WO2023155845A1 - 人源化抗cd28抗体及其与抗cd40的双特异性抗体 - Google Patents

人源化抗cd28抗体及其与抗cd40的双特异性抗体 Download PDF

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WO2023155845A1
WO2023155845A1 PCT/CN2023/076535 CN2023076535W WO2023155845A1 WO 2023155845 A1 WO2023155845 A1 WO 2023155845A1 CN 2023076535 W CN2023076535 W CN 2023076535W WO 2023155845 A1 WO2023155845 A1 WO 2023155845A1
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seq
variable region
chain variable
heavy chain
antibody
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PCT/CN2023/076535
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English (en)
French (fr)
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赵阳兵
刘晓军
曹庆娟
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上海优替济生生物医药有限公司
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Priority to CN202380022165.6A priority Critical patent/CN118696062A/zh
Publication of WO2023155845A1 publication Critical patent/WO2023155845A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application requires a PCT application with application number PCT/CN2022/076506 and a filing date of February 16, 2022, a PCT application with application number PCT/CN2022/076503 and a filing date of February 16, 2022, and a PCT application with application number of PCT/CN2022/076504, the priority of the PCT application with a filing date of February 16, 2022, is hereby incorporated by reference in its entirety.
  • the present invention relates to antibodies, specifically, the present invention relates to a humanized anti-CD28 antibody and its bispecific antibody with anti-CD40.
  • T helper cells T-helper cells, T H cells
  • B cell clones activate certain B cell clones to proliferate and differentiate into plasma cells, which then produce and secrete antibodies against foreign proteins.
  • Antibodies bind to cells expressing foreign proteins, targeting these cells for killing by immune effector cells [1].
  • T lymphocytes for activation, proliferation and functional differentiation requires binding to two receptors: (1) receptors that can specifically recognize antigens; (2) CD28 molecules.
  • Cluster of differentiation 28 (CD28) is a membrane protein expressed on most resting T cells, 44kDa, which can bind to CD80 (B7.1) and CD86 (B7.2) proteins on the surface of APC cells[2], for T cell activation and proliferation provide co-stimulatory signals.
  • CD80 B7.1
  • CD86 B7.2
  • CD28 binding lowers the T cell activation threshold and leads to enhanced TCR signaling events that are required for efficient cytokine production (through enhanced transcriptional activity and messenger RNA stability), cell cycle progression, survival, metabolic regulation, and T cell responses. required.
  • CD28 is a key factor in the organization of the immune synapse (IS), where CD28 enhances intimate contacts between T cells and APCs.
  • the CD28-specific monoclonal antibody (mAb) 9.3 can induce T cell proliferation without co-stimulation, that is, CD28-specific mAb activates resting T lymphocytes independent of antigen receptor binding [3,4].
  • 9.3 Antibodies are derived from mouse hybridoma cells [4], and are highly immunogenic in humans. Philip Tan et al. used CDR transplantation and the method of pairing the highly variable loop region and the human V gene to humanize the 9.3 antibody [5], and grafted the CDR of the mouse antibody into the human framework, which greatly reduced the Immunogenicity of chimeric antibodies. The antigen-binding ability of the humanized 9.3 chimeric antibody was reduced.
  • T helper cells T-helper cells, T H cells
  • B cell clones activate certain B cell clones to proliferate and differentiate into plasma cells, which then produce and secrete antibodies against foreign proteins.
  • Antibodies bind to cells expressing foreign proteins, targeting these cells for killing by immune effector cells [1].
  • T H cells and B cells will stimulate B cell proliferation and immunoglobulin (Ig) switching from IgM to IgG, IgA or IgE.
  • Ig immunoglobulin
  • the CD40/CD40L receptor-ligand interaction plays an important role in mediating the contact between T H cells and B cells.
  • CD40 is a costimulatory member of the tumor necrosis factor receptor (TNFR) superfamily.
  • Human CD40 is a type I transmembrane glycoprotein consisting of 277 amino acids with a molecular weight of 30,600. It is structurally divided into signal peptide, extracellular segment, transmembrane region and intracellular segment. Like other members of the TNFR superfamily, CD40 forms trimers with higher-order protein clustering properties required for optimal signaling.
  • Human CD40L is a member of the tumor necrosis factor (tumor necrosis factor, TNF) superfamily and belongs to type II transmembrane glycoproteins. CD40L was originally found on the surface of activated CD4+ T cells, and the CD40-CD40L interaction between B and T cells is also critical for germinal center (GC) responses.
  • GC germinal center
  • CD40 Function of CD40 on B cells.
  • CD40 promotes the activation and proliferation of B cells together with cytokines or other stimuli.
  • CD40 stimulation results in upregulation of CD80 and CD86 on human peripheral blood B cells, naive, memory and germinal center B cells.
  • CD40 signaling also induces upregulation of CD95/Fas and major histocompatibility complex class II (MHCII) on human B cells.
  • MHCII major histocompatibility complex class II
  • CD40 signaling promotes the next progression of activation through the cell cycle and B cell expansion.
  • CD40 also stimulates rapid proliferation of circulating and tissue-resident B cells in combination with anti-CD20, IL-4, or IL-21.
  • CD40 is also expressed on other hematopoietic cells, such as monocytes and dendritic cells, which can promote the growth of these cells and the release of cytokines, and can also release signals to induce the activation of CD4+ T cells expressing CD40L , proliferation and cytokine production.
  • CD40 signaling also affects the function of non-hematopoietic cells, including endothelial cells, epithelial cells, fibroblasts, and neurons.
  • CD40L is also widely expressed on a variety of cells, including mast cells, basophils, B cells, NK cells, macrophages, megakaryocytes, and platelets [6].
  • the CD40 signaling pathway has a wide range of roles in cell biology. Although CD40 is considered the most important binding protein of CD40L, CD40L also binds other proteins of the integrin family.
  • CD40 plays a broad role in immune regulation by mediating the interaction of T cells with B cells and other cells.
  • Agonistic CD40 antibodies can be used as immunostimulants to enhance an individual's immune response.
  • Several anti-CD40 antibodies are currently in development with less activity.
  • the tumor microenvironment is composed of blood vessels, myeloid-derived suppressor cells (MDSCs), antigen-presenting cells (APCs), lymphocytes, neutrophils, tumor-associated macrophages (TAMs), fibroblasts, extracellular It is composed of stroma, cytokines and growth factors, and is closely related to the occurrence and metastasis of tumors.
  • the tumor microenvironment participates in the expulsion of T cells out of the tumor margin, inhibits the accumulation of T cells in the tumor, and inhibits the proliferation of T cells [7].
  • Bispecific antibody (bispecific antibody, referred to as double antibody, BsAb) can simultaneously bind two different antigen epitopes, bridge two types of cells carrying different antigens, redirect effector T cells to tumor cells, and promote the formation of effector T cells. Function.
  • the double antibody in this article is composed of CD28-targeting scFv and CD40-targeting scFv, which is also called BiTE (bispecific T-cell engagers, bispecific T cell repositioning antibody).
  • CD28-scFv acts as an agonist for the CD28 molecule
  • CD40-scFv acts as an agonist for the CD40 molecule. Therefore, the CD28-CD40 double antibody can simultaneously activate the CD28 and CD40 signaling pathways and play an important role in the immune response.
  • CD40 signaling affects the differentiation of CD4 + T cells into effector cells.
  • CD4 + T cells transiently express CD40L (CD40 ligand), CD40L interacts with CD40 on other cells to cause CD40L degradation, and soluble CD40L is released outside the cell.
  • the expression of CD40L depends on the co-stimulatory molecule CD28.
  • the combination of CD28 and its ligands CD80 and CD86 can induce the complete activation and differentiation of T cells.
  • the combination of CD80 and CD86 on APCs is promoted.
  • the combination of CD28 and CD80/CD86 will upregulate CD40L, thereby producing a positive feedback effect, and the two synergistically drive T cell activation and dendritic cell maturation [6].
  • CD40 signaling affects dendritic cells and macrophages.
  • Dendritic cells as antigen-presenting cells, present antigens to T cells through the interaction between major histocompatibility complex (MHC) and T cell receptor (TCR), causing effector T cells to function.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • CD28-CD40 double antibody activates CD40 on dendritic cells, causing the expression of MHC and co-stimulatory molecules to up-regulate, thereby making dendritic cells mature, activate and pre-activate CD8 T cells.
  • the CD40 downstream signaling pathway activates the MAPK and NFKb signaling pathways through TRAF6, thereby promoting the survival, maturation and cytokine release of dendritic cells.
  • macrophages may enhance the immune response by secreting IL-10, IDO and TGF-beta.
  • CD40 signaling affects NK cells.
  • NK cells can be directly activated through their own CD40L/CD40 interaction, thereby promoting NK cell proliferation, activation and killing of target cells.
  • CD40 pathway activation can increase the killing function of NK cells [8].
  • CD40 signaling pathway in granulocytes also express CD40 under different circumstances.
  • the CD40 pathway can promote the survival of human peripheral blood eosinophils and the release of macrophage colony-stimulating factor (GMCSF).
  • Eosinophils can also act as antigen-presenting cells, activating numerous T-cell helper responses (pro-inflammatory and anti-inflammatory).
  • the CD28-CD40 double antibody not only provides co-stimulatory signals for T cell activation, but also stimulates the activation and function of various immune cells (such as dendritic cells, macrophages, NK cells, granulocytes, etc.), strongly enhancing immune response.
  • various immune cells such as dendritic cells, macrophages, NK cells, granulocytes, etc.
  • One aspect of the present invention provides a humanized anti-CD28 antibody or an antigen-binding fragment thereof, the humanized anti-CD28 antibody
  • the CD28 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein,
  • HCDR1 comprises SEQ ID NO: 1,
  • HCDR2 comprises a sequence selected from SEQ ID NO: 2, 17 and 18,
  • HCDR3 comprises SEQ ID NO: 3,
  • LCDR1 comprises SEQ ID NO:9
  • LCDR2 comprises SEQ ID NO: 10, and
  • LCDR3 comprises SEQ ID NO: 11;
  • the EC50 of the humanized anti-CD28 antibody binding to human CD28 determined by ELISA is in the range of 0.008 ⁇ g/mL to 0.016 ⁇ g/mL; or the humanized anti-CD28 antibody is detected by biofilm layer interference (BLI)
  • the determined equilibrium dissociation constant KD for binding to human CD28 is in the range of 6.1 ⁇ 10 -9 M to 1.2 ⁇ 10 -8 M.
  • the heavy chain variable region of the humanized anti-CD28 antibody includes HFR1, HFR2, HFR3, and HFR4, and the light chain variable region of the humanized anti-CD28 antibody includes LFR1, LFR2, LFR3, and LFR4, where,
  • HFR1 comprises a sequence selected from SEQ ID NO: 19-22,
  • HFR2 comprises a sequence selected from SEQ ID NO: 23-26,
  • HFR3 comprises selected from SEQ ID NO:27-30,
  • HFR4 comprises the sequence shown in SEQ ID NO:7,
  • LFR1 comprises a sequence selected from SEQ ID NO: 36-37,
  • LFR2 comprises a sequence selected from SEQ ID NO: 38-39,
  • LFR3 comprises a sequence selected from SEQ ID NO: 40-42, and
  • LFR4 comprises the sequence shown as SEQ ID NO: 15.
  • HFR1, HFR2, HFR3, and HFR4 included in the heavy chain variable region of the humanized anti-CD28 antibody are selected from the group consisting of:
  • HFR1 comprising a sequence as shown in SEQ ID NO:19
  • HFR2 comprising a sequence as shown in SEQ ID NO:23
  • HFR3 comprising a sequence as shown in SEQ ID NO:27, and comprising such as SEQ ID HFR4 of the sequence shown in NO:7;
  • HFR1 comprising a sequence as shown in SEQ ID NO:20
  • HFR2 comprising a sequence as shown in SEQ ID NO:24
  • HFR3 comprising a sequence as shown in SEQ ID NO:28, and comprising such as SEQ ID HFR4 of the sequence shown in NO:7;
  • HFR1 comprising a sequence as shown in SEQ ID NO:21
  • HFR2 comprising a sequence as shown in SEQ ID NO:25
  • HFR3 comprising a sequence as shown in SEQ ID NO:29, and comprising such as SEQ ID HFR4 of the sequence shown in NO:7;
  • HFR1 comprising the sequence shown in SEQ ID NO:21, comprising the sequence shown in SEQ ID NO:26 HFR2 of the sequence shown, HFR3 comprising the sequence shown in SEQ ID NO:30, and HFR4 comprising the sequence shown in SEQ ID NO:7;
  • HFR1 comprising a sequence as shown in SEQ ID NO:22
  • HFR2 comprising a sequence as shown in SEQ ID NO:26
  • HFR3 comprising a sequence as shown in SEQ ID NO:30
  • SEQ ID HFR4 of the sequence shown in NO:7.
  • the light chain variable region of the humanized anti-CD28 antibody includes LFR1, LFR2, LFR3, and LFR4 selected from the group consisting of:
  • LFR1 comprising a sequence as shown in SEQ ID NO:36
  • LFR2 comprising a sequence as shown in SEQ ID NO:38
  • LFR3 comprising a sequence as shown in SEQ ID NO:40, and comprising such as SEQ ID LFR4 of the sequence shown in NO:15;
  • LFR1 comprising the sequence shown in SEQ ID NO:37
  • LFR2 comprising the sequence shown in SEQ ID NO:39
  • LFR3 comprising the sequence shown in SEQ ID NO:41, and comprising such as SEQ ID LFR4 of the sequence shown in NO:15;
  • LFR1 comprising the sequence shown in SEQ ID NO:37
  • LFR2 comprising the sequence shown in SEQ ID NO:39
  • LFR3 comprising the sequence shown in SEQ ID NO:42
  • SEQ ID LFR4 of the sequence shown in NO:15.
  • HFR1, HFR2, HFR3, HFR4 included in the heavy chain variable region of the humanized anti-CD28 antibody and LFR1, LFR2, LFR3, LFR4 included in the light chain variable region are selected from the group consisting of:
  • HFR1 comprising the sequence shown in SEQ ID NO:19, comprising HFR2 of the sequence shown in SEQ ID NO:23, comprising HFR3 of the sequence shown in SEQ ID NO:27, comprising such as SEQ ID NO HFR4 of the sequence shown in: 7, LFR1 comprising the sequence shown in SEQ ID NO:36, LFR2 comprising the sequence shown in SEQ ID NO:38, comprising LFR3 of the sequence shown in SEQ ID NO:40 , and LFR4 comprising the sequence shown in SEQ ID NO:15;
  • HFR1 comprising the sequence shown in SEQ ID NO:19, comprising HFR2 of the sequence shown in SEQ ID NO:23, comprising HFR3 of the sequence shown in SEQ ID NO:27, comprising such as SEQ ID NO HFR4 of the sequence shown in: 7, LFR1 comprising the sequence shown in SEQ ID NO:37, LFR2 comprising the sequence shown in SEQ ID NO:39, comprising LFR3 of the sequence shown in SEQ ID NO:41 , and LFR4 comprising the sequence shown in SEQ ID NO:15;
  • HFR1 comprising the sequence shown in SEQ ID NO:20, comprising HFR2 of the sequence shown in SEQ ID NO:24, comprising HFR3 of the sequence shown in SEQ ID NO:28, comprising such as SEQ ID NO HFR4 of the sequence shown in: 7, LFR1 comprising the sequence shown in SEQ ID NO:37, LFR2 comprising the sequence shown in SEQ ID NO:39, comprising LFR3 of the sequence shown in SEQ ID NO:41 , and LFR4 comprising the sequence shown in SEQ ID NO:15;
  • HFR1 comprising a sequence as shown in SEQ ID NO:20, HFR2 comprising a sequence as shown in SEQ ID NO:24, HFR3 comprising a sequence as shown in SEQ ID NO:28, comprising as SEQ ID NO HFR4 of the sequence shown in: 7, comprising LFR1 of the sequence shown in SEQ ID NO: 37, comprising such as LFR2 of the sequence shown in SEQ ID NO:39, LFR3 comprising the sequence shown in SEQ ID NO:42, and LFR4 comprising the sequence shown in SEQ ID NO:15;
  • HFR1 comprising the sequence shown in SEQ ID NO:21
  • HFR2 comprising the sequence shown in SEQ ID NO:25
  • HFR3 of the sequence shown in SEQ ID NO:29 comprising such as SEQ ID NO HFR4 of the sequence shown in: 7
  • LFR1 comprising the sequence shown in SEQ ID NO:36
  • LFR2 comprising the sequence shown in SEQ ID NO:38
  • LFR4 comprising the sequence shown in SEQ ID NO:15;
  • HFR1 comprising a sequence as shown in SEQ ID NO:21, comprising HFR2 as a sequence as shown in SEQ ID NO:25, comprising HFR3 as a sequence as shown in SEQ ID NO:29, comprising as SEQ ID NO HFR4 of the sequence shown in: 7, LFR1 comprising the sequence shown in SEQ ID NO:37, LFR2 comprising the sequence shown in SEQ ID NO:39, comprising LFR3 of the sequence shown in SEQ ID NO:41 , and LFR4 comprising the sequence shown in SEQ ID NO:15;
  • HFR1 comprising a sequence as shown in SEQ ID NO:21
  • HFR2 comprising a sequence as shown in SEQ ID NO:25
  • HFR3 of a sequence as shown in SEQ ID NO:29 comprising such as SEQ ID NO HFR4 of the sequence shown in: 7
  • LFR1 comprising the sequence shown in SEQ ID NO:37
  • LFR2 comprising the sequence shown in SEQ ID NO:39
  • LFR4 comprising the sequence shown in SEQ ID NO:15;
  • HFR1 comprising the sequence shown in SEQ ID NO:21, comprising HFR2 of the sequence shown in SEQ ID NO:26, comprising HFR3 of the sequence shown in SEQ ID NO:30, comprising such as SEQ ID NO HFR4 of the sequence shown in: 7,
  • LFR1 comprising the sequence shown in SEQ ID NO: 37, comprising LFR2 of the sequence shown in SEQ ID NO: 39, comprising LFR2 of the sequence shown in SEQ ID NO: 41 LFR3 of the sequence, and LFR4 comprising the sequence shown in SEQ ID NO: 15;
  • HFR1 comprising the sequence shown in SEQ ID NO:21
  • HFR2 comprising the sequence shown in SEQ ID NO:26
  • HFR3 of the sequence shown in SEQ ID NO:30 comprising such as SEQ ID NO HFR4 of the sequence shown in: 7
  • LFR1 comprising the sequence shown in SEQ ID NO:37
  • LFR2 comprising the sequence shown in SEQ ID NO:39
  • LFR4 comprising the sequence shown in SEQ ID NO:15;
  • HFR1 comprising the sequence shown in SEQ ID NO:22, comprising HFR2 of the sequence shown in SEQ ID NO:26, comprising HFR3 of the sequence shown in SEQ ID NO:30, comprising such as SEQ ID NO HFR4 of the sequence shown in: 7, LFR1 comprising the sequence shown in SEQ ID NO:37, LFR2 comprising the sequence shown in SEQ ID NO:39, comprising LFR3 of the sequence shown in SEQ ID NO:41 , and LFR4 comprising the sequence shown in SEQ ID NO: 15; and
  • HFR1 comprising the sequence shown in SEQ ID NO:22
  • HFR2 comprising the sequence shown in SEQ ID NO:26
  • HFR3 comprising the sequence shown in SEQ ID NO:30
  • LFR1 comprising the sequence shown in SEQ ID NO: 37
  • LFR2 comprising the sequence shown in SEQ ID NO: 39
  • LFR3 comprising the sequence shown in SEQ ID NO: 42
  • LFR4 comprising the sequence shown in SEQ ID NO:15.
  • the heavy chain variable region of the humanized anti-CD28 antibody comprises a sequence selected from SEQ ID NO: 31-35.
  • the light chain variable region of the humanized anti-CD28 antibody comprises a sequence selected from SEQ ID NO: 43-45.
  • the heavy chain variable region and the light chain variable region of the humanized anti-CD28 antibody are selected from the following combinations:
  • the heavy chain variable region of the humanized anti-CD28 antibody is fused to an immunoglobulin heavy chain constant region, and/or the light chain variable region of the humanized anti-CD28 antibody is fused to an immunoglobulin Globulin light chain constant region.
  • the immunoglobulin heavy chain constant region comprises SEQ ID NO:81 and the immunoglobulin light chain constant region comprises SEQ ID NO:82.
  • the humanized anti-CD28 antibody is a scFv.
  • the humanized anti-CD28 scFv comprises a heavy chain variable chain linked by a peptide linker. region and light chain variable region.
  • sequence of the peptide linker in the humanized anti-CD28 scFv comprises SEQ ID NO:80.
  • variable region of the heavy chain in the humanized anti-CD28 scFv is located at the N-terminus or C-terminus of the variable region of the light chain.
  • Another aspect of the present invention provides a multispecific antibody comprising at least a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises the aforementioned humanized anti-CD28 antibody or an antigen-binding fragment thereof, and the second antigen-binding portion
  • the binding moiety binds a different antigen than the first antigen binding moiety.
  • the second antigen binding moiety specifically binds a tumor antigen or is capable of stimulating immune cells.
  • the second antigen binding moiety is an anti-CD40 antibody.
  • the first antigen binding moiety is a scFv and the second antigen binding moiety is a scFv.
  • said first antigen binding moiety is linked to said second antigen binding moiety via a peptide linker.
  • the peptide linker comprises the sequence of SEQ ID NO:79.
  • the first antigen binding moiety is N- or C-terminal to the second antigen binding moiety.
  • the multispecific antibody is a bispecific antibody.
  • the bispecific antibody comprises the first antigen-binding portion and the second antigen-binding portion, wherein the second antigen-binding portion is an anti-CD40 antibody or an antigen-binding fragment thereof, and the anti-CD40 antibody
  • the EC50 of binding to human CD40 determined by ELISA is less than 0.04919 ⁇ g/mL, or the EC50 of the anti-CD40 antibody binding to human CD40 determined by ELISA is less than 3.44 ⁇ 10 ⁇ 7 M.
  • the anti-CD40 antibody is obtained by affinity maturation of the parent antibody, and exhibits a stronger binding affinity to CD40 than the parent antibody;
  • the parent antibody has the following heavy chain variable region and A light chain variable region, the heavy chain variable region comprising: HCDR1 comprising SEQ ID NO:46, HCDR2 comprising SEQ ID NO:47 and HCDR3 comprising SEQ ID NO:48, the light chain variable region comprising : LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55 and LCDR3 comprising SEQ ID NO:56.
  • the heavy chain variable region of the anti-CD40 antibody comprises: HCDR1 comprising a sequence selected from SEQ ID NO:46 and SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47, and comprising SEQ ID NO:47 NO: 48 HCDR3.
  • the heavy chain variable region of the anti-CD40 antibody comprises:
  • HCDR1 comprising SEQ ID NO:46
  • HCDR2 comprising SEQ ID NO:47
  • HCDR3 comprising SEQ ID NO:48
  • HCDR1 comprising SEQ ID NO:62
  • HCDR2 comprising SEQ ID NO:47
  • HCDR3 comprising SEQ ID NO:48.
  • the light chain variable region of the anti-CD40 antibody comprises: LCDR1 comprising SEQ ID NO: 54, LCDR2 comprising a sequence selected from SEQ ID NO: 55, 64-66 and comprising a sequence selected from SEQ ID NO: 55, 64-66 LCDR3 of NO:56,67-68 sequence.
  • the light chain variable region of the anti-CD40 antibody comprises:
  • LCDR1 comprising SEQ ID NO:54
  • LCDR2 comprising SEQ ID NO:64
  • LCDR3 comprising SEQ ID NO:56;
  • LCDR1 comprising SEQ ID NO:54
  • LCDR2 comprising SEQ ID NO:55
  • LCDR3 comprising SEQ ID NO:67
  • LCDR1 comprising SEQ ID NO:54
  • LCDR2 comprising SEQ ID NO:55
  • LCDR3 comprising SEQ ID NO:68
  • LCDR1 comprising SEQ ID NO:54
  • LCDR2 comprising SEQ ID NO:65
  • LCDR3 comprising SEQ ID NO:56;
  • LCDR1 comprising SEQ ID NO:54
  • LCDR2 comprising SEQ ID NO:66
  • LCDR3 comprising SEQ ID NO:56.
  • the heavy chain variable region and light chain variable region of the anti-CD40 antibody comprise:
  • HCDR1 comprising SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47, HCDR3 comprising SEQ ID NO:48, LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:64 and comprising SEQ ID NO:47 LCDR3 with ID NO:56;
  • HCDR1 comprising SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47, HCDR3 comprising SEQ ID NO:48, LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55 and comprising SEQ ID NO:55 LCDR3 with ID NO:67;
  • HCDR1 comprising SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47, HCDR3 comprising SEQ ID NO:48, LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55 and comprising SEQ ID NO:55 LCDR3 with ID NO:68;
  • HCDR1 comprising SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47, HCDR3 comprising SEQ ID NO:48, LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:65 and comprising SEQ ID NO:47 LCDR3 with ID NO:56;
  • HCDR1 comprising SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47, HCDR3 comprising SEQ ID NO:48, LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:66 and comprising SEQ ID NO:47 LCDR3 with ID NO:56.
  • the heavy chain variable region of the anti-CD40 antibody comprises:
  • HFR1 comprising SEQ ID NO:49, HFR2 comprising SEQ ID NO:50, HFR3 comprising SEQ ID NO:51 and HFR4 comprising SEQ ID NO:52;
  • HFR1 comprising SEQ ID NO:63
  • HFR2 comprising SEQ ID NO:50
  • HFR3 comprising SEQ ID NO:51
  • HFR4 comprising SEQ ID NO:52.
  • the light chain variable region of the anti-CD40 antibody comprises: LFR1 comprising SEQ ID NO:57, LFR2 comprising SEQ ID NO:58, LFR3 comprising SEQ ID NO:59, and LFR3 comprising SEQ ID NO:57 :60 LFR4.
  • the heavy chain variable region of the anti-CD40 antibody has at least 70% sequence identity to a sequence selected from SEQ ID NOs: 69-73.
  • the heavy chain variable region of the anti-CD40 antibody comprises a sequence selected from SEQ ID NOs: 69-73.
  • the light chain variable region of the anti-CD40 antibody has at least 70% sequence identity to a sequence selected from SEQ ID NOs: 74-78.
  • the light chain variable region of the anti-CD40 antibody comprises a sequence selected from SEQ ID NOs: 74-78.
  • the anti-CD40 antibody is a scFv.
  • the anti-CD40 scFv comprises a heavy chain variable region and a light chain variable region connected by a peptide linker.
  • sequence of the peptide linker in the anti-CD40 scFv comprises SEQ ID NO:80.
  • the heavy chain variable region of the anti-CD40 scFv is located at the N-terminal or C-terminal of the light chain variable region.
  • Another aspect of the present invention provides a polynucleotide encoding the aforementioned humanized anti-CD28 antibody or an antigen-binding fragment thereof, or the aforementioned multispecific antibody or bispecific antibody.
  • Another aspect of the present invention provides a vector comprising the aforementioned polynucleotide.
  • the vector is a plasmid vector, a viral vector, a circular RNA comprising the polynucleotide, a precursor RNA capable of obtaining the circular RNA by circularization, or a vector comprising the precursor RNA.
  • IRS internal ribosome entry site
  • the polyA is at least 45 nucleotides in length; preferably, the polyA is at least 70 nucleotides in length.
  • Another aspect of the present invention provides a host cell comprising the aforementioned polynucleotide or the aforementioned vector.
  • Another aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned humanized anti-CD28 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned bispecific antibody, the aforementioned polynucleotide or the aforementioned carrier, and a pharmaceutically acceptable Carrier.
  • Another aspect of the present invention provides a pharmaceutical combination, comprising: a) the aforementioned humanized anti-CD28 antibody or an antigen-binding fragment thereof, the aforementioned multispecific antibody, the aforementioned bispecific antibody, or the aforementioned polynucleotide or the aforementioned carrier; and b) A combination of immune cells; or providing immune cells expressing the aforementioned humanized anti-CD28 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, and the aforementioned bispecific antibody.
  • the immune cells are T cells, NK cells, NKT cells, macrophages, neutrophils, or granulocytes.
  • the immune cells recombinantly express a chimeric antigen receptor (CAR), T cell receptor (TCR), or bispecific T cell engager (BiTE), wherein the CAR, TCR, or BiTE binds to a tumor Antigen or viral antigen.
  • the immune cells are CAR-T cells.
  • the CAR targets mesothelin, CD123, BCMA, HER2, IL13Ra2, CD19, or B7H3.
  • the CAR targeting mesothelin, CD123, BCMA, HER2, IL13Ra2, CD19 or B7H3 is as defined herein.
  • Another aspect of the present invention provides a method for enhancing the killing effect of immune cells on target cells in a subject, comprising administering the aforementioned humanized anti-CD28 antibody or an antigen-binding fragment thereof, the aforementioned The multispecific antibody, the aforementioned bispecific antibody, the aforementioned polynucleotide, the aforementioned carrier, or the aforementioned pharmaceutical composition.
  • the immune cells are as defined above.
  • the present invention also provides the aforementioned humanized anti-CD28 antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody, the aforementioned bispecific antibody, the aforementioned polynucleotide, the aforementioned carrier, or the aforementioned pharmaceutical composition in enhancing the effect of immune cells on the subject
  • the immune cells are as defined above.
  • Another aspect of the present invention provides a method for killing target cells in a subject, comprising administering the aforementioned drug combination or the aforementioned immune cells to the subject.
  • the target cells are tumor cells.
  • the tumor cells express mesothelin, CD123, BCMA, HER2, IL13Ra2, CD19, or B7H3.
  • the immune cells are CAR-T cells.
  • the present invention also provides the application of the above-mentioned drug combination or the above-mentioned immune cells in killing target cells in a subject, or the application in preparing target cells for killing in a subject.
  • Another aspect of the present invention provides a method for treating diseases in a subject in need, comprising administering a therapeutically effective amount of the aforementioned drug combination or the aforementioned immune cells to the subject.
  • the disease is a tumor, cancer, viral infection, or autoimmune disease.
  • the cancer expresses mesothelin, CD123, BCMA, HER2, IL13Ra2, or B7H3.
  • the cancer is a solid tumor or a hematological cancer.
  • the cancer is acute myelogenous leukemia (AML), B-type acute lymphoblastic leukemia (B-ALL), T-type acute lymphoblastic leukemia (T-ALL), B-cell precursor acute lymphoblastic leukemia (BCP-ALL) or blastic placytoid dendritic cell neoplasm (BPDCN).
  • AML acute myelogenous leukemia
  • B-ALL B-type acute lymphoblastic leukemia
  • T-ALL T-type acute lymphoblastic leukemia
  • BCP-ALL B-cell precursor acute lymphoblastic leukemia
  • blastic placytoid dendritic cell neoplasm BPDCN.
  • Non-Hodgkin's lymphoma chronic lymphocytic leukemia, acute lymphoblastic leukemia, human B-cell precursor leukemia, multiple myeloma, or malignant lymphoma.
  • the cancer is mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, breast cancer, gastric cancer, cervical cancer, urothelial cancer, esophageal cancer, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer , head and neck cancer, sarcoma, glioma, prostate cancer, thyroid cancer, or glioma.
  • Another aspect of the present invention provides the application of the aforementioned drug combination or the aforementioned immune cells in the treatment of the aforementioned diseases, or the application in the preparation of drugs for the treatment of the aforementioned diseases.
  • Another aspect of the present invention provides a method for inducing T cell proliferation, comprising combining the aforementioned humanized anti-CD28 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned bispecific antibody, the aforementioned polynucleotide, or the aforementioned carrier with T cell contact.
  • the method further includes introducing the aforementioned polynucleotide or the aforementioned vector into the T cells, and allowing the T cells to express the aforementioned humanized anti-CD28 antibody or an antigen-binding fragment thereof or the aforementioned multispecific antibody .
  • the T cells express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • Another aspect of the present invention provides the use of the aforementioned humanized anti-CD28 antibody or its antigen-binding fragment, the aforementioned multispecific antibody, the aforementioned bispecific antibody, the aforementioned polynucleotide or the aforementioned carrier in inducing T cell proliferation, or in Use in the preparation of medicines for inducing T cell proliferation.
  • Figure 1 A cartoon diagram of the structural model of the CD28 antibody 9.3 variable region.
  • the chain on the left represents the heavy chain and the chain on the right represents the light chain.
  • FIG. 1A Heavy chain mutant sequences of humanized antibodies. " ⁇ " represents the same amino acid, and the underline represents the CDR region.
  • Figure 2B Light chain mutated sequences of humanized antibodies. " ⁇ " represents the same amino acid, and the underline represents the CDR region.
  • Figure 3 SEC profiles of humanized antibodies.
  • Figure 4 ELISA results of humanized antibodies.
  • Figure 5 Sensorgrams of humanized antibodies.
  • Figure 6 Expression level of CD40 on A549 cells. 10 ⁇ g CD40 mRNA was electroporated in A549 cells, and after 24 hours, the expression level of CD40 was detected by flow cytometry and PE-anti-CD40 antibody.
  • Figure 7 Expression levels of bis-antibodies. 15 ⁇ g of double antibody mRNA and 0.5 ⁇ g of 4D5.BBZ mRNA were electroporated in T cells. After 24 hours, the expression level of double antibody was detected by flow cytometry, CD40-Fc and PE-anti-IgG-Fc antibodies.
  • Figure 8 Expression levels of Her2-CAR. 15 ⁇ g of double antibody mRNA and 0.5 ⁇ g of 4D5.BBZ mRNA were electroporated in T cells. After 24 hours, the expression level of Her2-CAR was detected by flow cytometry, Her2-Fc and PE-anti-Fc antibodies.
  • Figure 10 Pro-killing function.
  • Figure 11 Expression level of CD40 on A549 cells. 10 ⁇ g CD40 mRNA was electroporated in A549 cells, and after 24 hours, the expression level of CD40 was detected by flow cytometry and PE-anti-CD40 antibody.
  • FIG. 12 Meso expression levels on A549 cells. 10 ⁇ g Meso mRNA was electroporated in A549 cells, and after 24 hours, the expression level of Meso was detected by flow cytometry, Meso-biotin and PE-streptavidin antibodies.
  • Figure 13 Expression levels of bis-antibodies. 15 ⁇ g of double antibody mRNA and 2 ⁇ g of M12 mRNA were electroporated in T cells. After 24 hours, the expression level of double antibody was detected by flow cytometry, CD40-Fc and PE-anti-Fc antibody.
  • Figure 14 Expression levels of Meso-CAR. 15 ⁇ g of double antibody mRNA and 2 ⁇ g of M12 mRNA were electroporated in T cells. After 24 hours, the expression level of Meso-CAR was detected by flow cytometry, Meso-Fc and PE-anti-Fc antibody.
  • Figure 17 Detection of gene overexpression levels of CD40, Her2-CAR and double antibodies.
  • A CD40 expression level on A549 cells.
  • B Expression levels of Her2-CAR and bsAbs.
  • Figure 18 ELISA detection of IL-2 and IFN-gamma release levels of target cells under the action of Her2-CAR-T (with or without co-electroporation of BsAb).
  • Figure 19 The incucyte S3 live cell dynamic imaging and analysis system detects the killing function of Her2-CAR-T (with or without co-electroporation of BsAb) on target cells.
  • Figure 20 Detection of gene overexpression levels of CD40, Meso-CAR and double antibodies.
  • A Expression levels of Meso and CD40 on A549 cells.
  • B Expression levels of Meso-CAR and bsAbs.
  • Figure 21 ELISA detection of IL-2 and IFN-gamma release levels of target cells under the action of Meso-CAR-T (co-electroporation of BsAb).
  • Figure 22 The incucyte S3 live cell dynamic imaging and analysis system detects the killing effect of Meso-CAR-T (co-electroporated BsAb) on target cells.
  • Figure 23 Detection of gene overexpression levels of CD40, Her2-CAR and 9.3-40.52 optimized double antibody.
  • A CD40 expression level on A549 cells.
  • B Expression levels of Her2-CAR.
  • C 9.3-40.52 optimized the expression level of double antibody BsAb.
  • Figure 24 ELISA detection of IFN-gamma release level of target cells under the action of Her2-CAR-T (with or without co-electroporation 9.3-40.52 optimized BsAb).
  • Figure 25 The incucyte S3 live cell dynamic imaging and analysis system detects the killing function of Her2-CAR-T (with or without co-electroporation 9.3-40.52 optimized BsAb) on target cells.
  • Figure 26 Detection of gene overexpression levels of CD40, Meso-CAR and 9.3-40.52 optimized double antibody.
  • A Expression levels of Meso and CD40 on A549 cells.
  • B Expression levels of Meso-CAR and 9.3-40.52 optimized bis-antibodies.
  • Figure 27 IL-2 and IFN-gamma release levels of target cells under the action of Meso-CAR-T (with or without co-electroporation 9.3-40.52 optimized BsAb).
  • Figure 28 The incucyte S3 live cell dynamic imaging and analysis system detects the pro-killing function of Meso-CAR-T (with or without co-electroporation 9.3-40.52 optimized BsAb) on target cells.
  • Figure 29 Detection of gene overexpression levels of CD40, Her2-CAR and 9.3-A40C optimized double antibodies.
  • A CD40 expression level on A549 cells.
  • B Expression levels of Her2-CAR and 9.3-A40C optimized bis-antibodies.
  • Figure 30 The incucyte S3 live cell dynamic imaging and analysis system detects the killing function of Her2-CAR-T (with or without co-electroporation 9.3-A40C optimized BsAb) on target cells.
  • Figure 31 Detection of gene overexpression levels of CD40, Meso-CAR and 9.3-A40C optimized double antibodies.
  • A Expression levels of Meso and CD40 on A549 cells.
  • B Expression levels of Meso-CAR and 9.3-A40C optimized bis-antibodies.
  • Figure 32 IFN-gamma release level of target cells under the action of Meso-CAR-T (with or without co-electroporation 9.3-A40C optimized BsAb).
  • Figure 33 The incucyte S3 live cell dynamic imaging and analysis system detects the pro-killing function of the double antibody of Meso-CAR-T (with or without co-electroporation 9.3-A40C optimized BsAb) on target cells.
  • Figure 34 Affinity ELISA results of F2.103 ⁇ 40.52 ⁇ A40C-CD40 antibody.
  • Figure 35 Sequences of affinity matured antibodies. " ⁇ " represents the same amino acid, and the underline represents the CDR region. (A) Sequence alignment results of VH regions of affinity matured antibodies. (B) Sequence alignment of VL regions of affinity matured antibodies.
  • Figure 36 Results of phage ELISA.
  • Figure 37-A Expression level of CD40 on A549 cells.
  • Figure 37(B-D) Expression levels of Her2-CAR and AM40.52-9.3h11 bis-antibody.
  • Figure 38 ELISA detection of IL-2 and IFN-gamma release levels of target cells under the action of Her2-CAR-T (with or without co-electroporation of AM40.52-9.3h11BsAb).
  • Figure 40 Detection of gene overexpression levels.
  • A Expression of Meso and CD40 on Molm14 cells level.
  • B Expression levels of Meso-CAR and AM40.52-9.3h11 double antibody.
  • Figure 41 ELISA detection of IL-2 and IFN-gamma release levels of target cells under the action of Meso-CAR-T (with or without co-electroporation of AM40.52-9.3h11BsAb).
  • Figure 42 The incucyte S3 live cell dynamic imaging and analysis system detects the killing function of Meso-CAR-T (with or without co-electroporation AM40.52-9.3h11BsAb) on target cells.
  • Figure 43 ELISA detection of IL-2 and IFN-gamma release levels of target cells under the action of Meso-CAR-T (with or without co-electroporation of AM40.52-9.3h11BsAb).
  • Figure 44 The incucyte S3 live cell dynamic imaging and analysis system detects the specificity of bis-antibodies, and the expression abundance of Mesothelin and CD40 on the surface of different tumor cells (F).
  • Figure 45 SDS-PAGE profiles of affinity matured antibodies under non-reducing (A) and reducing (B) conditions.
  • Figure 46 ELISA results for affinity matured antibodies.
  • Figure 47 SPR fitting curves for 40.52 antibody (A) and 4052.17 antibody (B).
  • Figure 48 Transduction efficiency of lentivirus in CAR-T cells.
  • Figure 49 Killing results of lentivirus-transduced CAR-T cells.
  • Figure 50 Cytokines released by lentivirus-transduced CAR-T cells.
  • Figure 51 The killing results of lentivirus-transduced CAR-T cells on different tumor cells.
  • Figure 52 Cytokines released by lentivirus-transduced CAR-T cells to different tumor cells.
  • FIG. 53 Animal experiment results of low-dose CAR-T reinfusion.
  • A Quantitative map of tumors in mice
  • B statistical tumor fluorescence value
  • C tumor size in mice
  • D body weight changes in mice
  • E survival curve of mice.
  • FIG. 54 Animal experiment results of high-dose CAR-T reinfusion.
  • A Quantitative map of tumors in mice
  • B statistical tumor fluorescence value
  • C tumor size in mice
  • D body weight changes in mice
  • E survival curve of mice.
  • Figure 55 provides 96-well plate reads for two anti-human mesothelin-Fc monoclonal phage ELISAs.
  • Figure 56 provides a schematic of the pDA-CAR vector used to generate anti-mesothelin CAR mRNA.
  • Figure 57 provides the results of FACS staining showing the binding of anti-mesothelin scFv expressed in CART cells to mesothelin-Fc protein.
  • Figure 58 provides FACS staining by anti-mesothelin antibody of A549 cells electroporated with different amounts of mesothelin mRNA.
  • Figure 63 provides FACS staining of isotype control and anti-mesothelin mAbs for OVCAR3, H226, ASPCl, A549 and HCC70.
  • Figure 64 provides CD107a staining of anti-mesothelin M12 and M32 CART cells in co-culture and killing assays with OVCAR3, H226, ASPC1, A549 and HCC70 tumor cell lines.
  • Figures 65A-65B provide flow cytometry data of anti-BCMA CAR-T cells stained with CD19-Fc ( Figure 65A) or BCMA-Fc ( Figure 65B).
  • Figure 66A provides the frequency of CAR+ cells among T cells transduced with the indicated BCMA CARs.
  • Figure 66B provides mean fluorescence intensities (MFI) of CAR expression in T cells transduced with the indicated BCMA CARs.
  • Figure 67 provides the frequency of CAR+CD8 cells in T cells transduced with the indicated BCMA CARs.
  • Figure 68 provides the phenotypes of the indicated CART cells characterized by CCR7 expression and CD45RO expression.
  • Figures 69A-69B provide expression of BCMA in tumor cell lines.
  • Figure 69A provides FACS results.
  • Figure 69B provides relative expression levels compared to A549 cells.
  • Figures 70A-70B provide ELISA results showing INF- ⁇ and IL-2 production by indicated CART cells.
  • Figure 70A shows the production of INF- ⁇ .
  • Figure 70B shows IL-2 production.
  • Figures 71A-71D provide the results of a tumor killing assay showing the cytolytic activity of indicated CART cells against Jeko-1 cells at different E(T cell):T(tumor cell) ratios.
  • Figure 71A: E:T 0.1:1;
  • Figure 71B: E:T 0.5:1;
  • Figure 71C: E:T 2:1;
  • Figure 71D: E:T 2:1 (enlarged view).
  • Figures 72A-72E provide the results of a tumor killing assay showing the cytolytic activity of the indicated CART cells against RPMI-8226 cells.
  • Fig. 72E :E:T 0.5:1 (enlarged view).
  • Figure 73 provides the readout of two 96-well plates of the anti-human CD123-Fc monoclonal phage ELISA.
  • Figure 74 provides a schematic diagram of the pDA-CAR vector used for CAR mRNA production.
  • Figure 75 provides FACS staining results showing the binding of anti-CD123 scFv expressed in CART cells to CD123-Fc protein.
  • Figure 76 provides the results of FACS staining of isotypes and anti-CD123 antibodies on A549 cells electroporated with different amounts of CD123 mRNA.
  • Figure 79 provides the killing curves of different mRNA-based anti-CD123 CART cells at an E/T ratio of 10:1 to A549-GFP tumor cells electroporated with 10 ⁇ g of CD123 mRNA.
  • Figure 80 provides the killing curves of different mRNA-based anti-CD123 CART cells at an E/T ratio of 3:1 to A549-GFP tumor cells electroporated with 10 ⁇ g of CD123 mRNA.
  • Figure 81 shows the results of FACS staining of PE-isotype control and PE-anti-CD123 mAb on A549, SK-OV3, Jeko-1, Molm-14, SupT-1, 293T, Nalm-6 and PC-3 cells .
  • Figure 82 shows CD107a staining of anti-CD123-C5, anti-CD123-C7, anti-CD123-C11 CART cells in co-culture and killing assays with different tumor cells.
  • Numerical ranges described herein such as temperature ranges, time ranges, composition or concentration ranges, or other numerical ranges, etc., include end values, all intermediate ranges, and subranges (such as between an intermediate value and a certain ranges between end values), and all individual values, especially intermediate ranges, subranges, and individual integer values that end at integer values. Also, any intermediate ranges, subranges, and all individual values stated within a stated numerical range may be excluded from stated numerical ranges.
  • nucleic acid sequence and “polynucleotide” are used interchangeably to refer to a sequence composed of bases, sugars, and phosphate backbone linkages.
  • the nucleic acid sequence of the present invention may be deoxyribonucleic acid sequence (DNA) or ribonucleic acid sequence (RNA), and may include natural bases or unnatural bases, which may be single-stranded or double-stranded, and may be coding sequences or non-coding sequence.
  • polypeptide and “protein” are used interchangeably and refer to a polymer of amino acid residues. Such polymers may contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed within this definition.
  • the term also includes polypeptides with post-expression modifications such as glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • isolated protein or “isolated polypeptide” refers to a protein or polypeptide that: (1) is not associated with naturally associated components in its native state; (2) does not contain other proteins from the same species; The protein; (3) is expressed by cells from a different species; or (4) does not occur naturally.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell of its natural origin will be “isolated” from the components with which it is naturally associated.
  • a protein can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • the proteins or polypeptides mentioned in the present invention such as antibodies or antigen-binding fragments thereof, are preferably isolated.
  • isolated nucleic acid refers to a nucleic acid molecule of genomic, cDNA or synthetic origin, or combinations thereof, which is separated from other nucleic acid molecules present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules that are chromosomally separated from the genomic DNA with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid does not include sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid of interest).
  • the polynucleotides or nucleic acid sequences mentioned in the present invention are preferably isolated.
  • nucleic acid sequence is usually from 5' end to 3' end
  • amino acid sequence is usually from N-terminus to C-terminus
  • antigenic epitope may also be referred to as “antigenic determinant”, and refers to a chemical functional group on the surface of an antigen that determines antigen specificity. An antigenic epitope can be recognized and bound by an antigen-binding site in an antibody.
  • antibody is used in its broadest sense to include an immunoglobulin or other type of molecule comprising one or more antigen-binding domains that specifically bind an antigen, a protein or peptide.
  • Specific examples of antibodies may include (eg, classical four-chain antibody molecules), single chain antibodies, single domain antibodies, multispecific antibodies, and the like.
  • Intact antibodies also referred to as full-length antibodies or classical antibody molecules in the present invention, are immunoglobulin molecules (such as IgG) or multimers (such as IgA or IgM) composed of four polypeptide chains.
  • the four polypeptide chains include two identical heavy chains (H) and two identical light chains (L), which are interconnected by disulfide bonds to form a tetramer.
  • Each heavy chain consists of a heavy chain variable region ("HCVR” or "VH") and a heavy chain constant region (CH, including domains CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region ("LCVR or "VL”) and a light chain constant region (CL).
  • the constant regions (CH and CL) of the heavy and light chains are not directly involved in the binding of the antibody to the antigen, But exhibit multiple effector functions, such as can mediate the binding of antibodies to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • Heavy chain VH and VL form the antigen-binding site with the variable regions (VH and VL) of the light chain.
  • VH and VL have hypervariable regions called complementarity-determining regions (CDRs) respectively, and their amino acid composition and arrangement sequence have a high degree of variation.
  • Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to hydroxyl-terminus in the following order : FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 the three heavy chain CDRs are called HCDR1 (CDR-H1), HCDR2 (CDR-H2) and HCDR3 (CDR-H3), respectively.
  • the three heavy chain framework regions are called HFR1 (FR-H1), HFR2 (FR-H2), HFR3 (FR-H3) and HFR4 (FR-H4); the three light chain complementarity determining regions are called LCDR1 (CDR-L1) , LCDR2 (CDR-L2) and LCDR3 (CDR-L3), the four light chain framework regions are called LFR1 (FR-L1), LFR2 (FR-L2), LFR3 (FR-L3) and LFR4 (FR-L4 ).
  • CDR complementarity determining region
  • the precise boundaries of the CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
  • framework region or "FR” as used herein refers to those amino acid sequences in the variable region of an antibody other than the CDR sequences as defined above.
  • antibodies can be classified into five main different classes: IgA, IgD, IgE, IgG, and IgM. These antibody types can be further divided into subclasses according to the size of the hinge region, the position of the interchain disulfide bonds, and the molecular weight, for example, IgG1, IgG2a, IgG2b, IgG3, and IgG4. According to the amino acid composition and arrangement of the antibody light chain constant region, the light chain can be divided into two types: kappa and lambda. Antibodies of the invention include antibodies of any of the foregoing classes or subclasses.
  • monoclonal antibody refers to a homogeneous antibody directed only against a specific antigenic epitope. Each monoclonal antibody is directed against a single epitope on the antigen, in contrast to typical common polyclonal antibody preparations that include different antibodies directed against different epitopes. Although monoclonal antibodies can be obtained by culturing hybridomas, in the present invention, the modifier "monoclonal” indicates the uniform characteristics of the antibody and should not be interpreted as requiring Antibodies produced by any particular method. The monoclonal antibodies of the present invention can be produced by recombinant DNA methods, or obtained by other screening methods.
  • antigen-binding fragment refers to one or more portions of an intact antibody that retain the ability to specifically bind to the epitope against which the antibody is directed, see, for example, Fundamental Immunology, Ch.7 (Paul, W ., ed., 2th Edition, Raven Press, NY (1989).
  • antigen-binding fragments include: (1) Fab fragments: antigen-binding fragments produced by papain digestion of intact antibodies, composed of complete light chains of full-length antibodies (including the VL domain and CL domain) and the heavy chain variable region VH and heavy chain constant region CH1 of the full-length antibody, without the hinge region; (2) F(ab') 2 fragment: composed of the complete antibody Antigen-binding fragment produced by pepsin digestion, which contains the entire light chain (including VL domain and CL domain) of two full-length antibodies and the heavy chain variable region VH and heavy chain constant region structures of two full-length antibodies domain CH1 and the hinge region, which can be viewed as a paired fragment of two Fab' fragments with a disulfide bond in between; (3) Fab' fragments, which can break the F(ab') 2 hinge by treatment with a reducing agent (4) Fd fragment composed of VH domain and CH1 domain; (5) Fv fragment composed of VL domain and VH domain of antibody single arm.
  • Fab fragments antigen
  • antibody usually at least comprises either or both of the heavy chain variable region and the light chain variable region of an intact antibody, and may also comprise a part or all of the light chain constant region and/or the heavy chain constant region of an intact antibody, For example, one or two or more of CL domain, CH1 domain, CH2 domain, and CH3 domain may be included.
  • the term "Fc region” refers to the C-terminal region of an immunoglobulin heavy chain, i.e., the fragment crystallizable (Fc) region that constitutes the handle region of a Y-shaped classical antibody molecule, including the second and third regions of the heavy chain. Constant domains (CH2 and CH3 domains).
  • the Fc region can be obtained by hydrolysis of intact antibody molecules using papain.
  • the Fc region can comprise a hinge, CH2 and CH3. Dimerization between two Fc-containing polypeptides can be mediated when the Fc region comprises a hinge.
  • the Fc fragment can be from IgG, IgM, IgD, IgE or IgA.
  • the Fc region is from IgGl, IgG2, IgG3 or IgG4.
  • Fc fragment also includes variant Fc fragments derived from native Fc fragments that have been altered but still retain their effector functions.
  • a "variant Fc fragment” comprises an amino acid sequence having at least one amino acid substitution on the amino acid sequence of a native Fc fragment.
  • the variant Fc fragment has at least one amino acid substitution compared to the parental Fc fragment (native Fc fragment), for example about 1 to about 10 amino acids are substituted in the parental Fc fragment, and preferably about 1 to about 5 amino acids are substituted Amino acid substitutions.
  • the variant Fc fragment Fc region has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98% sequence identity to the parental Fc fragment. % or at least about 99% sequence identity.
  • the effector functions of the "Fc fragment” may include binding to Fc receptors, Clq binding and complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediation of phagocytosis, and the like.
  • single domain antibody refers to an antigen binding polypeptide having a single variable domain comprising three complementarity determining regions (CDRs). Single domain antibodies are capable of binding antigen alone without pairing with another CDR-containing polypeptide. Common single domain antibodies comprise a heavy chain but lack the light chain normally found in antibodies, such as camelid sdAbs (see eg, Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374 : 168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8: 1013-26 (2013)).
  • Single domain antibodies can be artificially derived from camelid heavy chain antibodies Engineered, called V H H antibody, V H H antibody has the following structure from N-terminal to C-terminal: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies can also be considered a special "antigen-binding fragment" of a full-length antibody.
  • single-chain antibody refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, 113, Roseburg & Moore, Springer-Verlag, New York, 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • a suitable linker may consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 or a variant thereof (Holliger et al., (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) can be used.
  • the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.
  • antibodies can be produced by various techniques, such as hybridoma technology (see, for example, Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, e.g., Clackson et al., Nature, 352:624-628, 1991, or Marks et al., J. Mol. Biol. 222:581-597, 1991).
  • Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, as known to those skilled in the art. Subsequently or alternatively, the specific antigen (the target molecule recognized by the antibody) or an epitope thereof can be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography.
  • the purification of antibodies can refer to, for example, D. Wilkinson, The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol.14, No.8 (Apr.17, 2000), pp.25-28).
  • Chimeric antibody refers to an antibody whose light chain and/or heavy chain are partly derived from one antibody (which may be derived from a specific species or belong to a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but still retains the target antigen. Binding activity (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • the term “chimeric antibody” may refer to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, for example having a human constant region and a murine heavy chain variable region sequence. region and light chain variable region antibodies.
  • human antibody refers to an antibody whose entire sequence (such as variable and constant regions) of the antibody is derived from human immunoglobulin.
  • Non-human antibody refers to an antibody whose entire sequence (such as variable and constant regions) of the antibody is derived from an immunoglobulin of a non-human species, for example, an antibody derived from a mouse immunoglobulin may be called a murine antibody.
  • humanized antibody refers to a non-human antibody, such as an antibody whose variable and constant regions (if any) are not derived from human immunoglobulin, has been engineered to match the sequence of a human antibody. Homology is improved, resulting in chimeric antibodies.
  • all or part of the CDR regions of humanized antibodies come from All or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from a human immunoglobulin (recipient antibody) from a non-human antibody (donor antibody).
  • Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, and the like.
  • Donor antibodies can be mouse, rat, rabbit or non-human primate with desired properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and/or ability to enhance immune response) Animal-like (eg, cynomolgus monkey) antibodies.
  • Humanized antibodies can not only retain the expected properties of non-human donor antibodies (such as murine antibodies), but also effectively reduce the immunogenicity of non-human donor antibodies (such as murine antibodies) in human subjects, It is therefore particularly advantageous.
  • the desired properties of the humanized antibody e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and/or The ability to enhance the immune response
  • non-human donor antibodies such as murine antibodies
  • the framework region (FR) of the humanized antibody may contain both the amino acid residues of the human recipient antibody and the corresponding amino acid residues of the non-human donor antibody, for example, the humanized antibody may contain back mutations.
  • back mutation is a mutation introduced in an amino acid of a humanized antibody that results in an amino acid that corresponds to an amino acid in the parental antibody (eg, a donor antibody, eg, a murine antibody).
  • the parent antibody is a murine antibody.
  • backmutation changes human framework residues to parental murine residues.
  • framework residues that can be back mutated include, but are not limited to, canonical residues, interface packing residues, unusual parental residues adjacent to the binding site, the "Vernier Zone” (which Form the platform for docking the CDR) (Foote & Winter, 1992, J. Mol. Biol. 224, 487-499) and those adjacent to CDRH3.
  • affinity matured antibody refers to an antibody that binds to the same antigen as a reference antibody, but with a higher binding affinity for that antigen than the reference antibody.
  • Affinity matured antibodies can be obtained by mutation of a reference antibody, in which case the reference antibody can also be called a parent antibody.
  • Affinity matured antibodies typically have one or more amino acid changes relative to the parent antibody in one or more CDRs that result in an increase in the affinity of the antibody for the target antigen compared to a parent antibody without these changes.
  • Exemplary affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Methods for generating affinity matured antibodies are known in the art.
  • Affinity matured variants can then be screened by phage display libraries and phage ELISA.
  • multispecific antibody used in the present invention refers to an anti-antibody capable of specifically binding to multiple antigenic epitopes. Antibodies, including bispecific, trispecific, or more specific antibodies. The multiple antigen epitopes may be different epitopes on the same antigen, or different epitopes on different antigens.
  • a multispecific antibody comprises a plurality of antigen-binding moieties, each antigen-binding moiety targeting one epitope and the epitopes targeted by the plurality of antigen-binding moieties are different from each other.
  • Each antigen binding portion may be independently selected from monoclonal antibodies (such as IgG immunoglobulins), Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments, Fd fragments, scFv, dsFv, single domain antibodies, Chimeric antibodies, humanized antibodies, affinity matured antibodies, etc.
  • One antigen-binding moiety can be functionally linked (eg, by chemical conjugation, genetic fusion, non-covalent association, or otherwise) to one or more additional antigen-binding moieties to form multispecific antibodies.
  • the terms "target”, “direct” or “specifically bind” mean that the antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions, and is preferably non-specific for other The desired antigen showed no appreciable binding.
  • a molecule may target, be directed against, or specifically bind more than one molecule, for example a bispecific antibody may have a higher binding affinity for two different antigens relative to other molecules.
  • Specific binding can be characterized by the equilibrium dissociation constant KD of antigen-antibody binding (a smaller KD indicates tighter binding), and can also be characterized by the EC50 value of antibody-antigen binding. Methods for determining whether two molecules are specifically bound are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
  • EC50 concentration for 50% of maximal effect
  • concentration concentration that causes 50% of the maximal effect.
  • ELISA enzyme-linked immunosorbent assay
  • concentration of antibody molecules that produces half of the maximum detection signal such as colorimetric or fluorescence intensity. The lower the EC50 value, the greater the binding affinity of the antibody to the antigen.
  • ka refers to the association rate constant of a specific antibody-antigen interaction, indicating the binding rate of an antibody to its target antigen, or the rate of complex formation between an antibody and an antigen, in units of M -1 s -1
  • the term “kd” refers to the dissociation rate constant of a specific antibody-antigen interaction, indicating the dissociation rate of an antibody from its target antigen, or the dissociation rate of an antigen-antibody complex over time into free antibody and antigen, in seconds - 1 .
  • KD refers to the equilibrium dissociation constant for a particular antibody-antigen interaction, which is derived from the ratio of kd to ka (ie, kd/ka), expressed in molarity (M). KD can be used to measure the affinity with which an antibody binds to its binding partner, such as an antigen. A smaller KD indicates tighter binding between antibody and antigen, or higher affinity between antibody and antigen. For example, an antibody with an equilibrium dissociation constant on the nanomolar (nM) scale binds more tightly to a particular antigen than an antibody with a dissociation constant on the micromolar ( ⁇ ) scale. KD values for antibodies can be determined using methods well known in the art.
  • One method of determining antibody ka, kd and KD values is by using surface plasmon resonance, typically measured using a biosensor system such as the Biacore TM system.
  • Another method for determining the KD value of an antibody is biolayer interferometry (BLI).
  • chimeric antigen receptor refers to a fusion protein comprising an extracellular domain capable of binding to an antigen (i.e., a binding domain), a transmembrane domain, and one or more Intracellular domain of the intracellular signaling domain of guidelin. These intracellular signaling domains are often different from the polypeptide from which the extracellular domain is derived.
  • the extracellular domain can be any protein molecule or part thereof capable of interacting with a predetermined antigen-specific binding. In some embodiments, the extracellular domain comprises an antibody or antigen-binding fragment thereof.
  • the intracellular domain can be any known oligopeptide or polypeptide domain whose function is to transmit signals leading to the activation or inhibition of intracellular biological processes, for example, activation of immune cells such as T cells or NK cells .
  • Intracellular domains often include immunoreceptor tyrosine activation motifs (ITAMs), such as the signaling domain from the CD3 ⁇ molecule, responsible for activating immune effector cells and producing killing effects.
  • chimeric antigen receptors can also include a signal peptide at the N-terminus, responsible for the intracellular localization of the fusion protein, and a hinge region between the extracellular domain and the transmembrane domain.
