L3和14C12H1L1进行比较。
图60：抗体BiAb004对PD-1HuGEMM MC38荷瘤鼠的肿瘤体积的影响。
关于生物材料保藏的说明：
杂交瘤细胞株LT002(CTLA4-4G10)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C201587，保藏地址为中国.武汉.武汉大学，邮编：430072。
杂交瘤细胞株LT003(PD-1-14C12)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015105，保藏地址为中国.武汉.武汉大学，邮编：430072。
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。
在下面的实施例中，使用的T细胞来自中山康方生物医药有限公司；使用的BALB/C小鼠购自广东省医学实验动物中心；使用的PD-1HuGEMM小鼠来源于南京银河生物医药有限公司；使用的MC38细胞来源于上海复旦IBS细胞资源中心。
实施例1：抗CTLA4的抗体4G10的制备
1.杂交瘤细胞株LT002的制备
制备抗CTLA4抗体所用的抗原CTLA4-mFc是人CTLA4(GenbankID：NP_005205.2)胞外区与鼠IgG1Fc的融合蛋白。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，参考目前已确立的方法(例如，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TEV蛋白酶酶切融合蛋白CTLA4-mFc，并过柱纯化获得CTLA4蛋白。用CTLA4蛋白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杂交瘤细胞株LT002(CTLA4-4G10)，其分泌的单克隆抗体命名为4G10。
杂交瘤细胞株LT002(CTLA4-4G10)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C201587，保藏地址为中国.武汉.武汉大学，邮编：430072。
2.抗CTLA4的抗体4G10的制备
用含10％的低IgG胎牛血清的IMDM培养基对上面制得的LT002细胞株进行培养(IMDM培养基，内含1％青链霉素，于5％CO2，37℃细胞培养箱中进行培养)，7天后收集细胞培养上清，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，制得抗体4G10。纯化后4G10样品进行SDS-PAGE电泳检测，结果如图1所示。
实施例2：抗CTLA4的抗体4G10的序列分析
抗体4G10的序列分析
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中培养的LT002细胞株中提取mRNA。
按照Invitrogen
Figure PCTCN2017098466-appb-000001
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，具体操作参考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：
重链可变区的核酸序列：(372bp)
Figure PCTCN2017098466-appb-000002
Figure PCTCN2017098466-appb-000003
其编码的氨基酸序列：(124aa)
Figure PCTCN2017098466-appb-000004
轻链可变区的核酸序列：(378bp)
Figure PCTCN2017098466-appb-000005
其编码的氨基酸序列：(126aa)
Figure PCTCN2017098466-appb-000006
实施例3：抗CTLA4的人源化抗体4G10H1L1、4G10H3L3和4G10H4L3的设计 和制备
1.抗CTLA4人源化抗体4G10H1L1、4G10H3L3和4G10H4L3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4G10的序列，通过计算机模拟抗体模型，根据模型设计突变，得到抗体4G10H1L1、4G10H3L3和4G10H4L3的可变区序列(抗体恒定区序列，来自NCBI的数 据库，重链恒定区为Ig gamma-1chain C region，ACCESSION：P01857，轻链恒定区为Ig kappa chain C region，ACCESSION：P01834)。
设计的可变区序列如下：
(1)人源化单克隆抗体4G10H1L1的重链和轻链序列
重链可变区的核酸序列：(345bp)
Figure PCTCN2017098466-appb-000007
其编码的氨基酸序列：(115aa)
Figure PCTCN2017098466-appb-000008
轻链可变区的核酸序列：(327bp)
Figure PCTCN2017098466-appb-000009
其编码的氨基酸序列：(109aa)
Figure PCTCN2017098466-appb-000010
(2)人源化单克隆抗体4G10H3L3的重链和轻链序列
重链可变区的核酸序列：(345bp)
Figure PCTCN2017098466-appb-000011
其编码的氨基酸序列：(115aa)
Figure PCTCN2017098466-appb-000012
轻链可变区的核酸序列：(327bp)
Figure PCTCN2017098466-appb-000013
其编码的氨基酸序列：(109aa)
Figure PCTCN2017098466-appb-000014
(3)人源化单克隆抗体4G10H4L3的重链和轻链序列
重链可变区的核酸序列：(345bp)
Figure PCTCN2017098466-appb-000015
Figure PCTCN2017098466-appb-000016
其编码的氨基酸序列：(115aa)
Figure PCTCN2017098466-appb-000017
轻链可变区的核酸序列同4G10H3L3的轻链可变区序列。
2.人源化抗体4G10H1L1、4G10H3L3和4G10H4L3的制备
重链恒定区均采用Ig gamma-1chain C region，ACCESSION：P01857；轻链恒定区均采用Ig kappa chain C region，ACCESSION：P01834。
将4G10H1L1重链cDNA和轻链的cDNA、4G10H3L3的重链cDNA和轻链的cDNA、以及4G10H4L3重链cDNA和轻链的cDNA，分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-4G10H1、pUC57simple-4G10L1；pUC57simple-4G10H3、pUC57simple-4G10L3；和pUC57simple-4G10H4、pUC57simple-4G10L3。并分别亚克隆到pcDNA3.1载体中。将重组质粒转染293F细胞后收集培养液进行纯化获得人源化抗体4G10H1L1、4G10H3L3和4G10H4L3。纯化后4G10H1L1样品进行SDS-PAGE电泳检测，结果如图2所示。纯化后4G10H3L3样品进行SDS-PAGE电泳检测，结果如图3所示。
实施例4：抗PD-1的抗体14C12的制备
1.杂交瘤细胞株LT003的制备
用PD-1-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，参考目前已确立的方法(例如，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PD-1-mFc作为抗原包被酶标板，进行间接ELISA法筛选，得到分泌与PD-1特异性结合的新的抗体的杂交瘤细胞。
通过竞争ELISA筛选出能够分泌与配体PDL1-hFc(PDL1Genbank ID：NP_054862.1)竞争结合PD-1的单克隆抗体的杂交瘤细胞株，经过有限稀释法得到稳定的杂交瘤细胞株，并经过有限稀释法得到LT003稳定细胞株(PD-1-14C12)，其分泌的单克隆抗体命名为14C12。
杂交瘤细胞株LT003(PD-1-14C12)，其于2015年6月16日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015105，保藏地址为中国.武汉.武汉大学，邮编：430072。
2.抗PD-1的抗体14C12的制备
用含10％的低IgG胎牛血清的IMDM培养基对上面制得的LT003细胞株进行培养(IMDM培养基，内含1％青链霉素，于5％CO2，37℃细胞培养箱中进行培养)，7天后收集细胞培养上清进行纯化，制得抗体14C12。
实施例5：抗体14C12的序列的获得
抗体14C12的序列的获得
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4制得的杂交瘤细胞株LT003中提取mRNA。
按照Invitrogen
Figure PCTCN2017098466-appb-000018
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，具体操作参考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：
重链可变区的核酸序列：(354bp)
Figure PCTCN2017098466-appb-000019
Figure PCTCN2017098466-appb-000020
其编码的氨基酸序列：(118aa)
Figure PCTCN2017098466-appb-000021
轻链可变区的核酸序列：(318bp)
Figure PCTCN2017098466-appb-000022
其编码的氨基酸序列：(106aa)
Figure PCTCN2017098466-appb-000023
实施例6：抗PD-1的人源化抗体14C12H1L1的设计、制备和检测
1.人源化抗体14C12H1L1的轻链和重链序列的设计
根据PD-1蛋白的三维晶体结构(Shinohara T，et al.，Structure and chromosomal localization of the human PD-1 gene(PDCD1).Genomics 1995，23(3)：704-6)以及实施例5获得的抗体14C12的序列，通过计算机模拟抗体模型，根据模型设计突变，得到抗体14C12H1L1的可变区序列。
设计的可变区序列如下：
重链可变区的核酸序列：(354bp)
Figure PCTCN2017098466-appb-000024
Figure PCTCN2017098466-appb-000025
其编码的氨基酸序列：(118aa)
Figure PCTCN2017098466-appb-000026
轻链可变区的核酸序列：(321bp)
Figure PCTCN2017098466-appb-000027
其编码的氨基酸序列：(107aa)
Figure PCTCN2017098466-appb-000028
2.人源化抗体14C12H1L1的制备和SDS-PAGE电泳检测
重链恒定区采用Ig gamma-1chain C region，ACCESSION：P01857；轻链恒定区采用Ig kappa chain C region，ACCESSION：P01834。
将14C12H1L1的重链eDNA和轻链的eDNA分别克隆到pcDNA3.1载体中，获得抗体14C12H1L1的重组表达质粒。将重组质粒转染293F细胞。将293F细胞培养液纯化后进行检测。结果如图4所示，还原型蛋白样品目标蛋白大约在24.5kD和49KD处，非 还原型蛋白样品目标蛋白大约在147kD处。
实施例7：双功能抗体BiAb001、BiAb002、BiAb003、BiAb004、BiAb007及BiAb010 的重链和轻链的序列设计、表达和检测
1.序列设计