  • Intracellular signaling domains may also include co-stimulatory domains from molecules such as 4-1BB or CD28.
  • CART or "CART cell” refers to a T cell capable of expressing or producing a CAR.
  • CART cells are usually obtained by engineering T cells to contain nucleic acid molecules encoding CARs.
  • CART cells can be obtained through the modification of autologous T cells or allogeneic T cells.
  • recombinant refers to expression of an antibody or antigen-binding fragment thereof of the invention by recombinant DNA techniques (which include, for example, DNA splicing and transgenic expression), for example using transfection into a non-human mammal (such as a mouse) or cells (for example, recombinant expression vector expression systems in CHO cells) express antibodies or antigen-binding fragments thereof, which antibodies may be or are isolated from recombinant or combinatorial human antibody libraries.
  • DNA techniques which include, for example, DNA splicing and transgenic expression
  • transfection into a non-human mammal such as a mouse
  • cells for example, recombinant expression vector expression systems in CHO cells express antibodies or antigen-binding fragments thereof, which antibodies may be or are isolated from recombinant or combinatorial human antibody libraries.
  • sequence identity refers to the nucleotide or amino acid residues at corresponding positions when two or more sequences are aligned for maximum matching, taking into account gaps and insertions percentage. Alignment of sequences and calculation of percent sequence identity can be performed using suitable computer programs known in the art. Such programs include local alignment programs and global alignment programs, including but not limited to BLAST, ALIGN, ClustalW, EMBOSS Needle, and the like.
  • BLAST Basic Local Alignment Search Tool
  • vector refers to any molecule capable of transporting heterologous nucleic acid contained therein into a host cell (existing in episomal form or integrated into the genome of the host cell), such as nucleic acid, plasmid or virus.
  • Vectors introduced into host cells can exist independently and replicate autonomously (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors), or be integrated into the host cell genome and replicate together with the host genome (e.g., non-episomal mammalian vectors). animal vector).
  • a vector used to express a gene of interest in a host cell is called an "expression vector” or a "recombinant expression vector”.
  • the vector may contain regulatory elements operably linked to the gene of interest, such as an origin of replication, expression control sequences (e.g., promoters and/or enhancers), and/or selectable marker genes (e.g., antibiotic resistance genes and genes useful in colorimetric assays). Genes in , such as ⁇ -galactose).
  • expression vectors encoding the heavy and light chains can be transfected into host cells by standard techniques.
  • transfection is intended to include various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.
  • the antibodies of the invention are expressed in either prokaryotic or eukaryotic host cells
  • expression of the antibodies in eukaryotic cells is most preferably mammalian host cells because such eukaryotic cells, especially mammalian cells, are more sensitive than prokaryotic cells Immunologically active antibodies that are correctly folded are more likely to be assembled and secreted.
  • host cell refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide.
  • Host cells can be prokaryotic or eukaryotic.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells.
  • Non-limiting exemplary mammalian cells include, but are not limited to, CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells can also be immune effector cells, such as T cells.
  • a host cell includes progeny of a single host cell, and the progeny may not necessarily be completely identical to the original parent cell, eg, in morphology or genomic DNA complementation, as a result of natural, accidental, or deliberate mutation.
  • a host cell can be an isolated cell or cell line, and also includes a cell transfected with a nucleic acid molecule or expression vector provided herein in vivo.
  • the present invention humanizes the mouse-derived 9.3 antibody by means of homology modeling and reverse mutation, which solves the shortcoming of reduced affinity, and at the same time, the humanized 9.3 antibody has stronger functionality.
  • the present invention addresses the weakness of CD40 antibody's low affinity.
  • CD28 used herein may also be referred to as cluster of differentiation 28, Tp44, which is an antigen expressed on the surface of T cells as a co-stimulatory receptor, which can provide co-stimulatory signals required for T cell activation and survival.
  • CD28 herein refers to any CD28 protein from any vertebrate source, including mammals such as primates (eg, humans), non-human primates (eg, rhesus monkeys), and rodents (eg, mice and rats).
  • the present invention provides a humanized anti-CD28 antibody.
  • the humanized anti-CD28 antibody is obtained by humanizing a murine anti-CD28 antibody.
  • the murine anti-CD28 antibody can be antibody 9.3, for example.
  • the humanized anti-CD28 antibody of the present invention can specifically recognize CD28, especially human CD28, including soluble CD28 or CD28 expressed on the cell surface.
  • Soluble CD28 includes native CD28 protein as well as recombinant CD28 protein or variants thereof, monomeric or dimeric CD28 constructs, CD28 lacking a transmembrane domain, and the like.
  • the EC50 of the humanized anti-CD28 antibody of the present invention binding to human CD28 is in the range of about 0.008 ⁇ g/mL to about 0.016 ⁇ g/mL, for example in the range of about 0.008 ⁇ g/mL to about 0.013 ⁇ g/mL, in the range of about In the range of 0.008 ⁇ g/mL to about 0.011 ⁇ g/mL, in the range of about 0.008 ⁇ g/mL to about 0.010 ⁇ g/mL or in the range of about 0.008 ⁇ g/mL to about 0.009 ⁇ g/mL; further, in In the range of about 8.00 ng/mL to about 15.50 ng/mL, for example in the range of about 8.27 ng/mL to about 15.09 ng/mL, in the range of about 8.27 ng/mL to about 15.07 ng/mL, in the range of about 8.27 ng/mL to about 12.67 ng/mL, in the
  • the humanized anti-CD28 antibodies of the invention have an EC50 for binding to human CD28 of about 8.27 ng/mL, about 9.14 ng/mL, about 10.40 ng/mL, about 10.90 ng/mL, about 12.11 ng /mL, about 12.54 ng/mL, about 12.59 ng/mL, about 12.65 ng/mL, about 12.67 ng/mL, about 15.07 ng/mL, or about 15.09 ng/mL.
  • the EC50 is determined by ELISA, and the ELISA can be used to determine the binding activity of the humanized antibody to human CD28 (such as human CD28 containing 6 histidines at the N-terminal), and the concentration of human CD28 used for the determination of the binding activity For example, it can be 2 ⁇ g/ml.
  • the binding equilibrium dissociation constant KD of the humanized anti-CD28 antibody of the present invention to human CD28 is in the range of about 6.1 ⁇ 10 -9 M to about 1.2 ⁇ 10 -8 M, for example, in the range of about 6.1 ⁇ 10 -9 M to about in the range of 1.1 ⁇ 10 -8 M, in the range of about 6.1 ⁇ 10 -9 M to about 9.6 ⁇ 10 -9 M, in the range of about 6.1 ⁇ 10 -9 M to about 9.2 ⁇ 10 -9 M, In the range of about 6.1 ⁇ 10 -9 M to about 8.4 ⁇ 10 -9 M, in the range of about 6.1 ⁇ 10 -9 M to about 7.5 ⁇ 10 -9 M, in the range of about 6.1 ⁇ 10 -9 M to about In the range of 6.6 ⁇ 10 -9 M, or in the range of about 6.1 ⁇ 10 -9 M to about 6.2 ⁇ 10 -9 M; further, in the range of about 6.13 ⁇ 10 -9 M to about 1.15 ⁇ 10 -8 M In the range of about 6.13 ⁇ 10 -9 M to about 1.12
  • the equilibrium dissociation constant KD for the binding of the humanized humanized anti-CD28 antibody of the present invention to human CD28 is about 6.13 ⁇ 10 -9 M, about 6.54 ⁇ 10 -9 M, about 6.59 ⁇ 10 -9 9 M, about 7.44 ⁇ 10 -9 M, about 8.36 ⁇ 10 -9 M, about 9.13 ⁇ 10 -9 M, about 9.59 ⁇ 10 -9 M, about 1.08 ⁇ 10 -8 M about 1.09 ⁇ 10 -8 M ,, about 1.12 ⁇ 10 -8 M or about 1.15 ⁇ 10 -8 M.
  • the binding equilibrium dissociation constant KD is determined by biolayer interferometry (BLI).
  • the three heavy chain CDRs of a murine anti-CD28 antibody can be linked to the four heavy chain FRs of a human antibody (recipient antibody) to humanize the heavy chain variable region of the antibody, and /Or link the three light chain CDRs of the mouse anti-CD28 antibody with the four light chain FRs of the human antibody to humanize the light chain variable region of the antibody.
  • a humanized anti-CD28 antibody can comprise a humanized heavy chain variable region and/or a humanized light chain variable region.
  • the heavy chain variable region of the murine anti-CD28 antibody comprises: HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:2 and HCDR3 comprising SEQ ID NO:3.
  • the light chain variable region of the murine anti-CD28 antibody comprises: LCDR1 comprising SEQ ID NO:9, LCDR2 comprising SEQ ID NO:10 and LCDR3 comprising SEQ ID NO:10.
  • any human germline antibody sequence can be used as the recipient antibody in the humanization transformation, and the human germline antibody sequence with the highest sequence identity with the heavy chain variable region of the donor antibody can be selected as the antibody heavy chain sequence.
  • the human germline IGHV4 sequence was used as the acceptor antibody for the humanization of the heavy chain variable region of the murine anti-CD28 antibody.
  • human germline IGKV1 sequences are used as recipient antibodies for humanization of the light chain variable region of murine anti-CD28 antibodies.
  • the humanized anti-CD28 antibody of the present invention may further comprise amino acid substitutions in the CDRs of the murine anti-CD28 antibody, for example, the humanized anti-CD28 antibody may comprise amino acid substitutions in HCDR1, HCDR2 and/or HCDR3, Amino acid substitutions in LCDR1, LCDR2 and/or LCDR3 may also be included.
  • the heavy chain variable region of the humanized anti-CD28 antibody comprises HCDR2 with amino acid substitutions at one or more of positions S12, A12 and M15 relative to SEQ ID NO: 2 (amino acid residue Numbering is based on SEQ ID NO:2, with the first amino acid residue of SEQ ID NO:2 as position 1).
  • the HCDR2 contained in the heavy chain variable region of the humanized anti-CD28 antibody has an amino acid substitution at position M15 relative to SEQ ID NO: 2, and the amino acid substitution is preferably M15K.
  • the HCDR2 contained in the heavy chain variable region of the humanized anti-CD28 antibody has amino acid substitutions at positions S12, A12 and M15 relative to SEQ ID NO: 2, and the amino acid substitutions are preferably S12P, A13S and M15K.
  • the heavy chain variable region of the humanized anti-CD28 antibody comprises: HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:17 or 18 and comprising SEQ ID NO:3 HCDR3.
  • the heavy chain variable region and the light chain variable region of the humanized anti-CD28 antibody comprise: (1) HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:2, comprising HCDR3 of SEQ ID NO:3, LCDR1 comprising SEQ ID NO:9, LCDR2 comprising SEQ ID NO:10 and LCDR3 comprising SEQ ID NO:10; (2) HCDR1 comprising SEQ ID NO:1, comprising SEQ ID HCDR2 of NO:17, HCDR3 comprising SEQ ID NO:3, LCDR1 comprising SEQ ID NO:9, LCDR2 comprising SEQ ID NO:10 and LCDR3 comprising SEQ ID NO:10; or (3) comprising SEQ ID NO HCDR1 comprising SEQ ID NO:18, HCDR3 comprising SEQ ID NO:3, LCDR1 comprising SEQ ID NO:9, LCDR2 comprising SEQ ID NO:10 and LCDR3 comprising SEQ ID NO:10;
  • the humanized anti-CD28 antibody of the present invention may also contain back mutations in the FR region derived from the human germline antibody sequence, that is, some amino acid residues in the FR region are replaced by corresponding residues from the murine antibody to restore Or improve the specificity and affinity of antibodies.
  • the heavy chain variable region of the humanized anti-CD28 antibody of the present invention comprises: HFR1 comprising a sequence selected from SEQ ID NO: 19-22, comprising a sequence selected from SEQ ID NO: 23-26 HFR2, HFR3 comprising a sequence selected from SEQ ID NO:27-30 and HFR4 comprising SEQ ID NO:7.
  • the light chain variable region of the humanized anti-CD28 antibody of the present invention comprises: LFR1 comprising a sequence selected from SEQ ID NO: 36-37, LFR2 comprising a sequence selected from SEQ ID NO: 38-39, comprising a sequence selected from LFR3 of the sequence of SEQ ID NO:40-42 and LFR4 comprising SEQ ID NO:15.
  • the heavy chain variable region of the humanized anti-CD28 antibody of the present invention comprises: (1) HFR1 comprising SEQ ID NO:19, HFR2 comprising SEQ ID NO:23, comprising SEQ ID NO:27 HFR3, and HFR4 comprising SEQ ID NO:7; (2) HFR1 comprising SEQ ID NO:20, comprising HFR2 comprising SEQ ID NO:24, HFR3 comprising SEQ ID NO:28, and HFR4 comprising SEQ ID NO:7; (3) HFR1 comprising SEQ ID NO:21, HFR2 comprising SEQ ID NO:25, comprising HFR3 of SEQ ID NO:29, and HFR4 comprising SEQ ID NO:7; (4) HFR1 comprising SEQ ID NO:21, HFR2 comprising SEQ ID NO:26, HFR3 comprising SEQ ID NO:30, and comprising HFR4 of SEQ ID NO:7; or (5) HFR1 comprising SEQ ID NO:22, HFR2 comprising SEQ ID NO:26
  • the light chain variable region of the humanized anti-CD28 antibody of the present invention comprises: (1) LFR1 comprising SEQ ID NO:36, LFR2 comprising SEQ ID NO:38, comprising SEQ ID NO:40 LFR3 comprising SEQ ID NO:15; (2) LFR1 comprising SEQ ID NO:37, LFR2 comprising SEQ ID NO:39, LFR3 comprising SEQ ID NO:41, and comprising SEQ ID NO:15 or (3) LFR1 comprising SEQ ID NO:37, LFR2 comprising SEQ ID NO:39, LFR3 comprising SEQ ID NO:42, and LFR4 comprising SEQ ID NO:15.
  • the heavy chain variable region and the light chain variable region of the humanized anti-CD28 antibody of the present invention comprise: (1) HFR1 comprising SEQ ID NO: 19, HFR2 comprising SEQ ID NO: 23, HFR3 comprising SEQ ID NO:27, HFR4 comprising SEQ ID NO:7, LFR1 comprising SEQ ID NO:36, LFR2 comprising SEQ ID NO:38, LFR3 comprising SEQ ID NO:40, and comprising SEQ ID NO (2) HFR1 comprising SEQ ID NO:19, HFR2 comprising SEQ ID NO:23, HFR3 comprising SEQ ID NO:27, HFR4 comprising SEQ ID NO:7, comprising SEQ ID NO:37 LFR1 comprising SEQ ID NO:39, LFR2 comprising SEQ ID NO:41, and LFR4 comprising SEQ ID NO:15; (3) HFR1 comprising SEQ ID NO:20, comprising SEQ ID NO:24 HFR2, HFR3 comprising SEQ ID NO:28, HFR4 comprising SEQ ID
  • the heavy chain variable region of a humanized anti-CD28 antibody of the invention comprises a sequence selected from SEQ ID NO: 31-35. In some embodiments, the light chain variable region of a humanized anti-CD28 antibody of the invention comprises a sequence selected from SEQ ID NO: 43-45.
  • the heavy chain variable region and the light chain variable region of the humanized anti-CD28 antibody of the present invention are selected from the following combinations: (1) a heavy chain variable region comprising SEQ ID NO: 31 and comprising A light chain variable region comprising SEQ ID NO:43; (2) a heavy chain variable region comprising SEQ ID NO:31 and a light chain variable region comprising SEQ ID NO:44; (3) comprising SEQ ID NO:32 A heavy chain variable region comprising SEQ ID NO:44 and a light chain variable region comprising SEQ ID NO:44; (4) a heavy chain variable region comprising SEQ ID NO:32 and a light chain variable region comprising SEQ ID NO:45; ( 5) heavy chain variable region comprising SEQ ID NO:33 and light chain variable region comprising SEQ ID NO:43; (6) heavy chain variable region comprising SEQ ID NO:33 and comprising SEQ ID NO:44 (7) a heavy chain variable region comprising SEQ ID NO:33 and a light chain variable region comprising SEQ ID NO:45; (8)
  • the humanized anti-CD28 antibody of the present invention can be any antibody form comprising the above-mentioned humanized heavy chain variable region and/or humanized light chain variable region, such as a full-length antibody (such as IgG), a single chain antibody or single domain antibodies, etc.
  • a humanized anti-CD28 antibody of the invention comprises a constant region of an immunoglobulin (such as an IgG, such as IgG1, IgG2, IgG3 or IgG4), such as an IgG1, IgG2, IgG3 or IgG4 constant region.
  • the constant region is a constant region of a human IgG, eg, a constant region of a human IgGl, human IgG2, human IgG3, or human IgG4.
  • the humanized heavy chain variable region described above is fused to an immunoglobulin heavy chain constant region (including CH1, CH2 and/or CH3).
  • the humanized light chain variable region described above is fused to an immunoglobulin light chain constant region (CL).
  • the immunoglobulin heavy chain constant region comprises SEQ ID NO:81.
  • the immunoglobulin light chain constant region comprises SEQ ID NO:82.
  • the humanized anti-CD28 antibody of the present invention is a single-chain antibody (scFv), wherein the humanized heavy chain variable region and the humanized light chain variable region are fused together through a peptide linker (Linker) .
  • the sequence of linkage of the humanized heavy chain variable region and the humanized light chain variable region can be different, for example, the humanized heavy chain
  • the C-terminus of the variable region is fused to the N-terminus of the humanized light chain variable region via a peptide linker, or the N-terminus of the humanized heavy chain variable region is fused to the N-terminus of the humanized light chain variable region via a peptide linker fusion.
  • the peptide linker used to link the variable region of the humanized heavy chain and the variable region of the humanized light chain is well known to those skilled in the art, for example, it can be a flexible linker such as a glycine-serine polymer.
  • the peptide linker used to join the humanized heavy chain variable region to the humanized light chain variable region is GGGGSGGGGSGGGGS (SEQ ID NO: 80).
  • the humanized anti-CD28 antibody of the present invention can also be a single domain antibody, eg, a single domain antibody comprising a heavy chain variable region but not a light chain variable region.
  • a single domain antibody comprising a heavy chain variable region but not a light chain variable region.
  • the present invention also relates to antigen-binding fragments of the humanized anti-CD28 antibody, such as Fab fragments, F(ab') 2 fragments, and Fab' fragments of full-length antibodies.
  • humanized anti-CD28 antibody or its binding fragment of the present invention can also be used as a part of fusion protein, fused with other functional polypeptides.
  • CD40 is a glycoprotein expressed on the cell surface, which belongs to the tumor necrosis factor receptor (TNFR) superfamily and plays a central role in the immune system. It is expressed in various immune cells such as B cells, dendritic cells , monocytes and macrophages.
  • TNFR tumor necrosis factor receptor
  • CD40 herein refers to any CD40 protein from any vertebrate source, including mammals such as primates (eg, humans), non-human primates (eg, rhesus monkeys), and rodents (eg, mice and rats).
  • the present invention provides affinity matured anti-CD40 antibodies.
  • the affinity matured CD40 antibodies were identified by a phage display library as having increased binding affinity to CD40.
  • Affinity maturation using phage display libraries has been described, for example, Lowman et al., Biochemistry 30(45):10832-10838 (1991), see also Hawkins et al., J. Mol Biol. 254:889-896( 1992), and the examples below. Without being strictly limited to the following description, this process can be briefly described as follows: One or more positions within a predetermined region of a parental antibody are mutated to generate possible amino acid substitutions at each position.
  • the antibody mutants thus generated were displayed from filamentous phage particles in a monovalent fashion as fusions to the gene III product of M13 packaged within each particle. Phage expressing various mutants can be cycled through multiple rounds of binding selection (eg, by ELISA to determine the binding affinity of the mutants to the antigen), followed by isolation and sequencing of those mutants displaying high affinity. It should be understood that although the anti-CD40 antibody variants of the invention were identified using phage display technology, other techniques can also be used to identify anti-CD40 antibody variants with improved binding affinity, including affinity matured anti-CD40 antibody variants.
  • the parent antibody may be, for example, an anti-CD40 antibody having a heavy chain variable region and a light chain variable region comprising: HCDR1 comprising SEQ ID NO: 46, HCDR2 comprising SEQ ID NO:47 and HCDR3 comprising SEQ ID NO:48, said light chain variable region comprising: LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55 and comprising SEQ ID NO: 56 LCDR3.
  • the VH of the parent antibody comprises SEQ ID NO:53, and/or the VL comprises SEQ ID NO:61.
  • the parent antibody comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, A VH having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and/or comprising a sequence identical to SEQ ID NO :61 have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , VL with at least 98% or at least 99% sequence identity.
  • the parent antibody may be a full length antibody, such as an immunoglobulin (such as an IgG, such as IgG1, IgG2, IgG3 or IgG4), a scFv or a single domain antibody, or an antigen-binding fragment thereof, such as a Fab fragment, F(ab') 2 Fragment, Fab' fragment.
  • an immunoglobulin such as an IgG, such as IgG1, IgG2, IgG3 or IgG4
  • a scFv or a single domain antibody such as a Fab fragment, F(ab') 2 Fragment, Fab' fragment.
  • the affinity matured anti-CD40 antibodies of the invention have a higher binding affinity to CD40 (eg, human CD40) than the parental antibody. In some embodiments, the affinity matured anti-CD40 antibodies of the invention have a lower EC50 value for binding to CD40 (eg, human CD40) than the parental antibody. In some embodiments, an affinity matured anti-CD40 antibody of the invention binds human CD40 with an EC50 of less than about 0.04919 ⁇ g/mL. In some embodiments, an affinity matured anti-CD40 antibody of the invention binds human CD40 with an EC50 of less than about 3.44 x 10 -7 M. The EC50 is determined by ELISA. The ELISA can be used to measure the binding activity of the antibody to human CD40 (such as human CD40-His). The concentration of human CD28 used for the determination of the binding activity can be 2 ⁇ g/ml, for example.
  • an affinity matured anti-CD40 antibody of the invention comprises a VH and a VL and has one, two or more amino acid substitutions relative to the parental antibody in at least one CDR.
  • the affinity matured anti-CD40 antibodies of the invention comprise VH and VL, and have one, two or more in at least one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 relative to the parental antibody. amino acid substitutions.
  • the VH of an affinity matured anti-CD40 antibody of the invention has one, two or more amino acid substitutions relative to the parental antibody in at least one of HCDR1, HCDR2, and HCDR3.
  • the VL of an affinity matured anti-CD40 antibody of the invention has one, two or more amino acid substitutions relative to the parental antibody in at least one of LCDR1, LCDR2, and LCDR3.
  • the VH of the affinity matured anti-CD40 antibody of the present invention comprises: HCDR1 comprising a sequence selected from SEQ ID NO:46 and SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47 and comprising SEQ ID NO:47 NO: 48 HCDR3.
  • the VL of the affinity matured anti-CD40 antibody of the invention comprises: LCDR1 comprising SEQ ID NO:54, LCDR2 comprising a sequence selected from SEQ ID NO:55,64-66 and comprising a sequence selected from SEQ ID NO:55,64-66 LCDR3 of NO:56,67-68 sequence.
  • the VH of an affinity matured anti-CD40 antibody of the invention comprises: (1) HCDR1 comprising SEQ ID NO:46, HCDR2 comprising SEQ ID NO:47, and HCDR3 comprising SEQ ID NO:48; or (2) HCDR1 comprising SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47 and HCDR3 comprising SEQ ID NO:48.
  • the VL of an affinity matured anti-CD40 antibody of the invention comprises: (1) LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:64, and LCDR3 comprising SEQ ID NO:56;( 2) LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55 and LCDR3 comprising SEQ ID NO:67; (3) LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55 and LCDR3 comprising SEQ ID NO:68; (4) comprising SEQ LCDR1 comprising ID NO:54, LCDR2 comprising SEQ ID NO:65 and LCDR3 comprising SEQ ID NO:56; (5) LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:66 and comprising SEQ ID NO :56 LCDR3.
  • the VH and VL of the affinity matured anti-CD40 antibody of the present invention comprise: (1) HCDR1 comprising SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47, HCDR3 comprising SEQ ID NO:48 , LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:64 and LCDR3 comprising SEQ ID NO:56; (2) HCDR1 comprising SEQ ID NO:62, HCDR2 comprising SEQ ID NO:47, comprising HCDR3 comprising SEQ ID NO:48, LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55 and LCDR3 comprising SEQ ID NO:67; (3) HCDR1 comprising SEQ ID NO:62, comprising SEQ ID HCDR2 of NO:47, HCDR3 comprising SEQ ID NO:48, LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55 and LCDR3 comprising SEQ ID NO:67;
  • the affinity matured anti-CD40 antibodies of the invention do not include a VH comprising: HCDR1 comprising SEQ ID NO:46, HCDR2 comprising SEQ ID NO:47, and comprising SEQ ID NO: 48 HCDR3.
  • an affinity matured anti-CD40 antibody of the invention does not include a VL comprising: LCDR1 comprising SEQ ID NO:54, LCDR2 comprising SEQ ID NO:55, and comprising SEQ ID NO: 56 LCDR3.
  • the HFR regions and/or LFR regions of the affinity matured anti-CD40 antibodies of the invention are identical to the parent antibody.
  • the VH of the affinity matured anti-CD40 antibody of the present invention may comprise: HFR1 comprising SEQ ID NO:49, HFR2 comprising SEQ ID NO:50, HFR3 comprising SEQ ID NO:51 and HFR3 comprising SEQ ID NO:52 HFR4.
  • the VL of the affinity matured anti-CD40 antibody of the present invention can comprise: LFR1 comprising SEQ ID NO:57, LFR2 comprising SEQ ID NO:58, LFR3 comprising SEQ ID NO:59 and LFR3 comprising SEQ ID NO:60 LFR4.
  • the affinity matured anti-CD40 antibodies of the invention may have one or more amino acid substitutions in the FR regions relative to the parental antibody.
  • HFR1 of the affinity-matured anti-CD40 antibody of the invention has an amino acid substitution at position E10 relative to SEQ ID NO:49 (amino acid residue numbering is based on SEQ ID NO:49, based on SEQ ID NO:49 The first amino acid residue of is taken as position 1).
  • HFR1 of an affinity matured anti-CD40 antibody of the invention has the amino acid substitution E10Q relative to SEQ ID NO:49.
  • the VH of an affinity matured anti-CD40 antibody of the invention comprises: HFR1 comprising SEQ ID NO: 63, HFR2 comprising SEQ ID NO: 50, HFR3 comprising SEQ ID NO: 51 and comprising SEQ ID NO :52 HFR4.
  • the VH of the affinity matured anti-CD40 antibody of the invention comprises a sequence selected from SEQ ID NO: 69-73, and/or the VL comprises a sequence selected from SEQ ID NO: 74-78.
  • the affinity matured anti-CD40 antibodies of the invention comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least A VH having a sequence identity of 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, and/or comprises a sequence identity selected from SEQ ID
  • the sequence of NO:74-78 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or at least 99% sequence identity of the VL.
  • the affinity-matured anti-CD40 antibody of the present invention is any antibody format comprising the above-mentioned heavy chain variable region and/or light chain variable region, such as a full-length antibody (such as IgG, such as IgG1, IgG2, IgG3 or IgG4), a single chain Antibodies or single domain antibodies, etc.
  • the affinity matured anti-CD40 antibodies of the invention comprise a constant region of an immunoglobulin (eg, IgG), eg, a constant region of IgGl, IgG2, IgG3, or IgG4.
  • the constant region is a constant region of a human IgG, eg, a constant region of a human IgGl, human IgG2, human IgG3, or human IgG4.
  • the heavy chain variable regions described above are fused to an immunoglobulin heavy chain constant region (including CH1, CH2 and/or CH3).
  • the light chain variable regions described above are fused to an immunoglobulin light chain constant region (CL).
  • the affinity-matured anti-CD40 antibody of the present invention is a single-chain antibody (scFv), wherein the variable region of the heavy chain and the variable region of the light chain are fused together through a peptide linker (Linker).
  • the order of joining the heavy chain variable region to the light chain variable region can be different, for example, the C-terminal of the heavy chain variable region is fused to the N-terminal of the light chain variable region via a peptide linker, or the N-terminal of the heavy chain variable region Fused to the N-terminus of the light chain variable region via a peptide linker.
  • the peptide linker used to connect the variable region of the heavy chain and the variable region of the light chain is well known to those skilled in the art, for example, it can be a flexible linker, such as a glycine-serine polymer.
  • the peptide linker used to connect the variable region of the heavy chain to the variable region of the light chain is GGGGSGGGGSGGGGS (SEQ ID NO: 80).
  • the affinity matured anti-CD40 antibody of the present invention can also be a single domain antibody, eg, a single domain antibody comprising a heavy chain variable region but not a light chain variable region.
  • a single domain antibody comprising a heavy chain variable region but not a light chain variable region.
  • the present invention also relates to antigen-binding fragments of the affinity-matured anti-CD40 antibody, such as Fab fragments, F(ab') 2 fragments, and Fab' fragments of full-length antibodies.
  • affinity-matured anti-CD40 antibody or its binding fragment of the present invention can also be fused with other functional polypeptides as a part of fusion protein.
  • the invention also provides multispecific antibodies, including but not limited to bispecific, trispecific, or tetraspecific antibodies.
  • the multispecific antibody comprises at least two antigen binding portions: a first antigen binding portion and a second antigen binding portion.
  • the first antigen-binding portion and the second antigen-binding portion specifically bind to different antigens or different epitopes of the same antigen, respectively.
  • the first antigen binding moiety and the second The antigen-binding portion can independently be a full-length antibody, a single-chain antibody, a single-domain antibody, or an antigen-binding fragment thereof, such as a Fab fragment, F(ab') 2 fragment, Fab' fragment, or Fv fragment of a full-length antibody.
  • “first” and “second” are for the purpose of distinction only, and do not imply a specific order.
  • the first antigen-binding portion is an anti-CD28 antibody, such as the murine anti-CD28 antibody or humanized anti-CD28 antibody of the present invention, and the second antigen-binding portion can specifically bind to an antibody different from CD28.
  • Second antigen In some embodiments, the second antigen binding moiety can specifically bind a tumor antigen or be capable of stimulating immune cells. In some embodiments, the second antigen binding moiety is an anti-CD40 antibody, such as a parental anti-CD40 antibody or an affinity matured anti-CD40 antibody described herein.
  • the first antigen-binding moiety is an anti-CD40 antibody, such as a parental anti-CD40 antibody or an affinity-matured anti-CD40 antibody of the present invention
  • the second antigen-binding moiety is capable of specifically binding to an antibody different from CD40.
  • Second antigen can specifically bind a tumor antigen or be capable of stimulating immune cells.
  • the second antigen binding moiety is an anti-CD28 antibody, such as a murine anti-CD28 antibody or a humanized anti-CD28 antibody described herein.
  • the first antigen-binding moiety can be any anti-CD28 antibody, such as the murine anti-CD28 antibody or humanized anti-CD28 antibody described in the present invention.
  • the second antigen-binding moiety can be any anti-CD40 antibody, such as the parental anti-CD40 antibody or affinity matured anti-CD40 antibody described in the present invention.
  • the first and second antigen-binding portions of the bispecific antibody are both scFvs linked by a peptide linker.
  • the first antigen binding moiety is an anti-CD28 scFv and the second antigen binding moiety is an anti-CD40 scFv.
  • the C-terminus of the first antigen-binding moiety is linked to the N-terminus of the second antigen-binding moiety via a peptide linker.
  • the N-terminus of the first antigen-binding moiety is linked to the C-terminus of the second antigen-binding moiety via a peptide linker.
  • the peptide linker used to link the first antigen-binding portion and the second antigen-binding portion of the bispecific antibody is well known to those skilled in the art, for example, it can be a flexible linker, such as a glycine-serine polymer.
  • the peptide linker used to link the first and second antigen binding portions of the bispecific antibody is GGGGS (SEQ ID NO:79).
  • the present invention also provides the humanized anti-CD28 antibody or its antigen-binding fragment encoding the present invention, the affinity matured anti-CD40 antibody or its antigen-binding fragment, or comprising the anti-CD28 antibody or its antigen-binding fragment and/or the anti-CD40 antibody
  • a polynucleotide of a multispecific antibody such as a bispecific antibody, hereinafter referred to as a CD28-CD40 double antibody
  • the polynucleotide may be DNA or RNA (eg, mRNA).
  • the present invention also provides a vector comprising the above polynucleotide, which can be used to express the above antibody or antigen-binding fragment thereof in a host cell.
  • the vector can be an expression vector, such as a plasmid vector or a virus Toxic carrier.
  • the viral vector may be a retroviral vector, lentiviral vector, adenoviral vector or adeno-associated viral vector.
  • the vector may be a circular RNA of the above-mentioned polynucleotide, and the circular RNA sequentially includes: an internal ribosome entry site (IRES) element, the above-mentioned polynucleotide, and polyA.
  • IRS internal ribosome entry site
  • the circular RNA can be used to express the above-mentioned antibody.
  • IRES can be derived from viruses.
  • IRES sequences are derived from coxsackievirus B3 (CVB3) or coxsackievirus A (CVB1/2), encephalomyositis virus (EMCV), Taura syndrome virus, triad virus, Seiler's encephalomyelitis virus, Simian virus 40, red fire ant virus 1, constrictor aphid virus, reticuloendotheliosis virus, human poliovirus 1, Plautia stali enterovirus, Kashmir bee virus.
  • CVB3 coxsackievirus B3
  • CVB1/2 coxsackievirus A
  • EMCV encephalomyositis virus
  • Taura syndrome virus encephalomyositis virus
  • triad virus Seiler's encephalomyelitis virus
  • Simian virus 40 red fire ant virus 1 constrictor aphid virus
  • reticuloendotheliosis virus human poliovirus 1
  • Human Rhinovirus 2 Leafhopper Virus-1, Human Immunodeficiency Virus Type 1, Lice P Virus, Hepatitis C Virus, Hepatitis A Virus, GB Hepatitis Virus, HFMD Virus, Human Enterovirus 71, Equine Rhinitis Virus , tea geometrid small RNA-like virus, Drosophila C virus, cruciferous tobacco mosaic virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute Bee paralysis virus, hibiscus green ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AMLURUNX1, Drosophila antennae, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c -IAP1, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1alpha, human n
  • the circular RNA of the present invention can be prepared by general strategies of RNA circularization methods, such as enzymatic method using RNA or DNA ligase, or ribozyme method using self-splicing intron. See, Petkovic, S. & Muller, S., "RNA circularization strategies in vivo and in vitro", Nucleic Acids Research, 43(4):2454-2465 (2015); Beadudry, D.
  • the present invention also provides a precursor RNA for preparing the circular RNA, the precursor RNA can be cyclized to obtain the circular RNA, and the precursor RNA can also be used as a Acid carriers are used.
  • the precursor RNA includes a circularization element, an internal ribosome entry site (IRES) element, the polynucleotides described above, and polyA.
  • the cyclization element comprises a first intron sequence located 5' to an internal ribosome entry site (IRES) element and a second intron sequence located 3' to polyA.
  • the first intron sequence and the second intron sequence are from Group I or Group II intron self-splicing sequences.
  • the first intron sequence comprises a 3' Group I intron fragment comprising a 3' splice site dinucleotide
  • the second intron element comprises a 5' splice site dinucleotide comprising The 5'Group I intron fragment of nucleotides.
  • the precursor RNA further comprises a 5' spacer sequence between the first intron sequence and an internal ribosome entry site (IRES) element, and a 5' spacer sequence between polyA and the second intron element. 3' spacing sequence.
  • the precursor RNA further comprises a 5' homology arm external to the first intronic element and a 3' homology arm external to the second intronic element.
  • the vector comprising the polynucleotide encoding the above-mentioned antibody or antigen-binding fragment can also be a vector for producing the above-mentioned precursor RNA, the vector comprising the DNA template of the precursor RNA, and the DNA template can be transcribed The precursor RNA is obtained.
  • the transcription can be in vitro or in a cell.
  • polyA as used herein is an abbreviation for polyadenylation and refers to a sequence consisting of contiguous adenine nucleotides with a length of at least 30.
  • the polyA sequence can be a ribonucleic acid sequence or a deoxyribonucleic acid sequence.
  • the length of consecutive adenine nucleotides in the polyA sequence can be at least 30 nucleotides, for example.
  • the length of consecutive adenine nucleotides in the polyA sequence can be in the range of 30-240 nucleotides, such as 40-230 nucleotides, 45-220 nucleotides, 50-210 nucleotides, 60-200 nucleotides, 70-190 nucleotides, 80-180 nucleotides, 90-170 nucleotides, 100-160 nucleotides, 110-150 nucleotides, 120- 140 nucleotides.
  • a polyA sequence may consist only of contiguous adenine nucleotides.
  • the polyA is at least 45 nucleotides in length.
  • the polyA is at least 70 nucleotides in length.
  • the present invention also provides a method for producing the above-mentioned circular RNA, comprising circularizing the above-mentioned precursor RNA to obtain the circular RNA.
  • the method further comprises transcribing a DNA template of the precursor RNA contained in a vector to obtain the precursor RNA.
  • the method further comprises purifying the circular RNA, eg, by the interaction between oligo-dT and polyA using an oligo-dT-based capture method.
  • the humanized anti-CD28 antibody or antigen-binding fragment thereof of the present invention can be used to induce the proliferation of T cells, activate T cells, and can be used to promote the killing effect of immune effector cells (such as T cells, such as CAR-T cells).
  • immune effector cells such as T cells, such as CAR-T cells.
  • the humanized anti-CD28 antibody, affinity-matured anti-CD40 antibody or antigen-binding fragment thereof of the present invention can form bispecific antibodies with antibodies that specifically bind tumor cell surface antigens, such as BiTE (bispecific T-cell engager), or poly Specific antibodies, targeted activation of self-T cells to kill tumors cell.
  • the present invention provides a method for inducing T cell proliferation using the humanized anti-CD28 antibody of the present invention or an antigen-binding fragment thereof, or the use of the humanized anti-CD28 antibody of the present invention or an antigen-binding fragment thereof in the preparation of a drug for inducing T cell proliferation in the application.
  • the method comprises making the humanized anti-CD28 antibody or antigen-binding fragment thereof of the present invention, a multispecific antibody comprising the humanized anti-CD28 antibody or antigen-binding fragment thereof of the present invention, encoding the humanized anti-CD28 antibody of the present invention
  • a polynucleotide of an antibody or an antigen-binding fragment thereof, a carrier comprising the polynucleotide, or a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof, the multispecific antibody, the polynucleotide or the carrier Contacting with T cells, whereby the humanized anti-CD28 antibody or antigen-binding fragment thereof stimulates the activation of T cells and induces their proliferation.
  • Said polynucleotide or vector can be introduced into said T cell, and thus said humanized anti-CD28 antibody or antigen-binding fragment thereof is expressed in said T cell, so as to stimulate T cell activation and induce its proliferation.
  • the polynucleotide may be DNA or RNA, such as mRNA.
  • the T cells may be CAR-T cells.
  • a humanized anti-CD28 antibody or antigen-binding fragment thereof of the invention, or a multispecific antibody comprising a humanized anti-CD28 antibody or antigen-binding fragment thereof of the invention may be administered in combination with CAR-T cells Give to subjects to enhance the killing effect of CAR-T cells on target cells.
  • the affinity-matured anti-CD40 antibodies of the present invention can be used to stimulate immune cells (such as dendritic cells, macrophages, NK cells, granulocytes, etc.), and thus can be used to stimulate immune responses, such as activating antigen-presenting cells (APCs) such as dendritic cells.
  • APCs antigen-presenting cells
  • DC Dendritic cells
  • the humanized anti-CD28 antibody, affinity-matured anti-CD40 antibody or antigen-binding fragment thereof of the present invention can form bispecific antibodies or multispecific antibodies with antibodies that specifically bind to tumor cell surface antigens, and activate toxic T cells to kill tumor cells .
  • the present invention provides a method for enhancing the immune response of a subject by using the affinity matured anti-CD40 antibody or antigen-binding fragment thereof of the present invention, or the method of using the affinity matured anti-CD40 antibody or antigen-binding fragment thereof of the present invention for enhancing The application of the drug to the subject's immune response.
  • the method comprises administering to a subject an affinity matured anti-CD40 antibody or an antigen-binding fragment thereof of the invention, a multispecific antibody comprising an affinity-matured anti-CD40 antibody or an antigen-binding fragment thereof of the invention, an affinity-matured anti-CD40 antibody or an antigen-binding fragment thereof encoding the invention
  • the polynucleotide may be DNA or RNA, such as mRNA.
  • the term "enhancing an immune response” refers to any response that stimulates, elicits, increases, modifies or enhances a subject's immune system.
  • the immune response can be a cellular response (ie, cell-mediated, such as a cytotoxic T lymphocyte-mediated response) or a humoral response (ie, an antibody-mediated response), and can be a primary or secondary immune response.
  • Exemplary enhancement of the immune response includes induction of T cell (e.g. CD4+T cell, CD8+T cell) proliferation, stimulation of T cell activation (e.g.
  • CD4+T cell, CD8+T cell activation of resting T lymphocytes
  • B cells activate dendritic cells
  • NK cells increase the killing function of NK cells
  • eosinophils in human peripheral blood Preservation
  • GMCSF macrophage colony-stimulating factor
  • T cells and/or B cells improve antigen presentation by antigen-presenting cells (such as dendritic cells), improve antigen clearance, increase cytokines ( For example interleukin-2, IFN-gamma) production etc.
  • cytokines For example interleukin-2, IFN-gamma
  • the immune response of a subject administered an antibody of the invention is enhanced when compared to the immune response of a subject not administered an antibody of the invention.
  • An anti-CD28 antibody e.g., a humanized anti-CD28 antibody of the invention
  • an anti-CD40 antibody e.g., an affinity matured anti-CD40 antibody of the invention
  • an antigen-binding fragment thereof to form a multispecific antibody, e.g., a bis Specific antibody (hereinafter referred to as CD28-CD40 double antibody).
  • Anti-CD28 antibody or its antigen-binding fragment can be used as an agonist of CD28 molecule, which can provide co-stimulatory signal for T cell activation.
  • Anti-CD40 antibody or its antigen-binding fragment is an agonist of CD40 molecule, which can stimulate various immune cells (such as tree CD28-CD40 double antibody can play an important role in the immune response, for example, it can enhance the immune response of the subject and improve the efficacy of cellular immunotherapy effects, especially promoting the killing effect of CAR-T cells.
  • cellular immunotherapy refers to the use of cells contained in the immune system that fight disease, or cells that help the immune system fight disease, to treat diseases, such as tumors, autoimmune diseases or infectious diseases.
  • Cells that can be used in cellular immunotherapy include but are not limited to T cells, NK cells, dendritic cells, regulatory T cells, cytotoxic T cells (CTL), macrophages and the like.
  • CTL cytotoxic T cells
  • Cellular immunotherapy can isolate autologous immune cells from the subject's blood or tumor, culture these cells in vitro, and then infuse them into the subject to kill tumor cells.
  • immune cells can also be genetically engineered to express tumor-specific receptors, and then cultured and infused back into the patient, such as CAR-T cells.
  • the present invention provides a pharmaceutical combination of the CD28-CD40 bisantibody of the present invention and immune cells, and the pharmaceutical combination can be used to kill target cells in a subject.
  • the CD28-CD40 double antibody of the present invention can enhance the killing effect of the immune cells on target cells.
  • the present invention also provides a method for enhancing the killing effect of immune cells on target cells in a subject, comprising administering the CD28-CD40 double antibody of the present invention and the polynucleotide encoding the double antibody to the subject receiving the immune cells , a carrier comprising the polynucleotide or a pharmaceutical composition comprising the double antibody, the polynucleotide or the carrier.
  • the polynucleotide encoding the CD28-CD40 double antibody of the present invention or the vector comprising the polynucleotide can be introduced into immune cells, so that the immune cells can express the double antibody.
  • the polynucleotide may be DNA or RNA, such as mRNA.
  • the vector may be a plasmid vector or a viral vector, such as a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and the like.
  • the carrier can also be the aforementioned circular RNA, precursor RNA or a DNA template carrier comprising the precursor RNA.
  • the "introduction" can be understood as "transfection".
  • the polynucleotide or vector can be transfected into immune cells by known means such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, virus-like particle infection and the like. Therefore, in some embodiments, immune cells capable of expressing the CD28-CD40 double antibody of the present invention can be administered to a subject to kill target cells in the subject.
  • the present invention also provides immune cells capable of expressing the CD28-CD40 double antibody of the present invention.
  • the term "immune cell” or “immune effector cell” refers to an immune cell that is used in cellular immunotherapy and has a therapeutic effect, such as an immune cell that can kill target cells in cellular immunotherapy, such as T cells, NK cells, dendritic cells, regulatory T cells, cytotoxic T cells (CTL), macrophages, etc.
  • the "target cell” refers to the target cell targeted by cellular immunotherapy.
  • the immune cell comprises or is capable of expressing a CAR.
  • the immune cells are CAR-T cells.
  • the CAR-T cells contain binding domains that can specifically bind target cell surface antigens, and thus can specifically kill target cells.
  • the method of the present invention can enhance the killing effect of immune cells on target cells.
  • the killing effect can be measured, for example, by a reduction in the number of target cells.
  • the target cells are tumor cells.
  • the present invention also provides the CD28-CD40 double antibody of the present invention, the polynucleotide encoding the double antibody, and the vector containing the polynucleotide in the preparation of a drug for enhancing the killing effect of immune cells on target cells in a subject the use of.
  • the present invention also provides genetically engineered immune effector cells capable of expressing the CD28-CD40 double antibody of the present invention, and the immune effector cells can be T cells, NK cells, NKT cells, macrophages , neutrophils or granulocytes.
  • the immune effector cells may comprise the polynucleotide encoding the CD28-CD40 double antibody of the present invention or the vector comprising the polynucleotide.
  • the genetically engineered immune effector cells further recombinantly express a chimeric antigen receptor (CAR), a T cell receptor (TCR) or a bispecific T cell engager (BiTE), wherein the CAR, TCR or BiTE may be a CAR, TCR or BiTE that binds a tumor antigen or a viral antigen.
  • the viral antigens may be selected from HPV, EBV and HIV.
  • the tumor antigen can be selected from Her2, NY-ESO-1, CD19, CD20, CD22, PSMA, c-Met, GPC3, IL13ra2, EGFR, CD123, CD7, GD2, PSCA, EBV16-E7, H3.3, EGFRvIII , BCMA and mesothelin.
  • the CAR, TCR or BiTE binds mesothelin (MESO), CD123, BCMA, HER2, IL13Ra2, B7H3 or CD19.
  • the CAR, TCR or BiTE can bind TSHR, CD19; CD123; CD22; CD30; CD171; CS-1; (EGFRvIII) binding; ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member; B cell maturation antigen; Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); prostate specific Sex membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit ⁇ -2; Mesothelin; Interleukin 11 receptor ⁇ (IL- l l)
  • the genetically engineered immune effector cells comprise a first polynucleotide encoding the CD40-CD28 double antibody of the present invention and a second polynucleotide encoding CAR, TCR or BiTE (CAR/TCR/BiTE). polynucleotide.
  • the first polynucleotide and the second polynucleotide can be in separate tables Expressed in the cassette, or co-expressed in the same cassette. When co-expressed in the same expression cassette, there may be a linker between the first polynucleotide and the second polynucleotide.
  • the linker may be a self-cleaving linker, such as a 2A linker, eg T2A, P2A or F2A.
  • the polynucleotide encoding the CD28-CD40 double antibody of the present invention or the vector containing the polynucleotide can be introduced into immune cells, so that the immune cells can express the double antibody.
  • the first polynucleotide and the second polynucleotide may be DNA or RNA, such as mRNA.
  • the first polynucleotide and the second polynucleotide can be introduced into immune effector cells by any means known in the art, for example, they can be introduced into immune effector cells by vector or mRNA transfection. In effector cells.
  • the vector may be a plasmid vector or a viral vector, such as a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and the like.
  • the carrier can also be the aforementioned circular RNA, precursor RNA or a DNA template carrier comprising the precursor RNA.
  • the CAR-T cell comprises a CAR with a HER2-binding molecule (such as an anti-HER2 antibody) as the binding domain, and targets cells expressing HER2.
  • the CAR-T cells comprise a CAR with a mesothelin-binding molecule (such as an anti-mesothelin antibody) as the binding domain, and target cells expressing mesothelin.
  • the target cells of the CAR-T cells are tumor cells expressing HER2 or mesothelin.
  • a CAR can include a signal peptide, a hinge region, a transmembrane domain, and an intracellular signaling domain.
  • the intracellular signaling domain may further include a co-stimulatory domain.
  • the signal peptide may include a CD8 signal peptide or a GM-CSF signal peptide.
  • the hinge region of the CAR may include CD28, CD8, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8A, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA,
  • the hinge region of GITR, DAP10, TIM1, SLAM, CD30 or LIGHT is preferably the hinge region of CD8.
  • the transmembrane domain of the CAR may include CD8, CD28, CD3 ⁇ (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CTLA-4, LAG-3 , CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, The transmembrane domain of CD9, CD16, CD22, CD33, CD37, CD64 or SLAM, preferably the CD8 transmembrane (TM) domain.
  • TM transmembrane
  • the intracellular signaling domain of the CAR may comprise the intracellular signaling domain of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, DAP10 or DAP-12, preferably CD3 ⁇ Inner signaling domain.
  • the intracellular signaling domain of the CAR may further include costimulatory domains, such as CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, A costimulatory domain of CD100, ICOS, CD40 or MyD88, preferably a 4-1BB costimulatory domain.
  • costimulatory domains such as CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, F
  • a CAR with a "BBZ” structure refers to a CAR with a 4-1BB co-stimulatory molecule, usually including a CD8 hinge domain, a CD8 transmembrane (TM) domain, a 4-1BB co-stimulatory domain, and a CD3 ⁇ domain .
  • the CAR used in the invention has a BBZ structure
  • the CAR comprises an antibody or antigen-binding fragment thereof as a binding domain.
  • the antibody or antigen-binding fragment specifically binds mesothelin (MESO), CD123, BCMA, HER2, IL13Ra2, or B7H3.
  • the antibody is a scFv.
  • the scFv comprises a heavy chain variable region (VH) fused to the N- or C-terminus of the light chain variable region (VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • an antibody that specifically binds mesothelin also known as an anti-mesothelin (or anti-MESO or anti-MSLN) antibody, as in PCT/CN2021/112767 (herein incorporated by reference in its entirety) Described, it comprises light chain variable region and heavy chain variable region, and light chain variable region comprises LCDR1, LCDR2 and LCDR3, and heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 are selected from the following groups:
  • LCDR1 as shown in SEQ ID NO:89
  • LCDR2 as shown in SEQ ID NO:104
  • LCDR3 as shown in SEQ ID NO:118
  • HCDR1 as shown in SEQ ID NO:133
  • SEQ ID HCDR2 shown in NO:146
  • HCDR3 shown in SEQ ID NO:159;
  • LCDR1 as shown in SEQ ID NO:90
  • LCDR2 as shown in SEQ ID NO:105
  • LCDR3 as shown in SEQ ID NO:119
  • HCDR1 as shown in SEQ ID NO:134
  • SEQ ID HCDR2 shown in NO:147
  • HCDR3 shown in SEQ ID NO:160;
  • LCDR1 as shown in SEQ ID NO:91
  • LCDR2 as shown in SEQ ID NO:106
  • LCDR3 as shown in SEQ ID NO:120
  • HCDR1 as shown in SEQ ID NO:135, as SEQ ID HCDR2 shown in NO:148 and HCDR3 shown in SEQ ID NO:161;
  • LCDR1 as shown in SEQ ID NO:92
  • LCDR2 as shown in SEQ ID NO:107
  • LCDR3 as shown in SEQ ID NO:121
  • HCDR1 as shown in SEQ ID NO:136
  • SEQ ID HCDR2 shown in NO:149
  • HCDR3 shown in SEQ ID NO:162;
  • LCDR1 as shown in SEQ ID NO:93
  • LCDR2 as shown in SEQ ID NO:108
  • LCDR3 as shown in SEQ ID NO:122
  • HCDR1 as shown in SEQ ID NO:137
  • SEQ ID HCDR2 shown in NO:150
  • HCDR3 shown in SEQ ID NO:163;
  • LCDR1 as shown in SEQ ID NO:94 LCDR2 as shown in SEQ ID NO:109, LCDR3 as shown in SEQ ID NO:123, HCDR1 as shown in SEQ ID NO:138, as SEQ ID HCDR2 shown in NO:151 and HCDR3 shown in SEQ ID NO:164;
  • LCDR1 as shown in SEQ ID NO:95
  • LCDR2 as shown in SEQ ID NO:110
  • LCDR3 as shown in SEQ ID NO:124
  • HCDR1 as shown in SEQ ID NO:139
  • SEQ ID HCDR2 shown in NO:152
  • HCDR3 shown in SEQ ID NO:165;
  • LCDR1 as shown in SEQ ID NO:97
  • LCDR2 as shown in SEQ ID NO:112
  • LCDR3 as shown in SEQ ID NO:126
  • HCDR1 as shown in SEQ ID NO:140
  • SEQ ID HCDR2 shown in NO:153
  • HCDR3 shown in SEQ ID NO:167;
  • LCDR1 as shown in SEQ ID NO:98
  • LCDR2 as shown in SEQ ID NO:113
  • LCDR3 as shown in SEQ ID NO:127
  • HCDR1 as shown in SEQ ID NO:139
  • SEQ ID HCDR2 shown in NO:152
  • HCDR3 shown in SEQ ID NO:168;
  • LCDR1 as shown in SEQ ID NO:99
  • LCDR2 as shown in SEQ ID NO:114
  • LCDR3 as shown in SEQ ID NO:128,
  • HCDR1 as shown in SEQ ID NO:141, as SEQ ID HCDR2 shown in NO:154 and HCDR3 shown in SEQ ID NO:169;
  • LCDR1 as shown in SEQ ID NO:100
  • LCDR2 as shown in SEQ ID NO:115
  • LCDR3 as shown in SEQ ID NO:129
  • HCDR1 as shown in SEQ ID NO:142
  • SEQ ID HCDR2 shown in NO: 155 and HCDR3 shown in SEQ ID NO: 170
  • LCDR1 as shown in SEQ ID NO:100
  • LCDR2 as shown in SEQ ID NO:115
  • LCDR3 as shown in SEQ ID NO:129
  • HCDR1 as shown in SEQ ID NO:142
  • SEQ ID HCDR2 shown in NO: 155 and HCDR3 shown in SEQ ID NO: 170
  • LCDR1 as shown in SEQ ID NO:101
  • LCDR2 as shown in SEQ ID NO:104
  • LCDR3 as shown in SEQ ID NO:130
  • HCDR1 as shown in SEQ ID NO:143
  • SEQ ID HCDR2 shown in NO: 156
  • HCDR3 shown in SEQ ID NO: 171.
  • an anti-MESO antibody comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:
  • the anti-MESO antibody is an anti-MESO scFv, which can include an amino acid sequence selected from SEQ ID NOs: 202-216.
  • the CAR includes a binding domain that can specifically bind to mesothelin, which can be referred to as a mesothelin-targeted CAR.
  • the CAR targeting mesothelin may be a CAR described in PCT/CN2021/112767 (herein incorporated by reference in its entirety).
  • the mesothelin-targeted CAR can include any of the anti-mesothelin antibodies described above.
  • the mesothelin-targeted CAR may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 217-231.
  • the antibody that specifically binds CD123 is an antibody described in PCT/CN2021/112748 (the entire contents of which are incorporated herein by reference), comprising a light chain variable region and a heavy chain variable region.
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:
  • LCDR1 as shown in SEQ ID NO:349, LCDR2 as shown in SEQ ID NO:379, LCDR3 as shown in SEQ ID NO:407, HCDR1 as shown in SEQ ID NO:437, as SEQ ID HCDR2 shown in NO:458 and HCDR3 shown in SEQ ID NO:482;
  • LCDR1 as shown in SEQ ID NO:363, LCDR2 as shown in SEQ ID NO:391, LCDR3 as shown in SEQ ID NO:421, HCDR1 as shown in SEQ ID NO:435, as SEQ ID HCDR2 shown in NO:468 and HCDR3 shown in SEQ ID NO:495;
  • LCDR1 as shown in SEQ ID NO:353, LCDR2 as shown in SEQ ID NO:383, LCDR3 as shown in SEQ ID NO:412, HCDR1 as shown in SEQ ID NO:441, as SEQ ID HCDR2 shown in NO:462 and HCDR3 shown in SEQ ID NO:486;
  • LCDR1 as shown in SEQ ID NO:364, LCDR2 as shown in SEQ ID NO:378, LCDR3 as shown in SEQ ID NO:422, HCDR1 as shown in SEQ ID NO:446, HCDR2 as shown in SEQ ID NO:469 and HCDR3 as shown in SEQ ID NO:496;
  • LCDR1 as shown in SEQ ID NO:369
  • LCDR2 as shown in SEQ ID NO:39
  • LCDR3 as shown in SEQ ID NO:428,
  • HCDR1 as shown in SEQ ID NO:445, as SEQ ID HCDR2 shown in NO:475 and HCDR3 shown in SEQ ID NO:503;
  • LCDR1 as shown in SEQ ID NO:370
  • LCDR2 as shown in SEQ ID NO:397
  • LCDR3 as shown in SEQ ID NO:429
  • HCDR1 as shown in SEQ ID NO:452
  • SEQ ID HCDR2 shown in NO:474
  • HCDR3 shown in SEQ ID NO:504;
  • LCDR1 as shown in SEQ ID NO:354, LCDR2 as shown in SEQ ID NO:384, LCDR3 as shown in SEQ ID NO:413, HCDR1 as shown in SEQ ID NO:442, as SEQ ID HCDR2 shown in NO:463 and HCDR3 shown in SEQ ID NO:487;
  • LCDR1 as shown in SEQ ID NO:350
  • LCDR2 as shown in SEQ ID NO:380
  • LCDR3 as shown in SEQ ID NO:408,
  • HCDR1 as shown in SEQ ID NO:435, as SEQ ID HCDR2 shown in NO:456 and HCDR3 shown in SEQ ID NO:480;
  • LCDR1 as shown in SEQ ID NO:371, LCDR2 as shown in SEQ ID NO:398, LCDR3 as shown in SEQ ID NO:430, HCDR1 as shown in SEQ ID NO:453, as SEQ ID HCDR2 shown in NO:476 and HCDR3 shown in SEQ ID NO:505;
  • LCDR1 as shown in SEQ ID NO:372, LCDR2 as shown in SEQ ID NO:399, LCDR3 as shown in SEQ ID NO:431, HCDR1 as shown in SEQ ID NO:441, as SEQ ID HCDR2 shown in NO:462 and HCDR3 shown in SEQ ID NO:506;
  • LCDR1 as shown in SEQ ID NO:348, LCDR2 as shown in SEQ ID NO:378, LCDR3 as shown in SEQ ID NO:406, HCDR1 as shown in SEQ ID NO:436, as SEQ ID HCDR2 shown in NO:457 and HCDR3 shown in SEQ ID NO:481;
  • LCDR1 as shown in SEQ ID NO:363, LCDR2 as shown in SEQ ID NO:392, LCDR3 as shown in SEQ ID NO:423, HCDR1 as shown in SEQ ID NO:447, as SEQ ID HCDR2 shown in NO:470 and HCDR3 shown in SEQ ID NO:497;
  • LCDR1 as shown in SEQ ID NO:373, LCDR2 as shown in SEQ ID NO:400, LCDR3 as shown in SEQ ID NO:432, HCDR1 as shown in SEQ ID NO:451, as SEQ ID HCDR2 shown in NO:477 and HCDR3 shown in SEQ ID NO:507;
  • LCDR1 as shown in SEQ ID NO:355, LCDR2 as shown in SEQ ID NO:385, LCDR3 as shown in SEQ ID NO:414, HCDR1 as shown in SEQ ID NO:443, as SEQ ID HCDR2 shown in NO:464 and HCDR3 shown in SEQ ID NO:488;
  • LCDR1 as shown in SEQ ID NO:356, LCDR2 as shown in SEQ ID NO:386, LCDR3 as shown in SEQ ID NO:415, HCDR1 as shown in SEQ ID NO:444, as SEQ ID HCDR2 shown in NO:465 and HCDR3 shown in SEQ ID NO:489;
  • LCDR1 as shown in SEQ ID NO:368, LCDR2 as shown in SEQ ID NO:395, LCDR3 as shown in SEQ ID NO:427, HCDR1 as shown in SEQ ID NO:451, HCDR2 as shown in SEQ ID NO:474 and HCDR3 as shown in SEQ ID NO:502;
  • LCDR1 as shown in SEQ ID NO:351, LCDR2 as shown in SEQ ID NO:381, LCDR3 as shown in SEQ ID NO:409, HCDR1 as shown in SEQ ID NO:438, as SEQ ID HCDR2 shown in NO:459 and HCDR3 shown in SEQ ID NO:483;
  • LCDR1 as shown in SEQ ID NO:347, LCDR2 as shown in SEQ ID NO:376, LCDR3 as shown in SEQ ID NO:410, HCDR1 as shown in SEQ ID NO:439, as SEQ ID HCDR2 shown in NO:460 and HCDR3 shown in SEQ ID NO:484;
  • LCDR1 as shown in SEQ ID NO:357
  • LCDR2 as shown in SEQ ID NO:387
  • LCDR3 as shown in SEQ ID NO:416, HCDR1 as shown in SEQ ID NO:437, as SEQ ID HCDR2 shown in NO:466 and HCDR3 shown in SEQ ID NO:490;
  • LCDR1 as shown in SEQ ID NO:347, LCDR2 as shown in SEQ ID NO:377, LCDR3 as shown in SEQ ID NO:405, HCDR1 as shown in SEQ ID NO:435, as SEQ ID HCDR2 shown in NO:456 and HCDR3 shown in SEQ ID NO:480;
  • LCDR1 as shown in SEQ ID NO:374, LCDR2 as shown in SEQ ID NO:401, LCDR3 as shown in SEQ ID NO:433, HCDR1 as shown in SEQ ID NO:454, as SEQ ID HCDR2 shown in NO:478 and HCDR3 shown in SEQ ID NO:508;
  • LCDR1 as shown in SEQ ID NO:358, LCDR2 as shown in SEQ ID NO:385, LCDR3 as shown in SEQ ID NO:417, HCDR1 as shown in SEQ ID NO:442, as SEQ ID HCDR2 shown in NO:463 and HCDR3 shown in SEQ ID NO:491;
  • LCDR1 as shown in SEQ ID NO:359
  • LCDR2 as shown in SEQ ID NO:38
  • LCDR3 as shown in SEQ ID NO:41
  • HCDR1 as shown in SEQ ID NO:441, as SEQ ID HCDR2 shown in NO:462 and HCDR3 shown in SEQ ID NO:492;
  • LCDR1 as shown in SEQ ID NO:366, LCDR2 as shown in SEQ ID NO:394, LCDR3 as shown in SEQ ID NO:425, HCDR1 as shown in SEQ ID NO:449, as SEQ ID HCDR2 shown in NO:472 and HCDR3 shown in SEQ ID NO:499;
  • LCDR1 as shown in SEQ ID NO:360
  • LCDR2 as shown in SEQ ID NO:389
  • LCDR3 as shown in SEQ ID NO:413,
  • HCDR1 as shown in SEQ ID NO:442
  • SEQ ID HCDR2 shown in NO:463
  • HCDR3 shown in SEQ ID NO:487;
  • LCDR1 as shown in SEQ ID NO:347, LCDR2 as shown in SEQ ID NO:376, LCDR3 as shown in SEQ ID NO:404, HCDR1 as shown in SEQ ID NO:435, HCDR2 as shown in SEQ ID NO:456 and HCDR3 as shown in SEQ ID NO:480;
  • LCDR1 as shown in SEQ ID NO:348, LCDR2 as shown in SEQ ID NO:378, LCDR3 as shown in SEQ ID NO:406, HCDR1 as shown in SEQ ID NO:436, as SEQ ID HCDR2 shown in NO:457 and HCDR3 shown in SEQ ID NO:481;
  • LCDR1 as shown in SEQ ID NO:361, LCDR2 as shown in SEQ ID NO:390, LCDR3 as shown in SEQ ID NO:419, HCDR1 as shown in SEQ ID NO:445, as SEQ ID HCDR2 shown in NO:467 and HCDR3 shown in SEQ ID NO:493;
  • LCDR1 as shown in SEQ ID NO:375
  • LCDR2 as shown in SEQ ID NO:402
  • LCDR3 as shown in SEQ ID NO:434
  • HCDR1 as shown in SEQ ID NO:455, as SEQ ID HCDR2 shown in NO:479 and HCDR3 shown in SEQ ID NO:509;
  • LCDR1 as shown in SEQ ID NO:368, LCDR2 as shown in SEQ ID NO:395, LCDR3 as shown in SEQ ID NO:427, HCDR1 as shown in SEQ ID NO:450, as SEQ ID HCDR2 shown in NO:473 and HCDR3 shown in SEQ ID NO:5015;
  • LCDR1 as shown in SEQ ID NO:362, LCDR2 as shown in SEQ ID NO:388, LCDR3 as shown in SEQ ID NO:420, HCDR1 as shown in SEQ ID NO:441, as SEQ ID HCDR2 shown in NO:462 and HCDR3 shown in SEQ ID NO:494;
  • LCDR1 as shown in SEQ ID NO:352
  • LCDR2 as shown in SEQ ID NO:382
  • LCDR3 as shown in SEQ ID NO:411, HCDR1 as shown in SEQ ID NO:440, as SEQ ID HCDR2 shown in NO:461 and HCDR3 shown in SEQ ID NO:485;
  • LCDR1 as shown in SEQ ID NO:367
  • LCDR2 as shown in SEQ ID NO:391
  • LCDR3 as shown in SEQ ID NO:426,
  • HCDR1 as shown in SEQ ID NO:435, as SEQ ID HCDR2 shown in NO:468 and HCDR3 shown in SEQ ID NO:500.
  • an anti-CD123 antibody comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:
  • (21) a light chain variable region as shown in SEQ ID NO:530 and a heavy chain variable region as shown in SEQ ID NO:565;
  • the anti-CD123 antibody is an anti-CD123 scFv, which may include any amino acid sequence selected from SEQ ID NO:583-617.
  • the CAR includes a binding domain that can specifically bind to CD123, which can be referred to as a CD123-targeting CAR.
  • the CAR targeting CD123 may be a CAR described in PCT/CN2021/112748 (herein incorporated by reference in its entirety).
  • the CD123-targeting CAR can include any of the anti-CD123 antibodies described above.
  • the CAR targeting CD123 may comprise an amino acid sequence selected from SEQ ID NOs: 580-582.
  • an antibody that specifically binds BCMA also referred to as an anti-BCMA antibody, as described in PCT/CN2021/112798 (which is incorporated herein by reference in its entirety), comprises a light chain variable region and a heavy chain variable region , the light chain variable region includes LCDR1, LCDR2 and LCDR3, the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, wherein LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 are selected from the group consisting of:
  • LCDR1 as shown in SEQ ID NO:232
  • LCDR2 as shown in SEQ ID NO:242
  • LCDR3 as shown in SEQ ID NO:253
  • HCDR1 as shown in SEQ ID NO:264
  • SEQ ID HCDR2 shown in NO:275
  • HCDR3 shown in SEQ ID NO:287;
  • LCDR1 as shown in SEQ ID NO:234, LCDR2 as shown in SEQ ID NO:244, LCDR3 as shown in SEQ ID NO:255, HCDR1 as shown in SEQ ID NO:266, as SEQ ID HCDR2 shown in NO:277 and HCDR3 shown in SEQ ID NO:289;
  • LCDR1 as shown in SEQ ID NO:235, LCDR2 as shown in SEQ ID NO:245, LCDR3 as shown in SEQ ID NO:255, HCDR1 as shown in SEQ ID NO:267, as SEQ ID HCDR2 shown in NO:278 and HCDR3 shown in SEQ ID NO:290;
  • LCDR1 as shown in SEQ ID NO:236, LCDR2 as shown in SEQ ID NO:246, LCDR3 as shown in SEQ ID NO:256, HCDR1 as shown in SEQ ID NO:268, as SEQ ID HCDR2 shown in NO:279 and HCDR3 shown in SEQ ID NO:291;
  • LCDR1 as shown in SEQ ID NO:237, LCDR2 as shown in SEQ ID NO:247, LCDR3 as shown in SEQ ID NO:257, HCDR1 as shown in SEQ ID NO:269, as SEQ ID HCDR2 shown in NO:280 and HCDR3 shown in SEQ ID NO:292;
  • LCDR1 as shown in SEQ ID NO:238, LCDR2 as shown in SEQ ID NO:248, LCDR3 as shown in SEQ ID NO:258, HCDR1 as shown in SEQ ID NO:266, as SEQ ID HCDR2 shown in NO:281 and HCDR3 shown in SEQ ID NO:293;
  • LCDR1 as shown in SEQ ID NO:239, LCDR2 as shown in SEQ ID NO:249, LCDR3 as shown in SEQ ID NO:259, HCDR1 as shown in SEQ ID NO:270, as SEQ ID HCDR2 shown in NO:282 and HCDR3 shown in SEQ ID NO:294;
  • LCDR1 as shown in SEQ ID NO:240, LCDR2 as shown in SEQ ID NO:252, LCDR3 as shown in SEQ ID NO:262, HCDR1 as shown in SEQ ID NO:273, as SEQ ID HCDR2 shown in NO:285 and HCDR3 shown in SEQ ID NO:297;
  • LCDR1 as shown in SEQ ID NO:241, LCDR2 as shown in SEQ ID NO:245, LCDR3 as shown in SEQ ID NO:263, HCDR1 as shown in SEQ ID NO:274, as SEQ ID NO:274 HCDR2 as shown in ID NO:286 and HCDR3 as shown in SEQ ID NO:298.
  • an anti-BCMA antibody comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:
  • the anti-BCMA antibody is an anti-BCMA scFv, which may comprise an amino acid sequence selected from SEQ ID NOs: 323-334.
  • the CAR includes a binding domain that can specifically bind to BCMA, which can be referred to as a BCMA-targeted CAR.
  • the CAR targeting BCMA may be a CAR described in PCT/CN2021/112798 (herein incorporated by reference in its entirety).
  • the BCMA-targeted CAR may comprise any of the anti-BCMA antibodies described above.
  • the BCMA-targeted CAR may comprise an amino acid sequence selected from SEQ ID NOs: 335-346.
  • an antibody that specifically binds CD19 also known as an anti-CD19 antibody, such as Chinese patent application CN202210274255.1 (published as CN114349863A, which is hereby incorporated by reference in its entirety), includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 , the heavy chain variable region includes HCDR1, HCDR2 and HCDR3.
  • LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 can be LCDR1 as shown in SEQ ID NO:628, LCDR2 as shown in SEQ ID NO:629, LCDR3 as shown in SEQ ID NO:630, such as SEQ ID HCDR1 as shown in NO:631, HCDR2 as shown in SEQ ID NO:632 and HCDR3 as shown in SEQ ID NO:633.
  • an anti-CD19 antibody comprises a light chain variable region set forth in SEQ ID NO:635 and a heavy chain variable region set forth in SEQ ID NO:634.
  • the anti-CD19 antibody is an anti-CD19 scFv, which can include the amino acid sequence set forth in SEQ ID NO:636.
  • the CAR includes a binding domain that can specifically bind to CD19, which can be referred to as a CD19-targeting CAR.
  • the CAR targeting CD19 may be the CAR described in Chinese patent application CN202210274255.1 (publication number CN114349863A, which is hereby incorporated by reference in its entirety).
  • the CD19-targeting CAR can include any of the anti-CD19 antibodies described above.
  • the CAR targeting CD19 may comprise the amino acid sequence shown in SEQ ID NO: 637.
  • antibodies specifically binding to HER2 also known as anti-HER2 antibodies, as described in Chinese patent application CN202210750853.1 (publication number CN114805584A, which is hereby incorporated by reference in its entirety), include light Chain variable region and heavy chain variable region, light chain variable region includes LCDR1, LCDR2 and LCDR3, heavy chain variable region includes HCDR1, HCDR2 and HCDR3.
  • LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 can be LCDR1 as shown in SEQ ID NO:618, LCDR2 as shown in SEQ ID NO:619, LCDR3 as shown in SEQ ID NO:620, as SEQ ID HCDR1 as shown in NO:621, HCDR2 as shown in SEQ ID NO:622 and HCDR3 as shown in SEQ ID NO:623.
  • an anti-HER2 antibody comprises a light chain variable region set forth in SEQ ID NO:624 and a heavy chain variable region set forth in SEQ ID NO:625.
  • the anti-HER2 antibody is an anti-HER2 scFv, which may include the amino acid sequence set forth in SEQ ID NO:626.
  • the CAR includes a binding domain that can specifically bind to HER2, which can be referred to as a HER2-targeted CAR.
  • the CAR targeting HER2 may be the CAR described in Chinese patent application CN202210750853.1 (publication number CN114805584A, which is hereby incorporated by reference in its entirety).
  • the HER2-targeted CAR may include any of the anti-HER2 antibodies described above.
  • the HER2-targeted CAR may comprise the amino acid sequence shown in SEQ ID NO: 627.
  • antibodies specifically binding to IL13Ra2 also known as anti-IL13Ra2 antibodies, as described in Chinese patent application CN202210743595.4 (published as CN114805581A, which is hereby incorporated by reference in its entirety), include light Chain variable region and heavy chain variable region, light chain variable region includes LCDR1, LCDR2 and LCDR3, heavy chain variable region includes HCDR1, HCDR2 and HCDR3.
  • That LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 can be LCDR1 as shown in SEQ ID NO:638, LCDR2 as shown in SEQ ID NO:639, LCDR3 as shown in SEQ ID NO:640, such as SEQ ID HCDR1 shown in NO:641, HCDR2 shown in SEQ ID NO:642 and HCDR3 shown in SEQ ID NO:642.
  • an anti-IL13Ra2 antibody comprises a light chain variable region set forth in SEQ ID NO:644 and a heavy chain variable region set forth in SEQ ID NO:645.
  • the anti-IL13Ra2 antibody is an anti-IL13Ra2 scFv, which can include the amino acid sequence set forth in SEQ ID NO:646.
  • the CAR includes a binding domain that can specifically bind IL13Ra2, which can be referred to as a CAR targeting IL13Ra2.
  • the CAR targeting IL13Ra2 may be the CAR described in Chinese patent application CN202210743595.4 (publication number CN114805581A, which is hereby incorporated by reference in its entirety).
  • the CAR targeting IL13Ra2 may comprise any of the anti-IL13Ra2 antibodies described above.
  • the CAR targeting IL13Ra2 may comprise the amino acid sequence shown in SEQ ID NO: 647.
  • antibodies specifically binding to B7H3, also known as anti-B7H3 antibodies include light Chain variable region and heavy chain variable region, light chain variable region includes LCDR1, LCDR2 and LCDR3, heavy chain variable region includes HCDR1, HCDR2 and HCDR3.
  • LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 can be LCDR1 as shown in SEQ ID NO:648, LCDR2 as shown in SEQ ID NO:649, LCDR3 as shown in SEQ ID NO:650, as SEQ ID HCDR1 as shown in NO:651, HCDR2 as shown in SEQ ID NO:652 and HCDR3 as shown in SEQ ID NO:653.
  • an anti-B7H3 antibody comprises a light chain variable region set forth in SEQ ID NO:654 and a heavy chain variable region set forth in SEQ ID NO:655.
  • the anti-B7H3 antibody is an anti-B7H3 scFv, which may include the amino acid sequence set forth in SEQ ID NO:656.
  • the CAR includes a binding domain that can specifically bind to B7H3, which can be referred to as a B7H3-targeted CAR.
  • the CAR targeting B7H3 may be the CAR described in Chinese patent application CN202210714289.8 (publication number CN114773477A, which is hereby incorporated by reference in its entirety).
  • the CAR targeting B7H3 may include any of the anti-B7H3 antibodies described above.
  • the CAR targeting B7H3 may comprise the amino acid sequence shown in SEQ ID NO: 657.
  • the carrier of the polynucleotide or the immune effector cells expressing the CD28-CD40 double antibody can be used as medicine for treating tumor, cancer, virus infection or autoimmune disease, etc., and can be prepared as a pharmaceutical composition.
  • the humanized anti-CD28 antibody, affinity-matured anti-CD40 antibody or antigen-binding fragment thereof, multispecific antibody comprising them, CD28-CD40 double antibody, polynucleotides encoding them or vectors comprising the polynucleotides may be Used in combination with immune effector cells.
  • the immune effector cells Can be selected from CAR T cells, TCR T cells, TIL, CIK, LAK and MIL.
  • the present invention provides methods for treating diseases, comprising humanized anti-CD28 antibodies, affinity-matured anti-CD40 antibodies or antigen-binding fragments thereof, multispecific antibodies comprising them, CD28-CD40 double antibodies, encoding Their polynucleotides, vectors containing the polynucleotides or immune effector cells expressing CD28-CD40 double antibodies are administered to subjects in need of treatment.
  • the disease may be tumor, cancer, viral infection or autoimmune disease, etc.
  • the treatment method may include: (a) humanized anti-CD28 antibody, affinity matured anti-CD40 antibody or antigen-binding fragment thereof, multispecific antibody comprising them, CD28-CD40 double antibody, Polynucleotides encoding them, vectors comprising the polynucleotides are administered to a subject in need of treatment, and (b) immune cell therapy is administered to the subject.
  • steps (a) and (b) can be carried out simultaneously or sequentially, wherein step (a) can be carried out before or after step (b).
  • the immune cell treatment in the step (b) may include CAR T treatment, TCRT treatment, TIL treatment, CIK treatment, LAK treatment and MIL treatment.
  • the cancer is a solid tumor or a hematological cancer (eg, leukemia).
  • the cancer is acute myelogenous leukemia (AML), B-type acute lymphoblastic leukemia (B-ALL), T-type acute lymphoblastic leukemia (T-ALL), B-cell precursor acute lymphoblastic leukemia (BCP -ALL) or blastic plasmacytoid dendritic cell neoplasm (BPDCN).
  • the disease eg, cancer
  • the disease is characterized by disease cells expressing mesothelin, CD123, BCMA, HER2, CD19, IL13Ra2, and/or B7H3.
  • the cancer is a CD123 expressing cancer.
  • the cancer is CD123 expressing AML.
  • the cancer is mesothelioma.
  • the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, or pericardial mesothelioma.
  • the cancer is pancreatic cancer.
  • the pancreatic cancer is pancreatic ductal carcinoma.
  • the cancer is ovarian cancer.
  • the cancer is epithelial ovarian cancer.
  • the cancer is lung cancer.
  • the cancer is non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, human B cell precursor leukemia, multiple myeloma, malignant lymphoma.
  • the cancer is breast cancer, gastric cancer, ovarian cancer, cervical cancer, urothelial cancer, esophageal cancer, bladder cancer, colorectal cancer, endometrial cancer, kidney cancer, lung cancer, pancreatic cancer, head and neck cancer, Sarcoma, glioblastoma, prostate cancer, or thyroid cancer.
  • the cancer is glioma or head and neck cancer.
  • subject refers to any organism to which an antibody or antigen-binding fragment thereof of the invention may be administered, eg, for experimental, diagnostic, prophylactic and/or therapeutic purposes.
  • Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates such as chimpanzees and other apes, and humans).
  • a subject can be a mammal, particularly a human, including female (female) or male (male), and includes newborns, infants, juveniles, youth, adults, or elderly, and further includes various races and nationalities.
  • a subject refers to an individual in need of diagnosis, treatment, or prevention of a disease or disorder that the subject may have, or be at risk of.
  • treating means providing a beneficial or desired clinical result for a disease, such as eliminating the disease, alleviating symptoms, reducing the extent of the disease, stabilizing, ameliorating or relieving the state of the disease, or slowing the progression of the disease.
  • Measures of treatment outcome can be based, for example, on the results of physical examinations, pathological tests, and/or diagnostic tests known in the art.
  • Treatment can also refer to prolonging the survival of a subject as compared to the expected survival of a subject if not receiving treatment. Treating can also refer to reducing the incidence or incidence of a disease, or its recurrence, as compared to what would have occurred in the absence of the measure. Clinically, this treatment can also be called prevention.
  • a pharmaceutical composition may contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to any carrier contained in a pharmaceutical composition as an inactive ingredient, which imparts to the pharmaceutical composition an appearance and properties suitable for administration.
  • Pharmaceutically acceptable carriers such as stabilizers, diluents, additives, adjuvants, excipients, etc., basically have no long-term or permanent adverse effects when administered to a subject.
  • pharmaceutically acceptable carrier shall be a pharmaceutically inert material, substantially devoid of biological activity, and constituting the major part of the formulation.
  • the pharmaceutical composition, pharmaceutical combination, and immune cells (including CAR-T cells) of the present invention can be formulated into various administration methods according to known techniques. See, for example, Remington, The Science and Practice of Pharmacy (9th Ed. 1995).
  • the active agent is usually mixed with a pharmaceutically acceptable carrier and the like.
  • a pharmaceutically acceptable carrier must be acceptable, ie compatible with any other ingredients of the formulation, and must not be injurious to the subject.
  • Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers, preservatives, wetting agents, surfactants, emulsifying agents, or combinations thereof.
  • buffers include, but are not limited to, acetic acid, citric acid, histidine, boric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphoric acid Salt buffered saline etc.
  • the pharmaceutical composition, pharmaceutical combination, and immune cells (including CAR-T cells) of the present invention can be administered in any manner suitable for the disease to be treated (or prevented) and the subject.
  • modes of administration may include, but are not limited to, parenteral or non-parenteral routes, including oral, sublingual, buccal, transdermal, rectal, vaginal, intradermal, intranasal or parenteral routes, such as Intravenous (i.v.), intraperitoneal, intradermal, subcutaneous, intramuscular, intracranial, intrathecal, intratumoral, percutaneous, intramucosal, intraarticular, intrathecal, intrathecal, intrahepatic, intraneural, or intracranial Injection or infusion.
  • the pharmaceutical composition can be injected directly into a tumor, lymph node, tissue, organ or site of infection.
  • Dosage forms suitable for oral administration include, but are not limited to, tablets, capsules, powders, pills, granules, suspensions, solutions or pre-concentrates of solutions, emulsions or pre-concentrates of emulsions.
  • Pharmaceutically acceptable carriers that can be used in oral dosage forms include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like. Carriers such as starch, sugar, microcrystalline cellulose, diluents, fillers, lubricants, granulating agents, lubricants, binders, stabilizers, disintegrants, etc. can be used to prepare oral solid preparations, such as powders, capsules or tablets agent.
  • Dosage forms suitable for parenteral administration include, but are not limited to, sterile liquid preparations, such as isotonic aqueous solutions, emulsions, suspensions, dispersions or viscous compositions, buffered to the desired pH.
  • Parenteral dosage forms can be either ready to use or a dry product ready to be dissolved or suspended in a pharmaceutically acceptable carrier.
  • Parenteral dosage forms can be formulated sterile, or capable of being sterilized prior to administration to a subject.
  • Pharmaceuticals that can be used to provide parenteral dosage forms Acceptable carriers include, but are not limited to, water for injection; aqueous carriers, such as but not limited to, Sodium Chloride Injection, Ringer's Injection, and Dextrose Injection.
  • Water-soluble carriers such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol
  • non-aqueous carriers such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate esters
  • solubilizers such as cyclodextrins.
  • the pharmaceutical composition, pharmaceutical combination, and immune cells (including CAR-T cells) of the present invention are administered to a subject in a therapeutically effective amount.
  • therapeutically effective amount and “effective amount” are used interchangeably and refer to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic effect.
  • a therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and body weight of the individual, as well as the ability of a treatment or combination of treatments to elicit a desired response in the individual.
  • An effective amount can refer to an amount that causes a detectable change in biological or chemical activity. Detectable changes can be detected and/or further quantified by one in the relevant art. Additionally, an "effective amount” can designate an amount that maintains a desired physiological state, ie reduces or prevents a significant decline and/or promotes improvement of the condition.
  • the amount and frequency of administration will be determined by factors such as the subject's condition (such as age, weight, sex, and subject's response to the drug) and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials .
  • the pharmaceutical composition, drug combination, and immune cells (including CAR-T cells) of the present invention can be administered to a subject at a therapeutically effective amount of about 0.5 to about 250 mg/kg, for example, about 1 to about 250 mg/kg, about 2 to about 250 mg/kg. About 200 mg/kg, about 3 to about 120 mg/kg, about 5 to about 250 mg/kg, about 10 to about 200 mg/kg, or about 20 to about 120 mg/kg.
  • the effective amount of immune cells can be, for example, 5 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 5 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 5 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 or 5 ⁇ 10 9 cells.
  • the pharmaceutical composition, drug combination, and immune cells (including CAR-T cells) of the present invention can be administered once or twice a day; or once every 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, Every 1, 2, 3, 4, 5, or 6 weeks, or every 1, 2, 3, 4, 5, or 6 months or more.
  • the pharmaceutical composition can also be administered several times a week (such as 1, 2, 3, 4 or 5 times) or several times a month (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times) regimen of dosing.
  • the pharmaceutical composition may be administered once a day for 5 consecutive days, followed by two consecutive days of rest.
  • drug combination refers to a combination of two or more drugs administered to a subject within a period of time, such as at the same time, or with a difference of minutes to hours, or even a difference of days or weeks. combination.
  • the two or more drugs may be formulated into the same pharmaceutical preparation, or may also be formulated into different pharmaceutical preparations respectively.
  • the pharmaceutical composition, pharmaceutical combination, and immune cells (including CAR-T cells) of the present invention can be used in combination with other drugs and treatment methods, such as other anti-tumor drugs, chemotherapy or radiotherapy.
  • “Used in combination” as used herein refers to administering two (or more) different drugs and/or therapies to a subject during the treatment of the subject.
  • the two or more drugs and/or therapies in the combination may be administered by different routes and schedules.
  • Two or more drugs and/or treatments may be administered to the subject simultaneously or sequentially. in some In embodiments, administration of one drug or therapy is still in progress when administration of a second drug or therapy begins, and thus there is overlap in administration.
  • Such a scheme may be referred to herein as "simultaneously.”
  • two or more drugs and/or therapies may be formulated together in a single dosage form, or separately as two or more separate dosage forms.
  • the CDRs of the antibody heavy chain variable region and light chain variable region are delineated by Kabat nomenclature. Unless otherwise specified, the methods and materials in the examples described below are conventional products that can be purchased from the market.
  • Homology modeling also known as “comparative modeling” is based on the principle that "if two proteins have a sufficiently high sequence similarity, they are likely to have very similar three-dimensional structures", predicting unknowns based on the sequence homology of known structures protein structure.
  • the accuracy of the prediction model can reach 80%; if the homology reaches 50%, the accuracy of the model can reach 95%; if the homology reaches more than 70% , the prediction model is considered to fully represent the real structure of the protein. But there are also special cases where the structures are different although the sequence homology is high.
  • the structure of the murine CD28 antibody 9.3 was predicted by means of homology modeling. For some antibody structures with low homology, ab initio modeling is used to build the model. The approximate experimental steps are as follows:
  • Use Discovery Studio and The Antibody Modeling software uses homology modeling to model the murine CD28 antibody 9.3, and selects 5-10 optimal structural analysis models from the prediction models.
  • the structure of the Loop region of the antibody is first predicted by the method of homology modeling. If the amino acid sequence homology between the CDR region of the 9.3 antibody and the CDR region of the template antibody is less than 50%, the method of de novo modeling is used to build the CDR region structure model.
  • the optimal structure model is shown in Figure 1.
  • the sequence of the light and heavy chains of the 9.3 antibody has more than 90% homology with the template sequence in the PDB structure database, which is a common antibody structure, and the reliability of the prediction model exceeds 95%.
  • the charge distribution on the antibody surface is uniform and the possibility of forming aggregates is low.
  • the heavy chain VH selects IGHV4 with the highest degree of homology as the humanized design template, and designs 5 sequences: VH1-VH5.
  • IGKV1 was selected as the humanized design template, and three sequences were designed: VL1-VL3 ( Figure 2A and B, where Parental is the VH and VL sequences of the 9.3 antibody, that is, the mVH and mVL sequences in Table 1).
  • IGHV4-KV1 is the most common heavy chain-light chain pairing method, IGHV4-KV1 is selected as the corresponding light and heavy chain pairing method.
  • VH is predicted to have low immunogenicity, and the peptide with immunogenicity is: FLKMNSLQA. It is predicted that VL has low immunogenicity, and the immunogenic peptide is: LLIFAASNV.
  • Embodiment 3 SDS-PAGE (SDS-polyacrylamide gel electrophoresis) identification humanized anti Molecular weight and purity of body
  • the molecular weight and purity of the full-length humanized antibody (IgG1) under non-reducing and reducing conditions were detected by SDS-PAGE electrophoresis, so as to facilitate the development of later production processes.
  • Sample preparation first measure the A280 of the protein sample with a Nano-300 micro-spectrophotometer (Hangzhou Aosheng Instrument Co., Ltd.), and calculate the protein concentration.
  • Non-reducing samples take 1 ⁇ g of protein, add 4 ⁇ LDS sample buffer, 40mM iodoacetamide, and incubate at 75°C for 10min.
  • Reduction sample Take 2 ⁇ g protein, add 4 ⁇ LDS sample buffer, 5mM DTT (Dithiothreitol, dithiothreitol), and incubate at 100°C for 10min. Electrophoresis at 140V for 50min.
  • the gel was stained with Coomassie Brilliant Blue R250, and scanned with EPSON V550 color scanner after decolorization.
  • Embodiment 4 SEC (Size exclusion chromatography, molecular sieve chromatography) analysis humanization Antibody purity
  • the purity of the antibody was identified by detecting the ratio of monomers and aggregates of the humanized antibody under non-reducing conditions by SEC.
  • the purity of the reference product Herceptin monomer is greater than 95%, and the separation degree of BSA monomer and dimer is greater than 2, which is regarded as a stable baseline.
  • Embodiment 5 ELISA (enzyme linked immunosorbent assay, enzyme-linked immunosorbent assay) Detection of binding activity of humanized antibodies
  • the binding activity of the humanized antibody to the antigen CD28 was detected by ELISA method.
  • the experimental steps are as follows:
  • Embodiment 6 BLI detects the affinity of humanized antibody
  • Biolayer interferometry was used to detect the affinity kinetics of humanized antibodies.
  • the experimental steps are as follows:
  • Embodiment 7 Auxiliary killing function detection
  • the humanized antibody huVH1huVL1 (abbreviated as 9.3h11) with the highest affinity was selected, and its VH and VL were combined with CD40 antibody 40.52 to form a bispecific antibody (bispecific antibody) in the form of scFv series. , referred to as double antibody or BsAb), two different scFvs are connected by GGGGS (abbreviated as G 4 S). VH and VL in each scFv are linked by 3 G 4 S (GGGGSGGGGSGGGGS). According to the front and rear positions of VH-VL and the difference between the front and rear positions of CD28 antibody and CD40 antibody, there are 8 construction modes of double antibodies (see Table 7), and these 8 double antibodies are named 4.1-4.8 respectively.
  • the anti-killing function of the double antibody 4.1-4.8 was detected by Her2-CAR-T cells, and compared with the parental 8.6 (see Table 7 for the construction mode, which is also in scFv tandem form), 1412-4D11 double antibody and A40C28 (LACO) Compare.
  • the experimental steps for the preparation of Her2-CAR-T cells and the anti-killing function of double antibodies are as follows:
  • CAR-T cells co-electroporate 0.5 ⁇ g Her2-CAR (4D5.BBZ) mRNA and 15 ⁇ g BsAb mRNA, or 5 ⁇ g A40C28 (see Table 8) to CD3 + T cells, and the electroporation conditions are 500V, 0.7ms, as Her2-CAR-T cells.
  • Her2-CAR (4D5.BBZ) is a chimeric antigen receptor (CAR), which contains anti-HER2 scFv, CD8 hinge domain, CD8 transmembrane (TM) domain, 4-1BB co-stimulatory domain and CD3 ⁇ , wherein The VH and VL in the anti-HER2 scFV are the same as the well-known anti-HER2 murine antibody 4D5.
  • the expression level of the double antibody was detected by flow cytometry, CD40-Fc and PE-anti-Fc antibody ( Figure 7); the expression level of Her2-CAR was detected by Her2-Fc and PE-anti-Fc antibody ( Figure 7). 8).
  • the experimental results showed that the expression levels of the double antibody and Her2-CAR were relatively high.
  • A549-no EP A549-CBG cells
  • A549-Meso electroporate 10 ⁇ g Meso (mesothelin) mRNA
  • A549-Meso+CD40 10 ⁇ g Meso and 10 ⁇ g CD40 mRNA
  • the electroporation condition is 300V, 0.5ms.
  • CD40 expression levels were detected by flow cytometry and PE-anti-CD40 antibody.
  • Figure 11 shows that the expression level of CD40 on the surface of A549 cells is higher.
  • the expression level of Meso was detected by flow cytometry analysis, Meso-biotin and PE-streptavidin antibodies.
  • Figure 12 shows that the expression level of Meso on the surface of A549 cells is relatively high, and the high expression of Meso also promotes the upregulation of the expression of CD40.
  • CAR-T cells co-electrotransfer 2 ⁇ g Meso-CAR (M12) to CD3 + T cells mRNA and 15 ⁇ g of BsAb mRNA (see Table 9), electroporation conditions of 500V, 0.7ms, as Meso-CAR-T cells.
  • Meso-CAR (M12) is a chimeric antigen receptor (CAR), which contains anti-mesothelin scFV (M12), CD8 hinge domain, CD8 transmembrane (TM) domain, 4-1BB co-stimulatory domain and
  • CAR chimeric antigen receptor
  • Bispecific antibody refers to a genetically engineered antibody that can specifically target two different epitopes on the same molecule or different molecules at the same time.
  • the double antibody herein is composed of scFv antibodies targeting CD28 and CD40 respectively, that is, the VH and VL of the antibody targeting CD28 (IgG1 or other forms) form the scFv antibody targeting CD28, and the CD40 targeting CD40.
  • the VH and VL of the antibody (Fab or other format) form a scFv antibody targeting CD40, and the two scFv antibodies are connected in series. Two different scFvs are linked by GGGGS (G 4 S for short).
  • VH and VL in each scFv are linked by 3 G 4 S (GGGGSGGGGSGGGGS) (see Table 10).
  • 1412 and 9.3 are CD28 antibodies, 4D11, F2.103, F5.77, 40.37, 40.38, 40.45, 40.47, 40.52 are all CD40 antibodies.
  • 9.3h11 is the 9.3 humanized antibody huVH1VL1, 4052.12, 4052.17, 4052.25, 4052.28, 4052.36 are 40.52 affinity matured antibodies.
  • A40C28 contains scFv antibody against CD40, transmembrane region and intracellular region of CD28 protein.
  • Some tumor cells specifically express CD40 antigen on the surface.
  • the CD40 antibody at one end of the double antibody can recognize and bind to CD40 on the tumor surface; the CD28 antibody at the other end can bind to and activate the CD28 co-stimulatory signaling pathway on T cells to enhance the function of T cells. . Therefore, the double antibody can simultaneously bind to CD40 on tumor cells and CD28 on T cells, and shorten the distance between tumor cells and T cells by bridging, which is more conducive to the function of T cells to kill tumors.
  • the plasmid construction method of the bispecific antibody is as follows:
  • VH and VL fragments of the antibody were amplified by PCR, and then subcloned into the pDA vector by Gibbson assembly homology arm recombination. The correctness of the plasmid was verified by Sanger sequencing.
  • TM transmembrane region
  • cytoplasmic intracellular region
  • Embodiment 9 mRNA preparation in vitro
  • Plasmid linearization The sequenced correct single clones were extracted with a plasmid mini-extraction kit (Tiangen Biochemical Technology Co., Ltd., DP103). The plasmid was linearized by cutting with SpeI endonuclease at 37°C for 4h or overnight. Take 2 ⁇ l of undigested and enzyme-digested plasmids and run 1% DNA nucleic acid gel to detect the degree of enzyme digestion. The digested product was purified with a PCR product recovery kit (Qiagen, 28104).
  • the anti-killing function of the double antibody was detected by Her2-CAR-T cells.
  • the experimental steps for the preparation of Her2-CAR-T cells and the anti-killing function of double antibodies are as follows:
  • A40C2828 is a fusion protein of anti-CD40 antibody and CD28 hinge region, its sequence can be found in SEQ ID NO: 76 in PCT/CN2022/126461 (which is incorporated herein by reference in its entirety).
  • the sequence of A40C2828 in the present invention is numbered as SEQ ID NO:658.
  • A40C2828 is also referred to as "new A40C28" in the figures of this disclosure.
  • A549-no EP A549-CBG cells
  • A549-Meso electroporate 10 ⁇ g Meso (mesothelin) mRNA
  • A549-Meso+CD40 10 ⁇ g Meso and 10 ⁇ g CD40 mRNA
  • the electroporation condition is 300V, 0.5ms.
  • CD40 expression levels were detected by flow cytometry and PE-anti-CD40 antibody.
  • CD28 antibody 9.3 and CD40 antibody (4D11, F2.103, F5.77, 40.37, 40.38, 40.45, 40.47, and 40.52) constitute a double antibody (see Table 10), and the mRNA of these double antibodies and CAR mRNA are co-electrotransferred into T cells middle.
  • BsAbs can be expressed by T cells and secreted extracellularly to form a free state of BsAbs.
  • Her2-CAR-T cells secreting double antibodies were co-cultured with target cells A549-no EP (or CD40), compared with Her2-CAR-T cells alone, double antibodies 9.3-F2.103 and 9.3-40.52 could promote More IL-2 and IFN-gamma cytokines are released (Fig.
  • the CD40 antibody (A40C) in A40C28 was combined with the 9.3 antibody to form a double antibody, which was named "3.1, 3.2...3.8” (see Table 10).
  • Her2-CAR-T cells secreting 3.1-3.8 double antibodies were co-cultured with target cells After raising, the 3.4 and 3.7 double antibodies can promote CAR-T cells to kill more target cells, and their killing ability is stronger than that of the 8.5 and 8.6 double antibodies ( Figure 29 and 30).
  • the Meso-CAR-T cells secreting the 3.1-3.8 double antibody were co-cultured with the target cells, similar experimental results were obtained ( Figures 31, 32 and 33).
  • Antibody purification Incubate the protein supernatant and protein A purification medium on ice for 2 h, and then separate the supernatant and purification medium through a gravity column. Rinse the purification medium with 100ml of pre-cooled PBS to completely remove foreign proteins, and finally use 5-10ml 0.1M Glycine-HCl (pH 2.7) to elute the target protein from the purification medium, and immediately add 1/10 volume of 1M Tris-HCl ( pH 9.0) neutralizing antibodies. The antibody buffer was replaced with PBS through an ultrafiltration tube, and after the protein concentration was measured with A280, the antibody was aliquoted and stored at -80°C.
  • Antigen coating 50 ⁇ l of recombinant human CD40-His antigen protein (ACRO, CD0-H5228) at a concentration of 2 ⁇ g/ml was added to each well of the ELISA plate, and incubated overnight at 4°C.
  • ACRO recombinant human CD40-His antigen protein
  • Blocking block with blocking solution (5% skimmed milk powder) at room temperature for 1-2 hours. Discard blocking solution completely.
  • a mutant Fab antibody library was constructed by using random point mutations in the CDR region of the 40.52 Fab antibody, and high-affinity antibodies were screened from the mutant library.
  • the 40.52 antibody sequence was analyzed by abYsis software, and the CDR region was delineated by Kabat, AbM, and Chothia antibody nomenclature respectively.
  • helper phage M13KO7 add helper phage M13KO7 to the phage library liquid, mix well, let stand at 37°C for 30 minutes, and then culture with shaking for 30 minutes.
  • Glucose removal centrifuge the infected bacterial liquid at 4000rpm for 10min, and discard the supernatant completely. clear to remove the inhibitory effect of glucose.
  • Phage precipitation After 16-20 hours, the bacterial solution was collected and centrifuged at 4000 rpm for 10 min at 4°C. Transfer 40ml supernatant to a new 50ml centrifuge tube, then add 10ml PEG/NaCl solution (20% PEG-8000, 2.5M NaCl), and mix well. Place on ice for 1 hour.
  • Resuspend the phage pellet Resuspend the phage pellet with 2ml PBS to form the phage library.
  • Coating antigen Dilute recombinant human CD40-Fc antigen protein to 1 ⁇ g/ml with PBS (1 ⁇ g/ml for the first round of screening, 0.5 ⁇ g/ml for the second round of screening, 0.1 ⁇ g/ml for the third round of screening) , Add 300 ⁇ l of diluted CD40-Fc to 6 wells of the ELISA plate. Incubate overnight at 4°C.
  • Blocking block with blocking solution (2% skimmed milk powder + 1% BSA) at room temperature for 1-2 hours. Discard blocking solution completely. While blocking the ELISA plate, the phages were also blocked. Add 1/10 volume of blocking solution to the phage solution, and incubate with rotation at room temperature for 1-2h.
  • blocking solution 2% skimmed milk powder + 1% BSA
  • Phage incubation the blocked phages were evenly added to the wells containing the antigen, 300 ⁇ l per well, and cultured with shaking at room temperature for 2 hours.
  • Rinse Discard the phage completely, and wash the ELISA plate 5 times with PBST.
  • Antigen coating Add 50 ⁇ l of recombinant human CD40-His antigen protein (ACRO, CD0-H5228) at a concentration of 2 ⁇ g/ml to each well of the ELISA plate and incubate overnight at 4°C.
  • ACRO recombinant human CD40-His antigen protein
  • Blocking block with blocking solution (2% skimmed milk powder + 1% BSA) at room temperature for 1-2 hours. Discard blocking solution completely.
  • Phage incubation add 50 ⁇ l of monoclonal phage to each well and incubate at room temperature for 2 hours. Rinse 5 times with PBST.
  • the experimental results are shown in Figure 36.
  • the H1 wells in the P1 and P8 plates are all female controls, and the single clones with an OD450 value greater than the female parent (the gray wells in Figure 36) were selected for colony PCR and identified by Sanger sequencing. DNA sequences were used to obtain multiple affinity-matured anti-CD40 Fab antibodies, among which the sequences of antibodies 4052.12, 4052.17, 4052.25, 4052.28 and 4052.36 are shown in Figure 35 and the "Sequence" section below.
  • the DNA sequence of the positive single clone with correct sequencing was subcloned into pDA vector for preparing mRNA in vitro.
  • the structural form of the bispecific antibody (referred to as double antibody) composed of CD40 antibody parent 40.52 and the VH and VL of CD28 antibody (9.3h11) is: CD40 antibody (VH)—(G 4 S) 3 linker— CD40 Antibody (VH)—(G 4 S) Linker—CD28 Antibody (VH)—(G 4 S) 3 Linker—CD28 Antibody (VL).
  • Plasmid linearization The sequenced correct single clones were extracted with a plasmid mini-extraction kit (Tiangen Biochemical Technology Co., Ltd., DP103). The plasmid was linearized by cutting with SpeI endonuclease at 37°C for 4h or overnight. Take 2 ⁇ l of undigested and enzyme-digested plasmids and run 1% DNA nucleic acid gel to detect the degree of enzyme digestion. The digested product was purified with a PCR product recovery kit (Qiagen, 28104).
  • mMESSAGE mMACHINE TM T7 transcription kit (Invitrogen, AM1344) was used to prepare mRNA in vitro. Then add 1 ⁇ l DNase I to this system to digest the template DNA at 37°C for 30min.
  • the mRNA prepared in vitro was used for subsequent auxiliary function killing experiments to detect the function of the CD40 affinity matured antibody.
  • Embodiment 16 detection of auxiliary killing function
  • CD40 affinity matured candidate antibodies obtained by phage screening.
  • Her2-CAR-T cells or Meso-CAR-T cells
  • the anti-killing function of 5.12, 5.17, 5.25, 5.28 and 5.36 is not only stronger than that of the parental double antibody 4.6, but also stronger than that of 1412-4D11, which is very close to the function of the new A40C28 (ie A40C2828) ( Figure 39F).
  • the killing function of the mature double antibody was detected by Mesothelin (Meso for short)-CAR-T cells, and compared with the maternal double antibody 4.6.
  • the experimental steps for the preparation of Meso-CAR-T cells and the anti-killing function of double antibodies are as follows:
  • Recombinant antibodies were expressed in vitro by 293F suspension cells and purified by protein A media.
  • the experimental steps are as follows: the V region of the antibody is inserted into the expression vector containing the IgG1 framework.
  • the expression plasmid was transiently transfected into 293F suspension cells by PEI, and the supernatant was collected after 4 days of culture. Protein A was added to the supernatant to capture the antibody, and after elution with the eluent, the purified antibody was obtained.
  • the antibody yield was calculated by measuring A280, and the purity of the antibody was detected by SDS-PAGE electrophoresis.
  • Figure 45 Under non-reducing conditions (Figure 45-A), the antibody has a single band and good purity; under reducing conditions (Figure 45-B), the heavy chain is relatively complete, and the light chain is somewhat diffusely degraded. The yields of different antibodies varied greatly. As shown in Table 13, the expression level of the affinity matured antibody 4052.17 was closest to that of the parental antibody 40.52.
  • Embodiment 19 ELISA (enzyme linked immunosorbent assay, enzyme-linked immunosorbent assay) Detection of Binding Activity of Affinity Matured Antibodies
  • the binding activity of the affinity matured antibody to the antigen CD40 was detected by ELISA method.
  • the experimental steps are as follows:
  • Embodiment 20 SPR detects the affinity of affinity maturation antibody
  • the experimental steps are as follows: Biacore 2000 (Stuofan, Sweden) was used to detect the binding affinity between the antibody and the CD40 antigen by SPR technology. All experiments were performed at 25°C. First equilibrate the machine with 10mM Glycine-HCl (pH 1.5) for 30 seconds, and use the protein A chip to capture the 40.52 or 4052.17 antibody at a concentration of 2 ⁇ g/ml at a flow rate of 10 ⁇ L/min PBST (pH 7.4, containing 0.05% Tween-20) . CD40 (3.125–100 nM) diluted in a 2-fold gradient was then injected into the mobile phase. Set the binding time to 90 seconds and the dissociation time to 300 seconds.
  • the chip surface was regenerated by incubating with 10mM Glycine-HCl (pH 1.5) for 30 seconds to remove bound CD40 and antibodies.
  • the affinity of the antibody was simulated by the heterogenerous ligand mode in the Biacore Evaluation software that comes with the machine.
  • the double antibody 5.17 is composed of affinity matured CD40 antibody 4052.17 and humanized CD28 antibody 9.3h11. 5.17 and mesothelin CAR (M12) were combined into: M12-5.17 and 5.17-M12.
  • the experimental steps are as follows: the lentiviral plasmid and helper plasmid containing CAR structure (M12), or CAR and double antibody (M12-5.17 or 5.17-M12), or CAR and LACO molecule (A40C2828-M12) were transiently transfected into The virus was packaged in 293T cells, the supernatant was collected, and the virus was concentrated after high-speed centrifugation. The virus titer was measured, and the infection had been infected with CD3/CD28 Dynabeads (Thermo, #11161D) activated human T cells. On the 5th day after infection, the magnetic beads were removed, and the CAR-T cells were continued to be cultured until the 13th day. The transduction efficiency of CAR-T cells was detected, and the CAR-T cells were cryopreserved.
  • CD3/CD28 Dynabeads Thermo, #11161D
  • CAR-T cell transduction efficiency is as follows: Take 5E6 CAR-T cells into each well of a 96-well plate, wash once with PBS, and add 50 ⁇ l of Meso-Fc or CD40-Fc with a final concentration of 2 ⁇ g/ml to each well. For Fc protein, incubate at 4°C for 30min. Wash the cells 3 times with PBS, add 50 ⁇ l PE-anti-Fc antibody with a final concentration of 1 ⁇ g/ml to each well, and incubate at 4°C for 30 minutes. After 2 times with PBS, the cells were detected by flow cytometry.
  • Example 22 Detection of auxiliary killing function of CAR-T cells transduced by lentivirus
  • the lentivirus-transduced CAR-T cells obtained in Example 21 were co-cultured with the target cells, the killing curve of the target cells was observed, and the release of cytokines in the co-culture supernatant was detected. At the same time, compare the functional differences with M12 and A40C2828-M12.
  • the experimental results FIG. 49 ) showed that, compared with only M12 and A40C2828-M12, M12-5.17 and 5.17-M12 had stronger killing functions and could reduce the fluorescence value of target cells to a lower level.
  • Example 23 Specific detection of lentivirus-transduced CAR-T cells
  • the lentivirus-transduced CAR-T cells obtained in Example 21 were co-cultured with different target cells, the killing curve of the target cells was observed, and the release of cytokines in the co-culture supernatant was detected.
  • the release levels of IL-2 and IFN-gamma in the supernatant were detected by Cisbio bioassays ⁇ human IL-2assay and human IFN- ⁇ assay.
  • the experimental results (Fig. 52) also showed that the specificity of M12-5.17 was better than that of 5.17-M12.
  • Example 24 In vivo efficacy of lentiviral-transduced CAR-T cells
  • the CAR-T cells transduced by the lentivirus obtained in Example 21 were reinfused into tumor-bearing NSG mice, the tumor changes were observed, and the in vivo drug efficacy of the CAR-T cells was detected.
  • Tumor-bearing 5E6 SKOV3-Meso+CD40-CBG cells were subcutaneously inoculated into the upper back of NSG mice, and the tumor was allowed to grow for 11 days.
  • CAR-T cells low-dose (5E5 CAR-T cells per mouse) and high-dose (3E6 CAR-T cells per mouse) were reinfused into tumor-bearing NSG mice, respectively. CAR-T cells. Every 7 days, the tumor size was measured with a vernier caliper, the body weight of the mice was weighed, the fluorescence value of the tumor was measured with a small animal in vivo imager, and the death of the mice was recorded.
  • the following examples 25-29 relate to antibodies targeting mesothelin (Meso), CAR and CAR-T
  • the pDA vector was digested with Xba1 and Sal1 enzymes, and purified by gel purification.
  • the scFv fragment and CAR fragment obtained in the above-mentioned Example 25 were amplified by PCR and purified by gel method for purification.
  • the scFv fragment, CAR fragment (from hinge domain to CD3-zeta domain) and pDA vector were ligated by Gibson assembly method and transformed into competent cells. Colonies with the correct construct were identified by Sanger sequencing and selected for further experiments.
  • Figure 56 provides a schematic diagram of the pDA-CAR vector used to generate CAR mRNA.
  • the pDA-CAR plasmid was linearized with Spe1 digestion.
  • the linearized vector was purified using a PCR Cleanup kit and eluted with RNase-free water. The concentration of DNA was determined by nanodrop and checked by agarose DNA gel. Then, in vitro transcription (IVT) was performed according to the protocol of the manufacturer (Thermofisher, catalog number: AM13455). Briefly, 20 ⁇ l of the system containing 1 ⁇ g of template DNA, NTP/ARCA buffer, T7 buffer, GTP, T7 enzyme, and RNase-free H2O was added to a 0.2 mL PCR tube and incubated at 37°C for 3 hours.
  • RNA concentration was determined by nanodrop, and checked by PAGE gel.
  • Tumor cell lines including A549-CBG (human lung cancer (cancer) cells), H226-CBG (human lung cancer (malignant epithelial tumor) cells), MOLM14-CBG (human leukemia cells), ASPC1-CBG (human pancreatic tumor cells), HCC70-CBG (human breast cancer cells) and OVCAR3-CBG (human ovarian cancer cells).
  • Primary lymphocytes from normal donors were stimulated with anti-CD3/CD28 immunomagnetic beads (Life Technologies), and the cells were cultured in R10 medium (added with 10% FCS, penicillin-streptomycin (100x), HEPES ( 100x), sodium pyruvate (100x), Glutamax (100x), RPMI-1640 of NEAA (100x)).
  • T cells were cryopreserved at 1 ⁇ 10 8 cells/flask in a solution of 90% FCS and 10% DMSO.
  • the mesothelin CAR mRNA obtained in Example 26 was introduced into A549 tumor cells and T cells by electroporation, and the steps were as follows: A549 tumor cells and T cells were collected and washed 3 times with Opti-MEM medium. Resuspend the cell pellet with Opti-MEM medium, and adjust the cell concentration to 1 ⁇ 10e7 cells/ml. Aliquot 10 ⁇ g RNA into 1.5mL EP tubes, add 100 ⁇ l T cells or A549 cells, and mix well. Add 100 ⁇ l of cells mixed with RNA to the BTX electroporation cuvette and tap gently to avoid air bubbles.
  • Electroporation was performed using a BTX instrument under the following parameters: for T cells: 500 V voltage, 0.7 ms; for A549 tumor cells: 300 V voltage, 0.5 ms. Cells were then transferred to pre-warmed medium and incubated at 37°C.
  • T cells without the CAR molecule served as a control ("Mock").
  • the level of ectopic expression of mesothelin correlated with the amount of mesothelin mRNA introduced into A549 cells by electroporation.
  • cytotoxicity of mesothelin CART cells to tumor cells was measured in an in vitro cytotoxicity assay.
  • EGFP-expressing tumor cell lines or EGFP-A549 cells electroporated with different amounts of tumor antigens were seeded on flat-bottomed 96-well plates at 3000 cells/100 ⁇ l/well. Dilute CART cells to an appropriate concentration, inoculate at 100 ⁇ l/well, and use different E/T ratios with tumor cells, such as 10:1, 3:1, 1:1, place the co-culture plate in the IncuCyte S3 instrument, and set the scan parameter. After 3 days of scanning, analyze the total integral intensity of the green image (GCU x ⁇ m2/well), calculate the killing efficiency.
  • A549 cells express low levels of mesothelin. As shown in Figure 59, CART cells expressing anti-mesothelin scFv-M4, -M22, -M28, and -M31 effectively blocked the growth of A549 cells, indicating that these scFv-based CART cells had a rather high cytotoxicity against tumor cells. toxicity.
  • CART cells expressing anti-mesothelin scFv-M4, -M6, -M13, -M20, -M27, -M31 and -M37CARs had less ectopic expression of mesothelin to A549 tumor cells ( Electroporation with 2 ⁇ g of mesothelin mRNA) maintained a strong killing effect.
  • CART cells expressing anti-mesothelin scFv-M7, -M8, -M9, -M10, -M11, -M12, -M15, -M23, -M24, -M32, -M35, and -M38 selectively target only high Tumor cells expressing mesothelin exhibit high cytotoxicity, which is not true for tumor cells expressing mesothelin low, while having a superior safety profile because mesothelin is also expressed in some normal tissues.
  • the anti-mesothelin scFv-M12, -M24 and -M32 CART cells had a mild killing effect on A549 tumor cells with low expression of mesothelin (2 ⁇ g group), but had a mild killing effect on A549 tumor cells with high expression of mesothelin ( 10 ⁇ g group) had a strong killing effect.
  • the results showed that these CART cells specifically targeted tumor cells with high expression of mesothelin, and did not kill normal tissues with low expression of mesothelin.
  • Figure 63 shows the effects of isotype control and anti-mesothelin mAbs on OVCAR3 (human ovarian cancer cells), H226 (human lung cancer (malignant epithelial tumor) cells), ASPC1 (human pancreatic tumor cells), A549 (human lung cancer (cancer) cells) and HCC70 (human breast cancer cells) by FACS staining.
  • OVCAR3 human ovarian cancer cells
  • H226 human lung cancer (malignant epithelial tumor) cells
  • ASPC1 human pancreatic tumor cells
  • A549 human lung cancer (cancer) cells
  • HCC70 human breast cancer cells
  • Example 29 Cancer cells expressing mesothelin specifically activate CART cells
  • CD107a is an early activation marker of T cells.
  • the activation of mesothelin CART by mesothelin-expressing tumor cells was measured by CD107a staining, and the steps were as follows: 20 ⁇ l PE-CD107a mAb was added to each well of a 96-well plate; the tumor cells were diluted to 2 ⁇ 10e6 cells/ml, Inoculate on a 96-well circular plate (100 ⁇ l/well); Dilute the CAR-T cells obtained in Example 28 to 1 ⁇ 10e6 cells/ml, inoculate on a 96-well circular plate (100 ⁇ l/well), 500 rpm ⁇ Centrifuge the plate for 5 minutes to make the cells adhere to the wall, incubate at 37°C for 1 hour, dilute GolgiStop 1500 times with medium and add it to each well (20 ⁇ l/well); APC and anti-CD8-FITC antibodies were stained for 30 min, washed and analyzed by flow cytometry.
  • Figure 64 shows CD107a staining of anti-mesothelin M12 and M32 CAR-T cells in co-culture and killing assays with OVCAR3, H226, ASPC1, A549 and HCC70. These data demonstrate that anti-mesothelin M12 and M32 CART cells are specifically activated by OVCAR3, H226, and ASPC1 (tumor cells with high mesothelin expression levels), but not by A549 and HCC70 (low or no mesothelin expression cells). tumor cell lines) activated.
  • the following examples 30-33 relate to antibodies targeting BCMA, CAR and CAR-T
  • acid elution buffer pH 2.2
  • mpELISA screening After three rounds of screening, 480 phage-infected colony clones were selected for monoclonal phage ELISA (mpELISA) screening. Phage supernatants were cloned from individual colonies and tested for binding to BCMA-Fc protein. The supernatant was incubated with pre-blocked Maxisorp plates coated with 2 ⁇ g/ml BCMA-6His protein. After washing three times, add 100 ⁇ l/well of HRP-conjugated anti-M13 antibody diluted 1:5000 in blocking buffer (1 ⁇ PBS containing 5% milk and 1% BSA), and then Incubate at room temperature for 60 min. After washing the plate 5 times with PBST, add 100 ⁇ l/well of TMB substrate solution and incubate for 10-30 minutes until blue color appears. The reaction was stopped by adding 50 ⁇ l/well of stop solution (2N H2SO4).
  • the anti-BCMA scFv was constructed into a bicistronic lentiviral CAR expression vector containing an IRES truncated EGFR (tEGFR) expression cassette.
  • Lentivirus was generated by transient transfection of 293T cells, followed by purification and concentration by ultracentrifugation.
  • T cells were transduced with CAR lentivirus to generate CAR-T cells, and then cultured for another 10 days. 10 days after lentiviral transduction, CAR-T cells were collected and stained with 5 ⁇ g/ml CD19-Fc protein (Ctrl Fc protein) or BCMA-Fc recombinant protein for 30 minutes at 4°C.
  • CAR-T cells were stained with anti-human IgG Fc and anti-EGFR mAb. Samples were analyzed by flow cytometry. As shown in Figure 65A and Figure 65B, T cells expressing CAR (containing the following anti-BCMA scFv) showed binding to BCMA-Fc ( Figure 65B) and were selected for further study: BCMA21, BCMA22, BCMA23, BCMA24, BCMA27 , BCMA28, BCMA30, BCMA31, BCMA32, BCMA33, BCMA34, and BCMA35.
  • T cells were transduced by lentiviral vectors to express different BCMA CARs.
  • Table 19 above shows the CAR T cells used in the studies disclosed in the present invention, the percentage of cells expressing CAR and their respective expression levels.
  • Figures 66A and 66B show the frequency of CAR+ T cells and their expression levels ("MFI" is mean fluorescence intensity), respectively.
  • MFI is mean fluorescence intensity
  • Figure 67 shows comparable frequencies of CAR+CD8 cells in the tested CART.
  • Figure 68 shows the phenotype of CART cells. Naive T cell populations (CD45RO-; CCR7+) were more frequently present in BCMA27(#7), BCMA31(#10) and BCMA33(#12) T cells than in other samples, suggesting that these T cells are less differentiated .
  • Example 33 BCMA CART is cytotoxic to tumor cells
  • the CART cells obtained in the foregoing Example 31 were co-cultured with Jeko-1 cells and RPMI-8226 tumor cells. The production of INF- ⁇ and IL-2 was detected.
  • Figure 70A (INF- ⁇ ) and 70B (IL-2) As shown in Figure 70A (INF- ⁇ ) and 70B (IL-2), Among the 12 CARTs produced by the present invention, BCMA23 (#5; SEQ ID NO:337), BCMA24 (#6; SEQ ID NO:338), BCMA27 (#7; SEQ ID NO:339), BCMA31 (#10 ; SEQ ID NO:342) and BCMA33 (#12; SEQ ID NO:344), were able to produce more cytokines than other CAR T cells (including NBC10 and B38M CAR T cells).
  • BCMA23(#5), BCMA24(#6), BCMA31(#10) and BCMA33(#12) CART cells showed different degrees of cytotoxicity to Jeko-1 cells, among which BCMA31(#10) had the highest cytotoxicity and could effectively Eliminate Jeko-1.
  • BCMA21 (#3; SEQ ID NO: 335), BCMA22 (#4; SEQ ID NO: 336), BCMA23 (#5; SEQ ID NO: 337), BCMA24 (#6; SEQ ID NO: 338), BCMA27 (#7; SEQ ID NO: 339), BCMA31 (#10; SEQ ID NO: 342), BCMA33 (#12; SEQ ID NO: 344), BCMA34 (#13; SEQ ID NO: 345) and BCMA35 ( #14; SEQ ID NO: 346) showed different levels of cytotoxicity to RPMI-8226 cells, wherein BCMA21 (#3), BCMA23 (#5), BCMA24 (#6), BCMA27 (#7), BCMA31 (# 10) and BCMA33 (#12) efficiently eliminated RPMI-8226 cells.
  • the following examples 34-37 relate to antibodies targeting CD123, CAR and CAR-T
  • CD123-specific scFv-phage In the first round of selection, coat Maxisorp plates with 20 ⁇ g/ml of CD123-6His protein overnight at 4°C, wash with PBS, and wash with 5% milk + 1% BSA was blocked in 1 ⁇ PBS, incubated in phage solution for 2 hours, and washed 10 times with PBST.
  • mpELISA monoclonal phage ELISA
  • the phage supernatant generated by single colony clone was incubated with the pre-blocked Maxisorp plate coated with 2 ⁇ g/mL CD123-6His protein. After washing three times, add 100 ⁇ l/ Wells of HRP-conjugated anti-M13 antibody diluted 1:5000 in blocking buffer (5% milk + 1% BSA, 1 ⁇ PBS), then incubated at room temperature for 60 minutes . After washing the plate 5 times with PBST, add 100 ⁇ l/well TMB substrate solution and incubate for 10-30 min until blue color appears.
  • Figure 73 shows the absorbance readings from the mpELISA screen. As shown, some positive colonies (450nm absorbance > 0.5) were identified as producing anti-CD123 antibodies capable of binding CD123-6His protein (Fig. 73).
  • RNA product was determined by nanodrop and checked by running a PAGE gel.
  • the CD123 CAR mRNA obtained in the foregoing Example 35 was introduced into T cells using electroporation through the following steps: T cells were collected, washed with Opti-MEM medium, and resuspended with Opti-MEM medium at 1 ⁇ 10e 7 /ml; 10 ⁇ g RNA was aliquoted with 100 ⁇ l T cells, mixed evenly and electroporated with the following parameters (BTX machine): 500V, 0.7ms; then the cells were transferred to the preheated medium and cultured at 37°C.
  • BTX machine 500V, 0.7ms
  • CD123 CART cells The binding of CD123 CART cells to CD123-Fc recombinant protein was measured by FACS staining. As shown in Figure 75, expression has anti-CD123 scFv-C1,-C2,-C3,-C4,-C5,-C6,-C7,-C9,-C10,-C11,-C13,-C14,-C15, -C16, -C17, -C18, -C19, -C21, -C23, -C24, -C25, -C26, -C27, -C28, -C29, -C30, -C32, -C33, -C34, and -C35
  • the CAR T cells can bind CD123-Fc recombinant protein.
  • Empty vector (Mock) is a control T cell without CAR molecule.
  • A549 tumor cells were electroporated with different amounts of CD123 mRNA.
  • the electroporation process was the same as above, only the parameters were changed, the parameters used were 300V, 0.5ms.
  • CD123 expression in A549 tumor cells was measured by FACS staining of A549 cells electroporated with isotype or anti-CD123 antibody and different amounts of CD123 mRNA. As shown in Figure 76, A549 cells weakly express endogenous CD123, and the level of ectopic expression of CD123 correlates with the amount of CD123 mRNA electroporated into A549 cells.
  • the cytotoxicity of CD123 CAR T cells to tumor cells was measured in an in vitro cytotoxicity assay.
  • Seed EGFP-expressing tumor cells or EGFP-A549 cells electroporated with different amounts of tumor antigens on a flat-bottomed 96-well plate at 3000 cells/100ul/well; (e.g., 10:1, 3:1, 1:1) inoculate 100 ⁇ l/well of tumor cells, then place the co-culture plate into the IncuCyte S3 machine and set the scanning parameters. After 3 days of scanning, the total green object integrated intensity (GCU x ⁇ m2/well) was analyzed to calculate the killing efficiency.
  • GCU x ⁇ m2/well the total green object integrated intensity
  • Figure 77 and Figure 78 show the killing curves of different mRNA-based anti-CD123 CART cells on A549-GFP tumor cells when the E/T ratio is 10:1 ( Figure 77) or 3:1 ( Figure 78).
  • Figure 79 and Figure 80 show the effect of different mRNA-based anti-CD123 CART cells expressing exogenous CD123 (with 10 ⁇ g CD123 mRNA) at an E/T ratio of 10:1 ( Figure 79) or 3:1 ( Figure 80).
  • FIG 81 shows the results of FACS staining of A549, SK-OV3, Jeko-1, Molm-14, SupT-1, 293T, Nalm-6 and PC-3 cells with PE-isotype control and PE-anti-CD123 mAb . As shown in Figure 81, most of the tested tumor cell lines did not express CD123, only Molm-14 expressed relatively high levels of CD123.
  • CD107a is an early activation marker of T cells. Activation of CD123 CARTs by CD123-expressing tumor cells was measured using CD107a staining by the following steps: Add 20 ⁇ l of PE-CD107a mAb to each well of a 96-well plate; dilute tumor cells to 2 ⁇ 10e 6 /ml, and seed in 96-well circular plate (100 ⁇ l/well); Dilute the CART cells obtained in Example 36 to 1 ⁇ 10e 6 /ml, inoculate in a 96-well circular plate (100 ⁇ l/well); centrifuge the plate at 500 rpm ⁇ 5 minutes , to make the cells attach well, and incubate at 37°C for 1 hour; dilute Golgi stop 1500 times with medium, add it to each well (20 ⁇ l/well); incubate the cells at 37°C for another 2.5 hours, and use CD3-APC and anti-CD8-FITC antibodies were stained at 37°C for 30 minutes, washed and analyzed by flow cyto
  • CD123-expressing tumor cells were measured by CD107a staining.
  • the cells tested included A549 electroporated with 10 ⁇ g, 0.1 ⁇ g and 0 ⁇ g CD123 mRNA, SK-OV3, PC-3, cord blood-derived CD34 hematopoietic stem cells (CD34+cord), bone marrow-derived hematopoietic stem cells (CD34+M), Molm-14, Nalm6, Jeko-1 tumor cell lines and freshly isolated patient AML tumor cells (CD123+).
  • CART cells expressing anti-CD123-C5, -C7 and -C11 were specifically activated by tumor cells with relatively high CD123 expression, especially CD123+AML tumor cells, but not by tumors with low CD123 expression Cell line-specific activation. These results suggest that tumor cells expressing CD123 can activate CD123 CARTs.
  • sequence numbers corresponding to the antibody VH, VL, scFv and CAR sequences involved in the above Examples 25-37 are shown in Table 21 below.
  • HCDR1 DYGVH (SEQ ID NO: 1)
  • HCDR2 VIWAGGGTNYNSALMS (SEQ ID NO: 2)
  • HCDR3 DKGYSYYYSMDY (SEQ ID NO: 3)
  • HFR1 QVKLQQSGPGLVTPSQSLSITCTVSGFSLS (SEQ ID NO: 4)
  • HFR2 WVRQSPGQGLEWLG (SEQ ID NO:5)
  • HFR3 RKSISKDNSKSQVFLKMNSLQADDTAVYYCAR (SEQ ID NO:6)
  • HFR4 WGQGTTVTVSS (SEQ ID NO: 7)
  • LCDR1 RASESVEYYVTSLMQ (SEQ ID NO:9)
  • LCDR2 AASNVES (SEQ ID NO: 10)
  • LCDR3 QQSRKVPYT (SEQ ID NO: 11)
  • LFR1 DIVLTQSPASLAVSLGQRATISC (SEQ ID NO: 12)
  • LFR2 WYQQKPGQPPKLLIF (SEQ ID NO: 13)
  • LFR3 GVPARFSGSGSGTNFSLNIHPVDEDDVAMYFC (SEQ ID NO: 14)
  • LFR4 FGGGTKLEIK (SEQ ID NO: 15)
  • HCDR1 DYGVH (SEQ ID NO: 1)
  • HCDR2 VIWAGGGTNYNSALMS (SEQ ID NO: 2)
  • HCDR3 DKGYSYYYSMDY (SEQ ID NO: 3)
  • HFR1 QVKLQESGPGLVKPSETLSITCTVSGFSLS (SEQ ID NO: 19)
  • HFR2 WVRQSPGKGLEWLG (SEQ ID NO: 23)
  • HFR3 RKTISKDNSKSQVFLKMSSLTAADTAVYYCAR (SEQ ID NO:27)
  • HFR4 WGQGTTVTVSS (SEQ ID NO: 7)
  • HCDR1 DYGVH (SEQ ID NO: 1)
  • HCDR2 VIWAGGGTNYNSALMS (SEQ ID NO: 2)
  • HCDR3 DKGYSYYYSMDY (SEQ ID NO: 3)
  • HFR1 QVQLQESGPGLVKPSETLSITCTVSGFSLS (SEQ ID NO: 20)
  • HFR2 WVRQPPGKGLEWLG (SEQ ID NO: 24)
  • HFR3 RKTISKDTSKNQVSLKMSSLTAADTAVYYCAR (SEQ ID NO: 28)
  • HFR4 WGQGTTVTVSS (SEQ ID NO: 7)
  • HCDR1 DYGVH (SEQ ID NO: 1)
  • HCDR2 VIWAGGGTNYNSALKS (SEQ ID NO: 17)
  • HCDR3 DKGYSYYYSMDY (SEQ ID NO: 3)
  • HFR1 QVQLQESGPGLVKPSETLSLTCTVSGFSLS (SEQ ID NO: 21)
  • HFR2 WVRQSPGKGLEWIG (SEQ ID NO: 25)
  • HFR3 RKTISKDNSKSQVSLKLSSVTAADTAVYYCAR (SEQ ID NO:29)
  • HFR4 WGQGTTVTVSS (SEQ ID NO: 7)
  • HCDR1 DYGVH (SEQ ID NO: 1)
  • HCDR2 VIWAGGGTNYNSALKS (SEQ ID NO: 17)
  • HCDR3 DKGYSYYYSMDY (SEQ ID NO: 3)
  • HFR1 QVQLQESGPGLVKPSETLSLTCTVSGFSLS (SEQ ID NO: 21)
  • HFR2 WIRQPPGKGLEWIG (SEQ ID NO: 26)
  • HFR3 RVTISVDTSKNQVSLKLSSVTAADTAVYYCAR (SEQ ID NO: 30)
  • HFR4 WGQGTTVTVSS (SEQ ID NO: 7)
  • HCDR1 DYGVH (SEQ ID NO: 1)
  • HCDR2 VIWAGGGTNYNPSLKS (SEQ ID NO: 18)
  • HCDR3 DKGYSYYYSMDY (SEQ ID NO: 3)
  • HFR1 QVQLQESGPGLVKPSETLSLTCTVSGFSIS (SEQ ID NO: 22)
  • HFR2 WIRQPPGKGLEWIG (SEQ ID NO: 26)
  • HFR3 RVTISVDTSKNQVSLKLSSVTAADTAVYYCAR (SEQ ID NO: 30)
  • HFR4 WGQGTTVTVSS (SEQ ID NO: 7)
  • LCDR1 RASESVEYYVTSLMQ (SEQ ID NO:9)
  • LCDR2 AASNVES (SEQ ID NO: 10)
  • LCDR3 QQSRKVPYT (SEQ ID NO: 11)
  • LFR1 DIQLTQSPSSLSVSVGDRVTISC (SEQ ID NO: 36)
  • LFR3 GVPSRFSGSGSGTNFTLTISSVQEEDFAMYFC (SEQ ID NO: 40)
  • LFR4 FGGGTKLEIK (SEQ ID NO: 15)
  • LCDR1 RASESVEYYVTSLMQ (SEQ ID NO:9)
  • LCDR2 AASNVES (SEQ ID NO: 10)
  • LCDR3 QQSRKVPYT (SEQ ID NO: 11)
  • LFR1 DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 37)
  • LFR3 GVPSRFSGSGSGTNFTLTISSVQPEDFATYFC (SEQ ID NO: 41)
  • LFR4 FGGGTKLEIK (SEQ ID NO: 15)
  • LCDR1 RASESVEYYVTSLMQ (SEQ ID NO:9)
  • LCDR2 AASNVES (SEQ ID NO: 10)
  • LCDR3 QQSRKVPYT (SEQ ID NO: 11)
  • LFR1 DIQLTQSPSSLSASVGDRVTITC (SEQ ID NO: 37)
  • LFR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 42)
  • LFR4 FGGGTKLEIK (SEQ ID NO: 15)
  • Heavy chain constant region CH of humanized anti-CD28 IgG1 antibody isomitted from humanized anti-CD28 IgG1 antibody.
  • HCDR1 SYAIS (SEQ ID NO: 46)
  • HCDR2 MINPSGGTTSYAQKFQG (SEQ ID NO: 47)
  • HCDR3 GFRMDQ (SEQ ID NO: 48)
  • HFR1 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 49)
  • HFR2 WVRQAPGQGLEWMG (SEQ ID NO: 50)
  • HFR3 RVTMTRDTSTSTVYMELSSLRPEDTAVYYCAR (SEQ ID NO:51)
  • HFR4 WGQGTLVTVSS (SEQ ID NO: 52)
  • LCDR2 DVRKRPS (SEQ ID NO: 55)
  • LFR1 QSALTQPPSASGSPGQSVTISC (SEQ ID NO: 57)
  • LFR2 WYQQHPGNAPKLIIY (SEQ ID NO:58)
  • LFR3 GIPDRFSASSKSGNTASLTVSGLQADDEADYYC (SEQ ID NO: 59)
  • LFR4 FGGGTKLTVL (SEQ ID NO: 60)
  • HCDR1 SYAVS (SEQ ID NO: 62)