本发明中之双功能抗体BiAb001、BiAb002、BiAb003、BiAb004、BiAb007以及BiAb010的结构模式属于Morrison模式(IgG-scFv)，即在一个IgG抗体的两条重链的C端均连接另一个抗体的seFv片段，其重链和轻链的主要组成设计如下面的表1。
表1：BiAb001、BiAb002、BiAb003、BiAb004、BiAb007及BiAb010的重链和轻链的组成设计
Figure PCTCN2017098466-appb-000029
上面的表1中：
(1)右下角标注“V”的，是指相应重链的可变区或者相应轻链的可变区。没有标注“V”的，相应重链或者轻链为包含恒定区的全长。这些可变区或者全长的氨基酸序列及其编码核酸序列均参照上面的实施例中记载的相应序列。
(2)Linker1的氨基酸序列为(GGGGS)3(SEQ ID NO：23)
Linker2的氨基酸序列为(GGGGS)4(SEQ ID NO：24)
(3)8D2H14L2的重链可变区(8D2H14V)的氨基酸序列：
Figure PCTCN2017098466-appb-000030
编码8D2H14V的核酸序列：
Figure PCTCN2017098466-appb-000031
(4)8D2H14L2的轻链可变区(8D2L2V)的氨基酸序列：
Figure PCTCN2017098466-appb-000032
编码8D2L2V的核酸序列：
Figure PCTCN2017098466-appb-000033
2.抗体BiAb001的表达和纯化
分别将BiAb001的重链cDNA序列和轻链的cDNA序列克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-BiAb001H和pUC57simple-BiAb001L质粒。
分别将质粒pUC57simple-BiAb001H和pUC57simple-BiAb001L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、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS。
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。BiAb001的电泳图如图5所示，还原型蛋白样品目标蛋白在23.6kD和75.8kD处，非还原型蛋白样品(单个抗体)目标蛋白在199kD处。
3.抗体BiAb002、BiAb003、BiAb004、BiAb007和BiAb010的表达和纯化
按照上述BiAb001的表达和纯化方法获得纯化的抗体BiAb002，BiAb003、BiAb004、BiAb007和BiAb010。
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。BiAb002、BiAb003、BiAb004、BiAb007和BiAb010的电泳图分别如图6、图7、图8、图9和图10所示，还原型蛋白样品目标蛋白在23.6kD和75.8kD处，非还原型蛋白样品(单个抗体)目标蛋白在199kD处。
实施例8：抗体的动力学参数测定
使用Fortebio分子相互作用仪测定抗原抗体结合的动力学参数。
1.抗体4G10及其人源化抗体4G10H1L1、4G10H3L3、4G10H4L3与抗原CTLA4结合的动力学参数测定
1.1.用TEV蛋白酶酶切CTLA4-mFc蛋白，并过柱纯化获得CTLA4抗原。
1.2.抗体4G10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268.1，134.1，67，33.5，16.8，8.38，4.19，0nM，于PBST中解离。人源化4G10H1L1、4G10H3L3、4G10H4L3的检测方法与4G10类似，抗原浓度为180、90、45、22.5、11.25、5.625、2.813、0nM。
1.3抗体4G10及其人源化抗体4G10H1L1、4G10H3L3、4G10H4L3与抗原结合的动力学参数检测结果如下表1，动力学特征参数检测结果分别如图11、图12、图13和图14所示。
2.抗体14C12及其人源化抗体14C12H1L1与抗原PD-1结合的动力学参数测定
2.1.用TEV蛋白酶酶切PD-1-mFc蛋白，并过柱纯化获得PD-1抗原。
2.2.抗原PD-1(抗原浓度为1μg/ml)经生物素标记后固定于SA传感器表面，于PBST中平衡后，分别与抗体14C12和14C12H1L1结合，抗体用PBST从200nM往下三倍稀释，于PBST中解离。
2.3抗体14C12和14C12H1L1与抗原结合的动力学参数检测结果如下表1，动力学特征参数检测结果分别如图15，图16所示。
3.抗体BiAb001、BiAb002、BiAb003、BiAb004、BiAb007和BiAb010与抗原CTLA4结合的动力学参数测定
3.1使用抗原CTLA4(抗原浓度为1μg/ml)经生物素标记后固定于SA传感器表面，于PBST中平衡后，分别与抗体BiAb001、BiAb002、BiAb003、BiAb004、BiAb007和BiAb010结合，抗体用PBST从200nM往下三倍稀释，于PBST中解离。
3.2抗体BiAb001、BiAb002、BiAb003、BiAb004、BiAb007和BiAb010与抗原CTLA4结合的动力学参数检测结果如下表1，动力学特征参数检测结果分别如图17-图21所示。
4.抗体BiAb001、BiAb002、BiAb003、BiAb004、BiAb007和BiAb010与抗原PD-1结合的动力学参数测定
4.1使用抗原PD-1(抗原浓度为1μg/ml)经生物素标记后固定于SA传感器表面，于PBST中平衡后，分别与抗体BiAb001、BiAb002、BiAb003、BiAb004、BiAb007和BiAb010结合，抗体用PBST从200nM往下三倍稀释，于PBST中解离。