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Abstract

本发明涉及人源化抗CD28抗体、包含该人源化抗CD28抗体的多特异性抗体、尤其是包含该人源化抗CD28抗体和抗CD40的双特异性抗体、编码这些抗体的多核苷酸、包含该多核苷酸的载体、包含该多核苷酸或载体的宿主细胞以及包含它们的药物组合物。本发明还涉及所述人源化抗CD28抗体在诱导T细胞增殖中的应用以及所述双特异性抗体在在增强免疫细胞对靶细胞杀伤作用中的应用。

Description

人源化抗CD28抗体及其与抗CD40的双特异性抗体
相关申请的交叉引用
本申请要求申请号为PCT/CN2022/076506、申请日为2022年2月16日的PCT申请，申请号为PCT/CN2022/076503、申请日为2022年2月16日的PCT申请以及申请号为PCT/CN2022/076504、申请日为2022年2月16日的PCT申请的优先权，在此通过引用将它们的全文并入本文。
技术领域
本发明涉及抗体，具体而言，本发明涉及人源化抗CD28抗体及其与抗CD40的双特异性抗体。
背景技术
当组织损伤或感染时，免疫系统被激活，B和T淋巴细胞、巨噬细胞、嗜酸性粒细胞、中性粒细胞等被募集和激活。抗原递呈细胞(antigen presenting cells，APC)如巨噬细胞或树突状细胞捕获外源蛋白并展示在APC细胞的表面。T细胞辅助细胞(T-helper cells，TH细胞)表面表达免疫球蛋白，在识别APC展示的抗原后被激活。激活的TH细胞又反过来激活某些B细胞克隆进行增殖并分化为浆细胞，然后产生和分泌针对外源蛋白的抗体。抗体与表达外源蛋白的细胞结合，靶向这些细胞以被免疫效应细胞杀伤[1]。
刺激静息状态的T淋巴细胞进行激活、增殖和功能性的分化需要结合2种受体：(1)能特异性识别抗原的受体；(2)CD28分子。分化簇28(CD28)是一种在绝大多数静息T细胞上表达的膜蛋白，44kDa，能结合APC细胞表面的CD80(B7.1)和CD86(B7.2)蛋白[2]，为T细胞激活和增殖提供共刺激信号。在经典的共刺激系统中，单独的抗原受体结合或CD28分子结合都不能诱导T细胞增殖。CD28是T细胞克隆扩增、分化和效应功能的必需的共刺激受体。CD28结合降低了T细胞激活阈值并导致TCR信号事件增强，其中所述TCR信号为高效细胞因子产生(通过增强转录活性和信使RNA稳定性)、细胞周期进程、存活、代谢调节和T细胞应答所必需。CD28是免疫突触(IS)组织的关键因素，在其中CD28增强T细胞和APC之间的密切接触。
然而CD28特异性单克隆抗体(mAb)9.3可以在没有共刺激的情况下诱导T细胞增殖，即不依赖于抗原受体结合的CD28特异性mAb激活静息T淋巴细胞[3,4]。9.3抗体来源于小鼠的杂交瘤细胞[4]，在人体内的免疫原性大。Philip Tan等人曾利用CDR移植和高变异loop区与人V基因相互配对的方法对9.3抗体进行人源化改造[5]，将鼠源抗体的CDR嫁接到人源的框架中，大大降低了嵌合抗体的免疫原性。经人源化改造的9.3嵌合抗体的结合抗原的能力有所降低。
当组织损伤或感染时，免疫系统被激活，B和T淋巴细胞、巨噬细胞、嗜酸性粒细胞、中性粒细胞等被募集和激活。抗原递呈细胞(antigen presenting cells，APC)如巨噬细胞或树突状细胞捕获外源蛋白并展示在APC细胞的表面。T细胞辅助细胞(T-helper cells，TH细胞)表面表达免疫球蛋白，在识别APC展示的抗原后被激活。激活的TH细胞又反过来激活某些B细胞克隆进行增殖并分化为浆细胞，然后产生和分泌针对外源蛋白的抗体。抗体与表达外源蛋白的细胞结合，靶向这些细胞以被免疫效应细胞杀伤[1]。TH细胞与B细胞接触会刺激B细胞增殖和免疫球蛋白(immunoglobulin，Ig)从IgM转换为IgG、IgA或IgE。其中CD40/CD40L受体-配体相互作用在介导TH细胞和B细胞的接触中发挥着重要作用。
CD40是肿瘤坏死因子受体(tumor necrosis factor receptor，TNFR)超家族的共刺激成员。人CD40是I型跨膜糖蛋白，由277个氨基酸组成，分子量为30,600，结构上分为信号肽、胞外段、跨膜区和胞内段。与TNFR超家族的其他成员一样，CD40形成三聚体，具有最佳信号传导所需的高阶蛋白质聚类特性。人CD40L是肿瘤坏死因子(tumor necrosis factor，TNF)超家族成员，属于II型跨膜糖蛋白，从C端到N端分别为：胞外段、跨膜区和胞内段。CD40L最初在活化的CD4+T细胞表面被发现，B和T细胞之间的CD40-CD40L相互作用对生发中心(germinal center，GC)反应也至关重要。
CD40在B细胞上的功能。CD40作为共刺激因子，和细胞因子或其他刺激共同促进B细胞的激活和增殖。CD40刺激致使人外周血B细胞、幼稚、记忆和生发中心的B细胞上的CD80和CD86上调。CD40信号还诱导人B细胞上CD95/Fas和主要组织相容性复合物II类(MHCII)的上调。除了早期激活，CD40信号还通过细胞周期和B细胞扩增促进激活的下一步进展。与抗CD20、IL-4或IL-21结合、CD40还能刺激循环和组织驻留的B细胞快速增殖。
除B细胞外，CD40也在其他造血细胞上表达，例如单核细胞和树突细胞，能促进这些细胞的生长和细胞因子的释放，也能释放信号来诱导表达CD40L的CD4+T细胞进行激活、增殖和细胞因子产生。CD40信号通路也影响非造血细胞的功能，包括内皮细胞、上皮细胞、成纤维细胞和神经细胞。CD40L也广泛表达在多种细胞中，包括肥大细胞、嗜碱性粒细胞、B细胞、NK细胞、巨噬细胞、巨核细胞和血小板上[6]。CD40信号通路在细胞生物学中具有广泛的作用。尽管CD40被认为是CD40L最重要的结合蛋白，CD40L也结合整合素家族的其他蛋白。
CD40通过介导T细胞与B细胞以及其他细胞的相互作用，在免疫调节中发挥着广泛的作用。激动性CD40抗体可用作免疫刺激剂，增强个体的免疫反应。目前正在开发几种抗CD40抗体活性较低。
肿瘤微环境(TME)由血管、髓源性抑制细胞(MDSC)、抗原提呈细胞(APC)、淋巴细胞、嗜中性粒细胞、肿瘤相关巨噬细胞(TAMs)、成纤维细胞、细胞外 基质、细胞因子和生长因子等组成，与肿瘤的发生、转移密切相关。肿瘤微环境参与驱逐T细胞出肿瘤边缘，抑制T细胞在肿瘤内聚集、抑制T细胞增殖[7]。
双特异性抗体(bispecific antibody,简称双抗、BsAb)能同时结合2个不同的抗原表位，桥联携带不同抗原的2种细胞，将效应T细胞重定向到肿瘤细胞，促进效应T细胞的功能。本文中的双抗由靶向CD28的scFv和靶向CD40的scFv串联而成，这种双抗也被称为BiTE(bispecific T-cell engagers，双特异性T细胞重定位抗体)。CD28-scFv作为CD28分子的激动剂，CD40-scFv是CD40分子的激动剂。因此，CD28-CD40双抗能同时激活CD28和CD40信号通路，在免疫反应中发挥重要作用。
CD40信号通路影响CD4+T细胞分化成效应细胞。在CD4+T细胞早期激活阶段，CD4+T细胞瞬时表达CD40L(CD40配体)，CD40L和其他细胞上CD40相互作用后引起CD40L降解，可溶性的CD40L释放到细胞外。激活后期，CD40L的表达依赖共刺激分子CD28。CD28与其配体CD80和CD86结合后才能诱导T细胞完全活化和分化。APCs表达的CD40与T细胞的CD40L结合后，促进APCs上CD80和CD86的表达。同时，CD28与CD80/CD86结合会上调CD40L，从而产生正反馈作用，二者协同驱动T细胞活化和树突状细胞成熟[6]。
CD40信号通路影响树突状细胞和巨噬细胞。树突状细胞作为抗原提呈细胞通过主要组织相容性复合物(MHC)和T细胞受体(TCR)之间相互作用将抗原提呈给T细胞，引起效应T细胞发挥功能。CD28-CD40双抗激活树突状细胞上的CD40后引起MHC和共刺激分子表达上调，从而使树突状细胞成熟，激活和CD8T细胞预激活。CD40下游信号通路又通过TRAF6激活MAPK和NFKb信号通路，从而促进树突状细胞的生存、成熟和细胞因子释放。在肿瘤微环境中，巨噬细胞因分泌IL-10，IDO和TGF-beta，可能增强免疫反应。
CD40信号通路影响NK细胞。NK细胞能通过自身的CD40L/CD40相互作用被直接激活，从而促进NK细胞增殖、激活和促进靶细胞杀伤。在肿瘤微环境中，CD40通路激活能增加NK细胞的杀伤功能[8]。
CD40信号通路粒细胞。粒细胞在不同的环境下也表达CD40。在中，CD40通路能促进人外周血嗜酸性粒细胞生存、巨噬细胞集落刺激因子(GMCSF)释放。嗜酸性粒细胞也能作为抗原提呈细胞，激活众多的T细胞辅助反应(促炎症和抑制炎症)。
因此，CD28-CD40双抗不仅提供T细胞活化的共刺激信号，还能刺激多种免疫细胞(如树突状细胞、巨噬细胞、NK细胞、粒细胞等)进行活化和发挥功能，强烈增强了免疫反应。
发明内容
本发明一方面提供人源化抗CD28抗体或其抗原结合片段，所述人源化抗 CD28抗体包括重链可变区和轻链可变区，所述重链可变区包括HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3；其中，
HCDR1包含SEQ ID NO:1，
HCDR2包含选自SEQ ID NO:2、17和18的序列，
HCDR3包含SEQ ID NO:3，
LCDR1包含SEQ ID NO:9，
LCDR2包含SEQ ID NO:10，和
LCDR3包含SEQ ID NO:11；
所述人源化抗CD28抗体通过ELISA测定的与人CD28结合的EC50在0.008μg/mL至0.016μg/mL的范围内；或所述人源化抗CD28抗体通过生物膜层干涉技术(BLI)测定的与人CD28的结合平衡解离常数KD在6.1×10-9M至1.2×10-8M的范围内。
在一些实施方案中，所述人源化抗CD28抗体的重链可变区包括HFR1、HFR2、HFR3和HFR4，所述人源化抗CD28抗体的轻链可变区包括LFR1、LFR2、LFR3和LFR4，其中，
HFR1包含选自SEQ ID NO:19-22的序列，
HFR2包含选自SEQ ID NO:23-26的序列，
HFR3包含选自SEQ ID NO:27-30，
HFR4包含如SEQ ID NO:7所示的序列，
LFR1包含选自SEQ ID NO:36-37的序列，
LFR2包含选自SEQ ID NO:38-39的序列，
LFR3包含选自SEQ ID NO:40-42的序列，和
LFR4包含如SEQ ID NO:15所示的序列。
在一些实施方案中，所述人源化抗CD28抗体的重链可变区包括的HFR1、HFR2、HFR3、HFR4选自下组：
(1)包含如SEQ ID NO:19所示的序列的HFR1，包含如SEQ ID NO:23所示的序列的HFR2，包含如SEQ ID NO:27所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4；
(2)包含如SEQ ID NO:20所示的序列的HFR1，包含如SEQ ID NO:24所示的序列的HFR2，包含如SEQ ID NO:28所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4；
(3)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:25所示的序列的HFR2，包含如SEQ ID NO:29所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4；
(4)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:26所 示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4；和
(5)包含如SEQ ID NO:22所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4。
在一些实施方案中，所述人源化抗CD28抗体的轻链可变区包括的LFR1、LFR2、LFR3、LFR4选自下组：
(1)包含如SEQ ID NO:36所示的序列的LFR1，包含如SEQ ID NO:38所示的序列的LFR2，包含如SEQ ID NO:40所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(2)包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；和
(3)包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4。
在一些实施方案中，所述人源化抗CD28抗体的重链可变区包括的HFR1、HFR2、HFR3、HFR4和轻链可变区包括的LFR1、LFR2、LFR3、LFR4选自下组：
(1)包含如SEQ ID NO:19所示的序列的HFR1，包含如SEQ ID NO:23所示的序列的HFR2，包含如SEQ ID NO:27所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:36所示的序列的LFR1，包含如SEQ ID NO:38所示的序列的LFR2，包含如SEQ ID NO:40所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(2)包含如SEQ ID NO:19所示的序列的HFR1，包含如SEQ ID NO:23所示的序列的HFR2，包含如SEQ ID NO:27所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(3)包含如SEQ ID NO:20所示的序列的HFR1，包含如SEQ ID NO:24所示的序列的HFR2，包含如SEQ ID NO:28所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(4)包含如SEQ ID NO:20所示的序列的HFR1，包含如SEQ ID NO:24所示的序列的HFR2，包含如SEQ ID NO:28所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如 SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(5)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:25所示的序列的HFR2，包含如SEQ ID NO:29所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:36所示的序列的LFR1，包含如SEQ ID NO:38所示的序列的LFR2，包含如SEQ ID NO:40所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(6)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:25所示的序列的HFR2，包含如SEQ ID NO:29所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(7)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:25所示的序列的HFR2，包含如SEQ ID NO:29所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(8)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，(2)包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(9)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
(10)包含如SEQ ID NO:22所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；和
(11)包含如SEQ ID NO:22所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4。
在一些实施方案中，所述人源化抗CD28抗体的重链可变区包含选自SEQ ID NO:31-35的序列。
在一些实施方案中，所述人源化抗CD28抗体的轻链可变区包含选自SEQ ID NO:43-45的序列。
在一些实施方案中，所述人源化抗CD28抗体的重链可变区和轻链可变区选自下述组合：
(1)包含如SEQ ID NO:31所示的序列的重链可变区和包含如SEQ ID NO:43所示的序列的轻链可变区；
(2)包含如SEQ ID NO:31所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；
(3)包含如SEQ ID NO:32所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；
(4)包含如SEQ ID NO:32所示的序列的重链可变区和包含如SEQ ID NO:45所示的序列的轻链可变区；
(5)包含如SEQ ID NO:33所示的序列的重链可变区和包含如SEQ ID NO:43所示的序列的轻链可变区；
(6)包含如SEQ ID NO:33所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；
(7)包含如SEQ ID NO:33所示的序列的重链可变区和包含如SEQ ID NO:45所示的序列的轻链可变区；
(8)包含如SEQ ID NO:34所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；
(9)包含如SEQ ID NO:34所示的序列的重链可变区和包含如SEQ ID NO:45所示的序列的轻链可变区；
(10)包含如SEQ ID NO:35所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；和
(11)包含如SEQ ID NO:35所示的序列的重链可变区和包含如SEQ ID NO:45所示的序列的轻链可变区。
在一些实施方案中，所述人源化抗CD28抗体的重链可变区融合至免疫球蛋白重链恒定区，和/或所述人源化抗CD28抗体的轻链可变区融合至免疫球蛋白轻链恒定区。
在一些实施方案中，所述免疫球蛋白重链恒定区包含SEQ ID NO:81，所述免疫球蛋白轻链恒定区包含SEQ ID NO:82。
在一些实施方案中，所述人源化抗CD28抗体是scFv。
在一些实施方案中，人源化抗CD28 scFv包含通过肽接头连接的重链可变 区和轻链可变区。
在一些实施方案中，人源化抗CD28 scFv中的肽接头的序列包含SEQ ID NO:80。
在一些实施方案中，人源化抗CD28 scFv中中重链可变区位于轻链可变区的N端或C端。
本发明的另一方面提供多特异性抗体，其至少包含第一抗原结合部分和第二抗原结合部分，其中第一抗原结合部分包含前述人源化抗CD28抗体或其抗原结合片段，第二抗原结合部分与第一抗原结合部分结合不同的抗原。
在一些实施方案中，第二抗原结合部分特异性结合肿瘤抗原或能够刺激免疫细胞。
在一些实施方案中，第二抗原结合部分是抗CD40抗体。
在一些实施方案中，所述第一抗原结合部分是scFv，且所述第二抗原结合部分是scFv。
在一些实施方案中，所述第一抗原结合部分与所述第二抗原结合部分通过肽接头连接。
在一些实施方案中，所述肽接头包含SEQ ID NO:79的序列。
在一些实施方案中，所述第一抗原结合部分位于所述第二抗原结合部分的N端或C端。
在一些实施方案中，所述多特异性抗体是双特异性抗体。
在一些实施方案中，所述双特异性抗体包含所述第一抗原结合部分与所述第二抗原结合部分，其中第二抗原结合部分是抗CD40抗体或其抗原结合片段，所述抗CD40抗体通过ELISA测定的与人CD40结合的EC50小于0.04919μg/mL，或者所述抗CD40抗体通过ELISA测定的与人CD40结合的EC50小于3.44×10-7M。
在一些实施方案中，所述抗CD40抗体通过对亲本抗体进行亲和力成熟获得，并且显示出比所述亲本抗体更强的与CD40的结合亲和力；所述亲本抗体具有下述重链可变区和轻链可变区，所述重链可变区包括：包含SEQ ID NO:46的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3，所述轻链可变区包括：包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:56的LCDR3。
在一些实施方案中，所述抗CD40抗体的重链可变区包括：包含选自SEQ ID NO:46和SEQ ID NO:62的序列的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3。
在一些实施方案中，所述抗CD40抗体的重链可变区包括：
(1)包含SEQ ID NO:46的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3；或
(2)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3。
在一些实施方案中，所述抗CD40抗体的轻链可变区包括：包含SEQ ID NO:54的LCDR1、包含选自SEQ ID NO:55,64-66的序列的LCDR2和包含选自SEQ ID NO:56,67-68序列的LCDR3。
在一些实施方案中，所述抗CD40抗体的轻链可变区包括：
(1)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:64的LCDR2和包含SEQ ID NO:56的LCDR3；
(2)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:67的LCDR3；
(3)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:68的LCDR3；
(4)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:65的LCDR2和包含SEQ ID NO:56的LCDR3；
(5)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:66的LCDR2和包含SEQ ID NO:56的LCDR3。
在一些实施方案中，所述抗CD40抗体的重链可变区和轻链可变区包括：
(1)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:64的LCDR2和包含SEQ ID NO:56的LCDR3；
(2)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:67的LCDR3；
(3)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:68的LCDR3；
(4)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:65的LCDR2和包含SEQ ID NO:56的LCDR3；
(5)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:66的LCDR2和包含SEQ ID NO:56的LCDR3。
在一些实施方案中，所述抗CD40抗体的重链可变区包括：
(1)包含SEQ ID NO:49的HFR1、包含SEQ ID NO:50的HFR2、包含SEQ ID NO:51的HFR3和包含SEQ ID NO:52的HFR4；
(2)包含SEQ ID NO:63的HFR1、包含SEQ ID NO:50的HFR2、包含SEQ  ID NO:51的HFR3和包含SEQ ID NO:52的HFR4。
在一些实施方案中，所述抗CD40抗体的轻链可变区包括：包含SEQ ID NO:57的LFR1、包含SEQ ID NO:58的LFR2、包含SEQ ID NO:59的LFR3和包含SEQ ID NO:60的LFR4。
在一些实施方案中，所述抗CD40抗体的重链可变区与选自SEQ ID NO:69-73的序列具有至少70％的序列一致性。
在一些实施方案中，所述抗CD40抗体的重链可变区包含选自SEQ ID NO:69-73的序列。
在一些实施方案中，所述抗CD40抗体的轻链可变区与选自SEQ ID NO:74-78的序列具有至少70％的序列一致性。
在一些实施方案中，所述抗CD40抗体的轻链可变区包含选自SEQ ID NO:74-78的序列。
在一些实施方案中，所述抗CD40抗体是scFv。
在一些实施方案中，所述抗CD40 scFv包含通过肽接头连接的重链可变区和轻链可变区。
在一些实施方案中，所述抗CD40 scFv中的肽接头的序列包含SEQ ID NO:80。
在一些实施方案中，所述抗CD40 scFv中中重链可变区位于轻链可变区的N端或C端。
本发明的另一方面提供编码前述人源化抗CD28抗体或其抗原结合片段，或前述多特异性抗体或双特异性抗体的多核苷酸。
本发明的另一方面提供包含前述多核苷酸的载体。
在一些实施方案中，所述载体是质粒载体、病毒载体、包含所述多核苷酸的环状RNA、能够通过环化获得所述环状RNA的前体RNA、或包含所述前体RNA的DNA模板的载体；其中所述环状RNA依序包括：内部核糖体进入位点(IRES)元件、所述多核苷酸和polyA。
在一些实施方案中，所述polyA的长度为至少45个核苷酸；优选地，所述polyA的长度为至少70个核苷酸。
本发明的另一方面提供包含前述多核苷酸或前述载体的宿主细胞。
本发明的另一方面提供药物组合物，其包含前述人源化抗CD28抗体或其抗原结合片段、前述多特异性抗体、前述双特异性抗体、前述多核苷酸或前述载体，以及药学可接受的载体。本发明的另一方面提供药物组合，包括：a)前述人源化抗CD28抗体或其抗原结合片段、前述多特异性抗体、前述双特异性抗体或前述多核苷酸或前述载体；和b)免疫细胞的组合；或者提供表达前述人源化抗CD28抗体或其抗原结合片段、前述多特异性抗体、前述双特异性抗体的免疫细胞。
在一些实施方案中，所述免疫细胞是T细胞、NK细胞、NKT细胞、巨噬细胞、中性粒细胞或粒细胞。
在一些实施方案中，所述免疫细胞重组表达嵌合抗原受体(CAR)、T细胞受体(TCR)或双特异性T细胞接合子(BiTE)，其中所述CAR、TCR或BiTE结合肿瘤抗原或病毒抗原。在一些实施方案中，所述免疫细胞是CAR-T细胞。
在一些实施方案中，所述CAR靶向间皮素、CD123、BCMA、HER2、IL13Ra2、CD19或B7H3。
在一些实施方案中，靶向间皮素、CD123、BCMA、HER2、IL13Ra2、CD19或B7H3的CAR如本文中所限定。
本发明的另一方面提供增强免疫细胞对受试者中的靶细胞的杀伤作用的方法，包括给接受所述免疫细胞的受试者施用前述人源化抗CD28抗体或其抗原结合片段、前述多特异性抗体、前述双特异性抗体、前述多核苷酸、前述载体或如前述药物组合物。在一些实施方案中，所述免疫细胞如前文所限定。
本发明还提供前述人源化抗CD28抗体或其抗原结合片段、前述多特异性抗体、前述双特异性抗体、前述多核苷酸、前述载体或如前述药物组合物在增强免疫细胞对受试者中的靶细胞的杀伤作用中的应用，或在制备用于增强免疫细胞对受试者中的靶细胞的杀伤作用的药物中的应用。所述免疫细胞如前文所限定。
本发明的另一方面提供杀伤受试者中的靶细胞的方法，包括给所述受试者施用前述的药物组合或前述的免疫细胞。
在一些实施方案中，所述靶细胞是肿瘤细胞。在一些实施方案中，所述肿瘤细胞表达间皮素、CD123、BCMA、HER2、IL13Ra2、CD19或B7H3。
在一些实施方案中，所述免疫细胞是CAR-T细胞。
本发明还提供前述的药物组合或前述的免疫细胞在杀伤受试者中的靶细胞中的应用，或在制备用于杀伤受试者中的靶细胞中的应用。
本发明的另一方面提供在有需要的受试者中治疗疾病的方法，包括给受试者施用治疗有效量的前述的药物组合或前述的免疫细胞。
在一些实施方案中，所述疾病是肿瘤、癌症、病毒感染或自身免疫性疾病。
在一些实施方案中，所述癌症表达间皮素、CD123、BCMA、HER2、IL13Ra2或B7H3。
在一些实施方案中，所述癌症是实体瘤或血液学癌症。
在一些实施方案中，所述癌症是急性骨髓性白血病(AML)、B型急性淋巴细胞白血病(B-ALL)、T型急性淋巴细胞白血病(T-ALL)、B细胞前体急性淋巴细胞白血病(BCP-ALL)或blastic placytoid dendritic cell neoplasm(BPDCN)。非霍奇金淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、人类B细胞前体白血病、多发性骨髓瘤或恶性淋巴瘤。
在一些实施方案中，癌症是间皮瘤、胰腺癌、卵巢癌、肺癌、乳腺癌、胃癌、宫颈癌、尿路上皮癌、食道癌、膀胱癌、结直肠癌、子宫内膜癌、肾癌、头颈癌、肉瘤、胶质细胞瘤、前列腺癌、甲状腺癌或胶质瘤。
本发明的另一方面提供前述的药物组合或前述的免疫细胞在治疗前述疾病中的应用，或者在制备用于治疗前述疾病的药物中的应用。
本发明的另一方面提供诱导T细胞增殖的方法，包括使前述人源化抗CD28抗体或其抗原结合片段、前述多特异性抗体、前述双特异性抗体、前述多核苷酸或前述载体与T细胞接触。
在一些实施方案中，所述方法进一步包括将前述多核苷酸或前述载体引入所述T细胞，并使所述T细胞表达前述人源化抗CD28抗体或其抗原结合片段或前述多特异性抗体。
在一些实施方案中，所述T细胞表达嵌合抗原受体(CAR)。
本发明的另一方面提供前述人源化抗CD28抗体或其抗原结合片段、前述多特异性抗体、前述双特异性抗体、前述多核苷酸或前述载体在诱导T细胞增殖中的用途，或在制备用于诱导T细胞增殖的药物中的用途。
附图说明
图1：CD28抗体9.3可变区的结构模型卡通图。左边链代表重链，右边链代表轻链。
图2A：人源化抗体的重链突变序列。“·”代表相同的氨基酸，下划线表示CDR区域。
图2B：人源化抗体的轻链突变序列。“·”代表相同的氨基酸，下划线表示CDR区域。
图3：人源化抗体的SEC图谱。
图4：人源化抗体的ELISA结果。
图5：人源化抗体的传感图。
图6：CD40在A549细胞上的表达水平。在A549细胞中电转10μg CD40 mRNA，24小时后，用流式细胞术和PE-抗-CD40抗体检测CD40的表达水平。
图7：双抗的表达水平。在T细胞中电转15μg双抗的mRNA和0.5μg 4D5.BBZ mRNA，24小时后，用流式细胞术、CD40-Fc和PE-抗-IgG-Fc抗体检测双抗的表达水平。
图8：Her2-CAR的表达水平。在T细胞中电转15μg双抗的mRNA和0.5μg 4D5.BBZ mRNA，24小时后，用流式细胞术、Her2-Fc和PE-抗-Fc抗体检测Her2-CAR的表达水平。
图9：IFN-gamma释放水平。将CAR-T细胞和靶细胞按E:T＝1:1共培养24小时后，通过ELISA检测上清中IFN-gamma的水平。
图10：促杀伤功能。将CAR-T细胞和靶细胞按E:T＝10:1共培养72小时，通过incucyte观察细胞杀伤，图示纵坐标TGOII(GCU×μm2/well)为“Total Green object integrated intensity(GCU×μm2/well)”的缩写，意为“总绿色对象集成强度(GCU×μm2/孔)”，下同。
图11：CD40在A549细胞上的表达水平。在A549细胞中电转10μg CD40 mRNA，24小时后，用流式细胞术和PE-抗-CD40抗体检测CD40的表达水平。
图12：Meso在A549细胞上的表达水平。在A549细胞中电转10μg Meso mRNA，24小时后，用流式细胞术、Meso-biotin和PE-streptavidin抗体检测Meso的表达水平。
图13：双抗的表达水平。在T细胞中电转15μg双抗的mRNA和2μg M12 mRNA，24小时后，用流式细胞术、CD40-Fc和PE-抗-Fc抗体检测双抗的表达水平。
图14：Meso-CAR的表达水平。在T细胞中电转15μg双抗的mRNA和2μg M12 mRNA，24小时后，用流式细胞术、Meso-Fc和PE-抗-Fc抗体检测Meso-CAR的表达水平。
图15：IL-2和IFN-gamma释放水平。将CAR-T细胞和靶细胞按E:T＝1:1共培养24小时后，通过ELISA检测上清中IL-2和IFN-gamma的水平。
图16：促杀伤功能。将CAR-T细胞和靶细胞按E:T＝10:1共培养72小时，通过incucyte观察细胞杀伤。
图17：CD40、Her2-CAR和双抗的基因过表达水平检测。(A)CD40在A549细胞上的表达水平。(B)Her2-CAR和双抗的表达水平。
图18：靶细胞在Her2-CAR-T(有\无共电转BsAb)作用下IL-2和IFN-gamma释放水平的ELISA检测。
图19：incucyte S3活细胞动态成像和分析系统检测Her2-CAR-T(有\无共电转BsAb)对靶细胞的促杀伤功能。
图20：CD40、Meso-CAR和双抗的基因过表达水平检测。(A)Meso和CD40在A549细胞上的表达水平。(B)Meso-CAR和双抗的表达水平。
图21：靶细胞在Meso-CAR-T(共电转BsAb)作用下IL-2和IFN-gamma释放水平的ELISA检测。
图22：incucyte S3活细胞动态成像和分析系统检测Meso-CAR-T(共电转BsAb)对靶细胞双抗的促杀伤功能。
图23：CD40、Her2-CAR和9.3-40.52优化双抗的基因过表达水平检测。(A)CD40在A549细胞上的表达水平。(B)Her2-CAR的表达水平。(C)9.3-40.52优化双抗BsAb的表达水平。
图24：靶细胞在Her2-CAR-T(有\无共电转9.3-40.52优化BsAb)作用下IFN-gamma释放水平的ELISA检测。
图25：incucyte S3活细胞动态成像和分析系统检测Her2-CAR-T(有\无共电转9.3-40.52优化BsAb)对靶细胞的促杀伤功能。
图26：CD40、Meso-CAR和9.3-40.52优化双抗的基因过表达水平检测。(A)Meso和CD40在A549细胞上的表达水平。(B)Meso-CAR和9.3-40.52优化双抗的表达水平。
图27：靶细胞在Meso-CAR-T(有\无共电转9.3-40.52优化BsAb)作用下IL-2和IFN-gamma释放水平。
图28：incucyte S3活细胞动态成像和分析系统检测Meso-CAR-T(有\无共电转9.3-40.52优化BsAb)对靶细胞的促杀伤功能。
图29：CD40、Her2-CAR和9.3-A40C优化双抗的基因过表达水平检测。(A)CD40在A549细胞上的表达水平。(B)Her2-CAR和9.3-A40C优化双抗的表达水平。
图30：incucyte S3活细胞动态成像和分析系统检测Her2-CAR-T(有\无共电转9.3-A40C优化BsAb)对靶细胞的促杀伤功能。
图31：CD40、Meso-CAR和9.3-A40C优化双抗的基因过表达水平检测。(A)Meso和CD40在A549细胞上的表达水平。(B)Meso-CAR和9.3-A40C优化双抗的表达水平。
图32：靶细胞在Meso-CAR-T(有\无共电转9.3-A40C优化BsAb)作用下IFN-gamma释放水平。
图33：incucyte S3活细胞动态成像和分析系统检测Meso-CAR-T(有\无共电转9.3-A40C优化BsAb)对靶细胞的双抗的促杀伤功能。
图34：F2.103\40.52\A40C-CD40抗体的亲和力ELISA结果。
图35：亲和力成熟抗体的序列。“·”代表相同的氨基酸，下划线表示CDR区域。(A)亲和力成熟抗体的VH区序列比对结果。(B)亲和力成熟抗体的VL区序列比对结果。
图36：噬菌体ELISA的结果。
图37-A：CD40在A549细胞上的表达水平。
图37(B-D)：Her2-CAR和AM40.52-9.3h11双抗的表达水平。
图38：靶细胞在Her2-CAR-T(有\无共电转AM40.52-9.3h11BsAb)作用下IL-2和IFN-gamma释放水平ELISA检测。
图39(A-F)：incucyte S3活细胞动态成像和分析系统检测Her2-CAR-T(有\无共电转AM40.52-9.3h11BsAb)对靶细胞促杀伤功能。
图40：基因过表达水平检测。(A)Meso和CD40在Molm14细胞上的表达 水平。(B)Meso-CAR和AM40.52-9.3h11双抗的表达水平。
图41：靶细胞在Meso-CAR-T(有\无共电转AM40.52-9.3h11BsAb)作用下IL-2和IFN-gamma释放水平ELISA检测。
图42(A-D)：incucyte S3活细胞动态成像和分析系统检测Meso-CAR-T(有\无共电转AM40.52-9.3h11BsAb)对靶细胞的促杀伤功能。
图43：靶细胞在Meso-CAR-T(有\无共电转AM40.52-9.3h11BsAb)作用下IL-2和IFN-gamma释放水平ELISA检测。
图44(A-F)：incucyte S3活细胞动态成像和分析系统检测双抗的特异性,Mesothelin和CD40在不同肿瘤细胞表面的表达丰度(F)。
图45：亲和力成熟抗体在非还原(A)和还原(B)条件下的SDS-PAGE图谱。
图46：亲和力成熟抗体的ELISA结果。
图47：40.52抗体(A)和4052.17抗体(B)的SPR拟合曲线。
图48：慢病毒在CAR-T细胞中的转导效率。
图49：慢病毒转导的CAR-T细胞的杀伤结果。
图50：慢病毒转导的CAR-T细胞释放的细胞因子。
图51：慢病毒转导的CAR-T细胞对不同肿瘤细胞的杀伤结果。
图52：慢病毒转导的CAR-T细胞对不同肿瘤细胞释放的细胞因子。
图53：低剂量CAR-T回输的动物实验结果。(A)小鼠体内肿瘤定量图，(B)统计肿瘤荧光值大小，(C)小鼠肿瘤大小，(D)小鼠体重变化，(E)小鼠生存曲线。
图54：高剂量CAR-T回输的动物实验结果。(A)小鼠体内肿瘤定量图，(B)统计肿瘤荧光值大小，(C)小鼠肿瘤大小，(D)小鼠体重变化，(E)小鼠生存曲线。
图55提供了两个抗人间皮素-Fc单克隆噬菌体ELISA的96孔板的读数。
图56提供了用于产生抗间皮素CAR mRNA的pDA-CAR载体的示意图。
图57提供了FACS染色结果，其显示了在CART细胞中表达的抗间皮素scFv与间皮素-Fc蛋白的结合。
图58提供了A549细胞通过抗间皮素抗体进行的FACS染色，所述A549细胞用不同量的间皮素mRNA电穿孔。
图59提供了，不同mRNA为基础的间皮素CART细胞在E/T比值＝10：1时，对A549-GFP肿瘤细胞的杀伤曲线。
图60提供了，不同mRNA为基础的间皮素CART细胞，在E/T比值＝10：1时，对A549-GFP肿瘤细胞的杀伤曲线，其中所述A549-GFP肿瘤细胞用10μg间皮素mRNA电穿孔。
图61提供了，不同mRNA为基础的间皮素CART细胞，在E/T比值＝10：1时，对A549-GFP肿瘤细胞的杀伤曲线，其中所述A549-GFP肿瘤细胞用2μg间皮素mRNA电穿孔。
图62提供了，不同mRNA为基础的间皮素CART细胞，在E/T比值＝10：1时，对A549-GFP肿瘤细胞的杀伤曲线，其中所述A549-GFP肿瘤细胞用0μg、2μg或10μg间皮素mRNA电穿孔。
图63提供了同种型对照和抗间皮素mAb对OVCAR3、H226、ASPC1、A549和HCC70的FACS染色。
图64提供了抗间皮素M12和M32 CART细胞在与OVCAR3、H226、ASPC1、A549和HCC70肿瘤细胞系共培养和杀伤试验中的CD107a染色。
图65A-65B提供了用CD19-Fc(图65A)或BCMA-Fc(图65B)染色的抗BCMA CAR-T细胞的流式细胞术数据。
图66A提供了用指定的BCMA CAR转导的T细胞中CAR+细胞的频率。
图66B提供了用指定的BCMA CAR转导的T细胞中CAR表达的平均荧光强度(MFI)。
图67提供了用指定的BCMA CAR转导的T细胞中CAR+CD8细胞的频率。
图68提供了以CCR7表达和CD45RO表达为特征的指定CART细胞的表型。
图69A-69B提供了BCMA在肿瘤细胞系中的表达。图69A提供了FACS结果。图69B提供了与相比于A549细胞的相对表达水平。
图70A-70B提供了ELISA结果，其显示了由指定的CART细胞产生INF-γ和IL-2。图70A显示了INF-γ的产生。图70B显示了IL-2的产生。
图71A-71D提供了肿瘤杀伤试验的结果，其显示了指定CART细胞在不同E(T细胞):T(肿瘤细胞)比例下对Jeko-1细胞的细胞溶解活性。图71A：E：T＝0.1：1；图71B：E：T＝0.5：1；图71C：E：T＝2：1；图71D：E：T＝2：1(放大图)。
图72A-72E提供了肿瘤杀伤试验结果，其显示了指定的CART细胞对RPMI-8226细胞的细胞溶解活性。图72A:E:T＝0.1:1；图72B:E:T＝0.5:1；图72C:E:T＝2:1(放大图)；图72D:E:T＝2:1；图72E:E:T＝0.5:1(放大视图)。
图73提供了抗人CD123-Fc单克隆噬菌体ELISA的两个96孔板的读数。
图74提供了用于CAR mRNA生成的pDA-CAR载体的示意图。
图75提供了FACS染色结果，显示了在CART细胞中表达的抗CD123 scFv与CD123-Fc蛋白的结合。
图76提供了同种型和抗CD123抗体对用不同量的CD123 mRNA电穿孔的A549细胞的FACS染色结果。
图77提供了不同的基于mRNA的抗CD123 CART细胞在E/T比值＝10:1时， 对A549-GFP肿瘤细胞的杀伤曲线。
图78提供了不同的基于mRNA的抗CD123 CART细胞在E/T比值＝3:1时，对A549-GFP肿瘤细胞的杀伤曲线。
图79提供了不同的基于mRNA的抗CD123 CART细胞在E/T比值＝10:1时，对用10μg CD123mRNA电穿孔的A549-GFP肿瘤细胞的杀伤曲线。
图80提供了不同的基于mRNA的抗CD123 CART细胞在E/T比值＝3:1时，对用10μg CD123mRNA电穿孔的A549-GFP肿瘤细胞的杀伤曲线。
图81显示了PE-同种型对照和PE-抗-CD123 mAb对A549、SK-OV3、Jeko-1、Molm-14、SupT-1、293T、Nalm-6和PC-3细胞的FACS染色结果。
图82显示了在与不同肿瘤细胞的共培养和杀伤试验中，抗-CD123-C5、抗-CD123-C7、抗-CD123-C11 CART细胞的CD107a染色。
具体实施方式
除非另外指出，本发明中所用的术语具有本领域通常所理解的含义，可以通过参考本领域技术人员已知的标准教科书，参考书、文献加以理解。本文所提到的所有出版物均通过全文引用的方式并入本文中。
应当理解，本发明实施例中所描述的具体方法和材料仅仅是出于描述具体实施例的目的，并非是是限制性的，在本发明的实践或测试中可以使用类似于或等同于本文所描述的方法和材料的任何方法和材料。本发明中对于相关理论或机理的解释仅仅是用于帮助理解发明，不应视为对本发明所保护的方案的限制。
本文中所使用的术语“包含”，或与其具有类似含义的其他术语形式“包括”、“含有”或“具有”等，应当理解为包括所列出的要素，但不排除其他要素的存在。这些术语也包括仅由所列出的要素组成的情况。术语“由......组成”是指仅由所列举的要素组成。术语“基本上由......组成”表示不排除对所涉及的方案没有显著影响的要素。
除非另外特别说明，本文中的“a”、“an”或“the”包括单数形式和复数形式。术语“至少一个”或“至少一种”或其它类似表述的含义等同于“一个或更多个”或“一种或更多种”的含义。
本文中所述的数值范围，例如温度范围，时间范围，组成或浓度范围，或其他数值范围等，包括该范围内的端值、所有中间范围、子范围(例如在某个中间值和某个端值之间的范围)以及所有单个数值，特别是以整数数值为端值的中间范围、子范围和单个的整数数值。并且，所述数值范围中所描述的任何中间范围、子范围以及所有单个数值可以从所述的数值范围中排除。
本文中所述的“约”、“大约”的意思为所述数值的±10％的范围。
本文中所述的“和/或”应当理解为由该术语连接的多个要素中的任何一个要素或任意几个要素的组合。
本文使用的术语“核酸序列”和“多核苷酸”可以互换使用，是指由碱基、糖和磷酸骨架连接构成的序列。本发明的核酸序列可以是脱氧核糖核酸序列(DNA)或核糖核酸序列(RNA)，且可包括天然碱基或非天然碱基，其可以是单链或双链，可以是编码序列或非编码序列。
术语“多肽”和“蛋白质”可互换使用且指氨基酸残基的聚合物。此类聚合物可含有天然或非天然氨基酸残基且包括但不限于氨基酸残基构成的肽、寡肽、二聚体、三聚体和多聚体。全长蛋白与其片段皆涵盖于该定义中。该术语亦包括具有表达后修饰，例如糖基化、唾液酸化、乙酰化、磷酸化和类似修饰的多肽。
术语“分离的蛋白质”或“分离的多肽”是指如这样的蛋白或多肽：(1)其与在天然状态中伴随的天然相关的成分不关联；(2)不含有没有来自相同物种的其他蛋白质；(3)由来自不同物种的细胞表达；或者(4)不是天然发生的。化学合成或在不同于其天然起源的细胞的细胞系统中合成的多肽将是与其天然结合的组分“分离的”。还可以通过分离，使用本领域众所周知的蛋白质纯化技术，使得蛋白质基本上不含天然结合的组分。本发明中提及的蛋白质或多肽，例如抗体或其抗原结合片段，优选是分离的。
术语“分离的核酸”是指基因组、cDNA或合成源或者其组合的核酸分子，其与所述核酸天然来源中存在的其他核酸分子分离。例如，关于基因组DNA，术语“分离的”包括与所述基因组DNA天然相关的染色体分离的核酸分子。优选地，“分离的”核酸不包括天然在核酸两侧的序列(即位于感兴趣的核酸的5’和3’末端的序列)。本发明提及的多核苷酸或核酸序列，优选是分离的。
本发明中，除非另有说明，核酸序列的描述通常是从5’端至3’端，氨基酸序列的描述通常是从N端至C端。
术语“抗原”具有本领域普通技术人员所通常理解的含义，包括多肽、多糖、糖蛋白、多核苷酸等。术语“抗原表位”也可被称为“抗原决定簇”，是指抗原表面上决定抗原特异性的化学官能团。抗原表位可被抗体中的抗原结合位点识别并结合。
本文使用的术语“抗体”以它的最广泛意义使用，包括包含一个或更多个特异性结合抗原的抗原结合结构域的免疫球蛋白或其他类型分子，为对特定抗原表现出结合特异性的蛋白质或多肽。抗体的具体实例可包括(例如经典四链抗体分子)、单链抗体、单域抗体、多特异性抗体等。
完整抗体，在本发明中也可以被称为全长抗体或经典抗体分子，是由四条多肽链组成的免疫球蛋白分子(如IgG)或其多聚体(如IgA或IgM)。所述的四条多肽链包括两条相同的重链(H)和两条相同的轻链(L)，它们通过二硫键相互连接形成四聚体。每条重链由重链可变区(“HCVR”或“VH”)和重链恒定 区(CH，包括结构域CH1、CH2和CH3)组成。每条轻链由轻链可变区(“LCVR或“VL”)和轻链恒定区(CL)组成。重链和轻链的恒定区(CH和CL)不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导抗体与宿主组织或因子，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。重链和轻链的可变区(VH和VL)形成抗原结合部位，VH和VL分别具有被称为互补决定区(CDR)的高变区，其氨基酸组成和排列顺序具有较高的变异程度，为抗体与抗原结合的关键位置，其间插有更保守的被称为框架区(FR)的序列。每个VH和VL由三个CDR和四个FR组成，以下列顺序从氨基末端至羟基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。在本文中，三个重链互补决定区分别称为HCDR1(CDR-H1)、HCDR2(CDR-H2)和HCDR3(CDR-H3)，四个重链框架区分别称为HFR1(FR-H1)、HFR2(FR-H2)、HFR3(FR-H3)和HFR4(FR-H4)；三个轻链互补决定区分别称为LCDR1(CDR-L1)、LCDR2(CDR-L2)和LCDR3(CDR-L3)，四个轻链链框架区分别称为LFR1(FR-L1)、LFR2(FR-L2)、LFR3(FR-L3)和LFR4(FR-L4)。重链和轻链的可变区(VH和VL)分别形成抗原结合部位。
本文使用的术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia et al.,(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别根据各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。在本发明的实施例中以及具体序列的描述中，使用Kabat、Chothia、AbM、Contact或IMGT编号系统定义CDR，但应当理解，本发明中的抗体还可以利用其他编号系统定义CDR。
本文使用的术语“框架区”或“FR”是指抗体可变区中除了如上定义的CDR序列以外的那些氨基酸序列。
根据抗体重链恒定区的氨基酸序列，可将抗体分为五种主要的不同类型：IgA、IgD、IgE、IgG和IgM。这些抗体类型根据铰链区的大小，链间二硫键的位置和分子量的不同可进一步分为亚类，例如，IgGl、IgG2a、IgG2b、IgG3和IgG4等。根据抗体轻链恒定区氨基酸组成和排列的不同，可将轻链分为κ和λ两种类型。本发明的抗体包括任何前述类别或亚类的抗体。
本文使用的术语“单克隆抗体”指均一的仅针对某一特定抗原表位的抗体。与典型的包括针对不同抗原表位的不同抗体的普通多克隆抗体制剂相比，每种单克隆抗体针对抗原上的单个抗原表位。虽然单克隆抗体可以通过杂交瘤培养获得，但在本发明中，修饰语“单克隆”表示抗体的均一特征，不应理解为需要通过 任何特定方法产生的抗体。本发明的单克隆抗体可以通过重组DNA方法产生，或通过其他筛选方法获得。
本文使用的术语“抗原结合片段”指完整抗体的一个或多个部分，所述部分保留特异性结合抗体所针对的抗原表位的能力，例如，可参见Fundamental Immunology,Ch.7(Paul,W.,ed.,2th Edition,Raven Press,N.Y.(1989)。抗原结合片段的实例包括：(1)Fab片段：由完整抗体经木瓜蛋白酶消化产生的抗原结合片段，由全长抗体的完整轻链(包括VL结构域和CL结构域)以及全长抗体的重链可变区VH和重链恒定区结构域CH1组成，不含铰链区；(2)F(ab’)2片段：由完整抗体经胃蛋白酶消化产生的抗原结合片段，其包含两个全长抗体的完整轻链(包括VL结构域和CL结构域)以及两个全长抗体的重链可变区VH和重链恒定区结构域CH1以及铰链区，其可视为在其间具有二硫键的两个Fab'片段配对而成的片段；(3)Fab’片段，可通过以还原剂处理断开F(ab’)2铰链区之间的二硫键而形成；(4)由VH结构域和CH1结构域组成的Fd片段；(5)由抗体单臂的VL结构域和VH结构域组成的Fv片段。术语“抗原结合片段”通常至少包含完整抗体的重链可变区和轻链可变区中的任一者或二者，还可以包含完整抗体的轻链恒定区和/或重链恒定区的一部分或全部，例如可以包含CL结构域、CH1结构域、CH2结构域、CH3结构域中的一种或两种或更多种。
本文使用的术语“Fc区”指免疫球蛋白重链的C末端区域，即构成Y形经典抗体分子的柄部区域的可结晶片段(fragment crystallizable，Fc)，包括重链的第二和第三恒定结构域(CH2和CH3结构域)。Fc区可通过使用木瓜蛋白酶水解完整抗体分子获得。在一些实例中，Fc区可包含铰链、CH2和CH3。当Fc区包含铰链时可介导两个含Fc的多肽之间的二聚化。Fc片段可来自IgG、IgM、IgD、IgE或IgA。在一些实例中，Fc区来自IgG1、IgG2、IgG3或IgG4。“Fc片段”还包括来自天然Fc片段，经改动但仍保持其效应功能的变体Fc片段。“变体Fc片段”包含在天然Fc片段的氨基酸序列上具有至少一个氨基酸取代的氨基酸序列。在一些实例中，变体Fc片段相比于亲本Fc片段(天然Fc片段)具有至少一个氨基酸取代，例如在亲本Fc片段中约1至约10个氨基酸被取代，且优选约1至约5个氨基酸取代。在一些实例中，变体Fc片段Fc区与亲本Fc片段具有至少约80％序列一致性、至少约90％序列一致性、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％序列一致性。“Fc片段”的效应功能可包括与Fc受体的结合、Clq结合和补体依赖性细胞毒性(CDC)、抗体依赖性细胞介导的细胞毒性(ADCC)、介导吞噬作用等。
本文使用的术语“单域抗体(sdAb)”是指具有单个可变结构域的抗原结合多肽，其包含三个互补决定区(CDR)。单域抗体能够单独结合抗原而不用与另外的包含CDR的多肽配对。常见的单域抗体包含重链但缺少通常见于抗体中的轻链，例如骆驼科sdAb(参见e.g.,Hamers-Casterman et al.,Nature 363:446-8(1993)；Greenberg et al.,Nature 374:168-73(1995)；Hassanzadeh-Ghassabeh et al.,Nanomedicine(Lond),8:1013-26(2013))。单域抗体可以从骆驼的重链抗体人为 工程化获得，称为VHH抗体，VHH抗体从N端到C端具有如下结构：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。单域抗体也可以被视为全长抗体的一种特殊的“抗原结合片段”。
本文使用的术语“单链抗体(scFv)”是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如，Bird et al.,Science 242:423-426(1988)；Huston et al.,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun,The Pharmacology of Monoclonal Antibodies,113,Roseburg&Moore,Springer-Verlag,New York,269-315(1994))。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。合适的接头可由重复的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，或其变体(Holliger et al.,(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。在一些情况下，scFv的VH与VL之间还可以存在二硫键。如本文中所使用的，术语“di-scFv”是指，由两个scFv连接形成的抗体片段。
如本领域技术人员所知，抗体可以通过多种技术进行制备，例如杂交瘤技术(参见，例如，Kohler et al.,Nature,256:495,1975)，重组DNA技术(参见，例如，美国专利申请4,816,567)，或噬菌体抗体库技术(参见，例如，Clackson et al.,Nature,352:624-628,1991，或Marks et al.,J.Mol.Biol.222:581-597,1991)。
如本领域技术人员所知，抗体可通过公知的技术，例如使用蛋白A或蛋白G的亲和层析进行纯化。随后或作为替代，可将特异性抗原(该抗体识别的靶分子)或其抗原表位固定在柱上，并通过免疫亲合层析法来纯化免疫特异性抗体。抗体的纯化可参考例如D.Wilkinson,TheScientist,published by The Scientist,Inc.,Philadelphia PA,Vol.14,No.8(Apr.17,2000),pp.25-28)。
本文使用的术语“嵌合抗体(Chimeric antibody)”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)，但仍保留对目标抗原的结合活性(Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。术语“嵌合抗体”可指包含来自一个物种的重链可变区和轻链可变区序列以及来自另一个物种的恒定区序列的抗体，例如具有与人恒定区连和鼠重链可变区和轻链可变区的抗体。
本文使用的术语“人源抗体”指抗体的全部序列(如可变区和恒定区)来自于人免疫球蛋白的抗体。“非人源抗体”指抗体的全部序列(如可变区和恒定区)来自于非人物种的免疫球蛋白的抗体，例如来自于鼠的免疫球蛋白的抗体可被称为鼠源抗体。
本文使用的术语“人源化抗体”是指对非人源抗体，例如可变区和恒定区(如果有的话)非衍生自人免疫球蛋白的抗体进行改造使其与人源抗体序列的同源性提高，由此获得的嵌合抗体。通常而言，人源化抗体的全部或部分CDR区来 自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)的小鼠、大鼠、兔或非人灵长类动物(例如，食蟹猴)抗体。人源化抗体既能够保留非人源供体抗体(例如鼠源抗体)的预期性质，又能够有效降低非人源供体抗体(例如鼠源抗体)在人受试者中的免疫原性，因此是特别有利的。然而，由于供体抗体的CDR与受体抗体的FR之间的匹配问题，人源化抗体的预期性质(例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)通常低于非人源供体抗体(例如鼠源抗体)。
在本发明中，为了使人源化抗体尽可能保留供体抗体的性质(包括例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)，人源化抗体中框架区(FR)可以既包含人源受体抗体的氨基酸残基，也包含相应的非人源供体抗体的氨基酸残基，例如，人源化抗体中可以包含回复突变。术语“回复突变(back mutation)”是在人源化抗体的氨基酸中引入的突变，所述突变所产生的氨基酸对应于亲代抗体(例如，供体抗体，例如，鼠源抗体)中的氨基酸。可在抗体的人源化期间保留来自亲代抗体的某些框架残基，以实质上保持亲代抗体的结合性质，而同时使所得抗体的潜在免疫原性降至最低。在本发明的一个实施方案中，亲代抗体是鼠源抗体。举例来说，回复突变将人框架残基变成亲代鼠源残基。可回复突变的框架残基的实例包括但不限于正规残基(canonical residue)、界面填充残基(interfacepacking residue)、邻近结合位点的罕见亲代残基、“游标区(Vernier Zone)”(其形成停靠CDR的平台)中的残基(Foote&Winter,1992,J.Mol.Biol.224,487-499)以及那些邻近CDRH3的残基。
本文使用的术语“亲和力成熟的”抗体是指与参照抗体结合相同抗原，但具有比参照抗体更高的针对该抗原的结合亲和力的抗体。亲和力成熟的抗体可由参照抗体突变获得，此时参照抗体也可被称为亲本抗体。亲和力成熟的抗体通常在一个或多个CDR中相对于亲本抗体具有一个或更多个氨基酸的改变，与没有这些改变的亲本抗体相比较，所述改变导致抗体对靶抗原的亲和力提高。示例性的亲和力成熟的抗体将对靶抗原具有纳摩尔或甚至皮摩尔的亲和力。用于产生亲和力成熟的抗体的方法是本领域已知的。例如，Marks et al.,BioTechnology,10:779-783(1992)描述了通过VH和VL结构域改组的亲和力成熟方法。还可以通过CDR和/或构架残基的随机诱变进行亲和力成熟，例如，参见：Barbas et al.,Proc.Nat.Acad.Sci.USA,91:3809-3813(1994)；Schier et al.,Gene,169:147-155(1995)；Yelton et al.,J.Immunol.,155:1994-2004(1995)；Jackson et al.,J.Immunol.,154(7):3310-3319(1995)；Hawkins et al.,J.Mol.Biol.,226:889-896(1992)。随后可以通过噬菌体展示文库和噬菌体ELISA筛选亲和力成熟的变体。
本发明使用的术语“多特异性抗体”是指能够特异性结合多个抗原表位的抗 体，包括双特异性、三特异性或更多特异性的抗体。所述多个抗原表位可以是同一抗原上的不同表位，也可以是不同抗原上的不同表位。多特异性抗体包含多个抗原结合部分，每个抗原结合部分靶向一种抗原表位且所述多个抗原结合部分靶向的抗原表位彼此不同。每个抗原结合部分可以独立地选自单克隆抗体(如IgG免疫球蛋白)，Fab片段、Fab’片段、F(ab’)2片段、Fv片段、Fd片段、scFv、dsFv、单域抗体、嵌合抗体、人源化抗体、亲和力成熟的抗体等。一个抗原结合部分可以功能性地(例如，通过化学偶联、基因融合、非共价结合或其他方式)连接至一个或更多个另外的抗原结合部分，以形成多特异性抗体。
对于抗体或其抗原结合片段来说，术语“靶向”、“针对”或“特异性结合”指抗体或其抗原结合片段与抗原形成在生理条件下相对稳定的复合物，并且优选对于其它不期望的抗原不表现出明显的结合。一种分子可以靶向、针对或特异性结合一种以上的分子，例如双特异性抗体可以对两种不同抗原具有相对于其他分子而言更高的结合亲和力。特异性结合可以通过抗原与抗体结合的平衡解离常数KD表征(更小的KD表示更紧密的结合)，还可以通过抗体与抗原结合的EC50值来表征。用于确定两分子是否特异性结合的方法为本领域熟知的，包括例如平衡透析、表面等离子体共振等。
术语“EC50(concentration for 50％of maximal effect)”指引起50％最大效应的浓度。在酶联免疫吸附测定(ELISA)中用于表示抗体分子与对应抗原的结合能力时，可指产生最大检测信号(如比色或荧光强度)一半时的抗体分子浓度。EC50值越低，则抗体与抗原的结合亲和力越大。
术语“ka”是指特定的抗体-抗原相互作用的结合速率常数，指示抗体与其靶抗原的结合速率、或抗体与抗原之间的复合物形成速率，单位为M-1s-1，术语“kd”是指特定的抗体-抗原相互作用的解离速率常数，指示抗体从其靶抗原的解离速率、或抗原抗体复合物随时间过去分离为游离抗体和抗原的解离速率，单位为s- 1。
术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，其得自kd与ka的比值(即kd/ka)，以摩尔浓度(M)表示。KD可用于衡量抗体与其结合配体(如抗原)结合的亲和力。较小的KD表示抗体与抗原之间更紧密的结合，或者抗体与抗原之间较高的亲和性。例如，具有纳摩尔(nM)级平衡解离常数的抗体与具有微摩尔(μΜ)级解离常数的抗体相比与特定抗原的结合更紧密。抗体的KD值可以使用本领域熟知的方法确定。确定抗体ka、kd和KD值的一种方法是使用表面等离子共振，典型地使用生物传感器系统如BiacoreTM系统测定。确定抗体KD值的另一种方法是生物膜层干涉技术(Biolayer interferometry，BLI)。
术语“嵌合抗原受体”或“CAR”是指一种融合蛋白，包括能与抗原结合的胞外结构域(即结合结构域)、跨膜结构域和包括一个或多个源自信号转导蛋白的细胞内信号结构域的胞内结构域。这些细胞内信号结构域通常与胞外结构域所来自的多肽不同。胞外结构域可以是任何蛋白质分子或其部分，能与预定 的抗原特异性结合。在一些实施方案中，胞外结构域包括抗体或其抗原结合片段。在一些实施方案中，胞内结构域可以是任何已知的寡肽或多肽域，其功能是传递信号，引起细胞内生物过程的激活或抑制，例如，激活免疫细胞，如T细胞或NK细胞。胞内结构域通常包括免疫受体酪氨酸激活基序(ITAM)，如来自CD3ζ分子的信号域，负责激活免疫效应细胞并产生杀伤作用。另外，嵌合抗原受体也可以在氨基端包括一个信号肽，负责融合蛋白的细胞内定位，以及胞外结构域和跨膜结构域之间的铰链区。胞内信号结构域还可以包括来自例如4-1BB或CD28分子的共刺激结构域。
术语“CART”或“CART细胞”是指能够表达或产生CAR的T细胞。CART细胞通常通过对T细胞进行改造使其包含编码CAR的核酸分子而获得。对于受试者而言，CART细胞可以是通过对自体T细胞或者异体T细胞的改造而获得。
本文使用的术语“重组”指通过重组DNA技术(其包括例如DNA剪接和转基因表达)表达本发明的抗体或其抗原结合片段，例如使用转染至非人哺乳动物(例如小鼠)或细胞(例如CHO细胞)中的重组表达载体表达系统表达抗体或其抗原结合片段，所述抗体可以是或分离自重组或组合性人抗体文库的抗体。
本文所使用的术语“序列同一性”是指当两个或多个序列以最大程度匹配的方式被排列，即考虑到间隙和插入的情况时，相应位置上相同的核苷酸或氨基酸残基的百分比。序列的比对和序列同一性百分比的计算可以用本领域已知的合适的计算机程序来进行。这样的程序包括局部比对程序和全局比对程序，包括但不限于BLAST、ALIGN、ClustalW、EMBOSS Needle等。局部比对程序的一个例子是BLAST(Basic Local Alignment Search Tool)(参见，例如，Altschul et al.,(1990)J.Mol.215；403-410)，它可以从美国国家生物技术信息中心(National Center for Biotechnology Information)的网页http://www.ncbi.nlm.nih.gov/上获得。全局比对程序(在全长序列上优化比对)的例子是基于Needleman-Wunsch算法的EMBOSS Needle和EMBOSS Stretcher程序(Needleman,Saul B.；and Wunsch,Christian D.(1970),"A general method applicable to the search for similarities in the amino acid sequence of two proteins",Journal of Molecular Biology 48(3):443-53)，它们都可以从http://www.ebi.ac.uk/Tools/psa/获得。
本文使用的术语“载体”是指能够将其中包含的异源核酸转运至宿主细胞内(以游离形式存在或整合到宿主细胞基因组上)的任一种分子，例如核酸、质粒或病毒等。载体被引入宿主细胞后可以独立存在并自主复制(例如具有细菌复制起点的细菌载体和游离型哺乳动物载体)，或被整合到宿主细胞基因组上，连同宿主基因组一起进行复制(例如非游离型哺乳动物载体)。用于在宿主细胞中表达目的基因的载体被称为“表达载体”或“重组表达载体”。载体可包含与目的基因可操作连接的调控元件，如复制起点、表达调控序列(例如启动子和/或增强子)和/或可选择标记物基因(例如抗生素抗性基因和可用于比色分析中的基因，例如β-半乳糖)。
为了表达本发明的抗体或其抗原结合片段，可以通过标准技术将编码重链和轻链的表达载体转染到宿主细胞中。术语“转染”旨在包括各种常用于将外源DNA引入原核或真核宿主细胞的技术，例如，电穿孔、磷酸钙沉淀、DEAE-葡聚糖转染等。尽管理论上可以在原核或真核宿主细胞中表达本发明的抗体，但在真核细胞中表达抗体，最优选哺乳动物宿主细胞，因为这种真核细胞，特别是哺乳动物细胞，比原核细胞更有可能组装和分泌正确折叠的免疫学活性抗体。
本文使用的术语“宿主细胞”指可为或已为载体或经分离多核苷酸的接受体的细胞。宿主细胞可为原核细胞或真核细胞。示例性真核细胞包括哺乳动物细胞，诸如灵长类动物或非灵长类动物细胞；真菌细胞，诸如酵母；植物细胞；以及昆虫细胞。非限制性示例性哺乳动物细胞包括但不限于CHO细胞、HEK-293细胞、BHK细胞或PER-C6细胞，以及其衍生细胞，诸如293-6E、CHO-DG44、CHO-K1、CHO-S和CHO-DS细胞，还可以是免疫效应细胞，如T细胞。宿主细胞包括单个宿主细胞的后代，且后代可能由于自然、偶然或故意突变而不一定与原始亲代细胞完全一致，例如在形态或基因组DNA互补方面。宿主细胞可以是分离的细胞或细胞系，也包括在活体内经本文提供的核酸分子或表达载体转染的细胞。
本发明通过同源建模和回复突变的方式对小鼠源9.3抗体进行人源化，解决了亲和力降低的缺点，同时人源化的9.3抗体功能性更强。本发明解决了CD40抗体低亲和力的弱点。
人源化抗CD28抗体
本文使用的术语“CD28”也可被称为分化簇28、Tp44，是作为共刺激受体在T细胞表面表达的抗原，可提供T细胞活化和存活所需的共刺激信号。本文的CD28指来自任何脊椎动物来源的任何CD28蛋白，包括哺乳动物诸如灵长类动物(例如人)、非人灵长类动物(例如猕猴)和啮齿动物(例如小鼠和大鼠)。
本发明提供人源化的抗CD28抗体，所述人源化抗CD28抗体由鼠源抗CD28抗体的基础上进行人源化改造获得，所述鼠源抗CD28抗体例如可以是抗体9.3。本发明的人源化抗CD28抗体可特异性识别CD28，特别是人CD28，包括可溶性CD28或在细胞表面表达的CD28。可溶性CD28包括天然CD28蛋白以及重组CD28蛋白或其变体、单体或二聚体CD28构建体、缺乏跨膜结构域的CD28等。
本发明的人源化抗CD28抗体与人CD28结合的EC50在约0.008μg/mL至约0.016μg/mL的范围内，例如在约0.008μg/mL至约0.013μg/mL的范围内，在约0.008μg/mL至约0.011μg/mL的范围内，在约0.008μg/mL至约0.010μg/mL的范围内或在约0.008μg/mL至约0.009μg/mL的范围内；进一步地，在约8.00ng/mL至约15.50ng/mL的范围内，例在如约8.27ng/mL至约15.09ng/mL的范围内，在如约8.27ng/mL至约15.07ng/mL的范围内，在如约8.27ng/mL至约12.67ng/mL的范围内，在如约8.27ng/mL至约12.65ng/mL的范围内，在如约8.27ng/mL至 约12.59ng/mL的范围内，在如约8.27ng/mL至约12.54ng/mL的范围内，在如约8.27ng/mL至约12.11ng/mL的范围内，在如约8.27ng/mL至约10.90ng/mL的范围内，在如约8.27ng/mL至约10.40ng/mL的范围内或在如约8.27ng/mL至约9.14ng/mL的范围内。在一些实施方案中，本发明的人源化抗CD28抗体的与人CD28结合的EC50为约8.27ng/mL、约9.14ng/mL、约10.40ng/mL、约10.90ng/mL、约12.11ng/mL、约12.54ng/mL、约12.59ng/mL、约12.65ng/mL、约12.67ng/mL、约15.07ng/mL或约15.09ng/mL。所述EC50通过ELISA测定，所述ELISA可以是测定人源化抗体与人CD28(例如在N端包含6个组氨酸的人CD28)的结合活性，用于测定的结合活性的人CD28的浓度例如可以是2μg/ml。
本发明的人源化抗CD28抗体与人CD28的结合平衡解离常数KD在约6.1×10-9M至约1.2×10-8M的范围内，例如在约6.1×10-9M至约1.1×10-8M的范围内，在约6.1×10-9M至约9.6×10-9M的范围内，在约6.1×10-9M至约9.2×10-9M的范围内，在约6.1×10-9M至约8.4×10-9M的范围内，在约6.1×10-9M至约7.5×10-9M的范围内，在约6.1×10-9M至约6.6×10-9M的范围内，或在约6.1×10-9M至约6.2×10-9M的范围内；进一步地，在约6.13×10-9M至约1.15×10-8M的范围内，在约6.13×10-9M至约1.12×10-8M的范围内，在约6.13×10-9M至约1.09×10-8M的范围内，在约6.13×10-9M至约1.08×10-8M的范围内，在约6.13×10-9M至约9.59×10-9M的范围内，在约6.13×10-9M至约9.13×10-9M的范围内，在约6.13×10-9M至约8.36×10-9M的范围内，在约6.13×10-9M至约7.44×10-9M的范围内，在约6.13×10-9M至约6.59×10-9M的范围内，或在约6.13×10-9M至约6.54×10-9M的范围内。在一些实施方案中，本发明的人源化人源化抗CD28抗体与人CD28的结合平衡解离常数KD为约6.13×10-9M、约6.54×10-9M、约6.59×10-9M、约7.44×10-9M、约8.36×10-9M、约9.13×10-9M、约9.59×10-9M、、约1.08×10-8M约1.09×10-8M、、约1.12×10-8M或约1.15×10-8M。所述结合平衡解离常数KD通过生物膜层干涉技术(BLI)测定。
可将鼠源抗CD28抗体(供体抗体)的三个重链CDR与人源抗体(受体抗体)的四个重链FR相连接，以进行抗体重链可变区的人源化，和/或将鼠源抗CD28抗体的三个轻链CDR与人源抗体的四个轻链FR相连接，以进行抗体轻链可变区的人源化。人源化抗CD28抗体可包含人源化重链可变区和/或人源化轻链可变区。在一些实施方案中，所述鼠源抗CD28抗体的重链可变区包括：包含SEQ ID NO:1的HCDR1，包含SEQ ID NO:2的HCDR2和包含SEQ ID NO:3的HCDR3。在一些实施方案中，所述鼠源抗CD28抗体的轻链可变区包括：包含SEQ ID NO:9的LCDR1，包含SEQ ID NO:10的LCDR2和包含SEQ ID NO:10的LCDR3。
在人源化改造中用作受体抗体的可以是任何人种系抗体序列，可以分别选择与供体抗体的重链可变区的序列一致性最高的人种系抗体序列作为抗体重链可变区人源化的受体抗体，以及选择与供体抗体的轻链可变区的序列一致性最高的人种系抗体序列作为抗体轻链可变区人源化的受体抗体。在一些实施方案 中，使用人种系IGHV4序列作为鼠源抗CD28抗体的重链可变区人源化的受体抗体。在一些实施方案中，使用人种系IGKV1序列作为鼠源抗CD28抗体的轻链可变区人源化的受体抗体。
本发明的人源化抗CD28抗体可以进一步包含与鼠源抗CD28抗体在CDR中的氨基酸取代，例如，所述人源化抗CD28抗体可以包含在HCDR1、HCDR2和/或HCDR3中的氨基酸取代，还可以包含在LCDR1、LCDR2和/或LCDR3中的氨基酸取代。在一些实施方案中，所述人源化抗CD28抗体重链可变区包含的HCDR2相对于SEQ ID NO:2在位置S12、A12和M15的一个或更多个上具有氨基酸取代(氨基酸残基编号以SEQ ID NO:2为基准，以SEQ ID NO:2的第一个氨基酸残基作为位置1)。在一些实施方案中，所述人源化抗CD28抗体重链可变区包含的HCDR2相对于SEQ ID NO:2在位置M15上具有氨基酸取代，所述氨基酸取代优选为M15K。在一些实施方案中，所述人源化抗CD28抗体重链可变区包含的HCDR2相对于SEQ ID NO:2在位置S12、A12和M15上具有氨基酸取代，所述氨基酸取代优选为S12P、A13S和M15K。因此，在一些实施方案中，所述人源化抗CD28抗体的重链可变区包括：包含SEQ ID NO:1的HCDR1，包含SEQ ID NO:17或18的HCDR2和包含SEQ ID NO:3的HCDR3。
在一些实施方案中，所述人源化抗CD28抗体的重链可变区和轻链可变区包括：(1)包含SEQ ID NO:1的HCDR1，包含SEQ ID NO:2的HCDR2，包含SEQ ID NO:3的HCDR3，包含SEQ ID NO:9的LCDR1，包含SEQ ID NO:10的LCDR2和包含SEQ ID NO:10的LCDR3；(2)包含SEQ ID NO:1的HCDR1，包含SEQ ID NO:17的HCDR2，包含SEQ ID NO:3的HCDR3，包含SEQ ID NO:9的LCDR1，包含SEQ ID NO:10的LCDR2和包含SEQ ID NO:10的LCDR3；或(3)包含SEQ ID NO:1的HCDR1，包含SEQ ID NO:18的HCDR2，包含SEQ ID NO:3的HCDR3，包含SEQ ID NO:9的LCDR1，包含SEQ ID NO:10的LCDR2和包含SEQ ID NO:10的LCDR3
本发明的人源化抗CD28抗体在源自于人种系抗体序列的FR区域上还可以包含回复突变，即FR区域上的一些氨基酸残基被来自鼠源抗体的相应残基取代，以恢复或提高抗体的特异性和亲和力。
在一些实施方案中，本发明的人源化抗CD28抗体的重链可变区包括：包含选自SEQ ID NO:19-22的序列的HFR1、包含选自SEQ ID NO:23-26的序列的HFR2、包含选自SEQ ID NO:27-30的序列的HFR3和包含SEQ ID NO:7的HFR4。本发明的人源化抗CD28抗体的轻链可变区包括：包含选自SEQ ID NO:36-37的序列的LFR1、包含选自SEQ ID NO:38-39的序列的LFR2、包含选自SEQ ID NO:40-42的序列的LFR3和包含SEQ ID NO:15的LFR4。
在一些实施方案中，本发明的人源化抗CD28抗体的重链可变区包含：(1)包含SEQ ID NO:19的HFR1，包含SEQ ID NO:23的HFR2，包含SEQ ID NO:27的HFR3，和包含SEQ ID NO:7的HFR4；(2)包含SEQ ID NO:20的HFR1，包 含SEQ ID NO:24的HFR2，包含SEQ ID NO:28的HFR3，和包含SEQ ID NO:7的HFR4；(3)包含SEQ ID NO:21的HFR1，包含SEQ ID NO:25的HFR2，包含SEQ ID NO:29的HFR3，和包含SEQ ID NO:7的HFR4；(4)包含SEQ ID NO:21的HFR1，包含SEQ ID NO:26的HFR2，包含SEQ ID NO:30的HFR3，和包含SEQ ID NO:7的HFR4；或(5)包含SEQ ID NO:22的HFR1，包含SEQ ID NO:26的HFR2，包含SEQ ID NO:30的HFR3，和包含SEQ ID NO:7的HFR4。在一些实施方案中，本发明的人源化抗CD28抗体的轻链可变区包含：(1)包含SEQ ID NO:36的LFR1，包含SEQ ID NO:38的LFR2，包含SEQ ID NO:40的LFR3，和包含SEQ ID NO:15的LFR4；(2)包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:41的LFR3，和包含SEQ ID NO:15的LFR4；或(3)包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:42的LFR3，和包含SEQ ID NO:15的LFR4。
在一些实施方案中，本发明的人源化抗CD28抗体的重链可变区和轻链可变区包含：(1)包含SEQ ID NO:19的HFR1，包含SEQ ID NO:23的HFR2，包含SEQ ID NO:27的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:36的LFR1，包含SEQ ID NO:38的LFR2，包含SEQ ID NO:40的LFR3，和包含SEQ ID NO:15的LFR4；(2)包含SEQ ID NO:19的HFR1，包含SEQ ID NO:23的HFR2，包含SEQ ID NO:27的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:41的LFR3，和包含SEQ ID NO:15的LFR4；(3)包含SEQ ID NO:20的HFR1，包含SEQ ID NO:24的HFR2，包含SEQ ID NO:28的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:41的LFR3，和包含SEQ ID NO:15的LFR4；(4)包含SEQ ID NO:20的HFR1，包含SEQ ID NO:24的HFR2，包含SEQ ID NO:28的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:42的LFR3，和包含SEQ ID NO:15的LFR4；(5)包含SEQ ID NO:21的HFR1，包含SEQ ID NO:25的HFR2，包含SEQ ID NO:29的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:36的LFR1，包含SEQ ID NO:38的LFR2，包含SEQ ID NO:40的LFR3，和包含SEQ ID NO:15的LFR4；(6)包含SEQ ID NO:21的HFR1，包含SEQ ID NO:25的HFR2，包含SEQ ID NO:29的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:41的LFR3，和包含SEQ ID NO:15的LFR4；(7)包含SEQ ID NO:21的HFR1，包含SEQ ID NO:25的HFR2，包含SEQ ID NO:29的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:42的LFR3，和包含SEQ ID NO:15的LFR4；(8)包含SEQ ID NO:21的HFR1，包含SEQ ID NO:26的HFR2，包含SEQ ID NO:30的HFR3，包含SEQ ID NO:7的HFR4，(2)包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:41的LFR3，和包含SEQ ID NO:15 的LFR4；(9)包含SEQ ID NO:21的HFR1，包含SEQ ID NO:26的HFR2，包含SEQ ID NO:30的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:42的LFR3，和包含SEQ ID NO:15的LFR4；(10)包含SEQ ID NO:22的HFR1，包含SEQ ID NO:26的HFR2，包含SEQ ID NO:30的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:41的LFR3，和包含SEQ ID NO:15的LFR4；或(11)包含SEQ ID NO:22的HFR1，包含SEQ ID NO:26的HFR2，包含SEQ ID NO:30的HFR3，包含SEQ ID NO:7的HFR4，包含SEQ ID NO:37的LFR1，包含SEQ ID NO:39的LFR2，包含SEQ ID NO:42的LFR3，和包含SEQ ID NO:15的LFR4。
在一些实施方案中，本发明的人源化抗CD28抗体的重链可变区包含选自SEQ ID NO:31-35的序列。在一些实施方案中，本发明的人源化抗CD28抗体的轻链可变区包含选自SEQ ID NO:43-45的序列。
在一些实施方案中，本发明的人源化抗CD28抗体的重链可变区和轻链可变区选自下述组合：(1)包含SEQ ID NO:31的重链可变区和包含SEQ ID NO:43的轻链可变区；(2)包含SEQ ID NO:31的重链可变区和包含SEQ ID NO:44的轻链可变区；(3)包含SEQ ID NO:32的重链可变区和包含SEQ ID NO:44的轻链可变区；(4)包含SEQ ID NO:32的重链可变区和包含SEQ ID NO:45的轻链可变区；(5)包含SEQ ID NO:33的重链可变区和包含SEQ ID NO:43的轻链可变区；(6)包含SEQ ID NO:33的重链可变区和包含SEQ ID NO:44的轻链可变区；(7)包含SEQ ID NO:33的重链可变区和包含SEQ ID NO:45的轻链可变区；(8)包含SEQ ID NO:34的重链可变区和包含SEQ ID NO:44的轻链可变区；(9)包含SEQ ID NO:34的重链可变区和包含SEQ ID NO:45的轻链可变区；(10)包含SEQ ID NO:35的重链可变区和包含SEQ ID NO:44的轻链可变区；和(11)包含SEQ ID NO:35的重链可变区和包含SEQ ID NO:45的轻链可变区。
本发明的人源化抗CD28抗体可以是任何包含上述人源化重链可变区和/或人源化轻链可变区的抗体形式，例如全长抗体(如IgG)、单链抗体或单域抗体等。在一些实施方案中，本发明的人源化抗CD28抗体包含免疫球蛋白(如IgG，例如IgG1、IgG2、IgG3或IgG4)的恒定区，例如IgG1、IgG2、IgG3或IgG4的恒定区。在一些实施方案中，所述恒定区是人IgG的恒定区，例如人IgG1、人IgG2、人IgG3或人IgG4的恒定区。在一些实施方案中，上述人源化重链可变区融合至免疫球蛋白重链恒定区(包括CH1、CH2和/或CH3)。在一些实施方案中，上述人源化轻链可变区融合至免疫球蛋白轻链恒定区(CL)。在一些实施方案中，所述免疫球蛋白重链恒定区包含SEQ ID NO:81。在一些实施方案中，所述免疫球蛋白轻链恒定区包含SEQ ID NO:82。
在一些实施方案中，本发明的人源化抗CD28抗体是单链抗体(scFv)，其中人源化重链可变区与人源化轻链可变区通过肽接头(Linker)融合在一起。人源化重链可变区与人源化轻链可变区的连接顺序可以不同，例如，人源化重链 可变区的C端通过肽接头与人源化轻链可变区的N端融合，或者人源化重链可变区的N端通过肽接头与人源化轻链可变区的N端融合。用于连接人源化重链可变区与人源化轻链可变区的肽接头是本领域技术人员熟知的，例如可以是柔性接头，如甘氨酸-丝氨酸聚合物。在一些实施方案中，用于连接人源化重链可变区与人源化轻链可变区的肽接头是GGGGSGGGGSGGGGS(SEQ ID NO:80)。
在一些实施方案中，本发明的人源化抗CD28抗体还可以是单域抗体，例如包含重链可变区，但不包含轻链可变区的单域抗体。制备单域抗体的方法是本领域技术人员熟知的。
本发明还涉及所述人源化抗CD28抗体的抗原结合片段，例如全长抗体的Fab片段、F(ab’)2片段、Fab’片段。
本发明的人源化抗CD28抗体或其结合片段还可以作为融合蛋白的一部分，与其他功能性多肽融合。
亲和力成熟的CD40抗体
本文使用的术语“CD40”是细胞表面表达的糖蛋白，属于肿瘤坏死因子受体(TNFR)超家族，并在免疫系统中起核心作用，其在多种免疫细胞，如B细胞、树突细胞、单核细胞和巨噬细胞上表达。本文的CD40指来自任何脊椎动物来源的任何CD40蛋白，包括哺乳动物诸如灵长类动物(例如人)、非人灵长类动物(例如猕猴)和啮齿动物(例如小鼠和大鼠)。
本发明提供亲和力成熟的抗CD40抗体。所述亲和力成熟的CD40抗体通过噬菌体展示文库被鉴定为对CD40具有提高的结合亲和力。使用噬菌体展示文库进行的亲和力成熟已有记载，例如Lowman et al.，Biochemistry 30(45):10832-10838(1991)，还可参见Hawkins et al.，J.Mol Biol.254:889-896(1992),及下文实施例。不严格限于下列描述，这种工艺可简单描述如下：突变亲本抗体预定区域内的一个或更多个个位点以生成每个位点处有可能的氨基酸取代。作为与每个颗粒内包装的M13基因III产物的融合物以单价形式自丝状噬菌体颗粒展示如此生成的抗体突变体。表达各种突变体的噬菌体可循环经历多轮结合选择(例如通过ELISA测定突变体与抗原的结合亲和力)，接着对那些展示高亲和力的突变体进行分离和测序。应当理解的是，虽然本发明的抗CD40抗体变体是使用噬菌体展示技术鉴定的，其它技术也可用于鉴定具有改进的结合亲和力的抗CD40抗体变体，包括亲和力成熟的抗CD40抗体变体。
在一些实施方案中，所述亲本抗体例如可以是具有下述重链可变区和轻链可变区的抗CD40抗体，所述重链可变区包括：包含SEQ ID NO:46的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3，所述轻链可变区包括：包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:56的LCDR3。在一些实施方案中，所述亲本抗体的VH包含SEQ ID NO:53，和/或VL包含SEQ ID NO:61。在一些实施方案中，所述亲本抗体包含与SEQ ID NO:53具有至少70％、至少75％、至少80％、至少85％、至少90％、 至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列一致性的VH，和/或包含与SEQ ID NO:61具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列一致性的VL。所述亲本抗体可以是全长抗体，例如免疫球蛋白(如IgG，例如IgG1、IgG2、IgG3或IgG4)、scFv或单域抗体，或其抗原结合片段，例如Fab片段、F(ab’)2片段、Fab’片段。
本发明的亲和力成熟的抗CD40抗体具有与亲本抗体相比更高的与CD40(例如人CD40)的结合亲和力。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体具有与亲本抗体相比更低的与CD40(例如人CD40)结合的EC50值。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体与人CD40结合的EC50小于约0.04919μg/mL。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体与人CD40结合的EC50小于约3.44×10-7M。所述EC50通过ELISA测定，所述ELISA可以是测定抗体与人CD40(例如人CD40-His)的结合活性，用于测定的结合活性的人CD28的浓度例如可以是2μg/ml。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体包含VH和VL，并且在至少一个CDR中相对于亲本抗体具有一个、两个或更多个氨基酸的取代。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体包含VH和VL，并且在HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3中的至少一个中相对于亲本抗体具有一个、两个或更多个氨基酸的取代。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VH在HCDR1、HCDR2和HCDR3中的至少一个中相对于亲本抗体具有一个、两个或更多个氨基酸的取代。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VL在LCDR1、LCDR2和LCDR3中的至少一个中相对于亲本抗体具有一个、两个或更多个氨基酸的取代。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VH包括：包含选自SEQ ID NO:46和SEQ ID NO:62的序列的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VL包括：包含SEQ ID NO:54的LCDR1、包含选自SEQ ID NO:55,64-66的序列的LCDR2和包含选自SEQ ID NO:56,67-68序列的LCDR3。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VH包括：(1)包含SEQ ID NO:46的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3；或(2)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VL包括：(1)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:64的LCDR2和包含SEQ ID NO:56的LCDR3；(2)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:67的LCDR3；(3)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:68的LCDR3；(4)包含SEQ  ID NO:54的LCDR1、包含SEQ ID NO:65的LCDR2和包含SEQ ID NO:56的LCDR3；(5)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:66的LCDR2和包含SEQ ID NO:56的LCDR3。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VH和VL包括：(1)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:64的LCDR2和包含SEQ ID NO:56的LCDR3；(2)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:67的LCDR3；(3)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:68的LCDR3；(4)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:65的LCDR2和包含SEQ ID NO:56的LCDR3；(5)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:66的LCDR2和包含SEQ ID NO:56的LCDR3。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的不包括下述VH，所述VH包括：包含SEQ ID NO:46的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的不包括下述VL，所述VL包括：包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:56的LCDR3。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的HFR区和/或LFR区与亲本抗体相同。例如，本发明的亲和力成熟的抗CD40抗体的VH可包括：包含SEQ ID NO:49的HFR1、包含SEQ ID NO:50的HFR2、包含SEQ ID NO:51的HFR3和包含SEQ ID NO:52的HFR4。例如，本发明的亲和力成熟的抗CD40抗体的VL可包括：包含SEQ ID NO:57的LFR1、包含SEQ ID NO:58的LFR2、包含SEQ ID NO:59的LFR3和包含SEQ ID NO:60的LFR4。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体可以在FR区相对于亲本抗体具有一个或更多个氨基酸的取代。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的HFR1相对于SEQ ID NO:49在位置E10具有氨基酸取代(氨基酸残基编号以SEQ ID NO:49为基准，以SEQ ID NO:49的第一个氨基酸残基作为位置1)。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的HFR1相对于SEQ ID NO:49具有氨基酸取代E10Q。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VH包括：包含SEQ ID NO:63的HFR1、包含SEQ ID NO:50的HFR2、包含SEQ ID NO:51的HFR3和包含SEQ ID NO:52的HFR4。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体的VH包含选自SEQ ID NO:69-73的序列，和/或VL包含选自SEQ ID NO:74-78的序列。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体包含与选自SEQ ID NO:69-73的序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列一致性的VH，和/或包含与选自SEQ ID NO:74-78的序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列一致性的VL。
本发明的亲和力成熟的抗CD40抗体是任何包含上述重链可变区和/或轻链可变区的抗体形式，例如全长抗体(如IgG，例如IgG1、IgG2、IgG3或IgG4)、单链抗体或单域抗体等。在一些实施方案中，本发明的亲和力成熟的抗CD40抗体包含免疫球蛋白(如IgG)的恒定区，例如IgG1、IgG2、IgG3或IgG4的恒定区。在一些实施方案中，所述恒定区是人IgG的恒定区，例如人IgG1、人IgG2、人IgG3或人IgG4的恒定区。在一些实施方案中，上述重链可变区融合至免疫球蛋白重链恒定区(包括CH1、CH2和/或CH3)。在一些实施方案中，上述轻链可变区融合至免疫球蛋白轻链恒定区(CL)。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体是单链抗体(scFv)，其中重链可变区与轻链可变区通过肽接头(Linker)融合在一起。重链可变区与轻链可变区的连接顺序可以不同，例如，重链可变区的C端通过肽接头与轻链可变区的N端融合，或者重链可变区的N端通过肽接头与轻链可变区的N端融合。用于连接重链可变区与轻链可变区的肽接头是本领域技术人员熟知的，例如可以是柔性接头，如甘氨酸-丝氨酸聚合物。在一些实施方案中，用于连接重链可变区与轻链可变区的肽接头是GGGGSGGGGSGGGGS(SEQ ID NO:80)。
在一些实施方案中，本发明的亲和力成熟的抗CD40抗体还可以是单域抗体，例如包含重链可变区，但不包含轻链可变区的单域抗体。制备单域抗体的方法是本领域技术人员熟知的。
本发明还涉及所述亲和力成熟的抗CD40抗体的抗原结合片段，例如全长抗体的Fab片段、F(ab’)2片段、Fab’片段。
本发明的亲和力成熟的抗CD40抗体或其结合片段还可以作为融合蛋白的一部分，与其他功能性多肽融合。
多特异性抗体
本发明还提供多特异性抗体，包括但不限于双特异性抗体、三特异性抗体或四特异性抗体。所述多特异性抗体包括至少两个抗原结合部分：第一抗原结合部分和第二抗原结合部分。所述第一抗原结合部分和第二抗原结合部分分别特异性结合不同的抗原或同一抗原的不同表位。所述第一抗原结合部分和第二 抗原结合部分独立地可以分别是全长抗体、单链抗体、单域抗体，或其抗原结合片段，如全长抗体的Fab片段、F(ab’)2片段、Fab’片段、Fv片段。本文中，“第一”和“第二”仅仅是出于区分的目的，不意味着特定的顺序。
在一些实施方案中，所述第一抗原结合部分是抗CD28抗体，例如本发明所述的鼠源抗CD28抗体或人源化抗CD28抗体，第二抗原结合部分能够特异性结合与CD28不同的第二抗原。在一些实施方案中，第二抗原结合部分可以特异性结合肿瘤抗原或能够刺激免疫细胞。在一些实施方案中，第二抗原结合部分是抗CD40抗体，例如本发明所述的亲本抗CD40抗体或亲和力成熟的抗CD40抗体。
在一些实施方案中，所述第一抗原结合部分是是抗CD40抗体，例如本发明所述的亲本抗CD40抗体或亲和力成熟的抗CD40抗体，第二抗原结合部分能够特异性结合与CD40不同的第二抗原。在一些实施方案中，第二抗原结合部分可以特异性结合肿瘤抗原或能够刺激免疫细胞。在一些实施方案中，第二抗原结合部分是抗CD28抗体，例如本发明所述的鼠源抗CD28抗体或人源化抗CD28抗体。
在一些优选的实施方案中，所述第一抗原结合部分可以是任何抗CD28抗体，例如本发明所述的鼠源抗CD28抗体或人源化抗CD28抗体。所述第二抗原结合部分可以是任何抗CD40抗体，例如本发明所述的亲本抗CD40抗体或亲和力成熟的抗CD40抗体。
在一些实施方案中，双特异性抗体的第一抗原结合部分和第二抗原结合部分均是scFv，二者通过肽接头相连。在一些实施方案中，第一抗原结合部分是抗CD28 scFv，第二抗原结合部分是抗CD40 scFv。在一些实施方案中，第一抗原结合部分的C端通过肽接头与第二抗原结合部分的N端相连。在一些实施方案中，第一抗原结合部分的N端通过肽接头与第二抗原结合部分的C端相连。
在一些实施方案中，用于连接双特异性抗体的第一抗原结合部分和第二抗原结合部分的肽接头是本领域技术人员熟知的，例如可以是柔性接头，如甘氨酸-丝氨酸聚合物。在一些实施方案中，用于连接双特异性抗体的第一抗原结合部分和第二抗原结合部分的肽接头是GGGGS(SEQ ID NO:79)。
核酸和载体
本发明还提供编码本发明的人源化抗CD28抗体或其抗原结合片段、亲和力成熟的抗CD40抗体或其抗原结合片段、或包含该抗CD28抗体或其抗原结合片段和/或该抗CD40抗体或其抗原结合片段的多特异性抗体(例如双特异性抗体，以下可简称CD28-CD40双抗)的多核苷酸。所述多核苷酸可以是DNA或RNA(例如mRNA)。
本发明还提供包含上述多核苷酸的载体，所述载体可用于在宿主细胞中表达上述抗体或其抗原结合片段。所述载体可以是表达载体，例如质粒载体或病 毒载体。所述病毒载体可以是逆转录病毒载体、慢病毒载体、腺病毒载体或腺相关病毒载体。
在一些实施方案中，所述载体可以是上述多核苷酸的环状RNA，所述环状RNA依序包括：内部核糖体进入位点(IRES)元件、上述多核苷酸和polyA。该环状RNA可用于表达上述抗体。
其中IRES可以来自于病毒。IRES序列是来自柯萨奇病毒B3(CVB3)或柯萨奇病毒A(CVB1/2)、脑肌炎病毒(EMCV)、陶拉综合症病毒、三联体病毒、塞勒氏脑脊髓炎病毒、猿病毒40、红火蚁病毒1、缢管蚜病毒、网状内皮病病毒、人脊髓灰质炎病毒1、Plautia stali肠道病毒、克什米尔蜜蜂病毒。人类鼻病毒2、琉璃叶蝉病毒-1、人类免疫缺陷病毒1型、虱P病毒、丙型肝炎病毒、甲型肝炎病毒、GB肝炎病毒、手足口病病毒、人肠病毒71、马鼻炎病毒、茶尺蠖小RNA样病毒、果蝇C病毒、十字花科烟草花叶病毒、蟋蟀麻痹病毒、牛病毒性腹泻病毒1、黑皇后细胞病毒、蚜虫致死性麻痹病毒、禽脑脊髓炎病毒、急性蜜蜂麻痹病毒、芙蓉花绿环斑病毒、古典猪瘟病毒、人FGF2、人SFTPA1、人AMLURUNX1、果蝇触足角、人AQP4、人AT1R、人BAG-1、人BCL2、人BiP、人c-IAP1、人c-myc、人eIF4G、小鼠NDST4L、人LEF1、小鼠HIF1alpha、人n.myc、小鼠Gtx、人p27kip、人PDGF2/c-sis、人p53、人Pim-1小鼠Rbm3果蝇reaper、犬Scamper、果蝇Ubx、人UNR、小鼠UtrA、人VEGF-A、人XIAP、Salivirus、Cosavirus、Parechovirus、果蝇hairless、酿酒酵母TFIID、酿酒酵母YAP1、人c-src、人FGF-1、猿猴皮卡病毒、芜菁皱缩病毒或eIF4G的适体的IRES。
本发明的环状RNA可以通过RNA环化方法的一般策略来制备，如使用RNA或DNA连接酶的酶法，或者使用自剪接内含子的核酶法。参见，Petkovic,S.&Muller,S.,“RNA circularization strategies in vivo and in vitro”,Nucleic Acids Research,43(4):2454-2465(2015)；Beadudry,D.&Perreault,J.,“An efficient strategy for the synthesis of circular RNA molecules”,Nucleic Acids Research,23(15):3064-3066(1995)；Micura,R.,“Cyclic Oligoribonucleotides(RNA)by Solid-Phase Synthesis",Chemistry A European Journal,5(7):2077-2082(1999)。
在一些实施方案中，本发明还提供用于制备所述环状RNA的前体RNA，该前体RNA经环化可获得所述环状RNA，所述前体RNA也可以作为包含上述多核苷酸的载体使用。所述前体RNA包括环化元件、内部核糖体进入位点(IRES)元件、上述多核苷酸和polyA。在一些实施方案中，所述环化元件包括位于内部核糖体进入位点(IRES)元件的5’位置的第一内含子序列和位于polyA的3’位置的第二内含子序列。在一些实施方案中，所述第一内含子序列和第二内含子序列来自Group I或Group II内含子自剪接序列。在一些实施方案中，所述第一内含子序列包括含有3’剪接位点二核苷酸的3’Group I内含子片段，第二内含子元素包括含有5’剪接位点二核苷酸的5’Group I内含子片段。在一些实施方案中，所述前体RNA进一步包括第一内含子序列和内部核糖体进入位点(IRES)元件之间的5’间隔序列，以及polyA和第二内含子元件之间的3’间隔 序列。在一些实施方案中，所述前体RNA进一步包括第一内含子元件外部的5’同源臂和第二内含子元件外部的3’同源臂。
在一些实施方案中，包含编码上述抗体或抗原结合片段的多核苷酸的载体还可以是用于产生上述前体RNA的载体，该载体包含该前体RNA的DNA模板，该DNA模板经转录可获得所述前体RNA。所述转录可以是体外转录或在细胞内转录。
本文所用的术语“polyA”是多腺苷酸化的缩写，是指由连续的腺嘌呤核苷酸组成的序列，其长度至少为30。polyA序列可以是一个核糖核酸序列或一个脱氧核糖核酸序列。polyA序列中的连续腺嘌呤核苷酸的长度可以是至少30个核苷酸，例如。至少45个核苷酸，至少50个核苷酸，至少55个核苷酸，至少60个核苷酸，至少65个核苷酸，至少70个核苷酸，至少75个核苷酸，至少80个核苷酸，至少85个核苷酸，至少90个核苷酸。至少95个核苷酸，至少100个核苷酸，至少105个核苷酸，至少110个核苷酸，至少115个核苷酸，至少120个核苷酸，至少125个核苷酸，至少130个核苷酸，至少135个核苷酸，至少140个核苷酸。至少145个核苷酸，至少150个核苷酸，至少155个核苷酸，至少160个核苷酸，至少165个核苷酸，至少170个核苷酸，至少175个核苷酸，至少180个核苷酸，至少185个核苷酸，至少190个核苷酸。至少195个核苷酸，至少200个核苷酸，至少205个核苷酸，至少210个核苷酸，至少215个核苷酸，至少220个核苷酸，至少225个核苷酸，至少230个核苷酸，至少235个核苷酸，至少240个核苷酸。polyA序列中连续的腺嘌呤核苷酸的长度可以在30-240个核苷酸的范围内，例如40-230个核苷酸、45-220个核苷酸、50-210个核苷酸、60-200个核苷酸、70-190个核苷酸、80-180个核苷酸、90-170个核苷酸、100-160个核苷酸、110-150个核苷酸、120-140个核苷酸。在一些实施方案中，polyA序列可以只由连续的腺嘌呤核苷酸组成。在一些实施方案中，所述polyA的长度为至少45个核苷酸。在一些实施方案中，所述polyA的长度为至少70个核苷酸。
本发明还提供生产上述环状RNA的方法，包括将上述前体RNA环化获得所述环状RNA。在一些实施方案中，所述方法还包括使载体中包含的所述前体RNA的DNA模板转录获得所述前体RNA。在一些实施方案中，所述方法还包括纯化所述环状RNA，例如使用基于寡聚dT的捕获方法，通过寡聚dT与polyA之间的相互作用纯化。
抗体的应用
本发明的人源化抗CD28抗体或其抗原结合片段可用于诱导T细胞增殖、激活T细胞，可用于促进免疫效应细胞(如T细胞，如CAR-T细胞)的杀伤作用。本发明的人源化抗CD28抗体、亲和力成熟的抗CD40抗体或其抗原结合片段可与特异性结合肿瘤细胞表面抗原的抗体形成双特异性抗体，例如BiTE(bispecific T-cell engager)，或多特异性抗体，靶向性激活自身T细胞杀伤肿瘤 细胞。
因此，本发明提供使用本发明的人源化抗CD28抗体或其抗原结合片段诱导T细胞增殖的方法，或者本发明的人源化抗CD28抗体或其抗原结合片段在制备诱导T细胞增殖的药物中的应用。所述方法包括使本发明的人源化抗CD28抗体或其抗原结合片段、包含本发明的人源化抗CD28抗体或其抗原结合片段的多特异性抗体、编码本发明的人源化抗CD28抗体或其抗原结合片段的多核苷酸、包含该多核苷酸的载体、或者包含所述抗体或其抗原结合片段、所述多特异性抗体、所述多核苷酸或所述载体的药物组合物与T细胞接触，由此使得所述人源化抗CD28抗体或其抗原结合片段刺激T细胞活化、诱导其增殖。所述多核苷酸或载体可以被引入所述T细胞中，并由此在所述T细胞中表达所述人源化抗CD28抗体或其抗原结合片段，以刺激T细胞活化、诱导其增殖。所述多核苷酸可以是DNA或RNA，例如mRNA。
在一些实施方案中，所述T细胞可以是CAR-T细胞。在一些实施方案中，本发明的人源化抗CD28抗体或其抗原结合片段，或者包含本发明的人源化抗CD28抗体或其抗原结合片段的多特异性抗体可以与CAR-T细胞组合施用给受试者，以增强CAR-T细胞对靶细胞的杀伤作用。
本发明的亲和力成熟的抗CD40抗体可用于刺激免疫细胞(如树突状细胞、巨噬细胞、NK细胞、粒细胞等)，因而可用于刺激免疫应答，例如激活抗原呈递细胞(APC)如树突状细胞(DC)、刺激B细胞的激活和/或增殖、诱导效应T细胞的细胞毒性，还可以用于治疗肿瘤。本发明的人源化抗CD28抗体、亲和力成熟的抗CD40抗体或其抗原结合片段可与特异性结合肿瘤细胞表面抗原的抗体形成双特异性抗体或多特异性抗体，激活毒性T细胞杀伤肿瘤细胞。
因此，本发明提供使用本发明的亲和力成熟的抗CD40抗体或其抗原结合片段增强受试者免疫反应的方法，或者本发明的亲和力成熟的抗CD40抗体或其抗原结合片段在制备用于增强受试者免疫反应的药物中的应用。所述方法包括给受试者施用本发明的亲和力成熟的抗CD40抗体或其抗原结合片段、包含本发明的亲和力成熟的抗CD40抗体或其抗原结合片段的多特异性抗体、编码本发明的亲和力成熟的抗CD40抗体或其抗原结合片段的多核苷酸或包含该多核苷酸的载体、或者包含所述抗体或其抗原结合片段、所述多特异性抗体、所述多核苷酸或所述载体的药物组合物。所述多核苷酸可以是DNA或RNA，例如mRNA。
本文使用的术语“增强免疫反应”是指刺激、激发、增加、改良或增强受试者免疫系统的任何反应。免疫应答可以是细胞反应(即细胞介导的，如细胞毒性T淋巴细胞介导的反应)或体液应答(即抗体介导的反应)，及可以是初次或二次免疫反应。示例性的免疫反应增强包括包括诱导T细胞(例如CD4+T细胞、CD8+T细胞)增殖、刺激T细胞(例如CD4+T细胞、CD8+T细胞)的活化、激活静息T淋巴细胞、促进B细胞的激活和增殖、激活树突状细胞、促进NK细胞的增殖和激活、增加NK细胞的杀伤功能、促进人外周血嗜酸性粒细胞生 存、促进巨噬细胞集落刺激因子(GMCSF)释放、增加T细胞和/或B细胞的存活率、改善抗原呈递细胞(例如树突状细胞)的抗原呈递、改善抗原清除率、增加细胞因子(例如白介素-2、IFN-gamma)的产生等。典型地，当与未施用本发明的抗体的受试者的免疫反应相比时，施用本发明抗体的受试者的免疫反应增强。
抗CD28抗体(例如本发明的人源化抗CD28抗体)或其抗原结合片段可以与抗CD40抗体(例如本发明的亲和力成熟的抗CD40抗体)或其抗原结合片段形成多特异性抗体，例如双特异性抗体(以下可简称CD28-CD40双抗)。抗CD28抗体或其抗原结合片段可作为CD28分子的激动剂，能够提供T细胞活化的共刺激信号，抗CD40抗体或其抗原结合片段是CD40分子的激动剂，能刺激多种免疫细胞(如树突状细胞、巨噬细胞、NK细胞、粒细胞等)进行活化和发挥功能，CD28-CD40双抗能够在免疫反应中发挥重要作用，例如可以增强受试者的免疫反应、提高细胞免疫治疗的效果，特别是促进CAR-T细胞的杀伤作用。
本文使用的术语“细胞免疫治疗”或“细胞免疫疗法”是指使用免疫系统中包含的、能对抗疾病的细胞，或能够帮助免疫系统对抗疾病的细胞来治疗疾病，例如肿瘤、自身免疫性疾病或感染性疾病等。可用于细胞免疫治疗的细胞包括但不限于T细胞、NK细胞、树突状细胞、调节性T细胞、细胞毒性T细胞(CTL)、巨噬细胞等。细胞免疫治疗可以从受试者的血液或肿瘤中分离出自体免疫细胞，在将这些细胞体外培养后再输至受试者体内去杀伤肿瘤细胞。此外，也可以经由基因工程改造后使免疫细胞表达肿瘤特异性受体，再经培养后输回患者体内，例如CAR-T细胞。因此，本发明提供本发明的CD28-CD40双抗和免疫细胞的药物组合，该药物组合可用于杀伤受试者中的靶细胞。本发明的CD28-CD40双抗可以增强所述免疫细胞对靶细胞的杀伤作用。本发明还提供增强免疫细胞对受试者中的靶细胞的杀伤作用的方法，包括给接受所述免疫细胞的受试者施用本发明的CD28-CD40双抗、编码该双抗的多核苷酸、包含该多核苷酸的载体或包含该双抗、多核苷酸或载体的药物组合物。
在一些实施方案中，可将编码本发明的CD28-CD40双抗的多核苷酸或包含该多核苷酸的载体引入免疫细胞中，以使所述免疫细胞能够表达所述双抗。所述多核苷酸可以是DNA或RNA，例如mRNA。所述载体可以是质粒载体或病毒载体，例如慢病毒载体、腺病毒载体、腺相关病毒载体、逆转录病毒载体等。所述载体还可以是前述的环状RNA，前体RNA或包含所述前体RNA的DNA模板的载体。所述“引入”可被理解为“转染”。所述多核苷酸或载体可以通过例如，电穿孔、磷酸钙沉淀、DEAE-葡聚糖转染、病毒样颗粒感染等公知的方式转染至免疫细胞中。因此，在一些实施方案中，可以给受试者施用能够表达本发明的CD28-CD40双抗的免疫细胞，以杀伤受试者中的靶细胞。本发明还提供能够表达本发明的CD28-CD40双抗的免疫细胞。
术语“免疫细胞”或“免疫效应细胞”是指用于细胞免疫治疗并具有治疗效果的免疫细胞，例如在细胞免疫治疗中可以杀伤靶细胞的免疫细胞，例如T细胞、 NK细胞、树突状细胞、调节性T细胞、细胞毒性T细胞(CTL)、巨噬细胞等。所述“靶细胞”是指细胞免疫治疗所针对的靶细胞。在一些实施方案中，所述免疫细胞包含CAR或能表达CAR。在一些实施方案中，所述免疫细胞是CAR-T细胞。所述CAR-T细胞中包含能够特异性结合靶细胞表面抗原的结合结构域，因而能够对靶细胞起到特异性杀伤作用。本发明的方法与单独施用免疫细胞相比，能够增强免疫细胞对靶细胞的杀伤作用。所述杀伤作用例如可以通过靶细胞数量的降低来测定。在一些实施方案中，所述靶细胞是肿瘤细胞。
本发明还提供本发明的CD28-CD40双抗、编码该双抗的多核苷酸、包含该多核苷酸的载体在制备用于增强免疫细胞对受试者中的靶细胞的杀伤作用的药物中的用途。
在一些实施方案中，本发明还提供经基因工程改造的免疫效应细胞，其能够表达本发明的CD28-CD40双抗，所述免疫效应细胞可以是T细胞、NK细胞、NKT细胞、巨噬细胞、中性粒细胞或粒细胞。所述免疫效应细胞可以包含编码本发明的CD28-CD40双抗的多核苷酸或包含该多核苷酸的载体。在一些实施方案中，所述经基因工程改造的免疫效应细胞进一步重组表达嵌合抗原受体(CAR)、T细胞受体(TCR)或双特异性T细胞接合子(BiTE)，其中所述CAR、TCR或BiTE可以是结合肿瘤抗原或病毒抗原的CAR、TCR或BiTE。所述病毒抗原可以选自HPV、EBV和HIV。所述肿瘤抗原可以选自Her2、NY-ESO-1、CD19、CD20、CD22、PSMA、c-Met、GPC3、IL13ra2、EGFR、CD123、CD7、GD2、PSCA、EBV16-E7、H3.3、EGFRvIII、BCMA和间皮素。在一些实施方案中，所述CAR、TCR或BiTE结合间皮素(MESO)、CD123、BCMA、HER2、IL13Ra2、B7H3或CD19。
在一些实施方案中，所述CAR、TCR或BiTE可以结合TSHR、CD19；CD123；CD22；CD30；CD171；CS-1；C型凝集素样分子-1、CD33；表皮生长因子受体变体III(EGFRvIII)结合；神经节苷脂G2(GD2)；神经节苷脂GD3；TNF受体家族成员；B细胞成熟抗原；Tn抗原((Tn Ag)或(GalNAca-Ser/Thr))；前列腺特异性膜抗原(PSMA)；受体酪氨酸激酶样孤儿受体1(ROR1)；Fms-like酪氨酸激酶3(FLT3)；肿瘤相关糖蛋白72(TAG72)；CD38；CD44v6；癌胚抗原(CEA)；上皮细胞粘附分子(EPCAM)；B7H3(CD276)；KIT(CD117)；白细胞介素-13受体亚单位α-2；中皮素；白细胞介素11受体α(IL-l lRa)；前列腺干细胞抗原(PSCA)；蛋白酶丝氨酸21；血管内皮细胞生长因子受体2(VEGFR2)；刘易斯(Y)抗原；CD24；血小板生长因子受体β(PDGFR-β)；阶段性特异性胚胎抗原-4(SSEA-4)；CD20；叶酸受体α；受体酪氨酸蛋白激酶ERBB2(Her2/neu)；粘蛋白1，细胞表面相关(MUC1)；表皮生长因子受体(EGFR)；神经细胞粘附分子(NCAM)；前列腺酶；前列腺酸性磷酸酶(PAP)；延长因子2突变(ELF2M)；Ephrin B2；成纤维细胞激活蛋白α(FAP)；胰岛素样生长因子1受体(IGF-I受体)；碳酸酐酶IX(CAIX)；蛋白酶体(Prosome，Macropain)亚单位，β型，9(LMP2)；糖蛋白100(gp100)；由断点群区(BCR) 和艾贝尔森小鼠白血病病毒肿瘤基因同源物1(Abl)组成的肿瘤基因多肽(bcr-abl)；酪氨酸酶；肾上腺素-A型受体2(EphA2)；Fucosyl GM1；sialyl Lewis粘附分子(sLe)；神经节苷脂GM3；转谷氨酰胺酶5(TGS5)；高分子量黑色素瘤相关抗原(HMWMAA)；邻乙酰-GD2神经节苷脂(OAcGD2)；叶酸受体β；肿瘤内皮标志物1(TEM1/CD248)；肿瘤内皮标志物7相关(TEM7R)；克劳丁6(CLDN6)；促甲状腺激素受体(TSHR)；G蛋白偶联受体C类第5组，成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；无性淋巴瘤激酶(ALK)；聚糖酸；胎盘特异性1(PLAC1)；globoH糖酰胺的六糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；尿囊素2(UPK2)；甲肝病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物，位点K9(LY6K)；嗅觉受体51E2(OR51E2)；TCR伽马交替读框蛋白(TARP)；威尔姆斯肿瘤蛋白(WTl)；癌/睾丸抗原1(NY-ESO-1)；癌/睾丸抗原2(LAGE-la)；黑色素瘤相关抗原1(MAGE-A1)；ETS易位变异基因6，位于12p染色体上(ETV6-AML)；精子蛋白17(SPA 17)；X抗原家族，成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie 2)；黑色素瘤癌睾丸抗原-1(MAD-CT-1)；黑色素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；肿瘤蛋白p53(p53)；p53突变体；前列腺素；存活；端粒酶；前列腺癌肿瘤抗原-1；T细胞识别的黑色素瘤抗原1；大鼠肉瘤(Ras)突变体；人类端粒酶逆转录酶(hTERT)；肉瘤易位断点；黑色素瘤凋亡抑制剂(ML-IAP)；ERG(跨膜蛋白酶，丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；Cyclin Bl；v-myc禽髓细胞瘤病毒肿瘤基因神经母细胞瘤衍生同源物(MYCN)Ras同源家族成员C(RhoC)；酪氨酸酶相关蛋白2(TRP-2)；细胞色素P450 1B 1(CYP1B1)；CCCTC-结合因子(锌指蛋白)-Like；T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；原核蛋白结合蛋白sp32(OY-TES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；高级糖化终产物受体(RAGE-1)；肾脏泛在1(RU1)；肾脏泛在2(RU2)；豆蛋白；人乳头瘤病毒E6(HPV E6)；人乳头瘤病毒E7(HPV E7)；肠道羧酸酯酶；热休克蛋白70-2突变(mut hsp70-2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIRl)；IgA受体的Fc片段(FCAR或CD89)；白细胞免疫球蛋白样受体A亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；EGF样模块含粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；Glypican-3(GPC3)；Fc受体样5(FCRL5)；或免疫球蛋白λ样多肽1(IGLL1)结合。
在一些实施方案中，所述经基因工程改造的免疫效应细胞包含编码本发明的CD40-CD28双抗的第一多核苷酸和编码CAR、TCR或BiTE(CAR/TCR/BiTE)的第二多核苷酸。所述第一多核苷酸和所述第二多核苷酸可以分别在独立的表 达盒中表达，或者在同一个表达盒中共同表达。当在同一表达盒中共同表达时，第一多核苷酸和第二多核苷酸之间可以具有接头。所述接头可以是自裂解接头，如2A接头，例如T2A、P2A或F2A。可将编码本发明的CD28-CD40双抗的多核苷酸或包含该多核苷酸的载体引入免疫细胞中，以使所述免疫细胞能够表达所述双抗。所述第一多核苷酸和所述第二多核苷酸可以是DNA或RNA，例如mRNA。所述第一多核苷酸和所述第二多核苷酸可以通过本领域所知的任何方式被引入到免疫效应细胞内，例如可以通过载体或mRNA转染免疫效应细胞而将它们引入免疫效应细胞内。所述载体可以是质粒载体或病毒载体，例如慢病毒载体、腺病毒载体、腺相关病毒载体、逆转录病毒载体等。所述载体还可以是前述的环状RNA，前体RNA或包含所述前体RNA的DNA模板的载体。
在一些实施方案中，所述CAR-T细胞包含以HER2结合分子(如抗HER2抗体)为结合结构域的CAR，并靶向表达HER2的细胞。在一些实施方案中，所述CAR-T细胞包含以间皮素结合分子(如抗间皮素抗体)为结合结构域的CAR，并靶向表达间皮素的细胞。在一些实施方案中，所述CAR-T细胞的靶细胞是表达HER2或间皮素的肿瘤细胞。
在一些实施方案中，CAR可以包括信号肽、铰链区、跨膜结构域和胞内信号传导结构域。胞内信号传导结构域可进一步包括共刺激结构域。在一些实施方案中，信号肽可包括CD8信号肽或GM-CSF信号肽。在一些实施方案中，CAR的铰链区可以包括CD28、CD8、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8A、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、TIM1、SLAM、CD30或LIGHT的铰链区，优选为最好是CD8铰链区。在一些实施方案中，CAR的跨膜结构域可以包括CD8、CD28、CD3ε(CD3e)、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12、CD40L(CD154)、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64或SLAM的跨膜结构域，优选为CD8跨膜(TM)结构域。在一些实施方案中，CAR的胞内信号传导结构域可以包括CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FceRIγ、FceRIβ、FcγRIIa、DAP10或DAP-12的胞内信号传导结构域，优选CD3ζ胞内信号传导结构域。在一些实施方案中，CAR的胞内信号传导结构域可进一步包括共刺激结构域，如CD28、4-1BB(CD137)、CD27、CD2、CD7、CD8A、CD8B、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD40或MyD88的共刺激结构域，优选为4-1BB共刺激域。
本发明中，具有“BBZ”结构的CAR是指具有4-1BB共刺激分子的CAR，通常包括CD8铰链结构域、CD8跨膜(TM)结构域、4-1BB共刺激结构域和CD3ζ结构域。在一些实施方案中，本发明中使用的CAR具有BBZ结构
在一些实施方案中，所述CAR包含抗体或其抗原结合片段作为结合结构域。在一些实施方案中，所述抗体或抗原结合片段特异性结合间皮素(MESO)、CD123、BCMA、HER2、IL13Ra2或B7H3。在一些实施方案中，所述抗体是scFv。在一些实施方案中，scFv包括一个重链可变区(VH)与轻链可变区(VL)的N端或C端融合。在一些实施方案中，在scFv的VH和VL之间具有肽接头。在一些实施方案中，特异性结合间皮素的抗体，也被称为抗间皮素(或抗MESO或抗MSLN)抗体，如PCT/CN2021/112767(通过引用将其全文并入本文)中所描述，其包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3选自下列组：
(1)如SEQ ID NO:87所示的LCDR1，如SEQ ID NO:102所示的LCDR2，如SEQ ID NO:116所示的LCDR3，如SEQ ID NO:131所示的HCDR1，如SEQ ID NO:144所示的HCDR2和如SEQ ID NO:157所示的HCDR3；
(2)如SEQ ID NO:88所示的LCDR1，如SEQ ID NO:103所示的LCDR2，如SEQ ID NO:117所示的LCDR3，如SEQ ID NO:132所示的HCDR1，如SEQ ID NO:145所示的HCDR2和如SEQ ID NO:158所示的HCDR3；
(3)如SEQ ID NO:89所示的LCDR1，如SEQ ID NO:104所示的LCDR2，如SEQ ID NO:118所示的LCDR3，如SEQ ID NO:133所示的HCDR1，如SEQ ID NO:146所示的HCDR2和如SEQ ID NO:159所示的HCDR3；
(4)如SEQ ID NO:90所示的LCDR1，如SEQ ID NO:105所示的LCDR2，如SEQ ID NO:119所示的LCDR3，如SEQ ID NO:134所示的HCDR1，如SEQ ID NO:147所示的HCDR2和如SEQ ID NO:160所示的HCDR3；
(5)如SEQ ID NO:91所示的LCDR1，如SEQ ID NO:106所示的LCDR2，如SEQ ID NO:120所示的LCDR3，如SEQ ID NO:135所示的HCDR1，如SEQ ID NO:148所示的HCDR2和如SEQ ID NO:161所示的HCDR3；
(6)如SEQ ID NO:92所示的LCDR1，如SEQ ID NO:107所示的LCDR2，如SEQ ID NO:121所示的LCDR3，如SEQ ID NO:136所示的HCDR1，如SEQ ID NO:149所示的HCDR2和如SEQ ID NO:162所示的HCDR3；
(7)如SEQ ID NO:93所示的LCDR1，如SEQ ID NO:108所示的LCDR2，如SEQ ID NO:122所示的LCDR3，如SEQ ID NO:137所示的HCDR1，如SEQ ID NO:150所示的HCDR2和如SEQ ID NO:163所示的HCDR3；
(8)如SEQ ID NO:94所示的LCDR1，如SEQ ID NO:109所示的LCDR2，如SEQ ID NO:123所示的LCDR3，如SEQ ID NO:138所示的HCDR1，如SEQ ID NO:151所示的HCDR2和如SEQ ID NO:164所示的HCDR3；
(9)如SEQ ID NO:95所示的LCDR1，如SEQ ID NO:110所示的LCDR2，如SEQ ID NO:124所示的LCDR3，如SEQ ID NO:139所示的HCDR1，如SEQ ID NO:152所示的HCDR2和如SEQ ID NO:165所示的HCDR3；
(10)如SEQ ID NO:96所示的LCDR1，如SEQ ID NO:111所示的LCDR2，如SEQ ID NO:125所示的LCDR3，如SEQ ID NO:134所示的HCDR1，如SEQ ID NO:147所示的HCDR2和如SEQ ID NO:166所示的HCDR3；
(11)如SEQ ID NO:97所示的LCDR1，如SEQ ID NO:112所示的LCDR2，如SEQ ID NO:126所示的LCDR3，如SEQ ID NO:140所示的HCDR1，如SEQ ID NO:153所示的HCDR2和如SEQ ID NO:167所示的HCDR3；
(12)如SEQ ID NO:98所示的LCDR1，如SEQ ID NO:113所示的LCDR2，如SEQ ID NO:127所示的LCDR3，如SEQ ID NO:139所示的HCDR1，如SEQ ID NO:152所示的HCDR2和如SEQ ID NO:168所示的HCDR3；
(13)如SEQ ID NO:99所示的LCDR1，如SEQ ID NO:114所示的LCDR2，如SEQ ID NO:128所示的LCDR3，如SEQ ID NO:141所示的HCDR1，如SEQ ID NO:154所示的HCDR2和如SEQ ID NO:169所示的HCDR3；
(14)如SEQ ID NO:100所示的LCDR1，如SEQ ID NO:115所示的LCDR2，如SEQ ID NO:129所示的LCDR3，如SEQ ID NO:142所示的HCDR1，如SEQ ID NO:155所示的HCDR2和如SEQ ID NO:170所示的HCDR3；和
(15)如SEQ ID NO:101所示的LCDR1，如SEQ ID NO:104所示的LCDR2，如SEQ ID NO:130所示的LCDR3，如SEQ ID NO:143所示的HCDR1，如SEQ ID NO:156所示的HCDR2和如SEQ ID NO:171所示的HCDR3。
在一些实施方案中，抗MESO抗体包括选自下组的轻链可变区和重链可变区：
(1)如SEQ ID NO:172所示的轻链可变区和如SEQ ID NO:187所示的重链可变区；
(2)如SEQ ID NO:173所示的轻链可变区和如SEQ ID NO:188所示的重链可变区；
(3)如SEQ ID NO:174所示的轻链可变区和如SEQ ID NO:189所示的重链可变区；
(4)如SEQ ID NO:175所示的轻链可变区和如SEQ ID NO:190所示的重链可变区；
(5)如SEQ ID NO:176所示的轻链可变区和如SEQ ID NO:191所示的重链可变区；
(6)如SEQ ID NO:177所示的轻链可变区和如SEQ ID NO:192所示的重链可变区；
(7)如SEQ ID NO:178所示的轻链可变区和如SEQ ID NO:193所示的重链可变区；
(8)如SEQ ID NO:179所示的轻链可变区和如SEQ ID NO:194所示的重链可变区；
(9)如SEQ ID NO:180所示的轻链可变区和如SEQ ID NO:195所示的重链可变区；
(10)如SEQ ID NO:181所示的轻链可变区和如SEQ ID NO:196所示的重链可变区；
(11)如SEQ ID NO:182所示的轻链可变区和如SEQ ID NO:197所示的重链可变区；
(12)如SEQ ID NO:183所示的轻链可变区和如SEQ ID NO:198所示的重链可变区；
(13)如SEQ ID NO:184所示的轻链可变区和如SEQ ID NO:199所示的重链可变区；
(14)如SEQ ID NO:185所示的轻链可变区和如SEQ ID NO:200所示的重链可变区；和
(15)如SEQ ID NO:186所示的轻链可变区和如SEQ ID NO:201所示的重链可变区。
在一些实施方案中，抗MESO抗体是抗MESO scFv，可以包括选自SEQ ID NO:202-216的氨基酸序列。
在一些实施方案中，所述CAR包括可以特异性地与间皮素结合的结合域，其可称为靶向间皮素的CAR。在一些实施方案中，靶向间皮素的CAR可以是PCT/CN2021/112767(通过引用将其全文并入本文)中描述的CAR。在一些实施方案中，靶向间皮素的CAR可以包括上述的任何一种抗间皮素抗体。在一些实施方案中，靶向间皮素的CAR可包括选自SEQ ID NO:217-231的氨基酸序列。
在一些实施方案中，特异性结合CD123的抗体，也称为抗CD123抗体，是PCT/CN2021/112748中描述的抗体(其全部内容通过引用纳入本文)，包括轻链可变区和重链可变区，轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3选自以下组：
(1)如SEQ ID NO:349所示的LCDR1，如SEQ ID NO:379所示的LCDR2，如SEQ ID NO:407所示的LCDR3，如SEQ ID NO:437所示的HCDR1，如SEQ ID NO:458所示的HCDR2和如SEQ ID NO:482所示的HCDR3；
(2)如SEQ ID NO:363所示的LCDR1，如SEQ ID NO:391所示的LCDR2，如SEQ ID NO:421所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:468所示的HCDR2和如SEQ ID NO:495所示的HCDR3；
(3)如SEQ ID NO:353所示的LCDR1，如SEQ ID NO:383所示的LCDR2，如SEQ ID NO:412所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:486所示的HCDR3；
(4)如SEQ ID NO:364所示的LCDR1，如SEQ ID NO:378所示的LCDR2， 如SEQ ID NO:422所示的LCDR3，如SEQ ID NO:446所示的HCDR1，如SEQ ID NO:469所示的HCDR2和如SEQ ID NO:496所示的HCDR3；
(5)如SEQ ID NO:369所示的LCDR1，如SEQ ID NO:396所示的LCDR2，如SEQ ID NO:428所示的LCDR3，如SEQ ID NO:445所示的HCDR1，如SEQ ID NO:475所示的HCDR2和如SEQ ID NO:503所示的HCDR3；
(6)如SEQ ID NO:370所示的LCDR1，如SEQ ID NO:397所示的LCDR2，如SEQ ID NO:429所示的LCDR3，如SEQ ID NO:452所示的HCDR1，如SEQ ID NO:474所示的HCDR2和如SEQ ID NO:504所示的HCDR3；
(7)如SEQ ID NO:354所示的LCDR1，如SEQ ID NO:384所示的LCDR2，如SEQ ID NO:413所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:487所示的HCDR3；
(8)如SEQ ID NO:350所示的LCDR1，如SEQ ID NO:380所示的LCDR2，如SEQ ID NO:408所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
(9)如SEQ ID NO:371所示的LCDR1，如SEQ ID NO:398所示的LCDR2，如SEQ ID NO:430所示的LCDR3，如SEQ ID NO:453所示的HCDR1，如SEQ ID NO:476所示的HCDR2和如SEQ ID NO:505所示的HCDR3；
(10)如SEQ ID NO:372所示的LCDR1，如SEQ ID NO:399所示的LCDR2，如SEQ ID NO:431所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:506所示的HCDR3；
(11)如SEQ ID NO:348所示的LCDR1，如SEQ ID NO:378所示的LCDR2，如SEQ ID NO:406所示的LCDR3，如SEQ ID NO:436所示的HCDR1，如SEQ ID NO:457所示的HCDR2和如SEQ ID NO:481所示的HCDR3；
(12)如SEQ ID NO:363所示的LCDR1，如SEQ ID NO:392所示的LCDR2，如SEQ ID NO:423所示的LCDR3，如SEQ ID NO:447所示的HCDR1，如SEQ ID NO:470所示的HCDR2和如SEQ ID NO:497所示的HCDR3；
(13)如SEQ ID NO:373所示的LCDR1，如SEQ ID NO:400所示的LCDR2，如SEQ ID NO:432所示的LCDR3，如SEQ ID NO:451所示的HCDR1，如SEQ ID NO:477所示的HCDR2和如SEQ ID NO:507所示的HCDR3；
(14)如SEQ ID NO:355所示的LCDR1，如SEQ ID NO:385所示的LCDR2，如SEQ ID NO:414所示的LCDR3，如SEQ ID NO:443所示的HCDR1，如SEQ ID NO:464所示的HCDR2和如SEQ ID NO:488所示的HCDR3；
(15)如SEQ ID NO:356所示的LCDR1，如SEQ ID NO:386所示的LCDR2，如SEQ ID NO:415所示的LCDR3，如SEQ ID NO:444所示的HCDR1，如SEQ ID NO:465所示的HCDR2和如SEQ ID NO:489所示的HCDR3；
(16)如SEQ ID NO:368所示的LCDR1，如SEQ ID NO:395所示的LCDR2， 如SEQ ID NO:427所示的LCDR3，如SEQ ID NO:451所示的HCDR1，如SEQ ID NO:474所示的HCDR2和如SEQ ID NO:502所示的HCDR3；
(17)如SEQ ID NO:351所示的LCDR1，如SEQ ID NO:381所示的LCDR2，如SEQ ID NO:409所示的LCDR3，如SEQ ID NO:438所示的HCDR1，如SEQ ID NO:459所示的HCDR2和如SEQ ID NO:483所示的HCDR3；
(18)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2，如SEQ ID NO:410所示的LCDR3，如SEQ ID NO:439所示的HCDR1，如SEQ ID NO:460所示的HCDR2和如SEQ ID NO:484所示的HCDR3；
(19)如SEQ ID NO:357所示的LCDR1，如SEQ ID NO:387所示的LCDR2，如SEQ ID NO:416所示的LCDR3，如SEQ ID NO:437所示的HCDR1，如SEQ ID NO:466所示的HCDR2和如SEQ ID NO:490所示的HCDR3；
(20)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:377所示的LCDR2，如SEQ ID NO:405所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
(21)如SEQ ID NO:365所示的LCDR1，如SEQ ID NO:393所示的LCDR2，如SEQ ID NO:424所示的LCDR3，如SEQ ID NO:448所示的HCDR1，如SEQ ID NO:471所示的HCDR2和如SEQ ID NO:498所示的HCDR3；
(22)如SEQ ID NO:374所示的LCDR1，如SEQ ID NO:401所示的LCDR2，如SEQ ID NO:433所示的LCDR3，如SEQ ID NO:454所示的HCDR1，如SEQ ID NO:478所示的HCDR2和如SEQ ID NO:508所示的HCDR3；
(23)如SEQ ID NO:358所示的LCDR1，如SEQ ID NO:385所示的LCDR2，如SEQ ID NO:417所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:491所示的HCDR3；
(24)如SEQ ID NO:359所示的LCDR1，如SEQ ID NO:388所示的LCDR2，如SEQ ID NO:418所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:492所示的HCDR3；
(25)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2，如SEQ ID NO:403所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
(26)如SEQ ID NO:366所示的LCDR1，如SEQ ID NO:394所示的LCDR2，如SEQ ID NO:425所示的LCDR3，如SEQ ID NO:449所示的HCDR1，如SEQ ID NO:472所示的HCDR2和如SEQ ID NO:499所示的HCDR3；
(27)如SEQ ID NO:360所示的LCDR1，如SEQ ID NO:389所示的LCDR2，如SEQ ID NO:413所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:487所示的HCDR3；
(28)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2， 如SEQ ID NO:404所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
(29)如SEQ ID NO:348所示的LCDR1，如SEQ ID NO:378所示的LCDR2，如SEQ ID NO:406所示的LCDR3，如SEQ ID NO:436所示的HCDR1，如SEQ ID NO:457所示的HCDR2和如SEQ ID NO:481所示的HCDR3；
(30)如SEQ ID NO:361所示的LCDR1，如SEQ ID NO:390所示的LCDR2，如SEQ ID NO:419所示的LCDR3，如SEQ ID NO:445所示的HCDR1，如SEQ ID NO:467所示的HCDR2和如SEQ ID NO:493所示的HCDR3；
(31)如SEQ ID NO:375所示的LCDR1，如SEQ ID NO:402所示的LCDR2，如SEQ ID NO:434所示的LCDR3，如SEQ ID NO:455所示的HCDR1，如SEQ ID NO:479所示的HCDR2和如SEQ ID NO:509所示的HCDR3；
(32)如SEQ ID NO:368所示的LCDR1，如SEQ ID NO:395所示的LCDR2，如SEQ ID NO:427所示的LCDR3，如SEQ ID NO:450所示的HCDR1，如SEQ ID NO:473所示的HCDR2和如SEQ ID NO:5015所示的HCDR3；
(33)如SEQ ID NO:362所示的LCDR1，如SEQ ID NO:388所示的LCDR2，如SEQ ID NO:420所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:494所示的HCDR3；
(34)如SEQ ID NO:352所示的LCDR1，如SEQ ID NO:382所示的LCDR2，如SEQ ID NO:411所示的LCDR3，如SEQ ID NO:440所示的HCDR1，如SEQ ID NO:461所示的HCDR2和如SEQ ID NO:485所示的HCDR3；和
(35)如SEQ ID NO:367所示的LCDR1，如SEQ ID NO:391所示的LCDR2，如SEQ ID NO:426所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:468所示的HCDR2和如SEQ ID NO:500所示的HCDR3。
在一些实施方案中，抗CD123抗体包括选自下组的轻链可变区和重链可变区:
(1)如SEQ ID NO:510所示的轻链可变区和如SEQ ID NO:545所示的重链可变区；
(2)如SEQ ID NO:511所示的轻链可变区和如SEQ ID NO:546所示的重链可变区；
(3)如SEQ ID NO:512所示的轻链可变区和如SEQ ID NO:547所示的重链可变区；
(4)如SEQ ID NO:513所示的轻链可变区和如SEQ ID NO:548所示的重链可变区；
(5)如SEQ ID NO:514所示的轻链可变区和如SEQ ID NO:549所示的重链可变区；
(6)如SEQ ID NO:515所示的轻链可变区和如SEQ ID NO:550所示的重链 可变区；
(7)如SEQ ID NO:516所示的轻链可变区和如SEQ ID NO:551所示的重链可变区；
(8)如SEQ ID NO:517所示的轻链可变区和如SEQ ID NO:552所示的重链可变区；
(9)如SEQ ID NO:518所示的轻链可变区和如SEQ ID NO:553所示的重链可变区；
(10)如SEQ ID NO:519所示的轻链可变区和如SEQ ID NO:554所示的重链可变区；
(11)如SEQ ID NO:520所示的轻链可变区和如SEQ ID NO:555所示的重链可变区；
(12)如SEQ ID NO:521所示的轻链可变区和如SEQ ID NO:556所示的重链可变区；
(13)如SEQ ID NO:522所示的轻链可变区和如SEQ ID NO:557所示的重链可变区；
(14)如SEQ ID NO:523所示的轻链可变区和如SEQ ID NO:558所示的重链可变区；
(15)如SEQ ID NO:524所示的轻链可变区和如SEQ ID NO:559所示的重链可变区；
(16)如SEQ ID NO:525所示的轻链可变区和如SEQ ID NO:560所示的重链可变区；
(17)如SEQ ID NO:526所示的轻链可变区和如SEQ ID NO:561所示的重链可变区；
(18)如SEQ ID NO:527所示的轻链可变区和如SEQ ID NO:562所示的重链可变区；
(19)如SEQ ID NO:528所示的轻链可变区和如SEQ ID NO:563所示的重链可变区；
(20)如SEQ ID NO:529所示的轻链可变区和如SEQ ID NO:564所示的重链可变区；
(21)如SEQ ID NO:530所示的轻链可变区和如SEQ ID NO:565所示的重链可变区；
(22)如SEQ ID NO:531所示的轻链可变区和如SEQ ID NO:566所示的重链可变区；
(23)如SEQ ID NO:532所示的轻链可变区和如SEQ ID NO:567所示的重链可变区；
(24)如SEQ ID NO:533所示的轻链可变区和如SEQ ID NO:568所示的重链可变区；
(25)如SEQ ID NO:534所示的轻链可变区和如SEQ ID NO:569所示的重链可变区；
(26)如SEQ ID NO:535所示的轻链可变区和如SEQ ID NO:570所示的重链可变区；
(27)如SEQ ID NO:536所示的轻链可变区和如SEQ ID NO:571所示的重链可变区；
(28)如SEQ ID NO:537所示的轻链可变区和如SEQ ID NO:572所示的重链可变区；
(29)如SEQ ID NO:538所示的轻链可变区和如SEQ ID NO:573所示的重链可变区；
(30)如SEQ ID NO:539所示的轻链可变区和如SEQ ID NO:574所示的重链可变区；
(31)如SEQ ID NO:540所示的轻链可变区和如SEQ ID NO:575所示的重链可变区；
(32)如SEQ ID NO:541所示的轻链可变区和如SEQ ID NO:576所示的重链可变区；
(33)如SEQ ID NO:542所示的轻链可变区和如SEQ ID NO:577所示的重链可变区；
(34)如SEQ ID NO:543所示的轻链可变区和如SEQ ID NO:578所示的重链可变区；和
(35)如SEQ ID NO:544所示的轻链可变区域和如SEQ ID NO:579所示的重链可变区域。
在一些实施方案中，抗CD123抗体是抗CD123 scFv，可以包括选自SEQ ID NO:583-617中任一氨基酸序列。
在一些实施方案中，所述CAR包括可以特异性地与CD123结合的结合域，其可称为靶向CD123的CAR。在一些实施方案中，靶向CD123的CAR可以是PCT/CN2021/112748(通过引用将其全文并入本文)中描述的CAR。在一些实施方案中，靶向CD123的CAR可以包括上述的任何一种抗CD123抗体。在一些实施方案中，靶向CD123的CAR可包括选自SEQ ID NO:580-582的氨基酸序列。
在一些实施方案中，特异性结合BCMA的抗体，也称为抗BCMA抗体，如是PCT/CN2021/112798(其全部内容通过引用纳入本文)中描述，包括轻链可变区和重链可变区，轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2 和HCDR3选自下组:
(1)如SEQ ID NO:232所示的LCDR1，如SEQ ID NO:242所示的LCDR2，如SEQ ID NO:253所示的LCDR3，如SEQ ID NO:264所示的HCDR1，如SEQ ID NO:275所示的HCDR2和如SEQ ID NO:287所示的HCDR3；
(2)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:243所示的LCDR2，如SEQ ID NO:254所示的LCDR3，如SEQ ID NO:265所示的HCDR1，如SEQ ID NO:276所示的HCDR2和如SEQ ID NO:288所示的HCDR3；
(3)如SEQ ID NO:234所示的LCDR1，如SEQ ID NO:244所示的LCDR2，如SEQ ID NO:255所示的LCDR3，如SEQ ID NO:266所示的HCDR1，如SEQ ID NO:277所示的HCDR2和如SEQ ID NO:289所示的HCDR3；
(4)如SEQ ID NO:235所示的LCDR1，如SEQ ID NO:245所示的LCDR2，如SEQ ID NO:255所示的LCDR3，如SEQ ID NO:267所示的HCDR1，如SEQ ID NO:278所示的HCDR2和如SEQ ID NO:290所示的HCDR3；
(5)如SEQ ID NO:236所示的LCDR1，如SEQ ID NO:246所示的LCDR2，如SEQ ID NO:256所示的LCDR3，如SEQ ID NO:268所示的HCDR1，如SEQ ID NO:279所示的HCDR2和如SEQ ID NO:291所示的HCDR3；
(6)如SEQ ID NO:237所示的LCDR1，如SEQ ID NO:247所示的LCDR2，如SEQ ID NO:257所示的LCDR3，如SEQ ID NO:269所示的HCDR1，如SEQ ID NO:280所示的HCDR2和如SEQ ID NO:292所示的HCDR3；
(7)如SEQ ID NO:238所示的LCDR1，如SEQ ID NO:248所示的LCDR2，如SEQ ID NO:258所示的LCDR3，如SEQ ID NO:266所示的HCDR1，如SEQ ID NO:281所示的HCDR2和如SEQ ID NO:293所示的HCDR3；
(8)如SEQ ID NO:239所示的LCDR1，如SEQ ID NO:249所示的LCDR2，如SEQ ID NO:259所示的LCDR3，如SEQ ID NO:270所示的HCDR1，如SEQ ID NO:282所示的HCDR2和如SEQ ID NO:294所示的HCDR3；
(9)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:250所示的LCDR2，如SEQ ID NO:260所示的LCDR3，如SEQ ID NO:271所示的HCDR1，如SEQ ID NO:283所示的HCDR2和如SEQ ID NO:295所示的HCDR3；
(10)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:251所示的LCDR2，如SEQ ID NO:261所示的LCDR3，如SEQ ID NO:272所示的HCDR1，如SEQ ID NO:284所示的HCDR2和如SEQ ID NO:296所示的HCDR3；
(11)如SEQ ID NO:240所示的LCDR1，如SEQ ID NO:252所示的LCDR2，如SEQ ID NO:262所示的LCDR3，如SEQ ID NO:273所示的HCDR1，如SEQ ID NO:285所示的HCDR2和如SEQ ID NO:297所示的HCDR3；和
(12)如SEQ ID NO:241所示的LCDR1，如SEQ ID NO:245所示的LCDR2，如SEQ ID NO:263所示的LCDR3，如SEQ ID NO:274所示的HCDR1，如SEQ  ID NO:286所示的HCDR2和如SEQ ID NO:298所示的HCDR3。
在一些实施方案中，抗BCMA抗体包括选自下组的轻链可变区和重链可变区：
(1)如SEQ ID NO:299所示所示的轻链可变区和如SEQ ID NO:311所示的重链可变区；
(2)如SEQ ID NO:300所示的轻链可变区域和如SEQ ID NO:312所示的重链可变区域；
(3)如SEQ ID NO:301所示的轻链可变区域和如SEQ ID NO:313所示的重链可变区域；
(4)如SEQ ID NO:302所示的轻链可变区域和如SEQ ID NO:314所示的重链可变区域；
(5)如SEQ ID NO:303所示的轻链可变区域和如SEQ ID NO:315所示的重链可变区域；
(6)如SEQ ID NO:304所示的轻链可变区域和如SEQ ID NO:316所示的重链可变区域；
(7)如SEQ ID NO:305所示的轻链可变区域和如SEQ ID NO:317所示的重链可变区域；
(8)如SEQ ID NO:306所示的轻链可变区域和如SEQ ID NO:318所示的重链可变区域；
(9)如SEQ ID NO:307所示的轻链可变区和如SEQ ID NO:319所示的重链可变区；
(10)如SEQ ID NO:308所示的轻链可变区和如SEQ ID NO:320所示的重链可变区；
(11)如SEQ ID NO:309所示的轻链可变区和如SEQ ID NO:321所示的重链可变区；和
(12)如SEQ ID NO:310所示的轻链可变区域和如SEQ ID NO:322所示的重链可变区域。
在一些实施方案中，抗BCMA抗体是抗BCMA scFv，它可以包括选自SEQ ID NO:323-334的氨基酸序列。
在一些实施方案中，所述CAR包括可以特异性地与BCMA结合的结合域，其可称为靶向BCMA的CAR。在一些实施方案中，靶向BCMA的CAR可以是PCT/CN2021/112798(通过引用将其全文并入本文)中描述的CAR。在一些实施方案中，靶向BCMA的CAR可以包括上述的任何一种抗BCMA抗体。在一些实施方案中，靶向BCMA的CAR可包括选自SEQ ID NO:335-346的氨基酸序列。
在一些实施方案中，特异性结合CD19的抗体，也被称为抗CD19抗体，如 中国专利申请CN202210274255.1(公布号为CN114349863A，在此通过引用将其全文并入本文)中描述，包括轻链可变区和重链可变区，轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3。其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3可以是如SEQ ID NO:628所示的LCDR1，如SEQ ID NO:629所示的LCDR2，如SEQ ID NO:630所示的LCDR3，如SEQ ID NO:631所示的HCDR1，如SEQ ID NO:632所示的HCDR2和如SEQ ID NO:633所示的HCDR3。
在一些实施方案中，抗CD19抗体包括如SEQ ID NO:635所示的轻链可变区和如SEQ ID NO:634所示的重链可变区。在一些实施方案中，抗CD19抗体是抗CD19scFv，它可以包括如SEQ ID NO:636所示的氨基酸序列。
在一些实施方案中，所述CAR包括可以特异性地与CD19结合的结合域，其可称为靶向CD19的CAR。在一些实施方案中，靶向CD19的CAR可以是中国专利申请CN202210274255.1(公布号为CN114349863A，在此通过引用将其全文并入本文)中描述的CAR。在一些实施方案中，靶向CD19的CAR可以包括上述的任何一种抗CD19抗体。在一些实施方案中，靶向CD19的CAR可包括如SEQ ID NO:637所示的氨基酸序列。
在一些实施方案中，特异性结合HER2的抗体，也被称为抗HER2抗体，如中国专利申请CN202210750853.1(公布号为CN114805584A，在此通过引用将其全文并入本文)中描述，包括轻链可变区和重链可变区，轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3。其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3可以是如SEQ ID NO:618所示的LCDR1，如SEQ ID NO:619所示的LCDR2，如SEQ ID NO:620所示的LCDR3，如SEQ ID NO:621所示的HCDR1，如SEQ ID NO:622所示的HCDR2和如SEQ ID NO:623所示的HCDR3。
在一些实施方案中，抗HER2抗体包括如SEQ ID NO:624所示的轻链可变区和如SEQ ID NO:625所示的重链可变区。在一些实施方案中，抗HER2抗体是一种抗HER2 scFv，它可以包括如SEQ ID NO:626所示的氨基酸序列。
在一些实施方案中，所述CAR包括可以特异性地与HER2结合的结合域，其可称为靶向HER2的CAR。在一些实施方案中，靶向HER2的CAR可以是中国专利申请CN202210750853.1(公布号为CN114805584A，在此通过引用将其全文并入本文)中描述的CAR。在一些实施方案中，靶向HER2的CAR可以包括上述的任何一种抗HER2抗体。在一些实施方案中，靶向HER2的CAR可包括如SEQ ID NO:627所示的氨基酸序列。
在一些实施方案中，特异性结合IL13Ra2的抗体，也被称为抗IL13Ra2抗体，如中国专利申请CN202210743595.4(公布号为CN114805581A，在此通过引用将其全文并入本文)中描述，包括轻链可变区和重链可变区，轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3。其 中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3可以是如SEQ ID NO:638所示的LCDR1，如SEQ ID NO:639所示的LCDR2，如SEQ ID NO:640所示的LCDR3，如SEQ ID NO:641所示的HCDR1，如SEQ ID NO:642所示的HCDR2和如SEQ ID NO:642所示的HCDR3。
在一些实施方案中，抗IL13Ra2抗体包括如SEQ ID NO:644所示的轻链可变区和如SEQ ID NO:645所示的重链可变区。在一些实施方案中，抗IL13Ra2抗体是抗IL13Ra2 scFv，它可以包括如SEQ ID NO:646所示的氨基酸序列。
在一些实施方案中，所述CAR包括可以特异性地与IL13Ra2结合的结合域，其可称为靶向IL13Ra2的CAR。在一些实施方案中，靶向IL13Ra2的CAR可以是中国专利申请CN202210743595.4(公布号为CN114805581A，在此通过引用将其全文并入本文)中描述的CAR。在一些实施方案中，靶向IL13Ra2的CAR可以包括上述的任何一种抗IL13Ra2抗体。在一些实施方案中，靶向IL13Ra2的CAR可包括如SEQ ID NO:647所示的氨基酸序列。
在一些实施方案中，特异性结合B7H3的抗体，也被称为抗B7H3抗体，如是中国专利申请CN202210714289.8(公布号为CN114773477A，在此通过引用将其全文并入本文)中描述，包括轻链可变区和重链可变区，轻链可变区包括LCDR1、LCDR2和LCDR3，重链可变区包括HCDR1、HCDR2和HCDR3。其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3可以是如SEQ ID NO:648所示的LCDR1，如SEQ ID NO:649所示的LCDR2，如SEQ ID NO:650所示的LCDR3，如SEQ ID NO:651所示的HCDR1，如SEQ ID NO:652所示的HCDR2和如SEQ ID NO:653所示的HCDR3。
在一些实施方案中，抗B7H3抗体包括SEQ ID NO:654所示的轻链可变区和SEQ ID NO:655所示的重链可变区。在一些实施方案中，抗B7H3抗体是抗B7H3 scFv，它可以包括如SEQ ID NO:656所示的氨基酸序列。
在一些实施方案中，所述CAR包括可以特异性地与B7H3结合的结合域，其可称为靶向B7H3的CAR。在一些实施方案中，靶向B7H3的CAR可以是中国专利申请CN202210714289.8(公布号为CN114773477A，在此通过引用将其全文并入本文)中描述的CAR。在一些实施方案中，靶向B7H3的CAR可以包括上述的任何一种抗B7H3抗体。在一些实施方案中，靶向B7H3的CAR可包括如SEQ ID NO:657所示的氨基酸序列。
在一些实施方案中，本发明的人源化抗CD28抗体、亲和力成熟的抗CD40抗体或其抗原结合片段、包含它们的多特异性抗体、CD28-CD40双抗、编码它们的多核苷酸、包含该多核苷酸的载体或表达CD28-CD40双抗的免疫效应细胞可用作药物，用于治疗肿瘤、癌症、病毒感染或自身免疫性疾病等，其可被配制为药物组合物。所述人源化抗CD28抗体、亲和力成熟的抗CD40抗体或其抗原结合片段、包含它们的多特异性抗体、CD28-CD40双抗、编码它们的多核苷酸或包含该多核苷酸的载体可以和免疫效应细胞组合使用。所述免疫效应细胞 可以选自CAR T细胞、TCR T细胞、TIL、CIK、LAK和MIL。
在一些实施方案中，本发明提供治疗疾病的方法，包括将人源化抗CD28抗体、亲和力成熟的抗CD40抗体或其抗原结合片段、包含它们的多特异性抗体、CD28-CD40双抗、编码它们的多核苷酸、包含该多核苷酸的载体或表达CD28-CD40双抗的免疫效应细胞施用给需要治疗的受试者。所述疾病可以是肿瘤、癌症、病毒感染或自身免疫性疾病等。
在一些实施方案中，所述治疗方法可以包括：(a)将人源化抗CD28抗体、亲和力成熟的抗CD40抗体或其抗原结合片段、包含它们的多特异性抗体、CD28-CD40双抗、编码它们的多核苷酸、包含该多核苷酸的载体施用给需要治疗的受试者，并且(b)对受试者施用免疫细胞治疗。其中步骤(a)和(b)可以同时进行或先后进行，其中步骤(a)可以在步骤(b)之前或之后进行。所述步骤(b)中的免疫细胞治疗可以包括CAR T治疗、TCR T治疗、TIL治疗、CIK治疗、LAK治疗和MIL治疗。
在一些实施方案中，癌症是实体瘤或血液系统癌症(如白血病)。在一些实施方案中，癌症是急性骨髓性白血病(AML)、B型急性淋巴细胞白血病(B-ALL)、T型急性淋巴细胞白血病(T-ALL)、B细胞前体急性淋巴细胞白血病(BCP-ALL)或母细胞性浆细胞样树突细胞肿瘤(BPDCN)。在一些实施方案中，所述疾病(如癌症)的特征在于疾病细胞表达间皮素、CD123、BCMA、HER2、CD19、IL13Ra2和/或B7H3。在一些实施例中，癌症是表达CD123的癌症。在一些实施方案中，癌症是表达CD123的AML。在一些实施方案中，癌症是间皮瘤。在一些实施方案中，间皮瘤是胸膜间皮瘤，腹膜间皮瘤，或心包间皮瘤。在一些实施方案中，癌症是胰腺癌。在一些实施方案中，胰腺癌是胰腺导管癌。在一些实施方案中，癌症是卵巢癌。在一些实施方案中，癌症是卵巢上皮癌。在一些实施方案中，癌症是肺癌。在一些实施方案中，癌症是非霍奇金淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、人类B细胞前体白血病、多发性骨髓瘤、恶性淋巴瘤。在一些实施方案中，癌症是乳腺癌、胃癌、卵巢癌、宫颈癌、尿路上皮癌、食道癌、膀胱癌、结直肠癌、子宫内膜癌、肾癌、肺癌、胰腺癌、头颈癌、肉瘤、胶质母细胞瘤、前列腺癌或甲状腺癌。在一些实施方案中，癌症是胶质瘤或头颈部癌症。
术语"受试者"和"患者"在这里可以互换使用。本文所用的术语"受试者"是指可对其施用本发明的抗体或其抗原结合片段的任何生物体，例如，为了实验、诊断、预防和/或治疗目的。典型的受试者包括动物(例如，哺乳动物，如小鼠、大鼠、兔子、非人类灵长类动物，如黑猩猩和其他猿猴类，以及人类)。受试者可以是哺乳动物，特别是人类，包括雌性(女性)或雄性(男性)，并包括新生儿、婴儿、少年、青年、成人或老年，并进一步包括各种种族和民族。在一些实例中，受试者指需要诊断、治疗或预防疾病或病症的个体，所述受试者可能患有所述疾病或病症，或具有患所述疾病或病症的风险。
本文使用的术语“治疗”是指对疾病提供有益的或期望的临床结果，如消除疾病、减轻症状、减少疾病的程度、稳定、改善或缓解疾病的状态，或减缓疾病的进展。对治疗结果的测量可以基于，例如，本领域已知的体检、病理测试和/或诊断测试的结果。治疗也可以指与受试者不接受治疗时的预期生存期相比，延长生存期。治疗也可以指与不采取该措施的情况下会发生的疾病相比，减少疾病的发生率或发病率，或其复发。在临床上，这种治疗也可以称为预防。
本发明的术语“药物组合物”与“药物制剂”可以互换使用。药物组合物可包含药学可接受的载体。本文使用的术语“药学可接受的载体”是指作为非活性成分包含在药物组合物中的任何载体，它使药物组合物具有适合给药的外观和特性。药学上可接受的载体在给受试者施用时基本上没有长期或永久性的不利影响，如稳定剂、稀释剂、添加剂、辅助剂、赋形剂等。“药学可接受的载体”应该是一种药学上的惰性材料，基本上没有生物活性，并构成制剂的主要部分。
本发明的药物组合物、药物组合、免疫细胞(包括CAR-T细胞)可以按照已知技术配制成各种给药方式。例如，见Remington,The Science and Practice of Pharmacy(9th Ed.1995)。在制造药物组合物时，活性剂通常与药物可接受载体等混合。当然，药学上可接受的载体必须是可接受的，即与配方中的任何其他成分兼容，并且必须对受试者无害。药学上可接受的载体可以包括但不限于缓冲剂、赋形剂、稳定剂、防腐剂、润湿剂、表面活性剂、乳化剂或其组合。缓冲剂的例子包括但不限于醋酸、柠檬酸、组氨酸、硼酸、甲酸、琥珀酸、磷酸、碳酸、苹果酸、天冬氨酸、Tris缓冲剂、HEPPSO、HEPES、中性缓冲盐水、磷酸盐缓冲盐水等。
本发明的药物组合物、药物组合、免疫细胞(包括CAR-T细胞)可以以任何适合于待治疗(或预防)的疾病和受试者的方式给药。在某些实施方案中，给药方式可包括但不限于肠外或非肠外途径，包括口服、舌下、口腔、经皮、直肠、阴道、皮内、鼻内途径或肠外途径，如静脉注射(i.v.)、腹膜内、皮内、皮下、肌肉内、颅内、鞘内、瘤内、经皮、经粘膜内、关节内、鞘内、鞘内、肝内、神经内或颅内注射或输液。药物组合物可以直接注射到肿瘤、淋巴结、组织、器官或感染部位。
适合口服的剂型包括但不限于片剂、胶囊、粉末、药丸、颗粒、悬浮液、溶液或溶液的预浓缩物、乳剂或乳剂的预浓缩物。可用于口服剂型的药品可接受载体包括水、乙二醇、油、醇、调味剂、防腐剂、着色剂等。载体如淀粉、糖、微晶纤维素、稀释剂、填料、滑润剂、造粒剂、润滑剂、粘合剂、稳定剂、崩解剂等可用于制备口服固体制剂，如粉末、胶囊或片剂。
适合肠外给药的剂型包括但不限于无菌液体制剂，如等渗水溶液、乳剂、悬浮液、分散液或粘稠组合物，可缓冲到理想的pH值。肠外剂型可以是即用型，也可以是准备溶解或悬浮在药学上可接受的载体中的干燥产品。肠外剂型可以是无菌配方，或在给受试者服用前能够进行消毒。可用于提供肠外剂型的药学 上可接受的载体包括，但不限于，注射用水；水性载体，例如但不限于，氯化钠注射液、林格注射液、葡萄糖注射液。水溶性载体，例如但不限于乙醇、聚乙二醇和聚丙二醇；非水载体，例如但不限于玉米油、棉籽油、花生油、芝麻油、油酸乙酯、肉豆蔻酸异丙酯和苯甲酸苄酯；以及增溶剂，如环糊精。
本发明的药物组合物、药物组合、免疫细胞(包括CAR-T细胞)以治疗有效量被施用给受试者。本文使用的术语“治疗有效量”和“有效量”可以互换使用，指的是在必要的剂量和时间段内有效的量，以达到预期的治疗效果。治疗上的有效量可根据不同的因素而变化，如疾病状态、年龄、性别和个人体重，以及一种治疗方法或治疗方法的组合在个人身上引起所需反应的能力。有效量可以指引起生物或化学活性可检测变化的量。可检测的变化可由相关领域的人员检测和/或进一步量化。此外，“有效量”可以指定保持所需生理状态的量，即减少或防止显著下降和/或促进病情的改善。
给药的数量和频率将由受试者的状况(如年龄、体重、性别和受试者对药物的反应)以及受试者疾病的类型和严重程度等因素决定，尽管适当的剂量可由临床试验决定。
本发明的药物组合物、药物组合、免疫细胞(包括CAR-T细胞)可以以约0.5至约250mg/kg的治疗有效量给予受试者，例如，约1至约250mg/kg，约2至约200mg/kg，约3至约120mg/kg，约5至约250mg/kg，约10至约200mg/kg，或约20至约120mg/kg。
在本发明中，免疫细胞(包括CAR-T细胞)的有效量可以是例如在单次给药中施用5×106、1×107、2×107、5×107、1×108、2×108、5×108、1×109、2×109或5×109个细胞。
本发明的药物组合物、药物组合、免疫细胞(包括CAR-T细胞)可以一天给药一次或两次；或每2、3、4、5、6、7、8、9或10天一次，每1、2、3、4、5或6周一次，或每1、2、3、4、5或6个月一次或更长时间。该药物组合物也可按每周数次(如1、2、3、4或5次)或每月数次(如1、2、3、4、5、6、7、8、9或10次)的方案给药。例如，在每周5次的方案中，该药物组合物可连续5天每天给药一次，然后连续两天休息。
本文使用的术语“药物组合”指在一个时间段内(例如同时，或相差几分钟到几小时，或甚至相差几天或几周)向受试者给药的两种或更多种药物的组合。所述两种或更多种药物可以被配制为同一药物制剂，或者也可以分别被配制成不同的药物制剂。
本发明的药物组合物、药物组合、免疫细胞(包括CAR-T细胞)可与其他药物和治疗方法联合使用，例如其他抗肿瘤药物、化学治疗或放射性治疗。这里所说的"联合使用"是指在受试者的治疗过程中对其施用两种(或更多)不同的药物和/或疗法。组合中的两种或多种药物和/或疗法可以通过不同的途径和方案给药。两种或多种药物和/或治疗方法可以同时或依次给受试者施用。在一些 实施方案中，当第二种药物或疗法的施用开始时，一种药物或疗法的施用仍在进行中，因此在施用方面存在重叠。这样的方案在此可称为"同时"。当同时给药时，两种或多种药物和/或疗法可以一起配制成单一的剂型，或分别配制成两种或多种独立的剂型。
实施例
下述实施例中，抗体重链可变区和轻链可变区的CDR均以Kabat命名法划定。除非另有说明，下文描述的实施例的方法和材料均为可以通过市场购买获得的常规产品。
实施例1：结构模拟
同源建模也称“比较建模”，基于“如果两个蛋白质具有足够高的序列相似性，它们很可能具有非常相似的三维结构”的原理，根据已知结构的序列同源性预测未知蛋白质的结构。
如果目标序列与模板序列同源性达到30％，则预测模型的准确度可达80％；如果同源性达到50％，则模型的准确度可达95％；如果同源性达到70％以上，则认为预测模型完全代表蛋白的真实结构。但也存在特殊情况，虽然序列同源性很高，但结构却不同。
因此，先采用同源建模的方式，预测鼠源CD28抗体9.3的结构。对于部分同源性较低的抗体结构，则采用从头建模的方式搭建模型。大概的实验步骤如下：
1.分别使用Discovery Studio和Antibody Modeling软件，对鼠源CD28抗体9.3采用同源建模的方法建模，并从预测模型中挑选5-10个最优的结构解析模型。
2.抗体的Loop区域先用同源建模的方法预测结构，如果9.3抗体的CDR区与模板抗体CDR区的氨基酸序列同源度低于50％时，则使用从头建模的方法搭建CDR区的结构模型。
3.使用PDB蛋白质结构数据库中的BLAST程序搜索与母本抗体9.3的氨基酸序列同源性最高的13个抗体晶体结构模型(结构分辨率高于2.5埃)，对比自动建模的模型，选取最优的结构模型。
最优的结构模型如图1所示，9.3抗体轻重链的序列与PDB结构数据库中的模板序列的同源度超过90％，为常见的抗体结构，预测模型的可信度超过95％。抗体表面的电荷分布均一，形成聚体的可能性低。
实施例2.人源化设计
依据预测的抗体结构模型，对9.3抗体进行人源化设计，选择小鼠源的氨基酸进行点突变和回复突变。简略的实验步骤如下：
1.使用NCBI的Ig-BLAST程序，将小鼠源的9.3抗体序列与人Germline序列数据库进行比对。搜索结果显示9.3抗体的重链可变区(VH)与人Germline IGHV4序列同源度最高，含24个鼠源氨基酸位点。9.3抗体的轻链可变区(VL)与人Germline IGKV1序列同源度最高，也含24个鼠源氨基酸位点。
2.重链VH选择同源度最高的IGHV4为人源化设计模板，设计5条序列：VH1-VH5。轻链VL选择IGKV1为人源化设计模板，设计3条序列：VL1-VL3(图2A和B，其中Parental为9.3抗体的VH和VL序列，也即表1中的mVH和mVL序列)。
3.由于IGHV4-KV1为最常见的重链-轻链配对方式，所以选择IGHV4-KV1为对应轻重链配对形式。
4.免疫原性分析：预测VH为低免疫原性，具有免疫原性肽段为：FLKMNSLQA。预测VL为低免疫原性，具有免疫原性肽段为：LLIFAASNV。
5.人源化抗体人源化程度计算：将设计好的人源化序列轻重链进行配对组合后与人源Germline序列进行比对，计算出每个部分及全长抗体的人源化程度百分比。依据轻重链配对的结构、剔除易被氧化和氨基化等位点的抗体、剔除具有糖基化、磷酸化等蛋白修饰位点的抗体，获得11个人源化抗体，汇总结果见表1，其中所有抗体样品均为IgG1形式，且具有相同的重链恒定区和轻链恒定区，“mVHmVL”是包含9.3抗体的鼠源重链可变区(mVH)和鼠源轻链可变区(mVL)的抗体，“huVH1huVL1”表示该抗体包含huVH1和huVL1，其他人源化抗体名称含义类似。所有抗体样品的具体序列参见实施例后所附“序列”部分。
表1.抗体人源化程度汇总表