4.2抗体BiAb001、BiAb002、BiAb003、BiAb004、BiAb007和BiAb010与抗原PD-1结合的动力学参数检测结果如下表2，动力学特征参数检测结果分别如图22-图27所示。
表2：抗原抗体结合动力学参数
Figure PCTCN2017098466-appb-000034
Figure PCTCN2017098466-appb-000035
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：
抗体4G10及其人源化抗体均与抗原有较好的亲和力。14C12和14C12H1L1与抗原PD-1均有较好的亲和力。
双功能抗体均具有与抗原CTLA4和PD-1较好的亲和力。
实施例9：ELISA方法检测抗体与抗原的结合活性
1.人源化抗体4G10H1L1、4G10H3L3与抗原CTLA4的结合活性
1.1采用间接ELISA方法分别测定人源化抗体4G10H1L1、4G10H3L3与CTLA4的结合活性。
酶标板中加入抗原孵育，4℃过夜，用1％的BSA 37℃封闭2h后，分别加入抗体，37℃孵育30min，加入HRP标记羊抗人IgG(H+L)二抗(Jackson，109-035-088)，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
检测结果如图28所示。由图可见，人源化抗体4G10H1L1、4G10H3L3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3。通过对结合的抗体进行荧光定量分析，曲线模拟计算获得抗体4G10H1L1、4G10H3L3的结合效率EC50分别为0.048和0.067nM。
表3：间接ELISA检测4G10H1L1、4G10H3L3与CTLA4的结合
Figure PCTCN2017098466-appb-000036
Figure PCTCN2017098466-appb-000037
1.2.采用竞争ELISA方法分别检测人源化抗体4G10H1L1，4G10H3L3与B7竞争结合抗原CTLA4的结合活性
用B7/1-hFc(B7/1Genbank ID：NP_005182.1)包被酶标板4℃过夜，1％BSA封闭2h后加入抗体分别和CTLA4-mFc的抗原抗体混合液(稀释浓度见表4)，37℃孵育30min后加入酶标二抗孵育1h，37℃孵育30min。在酶标仪上检测450nm的吸光值(见表4)。
检测抗体与与B7-1竞争结合抗原CTLA4的结果如图29所示。由图可见，抗体4G10H1L1和4G10H3L3能有效地与B7-1竞争结合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。通过对结合的抗体4G10H1L1和4G10H3L3进行荧光定量分析，曲线模拟结合效率EC50分别为1.297nM和1.229nM。
表4：竞争ELISA检测4G10H1L1、4G10H3L3与B7竞争结合人CTLA4的结合效率
Figure PCTCN2017098466-appb-000038
Figure PCTCN2017098466-appb-000039
2.ELISA方法检测单克隆抗体14C12及其人源化抗体14C12H1L1与抗原PD-1的结合活性
2.1.采用间接ELISA方法分别测定单克隆重组抗体14C12和14C12H1L1分别与PD-1的结合活性，方法具体如下：
酶标板中加入PD-1-mFc孵育，4℃过夜，用1％的BSA 37℃封闭2h后，分别加入抗体，37℃孵育30min，加入HRP标记羊抗人IgG(H+L)二抗(Jackson，109-035-088)，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
检测抗体14C12，14C12H1L1与抗原PD-1结合结果如图30所示。由图可见，抗体14C12和14C12H1L1均能有效地结合PD-1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5。通过对结合的抗体14C12和14C12H1L1进行荧光定量分析，曲线模拟14C12和14C12H1L1抗体的结合效率EC50分别为0.175nM和0.043nM。
表5：抗体14C12和14C12H1L1分别与PD-1的结合(间接ELISA)
Figure PCTCN2017098466-appb-000040
Figure PCTCN2017098466-appb-000041
2.2.采用竞争ELISA方法分别测定杂交瘤细胞产生的单克隆抗体14C12及人源化抗体14C12H1L1与PDL1的竞争结合抗原PD-1，方法具体如下：
用PD-1-hFc或PD-1-mFc包被酶标板4℃过夜，1％BSA封闭2h后，分别将不同浓度的抗体14C12和14C12H1L1与PDL1-hFc和PDL1-mFc混合10min(稀释浓度见表6)，37℃孵育30min后加入酶标二抗37℃孵育30min。在酶标仪上检测450nm的吸光值(见表6)。
检测抗体14C12及人源化抗体14C12H1L1与抗原PD-1结合结果如图31所示。由图可见，14C12及人源化抗体14C12H1L1均能有效地与PDL1竞争结合PD-1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6。通过对结合的抗体14C12及人源化抗体14C12H1L1进行荧光定量分析，曲线模拟抗体14C12及14C12H1L1的结合效率EC50分别为：0.853nM和0.37nM。
表6：抗体14C12及14C12H1L1与PDL1竞争结合PD-1ELISA
Figure PCTCN2017098466-appb-000042
Figure PCTCN2017098466-appb-000043
3.ELISA方法检测抗体BiAb001、BiAb002、BiAb003和BiAb004与抗原的结合活性
3.1.间接ELISA方法分别测定抗体BiAb001、BiAb002、BiAb003和BiAb004分别与抗原CTLA4的结合活性，方法参照本实施例中1.1。
检测抗体BiAb001、BiAb002、BiAb003和BiAb004与抗原PD-1结合结果如图32所示。由图可见，抗体BiAb001、BiAb002、BiAb003和BiAb004均能有效地结合PD-1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7。通过对结合的抗体BiAb001、BiAb002、BiAb003和BiAb004进行荧光定量分析，曲线模拟抗体的结合效率EC50，如下表7所示。
表7：双功能抗体分别与CTLA4的结合(间接ELISA)
Figure PCTCN2017098466-appb-000044
3.2.间接ELISA方法分别测定抗体BiAb001、BiAb002、BiAb003和BiAb004分别与抗原PD-1的结合活性，方法参照本实施例中2.1。
检测抗体BiAb001、BiAb002、BiAb003和BiAb004与抗原PD-1结合结果如图33所示。由图可见，抗体BiAb001、BiAb002、BiAb003和BiAb004均能有效地结合PD-1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7。通过对结合的抗体BiAb001、BiAb002、BiAb003和BiAb004进行荧光定量分析，曲线模拟抗体的结合效率EC50，如下表8所示。
表8：双功能抗体与CTLA4的结合(间接ELISA)
Figure PCTCN2017098466-appb-000045
3.3竞争ELISA方法分别测定抗体BiAb001、BiAb002、BiAb003和BiAb004与B7/1-hFc的竞争结合抗原CTLA4，方法参照本实施例中1.2。
检测结果如图34所示。由图可见，抗体BiAb001、BiAb002、BiAb003和BiAb004均能有效地结合抗原CTLA4，抑制CTLA4结合B7/1，并且抗体抑制CTLA4结合B7/1的效率呈剂量依赖关系，各剂量的吸光强度见表9。通过对结合的抗体BiAb001、BiAb002、BiAb003和BiAb004进行吸光强度定量分析，曲线模拟抗体的结合效率获得结合EC50(表9)。
表9：竞争ELISA检测抗体与B7/1-hFc竞争结合抗原CTLA4
Figure PCTCN2017098466-appb-000046
Figure PCTCN2017098466-appb-000047
3.4.竞争ELISA方法分别测定抗体BiAb001、BiAb002、BiAb003和BiAb004与PDL1的竞争结合抗原PD-1，方法同本实施例中2.2。
检测结果如图35所示。由图可见，抗体BiAb001、BiAb002、BiAb003和BiAb004均能有效地结合抗原PD-1，抑制PD-1结合其配体PDL1，并且抗体抑制PD-1结合其配体PDL1的效率呈剂量依赖关系，各剂量的吸光强度见表10。通过对结合的抗体BiAb001、BiAb002、BiAb003和BiAb004进行吸光强度定量分析，曲线模拟抗体的结合效率获得结合EC50(表10)。
表10：竞争ELISA检测双功能抗体与PDL1的竞争结合PD-1
Figure PCTCN2017098466-appb-000048
Figure PCTCN2017098466-appb-000049
实施例10：流式细胞仪方法检测抗体与细胞表面抗原的结合活性
首先分别构建表达CTLA4、PD-1抗原的宿主细胞293T，用本发明中制备的人源化抗体分别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对细胞表面天然构象的抗原特异性的结合能力。
1.分别表达CTLA4、PD-1抗原的宿主细胞293T的构建
CTLA4或PD-1的载体pLenti6.3-CTLA4、pLenti6.3-PD-1(载体pLenti6.3购自Invitrogen公司)转染293T细胞，经筛选分别获得稳定表达CTLA4的克隆群体293T-CTLA4细胞和稳定表达PD-1的克隆群体293T-PD-1细胞。
2.抗体对细胞表面抗原的结合检测方法
采用常规胰酶消化方法上述步骤获得的表达抗原的宿主细胞，并使每个收集管细胞数为2×105，用PBS(1％BSA)配制抗体浓度梯度稀释液，冰上与表达相应抗原的293T细胞孵育2小时，每管加入100μL FITC羊抗人IgG(1∶500)冰上孵育1小时用PBS洗3次后加入300μL PBS重悬细胞，在流式细胞仪上用FITC通道检测荧光信号。
2.1抗体对细胞表面抗原的结合检测结果
人源化抗体4G10H1L1和4G10H3L3与293T-CTLA4细胞的结合结果分别如图36、图37所示。由图可见，4G10H1L1和4G10H3L3抗体能有效地结合宿主细胞293T-CTLA4表面的靶标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11。通过对结合的4G10H1L1和4G10H3L3抗体进行荧光定量分析，曲线模拟4G10H1L1和4G10H3L3抗体的结合效率EC50分别为7.58nM、10.54nM。
表11：流式细胞仪检测4G10H1L1和4G10H3L3结合CTLA4宿主细胞293T-CTLA4表面抗原的荧光强度分析
Figure PCTCN2017098466-appb-000050
Figure PCTCN2017098466-appb-000051
2.2人源化抗体14C12H1L1与293T-PD-1细胞的结合结果如图38所示。由图可见，14C12H1L1抗体能有效地结合宿主细胞293T-PD-1表面的靶标PD-1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12。通过对结合的14C12H1L1抗体进行荧光定量分析，曲线模拟14C12H1L1抗体的结合效率EC50为1.89nM。
表12：流式细胞仪检测14C12H1L1结合PD-1宿主细胞293T-PD-1表面抗原的荧光强度分析
浓度(nM) 0.01 0.1 1 5 10 50
荧光强度 8.32 20.31 174.62 579.41 686.49 669.54