实施例3：SDS-PAGE(SDS-polyacrylamide gel electrophoresis)鉴定人源化抗 体的分子量和纯度
通过SDS-PAGE电泳检测全长人源化抗体(IgG1)在非还原和还原条件下的分子量和纯度，以利于后期的生产工艺开发。
1.样品制备：先用Nano-300微量分光光度计(杭州奥盛仪器有限公司)测定蛋白样品A280，并计算蛋白浓度。非还原样品：取1μg蛋白，加入4×LDS上样缓冲液，40mM碘代乙酰胺，75℃孵育10min。还原样品：取2μg蛋白，加入4×LDS上样缓冲液，5mM DTT(Dithiothreitol，二硫苏糖醇)，100℃孵育10min。在140V电泳50min。
2.凝胶用考马斯亮蓝R250染色，脱色后用EPSON V550彩色扫描仪扫描。
3.用Image J软件按照峰面积归一法计算还原条带纯度，或者还原的重链加轻链和的纯度。
实验结果(表2)表明人源化抗体的分子量均大约为146kDa，纯度也比较高。
表2.人源化抗体SDS-PAGE结果汇总表

实施例4：SEC(Size exclusion chromatography，分子筛色谱法)分析人源化 抗体的纯度
通过SEC方法检测非还原条件下人源化抗体的单体和聚体的比例，来鉴定抗体的纯度。
1.选用Agilent HPLC 1100，XBridgeSEC 3.5μm，7.8×300mm色谱柱。
2.以0.15M PB,pH 7.4为流动相，设置流速为0.8mL/min，上样体积为20μL，检测波长280nm，带宽16nm，参比波长360nm，带宽100nm，峰宽(响应时间)>0.1min(2s)；狭缝4nm；负吸光度基线100mAU。
3.参比品Herceptin单体纯度大于95％，BSA单体与二聚体的分离度大于2，视为基线平稳。
实验结果(表3和图3)表明人源化抗体的纯度均较好，单体占总体的比例高。除了低比例的高聚体外，无低分子量物质或抗体碎片存在。
表3.人源化抗体SEC结果汇总表
实施例5：ELISA(enzyme linked immunosorbent assay，酶联免疫吸附测定) 检测人源化抗体的结合活性
通过ELISA方法检测人源化抗体与抗原CD28的结合活性。实验步骤如下：
1.向ELISA板的每孔中加入30μL浓度为2μg/ml的recombinant Human/Cynomolgus CD28(N-6His)(苏州近岸蛋白科技有限公司，C406)，在4℃孵育过夜。
2.次日，用PBST(含0.05％Tween-20)洗涤ELISA板3次。
3.向ELISA板的每孔中加入250μl 5％脱脂奶粉(PBS溶解)，在室温下孵育2小时。
4.用PBST洗涤板3次，并在ELISA板中加入不同稀释比例的抗体，每孔30μL，在室温下孵育60分钟。
5.用PBST洗涤板3次，每孔加入30μL HRP-抗-hFc抗体，在室温下孵育60分钟。
6.用PBST洗涤板3次，每孔加入30μL TMB，室温显色后，每孔加30μL2M H2SO4终止反应，并在450nm读数。
实验结果(表4和图4)显示，和母本mVHmVL相比，只有4种人源化抗体huVH2huVL3、huVH3huVL2、huVH3huVL3和huVH5huVL3的结合活性提高，其余抗体的结合活性均不同程度的降低。
表4.人源化抗体ELISA结果汇总表
实施例6：BLI检测人源化抗体的亲和力
利用生物膜层干涉技术(Biolayer interferometry，BLI)检测人源化抗体的亲和动力学。实验步骤如下：
1.运行GATOR仪器，选择Kinetics实验模式。
2.按表5运行分析程序
表5.GATOR分析程序
实验结果如表6和图5所示，在所得到的人源化抗体中，huVH1VL1的KD值最低，亲和力最高。
表6.人源化抗体的亲和动力学检测结果汇总表
实施例7：辅助杀伤功能检测
为了检测CD28人源化抗体在细胞水平的功能，选取亲和力最高的人源化抗体huVH1huVL1(简称9.3h11)，将其VH和VL与CD40抗体40.52通过scFv串联的形式组成双特异性抗体(bispecific antibody，简称双抗或BsAb)，两个不同scFv之间由GGGGS(简称G4S)连接。每个scFv内的VH与VL通过3个G4S(GGGGSGGGGSGGGGS)连接。依据VH-VL前后位置、以及CD28抗体与CD40抗体前后位置差异，双抗有8种构建模式(见表7)，并将这8种双抗分别命名为4.1-4.8。
1.通过Her2-CAR-T细胞检测双抗4.1-4.8的促杀伤功能，并与母本8.6(构建模式见表7，亦为scFv串联形式)以及1412-4D11双抗和A40C28(LACO)相比较。Her2-CAR-T细胞制备和双抗促杀伤功能的实验步骤如下：
(1)准备靶细胞：用BTX ECM 830电穿孔仪向A549-CBG细胞中电转10μg CD40 mRNA(简称A549-CD40)或不电转任何mRNA(简称A549-无EP，无EP在本发明中的含义即为不电转任何mRNA，)，电转条件为300V，0.5ms。24小时后，通过流式分析和PE-抗-CD40抗体，检测CD40的表达水平。图6表明CD40在A549细胞表面的表达水平较高。
(2)准备CAR-T细胞：向CD3+T细胞中共电转0.5μg Her2-CAR(4D5.BBZ)mRNA和15μg BsAb mRNA，或5μg A40C28(见表8)，电转条件为500V，0.7ms，作为Her2-CAR-T细胞。其中Her2-CAR(4D5.BBZ)是嵌合抗原受体(CAR)，其包含抗HER2 scFV、CD8铰链结构域、CD8跨膜(TM)结构域、4-1BB共刺激结构域和CD3ζ，其中抗HER2 scFV中的VH和VL与公知的抗HER2鼠源抗体4D5相同。24小时后，通过流式分析、CD40-Fc和PE-抗-Fc抗体检测双抗的表达水平(图7)；通过Her2-Fc和PE-抗-Fc抗体检测Her2-CAR的表达水平(图8)。实验结果显示双抗和Her2-CAR的表达水平均较高。
(3)共培养：将Her2-CAR-T细胞和靶细胞A549-无EP或A549-CD40按照效应细胞(E):靶细胞(T)细胞数比例＝10:1共培养72小时，并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，并绘制杀伤曲线。实验结果表明在4.1-4.8双抗中，4.4和4.6双抗和Her2-CAR-T细胞共同作用，能使靶细胞的荧光值降至最低，4.4和4.6促杀伤功能最强(图10A和B)。同时4.4、4.6的促杀伤功能强于母本8.6和1412-4D11，但仍弱于A40C28(图10B和D)。
(4)细胞因子释放：将Her2-CAR-T细胞和靶细胞A549-无EP或A549-CD40按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IFN-γ assay来检测上清中IFN-gamma的释放水平。实验结果(图9)表明在4.1-4.8双抗中，4.4和4.6双抗能促使更多的IFN-gamma细胞因子释放，比8.6和1412-4D11双抗的促释放能力更强，但仍弱于A40C28。
表7.双抗的构建模式
表8.Her2-CAR-T细胞mRNA共电转表
2.通过Mesothelin(简称Meso)-CAR-T细胞检测双抗4.1-4.8的促杀伤功能，以及与母本8.6和1412-4D11双抗、A40C28(LACO)的差异。Meso-CAR-T细胞制备和双抗促杀伤功能的实验步骤如下：
(1)准备靶细胞：向A549-CBG细胞中不电转任何mRNA(简称A549-无EP)，电转10μg Meso(间皮素)mRNA(简称A549-Meso)，或10μg Meso和10μg CD40 mRNA(简称A549-Meso+CD40)，电转条件为300V，0.5ms。24小时后，通过流式分析和PE-抗-CD40抗体，检测CD40表达水平。图11表明CD40在A549细胞表面的表达水平较高。通过流式分析、Meso-biotin和PE-streptavidin抗体，检测Meso的表达水平。图12表明Meso在A549细胞表面的表达水平较高，同时Meso高表达也促使CD40的表达上调。
(2)准备CAR-T细胞：向CD3+T细胞中共电转2μg Meso-CAR(M12) mRNA和15μg BsAb mRNA(见表9)，电转条件为500V，0.7ms，作为Meso-CAR-T细胞。其中Meso-CAR(M12)是嵌合抗原受体(CAR)，其包含抗间皮素scFV(M12)、CD8铰链结构域、CD8跨膜(TM)结构域、4-1BB共刺激结构域和CD3ζ，其具体序列参见实施例后所附“序列表”部分。24小时后，通过流式分析、CD40-Fc和PE-抗-Fc抗体检测双抗的表达水平(图13)；通过Meso-Fc和PE-抗-Fc抗体检测Meso-CAR的表达水平(图14)。实验结果显示双抗和Meso-CAR的表达水平均较高。
(3)共培养：将Meso-CAR-T细胞和靶细胞A549-无EP、A549-Meso或A549-Meso+CD40按照E:T＝10:1共培养72小时，并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，并绘制杀伤曲线。实验结果表明在4.1-4.8双抗中，4.6和4.8双抗和Meso-CAR-T细胞共同作用，能使靶细胞的荧光值降至最低，4.6和4.8促杀伤功能最强(图16A和B)。同时4.6、4.8的促杀伤功能强于母本8.6和A40C28，但弱于1412-4D11(图16C和D)。
(4)细胞因子释放：将Meso-CAR-T细胞和靶细胞A549-无EP、A549-Meso或A549-Meso+CD40按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IL-2 assay和human IFN-γ assay来检测上清中IL-2和IFN-gamma的释放水平。实验结果表明在4.1-4.8双抗中，4.4和4.6双抗能促使更多的IL-2释放(图15A)和IFN-gamma释放(图15B)，比8.6双抗的促释放能力强，但仍弱于1412-4D11和A40C28。
表9.Meso-CAR-T细胞mRNA共电转表
实施例8：双特异性抗体的构建
双特异性抗体(bispecific antibody，简称双抗、BsAb)指能同时特异性靶向同一个分子或不同分子上2个不同表位的基因工程抗体。本文中的双抗由分别靶向CD28和CD40的scFv抗体串联组成，即由所述的靶向CD28的抗体(IgG1或其它形式)的VH和VL形成靶向CD28的scFv抗体，由靶向CD40的抗体(Fab或其它形式)的VH和VL形成靶向CD40的scFv抗体，并将两个scFv抗体串联连接。两个不同scFv之间由GGGGS(简称G4S)连接。每个scFv内的VH与VL通过3个G4S(GGGGSGGGGSGGGGS)连接(见表10)。1412和9.3是CD28抗体，4D11、F2.103、F5.77、40.37、40.38、40.45、40.47、40.52均是CD40抗体。9.3h11是9.3人源化抗体huVH1VL1，4052.12、4052.17、4052.25、4052.28、4052.36是40.52亲和力成熟的抗体。A40C28包含抗CD40的scFv抗体、跨膜区和CD28蛋白的胞内区。
一些肿瘤细胞表面特异性高表达CD40抗原，双抗一端的CD40抗体可识别并结合肿瘤表面的CD40；另一端的CD28抗体能结合并激活T细胞上的CD28共刺激信号通路，增强T细胞的功能。因此，双抗能同时结合肿瘤细胞上的CD40和T细胞上的CD28分子，通过桥联的方式将肿瘤细胞和T细胞的距离拉进，更有利于T细胞发挥杀伤肿瘤的功能。
双特异抗体的质粒构建方式如下：
通过PCR的方式，分别扩增抗体的VH和VL片段，再通过Gibbson assembly同源臂重组的连接方式，亚克隆到pDA载体中。通过桑格测序鉴定质粒的正确性。
表10.双抗的组成信息表

备注：TM：跨膜区；cytoplasmic:胞内区
实施例9：mRNA体外制备
在体外从DNA模板上转录mRNA，以便后续在细胞水平上检测双抗的功能。
(1)质粒线性化：测序正确的单克隆，通过质粒小量提取试剂盒(天根生化科技有限公司，DP103)抽提质粒。用SpeI内切酶在37℃切割4h或过夜，使质粒线性化。取2μl未酶切和酶切的质粒跑1％DNA核酸胶，检测酶切程度。用PCR产物回收试剂盒(Qiagen，28104)纯化酶切产物。
(2)体外mRNA制备：用mMESSAGE mMACHINETMT7转录试剂盒(Invitrogen,AM1344)在体外制备mRNA。再向此体系中加入1μl DNase I在37℃消化模板DNA 30min。
(3)用RNeasy Kit(Qiagen)纯化mRNA。
(4)用Nano-300测mRNA浓度后，将mRNA冻存于-80℃。
实施例10：双抗的辅助杀伤功能检测
通过将双抗、CAR-T细胞和靶细胞共培养的方式，检测双抗的促杀伤功能。
1.通过Her2-CAR-T细胞检测双抗的促杀伤功能。Her2-CAR-T细胞制备和双抗促杀伤功能的实验步骤如下：
(1)准备靶细胞：用BTX ECM 830电穿孔仪向A549-CBG细胞中电转(EP)10μg CD40 mRNA(简称A549-CD40)或不电转任何mRNA(简称A549-无EP)，电转条件为300V，0.5ms。24小时后，通过流式分析和PE-抗-CD40抗体，检测CD40的表达水平，检测结果如图17A所示。
(2)准备CAR-T细胞：向CD3+T细胞中单电转或共电转0.5μg Her2-CAR(4D5.BBZ)mRNA、15μg BsAb mRNA或10μg新A40C28(即A40C2828)mRNA，电转条件为500V，0.7ms。其中Her2-CAR(4D5.BBZ)与实施例7相同。24小时后，通过流式分析、CD40-Fc、Her2-Fc和PE-抗-Fc抗体检测双抗和Her2-CAR的表达水平，检测结果如图17B所示。其中A40C2828为抗CD40抗体与CD28铰链区的融合蛋白，其序列可参见PCT/CN2022/126461(通过引用将其全文并入本文)中的SEQ ID NO:76。本发明中A40C2828的序列被编号为SEQ ID NO:658。A40C2828在本发明的附图部分也被称为“新A40C28”。
(3)共培养：将Her2-CAR-T细胞和靶细胞A549-无EP或A549-CD40按照效应细胞(E):靶细胞(T)细胞数比例＝10:1共培养72小时，并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，并绘制杀伤曲线。
(4)细胞因子释放：将Her2-CAR-T细胞和靶细胞A549-无EP或A549-CD40按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IL-2 和IFN-γassay试剂盒来检测上清中IFN-gamma的释放水平。
2.通过Mesothelin(简称Meso)-CAR-T细胞检测双抗的功能。Meso-CAR-T细胞制备和双抗促杀伤功能的实验步骤如下：
(1)准备靶细胞：向A549-CBG细胞中不电转任何mRNA(简称A549-无EP)，电转10μg Meso(间皮素)mRNA(简称A549-Meso)，或10μg Meso和10μg CD40 mRNA(简称A549-Meso+CD40)，电转条件为300V，0.5ms。24小时后，通过流式分析和PE-抗-CD40抗体，检测CD40表达水平。
(2)准备CAR-T细胞：向CD3+T细胞中电转2μg Meso-CAR(M12)mRNA、15μg BsAb mRNA或5μg A40C28mRNA，电转条件为500V，0.7ms。其中Meso-CAR(M12)与实施例7相同。24小时后，通过流式分析、CD40-Fc和PE-抗-Fc、Meso-Fc和PE-抗-Fc抗体检测双抗和Meso-CAR的表达水平。
(3)共培养：将Meso-CAR-T细胞和靶细胞A549-无EP、A549-Meso或A549-Meso+CD40按照E:T＝10:1共培养72小时，并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，绘制杀伤曲线。
(4)细胞因子释放：将Meso-CAR-T细胞和靶细胞A549-无EP、A549-Meso或A549-Meso+CD40按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IL-2assay和human IFN-γassay来检测上清中IL-2和IFN-gamma的释放水平。
结果分析：
CD28抗体9.3与CD40抗体(4D11、F2.103、F5.77、40.37、40.38、40.45、40.47和40.52)组成双抗(见表10)，将这些双抗的mRNA和CAR mRNA共电转到T细胞中。双抗能被T细胞表达并分泌到细胞外，形成游离状态的双抗。当分泌双抗的Her2-CAR-T细胞与靶细胞A549-无EP(或CD40)共培养后，与只有Her2-CAR-T细胞相比，双抗9.3-F2.103和9.3-40.52能促使更多的IL-2和IFN-gamma细胞因子释放(图18)；能促使靶细胞的荧光值降更低，促进靶细胞杀伤的能力更强(图19)。分泌双抗的Meso-CAR-T细胞与靶细胞共培养后，细胞因子IL-2和IFN-gamma的释放和靶细胞杀伤情况与Her2-CAR-T细胞的共培养相似(图20、21和22)。
挑选9.3-40.52双抗进行后续的优化实验。将VH与VL、9.3与40.52前后位置重新组合，得到8种不同的双抗，分别命名为“8.1、8.2……8.8”(见表10)构建不同的细胞并检测基因过表达水平(图23)。当分泌8.1-8.8双抗的Her2-CAR-T细胞与靶细胞共培养后，8.5和8.6双抗能促使更多的IFN-gamma释放(图24)，能促使CAR-T细胞杀伤更多的靶细胞，但促杀伤能力仍弱于双抗1412-4D11(图25)。分泌8.1-8.8双抗的Meso-CAR-T细胞与靶细胞共培养后，得到相似的实验结果(图26、27和28)。
将A40C28中的CD40抗体(A40C)，与9.3抗体组成双抗，命名为“3.1、3.2……3.8”(见表10)。当分泌3.1-3.8双抗的Her2-CAR-T细胞与靶细胞共培 养后，3.4和3.7双抗能促使CAR-T细胞杀伤更多的靶细胞，其促杀伤能力强于8.5和8.6双抗(图29和30)。分泌3.1-3.8双抗的Meso-CAR-T细胞与靶细胞共培养后，得到相似的实验结果(图31、32和33)。
实施例11：结合ELISA
3.4和3.7双抗的促杀伤能力强于8.5和8.6双抗，可能是由于CD40抗体不同的亲和力引起的。因此，通过体外结合ELISA的方式检测CD40抗体(以人经典单克隆抗体IgG1的形式)的亲和力。简略的实验步骤如下：
(1)抗体表达质粒构建：将抗体VH区亚克隆到含人CH1-Fc抗体片段的pEF载体中，将抗体VL区亚克隆到含人CL抗体片段的pEF载体中。
(2)抗体表达：将33μg抗体重链表达质粒和67μg抗体轻链表达质粒通过PEI共转染到293F悬浮细胞中。培养4天后，通过离心收集上清。
(3)抗体纯化：将蛋白上清和protein A纯化介质在冰上孵育2h，然后通过重力柱分离上清和纯化介质。用100ml预冷的PBS漂洗纯化介质彻底去除杂蛋白，最后用5～10ml 0.1M Glycine-HCl(pH 2.7)从纯化介质上洗脱目的蛋白，并立即加入1/10体积的1M Tris-HCl(pH 9.0)中和抗体。通过超滤管将抗体的缓冲液置换成PBS，用A280测蛋白浓度后，将抗体分装冻存于-80℃。
(4)包被抗原：在ELISA板的每孔中加入50μl浓度为2μg/ml的重组的人CD40-His抗原蛋白(ACRO，CD0-H5228)，在4℃孵育过夜。
(5)次日，用PBST(含0.05％Tween-20)漂洗ELISA板5次。
(6)封闭：用封闭液(5％脱脂奶粉)在室温封闭1-2h。彻底弃掉封闭液。
(7)一抗孵育：每孔加入50μl不同稀释浓度的CD40抗体，在室温孵育2h。用PBST漂洗5次。
(8)二抗孵育：每孔加入50μl Goat Anti-Human IgG-Fc(HRP)抗体(义翘神州，SSA001)，在室温孵育1h。用PBST漂洗5次。
(9)显色：每孔加入50μl TMB显色液(Invitrogen，501129758)，在室温显色后，加50μl 2M HCl终止反应，在450nm读数。
实验结果(图34和表11)显示，CD40抗体的亲和力EC50排序为：40.52<F2.103<A40C。40.52抗体与CD40抗原的亲和力最低。提高40.52的亲和力，或许能提高8.5和8.6双抗的功能。
表11.CD40抗体亲和力汇总表

实施例12：突变文库构建
为了提高40.52抗体的亲和力，本实施例通过对40.52的Fab抗体的CDR区采用随机点突变的方式，构建突变Fab抗体文库，从突变文库中筛选高亲和力的抗体。
(1)用abYsis软件分析40.52抗体序列，分别以Kabat、AbM、Chothia抗体命名法来划定CDR区。
(2)采用引物NNK突变和PCR的方式，对CDR区相邻的2个氨基酸进行随机点突变。将突变片段通过酶切、连接的方式亚克隆到噬菌体载体pComb3XSS中。
(3)通过BIO-RAD Micropulser电穿孔仪，将噬菌体质粒电转到感受态细胞TG1中。取5μl噬菌体质粒加到50μl TG1电转感受态中，轻轻吹打混匀，将质粒—感受态混合物转移到间距为0.1cm电击杯(Biorad,1652089)中，在Ec1模式下电击(大约1800V，0.5ms)。电击完立即插入冰中静置2min。用1ml SOC(37℃预热)培养基重悬电击杯中的菌液，并转移到15ml离心管中，再向离心管中补加3ml SOC，在37℃，275rpm震荡培养1h。
(4)取一部分菌液用SOC梯度稀释后涂布在2xTY-AG平板(含100μg/mL氨苄抗生素,2％葡萄糖)上，其余菌液全部涂布在3～5个直径为15cm的2xTY-AG平板上。在37℃培养过夜。
(5)次日，计数克隆，计算文库大小。将大平板上的菌液用3ml 2xTY培养基刮下来，测OD600后，分装冻存在-80℃。
实施例13：噬菌体筛选
从突变文库中，通过噬菌体筛选的方法淘选到高亲和力Fab抗体。
1.噬菌体文库的制备
(1)噬菌体文库扩增：按照OD600＝0.1，取大约500μl噬菌体文库菌液加到100ml 2xTY/AG培养基中(含100μg/mL氨苄抗生素,2％葡萄糖)。将稀释的噬菌体文库在37℃，250rpm震荡培养2h，使OD600达到0.5～0.8之间。
(2)加辅助噬菌体：按照MOI＝20:1,向噬菌体文库菌液中加入辅助噬菌体M13KO7，混匀后在37℃静置30min，再震荡培养30min。
(3)去除葡萄糖：将感染后的菌液在4000rpm，离心10min，彻底弃去上 清，以去除葡萄糖的抑制作用。
(4)诱导表达：用100ml 2×YT/AKI培养基(含100μg/mL氨苄抗生素、50μg/mL卡纳抗生素和100μM IPTG)重悬菌体沉淀。在25℃,250rpm震荡培养过夜，避光。
(5)噬菌体沉淀：16-20小时后，收集菌液，在4℃，以4000rpm离心10min。转移40ml上清至新的50ml离心管，再加入10ml PEG/NaCl溶液(20％PEG-8000，2.5M NaCl)，混匀。冰上放置1个小时。
(6)对沉淀物在4℃，4000rpm离心30min，沉底弃掉上清。
(7)重悬噬菌体沉淀：用2ml PBS重悬噬菌体沉淀，即为噬菌体文库。
2.噬菌体筛选
(1)包被抗原：将重组的人CD40-Fc抗原蛋白用PBS稀释到1μg/ml(第一轮筛选1μg/ml，第二轮筛选0.5μg/ml，第三轮筛选0.1μg/ml)，取300μl稀释的CD40-Fc加到ELISA板的6个孔中。在4℃孵育过夜。
(2)次日，用PBST(含0.05％Tween-20)漂洗ELISA板5次。
(3)封闭：用封闭液(2％脱脂奶粉+1％BSA)在室温封闭1-2h。彻底弃掉封闭液。在封闭ELISA板的同时，对噬菌体也进行封闭。向噬菌体溶液中加入1/10体积的封闭液，在室温旋转孵育1-2h。
(4)噬菌体孵育：将已封闭的噬菌体均匀的加到含抗原的孔中，300μl每孔，在室温震荡培养2h。
(5)漂洗：彻底弃掉噬菌体，用PBST洗ELISA板5次。
(6)洗脱：每孔加入150μl洗脱液(0.1M甘氨酸，pH 2.7,0.5M NaCl)，吹打孔底和孔壁10min，并转移洗脱液到15ml离心管中。立即加入1/10体积的1M Tris-HCl(pH 9.0)来中和。
(7)噬菌体侵染：再向此离心管中加入8ml TG1(OD600＝0.5)菌液，在37℃静置30min，再37℃，250rpm震荡培养30min。
(8)涂平板：将菌液在4000rpm离心10min，弃掉绝大多数上清，留大约1ml上清重悬菌体沉淀，并将菌液均匀涂布在直径为15cm的2xTY-AG平板上。在37℃培养过夜。
(9)次日，用3ml 2x TY培养基将菌落从平板上刮下来，取200μl菌液用于下一次筛选，其余的菌液加入等体积的50％甘油，冻存在-80℃。
实施例14：噬菌体ELISA
通过噬菌体ELISA筛选特异性高亲和力的克隆。具体的实验步骤如下：
1.制备单克隆的噬菌体
(1)准备单克隆：取适量第三轮筛选得到的菌液，用2xTY培养基稀释后， 涂布在2xTY-AG平板上，在37℃培养过夜。
(2)挑单克隆菌落到含250μl 2xTY-AG培养基(含100μg/mL氨苄抗生素,2％葡萄糖)的深孔板中，在37℃，250rpm震荡培养过夜。
(3)转接：次日，转移10μl细菌过夜培养物到含300μl 2xTY-AG培养基的深孔板中，在37℃，250rpm震荡培养2h。
(4)辅助噬菌体侵染：按MOI＝20:1，向每孔中加入20μl M13KO7辅助噬菌体，轻轻混匀后，在37℃静置孵育30min，再在37℃，250rpm震荡培养30min。
(5)将菌液在4000rpm离心7min，彻底弃掉上清，用300μl 2xTY-AKI培养基(含100μg/mL氨苄抗生素、50μg/mL卡纳抗生素和100μM IPTG)重悬菌体沉淀，在25℃，250rpm震荡培养过夜。
(6)次日，将菌液在4000rpm离心10min。上清用于噬菌体ELISA。
2.噬菌体ELISA
(1)包被抗原：在ELISA板的每孔中加入50μl浓度为2μg/ml的重组的人CD40-His抗原蛋白(ACRO，CD0-H5228)到ELISA板中，在4℃孵育过夜。
(2)次日，用PBST(含0.05％Tween-20)漂洗ELISA板5次。
(3)封闭：用封闭液(2％脱脂奶粉+1％BSA)在室温封闭1-2h。彻底弃掉封闭液。
(4)噬菌体孵育：每孔加入50μl单克隆的噬菌体，在室温孵育2h。用PBST漂洗5次。
(5)二抗孵育：每孔加入50μl HRP-抗-M13抗体(义翘神州，11973-MM05T-H)，在室温孵育1h。用PBST漂洗5次。
(6)显色：每孔加入50μl TMB显色液(Invitrogen，501129758)，在室温显色后，加50μl 2M HCl终止反应，在450nm读数。
实验结果如图36所示，P1和P8板中H1孔均为母本对照，挑取OD450值大于母本的单克隆(图36中的灰色孔)，进行菌落PCR，通过桑格测序，鉴定DNA序列，获得多个亲和力成熟的抗CD40Fab抗体，其中抗体4052.12、4052.17、4052.25、4052.28和4052.36的序列如图35和下文“序列”部分所示。
实施例15：双抗的质粒构建
将测序正确的阳性单克隆的DNA序列，亚克隆到pDA载体中，用于体外制备mRNA。
(1)CD40抗体母本40.52与CD28抗体(9.3h11)的VH和VL组成的双特异性抗体(简称双抗)的结构形式为：CD40抗体(VH)—(G4S)3连接子—CD40抗体(VH)—(G4S)连接子—CD28抗体(VH)—(G4S)3连接子—CD28 抗体(VL)。
(2)以阳性单克隆的菌液为模板，通过PCR的方式，扩增CD40亲和力成熟抗体的VH和VL片段，再通过Gibson assembly同源臂重组的连接方式，亚克隆到含CD28(VH-VL)片段的pDA载体中。通过桑格测序鉴定质粒的正确性。
(3)质粒线性化：测序正确的单克隆，通过质粒小量提取试剂盒(天根生化科技有限公司，DP103)抽提质粒。用SpeI内切酶在37℃切割4h或过夜，使质粒线性化。取2μl未酶切和酶切的质粒跑1％DNA核酸胶，检测酶切程度。用PCR产物回收试剂盒(Qiagen，28104)纯化酶切产物。
(4)体外mRNA制备：用mMESSAGE mMACHINETMT7转录试剂盒(Invitrogen,AM1344)在体外制备mRNA。再向此体系中加入1μl DNase I在37℃消化模板DNA 30min。
(5)用RNeasy Kit(Qiagen)纯化mRNA。
(6)用Nano-300测mRNA浓度后，将mRNA冻存于-80℃。
体外制备的mRNA用于后续的辅助功能杀伤实验，检测CD40亲和力成熟抗体的功能。
实施例16：辅助杀伤功能检测
对于通过噬菌体筛选得到的多个CD40亲和力成熟的候选抗体。先使用CD40亲和力成熟抗体与CD28抗体的VH和VL组成双抗(简称成熟双抗)，再通过双抗和Her2-CAR-T细胞(或Meso-CAR-T细胞)和靶细胞共培养的方式，检测由亲和力成熟分子组成的双抗的促杀伤功能。
1.通过Her2-CAR-T细胞检测成熟双抗的促杀伤功能，并与母本双抗4.6相比较。Her2-CAR-T细胞制备和双抗促杀伤功能的实验步骤如下：
(1)准备靶细胞：用BTX ECM 830电穿孔仪向A549-CBG细胞中电转10μg CD40 mRNA(简称A549-CD40)或不电转任何mRNA(简称A549-无EP)，电转条件为300V，0.5ms。24小时后，通过流式分析和PE-抗-CD40抗体，检测CD40的表达水平。图37A表明CD40在A549细胞表面的表达水平较高。
(2)准备CAR-T细胞：向CD3+T细胞中单电转或共电转1μg Her2-CAR(4D5.BBZ)mRNA、10μg BsAb mRNA或10μg新A40C28(即A40C2828)mRNA，电转条件为500V，0.7ms，作为Her2-CAR-T细胞。其中Her2-CAR(4D5.BBZ)与实施例7相同。24小时后，通过流式分析、CD40-Fc、Her2-Fc和PE-抗-Fc抗体检测双抗的表达水平和Her2-CAR的表达水平(图37B,C,D)。实验结果显示双抗和Her2-CAR的表达水平均较高。
(3)共培养：将Her2-CAR-T细胞和靶细胞A549-无EP或A549-CD40按照效应细胞(E):靶细胞(T)细胞数比例＝10:1、3:1或1:1共培养72小时， 并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，并绘制杀伤曲线。实验结果(图39A,B,C,D,E)显示有多个成熟双抗的促杀伤功能强于母本双抗，能使靶细胞的荧光值降至更低。同时5.12、5.17、5.25、5.28和5.36双抗分子的促杀伤功能不仅强于母本双抗4.6，也强于1412-4D11，和新A40C28(即A40C2828)的功能非常接近(图39F)。
(4)细胞因子释放：将Her2-CAR-T细胞和靶细胞A549-无EP或A549-CD40按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IL-2和IFN-γassay试剂盒来检测上清中IFN-gamma的释放水平。实验结果(图38)表明5.12、5.17、5.25、5.28和5.36双抗分子能促使更多的IL-2和IFN-gamma细胞因子释放，促释放能力强于母本双抗4.6和1412-4D11，但仍弱于新A40C28(即A40C2828)。
2.通过Mesothelin(简称Meso)-CAR-T细胞检测成熟双抗的促杀伤功能，并与母本双抗4.6相比较。Meso-CAR-T细胞制备和双抗促杀伤功能的实验步骤如下：
(1)准备靶细胞：向Molm14-CBG细胞中不电转任何mRNA(简称Molm14-无EP)、电转10μg Meso(间皮素)mRNA(简称Molm14-Meso)、电转10μg CD40mRNA(简称Molm14-CD40)或同时共电转10μg Meso和10μg CD40 mRNA(简称Molm14-Meso+CD40)，电转条件为400V，0.5ms。24小时后，通过流式分析、Meso-biotin和PE-streptavidin抗体、A40C-IgG1和PE-抗-Fc抗体检测Meso和CD40的表达水平。图40A表明CD40在A549细胞表面的表达水平较高。通过流式分析、Meso-biotin和PE-streptavidin抗体，检测Meso的表达水平。图40A表明Meso和CD40在Molm14细胞表面的表达水平较高，同时Meso高表达也促使CD40的表达上调。
(2)准备CAR-T细胞：向CD3+T细胞中单电转或共电转1μg Meso-CAR(M12)mRNA、10μg BsAb mRNA或10μg新A40C28(即A40C2828)mRNA(见表12)，电转条件为500V，0.7ms，作为Meso-CAR-T细胞。其中Meso-CAR(M12)与实施例7相同。24小时后，通过流式分析、CD40-Fc和PE-抗-Fc抗体检测双抗的表达水平；通过Meso-Fc和PE-抗-Fc抗体检测Meso-CAR的表达水平。实验结果(图40B)显示双抗和Meso-CAR的表达水平均较高。
(3)共培养：将Meso-CAR-T细胞和靶细胞Molm14-无EP、Molm14-Meso、Molm14-CD40或Molm14-Meso+CD40按照E:T＝10:1或3:1共培养72小时，并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，并绘制杀伤曲线。实验结果(图42A,B,C,D)表明，和母本双抗4.6以及双抗1412-4D11相比，5.12、5.17、5.25、5.28和5.36双抗的促杀伤功能更强，和Meso-CAR-T细胞共同作用后，能使靶细胞的荧光值降至更低。其中5.17双抗的促杀伤功非常接近新A40C28(即A40C2828)(图42B和D)。
(4)细胞因子释放：将Meso-CAR-T细胞和靶细胞Molm14-无EP、 Molm14-Meso、Molm14-CD40或Molm14-Meso+CD40按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IL-2 assay和human IFN-γassay来检测上清中IL-2和IFN-gamma的释放水平。实验结果(图41)表明5.12、5.17、5.25、5.28和5.36双抗的促释放均强于母本双抗4.6，比较接近双抗1412-4D11和新A40C28(即A40C2828)。
表12.Meso-CAR-T细胞mRNA共电转表
实施例17：双特异性抗体的特异性检测
通过将Meso-CAR-T细胞与不同靶细胞共培养的方式，观察靶细胞的杀伤曲线，检测共培养上清中细胞因子的释放情况。
(1)Meso-CAR-T细胞制备详见实施例16中的2(2)条。
(2)将Meso-CAR-T细胞与不同的靶细胞(Jeko-1、Raji、THP1、Nolm6、HCC70、H226、SKOV3、Caski、PC3和A549)按E:T＝10:1共培养，并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，并绘制杀伤曲线(图44A-F)。实验结果(图44A、B、C、D和E)表明，5.12、5.17、5.25、5.28和5.36双抗的特异性比较好，好于母本4.6、双抗1412-4D11和新A40C28(即A40C2828)。
(3)细胞因子释放：将Meso-CAR-T细胞和不同的靶细胞(Jeko-1、Raji、THP1、Nolm6、HCC70、H226、SKOV3、Caski、PC3和A549)按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IL-2assay和human IFN-γassay来检测上清中IL-2和IFN-gamma的释放水平。实验结果(图43)也表明5.12、5.17、5.25、5.28和5.36双抗的特异性比较好，好于母本4.6、双抗1412-4D11和新A40C28(即A40C2828)。
实施例18：抗体的表达与纯化
通过293F悬浮细胞在体外表达重组的抗体，并通过蛋白A介质纯化。
实验步骤如下：将抗体的V区插入到含IgG1框架的表达载体中。通过PEI将表达质粒瞬时转染到293F悬浮细胞中，培养4天后收集上清。向上清液中加入蛋白A来捕获抗体，用洗脱液洗脱后，获得纯化的抗体。通过测A280计算抗体的产量，通过SDS-PAGE电泳，检测抗体的纯度。结果如图45所示，抗体在非还原条件(图45-A)下条带单一，纯度好；在还原条件(图45-B)下，重链比较完整，轻链有些弥散降解。不同抗体的产量差异较大，如表13所示，亲和力成熟抗体4052.17的表达量最接近母本抗体40.52。
表13.抗体的产量
实施例19：ELISA(enzyme linked immunosorbent assay，酶联免疫吸附测定) 检测亲和力成熟抗体的结合活性
通过ELISA方法检测亲和力成熟抗体与抗原CD40的结合活性。实验步骤如下：
1.向ELISA板的每孔中加入50μL浓度为2μg/ml的重组的CD40-His蛋白，在4℃孵育过夜。
2.次日，用PBST(含0.05％Tween-20)洗涤ELISA板3次。
3.向ELISA板的每孔中加入250μl 5％脱脂奶粉(PBS溶解)，在室温下孵育2小时。
4.用PBST洗涤板3次，并在ELISA板中加入不同稀释比例的抗体，每孔50μL，在室温下孵育60分钟。
5.用PBST洗涤板3次，每孔加入50μL HRP-抗-hFc抗体，在室温下孵育60分钟。
6.用PBST洗涤板3次，每孔加入50μL TMB，室温显色后，每孔加50μL 2M HCl终止反应，并在450nm读数。
实验结果(表14和图46)显示，和母本40.52相比，只有1种亲和力成熟抗体4052.17的结合活性高于母本，其余亲和力成熟抗体的结合活性均不同程度的降低。
表14.亲和力成熟抗体ELISA结果汇总表
实施例20：SPR检测亲和力成熟抗体的亲和力
利用表面等离子共振(Surface Plasmon Resonance，SPR)技术来检测亲和力成熟抗体的亲和动力学。
实验步骤如下：用Biacore 2000(思拓凡,瑞典)通过SPR技术来检测抗体和CD40抗原之间的结合亲和力。所有实验都是在25℃条件下进行的。先用10mM Glycine-HCl(pH 1.5)平衡机器30秒，用蛋白A芯片以10μL/min PBST(pH 7.4，含0.05％吐温-20)的流速来捕获浓度为2μg/ml的40.52或4052.17抗体。再把以2倍梯度稀释的CD40(3.125–100nM)注射到流动相中。设置结合时间90秒，解离时间300秒。芯片表面用10mM Glycine-HCl(pH 1.5)孵育30秒再生来除去结合的CD40和抗体。用机器自带的Biacore Evaluation软件中的双相配体模型(heterogenerous ligand mode)来模拟抗体的亲和力。
实验结果(表15和图47)显示，亲和力成熟抗体4052.17的KD1和KD2相对于40.52均升高，说明其亲和力升高。
表15.亲和力成熟抗体的亲和力动力学检测结果汇总表
实施例21：慢病毒转导的CAR-T细胞的制备
用含CAR结构的慢病毒感染T细胞，制备成具有功能的CAR-T细胞。双抗5.17由亲和力成熟的CD40抗体4052.17和人源化的CD28抗体9.3h11组成。5.17和间皮素CAR(M12)组合成：M12-5.17和5.17-M12。
实验步骤如下：将含CAR结构(M12)、或含CAR和双抗(M12-5.17或5.17-M12)、或含CAR和LACO分子(A40C2828-M12)的慢病毒质粒和辅助质粒瞬时转染到293T细胞中进行病毒包装，收集上清，高速离心后浓缩病毒。对病毒滴度进行测定，并按照MOI＝3比例感染已被CD3/CD28 Dynabeads(Thermo, #11161D)激活的人T细胞。感染后第5天去磁珠，继续培养CAR-T细胞到第13天。检测CAR-T细胞的转导效率，冻存CAR-T细胞。
CAR-T细胞转导效率的检测如下：取5E6个CAR-T细胞到96孔板的每个孔中，用PBS洗1次，每孔加入50μl终浓度为2μg/ml Meso-Fc或CD40-Fc蛋白，在4℃孵育30min。用PBS洗细胞3次，每孔加入50μl终浓度为1μg/ml PE-抗-Fc抗体，在4℃孵育30min。用PBS细胞2次后，用流式细胞仪检测。
实验结果(图48)显示，M12-5.17的转导效率偏低，其他CAR的转导效率均在30％左右。CAR的转导效率和LACO分子A40C2828的转导效率比较接近。
实施例22：慢病毒转导的CAR-T细胞的辅助杀伤功能检测
将实施例21获得的慢病毒转导的CAR-T细胞与靶细胞共培养，观察靶细胞的杀伤曲线，检测共培养上清中细胞因子的释放情况。同时与M12，A40C2828-M12比较功能差异。
共培养：将慢病毒转导的CAR-T细胞和靶细胞SKOV3,SKOV3-Meso,SKOV3-Meso+CD40按照E:T＝10:1、3:1或1:1共培养72小时，并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，并绘制杀伤曲线。实验结果(图49)表明，和只有M12，A40C2828-M12相比，M12-5.17和5.17-M12的杀伤功能更强，能使靶细胞的荧光值降至更低。
细胞因子释放：将慢病毒转导的CAR-T细胞和靶细胞SKOV3,SKOV3-Meso,SKOV3-Meso+CD40按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IL-2和IFN-γassay试剂盒来检测上清中IFN-gamma的释放水平。实验结果(图50)表明和只有M12，A40C2828-M12相比，M12-5.17和5.17-M12未能释放更多的IL-2；但能释放更多的细胞因子IFN-gamma。
实施例23：慢病毒转导的CAR-T细胞的特异性检测
将实施例21获得的慢病毒转导的CAR-T细胞与不同靶细胞共培养，观察靶细胞的杀伤曲线，检测共培养上清中细胞因子的释放情况。
将慢病毒转导的CAR-T细胞与不同的靶细胞(SKOV3、ASPC1、H226、786-O、A549、OVCAR3、U251、Caski、SH-SY5Y、Siha、HepG2、THP-1、Raji、Nolm6、和Molm14)按E:T＝3:1共培养，并用incucyte S3活细胞动态成像和分析系统观察细胞杀伤，并绘制杀伤曲线。实验结果(图51)表明，M12-5.17的特异性好于5.17-M12。
(3)细胞因子释放：将慢病毒转导的CAR-T细胞和不同的靶细胞(SKOV3、 ASPC1、H226、786-O、A549、OVCAR3、U251、Caski、SH-SY5Y、Siha、HepG2、THP-1、Raji、Nolm6、和Molm14)按照E:T＝1:1共培养24小时后，离心取上清。通过Cisbio bioassays`human IL-2assay和human IFN-γassay来检测上清中IL-2和IFN-gamma的释放水平。实验结果(图52)也表明M12-5.17特异性好于5.17-M12。
实施例24：慢病毒转导的CAR-T细胞的体内药效
将实施例21获得的慢病毒转导的CAR-T细胞回输到携带肿瘤的NSG小鼠中，观察肿瘤变化，检测CAR-T细胞的体内药效。
实验步骤如下：
(1)荷瘤：向NSG小鼠的上背部皮下接种5E6个SKOV3-Meso+CD40-CBG细胞，让肿瘤生长11天。
(2)回输CAR-T细胞：向携带肿瘤的NSG小鼠分别回输低剂量(5E5个CAR-T细胞每只小鼠)和高剂量(3E6个CAR-T细胞每只小鼠)的CAR-T细胞。每隔7天，用游标卡尺测量肿瘤大小，称量小鼠体重，用小动物活体成像仪测量肿瘤的荧光值变化，记录小鼠死亡情况等。
实验结果(图53和图54)显示，低剂量CAR-T回输后，和对照组(UTD)，只有M12 CAR组，A40C2828-M12组相比，M12-5.17CAR-T细胞能使肿瘤的平均荧光值降低的更快(图53-A和B)，肿瘤大小缩小的更快(图53-C)，小鼠体重未有显著变化(图53-D)，无任何小鼠死亡(图53-E)。
高剂量CAR-T回输后，和对照组(UTD)、只有M12 CAR组相比，M12-5.17和A40C2828-M12组肿瘤大小和平均荧光值均快速降低(图53-A、B和C)；除了A40C2828-M12组小鼠体重显著下降，其他组未有显著变化(图53-D)；A40C2828-M12组小鼠生存曲线最差，M12-5.17组的生存曲线好于只有M12CAR组，弱于对照组(图53-E)。
以下实施例25-29涉及靶向间皮素(Meso)的抗体、CAR和CAR-T
实施例25：抗间皮素抗体的制备
按照以下步骤，使用全人抗体噬菌体展示库制备抗间皮素抗体：
(1)噬菌体展示文库的表达和纯化：用新鲜解冻的M13K07辅助噬菌体感染对数期TG1文库培养物，多重感染率为20:1(噬菌体与细胞比率)，并用IPTG诱导过夜；用PEG/NaCl沉淀法纯化噬菌体文库，然后测定噬菌体效价。噬菌体储存在4℃条件下，稍后进行scFv选择。
(2)间皮素特异性scFv-噬菌体的选择：在第一轮选择中，在Maxisorp平 板上包被溶解于1×PBS中的20μg/ml间皮素-6His蛋白，并在4℃下孵育过夜。(在随后的几轮选择中，较低的蛋白质浓度用于更严格的选择，包括第二轮生物筛选中的2μg/ml，第三轮生物筛选中的0.5μg/ml。)然后用PBS洗涤平板三次后，向每个孔中加入封闭缓冲液(在1×PBS中含有5％牛奶和1％BSA)。在室温下孵育2小时后，丢弃封闭缓冲液，加入噬菌体溶液，用保鲜膜密封平板，轻轻摇动孵育2小时。在第一轮选择中，然后用PBST洗涤平板10次。(在接下来的几轮中，通过增加更多的洗涤循环来提高洗涤的严格性：第二轮20次洗涤循环，第三轮30次洗涤循环)。然后使用1mL酸洗脱缓冲液(pH 2.2)洗脱抗原结合scFv-噬菌体，中和，接种到15mL对数期TG1培养物(OD600＝0.5)中，在37℃下静置30min并摇动30min培养，接种于2xYT-GA琼脂板上，并在30℃下培养过夜以供后续选择。
(3)mpELISA筛选：经过三轮筛选后，选择288个阳性克隆用于单克隆噬菌体ELISA(mpELISA)筛选。从单个菌落克隆产生噬菌体上清液，并测试其与间皮素-Fc蛋白的结合。所述上清液与涂有2μg/ml间皮素-6His蛋白的预封闭Maxisorp平板一起孵育。三次洗涤后，加入100μl/孔HRP缀合抗M13抗体，所述HRP缀合抗M13抗体在封闭缓冲液(1×PBS中含有5％牛奶和1％BSA)中经1：5000稀释，然后在常温下孵育60min。用PBST洗涤平板5次后，添加100μl/孔TMB基质溶液，并孵育10-30分钟直到出现蓝色。通过添加50μl/孔的终止溶液(2N H2SO4)停止反应。在微孔板读取器中，在450nm处读取吸光度。图55显示了mpELISA筛选的所得吸光度读数。如图所示，鉴定出阳性菌落(450nm吸光度≥0.5)以产生能够结合间皮素-6His蛋白的抗间皮素抗体(图55)。
(4)克隆和序列分析：根据ELISA结果选择阳性克隆，并用作scFv序列的PCR克隆模板(正向引物序列如SEQ ID NO:659：tgcagctggcacgacaggtttc，反向引物序列如SEQ ID NO:660：cgtcagactgtagcacgtt)。然后通过sanger测序法对PCR产物进行测序(正向引物序列如SEQ ID NO:661：aacaattgaattcaggagga，反向引物序列如SEQ ID NO:662：cctcctaagaagcgtagtc)。通过abysis网站分析scFv的CDR区域(http://abysis.org/)，编号规则采用Kabat、Chothia、AbM、Contact或IMGT系统，见表16。
表16：CDR及其序列号(SEQ ID NO)。