2.3抗体BiAb001、BiAb002、BiAb003和BiAb004与293F-CTLA4细胞的结合结果分别如图39、图40、图41和图42所示。由图可见，抗体BiAb001、BiAb002、BiAb003和BiAb004均能有效地结合宿主细胞293F-CTLA4表面的靶标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13。通过对结合的抗体进行荧光定量分析，曲线模拟抗体的结合效率获得结合EC50(表13)。
表13：流式细胞仪检测抗体BiAb001、BiAb002、BiAb003和BiAb004结合293F-CTLA4细胞表面抗原的荧光强度分析及EC50
Figure PCTCN2017098466-appb-000052
Figure PCTCN2017098466-appb-000053
2.4抗体BiAb001、BiAb002、BiAb003和BiAb004与293T-PD-1细胞的结合结果分别如图43-图46所示。由图可见，抗体BiAb001、BiAb002、BiAb003和BiAb004均能有效地结合宿主细胞293T-PD-1表面的靶标PD-1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14。通过对结合的抗体进行荧光定量分析，曲线模拟抗体的结合效率获得结合EC50(表14)。
表14：流式细胞仪检测抗体BiAb001、BiAb002、BiAb003和BiAb004结合293T-PD-1细胞表面抗原的荧光强度分析
Figure PCTCN2017098466-appb-000054
Figure PCTCN2017098466-appb-000055
3.抗体与T细胞表面抗原CTLA4和PD-1的结合活性
采用Ficoll-Paque Plus(GE Healthcare LOT No.：171440-02)分离PBMC，从PBMC中分离得到CD4+细胞，用PHA(50μl/ml)刺激三天后用PBS洗一次，加入不同浓度的抗体，冰上孵育1.5h。孵育完成后，用PBS洗涤一次，加入FITC-标记抗人二抗IgG(Jackson immunoresearch lot.102155)。冰上避光孵育1h后，用PBS洗涤一次并用流式细胞仪检测。
其中所用对照抗体Nivolumab为抗PD-1的抗体，可以商购得到，其信息亦可参考http：//www.drugbank.ca/drugs/DB09035；
对照抗体Ipilimumab为抗CTLA4的抗体，可以商购得到，其信息亦可参考http：//www.drugbank.ca/drugs/DB06186。
3.1人源化抗体4G10H3L3与T细胞的结合结果如图47所示。由图可见，4G10H3L3抗体能有效地结合T细胞表面的靶标CTLA4蛋白，并且其结合效率呈剂量依赖关系。
3.2人源化抗体14C12H1L1与T细胞的结合结果如图48所示。由图可见，14C12H1L1抗体能有效地结合T细胞表面的靶标PD-1蛋白，并且其结合效率呈剂量依赖关系。
3.3抗体BiAb003和BiAb004与T细胞的结合与14C12H1L1和4G10H3L3对比结果如图49。由图可见，抗体BiAb003、BiAb004和14C12H1L1、4G10H3L3抗体均能有效地结合T细胞表面的靶标PD-1蛋白，并且其结合效率呈剂量依赖关系。并且抗体BiAb003、BiAb004和14C12H1L1与T细胞的结合强于抗体4G10H3L3，Nivolumab和Ipilimumab。荧光强度检测结果如表15中。
表15：抗体14C12H1L1、4G10H3L3和BiAb003和BiAb004与T细胞的结合活性
Figure PCTCN2017098466-appb-000056
Figure PCTCN2017098466-appb-000057
实施例11：混合淋巴细胞反应：细胞因子IFN-γ，IL-2的分泌
1.采用Ficoll-Paque Plus(GE Healthcare LOT No.：171440-02)分离PBMC，将分离出来的PBMC加入IL-4(Peprotech K2513，1000U/ml)和GM-CSF(Peprotech H1513，1000U/ml)诱导6天后，加入TNF-α(Peprotech G1513，200U/ml)诱导3天获得DC细胞。
PBMC中分离得到T细胞，将获得的DC细胞与T细胞按1∶10的比例混合培养，同时加入不同比例的抗体(hIgG做为对照)培养5-6天后，采用ELISA试剂盒检测IFN-γ(购自达科为公司)和IL-2(购自达科为公司)的分泌量。
DC细胞和T细胞混合培养后的IFN-γ的分泌检测结果分别如图50-图53所示。DC细胞和T细胞混合培养后的IL-2的分泌检测结果分别如图54-图56所示。
由图可见，4G10H1L1、4G10H3L3、14C12H1L1、以及双功能抗体BiAb001、BiAb002、BiAb003和BiAb004均能有效地诱导混合淋巴细胞分泌IFN-γ和IL-2。其中，抗PD-1抗体14C12H1L1在浓度为1nM和10nM时，对IFN-γ分泌的诱导效果与对照抗体Nivolumab在100nM时相当。CTLA4抗体4G10H1L1和4G10H3L3在浓度为100nM时对IFN-γ分泌的诱导效果强于对照抗体Ipilimumab(图52)。
实施例12：IL-2的分泌
将分离得到的PBMC(方法同实施例10)用PHA(上海疾控生物科技有限公司，50μl/mL)刺激3天后，96孔板中加入刺激成熟的PBMC(来源于志愿献血者，5×104cells/孔)，Raji细胞(来源于ATCC，5×104cells/孔)和MDA-MB-231细胞(来源于ATCC)(1×104cells/孔)，同时加入抗体(100nM)混合均匀共培养。3天后，采用ELISA试剂盒检测IL-2(购自达科为公司)的分泌量，具体操作按照试剂盒说明书进行。
细胞混合培养后的IL-2的分泌检测结果分别如图57、图58和图59所示。由图可见， 4G10H1L1、4G10H3L3、14C12H1L1、以及双功能抗体BiAb001、BiAb002、BiAb003和BiAb004均能有效地诱导PBMC分泌IL-2。其中，抗PD-1抗体14C12H1L1对IL-2分泌的诱导效果强于对照抗体Nivolumab(图58)，双功能抗体BiAb001、BiAb002、BiAb003和BiAb004对IL-2分泌的诱导效果与14C12H1L1+4G10H1L1或者14C12H1L1+4G10H3L3的效果相当(图59)。
实例13：抗体BiAb004对PD-1 HuGEMM小鼠MC38肿瘤模型的肿瘤生长影响
在PD-1HuGEMM小鼠(人PD-1转基因小鼠，右侧皮下接种MC38肿瘤细胞1×106/只)，当平均肿瘤体积达到约144mm3时，根据肿瘤体积将小鼠随机分入4个实验组并进行腹腔给药，每组小鼠均为8只。具体分组和给药剂量如下：
Isotype Control组(给药剂量2.67mg/kg)，
BiAb004高剂量组(给药剂量2.67mg/kg)，
BiAb004低剂量组(给药剂量0.267mg/kg)，
以上3组均按照每周给药2次，共给药5次。分组给药后，每周两次测量肿瘤大小。
结果如图60所示。
结果显示：
BiAb004高剂量组和BiAb004低剂量组的肿瘤体积在统计学上均显著小于Isotype control组(P＜0.001、＜0.05)。BiAb004低剂量组在PD-1HuGEMM小鼠MC38肿瘤模型中显示有统计学显著意义的抗肿瘤作用。
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 (25)

  1. 一种双特异性抗体，其包括：
    靶向PD-1的第一蛋白功能区，和
    靶向CTLA4的第二蛋白功能区。
  2. 根据权利要求1所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区直接连接或者通过连接片段连接；优选地，所述连接片段为(GGGGS)n，n为正整数，例如1、2、3、4、5或6。
  3. 根据权利要求1或2所述的双特异性抗体，其中，
    所述第一蛋白功能区和第二蛋白功能区独立地为免疫球蛋白或其抗原结合片段，例如半抗体、Fab、F(ab’)2或单链抗体；
    优选地，所述第一蛋白功能区为免疫球蛋白，所述第二蛋白功能区为单链抗体；或者
    优选地，所述第一蛋白功能区为单链抗体，所述第二蛋白功能区为免疫球蛋白。
  4. 根据权利要求1至3中任一权利要求所述的双特异性抗体，其中，所述第一蛋白功能区和第二蛋白功能区独立地为1个、2个或者2个以上。
  5. 根据权利要求3或4所述的双特异性抗体，其中，所述免疫球蛋白为IgG、IgA、IgD、IgE或IgM；优选为IgG。
  6. 根据权利要求3至5中任一权利要求所述的双特异性抗体，其中，所述单链抗体连接在免疫球蛋白的重链的C末端。
  7. 根据权利要求3至6中任一权利要求所述的双特异性抗体，其中，
    在本发明的一个实施方案中：
    所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：29-31的CDR， 其轻链可变区包含氨基酸序列为SEQ ID NO：32-34的CDR；和/或
    所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：35-37的CDR或者包含氨基酸序列为SEQ ID NO：35、SEQ ID NO：41和SEQ ID NO：37的CDR或者包含氨基酸序列为SEQ ID NO：42-44的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：38-40的CDR或者包含氨基酸序列为SEQ ID NO：45-47的CDR；
    或者
    在本发明的另一个实施方案中：
    所述的免疫球蛋白，其重链可变区包含氨基酸序列为SEQ ID NO：35-37的CDR或者包含氨基酸序列为SEQ ID NO：35、SEQ ID NO：41和SEQ ID NO：37的CDR或者包含氨基酸序列为SEQ ID NO：42-44的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：38-40的CDR或者包含氨基酸序列为SEQ ID NO：45-47的CDR；和/或
    所述的单链抗体，其重链可变区包含氨基酸序列为SEQ ID NO：29-31的CDR，其轻链可变区包含氨基酸序列为SEQ ID NO：32-34的CDR。
  8. 根据权利要求3至7中任一权利要求所述的双特异性抗体，其中，
    在本发明的一个实施方案中：
    所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO：16和SEQ ID NO：20；所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO：18和SEQ ID NO：22；和/或
    所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6、SEQ ID NO：10、SEQ ID NO：14和SEQ ID NO：25；所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12和SEQ ID NO：27；
    或者
    在本发明的另一个实施方案中：
    所述免疫球蛋白的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6、SEQ ID NO：10、SEQ ID NO：14和SEQ ID NO：25；所述单链抗体的轻链可变区的氨基酸序列选自SEQ ID NO：4、SEQ ID NO：8、SEQ ID NO：12和SEQ ID NO：27；和/或
    所述单链抗体的重链可变区的氨基酸序列选自SEQ ID NO：16和SEQ ID NO：20；所述免疫球蛋白的轻链可变区的氨基酸序列选自SEQ ID NO：18和SEQ ID NO：22。