实施例26：间皮素CAR的制备
构建用于产生抗间皮素CAR mRNA的载体。首先将pDA载体用Xba1和Sal1酶消化，通过凝胶纯化的方法进行纯化。将上述实施例25中获得的scFv片段和CAR片段(CD8铰链、CD8跨膜结构域、4-1BB共刺激结构域、CD3-zeta信号传导结构域)通过PCR进行扩增并通过凝胶纯化的方法进行纯化。将scFv片段、CAR片段(从铰链结构域至CD3-zeta结构域)和pDA载体通过Gibson组装方法连接，并转化到感受态细胞中。通过Sanger测序鉴定具有正确构建体的菌落，并选择用于进一步实验。图56提供了用于产生CAR mRNA的pDA-CAR载体的示意图。
然后，用Spe1酶切使pDA-CAR质粒线性化。使用PCR Cleanup试剂盒纯化线性化载体，并用无核糖核酸酶的水洗脱。通过nanodrop测定DNA的浓度，并通过琼脂糖DNA凝胶进行检验。然后，按照生产商(Thermofisher，目录号：AM13455)的实验方案进行体外转录(IVT)。简言之，将含有1μg模板DNA、NTP/ARCA缓冲液、T7缓冲液、GTP、T7酶和无RNase H2O的20μl体系加入到0.2mLPCR管中，并在37℃下孵育3小时。3小时后，每个反应加入2μl DNase，37℃孵育15min。根据生产商的建议进行结尾步骤。使用RNasy试剂盒(Qiagen) 纯化IVT mRNA，通过nanodrop测定RNA的浓度，并通过PAGE凝胶进行检验。
实施例27：肿瘤细胞系和人原代淋巴细胞
在添加有10％FCS的RPMI-1640培养基中培养肿瘤细胞系，包括A549-CBG(人肺癌(癌症)细胞)、H226-CBG(人肺癌(恶性上皮肿瘤)细胞)、MOLM14-CBG(人白血病细胞)、ASPC1-CBG(人胰腺肿瘤细胞)、HCC70-CBG(人乳腺癌细胞)和OVCAR3-CBG(人卵巢癌细胞)。用抗CD3/CD28免疫磁珠(Life Technologies)刺激来自正常供体的原代淋巴细胞，并将所述细胞在R10培养基(添加有10％FCS、青霉素-链霉素(100x)、HEPES(100x)、丙酮酸钠(100x)、Glutamax(100x)、NEAA(100x)的RPMI-1640)中培养。刺激后第10天，将T细胞以1×108个细胞/瓶在90％FCS和10％DMSO的溶液中冷冻保存。
实施例28：间皮素CART的制备和表征
通过电穿孔法将前述实施例26获得的间皮素CAR mRNA引入A549肿瘤细胞和T细胞中，步骤如下：收集A549肿瘤细胞和T细胞，用Opti-MEM培养基洗涤3次。用Opti-MEM培养基重悬细胞沉淀，并将细胞浓度调节至1×10e7个/ml。将10μg RNA等量分装至1.5mL EP管中，加入100μl T细胞或A549细胞，混匀。将100μl与RNA混合的细胞加入BTX电穿孔杯中，轻敲以避免气泡。使用BTX仪器在以下参数下进行电穿孔：对于T细胞：500V电压，0.7ms；对于A549肿瘤细胞：300V电压，0.5ms。然后将细胞转移至预热的培养基中，并在37℃下培养。
通过FACS染色测定间皮素CART细胞与间皮素-Fc重组蛋白的结合。如图57所示，抗间皮素scFv-M1、-M2、-M3、-M6、-M7、-M8、-M9、-M10、-M11、-M12、-M13、-M14、-M15、-M16、-M17、-M20、-M22、-M23、-M24、-M25、-M27、-M28、-M29、-M30、-M31、-M32、-M33、-M34、-M35、-M36和-M37结合至间皮素-Fc重组蛋白。不含CAR分子的T细胞作为对照(“Mock”)。如图58所示，间皮素的异位表达水平与通过电穿孔引入A549细胞中的间皮素mRNA的量相关。
在体外细胞毒性试验中测定了间皮素CART细胞对肿瘤细胞的细胞毒性。将用不同量肿瘤抗原电穿孔的表达EGFP的肿瘤细胞系或EGFP-A549细胞以3000个细胞/100μl/孔接种于平底96孔板上。将CART细胞稀释至适当浓度，以100μl/孔接种，与肿瘤细胞按不同的E/T比例，如10:1、3:1、1:1，共培养板置于IncuCyte S3仪器中，设置扫描参数。扫描3天后，分析绿色图像总积分强度 (GCU xμm2/孔)，计算杀伤效率。
A549细胞低水平表达间皮素。如图59所示，表达抗间皮素scFv-M4、-M22、-M28和-M31的CART细胞有效地阻碍了A549细胞的生长，表明这些基于scFv的CART细胞对肿瘤细胞具有相当高的细胞毒性。如图60所示，表达抗间皮素scFv-M4、-M6、-M7、-M8、-M9、-M10、-M11、-M12、-M13、-M15、-M20、-M22、-M23、-M24、-M27、-M28、-M31、-M32、-M35、-M37和-M38CAR的CART细胞对间皮素过表达的A549肿瘤细胞(用10μg间皮素mRNA电穿孔)显示出有效的杀伤作用。如图61所示，表达抗间皮素scFv-M4、-M6、-M13、-M20、-M27、-M31和-M37CAR的CART细胞对间皮素的异位表达较少的A549肿瘤细胞(用2μg间皮素mRNA电穿孔)保持强杀伤作用。表达抗间皮素scFv-M7、-M8、-M9、-M10、-M11、-M12、-M15、-M23、-M24、-M32、-M35和-M38的CART细胞选择性地仅对高表达间皮素的肿瘤细胞表现出高细胞毒性，对低表达间皮素的肿瘤细胞不是如此，而具有优越的安全性，因为间皮素还在某些正常组织中表达。
图62显示了不同基于mRNA的抗间皮素CART细胞的杀伤曲线，以用0(上图)、2μg(中图)或10μg(下图)间皮素mRNA电穿孔的A549-GFP肿瘤细胞为靶细胞(E/T比＝10:1)。如图所示，抗间皮素scFv-M12、-M24和-M32CART细胞对低表达间皮素的A549肿瘤细胞(2μg组)具有温和杀伤作用，但对高表达间皮素的A549肿瘤细胞(10μg组)具有较强的杀伤作用。结果表明，这些CART细胞会特异性地靶向高表达间皮素的肿瘤细胞，且不会杀伤低表达间皮素的正常组织。
图63显示了同种型对照和抗间皮素mAb对OVCAR3(人卵巢癌细胞)、H226(人肺癌(恶性上皮肿瘤)细胞)、ASPC1(人胰腺肿瘤细胞)、A549(人肺癌(癌症)细胞)和HCC70(人乳腺癌细胞)的FACS染色。如图所示，某些癌细胞，包括OVCAR3、H226和ASPC1，高水平表达间皮素；A549低水平表达间皮素，HCC70不表达间皮素。
实施例29：表达间皮素的癌细胞特异性激活CART细胞
CD107a是T细胞的早期激活标志物。通过CD107a染色测定表达间皮素的肿瘤细胞对间皮素CART的激活，步骤如下：向96孔板的每个孔中加入20μl PE-CD107a mAb；将肿瘤细胞稀释至2×10e6个/ml，接种于96孔圆形平板上(100μl/孔)；将前述实施例28获得的CAR-T细胞稀释至1×10e6个/ml，接种于96孔圆形平板上(100μl/孔)，500rpm×5min离心平板以使细胞贴壁，37℃培养1小时，将GolgiStop用培养基1500倍稀释后加入各孔中(20μl/孔)；37℃再培养细胞2.5小时，在37℃下用抗CD3-APC和抗CD8-FITC抗体染色30min，洗涤并通过流式细胞术进行分析。
图64显示了与OVCAR3、H226、ASPC1、A549和HCC70共培养和杀伤试验中抗间皮素M12和M32 CAR-T细胞的CD107a染色。这些数据表明，抗间皮素M12和M32 CART细胞被OVCAR3、H226和ASPC1(高间皮素表达水平的肿瘤细胞)特异性激活，但不被A549和HCC70(低间皮素表达或无表达的肿瘤细胞系)激活。
以下实施例30-33涉及靶向BCMA的抗体、CAR和CAR-T
实施例30：抗BCMA抗体的制备
按照以下步骤，使用全人抗体噬菌体展示文库制备抗BCMA抗体：
(1)噬菌体展示文库的表达和纯化：用新鲜解冻的M13K07辅助噬菌体感染对数期TG1文库培养物，多重感染率为20:1(噬菌体与细胞比率)，并用IPTG诱导过夜；用PEG/NaCl沉淀法纯化噬菌体文库，然后测定噬菌体效价。噬菌体储存在4℃条件下，稍后进行scFv选择。
(2)BCMA特异性scFv-噬菌体的选择：在第一轮选择中，在Maxisorp平板上包被溶解于1×PBS中的20μg/ml BCMA-6His蛋白，并在4℃下孵育过夜。(在随后的几轮选择中，使用较低的蛋白质浓度来进行更严格的选择，包括第二轮生物筛选中的2μg/ml，第三轮生物筛选中的0.5μg/ml。)然后用PBS洗涤平板三次后，向每个孔中加入封闭缓冲液(在1×PBS中含有5％牛奶和1％BSA)。在室温下孵育2小时后，丢弃封闭缓冲液，加入噬菌体溶液，用保鲜膜密封平板，轻轻摇动孵育2小时。在第一轮选择中，然后用PBST洗涤平板10次。(在接下来的几轮中，通过增加更多的洗涤循环来提高洗涤的严格性：第二轮20次洗涤循环，第三轮30次洗涤循环)。然后使用1mL酸洗脱缓冲液(pH 2.2)洗脱抗原结合scFv-噬菌体，中和，接种到15mL对数期TG1培养物(OD600＝0.5)中，在37℃下静置30min并摇动30min培养，接种于2xYT-GA琼脂板上，并在30℃下培养过夜以供后续选择。
(3)mpELISA筛选：经过三轮筛选后，选择480个噬菌体感染的菌落克隆用于单克隆噬菌体ELISA(mpELISA)筛选。从单个菌落克隆产生噬菌体上清液，并测试其与BCMA-Fc蛋白的结合。所述上清液与涂有2μg/ml BCMA-6His蛋白的预封闭Maxisorp平板一起孵育。三次洗涤后，加入100μl/孔的HRP缀合抗M13抗体，所述HRP缀合抗M13抗体在封闭缓冲液(1×PBS中含有5％牛奶和1％BSA)中经1：5000稀释，然后在常温下孵育60min。用PBST洗涤平板5次后，添加100μl/孔的TMB基质溶液，并孵育10-30分钟直到出现蓝色。通过添加50μl/孔的终止溶液(2N H2SO4)停止反应。
在微孔板读取器中，在450nm处读取吸光度。下表17提供了三种具有代表性的抗人BCMA-Fc单克隆噬菌体ELISA 96孔板的读数。带有灰色高光的克隆为阳性克隆。在噬菌体ELISA试验中，共选择36个阳性克隆，通过 PCR扩增scFv片段并进行测序。
表17：
平板-1
平板-2
平板-3
(4)克隆和序列分析：根据ELISA结果选择阳性克隆，并用作scFv序列的PCR克隆模板(正向引物序列如SEQ ID NO:659：tgcagctggcacgacaggtttc，反向引物序列如SEQ ID NO:660：cgtcagactgtagcacgtt)。然后通过sanger测序法对PCR产物进行测序(正向引物序列如SEQ ID NO:661：aacaattgaattcaggagga，反向引物序列如SEQ ID NO:662：cctcctaagaagcgtagtc)。通过abysis网站分析scFv的CDR区域(http://abysis.org/)，编号规则采用Kabat、Chothia、AbM、Contact或IMGT系统，见表18。
表18：CDR及其序列号(SEQ ID No)。


(5)T细胞中功能性抗BCMA scFv的筛选：将抗BCMA scFv构建到双顺反子慢病毒CAR表达载体中，该载体含有IRES截断的EGFR(tEGFR)表达盒。通过瞬时转染293T细胞产生慢病毒，然后通过超速离心纯化和浓缩。用CAR慢病毒转导T细胞以生成CAR-T细胞，然后再培养10天。慢病毒转导10天后，收集CAR-T细胞，并在4℃下用5μg/ml CD19-Fc蛋白(Ctrl Fc蛋白)或BCMA-Fc重组蛋白染色30分钟。洗涤后，用抗人IgG Fc和抗EGFR mAb对CAR-T细胞进行染色。采用流式细胞仪对样品进行分析。如图65A和图65B所示，表达CAR(含有以下抗BCMA scFv)的T细胞显示出与BCMA-Fc的结合(图65B)，并被选作进一步研究：BCMA21、BCMA22、BCMA23、BCMA24、BCMA27、BCMA28、BCMA30、BCMA31、BCMA32、BCMA33、BCMA34和BCMA35。
实施例31：BCMA-CART的制备与表征
我们使用上述抗BCMA scFv构建了12种不同的抗BCMA CAR。对3种其他CART产物进行平行测试，包括NBC10(Novartis AG and University of Pennsylvania,BMCA10.BBz)、FHVH33(National Institutes of Health,US)，和B38M(南京传奇生物科技)。所有测试的CAR都有41BBz共激活结构域。
表19：BCMA CART、CAR％和表达水平

通过慢病毒载体转导T细胞，以表达不同的BCMA CAR。上表19显示了本发明公开的研究中使用的CART细胞、表达CAR细胞的百分比及其各自的表达水平。图66A和66B分别显示了CAR+T细胞的频率及其表达水平(“MFI”为平均荧光强度)。在本发明产生的12种scFv中，BCMA31(#10；SEQ ID NO:342)和BCMA33(#12；SEQ ID NO:344)的表达水平高于其他scFv。图67显示了在测试CART中，CAR+CD8细胞的可比频率(comparable frequencies)。图68显示了CART细胞的表型。在BCMA27(#7)、BCMA31(#10)和BCMA33(#12)T细胞中幼稚T细胞群(CD45RO-；CCR7+)的频率高于在其他样本中的频率，表明这些T细胞分化程度较低。
实施例32：BCMA在肿瘤细胞中的表达
如图69A和69B所示，通过FACS染色(图69A)和RT-PCR(图69B)对不同肿瘤细胞系的BCMA表达进行检测。通过FACS染色检测BCMA在Jeko-1(低水平)、Raji(中等水平)和RPMI-8226细胞(高水平)中的表达。虽然通过FACS未检测到BCMA在Nalm6中的表达，但RT-PCR分析显示BCMA在Nalm6中有表达，尽管表达水平很低。
实施例33：BCMA CART对肿瘤细胞具有细胞毒性
将前述实施例31获得的CART细胞与Jeko-1细胞和RPMI-8226肿瘤细胞共培养。检测INF-γ和IL-2的产生。如图70A(INF-γ)和70B(IL-2)所示， 在本发明产生的12种CART中，BCMA23(#5；SEQ ID NO:337)、BCMA24(#6；SEQ ID NO:338)、BCMA27(#7；SEQ ID NO:339)、BCMA31(#10；SEQ ID NO:342)和BCMA33(#12；SEQ ID NO:344)，相比于其他CART细胞(包括NBC10和B38M CAR T细胞)，能够产生更多细胞因子。
我们还分别检测了这些CART细胞对Jeko-1(图71A-71D)和RPMI-8226细胞(图72A-72E)的细胞溶解活性。BCMA23(#5)、BCMA24(#6)、BCMA31(#10)和BCMA33(#12)CART细胞对Jeko-1细胞表现出不同程度的细胞毒性，其中BCMA31(#10)细胞毒性最高，可有效消除Jeko-1。此外，BCMA21(#3；SEQ ID NO：335)、BCMA22(#4；SEQ ID NO：336)、BCMA23(#5；SEQ ID NO：337)、BCMA24(#6；SEQ ID NO：338)、BCMA27(#7；SEQ ID NO：339)、BCMA31(#10；SEQ ID NO：342)、BCMA33(#12；SEQ ID NO：344)、BCMA34(#13；SEQ ID NO：345)和BCMA35(#14；SEQ ID NO：346)对RPMI-8226细胞表现出不同水平的细胞毒性，其中BCMA21(#3)、BCMA23(#5)、BCMA24(#6)、BCMA27(#7)、BCMA31(#10)和BCMA33(#12)有效消除了RPMI-8226细胞。
以下实施例34-37涉及靶向CD123的抗体、CAR和CAR-T
实施例34：人抗CD123抗体的制备
采取以下步骤制备全人抗CD123抗体:
1)噬菌体展示文库的表达和纯化：用新鲜解冻的M13K07辅助噬菌体感染对数期TG1文库培养物，多重感染率为20：1(噬菌体与细胞之比)，并用IPTG诱导过夜；用PEG/NaCl沉淀法纯化噬菌体文库，然后测定噬菌体效价。
2)CD123特异性scFv-噬菌体的选择：在第一轮选择中，在Maxisorp平板上在4℃下用20μg/ml的CD123-6His蛋白包被过夜，用PBS洗涤，用5％牛奶+1％BSA在1×PBS中封闭，在噬菌体溶液中孵育2小时，用PBST清洗10次。使用酸洗脱缓冲液(pH2.2)洗脱与抗原结合的scFv噬菌体，中和，接种于15毫升的对数期的TG1培养(OD600＝0.5)中，在37℃下静置30分钟并摇动30分钟培养，接种到2xYT-GA琼脂平板上，并在30℃下培养过夜以供后续选择。在随后的几轮选择中，使用了更严格的选择条件，包括降低包被的蛋白质浓度(第二轮为2μg/ml，第三轮为0.5μg/ml)和增加洗涤周期(第二轮为20次，第三轮为30次)。
3)mpELISA筛选。经过三轮筛选后，选出292个阳性菌落进行单克隆噬菌体ELISA(mpELISA)筛选。将单个菌落克隆生成的噬菌体上清液与包被有2μg/mL CD123-6His蛋白的预封闭Maxisorp板共同孵育。洗涤三次后，加入100μl/ 孔的HRP缀合抗M13抗体，所述HRP缀合抗M13抗体在封闭缓冲液(5％牛奶+1％BSA，1×PBS)中经1:5000的比例稀释，然后在常温下孵育60分钟。用PBST洗板5次后，添加100μl/孔的TMB底物溶液，并孵育10-30min，直至出现蓝色。通过添加50μl/孔的终止溶液(2N H2SO4)来终止反应。在微孔板读取器中读取450nm处的吸光度。图73显示了mpELISA筛选所得的吸光度读数。如图所示，一些阳性菌落(450nm吸光度≥0.5)被鉴定为产生了能够结合CD123-6His蛋白的抗CD123抗体(图73)。
4)克隆和序列分析：根据ELISA结果选择阳性克隆，并用作scFv序列的PCR克隆模板(正向引物序列如SEQ ID NO:659：tgcagctggcacgacaggtttc，反向引物序列如SEQ ID NO:660：cgtcagactgtagcacgtt)。然后通过sanger测序法对PCR产物进行测序(正向引物序列如SEQ ID NO:661：aacaattgaattcaggagga，反向引物序列如SEQ ID NO:662：cctcctaagaagcgtagtc)。通过abysis网站分析scFv的CDR区域(http://abysis.org/)，编号规则采用Kabat、Chothia、AbM、Contact或IMGT系统，见表20。
表20：CDR及其序列号(SEQ ID No)。




实施例35：CD123 CAR的制备
构建用于产生抗CD123 CAR的mRNA的载体。首先，通过PCR扩增前述实施例34中获得的scFv序列和CAR片段(从铰链结构域到CD3-zeta结构域)并克隆到pDA载体(Xba1/Sal1)。图74提供了用于生成CAR mRNA的pDA-CAR载体的示意图。其次，通过体外转录(IVT)制备CD123 CAR mRNA。pDA-CAR质粒通过Spe1酶切线性化，并通过PCR Cleanup试剂盒进行纯化。用nanodrop测量DNA浓度并通过运行琼脂糖DNA凝胶检查后，按照制造商的操作指南(Thermofisher,Cat No:AM13455)进行IVT。通过nanodrop测定RNA产物的浓度，并运行PAGE凝胶进行检查。
实施例36：CD123 CARTs的制备和表征
通过以下步骤使用电穿孔将前述实施例35中获得的CD123 CAR mRNA引入T细胞:收集T细胞，用Opti-MEM培养基清洗，并用Opti-MEM培养基以1×10e7/ml重悬；将10μg RNA与100μl T细胞等分，混合均匀进行电穿孔，参数如下(BTX机)：500V，0.7ms；然后将细胞转移到预热的培养基上，在37℃下培养。
通过FACS染色测量CD123 CART细胞与CD123-Fc重组蛋白的结合。如图75所示，表达具有抗CD123 scFv-C1,-C2，-C3，-C4，-C5，-C6，-C7，-C9，-C10，-C11，-C13，-C14，-C15，-C16，-C17，-C18，-C19、-C21、-C23、-C24、-C25、-C26、-C27、-C28、-C29、-C30、-C32、-C33、-C34和-C35的CAR的T细胞能够结合CD123-Fc重组蛋白。空载体(Mock)是没有CAR分子的对照T细胞。
用不同量的CD123 mRNA电穿孔A549肿瘤细胞。电穿孔过程与上述过程相同，仅改变了参数，使用的参数为300V，0.5ms。通过对同种型或抗CD123抗体、不同量CD123 mRNA电穿孔的A549细胞进行FACS染色，以测量A549肿瘤细胞中CD123的表达。如图76所示，A549细胞弱表达内源性CD123，并且CD123的异位表达水平与电穿孔到A549细胞中的CD123 mRNA的量相关。
在体外细胞毒性试验中测量了CD123 CART细胞对肿瘤细胞的细胞毒性。将表达EGFP的肿瘤细胞或用不同量肿瘤抗原电穿孔的EGFP-A549细胞以3000细胞/100ul/孔接种在平底96孔板上；将CART细胞稀释至适当浓度，并以不同的E/T比值(例如，10:1，3:1，1:1)接种于100μl/孔肿瘤细胞，然后将共培养板放入IncuCyte S3机器中，并设置扫描参数。扫描3天后，分析总绿色物体积分强度(GCU xμm2/孔)以计算杀灭效率。
图77和图78显示了不同的基于mRNA的抗CD123 CART细胞在E/T比值为10:1(图77)或3:1(图78)时对A549-GFP肿瘤细胞的杀伤曲线。如图所示，表达抗CD123 scFv-C2，-C3，-C4，-C6，-C9，-C11，-C13，-C14，-C15，-C16，-C17，-C19，-C21，-C23，-C24和-C32的CART细胞有效地阻止了A549 细胞生长，甚至消除了A549细胞，尽管A549细胞表达低水平的内源性CD123，这表明这些基于scFv的CART细胞对肿瘤细胞具有较高的细胞毒性。
图79和图80显示了不同的基于mRNA的抗CD123 CART细胞在对E/T比值为10:1(图79)或3:1(图80)时对表达外源性CD123(用10μg CD123 mRNA电穿孔)的A549-GFP肿瘤细胞的杀伤曲线。如图79、80所示，表达抗CD123scFv-C1，-C2，-C3，-C4，-C5，-C6，-C7，-C9，-C11，-C13，-C14，-C15，-C16，-C17，-C18，-C19，-C21，-C23，-C24，-C25，-C26，-C27，-C28，-C29，-C30，-C32，-C33，-C34和-C35的CART细胞有效地阻止了表达CD123的A549肿瘤细胞的生长，甚至减少了表达CD123的A549肿瘤细胞的数量，证实了它们杀死表达CD123的肿瘤细胞的能力。
实施例37：CART细胞CD107a染色
通过FACS染色测量不同肿瘤细胞系的CD123表达。图81显示了A549、SK-OV3、Jeko-1、Molm-14、SupT-1、293T、Nalm-6和PC-3细胞与PE-同种型对照和PE-抗-CD123 mAb的FACS染色结果。如图81所示，大多数测试的肿瘤细胞系不表达CD123，仅Molm-14表达相对高水平的CD123。
CD107a是T细胞的早期活化标记。通过以下步骤使用CD107a染色测量表达CD123的肿瘤细胞对CD123 CARTs的活化：将20μl PE-CD107a mAb添加到96孔板的每个孔中；将肿瘤细胞稀释至2×10e6/ml，并接种在96孔圆板上(100μl/孔)；将前述实施例36获得的CART细胞稀释至1×10e6/ml，接种于96孔圆板(100μl/孔)中；将平板以500rpm×5分钟离心，使细胞附着良好，并在37℃下培养1小时；将Golgi stop用培养基稀释1500倍后，添加到每个孔(20μl/孔)中；细胞在37℃下再培养2.5小时，用抗CD3-APC和抗CD8-FITC抗体在37℃下染色30分钟，洗净后用流式细胞仪进行分析。
在我们的研究中，通过CD107a染色测量了表达CD123的肿瘤细胞对CARTs(表达抗CD123-C5，抗CD123-C7，抗CD123-C11)的激活。测试的细胞包括用10μg，0.1μg和0μg CD123 mRNA电穿孔的A549，SK-OV3，PC-3，脐带血来源的CD34造血干细胞(CD34+cord)，骨髓来源的造血干细胞(CD34+M)，Molm-14，Nalm6，Jeko-1肿瘤细胞系和新鲜分离的患者AML肿瘤细胞(CD123+)。如图82所示，表达抗CD123-C5、-C7和-C11的CART细胞被具有相对高CD123表达的肿瘤细胞，尤其是CD123+AML肿瘤细胞特异性活化，但不被具有低CD123表达的肿瘤细胞系特异性活化。这些结果表明，表达CD123的肿瘤细胞可以活化CD123 CARTs。
上述实施例25-37中涉及的抗体VH、VL、scFv和CAR序列对应的序列号(SEQ ID NO)如下表21所示。
表21：

参考文献
1.Hillion,S.,et al.,The Innate Part of the Adaptive Immune System.Clin Rev Allergy Immunol,2020.58(2):p.151-154.
2.Bajorath,J.,R.J.Peach,and P.S.Linsley,Immunoglobulin fold characteristics of B7-1(CD80)and B7-2(CD86).Protein Sci,1994.3(11):p.2148-50.
3.Tacke,M.,et al.,CD28-mediated induction of proliferation in resting T cells in vitro and in vivo without engagement of the T cell receptor:evidence for functionally distinct forms of CD28.Eur J Immunol,1997.27(1):p.239-47.
4.Hansen,J.A.,P.J.Martin,and R.C.Nowinski,Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes.Immunogenetics,1980.10(1):p.247-260.
5.Tan,P.,et al.,"Superhumanized"antibodies:reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences:application to an anti-CD28.J Immunol,2002.169(2):p.1119-25.
6.Edner,N.M.,et al.,Targeting co-stimulatory molecules in autoimmune disease.Nat Rev Drug Discov,2020.19(12):p.860-883.
7.Joyce,J.A.and D.T.Fearon,T cell exclusion,immune privilege,and the tumor microenvironment.Science,2015.348(6230):p.74-80.
8.Terrazzano,G.,et al.,Interaction between natural killer and dendritic cells:the role of CD40,CD80and major histocompatibility complex class i molecules in cytotoxicity induction and interferon-gamma production.Scand J Immunol,2004.59(4):p.356-62.
序列：
抗CD28抗体9.3重链可变区(mVH)
HCDR1：DYGVH(SEQ ID NO:1)
HCDR2：VIWAGGGTNYNSALMS(SEQ ID NO:2)
HCDR3：DKGYSYYYSMDY(SEQ ID NO:3)
HFR1：QVKLQQSGPGLVTPSQSLSITCTVSGFSLS(SEQ ID NO:4)
HFR2：WVRQSPGQGLEWLG(SEQ ID NO:5)
HFR3：RKSISKDNSKSQVFLKMNSLQADDTAVYYCAR(SEQ ID NO:6)
HFR4：WGQGTTVTVSS(SEQ ID NO:7)
VH：
抗CD28抗体9.3轻链可变区(mVL)
LCDR1：RASESVEYYVTSLMQ(SEQ ID NO:9)
LCDR2：AASNVES(SEQ ID NO:10)
LCDR3：QQSRKVPYT(SEQ ID NO:11)
LFR1：DIVLTQSPASLAVSLGQRATISC(SEQ ID NO:12)
LFR2：WYQQKPGQPPKLLIF(SEQ ID NO:13)
LFR3：GVPARFSGSGSGTNFSLNIHPVDEDDVAMYFC(SEQ ID NO:14)
LFR4：FGGGTKLEIK(SEQ ID NO:15)
VL：
人源化抗CD28抗体重链可变区1(huVH1)
HCDR1：DYGVH(SEQ ID NO:1)
HCDR2：VIWAGGGTNYNSALMS(SEQ ID NO:2)
HCDR3：DKGYSYYYSMDY(SEQ ID NO:3)
HFR1：QVKLQESGPGLVKPSETLSITCTVSGFSLS(SEQ ID NO:19)
HFR2：WVRQSPGKGLEWLG(SEQ ID NO:23)
HFR3：RKTISKDNSKSQVFLKMSSLTAADTAVYYCAR(SEQ ID NO:27)
HFR4：WGQGTTVTVSS(SEQ ID NO:7)
VH：
人源化抗CD28抗体重链可变区2(huVH2)
HCDR1：DYGVH(SEQ ID NO:1)
HCDR2：VIWAGGGTNYNSALMS(SEQ ID NO:2)
HCDR3：DKGYSYYYSMDY(SEQ ID NO:3)
HFR1：QVQLQESGPGLVKPSETLSITCTVSGFSLS(SEQ ID NO:20)
HFR2：WVRQPPGKGLEWLG(SEQ ID NO:24)
HFR3：RKTISKDTSKNQVSLKMSSLTAADTAVYYCAR(SEQ ID NO:28)
HFR4：WGQGTTVTVSS(SEQ ID NO:7)
VH：
人源化抗CD28抗体重链可变区3(huVH3)
HCDR1：DYGVH(SEQ ID NO:1)
HCDR2：VIWAGGGTNYNSALKS(SEQ ID NO:17)
HCDR3：DKGYSYYYSMDY(SEQ ID NO:3)
HFR1：QVQLQESGPGLVKPSETLSLTCTVSGFSLS(SEQ ID NO:21)
HFR2：WVRQSPGKGLEWIG(SEQ ID NO:25)
HFR3：RKTISKDNSKSQVSLKLSSVTAADTAVYYCAR(SEQ ID NO:29)
HFR4：WGQGTTVTVSS(SEQ ID NO:7)
VH：
人源化抗CD28抗体重链可变区4(huVH4)
HCDR1：DYGVH(SEQ ID NO:1)
HCDR2：VIWAGGGTNYNSALKS(SEQ ID NO:17)
HCDR3：DKGYSYYYSMDY(SEQ ID NO:3)
HFR1：QVQLQESGPGLVKPSETLSLTCTVSGFSLS(SEQ ID NO:21)
HFR2：WIRQPPGKGLEWIG(SEQ ID NO:26)
HFR3：RVTISVDTSKNQVSLKLSSVTAADTAVYYCAR(SEQ ID NO:30)
HFR4：WGQGTTVTVSS(SEQ ID NO:7)
VH：
人源化抗CD28抗体重链可变区5(huVH5)
HCDR1：DYGVH(SEQ ID NO:1)
HCDR2：VIWAGGGTNYNPSLKS(SEQ ID NO:18)
HCDR3：DKGYSYYYSMDY(SEQ ID NO:3)
HFR1：QVQLQESGPGLVKPSETLSLTCTVSGFSIS(SEQ ID NO:22)
HFR2：WIRQPPGKGLEWIG(SEQ ID NO:26)
HFR3：RVTISVDTSKNQVSLKLSSVTAADTAVYYCAR(SEQ ID NO:30)
HFR4：WGQGTTVTVSS(SEQ ID NO:7)
VH：
人源化抗CD28抗体轻链可变区1(huVL1)
LCDR1：RASESVEYYVTSLMQ(SEQ ID NO:9)
LCDR2：AASNVES(SEQ ID NO:10)
LCDR3：QQSRKVPYT(SEQ ID NO:11)
LFR1：DIQLTQSPSSLSVSVGDRVTISC(SEQ ID NO:36)
LFR2：WYQQKPGQAPKLLIF(SEQ ID NO:38)
LFR3：GVPSRFSGSGSGTNFTLTISSVQEEDFAMYFC(SEQ ID NO:40)
LFR4：FGGGTKLEIK(SEQ ID NO:15)
VL：
人源化抗CD28抗体轻链可变区2(huVL2)
LCDR1：RASESVEYYVTSLMQ(SEQ ID NO:9)
LCDR2：AASNVES(SEQ ID NO:10)
LCDR3：QQSRKVPYT(SEQ ID NO:11)
LFR1：DIQLTQSPSSLSASVGDRVTITC(SEQ ID NO:37)
LFR2：WYQQKPGKAPKLLIF(SEQ ID NO:39)
LFR3：GVPSRFSGSGSGTNFTLTISSVQPEDFATYFC(SEQ ID NO:41)
LFR4：FGGGTKLEIK(SEQ ID NO:15)
VL：

人源化抗CD28抗体轻链可变区3(huVL3)
LCDR1：RASESVEYYVTSLMQ(SEQ ID NO:9)
LCDR2：AASNVES(SEQ ID NO:10)
LCDR3：QQSRKVPYT(SEQ ID NO:11)
LFR1：DIQLTQSPSSLSASVGDRVTITC(SEQ ID NO:37)
LFR2：WYQQKPGKAPKLLIF(SEQ ID NO:39)
LFR3：GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:42)
LFR4：FGGGTKLEIK(SEQ ID NO:15)
VL：
人源化抗CD28 IgG1抗体的重链恒定区CH：
人源化抗CD28 IgG1抗体的重链恒定区CL：
抗CD40抗体40.52重链可变区
HCDR1：SYAIS(SEQ ID NO:46)
HCDR2：MINPSGGTTSYAQKFQG(SEQ ID NO:47)
HCDR3：GFRMDQ(SEQ ID NO:48)
HFR1：QVQLVQSGAEVKKPGSSVKVSCKASGGTFS(SEQ ID NO:49)
HFR2：WVRQAPGQGLEWMG(SEQ ID NO:50)
HFR3：RVTMTRDTSTSTVYMELSSLRPEDTAVYYCAR(SEQ ID NO:51)
HFR4：WGQGTLVTVSS(SEQ ID NO:52)
VH：
抗CD40抗体40.52轻链可变区
LCDR1：TGTSSDVGAYNFVS(SEQ ID NO:54)
LCDR2：DVRKRPS(SEQ ID NO:55)
LCDR3：SSYAGNNNLV(SEQ ID NO:56)
LFR1：QSALTQPPSASGSPGQSVTISC(SEQ ID NO:57)
LFR2：WYQQHPGNAPKLIIY(SEQ ID NO:58)
LFR3：GIPDRFSASKSGNTASLTVSGLQADDEADYYC(SEQ ID NO:59)
LFR4：FGGGTKLTVL(SEQ ID NO:60)
VL：
40.52的Fab抗体重链恒定区(CH1)：
40.52的Fab抗体轻链恒定区(CL)：
亲和力成熟的抗CD40抗体4052.12重链可变区
HCDR1：SYAVS(SEQ ID NO:62)
HCDR2：MINPSGGTTSYAQKFQG(SEQ ID NO:47)
HCDR3：GFRMDQ(SEQ ID NO:48)
HFR1：QVQLVQSGAEVKKPGSSVKVSCKASGGTFS(SEQ ID NO:49)
HFR2：WVRQAPGQGLEWMG(SEQ ID NO:50)
HFR3：RVTMTRDTSTSTVYMELSSLRPEDTAVYYCAR(SEQ ID NO:51)
HFR4：WGQGTLVTVSS(SEQ ID NO:52)
VH：
亲和力成熟的抗CD40抗体4052.12轻链可变区
LCDR1：TGTSSDVGAYNFVS(SEQ ID NO:54)
LCDR2：DVRKRPR(SEQ ID NO:64)
LCDR3：SSYAGNNNLV(SEQ ID NO:56)
LFR1：QSALTQPPSASGSPGQSVTISC(SEQ ID NO:57)
LFR2：WYQQHPGNAPKLIIY(SEQ ID NO:58)
LFR3：GIPDRFSASKSGNTASLTVSGLQADDEADYYC(SEQ ID NO:59)
LFR4：FGGGTKLTVL(SEQ ID NO:60)
VL：
亲和力成熟的抗CD40抗体4052.17重链可变区
HCDR1：SYAIS(SEQ ID NO:46)
HCDR2：MINPSGGTTSYAQKFQG(SEQ ID NO:47)
HCDR3：GFRMDQ(SEQ ID NO:48)
HFR1：QVQLVQSGAEVKKPGSSVKVSCKASGGTFS(SEQ ID NO:49)
HFR2：WVRQAPGQGLEWMG(SEQ ID NO:50)
HFR3：RVTMTRDTSTSTVYMELSSLRPEDTAVYYCAR(SEQ ID NO:51)
HFR4：WGQGTLVTVSS(SEQ ID NO:52)
VH：
亲和力成熟的抗CD40抗体4052.17轻链可变区
LCDR1：TGTSSDVGAYNFVS(SEQ ID NO:54)
LCDR2：DVRKRPS(SEQ ID NO:55)
LCDR3：SSYAGNNEMV(SEQ ID NO:67)
LFR1：QSALTQPPSASGSPGQSVTISC(SEQ ID NO:57)
LFR2：WYQQHPGNAPKLIIY(SEQ ID NO:58)
LFR3：GIPDRFSASKSGNTASLTVSGLQADDEADYYC(SEQ ID NO:59)
LFR4：FGGGTKLTVL(SEQ ID NO:60)
VL：
亲和力成熟的抗CD40抗体4052.25重链可变区
HCDR1：SYAIS(SEQ ID NO:46)
HCDR2：MINPSGGTTSYAQKFQG(SEQ ID NO:47)
HCDR3：GFRMDQ(SEQ ID NO:48)
HFR1：QVQLVQSGAEVKKPGSSVKVSCKASGGTFS(SEQ ID NO:49)
HFR2：WVRQAPGQGLEWMG(SEQ ID NO:50)
HFR3：RVTMTRDTSTSTVYMELSSLRPEDTAVYYCAR(SEQ ID NO:51)
HFR4：WGQGTLVTVSS(SEQ ID NO:52)
VH：
亲和力成熟的抗CD40抗体4052.25轻链可变区
LCDR1：TGTSSDVGAYNFVS(SEQ ID NO:54)
LCDR2：DVRKRPS(SEQ ID NO:55)
LCDR3：SSYAGNNRLV(SEQ ID NO:68)
LFR1：QSALTQPPSASGSPGQSVTISC(SEQ ID NO:57)
LFR2：WYQQHPGNAPKLIIY(SEQ ID NO:58)
LFR3：GIPDRFSASKSGNTASLTVSGLQADDEADYYC(SEQ ID NO:59)
LFR4：FGGGTKLTVL(SEQ ID NO:60)
VL：
亲和力成熟的抗CD40抗体4052.28重链可变区
HCDR1：SYAIS(SEQ ID NO:46)
HCDR2：MINPSGGTTSYAQKFQG(SEQ ID NO:47)
HCDR3：GFRMDQ(SEQ ID NO:48)
HFR1：QVQLVQSGAEVKKPGSSVKVSCKASGGTFS(SEQ ID NO:49)
HFR2：WVRQAPGQGLEWMG(SEQ ID NO:50)
HFR3：RVTMTRDTSTSTVYMELSSLRPEDTAVYYCAR(SEQ ID NO:51)
HFR4：WGQGTLVTVSS(SEQ ID NO:52)
VH：
亲和力成熟的抗CD40抗体4052.28轻链可变区
LCDR1：TGTSSDVGAYNFVS(SEQ ID NO:54)
LCDR2：DTRKRPS(SEQ ID NO:65)
LCDR3：SSYAGNNNLV(SEQ ID NO:56)
LFR1：QSALTQPPSASGSPGQSVTISC(SEQ ID NO:57)
LFR2：WYQQHPGNAPKLIIY(SEQ ID NO:58)
LFR3：GIPDRFSASKSGNTASLTVSGLQADDEADYYC(SEQ ID NO:59)
LFR4：FGGGTKLTVL(SEQ ID NO:60)
VL：
亲和力成熟的抗CD40抗体4052.36重链可变区
HCDR1：SYAIS(SEQ ID NO:46)
HCDR2：MINPSGGTTSYAQKFQG(SEQ ID NO:47)
HCDR3：GFRMDQ(SEQ ID NO:48)
HFR1：QVQLVQSGAQVKKPGSSVKVSCKASGGTFS(SEQ ID NO:63)
HFR2：WVRQAPGQGLEWMG(SEQ ID NO:50)
HFR3：RVTMTRDTSTSTVYMELSSLRPEDTAVYYCAR(SEQ ID NO:51)
HFR4：WGQGTLVTVSS(SEQ ID NO:52)
VH：
亲和力成熟的抗CD40抗体4052.36轻链可变区
LCDR1：TGTSSDVGAYNFVS(SEQ ID NO:54)
LCDR2：DVRKRPI(SEQ ID NO:66)
LCDR3：SSYAGNNNLV(SEQ ID NO:56)
LFR1：QSALTQPPSASGSPGQSVTISC(SEQ ID NO:57)
LFR2：WYQQHPGNAPKLIIY(SEQ ID NO:58)
LFR3：GIPDRFSASKSGNTASLTVSGLQADDEADYYC(SEQ ID NO:59)
LFR4：FGGGTKLTVL(SEQ ID NO:60)
VL：
Linker：
GGGGS(SEQ ID NO:79)
GGGGSGGGGSGGGGS(SEQ ID NO:80)
抗间皮素scFv(M12)：
注：其中划线部分依序为LCDR1、LCDR2、LCDR3、HCDR1、HCDR2、HCDR3Meso-CAR(M12)：

Claims (124)