  9. 根据权利要求1至8中任一权利要求所述的双特异性抗体，其中，
    所述的免疫球蛋白包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  10. 根据权利要求1至9中任一权利要求所述的双特异性抗体，其中，所述的双特异性抗体以小于大约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蛋白和/或PD-1蛋白。
  11. 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，
    所述抗体的重链可变区包含：
    氨基酸序列为SEQ ID NO：29-31的CDR、
    氨基酸序列为SEQ ID NO：35-37的CDR或者氨基酸序列为SEQ ID NO：35、SEQ ID NO：41和SEQ ID NO：37的CDR或者氨基酸序列为SEQ ID NO：42-44的CDR，和
    氨基酸序列为SEQ ID NO：32-34的CDR或者氨基酸序列为SEQ ID NO：38-40的CDR或者氨基酸序列为SEQ ID NO：45-47的CDR。
  12. 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：
    氨基酸序列为SEQ ID NO：32-34的CDR，
    氨基酸序列为SEQ ID NO：38-40的CDR，或者
    氨基酸序列为SEQ ID NO：45-47的CDR。
  13. 一种载体，其包含权利要求11和/或12所述的分离的核酸分子。
  14. 一种宿主细胞，其包含权利要求11和/或12所述的分离的核酸分子，或者权利要求13所述的载体。
  15. 制备权利要求1至10中任一权利要求所述的双特异性抗体的方法，其包括在合 适的条件下培养权利要求14的宿主细胞，以及从细胞培养物中回收所述双特异性抗体的步骤。
  16. 偶联物，其包括双特异性抗体以及偶联部分，其中，所述双特异性抗体为权利要求1至10中任一权利要求所述的双特异性抗体，所述偶联部分为可检测的标记；优选地，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。
  17. 试剂盒，其包括权利要求1至10中任一权利要求所述的双特异性抗体，或者包括权利要求16所述的偶联物；
    优选地，所述试剂盒还包括第二抗体，其特异性识别所述双特异性抗体；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。
  18. 权利要求1至10中任一权利要求所述的双特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测CTLA4和/PD-1在样品中的存在或其水平。
  19. 一种药物组合物，其包含权利要求1至10中任一权利要求所述的双特异性抗体或者权利要求16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。
  20. 权利要求1至10中任一权利要求所述的双特异性抗体或者权利要求16所述的偶联物在制备预防和/或治疗肿瘤或者贫血病的药物中的用途，或者用于诊断肿瘤或者贫血病的药物中的用途；优选地，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、肝癌、非小细胞性肺癌、卵巢癌和白血病。
  21. 权利要求1至10中任一权利要求所述的双特异性抗体或者权利要求16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；
    和/或
    阻断PD-1与PDL1结合的药物，
    调节(例如下调)PD-1活性或水平的药物，
    解除PD-1对机体免疫抑制的药物，或者
    提高T淋巴细胞中IFN-γ表达的药物。
  22. 一种在体内或体外方法，包括施加细胞或者有需求的受试者以有效量的权利要求1至10中任一权利要求所述的双特异性抗体或者权利要求16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；
    和/或
    阻断PD-1与PDL1结合的方法，
    调节(例如下调)PD-1活性或水平的方法，
    解除PD-1对机体免疫抑制的方法，或者
    提高T淋巴细胞中IFN-γ表达的方法。
  23. 根据权利要求1至10中任一权利要求所述的双特异性抗体或者权利要求16所述的偶联物，其用于预防和/或治疗肿瘤或者贫血病，或者用于诊断肿瘤或者贫血病；优选地，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、肝癌、非小细胞性肺癌、卵巢癌和白血病。
  24. 根据权利要求1至10中任一权利要求所述的双特异性抗体或者权利要求16所述的偶联物，其用于：
    检测样品中的CTLA4水平，
    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，
    激活T淋巴细胞，或者
    提高T淋巴细胞中IL-2表达；
    和/或
    阻断PD-1与PDL1结合，
    调节(例如下调)PD-1活性或水平，
    解除PD-1对机体免疫抑制，或者
    提高T淋巴细胞中IFN-γ表达。
  25. 一种预防和/或治疗肿瘤或者贫血病的方法，或者一种用于诊断肿瘤或者贫血病的方法，包括给予有需求的受试者以有效量的权利要求1至10中任一权利要求所述的双特异性抗体或者权利要求16所述的偶联物的步骤；优选地，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、肝癌、非小细胞性肺癌、卵巢癌和白血病。
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