  1. 人源化抗CD28抗体或其抗原结合片段，所述人源化抗CD28抗体包括重链可变区和轻链可变区，所述重链可变区包括HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3；其中，
    HCDR1包含SEQ ID NO:1，
    HCDR2包含选自SEQ ID NO:2、17和18的序列，
    HCDR3包含SEQ ID NO:3，
    LCDR1包含SEQ ID NO:9，
    LCDR2包含SEQ ID NO:10，和
    LCDR3包含SEQ ID NO:11；
    所述人源化抗CD28抗体通过ELISA测定的与人CD28结合的EC50在0.008μg/mL至0.016μg/mL的范围内；或所述人源化抗CD28抗体通过生物膜层干涉技术(BLI)测定的与人CD28的结合平衡解离常数KD在6.1×10-9M至1.2×10-8M的范围内。
  2. 如权利要求1所述的人源化抗CD28抗体或其抗原结合片段，其中所述人源化抗CD28抗体的重链可变区包括HFR1、HFR2、HFR3和HFR4，所述人源化抗CD28抗体的轻链可变区包括LFR1、LFR2、LFR3和LFR4，其中，
    HFR1包含选自SEQ ID NO:19-22的序列，
    HFR2包含选自SEQ ID NO:23-26的序列，
    HFR3包含选自SEQ ID NO:27-30，
    HFR4包含如SEQ ID NO:7所示的序列，
    LFR1包含选自SEQ ID NO:36-37的序列，
    LFR2包含选自SEQ ID NO:38-39的序列，
    LFR3包含选自SEQ ID NO:40-42的序列，和
    LFR4包含如SEQ ID NO:15所示的序列。
  3. 如权利要求1或2所述的人源化抗CD28抗体或其抗原结合片段，其中所述人源化抗CD28抗体的重链可变区包括的HFR1、HFR2、HFR3、HFR4选自下组：
    (1)包含如SEQ ID NO:19所示的序列的HFR1，包含如SEQ ID NO:23所示的序列的HFR2，包含如SEQ ID NO:27所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4；
    (2)包含如SEQ ID NO:20所示的序列的HFR1，包含如SEQ ID NO:24所示的序列的HFR2，包含如SEQ ID NO:28所示的序列的HFR3，和包含如SEQ ID  NO:7所示的序列的HFR4；
    (3)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:25所示的序列的HFR2，包含如SEQ ID NO:29所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4；
    (4)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4；和
    (5)包含如SEQ ID NO:22所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，和包含如SEQ ID NO:7所示的序列的HFR4。
  4. 如权利要求1-3任一项所述的人源化抗CD28抗体或其抗原结合片段，其中所述人源化抗CD28抗体的轻链可变区包括的LFR1、LFR2、LFR3、LFR4选自下组：
    (1)包含如SEQ ID NO:36所示的序列的LFR1，包含如SEQ ID NO:38所示的序列的LFR2，包含如SEQ ID NO:40所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (2)包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；和
    (3)包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4。
  5. 如权利要求1-4任一项所述的人源化抗CD28抗体或其抗原结合片段，其中所述人源化抗CD28抗体的重链可变区包括的HFR1、HFR2、HFR3、HFR4和轻链可变区包括的LFR1、LFR2、LFR3、LFR4选自下组：
    (1)包含如SEQ ID NO:19所示的序列的HFR1，包含如SEQ ID NO:23所示的序列的HFR2，包含如SEQ ID NO:27所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:36所示的序列的LFR1，包含如SEQ ID NO:38所示的序列的LFR2，包含如SEQ ID NO:40所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (2)包含如SEQ ID NO:19所示的序列的HFR1，包含如SEQ ID NO:23所示的序列的HFR2，包含如SEQ ID NO:27所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (3)包含如SEQ ID NO:20所示的序列的HFR1，包含如SEQ ID NO:24所 示的序列的HFR2，包含如SEQ ID NO:28所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (4)包含如SEQ ID NO:20所示的序列的HFR1，包含如SEQ ID NO:24所示的序列的HFR2，包含如SEQ ID NO:28所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (5)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:25所示的序列的HFR2，包含如SEQ ID NO:29所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:36所示的序列的LFR1，包含如SEQ ID NO:38所示的序列的LFR2，包含如SEQ ID NO:40所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (6)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:25所示的序列的HFR2，包含如SEQ ID NO:29所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (7)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:25所示的序列的HFR2，包含如SEQ ID NO:29所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (8)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，(2)包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (9)包含如SEQ ID NO:21所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；
    (10)包含如SEQ ID NO:22所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如 SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:41所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4；和
    (11)包含如SEQ ID NO:22所示的序列的HFR1，包含如SEQ ID NO:26所示的序列的HFR2，包含如SEQ ID NO:30所示的序列的HFR3，包含如SEQ ID NO:7所示的序列的HFR4，包含如SEQ ID NO:37所示的序列的LFR1，包含如SEQ ID NO:39所示的序列的LFR2，包含如SEQ ID NO:42所示的序列的LFR3，和包含如SEQ ID NO:15所示的序列的LFR4。
  6. 如权利要求1-5任一项所述的人源化抗CD28抗体或其抗原结合片段，其中所述人源化抗CD28抗体的重链可变区包含选自SEQ ID NO:31-35的序列。
  7. 如权利要求1-6任一项所述的人源化抗CD28抗体或其抗原结合片段，其中所述人源化抗CD28抗体的轻链可变区包含选自SEQ ID NO:43-45的序列。
  8. 如权利要求1-7任一项所述的人源化抗CD28抗体或其抗原结合片段，其中所述人源化抗CD28抗体的重链可变区和轻链可变区选自下述组合：
    (1)包含如SEQ ID NO:31所示的序列的重链可变区和包含如SEQ ID NO:43所示的序列的轻链可变区；
    (2)包含如SEQ ID NO:31所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；
    (3)包含如SEQ ID NO:32所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；
    (4)包含如SEQ ID NO:32所示的序列的重链可变区和包含如SEQ ID NO:45所示的序列的轻链可变区；
    (5)包含如SEQ ID NO:33所示的序列的重链可变区和包含如SEQ ID NO:43所示的序列的轻链可变区；
    (6)包含如SEQ ID NO:33所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；
    (7)包含如SEQ ID NO:33所示的序列的重链可变区和包含如SEQ ID NO:45所示的序列的轻链可变区；
    (8)包含如SEQ ID NO:34所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；
    (9)包含如SEQ ID NO:34所示的序列的重链可变区和包含如SEQ ID NO:45所示的序列的轻链可变区；
    (10)包含如SEQ ID NO:35所示的序列的重链可变区和包含如SEQ ID NO:44所示的序列的轻链可变区；和
    (11)包含如SEQ ID NO:35所示的序列的重链可变区和包含如SEQ ID NO:45所示的序列的轻链可变区。
  9. 如权利要求1-8任一项所述的双特异性抗体，其中所述人源化抗CD28 抗体是scFv。
  10. 如权利要求9所述的人源化抗CD28抗体或其抗原结合片段，其中人源化抗CD28scFv包含通过肽接头连接的重链可变区和轻链可变区。
  11. 如权利要求10所述的人源化抗CD28抗体或其抗原结合片段，其中人源化抗CD28scFv中的肽接头的序列包含SEQ ID NO:80。
  12. 如权利要求9-11任一项所述的人源化抗CD28抗体或其抗原结合片段，其中人源化抗CD28scFv中中重链可变区位于轻链可变区的N端或C端。
  13. 多特异性抗体，其至少包含第一抗原结合部分和第二抗原结合部分，其中第一抗原结合部分包含权利要求1-12任一项所述的人源化抗CD28抗体或其抗原结合片段，第二抗原结合部分与第一抗原结合部分结合不同的抗原。
  14. 如权利要求13所述的多特异性抗体，其中所述第二抗原结合部分特异性结合肿瘤抗原或能够刺激免疫细胞。
  15. 如权利要求13-14任一项所述的多特异性抗体，其中所述第一抗原结合部分是scFv，且所述第二抗原结合部分是scFv。
  16. 如权利要求13-15任一项所述的多特异性抗体，其中所述第一抗原结合部分与所述第二抗原结合部分通过肽接头连接。
  17. 如权利要求13-16任一项所述的多特异性抗体，其中所述肽接头包含SEQ ID NO:79的序列。
  18. 如权利要求13-17任一项所述的多特异性抗体，其中所述第一抗原结合部分位于所述第二抗原结合部分的N端或C端。
  19. 如权利要求13-18任一项所述的多特异性抗体，其中所述第二抗原结合部分是抗CD40抗体。
  20. 如权利要求13-19任一项所述的多特异性抗体，其中所述多特异性抗体是双特异性抗体，其包含所述第一抗原结合部分和所述第二抗原结合部分。
  21. 如权利要求20所述的多特异性抗体，其中所述第二抗原结合部分是抗CD40抗体或其抗原结合片段，所述抗CD40抗体通过ELISA测定的与人CD40结合的EC50小于0.04919μg/mL，或者所述抗CD40抗体通过ELISA测定的与人CD40结合的EC50小于3.44×10-7M。
  22. 如权利要求13-21任一项所述的多特异性抗体，其中所述抗CD40抗体通过对亲本抗体进行亲和力成熟获得，并且显示出比所述亲本抗体更强的与CD40的结合亲和力；所述亲本抗体具有下述重链可变区和轻链可变区，所述重链可变区包括：包含SEQ ID NO:46的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3，所述轻链可变区包括：包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:56的LCDR3。
  23. 如权利要求13-22任一项所述的多特异性抗体，其中所述抗CD40抗体的重链可变区包括：包含选自SEQ ID NO:46和SEQ ID NO:62的序列的HCDR1、 包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3。
  24. 如权利要求13-23任一项所述的多特异性抗体，其中所述抗CD40抗体的重链可变区包括：
    (1)包含SEQ ID NO:46的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3；或
    (2)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2和包含SEQ ID NO:48的HCDR3。
  25. 如权利要求13-24任一项所述的多特异性抗体，其中所述抗CD40抗体的轻链可变区包括：包含SEQ ID NO:54的LCDR1、包含选自SEQ ID NO:55,64-66的序列的LCDR2和包含选自SEQ ID NO:56,67-68序列的LCDR3。
  26. 如权利要求13-25任一项所述的多特异性抗体，其中所述抗CD40抗体的轻链可变区包括：
    (1)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:64的LCDR2和包含SEQ ID NO:56的LCDR3；
    (2)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:67的LCDR3；
    (3)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:68的LCDR3；
    (4)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:65的LCDR2和包含SEQ ID NO:56的LCDR3；
    (5)包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:66的LCDR2和包含SEQ ID NO:56的LCDR3。
  27. 如权利要求13-26任一项所述的多特异性抗体，其中所述抗CD40抗体的重链可变区和轻链可变区包括：
    (1)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:64的LCDR2和包含SEQ ID NO:56的LCDR3；
    (2)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:67的LCDR3；
    (3)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:55的LCDR2和包含SEQ ID NO:68的LCDR3；
    (4)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:65的LCDR2和包含SEQ ID NO:56的LCDR3；
    (5)包含SEQ ID NO:62的HCDR1、包含SEQ ID NO:47的HCDR2、包含SEQ ID NO:48的HCDR3、包含SEQ ID NO:54的LCDR1、包含SEQ ID NO:66的LCDR2和包含SEQ ID NO:56的LCDR3。
  28. 如权利要求13-27任一项所述的多特异性抗体，所述抗CD40抗体的重链可变区包括：
    (1)包含SEQ ID NO:49的HFR1、包含SEQ ID NO:50的HFR2、包含SEQ ID NO:51的HFR3和包含SEQ ID NO:52的HFR4；
    (2)包含SEQ ID NO:63的HFR1、包含SEQ ID NO:50的HFR2、包含SEQ ID NO:51的HFR3和包含SEQ ID NO:52的HFR4。
  29. 如权利要求13-28任一项所述的多特异性抗体，所述抗CD40抗体的轻链可变区包括：包含SEQ ID NO:57的LFR1、包含SEQ ID NO:58的LFR2、包含SEQ ID NO:59的LFR3和包含SEQ ID NO:60的LFR4。
  30. 如权利要求13-29任一项所述的多特异性抗体，所述抗CD40抗体的重链可变区与选自SEQ ID NO:69-73的序列具有至少70％的序列一致性。
  31. 如权利要求30所述的多特异性抗体，所述抗CD40抗体的重链可变区包含选自SEQ ID NO:69-73的序列。
  32. 如权利要求13-31任一项所述的多特异性抗体，所述抗CD40抗体的轻链可变区与选自SEQ ID NO:74-78的序列具有至少70％的序列一致性。
  33. 如权利要求32所述的多特异性抗体，所述抗CD40抗体的轻链可变区包含选自SEQ ID NO:74-78的序列。
  34. 如权利要求13-33任一项所述的多特异性抗体，其中所述抗CD40抗体是scFv。
  35. 如权利要求34所述的多特异性抗体，其中抗CD40scFv包含通过肽接头连接的重链可变区和轻链可变区。
  36. 如权利要求35所述的多特异性抗体，其中抗CD40scFv中的肽接头的序列包含SEQ ID NO:80。
  37. 如权利要求34-36中任一项所述的多特异性抗体，其中抗CD40scFv中中重链可变区位于轻链可变区的N端或C端。
  38. 编码如权利要求1-12任一项所述的人源化抗CD28抗体或其抗原结合片段、或权利要求13-37任一项所述的多特异性抗体的多核苷酸。
  39. 包含如权利要求38所述的多核苷酸的载体。
  40. 如权利要求39所述的载体，其中所述载体是质粒载体、病毒载体、包含所述多核苷酸的环状RNA、能够通过环化获得所述环状RNA的前体RNA、或包含所述前体RNA的DNA模板的载体；其中所述环状RNA依序包括：内部核糖体进入位点(IRES)元件、所述多核苷酸和polyA。
  41. 如权利要求40所述的载体，其中所述polyA的长度为至少45个核苷 酸；优选地，所述polyA的长度为至少70个核苷酸。
  42. 包含如权利要求38所述的多核苷酸或如权利要求39-41任一项所述的载体的宿主细胞。
  43. 药物组合物，包含如权利要求1-12任一项所述的人源化抗CD28抗体或其抗原结合片段、如权利要求13-37任一项所述的多特异性抗体、如权利要求38所述的多核苷酸或如权利要求39-41任一项所述的载体，以及药学可接受的载体。
  44. 药物组合，包括：a)如权利要求1-12任一项所述的人源化抗CD28抗体或其抗原结合片段、如权利要求13-37任一项所述的多特异性抗体、如权利要求38所述的多核苷酸或如权利要求39-41任一项所述的载体；和b)免疫细胞的组合。
  45. 表达如权利要求1-12任一项所述的人源化抗CD28抗体或其抗原结合片段或如权利要求13-37任一项所述的多特异性抗体的免疫细胞。
  46. 如权利要求44所述的药物组合或如权利要求45所述的免疫细胞，其中所述免疫细胞是T细胞、NK细胞、NKT细胞、巨噬细胞、中性粒细胞或粒细胞。
  47. 如权利要求44-46任一项所述的药物组合或免疫细胞，其中所述免疫细胞重组表达嵌合抗原受体(CAR)、T细胞受体(TCR)或双特异性T细胞接合子(BiTE)，其中所述CAR、TCR或BiTE结合肿瘤抗原或病毒抗原。
  48. 如权利要求44-47任一项所述的药物组合或免疫细胞，其中所述免疫细胞是CAR-T细胞。
  49. 如权利要求47或48所述的药物组合或免疫细胞，其中所述CAR靶向间皮素、CD123、BCMA、HER2、IL13Ra2、CD19或B7H3。
  50. 如权利要求49所述的药物组合或免疫细胞，其中靶向间皮素的CAR包含抗间皮素抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3选自下列组：
    (1)如SEQ ID NO:87所示的LCDR1，如SEQ ID NO:102所示的LCDR2，如SEQ ID NO:116所示的LCDR3，如SEQ ID NO:131所示的HCDR1，如SEQ ID NO:144所示的HCDR2和如SEQ ID NO:157所示的HCDR3；
    (2)如SEQ ID NO:88所示的LCDR1，如SEQ ID NO:103所示的LCDR2，如SEQ ID NO:117所示的LCDR3，如SEQ ID NO:132所示的HCDR1，如SEQ ID NO:145所示的HCDR2和如SEQ ID NO:158所示的HCDR3；
    (3)如SEQ ID NO:89所示的LCDR1，如SEQ ID NO:104所示的LCDR2，如SEQ ID NO:118所示的LCDR3，如SEQ ID NO:133所示的HCDR1，如SEQ  ID NO:146所示的HCDR2和如SEQ ID NO:159所示的HCDR3；
    (4)如SEQ ID NO:90所示的LCDR1，如SEQ ID NO:105所示的LCDR2，如SEQ ID NO:119所示的LCDR3，如SEQ ID NO:134所示的HCDR1，如SEQ ID NO:147所示的HCDR2和如SEQ ID NO:160所示的HCDR3；
    (5)如SEQ ID NO:91所示的LCDR1，如SEQ ID NO:106所示的LCDR2，如SEQ ID NO:120所示的LCDR3，如SEQ ID NO:135所示的HCDR1，如SEQ ID NO:148所示的HCDR2和如SEQ ID NO:161所示的HCDR3；
    (6)如SEQ ID NO:92所示的LCDR1，如SEQ ID NO:107所示的LCDR2，如SEQ ID NO:121所示的LCDR3，如SEQ ID NO:136所示的HCDR1，如SEQ ID NO:149所示的HCDR2和如SEQ ID NO:162所示的HCDR3；
    (7)如SEQ ID NO:93所示的LCDR1，如SEQ ID NO:108所示的LCDR2，如SEQ ID NO:122所示的LCDR3，如SEQ ID NO:137所示的HCDR1，如SEQ ID NO:150所示的HCDR2和如SEQ ID NO:163所示的HCDR3；
    (8)如SEQ ID NO:94所示的LCDR1，如SEQ ID NO:109所示的LCDR2，如SEQ ID NO:123所示的LCDR3，如SEQ ID NO:138所示的HCDR1，如SEQ ID NO:151所示的HCDR2和如SEQ ID NO:164所示的HCDR3；
    (9)如SEQ ID NO:95所示的LCDR1，如SEQ ID NO:110所示的LCDR2，如SEQ ID NO:124所示的LCDR3，如SEQ ID NO:139所示的HCDR1，如SEQ ID NO:152所示的HCDR2和如SEQ ID NO:165所示的HCDR3；
    (10)如SEQ ID NO:96所示的LCDR1，如SEQ ID NO:111所示的LCDR2，如SEQ ID NO:125所示的LCDR3，如SEQ ID NO:134所示的HCDR1，如SEQ ID NO:147所示的HCDR2和如SEQ ID NO:166所示的HCDR3；
    (11)如SEQ ID NO:97所示的LCDR1，如SEQ ID NO:112所示的LCDR2，如SEQ ID NO:126所示的LCDR3，如SEQ ID NO:140所示的HCDR1，如SEQ ID NO:153所示的HCDR2和如SEQ ID NO:167所示的HCDR3；
    (12)如SEQ ID NO:98所示的LCDR1，如SEQ ID NO:113所示的LCDR2，如SEQ ID NO:127所示的LCDR3，如SEQ ID NO:139所示的HCDR1，如SEQ ID NO:152所示的HCDR2和如SEQ ID NO:168所示的HCDR3；
    (13)如SEQ ID NO:99所示的LCDR1，如SEQ ID NO:114所示的LCDR2，如SEQ ID NO:128所示的LCDR3，如SEQ ID NO:141所示的HCDR1，如SEQ ID NO:154所示的HCDR2和如SEQ ID NO:169所示的HCDR3；
    (14)如SEQ ID NO:100所示的LCDR1，如SEQ ID NO:115所示的LCDR2，如SEQ ID NO:129所示的LCDR3，如SEQ ID NO:142所示的HCDR1，如SEQ ID NO:155所示的HCDR2和如SEQ ID NO:170所示的HCDR3；和
    (15)如SEQ ID NO:101所示的LCDR1，如SEQ ID NO:104所示的LCDR2，如SEQ ID NO:130所示的LCDR3，如SEQ ID NO:143所示的HCDR1，如SEQ  ID NO:156所示的HCDR2和如SEQ ID NO:171所示的HCDR3。
  51. 如权利要求50所述的药物组合或免疫细胞，其中所述轻链可变区和重链可变区选自下列组：
    (1)如SEQ ID NO:172所示的轻链可变区和如SEQ ID NO:187所示的重链可变区；
    (2)如SEQ ID NO:173所示的轻链可变区和如SEQ ID NO:188所示的重链可变区；
    (3)如SEQ ID NO:174所示的轻链可变区和如SEQ ID NO:189所示的重链可变区；
    (4)如SEQ ID NO:175所示的轻链可变区和如SEQ ID NO:190所示的重链可变区；
    (5)如SEQ ID NO:176所示的轻链可变区和如SEQ ID NO:191所示的重链可变区；
    (6)如SEQ ID NO:177所示的轻链可变区和如SEQ ID NO:192所示的重链可变区；
    (7)如SEQ ID NO:178所示的轻链可变区和如SEQ ID NO:193所示的重链可变区；
    (8)如SEQ ID NO:179所示的轻链可变区和如SEQ ID NO:194所示的重链可变区；
    (9)如SEQ ID NO:180所示的轻链可变区和如SEQ ID NO:195所示的重链可变区；
    (10)如SEQ ID NO:181所示的轻链可变区和如SEQ ID NO:196所示的重链可变区；
    (11)如SEQ ID NO:182所示的轻链可变区和如SEQ ID NO:197所示的重链可变区；
    (12)如SEQ ID NO:183所示的轻链可变区和如SEQ ID NO:198所示的重链可变区；
    (13)如SEQ ID NO:184所示的轻链可变区和如SEQ ID NO:199所示的重链可变区；
    (14)如SEQ ID NO:185所示的轻链可变区和如SEQ ID NO:200所示的重链可变区；和
    (15)如SEQ ID NO:186所示的轻链可变区和如SEQ ID NO:201所示的重链可变区。
  52. 如权利要求50或51所述的药物组合或免疫细胞，其中所述抗间皮素抗体是scFv，其包括选自SEQ ID NO:202-216的氨基酸序列。
  53. 如权利要求50-52任一项所述的药物组合或免疫细胞，其中靶向间皮素的CAR包括选自SEQ ID NO:217-231的氨基酸序列。
  54. 如权利要求49所述的药物组合或免疫细胞，其中靶向CD123的CAR包含抗CD123抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3选自下列组：
    (1)如SEQ ID NO:349所示的LCDR1，如SEQ ID NO:379所示的LCDR2，如SEQ ID NO:407所示的LCDR3，如SEQ ID NO:437所示的HCDR1，如SEQ ID NO:458所示的HCDR2和如SEQ ID NO:482所示的HCDR3；
    (2)如SEQ ID NO:363所示的LCDR1，如SEQ ID NO:391所示的LCDR2，如SEQ ID NO:421所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:468所示的HCDR2和如SEQ ID NO:495所示的HCDR3；
    (3)如SEQ ID NO:353所示的LCDR1，如SEQ ID NO:383所示的LCDR2，如SEQ ID NO:412所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:486所示的HCDR3；
    (4)如SEQ ID NO:364所示的LCDR1，如SEQ ID NO:378所示的LCDR2，如SEQ ID NO:422所示的LCDR3，如SEQ ID NO:446所示的HCDR1，如SEQ ID NO:469所示的HCDR2和如SEQ ID NO:496所示的HCDR3；
    (5)如SEQ ID NO:369所示的LCDR1，如SEQ ID NO:396所示的LCDR2，如SEQ ID NO:428所示的LCDR3，如SEQ ID NO:445所示的HCDR1，如SEQ ID NO:475所示的HCDR2和如SEQ ID NO:503所示的HCDR3；
    (6)如SEQ ID NO:370所示的LCDR1，如SEQ ID NO:397所示的LCDR2，如SEQ ID NO:429所示的LCDR3，如SEQ ID NO:452所示的HCDR1，如SEQ ID NO:474所示的HCDR2和如SEQ ID NO:504所示的HCDR3；
    (7)如SEQ ID NO:354所示的LCDR1，如SEQ ID NO:384所示的LCDR2，如SEQ ID NO:413所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:487所示的HCDR3；
    (8)如SEQ ID NO:350所示的LCDR1，如SEQ ID NO:380所示的LCDR2，如SEQ ID NO:408所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
    (9)如SEQ ID NO:371所示的LCDR1，如SEQ ID NO:398所示的LCDR2，如SEQ ID NO:430所示的LCDR3，如SEQ ID NO:453所示的HCDR1，如SEQ ID NO:476所示的HCDR2和如SEQ ID NO:505所示的HCDR3；
    (10)如SEQ ID NO:372所示的LCDR1，如SEQ ID NO:399所示的LCDR2，如SEQ ID NO:431所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ  ID NO:462所示的HCDR2和如SEQ ID NO:506所示的HCDR3；
    (11)如SEQ ID NO:348所示的LCDR1，如SEQ ID NO:378所示的LCDR2，如SEQ ID NO:406所示的LCDR3，如SEQ ID NO:436所示的HCDR1，如SEQ ID NO:457所示的HCDR2和如SEQ ID NO:481所示的HCDR3；
    (12)如SEQ ID NO:363所示的LCDR1，如SEQ ID NO:392所示的LCDR2，如SEQ ID NO:423所示的LCDR3，如SEQ ID NO:447所示的HCDR1，如SEQ ID NO:470所示的HCDR2和如SEQ ID NO:497所示的HCDR3；
    (13)如SEQ ID NO:373所示的LCDR1，如SEQ ID NO:400所示的LCDR2，如SEQ ID NO:432所示的LCDR3，如SEQ ID NO:451所示的HCDR1，如SEQ ID NO:477所示的HCDR2和如SEQ ID NO:507所示的HCDR3；
    (14)如SEQ ID NO:355所示的LCDR1，如SEQ ID NO:385所示的LCDR2，如SEQ ID NO:414所示的LCDR3，如SEQ ID NO:443所示的HCDR1，如SEQ ID NO:464所示的HCDR2和如SEQ ID NO:488所示的HCDR3；
    (15)如SEQ ID NO:356所示的LCDR1，如SEQ ID NO:386所示的LCDR2，如SEQ ID NO:415所示的LCDR3，如SEQ ID NO:444所示的HCDR1，如SEQ ID NO:465所示的HCDR2和如SEQ ID NO:489所示的HCDR3；
    (16)如SEQ ID NO:368所示的LCDR1，如SEQ ID NO:395所示的LCDR2，如SEQ ID NO:427所示的LCDR3，如SEQ ID NO:451所示的HCDR1，如SEQ ID NO:474所示的HCDR2和如SEQ ID NO:502所示的HCDR3；
    (17)如SEQ ID NO:351所示的LCDR1，如SEQ ID NO:381所示的LCDR2，如SEQ ID NO:409所示的LCDR3，如SEQ ID NO:438所示的HCDR1，如SEQ ID NO:459所示的HCDR2和如SEQ ID NO:483所示的HCDR3；
    (18)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2，如SEQ ID NO:410所示的LCDR3，如SEQ ID NO:439所示的HCDR1，如SEQ ID NO:460所示的HCDR2和如SEQ ID NO:484所示的HCDR3；
    (19)如SEQ ID NO:357所示的LCDR1，如SEQ ID NO:387所示的LCDR2，如SEQ ID NO:416所示的LCDR3，如SEQ ID NO:437所示的HCDR1，如SEQ ID NO:466所示的HCDR2和如SEQ ID NO:490所示的HCDR3；
    (20)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:377所示的LCDR2，如SEQ ID NO:405所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
    (21)如SEQ ID NO:365所示的LCDR1，如SEQ ID NO:393所示的LCDR2，如SEQ ID NO:424所示的LCDR3，如SEQ ID NO:448所示的HCDR1，如SEQ ID NO:471所示的HCDR2和如SEQ ID NO:498所示的HCDR3；
    (22)如SEQ ID NO:374所示的LCDR1，如SEQ ID NO:401所示的LCDR2，如SEQ ID NO:433所示的LCDR3，如SEQ ID NO:454所示的HCDR1，如SEQ  ID NO:478所示的HCDR2和如SEQ ID NO:508所示的HCDR3；
    (23)如SEQ ID NO:358所示的LCDR1，如SEQ ID NO:385所示的LCDR2，如SEQ ID NO:417所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:491所示的HCDR3；
    (24)如SEQ ID NO:359所示的LCDR1，如SEQ ID NO:388所示的LCDR2，如SEQ ID NO:418所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:492所示的HCDR3；
    (25)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2，如SEQ ID NO:403所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
    (26)如SEQ ID NO:366所示的LCDR1，如SEQ ID NO:394所示的LCDR2，如SEQ ID NO:425所示的LCDR3，如SEQ ID NO:449所示的HCDR1，如SEQ ID NO:472所示的HCDR2和如SEQ ID NO:499所示的HCDR3；
    (27)如SEQ ID NO:360所示的LCDR1，如SEQ ID NO:389所示的LCDR2，如SEQ ID NO:413所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:487所示的HCDR3；
    (28)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2，如SEQ ID NO:404所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
    (29)如SEQ ID NO:348所示的LCDR1，如SEQ ID NO:378所示的LCDR2，如SEQ ID NO:406所示的LCDR3，如SEQ ID NO:436所示的HCDR1，如SEQ ID NO:457所示的HCDR2和如SEQ ID NO:481所示的HCDR3；
    (30)如SEQ ID NO:361所示的LCDR1，如SEQ ID NO:390所示的LCDR2，如SEQ ID NO:419所示的LCDR3，如SEQ ID NO:445所示的HCDR1，如SEQ ID NO:467所示的HCDR2和如SEQ ID NO:493所示的HCDR3；
    (31)如SEQ ID NO:375所示的LCDR1，如SEQ ID NO:402所示的LCDR2，如SEQ ID NO:434所示的LCDR3，如SEQ ID NO:455所示的HCDR1，如SEQ ID NO:479所示的HCDR2和如SEQ ID NO:509所示的HCDR3；
    (32)如SEQ ID NO:368所示的LCDR1，如SEQ ID NO:395所示的LCDR2，如SEQ ID NO:427所示的LCDR3，如SEQ ID NO:450所示的HCDR1，如SEQ ID NO:473所示的HCDR2和如SEQ ID NO:5015所示的HCDR3；
    (33)如SEQ ID NO:362所示的LCDR1，如SEQ ID NO:388所示的LCDR2，如SEQ ID NO:420所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:494所示的HCDR3；
    (34)如SEQ ID NO:352所示的LCDR1，如SEQ ID NO:382所示的LCDR2，如SEQ ID NO:411所示的LCDR3，如SEQ ID NO:440所示的HCDR1，如SEQ  ID NO:461所示的HCDR2和如SEQ ID NO:485所示的HCDR3；和
    (35)如SEQ ID NO:367所示的LCDR1，如SEQ ID NO:391所示的LCDR2，如SEQ ID NO:426所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:468所示的HCDR2和如SEQ ID NO:500所示的HCDR3。
  55. 如权利要求54所述的药物组合或免疫细胞，其中所述轻链可变区和重链可变区选自下列组：
    (1)如SEQ ID NO:510所示的轻链可变区和如SEQ ID NO:545所示的重链可变区；
    (2)如SEQ ID NO:511所示的轻链可变区和如SEQ ID NO:546所示的重链可变区；
    (3)如SEQ ID NO:512所示的轻链可变区和如SEQ ID NO:547所示的重链可变区；
    (4)如SEQ ID NO:513所示的轻链可变区和如SEQ ID NO:548所示的重链可变区；
    (5)如SEQ ID NO:514所示的轻链可变区和如SEQ ID NO:549所示的重链可变区；
    (6)如SEQ ID NO:515所示的轻链可变区和如SEQ ID NO:550所示的重链可变区；
    (7)如SEQ ID NO:516所示的轻链可变区和如SEQ ID NO:551所示的重链可变区；
    (8)如SEQ ID NO:517所示的轻链可变区和如SEQ ID NO:552所示的重链可变区；
    (9)如SEQ ID NO:518所示的轻链可变区和如SEQ ID NO:553所示的重链可变区；
    (10)如SEQ ID NO:519所示的轻链可变区和如SEQ ID NO:554所示的重链可变区；
    (11)如SEQ ID NO:520所示的轻链可变区和如SEQ ID NO:555所示的重链可变区；
    (12)如SEQ ID NO:521所示的轻链可变区和如SEQ ID NO:556所示的重链可变区；
    (13)如SEQ ID NO:522所示的轻链可变区和如SEQ ID NO:557所示的重链可变区；
    (14)如SEQ ID NO:523所示的轻链可变区和如SEQ ID NO:558所示的重链可变区；
    (15)如SEQ ID NO:524所示的轻链可变区和如SEQ ID NO:559所示的重链 可变区；
    (16)如SEQ ID NO:525所示的轻链可变区和如SEQ ID NO:560所示的重链可变区；
    (17)如SEQ ID NO:526所示的轻链可变区和如SEQ ID NO:561所示的重链可变区；
    (18)如SEQ ID NO:527所示的轻链可变区和如SEQ ID NO:562所示的重链可变区；
    (19)如SEQ ID NO:528所示的轻链可变区和如SEQ ID NO:563所示的重链可变区；
    (20)如SEQ ID NO:529所示的轻链可变区和如SEQ ID NO:564所示的重链可变区；
    (21)如SEQ ID NO:530所示的轻链可变区和如SEQ ID NO:565所示的重链可变区；
    (22)如SEQ ID NO:531所示的轻链可变区和如SEQ ID NO:566所示的重链可变区；
    (23)如SEQ ID NO:532所示的轻链可变区和如SEQ ID NO:567所示的重链可变区；
    (24)如SEQ ID NO:533所示的轻链可变区和如SEQ ID NO:568所示的重链可变区；
    (25)如SEQ ID NO:534所示的轻链可变区和如SEQ ID NO:569所示的重链可变区；
    (26)如SEQ ID NO:535所示的轻链可变区和如SEQ ID NO:570所示的重链可变区；
    (27)如SEQ ID NO:536所示的轻链可变区和如SEQ ID NO:571所示的重链可变区；
    (28)如SEQ ID NO:537所示的轻链可变区和如SEQ ID NO:572所示的重链可变区；
    (29)如SEQ ID NO:538所示的轻链可变区和如SEQ ID NO:573所示的重链可变区；
    (30)如SEQ ID NO:539所示的轻链可变区和如SEQ ID NO:574所示的重链可变区；
    (31)如SEQ ID NO:540所示的轻链可变区和如SEQ ID NO:575所示的重链可变区；
    (32)如SEQ ID NO:541所示的轻链可变区和如SEQ ID NO:576所示的重链可变区；
    (33)如SEQ ID NO:542所示的轻链可变区和如SEQ ID NO:577所示的重链可变区；
    (34)如SEQ ID NO:543所示的轻链可变区和如SEQ ID NO:578所示的重链可变区；和
    (35)如SEQ ID NO:544所示的轻链可变区域和如SEQ ID NO:579所示的重链可变区域。
  56. 如权利要求54或55所述的药物组合或免疫细胞，其中所述CD123抗体是scFv，其包括选自SEQ ID NO:583-617的氨基酸序列。
  57. 如权利要求54-56任一项所述的药物组合或免疫细胞，其中靶向CD123的CAR包括选自SEQ ID NO:580-582的氨基酸序列。
  58. 如权利要求49所述的药物组合或免疫细胞，其中靶向BCMA的CAR包含抗BCMA抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3选自下列组：
    (1)如SEQ ID NO:232所示的LCDR1，如SEQ ID NO:242所示的LCDR2，如SEQ ID NO:253所示的LCDR3，如SEQ ID NO:264所示的HCDR1，如SEQ ID NO:275所示的HCDR2和如SEQ ID NO:287所示的HCDR3；
    (2)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:243所示的LCDR2，如SEQ ID NO:254所示的LCDR3，如SEQ ID NO:265所示的HCDR1，如SEQ ID NO:276所示的HCDR2和如SEQ ID NO:288所示的HCDR3；
    (3)如SEQ ID NO:234所示的LCDR1，如SEQ ID NO:244所示的LCDR2，如SEQ ID NO:255所示的LCDR3，如SEQ ID NO:266所示的HCDR1，如SEQ ID NO:277所示的HCDR2和如SEQ ID NO:289所示的HCDR3；
    (4)如SEQ ID NO:235所示的LCDR1，如SEQ ID NO:245所示的LCDR2，如SEQ ID NO:255所示的LCDR3，如SEQ ID NO:267所示的HCDR1，如SEQ ID NO:278所示的HCDR2和如SEQ ID NO:290所示的HCDR3；
    (5)如SEQ ID NO:236所示的LCDR1，如SEQ ID NO:246所示的LCDR2，如SEQ ID NO:256所示的LCDR3，如SEQ ID NO:268所示的HCDR1，如SEQ ID NO:279所示的HCDR2和如SEQ ID NO:291所示的HCDR3；
    (6)如SEQ ID NO:237所示的LCDR1，如SEQ ID NO:247所示的LCDR2，如SEQ ID NO:257所示的LCDR3，如SEQ ID NO:269所示的HCDR1，如SEQ ID NO:280所示的HCDR2和如SEQ ID NO:292所示的HCDR3；
    (7)如SEQ ID NO:238所示的LCDR1，如SEQ ID NO:248所示的LCDR2，如SEQ ID NO:258所示的LCDR3，如SEQ ID NO:266所示的HCDR1，如SEQ ID NO:281所示的HCDR2和如SEQ ID NO:293所示的HCDR3；
    (8)如SEQ ID NO:239所示的LCDR1，如SEQ ID NO:249所示的LCDR2，如SEQ ID NO:259所示的LCDR3，如SEQ ID NO:270所示的HCDR1，如SEQ ID NO:282所示的HCDR2和如SEQ ID NO:294所示的HCDR3；
    (9)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:250所示的LCDR2，如SEQ ID NO:260所示的LCDR3，如SEQ ID NO:271所示的HCDR1，如SEQ ID NO:283所示的HCDR2和如SEQ ID NO:295所示的HCDR3；
    (10)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:251所示的LCDR2，如SEQ ID NO:261所示的LCDR3，如SEQ ID NO:272所示的HCDR1，如SEQ ID NO:284所示的HCDR2和如SEQ ID NO:296所示的HCDR3；
    (11)如SEQ ID NO:240所示的LCDR1，如SEQ ID NO:252所示的LCDR2，如SEQ ID NO:262所示的LCDR3，如SEQ ID NO:273所示的HCDR1，如SEQ ID NO:285所示的HCDR2和如SEQ ID NO:297所示的HCDR3；和
    (12)如SEQ ID NO:241所示的LCDR1，如SEQ ID NO:245所示的LCDR2，如SEQ ID NO:263所示的LCDR3，如SEQ ID NO:274所示的HCDR1，如SEQ ID NO:286所示的HCDR2和如SEQ ID NO:298所示的HCDR3。
  59. 如权利要求58所述的药物组合或免疫细胞，其中所述轻链可变区和重链可变区选自下列组：
    (1)如SEQ ID NO:299所示所示的轻链可变区和如SEQ ID NO:311所示的重链可变区；
    (2)如SEQ ID NO:300所示的轻链可变区域和如SEQ ID NO:312所示的重链可变区域；
    (3)如SEQ ID NO:301所示的轻链可变区域和如SEQ ID NO:313所示的重链可变区域；
    (4)如SEQ ID NO:302所示的轻链可变区域和如SEQ ID NO:314所示的重链可变区域；
    (5)如SEQ ID NO:303所示的轻链可变区域和如SEQ ID NO:315所示的重链可变区域；
    (6)如SEQ ID NO:304所示的轻链可变区域和如SEQ ID NO:316所示的重链可变区域；
    (7)如SEQ ID NO:305所示的轻链可变区域和如SEQ ID NO:317所示的重链可变区域；
    (8)如SEQ ID NO:306所示的轻链可变区域和如SEQ ID NO:318所示的重链可变区域；
    (9)如SEQ ID NO:307所示的轻链可变区和如SEQ ID NO:319所示的重链可变区；
    (10)如SEQ ID NO:308所示的轻链可变区和如SEQ ID NO:320所示的重链 可变区；
    (11)如SEQ ID NO:309所示的轻链可变区和如SEQ ID NO:321所示的重链可变区；和
    (12)如SEQ ID NO:310所示的轻链可变区域和如SEQ ID NO:322所示的重链可变区域。
  60. 如权利要求58或59所述的药物组合或免疫细胞，其中所述抗BCMA抗体是scFv，其包括选自SEQ ID NO:323-334的氨基酸序列。
  61. 如权利要求58-60任一项所述的药物组合或免疫细胞，其中靶向BCMA的CAR包括选自SEQ ID NO:335-346的氨基酸序列。
  62. 如权利要求49所述的药物组合或免疫细胞，其中靶向CD19的CAR包含抗CD19抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3是如SEQ ID NO:628所示的LCDR1，如SEQ ID NO:629所示的LCDR2，如SEQ ID NO:630所示的LCDR3，如SEQ ID NO:631所示的HCDR1，如SEQ ID NO:632所示的HCDR2和如SEQ ID NO:633所示的HCDR3。
  63. 如权利要求62所述的药物组合或免疫细胞，其中所述抗CD19抗体包括如SEQ ID NO:635所示的轻链可变区和如SEQ ID NO:634所示的重链可变区。
  64. 如权利要求62或63所述的药物组合或免疫细胞，其中所述抗CD19抗体是scFv，其包括如SEQ ID NO:636所示的氨基酸序列。
  65. 如权利要求62-64任一项所述的药物组合或免疫细胞，其中靶向CD19的CAR包括如SEQ ID NO:637所示的氨基酸序列。
  66. 如权利要求49所述的药物组合或免疫细胞，其中靶向HER2的CAR包含抗HER2抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3是如SEQ ID NO:618所示的LCDR1，如SEQ ID NO:619所示的LCDR2，如SEQ ID NO:620所示的LCDR3，如SEQ ID NO:621所示的HCDR1，如SEQ ID NO:622所示的HCDR2和如SEQ ID NO:623所示的HCDR3。
  67. 如权利要求66所述的药物组合或免疫细胞，其中抗HER2抗体包括如SEQ ID NO:624所示的轻链可变区和如SEQ ID NO:625所示的重链可变区。
  68. 如权利要求66或67所述的药物组合或免疫细胞，其中所述抗HER2抗体是scFv，其包括如SEQ ID NO:626所示的的氨基酸序列。
  69. 如权利要求66-68任一项所述的药物组合或免疫细胞，其中靶向HER2的CAR包括如SEQ ID NO:627所示的氨基酸序列。
  70. 如权利要求49所述的药物组合或免疫细胞，其中靶向IL13Ra2的CAR包含抗IL13Ra2抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3是如SEQ ID NO:638所示的LCDR1，如SEQ ID NO:639所示的LCDR2，如SEQ ID NO:640所示的LCDR3，如SEQ ID NO:641所示的HCDR1，如SEQ ID NO:642所示的HCDR2和如SEQ ID NO:642所示的HCDR3。
  71. 如权利要求70所述的药物组合或免疫细胞，其中所述抗IL13Ra2抗体包括如SEQ ID NO:644所示的轻链可变区和如SEQ ID NO:645所示的重链可变区。
  72. 如权利要求70或71所述的药物组合或免疫细胞，其中所述抗IL13Ra2抗体是scFv，其包括如SEQ ID NO:646所示的氨基酸序列。
  73. 如权利要求70-72任一项所述的药物组合或免疫细胞，其中靶向IL13Ra2的CAR包括如SEQ ID NO:647所示的氨基酸序列。
  74. 如权利要求49所述的药物组合或免疫细胞，其中靶向B7H3的CAR包含抗B7H3抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3是如SEQ ID NO:648所示的LCDR1，如SEQ ID NO:649所示的LCDR2，如SEQ ID NO:650所示的LCDR3，如SEQ ID NO:651所示的HCDR1，如SEQ ID NO:652所示的HCDR2和如SEQ ID NO:653所示的HCDR3。
  75. 如权利要求74所述的药物组合或免疫细胞，其中所述抗B7H3抗体包括SEQ ID NO:654所示的轻链可变区和SEQ ID NO:655所示的重链可变区。
  76. 如权利要求74或75所述的药物组合或免疫细胞，其中所述抗B7H3抗体是scFv，其包括如SEQ ID NO:656所示的氨基酸序列。
  77. 如权利要求74-76任一项所述的药物组合或免疫细胞，其中靶向B7H3的CAR包括如SEQ ID NO:657所示的氨基酸序列。
  78. 增强免疫细胞对受试者中的靶细胞的杀伤作用的方法，包括给接受所述免疫细胞的受试者施用如权利要求1-12任一项所述的人源化抗CD28抗体或其抗原结合片段或如权利要求13-37任一项所述的多特异性抗体、如权利要求38所述的多核苷酸、如权利要求39-41任一项所述的载体或如权利要求43所述的药物组合物。
  79. 如权利要求78所述的方法，其中所述免疫细胞是T细胞、NK细胞、NKT细胞、巨噬细胞、中性粒细胞或粒细胞。
  80. 如权利要求78或79所述的方法，其中所述免疫细胞重组表达嵌合抗原受体(CAR)、T细胞受体(TCR)或双特异性T细胞接合子(BiTE)，其中所述CAR、TCR或BiTE结合肿瘤抗原或病毒抗原。
  81. 如权利要求78-80任一项所述的方法，其中所述免疫细胞是CAR-T细胞。
  82. 如权利要求80或81任一项所述的方法，其中所述CAR靶向间皮素、CD123、BCMA、HER2、IL13Ra2、CD19或B7H3。
  83. 如权利要求82所述的方法，其中靶向间皮素的CAR包含抗间皮素抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3选自下列组：
    (1)如SEQ ID NO:87所示的LCDR1，如SEQ ID NO:102所示的LCDR2，如SEQ ID NO:116所示的LCDR3，如SEQ ID NO:131所示的HCDR1，如SEQ ID NO:144所示的HCDR2和如SEQ ID NO:157所示的HCDR3；
    (2)如SEQ ID NO:88所示的LCDR1，如SEQ ID NO:103所示的LCDR2，如SEQ ID NO:117所示的LCDR3，如SEQ ID NO:132所示的HCDR1，如SEQ ID NO:145所示的HCDR2和如SEQ ID NO:158所示的HCDR3；
    (3)如SEQ ID NO:89所示的LCDR1，如SEQ ID NO:104所示的LCDR2，如SEQ ID NO:118所示的LCDR3，如SEQ ID NO:133所示的HCDR1，如SEQ ID NO:146所示的HCDR2和如SEQ ID NO:159所示的HCDR3；
    (4)如SEQ ID NO:90所示的LCDR1，如SEQ ID NO:105所示的LCDR2，如SEQ ID NO:119所示的LCDR3，如SEQ ID NO:134所示的HCDR1，如SEQ ID NO:147所示的HCDR2和如SEQ ID NO:160所示的HCDR3；
    (5)如SEQ ID NO:91所示的LCDR1，如SEQ ID NO:106所示的LCDR2，如SEQ ID NO:120所示的LCDR3，如SEQ ID NO:135所示的HCDR1，如SEQ ID NO:148所示的HCDR2和如SEQ ID NO:161所示的HCDR3；
    (6)如SEQ ID NO:92所示的LCDR1，如SEQ ID NO:107所示的LCDR2，如SEQ ID NO:121所示的LCDR3，如SEQ ID NO:136所示的HCDR1，如SEQ ID NO:149所示的HCDR2和如SEQ ID NO:162所示的HCDR3；
    (7)如SEQ ID NO:93所示的LCDR1，如SEQ ID NO:108所示的LCDR2，如SEQ ID NO:122所示的LCDR3，如SEQ ID NO:137所示的HCDR1，如SEQ ID NO:150所示的HCDR2和如SEQ ID NO:163所示的HCDR3；
    (8)如SEQ ID NO:94所示的LCDR1，如SEQ ID NO:109所示的LCDR2，如SEQ ID NO:123所示的LCDR3，如SEQ ID NO:138所示的HCDR1，如SEQ ID NO:151所示的HCDR2和如SEQ ID NO:164所示的HCDR3；
    (9)如SEQ ID NO:95所示的LCDR1，如SEQ ID NO:110所示的LCDR2，如SEQ ID NO:124所示的LCDR3，如SEQ ID NO:139所示的HCDR1，如SEQ ID NO:152所示的HCDR2和如SEQ ID NO:165所示的HCDR3；
    (10)如SEQ ID NO:96所示的LCDR1，如SEQ ID NO:111所示的LCDR2，如SEQ ID NO:125所示的LCDR3，如SEQ ID NO:134所示的HCDR1，如SEQ ID NO:147所示的HCDR2和如SEQ ID NO:166所示的HCDR3；
    (11)如SEQ ID NO:97所示的LCDR1，如SEQ ID NO:112所示的LCDR2，如SEQ ID NO:126所示的LCDR3，如SEQ ID NO:140所示的HCDR1，如SEQ ID NO:153所示的HCDR2和如SEQ ID NO:167所示的HCDR3；
    (12)如SEQ ID NO:98所示的LCDR1，如SEQ ID NO:113所示的LCDR2，如SEQ ID NO:127所示的LCDR3，如SEQ ID NO:139所示的HCDR1，如SEQ ID NO:152所示的HCDR2和如SEQ ID NO:168所示的HCDR3；
    (13)如SEQ ID NO:99所示的LCDR1，如SEQ ID NO:114所示的LCDR2，如SEQ ID NO:128所示的LCDR3，如SEQ ID NO:141所示的HCDR1，如SEQ ID NO:154所示的HCDR2和如SEQ ID NO:169所示的HCDR3；
    (14)如SEQ ID NO:100所示的LCDR1，如SEQ ID NO:115所示的LCDR2，如SEQ ID NO:129所示的LCDR3，如SEQ ID NO:142所示的HCDR1，如SEQ ID NO:155所示的HCDR2和如SEQ ID NO:170所示的HCDR3；和
    (15)如SEQ ID NO:101所示的LCDR1，如SEQ ID NO:104所示的LCDR2，如SEQ ID NO:130所示的LCDR3，如SEQ ID NO:143所示的HCDR1，如SEQ ID NO:156所示的HCDR2和如SEQ ID NO:171所示的HCDR3。
  84. 如权利要求83所述的方法，其中所述轻链可变区和重链可变区选自下列组：
    (1)如SEQ ID NO:172所示的轻链可变区和如SEQ ID NO:187所示的重链可变区；
    (2)如SEQ ID NO:173所示的轻链可变区和如SEQ ID NO:188所示的重链可变区；
    (3)如SEQ ID NO:174所示的轻链可变区和如SEQ ID NO:189所示的重链可变区；
    (4)如SEQ ID NO:175所示的轻链可变区和如SEQ ID NO:190所示的重链可变区；
    (5)如SEQ ID NO:176所示的轻链可变区和如SEQ ID NO:191所示的重链可变区；
    (6)如SEQ ID NO:177所示的轻链可变区和如SEQ ID NO:192所示的重链可变区；
    (7)如SEQ ID NO:178所示的轻链可变区和如SEQ ID NO:193所示的重链可变区；
    (8)如SEQ ID NO:179所示的轻链可变区和如SEQ ID NO:194所示的重链可变区；
    (9)如SEQ ID NO:180所示的轻链可变区和如SEQ ID NO:195所示的重链可变区；
    (10)如SEQ ID NO:181所示的轻链可变区和如SEQ ID NO:196所示的重链可变区；
    (11)如SEQ ID NO:182所示的轻链可变区和如SEQ ID NO:197所示的重链可变区；
    (12)如SEQ ID NO:183所示的轻链可变区和如SEQ ID NO:198所示的重链可变区；
    (13)如SEQ ID NO:184所示的轻链可变区和如SEQ ID NO:199所示的重链可变区；
    (14)如SEQ ID NO:185所示的轻链可变区和如SEQ ID NO:200所示的重链可变区；和
    (15)如SEQ ID NO:186所示的轻链可变区和如SEQ ID NO:201所示的重链可变区。
  85. 如权利要求83或84所述的方法，其中所述抗间皮素抗体是scFv，其包括选自SEQ ID NO:202-216的氨基酸序列。
  86. 如权利要求83-85任一项所述的方法，其中靶向间皮素的CAR包括选自SEQ ID NO:217-231的氨基酸序列。
  87. 如权利要求82所述的方法，其中靶向CD123的CAR包含抗CD123抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3选自下列组：
    (1)如SEQ ID NO:349所示的LCDR1，如SEQ ID NO:379所示的LCDR2，如SEQ ID NO:407所示的LCDR3，如SEQ ID NO:437所示的HCDR1，如SEQ ID NO:458所示的HCDR2和如SEQ ID NO:482所示的HCDR3；
    (2)如SEQ ID NO:363所示的LCDR1，如SEQ ID NO:391所示的LCDR2，如SEQ ID NO:421所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:468所示的HCDR2和如SEQ ID NO:495所示的HCDR3；
    (3)如SEQ ID NO:353所示的LCDR1，如SEQ ID NO:383所示的LCDR2，如SEQ ID NO:412所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:486所示的HCDR3；
    (4)如SEQ ID NO:364所示的LCDR1，如SEQ ID NO:378所示的LCDR2，如SEQ ID NO:422所示的LCDR3，如SEQ ID NO:446所示的HCDR1，如SEQ ID NO:469所示的HCDR2和如SEQ ID NO:496所示的HCDR3；
    (5)如SEQ ID NO:369所示的LCDR1，如SEQ ID NO:396所示的LCDR2， 如SEQ ID NO:428所示的LCDR3，如SEQ ID NO:445所示的HCDR1，如SEQ ID NO:475所示的HCDR2和如SEQ ID NO:503所示的HCDR3；
    (6)如SEQ ID NO:370所示的LCDR1，如SEQ ID NO:397所示的LCDR2，如SEQ ID NO:429所示的LCDR3，如SEQ ID NO:452所示的HCDR1，如SEQ ID NO:474所示的HCDR2和如SEQ ID NO:504所示的HCDR3；
    (7)如SEQ ID NO:354所示的LCDR1，如SEQ ID NO:384所示的LCDR2，如SEQ ID NO:413所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:487所示的HCDR3；
    (8)如SEQ ID NO:350所示的LCDR1，如SEQ ID NO:380所示的LCDR2，如SEQ ID NO:408所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
    (9)如SEQ ID NO:371所示的LCDR1，如SEQ ID NO:398所示的LCDR2，如SEQ ID NO:430所示的LCDR3，如SEQ ID NO:453所示的HCDR1，如SEQ ID NO:476所示的HCDR2和如SEQ ID NO:505所示的HCDR3；
    (10)如SEQ ID NO:372所示的LCDR1，如SEQ ID NO:399所示的LCDR2，如SEQ ID NO:431所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:506所示的HCDR3；
    (11)如SEQ ID NO:348所示的LCDR1，如SEQ ID NO:378所示的LCDR2，如SEQ ID NO:406所示的LCDR3，如SEQ ID NO:436所示的HCDR1，如SEQ ID NO:457所示的HCDR2和如SEQ ID NO:481所示的HCDR3；
    (12)如SEQ ID NO:363所示的LCDR1，如SEQ ID NO:392所示的LCDR2，如SEQ ID NO:423所示的LCDR3，如SEQ ID NO:447所示的HCDR1，如SEQ ID NO:470所示的HCDR2和如SEQ ID NO:497所示的HCDR3；
    (13)如SEQ ID NO:373所示的LCDR1，如SEQ ID NO:400所示的LCDR2，如SEQ ID NO:432所示的LCDR3，如SEQ ID NO:451所示的HCDR1，如SEQ ID NO:477所示的HCDR2和如SEQ ID NO:507所示的HCDR3；
    (14)如SEQ ID NO:355所示的LCDR1，如SEQ ID NO:385所示的LCDR2，如SEQ ID NO:414所示的LCDR3，如SEQ ID NO:443所示的HCDR1，如SEQ ID NO:464所示的HCDR2和如SEQ ID NO:488所示的HCDR3；
    (15)如SEQ ID NO:356所示的LCDR1，如SEQ ID NO:386所示的LCDR2，如SEQ ID NO:415所示的LCDR3，如SEQ ID NO:444所示的HCDR1，如SEQ ID NO:465所示的HCDR2和如SEQ ID NO:489所示的HCDR3；
    (16)如SEQ ID NO:368所示的LCDR1，如SEQ ID NO:395所示的LCDR2，如SEQ ID NO:427所示的LCDR3，如SEQ ID NO:451所示的HCDR1，如SEQ ID NO:474所示的HCDR2和如SEQ ID NO:502所示的HCDR3；
    (17)如SEQ ID NO:351所示的LCDR1，如SEQ ID NO:381所示的LCDR2， 如SEQ ID NO:409所示的LCDR3，如SEQ ID NO:438所示的HCDR1，如SEQ ID NO:459所示的HCDR2和如SEQ ID NO:483所示的HCDR3；
    (18)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2，如SEQ ID NO:410所示的LCDR3，如SEQ ID NO:439所示的HCDR1，如SEQ ID NO:460所示的HCDR2和如SEQ ID NO:484所示的HCDR3；
    (19)如SEQ ID NO:357所示的LCDR1，如SEQ ID NO:387所示的LCDR2，如SEQ ID NO:416所示的LCDR3，如SEQ ID NO:437所示的HCDR1，如SEQ ID NO:466所示的HCDR2和如SEQ ID NO:490所示的HCDR3；
    (20)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:377所示的LCDR2，如SEQ ID NO:405所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
    (21)如SEQ ID NO:365所示的LCDR1，如SEQ ID NO:393所示的LCDR2，如SEQ ID NO:424所示的LCDR3，如SEQ ID NO:448所示的HCDR1，如SEQ ID NO:471所示的HCDR2和如SEQ ID NO:498所示的HCDR3；
    (22)如SEQ ID NO:374所示的LCDR1，如SEQ ID NO:401所示的LCDR2，如SEQ ID NO:433所示的LCDR3，如SEQ ID NO:454所示的HCDR1，如SEQ ID NO:478所示的HCDR2和如SEQ ID NO:508所示的HCDR3；
    (23)如SEQ ID NO:358所示的LCDR1，如SEQ ID NO:385所示的LCDR2，如SEQ ID NO:417所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:491所示的HCDR3；
    (24)如SEQ ID NO:359所示的LCDR1，如SEQ ID NO:388所示的LCDR2，如SEQ ID NO:418所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:492所示的HCDR3；
    (25)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2，如SEQ ID NO:403所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
    (26)如SEQ ID NO:366所示的LCDR1，如SEQ ID NO:394所示的LCDR2，如SEQ ID NO:425所示的LCDR3，如SEQ ID NO:449所示的HCDR1，如SEQ ID NO:472所示的HCDR2和如SEQ ID NO:499所示的HCDR3；
    (27)如SEQ ID NO:360所示的LCDR1，如SEQ ID NO:389所示的LCDR2，如SEQ ID NO:413所示的LCDR3，如SEQ ID NO:442所示的HCDR1，如SEQ ID NO:463所示的HCDR2和如SEQ ID NO:487所示的HCDR3；
    (28)如SEQ ID NO:347所示的LCDR1，如SEQ ID NO:376所示的LCDR2，如SEQ ID NO:404所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:456所示的HCDR2和如SEQ ID NO:480所示的HCDR3；
    (29)如SEQ ID NO:348所示的LCDR1，如SEQ ID NO:378所示的LCDR2， 如SEQ ID NO:406所示的LCDR3，如SEQ ID NO:436所示的HCDR1，如SEQ ID NO:457所示的HCDR2和如SEQ ID NO:481所示的HCDR3；
    (30)如SEQ ID NO:361所示的LCDR1，如SEQ ID NO:390所示的LCDR2，如SEQ ID NO:419所示的LCDR3，如SEQ ID NO:445所示的HCDR1，如SEQ ID NO:467所示的HCDR2和如SEQ ID NO:493所示的HCDR3；
    (31)如SEQ ID NO:375所示的LCDR1，如SEQ ID NO:402所示的LCDR2，如SEQ ID NO:434所示的LCDR3，如SEQ ID NO:455所示的HCDR1，如SEQ ID NO:479所示的HCDR2和如SEQ ID NO:509所示的HCDR3；
    (32)如SEQ ID NO:368所示的LCDR1，如SEQ ID NO:395所示的LCDR2，如SEQ ID NO:427所示的LCDR3，如SEQ ID NO:450所示的HCDR1，如SEQ ID NO:473所示的HCDR2和如SEQ ID NO:5015所示的HCDR3；
    (33)如SEQ ID NO:362所示的LCDR1，如SEQ ID NO:388所示的LCDR2，如SEQ ID NO:420所示的LCDR3，如SEQ ID NO:441所示的HCDR1，如SEQ ID NO:462所示的HCDR2和如SEQ ID NO:494所示的HCDR3；
    (34)如SEQ ID NO:352所示的LCDR1，如SEQ ID NO:382所示的LCDR2，如SEQ ID NO:411所示的LCDR3，如SEQ ID NO:440所示的HCDR1，如SEQ ID NO:461所示的HCDR2和如SEQ ID NO:485所示的HCDR3；和
    (35)如SEQ ID NO:367所示的LCDR1，如SEQ ID NO:391所示的LCDR2，如SEQ ID NO:426所示的LCDR3，如SEQ ID NO:435所示的HCDR1，如SEQ ID NO:468所示的HCDR2和如SEQ ID NO:500所示的HCDR3。
  88. 如权利要求87所述的方法，其中所述轻链可变区和重链可变区选自下列组：
    (1)如SEQ ID NO:510所示的轻链可变区和如SEQ ID NO:545所示的重链可变区；
    (2)如SEQ ID NO:511所示的轻链可变区和如SEQ ID NO:546所示的重链可变区；
    (3)如SEQ ID NO:512所示的轻链可变区和如SEQ ID NO:547所示的重链可变区；
    (4)如SEQ ID NO:513所示的轻链可变区和如SEQ ID NO:548所示的重链可变区；
    (5)如SEQ ID NO:514所示的轻链可变区和如SEQ ID NO:549所示的重链可变区；
    (6)如SEQ ID NO:515所示的轻链可变区和如SEQ ID NO:550所示的重链可变区；
    (7)如SEQ ID NO:516所示的轻链可变区和如SEQ ID NO:551所示的重链可变区；
    (8)如SEQ ID NO:517所示的轻链可变区和如SEQ ID NO:552所示的重链可变区；
    (9)如SEQ ID NO:518所示的轻链可变区和如SEQ ID NO:553所示的重链可变区；
    (10)如SEQ ID NO:519所示的轻链可变区和如SEQ ID NO:554所示的重链可变区；
    (11)如SEQ ID NO:520所示的轻链可变区和如SEQ ID NO:555所示的重链可变区；
    (12)如SEQ ID NO:521所示的轻链可变区和如SEQ ID NO:556所示的重链可变区；
    (13)如SEQ ID NO:522所示的轻链可变区和如SEQ ID NO:557所示的重链可变区；
    (14)如SEQ ID NO:523所示的轻链可变区和如SEQ ID NO:558所示的重链可变区；
    (15)如SEQ ID NO:524所示的轻链可变区和如SEQ ID NO:559所示的重链可变区；
    (16)如SEQ ID NO:525所示的轻链可变区和如SEQ ID NO:560所示的重链可变区；
    (17)如SEQ ID NO:526所示的轻链可变区和如SEQ ID NO:561所示的重链可变区；
    (18)如SEQ ID NO:527所示的轻链可变区和如SEQ ID NO:562所示的重链可变区；
    (19)如SEQ ID NO:528所示的轻链可变区和如SEQ ID NO:563所示的重链可变区；
    (20)如SEQ ID NO:529所示的轻链可变区和如SEQ ID NO:564所示的重链可变区；
    (21)如SEQ ID NO:530所示的轻链可变区和如SEQ ID NO:565所示的重链可变区；
    (22)如SEQ ID NO:531所示的轻链可变区和如SEQ ID NO:566所示的重链可变区；
    (23)如SEQ ID NO:532所示的轻链可变区和如SEQ ID NO:567所示的重链可变区；
    (24)如SEQ ID NO:533所示的轻链可变区和如SEQ ID NO:568所示的重链可变区；
    (25)如SEQ ID NO:534所示的轻链可变区和如SEQ ID NO:569所示的重链 可变区；
    (26)如SEQ ID NO:535所示的轻链可变区和如SEQ ID NO:570所示的重链可变区；
    (27)如SEQ ID NO:536所示的轻链可变区和如SEQ ID NO:571所示的重链可变区；
    (28)如SEQ ID NO:537所示的轻链可变区和如SEQ ID NO:572所示的重链可变区；
    (29)如SEQ ID NO:538所示的轻链可变区和如SEQ ID NO:573所示的重链可变区；
    (30)如SEQ ID NO:539所示的轻链可变区和如SEQ ID NO:574所示的重链可变区；
    (31)如SEQ ID NO:540所示的轻链可变区和如SEQ ID NO:575所示的重链可变区；
    (32)如SEQ ID NO:541所示的轻链可变区和如SEQ ID NO:576所示的重链可变区；
    (33)如SEQ ID NO:542所示的轻链可变区和如SEQ ID NO:577所示的重链可变区；
    (34)如SEQ ID NO:543所示的轻链可变区和如SEQ ID NO:578所示的重链可变区；和
    (35)如SEQ ID NO:544所示的轻链可变区域和如SEQ ID NO:579所示的重链可变区域。
  89. 如权利要求87或88所述的方法，其中所述CD123抗体是scFv，其包括选自SEQ ID NO:583-617的氨基酸序列。
  90. 如权利要求87-89任一项所述的方法，其中靶向CD123的CAR包括选自SEQ ID NO:580-582的氨基酸序列。
  91. 如权利要求82所述的方法，其中靶向BCMA的CAR包含抗BCMA抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3选自下列组：
    (1)如SEQ ID NO:232所示的LCDR1，如SEQ ID NO:242所示的LCDR2，如SEQ ID NO:253所示的LCDR3，如SEQ ID NO:264所示的HCDR1，如SEQ ID NO:275所示的HCDR2和如SEQ ID NO:287所示的HCDR3；
    (2)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:243所示的LCDR2，如SEQ ID NO:254所示的LCDR3，如SEQ ID NO:265所示的HCDR1，如SEQ ID NO:276所示的HCDR2和如SEQ ID NO:288所示的HCDR3；
    (3)如SEQ ID NO:234所示的LCDR1，如SEQ ID NO:244所示的LCDR2，如SEQ ID NO:255所示的LCDR3，如SEQ ID NO:266所示的HCDR1，如SEQ ID NO:277所示的HCDR2和如SEQ ID NO:289所示的HCDR3；
    (4)如SEQ ID NO:235所示的LCDR1，如SEQ ID NO:245所示的LCDR2，如SEQ ID NO:255所示的LCDR3，如SEQ ID NO:267所示的HCDR1，如SEQ ID NO:278所示的HCDR2和如SEQ ID NO:290所示的HCDR3；
    (5)如SEQ ID NO:236所示的LCDR1，如SEQ ID NO:246所示的LCDR2，如SEQ ID NO:256所示的LCDR3，如SEQ ID NO:268所示的HCDR1，如SEQ ID NO:279所示的HCDR2和如SEQ ID NO:291所示的HCDR3；
    (6)如SEQ ID NO:237所示的LCDR1，如SEQ ID NO:247所示的LCDR2，如SEQ ID NO:257所示的LCDR3，如SEQ ID NO:269所示的HCDR1，如SEQ ID NO:280所示的HCDR2和如SEQ ID NO:292所示的HCDR3；
    (7)如SEQ ID NO:238所示的LCDR1，如SEQ ID NO:248所示的LCDR2，如SEQ ID NO:258所示的LCDR3，如SEQ ID NO:266所示的HCDR1，如SEQ ID NO:281所示的HCDR2和如SEQ ID NO:293所示的HCDR3；
    (8)如SEQ ID NO:239所示的LCDR1，如SEQ ID NO:249所示的LCDR2，如SEQ ID NO:259所示的LCDR3，如SEQ ID NO:270所示的HCDR1，如SEQ ID NO:282所示的HCDR2和如SEQ ID NO:294所示的HCDR3；
    (9)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:250所示的LCDR2，如SEQ ID NO:260所示的LCDR3，如SEQ ID NO:271所示的HCDR1，如SEQ ID NO:283所示的HCDR2和如SEQ ID NO:295所示的HCDR3；
    (10)如SEQ ID NO:233所示的LCDR1，如SEQ ID NO:251所示的LCDR2，如SEQ ID NO:261所示的LCDR3，如SEQ ID NO:272所示的HCDR1，如SEQ ID NO:284所示的HCDR2和如SEQ ID NO:296所示的HCDR3；
    (11)如SEQ ID NO:240所示的LCDR1，如SEQ ID NO:252所示的LCDR2，如SEQ ID NO:262所示的LCDR3，如SEQ ID NO:273所示的HCDR1，如SEQ ID NO:285所示的HCDR2和如SEQ ID NO:297所示的HCDR3；和
    (12)如SEQ ID NO:241所示的LCDR1，如SEQ ID NO:245所示的LCDR2，如SEQ ID NO:263所示的LCDR3，如SEQ ID NO:274所示的HCDR1，如SEQ ID NO:286所示的HCDR2和如SEQ ID NO:298所示的HCDR3。
  92. 如权利要求91所述的方法，其中所述轻链可变区和重链可变区选自下列组：
    (1)如SEQ ID NO:299所示所示的轻链可变区和如SEQ ID NO:311所示的重链可变区；
    (2)如SEQ ID NO:300所示的轻链可变区域和如SEQ ID NO:312所示的重链可变区域；
    (3)如SEQ ID NO:301所示的轻链可变区域和如SEQ ID NO:313所示的重链可变区域；
    (4)如SEQ ID NO:302所示的轻链可变区域和如SEQ ID NO:314所示的重链可变区域；
    (5)如SEQ ID NO:303所示的轻链可变区域和如SEQ ID NO:315所示的重链可变区域；
    (6)如SEQ ID NO:304所示的轻链可变区域和如SEQ ID NO:316所示的重链可变区域；
    (7)如SEQ ID NO:305所示的轻链可变区域和如SEQ ID NO:317所示的重链可变区域；
    (8)如SEQ ID NO:306所示的轻链可变区域和如SEQ ID NO:318所示的重链可变区域；
    (9)如SEQ ID NO:307所示的轻链可变区和如SEQ ID NO:319所示的重链可变区；
    (10)如SEQ ID NO:308所示的轻链可变区和如SEQ ID NO:320所示的重链可变区；
    (11)如SEQ ID NO:309所示的轻链可变区和如SEQ ID NO:321所示的重链可变区；和
    (12)如SEQ ID NO:310所示的轻链可变区域和如SEQ ID NO:322所示的重链可变区域。
  93. 如权利要求91或92所述的方法，其中所述抗BCMA抗体是scFv，其包括选自SEQ ID NO:323-334的氨基酸序列。
  94. 如权利要求91-93任一项所述的方法，其中靶向BCMA的CAR包括选自SEQ ID NO:335-346的氨基酸序列。
  95. 如权利要求82所述的方法，其中靶向CD19的CAR包含抗CD19抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3是如SEQ ID NO:628所示的LCDR1，如SEQ ID NO:629所示的LCDR2，如SEQ ID NO:630所示的LCDR3，如SEQ ID NO:631所示的HCDR1，如SEQ ID NO:632所示的HCDR2和如SEQ ID NO:633所示的HCDR3。
  96. 如权利要求95所述的方法，其中所述抗CD19抗体包括如SEQ ID NO:635所示的轻链可变区和如SEQ ID NO:634所示的重链可变区。
  97. 如权利要求95或96所述的方法，其中所述抗CD19抗体是scFv，其包括如SEQ ID NO:636所示的氨基酸序列。
  98. 如权利要求95-97任一项所述的方法，其中靶向CD19的CAR包括如 SEQ ID NO:637所示的氨基酸序列。
  99. 如权利要求82所述的方法，其中靶向HER2的CAR包含抗HER2抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3是如SEQ ID NO:618所示的LCDR1，如SEQ ID NO:619所示的LCDR2，如SEQ ID NO:620所示的LCDR3，如SEQ ID NO:621所示的HCDR1，如SEQ ID NO:622所示的HCDR2和如SEQ ID NO:623所示的HCDR3。
  100. 如权利要求99所述的方法，其中抗HER2抗体包括如SEQ ID NO:624所示的轻链可变区和如SEQ ID NO:625所示的重链可变区。
  101. 如权利要求99或100所述的方法，其中所述抗HER2抗体是scFv，其包括如SEQ ID NO:626所示的的氨基酸序列。
  102. 如权利要求99-101任一项所述的方法，其中靶向HER2的CAR包括如SEQ ID NO:627所示的氨基酸序列。
  103. 如权利要求82所述的方法，其中靶向IL13Ra2的CAR包含抗IL13Ra2抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3是如SEQ ID NO:638所示的LCDR1，如SEQ ID NO:639所示的LCDR2，如SEQ ID NO:640所示的LCDR3，如SEQ ID NO:641所示的HCDR1，如SEQ ID NO:642所示的HCDR2和如SEQ ID NO:642所示的HCDR3。
  104. 如权利要求103所述的方法，其中所述抗IL13Ra2抗体包括如SEQ ID NO:644所示的轻链可变区和如SEQ ID NO:645所示的重链可变区。
  105. 如权利要求103或105所述的方法，其中所述抗IL13Ra2抗体是scFv，其包括如SEQ ID NO:646所示的氨基酸序列。
  106. 如权利要求103-105任一项所述的方法，其中靶向IL13Ra2的CAR包括如SEQ ID NO:647所示的氨基酸序列。
  107. 如权利要求82所述的方法，其中靶向B7H3的CAR包含抗B7H3抗体作为其结合结构域，所述抗间皮素抗体包括轻链可变区和重链可变区，轻链可变区包含LCDR1、LCDR2和LCDR3，重链可变区包含HCDR1、HCDR2和HCDR3，其中LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3是如SEQ ID NO:648所示的LCDR1，如SEQ ID NO:649所示的LCDR2，如SEQ ID NO:650所示的LCDR3，如SEQ ID NO:651所示的HCDR1，如SEQ ID NO:652所示的HCDR2和如SEQ ID NO:653所示的HCDR3。
  108. 如权利要求107所述的方法，其中所述抗B7H3抗体包括SEQ ID NO:654所示的轻链可变区和SEQ ID NO:655所示的重链可变区。
  109. 如权利要求107或108所述的方法，其中所述抗B7H3抗体是scFv，其包括如SEQ ID NO:656所示的氨基酸序列。
  110. 如权利要求107-109任一项所述的方法，其中靶向B7H3的CAR包括如SEQ ID NO:657所示的氨基酸序列。
  111. 杀伤受试者中的靶细胞的方法，包括给所述受试者施用如权利要求43-77任一项所述的药物组合或免疫细胞。
  112. 如权利要求78-111任一项所述的方法，其中所述靶细胞是肿瘤细胞。
  113. 如权利要求112所述的方法，其中所述肿瘤细胞表达间皮素、CD123、BCMA、HER2、IL13Ra2、CD19或B7H3。
  114. 在有需要的受试者中治疗疾病的方法，包括给受试者施用治疗有效量的如权利要求44-77任一项所述的药物组合或免疫细胞。
  115. 如权利要求114所述的方法，其中所述疾病是肿瘤、癌症、病毒感染或自身免疫性疾病。
  116. 如权利要求115所述的方法，其中所述癌症表达间皮素、CD123、BCMA、HER2、IL13Ra2或B7H3。
  117. 如权利要求115或116所述的方法，其中所述癌症是实体瘤或血液学癌症。
  118. 如权利要求115-117任一项所述的方法，其中所述癌症是急性骨髓性白血病(AML)、B型急性淋巴细胞白血病(B-ALL)、T型急性淋巴细胞白血病(T-ALL)、B细胞前体急性淋巴细胞白血病(BCP-ALL)或blastic placytoid dendritic cell neoplasm(BPDCN)。非霍奇金淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、人类B细胞前体白血病、多发性骨髓瘤或恶性淋巴瘤。
  119. 如权利要求115-117任一项所述的方法，其中癌症是间皮瘤、胰腺癌、卵巢癌、肺癌、乳腺癌、胃癌、宫颈癌、尿路上皮癌、食道癌、膀胱癌、结直肠癌、子宫内膜癌、肾癌、头颈癌、肉瘤、胶质细胞瘤、前列腺癌、甲状腺癌或胶质瘤。
  120. 如权利要求1-12任一项所述的人源化抗CD28抗体或其抗原结合片段，其中所述人源化抗CD28抗体的重链可变区融合至免疫球蛋白重链恒定区，和/或所述人源化抗CD28抗体的轻链可变区融合至免疫球蛋白轻链恒定区。
  121. 如权利要求120所述的人源化抗CD28抗体或其抗原结合片段，其中所述免疫球蛋白重链恒定区包含SEQ ID NO:81，所述免疫球蛋白轻链恒定区包含SEQ ID NO:82。
  122. 诱导T细胞增殖的方法，包括使如权利要求1-12任一项所述的人源化抗CD28抗体或其抗原结合片段或如权利要求13-37任一项所述的多特异性抗体、如权利要求38所述的多核苷酸、如权利要求39-41任一项所述的载体或如权利要求43所述的药物组合物与T细胞接触。
  123. 如权利要求122所述的方法，所述方法进一步包括将所述多核苷酸或载体引入所述T细胞，并使所述T细胞表达所述人源化抗CD28抗体或其抗原结合片段或所述多特异性抗体。
  124. 如权利要求122或123所述的方法，其中所述T细胞表达嵌合抗原受体(CAR)。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024077118A2 (en) 2022-10-06 2024-04-11 Bicara Therapeutics Inc. Multispecific proteins and related methods

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
CN107427573A (zh) * 2014-12-24 2017-12-01 耐克西缪恩有限公司 用于免疫疗法的纳米颗粒组合物和方法
CN109476732A (zh) * 2016-04-13 2019-03-15 赛诺菲 三特异性和/或三价结合蛋白
CN113286821A (zh) * 2018-12-21 2021-08-20 豪夫迈·罗氏有限公司 靶向肿瘤的激动性cd28抗原结合分子
CN113286822A (zh) * 2018-12-21 2021-08-20 豪夫迈·罗氏有限公司 靶向肿瘤的超激动性cd28抗原结合分子
CN114349863A (zh) 2022-03-21 2022-04-15 上海优替济生生物医药有限公司 一种抗cd19抗体及其制备方法和用途
CN114773477A (zh) 2022-06-23 2022-07-22 上海优替济生生物医药有限公司 抗b7-h3抗体及其用途
CN114805581A (zh) 2022-06-29 2022-07-29 上海优替济生生物医药有限公司 靶向il13ra2的抗体、嵌合抗原受体及其用途
CN114805584A (zh) 2022-06-30 2022-07-29 上海优替济生生物医药有限公司 抗原结合蛋白及其用途

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020253722A1 (en) * 2019-06-17 2020-12-24 Eucure (Beijing) Biopharma Co., Ltd Anti-cd40 antibodies and uses thereof
CA3158527A1 (en) * 2019-10-23 2021-04-29 Lyvgen Biopharma Holdings Limited Anti-cd40 binding molecules and bi-specific antibodies comprising such
US20210214454A1 (en) * 2020-01-10 2021-07-15 Symphogen A/S Anti-cd40 antibodies and compositions
EP4118118A1 (en) * 2020-03-09 2023-01-18 Bristol-Myers Squibb Company Antibodies to cd40 with enhanced agonist activity
KR20220160555A (ko) * 2020-03-30 2022-12-06 바이오션, 인코포레이티드 Cd40에 결합하는 항체 및 이의 용도

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
CN107427573A (zh) * 2014-12-24 2017-12-01 耐克西缪恩有限公司 用于免疫疗法的纳米颗粒组合物和方法
CN109476732A (zh) * 2016-04-13 2019-03-15 赛诺菲 三特异性和/或三价结合蛋白
CN113286821A (zh) * 2018-12-21 2021-08-20 豪夫迈·罗氏有限公司 靶向肿瘤的激动性cd28抗原结合分子
CN113286822A (zh) * 2018-12-21 2021-08-20 豪夫迈·罗氏有限公司 靶向肿瘤的超激动性cd28抗原结合分子
CN114349863A (zh) 2022-03-21 2022-04-15 上海优替济生生物医药有限公司 一种抗cd19抗体及其制备方法和用途
CN114773477A (zh) 2022-06-23 2022-07-22 上海优替济生生物医药有限公司 抗b7-h3抗体及其用途
CN114805581A (zh) 2022-06-29 2022-07-29 上海优替济生生物医药有限公司 靶向il13ra2的抗体、嵌合抗原受体及其用途
CN114805584A (zh) 2022-06-30 2022-07-29 上海优替济生生物医药有限公司 抗原结合蛋白及其用途

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Fundamental Immunology", 1989, RAVEN PRESS
"Remington, The Science and Practice of Pharmacy", 1995
ALTSCHUL ET AL., J. MOL., vol. 215, 1990, pages 403 - 410
BAJORATH, J.R.J. PEACHP. S. LINSLEY: "Immunoglobulin fold characteristics of B7-1 (CD80) and B7-2 (CD86)", PROTEIN SCI, vol. 3, no. 11, 1994, pages 2148 - 50, XP009012247
BARBAS ET AL., PROC. NAT. ACAD. SCI. USA, vol. 113, 1994, pages 3809 - 3813
BEADUDRY, D.PERREAULT, J.: "An efficient strategy for the synthesis of circular RNA molecules", NUCLEIC ACIDS RESEARCH, vol. 23, no. 15, 1995, pages 3064 - 3066, XP055710686, DOI: 10.1093/nar/23.15.3064
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CLACKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628
D.WILKINSON: "The Scientist", vol. 14, 17 April 2000, THE SCIENTIST, INC., pages: 25 - 28
EDNER, N.M.: "Targeting co-stimulatory molecules in autoimmune disease", NAT REV DRUG DISCOV, vol. 19, no. 12, 2020, pages 860 - 883, XP037320743, DOI: 10.1038/s41573-020-0081-9
GREENBERG ET AL., NATURE, vol. 374, 1995, pages 168 - 73
HAMERS-CASTERMAN ET AL., NATURE, vol. 363, 1993, pages 446 - 8
HANSEN, J.A., P.J. MARTIN, AND R.C. NOWINSKI: "Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes", IMMUNOGENETICS, vol. 10, no. 1, 1980, pages 247 - 260
HASSANZADEH-GHASSABEH ET AL., NANOMEDICINE, vol. 8, 2013, pages 1013 - 26
HAWKINS ET AL., J. MOL BIOL., vol. 254, 1992, pages 889 - 896
HAWKINS ET AL., J. MOL. BIOL., vol. 226, 1992, pages 889 - 896
HILLION, S. ET AL.: "The Innate Part of the Adaptive Immune System", CLIN REV ALLERGY IMMUNOL, vol. 58, no. 2, 2020, pages 151 - 154, XP037061044, DOI: 10.1007/s12016-019-08740-1
HOLLIGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
JACKSON ET AL., J. IMMUNOL., vol. 154, no. 7, 1995, pages 3310 - 3319
JOYCE, J.A.D.T. FEARON: "T cell exclusion, immune privilege, and the tumor microenvironment", SCIENCE, vol. 348, no. 6230, 2015, pages 74 - 80, XP055522170, DOI: 10.1126/science.aaa6204
KOHLER ET AL., NATURE, vol. 256, 1975, pages 495
LEFRANC ET AL., DEV. COMPARAT. IMMUNOL., vol. 27, 2003, pages 55 - 77
LOWMAN ET AL., BIOCHEMISTRY, vol. 30, no. 45, 1991, pages 10832 - 10838
MARKS ET AL., BIOTECHNOLOGY, vol. 10, 1992, pages 779 - 783
MARKS ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
MICURA, R.: "Cyclic Oligoribonucleotides (RNA) by Solid-Phase Synthesis", CHEMISTRY A EUROPEAN JOURNAL, vol. 5, no. 7, 1999, pages 2077 - 2082, XP055328566, DOI: 10.1002/(SICI)1521-3765(19990702)5:7<2077::AID-CHEM2077>3.0.CO;2-U
MORRISON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6851 - 6855
NEEDLEMAN, SAUL B.; AND WUNSCH, CHRISTIAN D.: "A general method applicable to the search for similarities in the amino acid sequence of two proteins", JOURNAL OF MOLECULAR BIOLOGY, vol. 48, no. 3, 1970, pages 443 - 53, XP024011703, DOI: 10.1016/0022-2836(70)90057-4
PETKOVIC, S.MULLER, S.: "RNA circularization strategies in vivo and in vitro", NUCLEIC ACIDS RESEARCH, vol. 43, no. 4, 2015, pages 2454 - 2465, XP055488942, DOI: 10.1093/nar/gkv045
SCHIER ET AL., GENE, vol. 169, 1995, pages 147 - 155
TACKE, M. ET AL.: "CD28-mediated induction of proliferation in resting T cells in vitro and in vivo without engagement of the T cell receptor: evidence for functionally distinct forms of CD28", EUR J IMMUNOL, vol. 27, no. 1, 1997, pages 239 - 47, XP071225800, DOI: 10.1002/eji.1830270136
TAN, P. ET AL.: "Superhumanized'' antibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28", J IMMUNOL, vol. 169, no. 2, 2002, pages 1119 - 25, XP008056579
TERRAZZANO, G. ET AL.: "Interaction between natural killer and dendritic cells: the role of CD40, CD80 and major histocompatibility complex class i molecules in cytotoxicity induction and interferon-gamma production", SCAND J IMMUNOL, vol. 59, no. 4, 2004, pages 356 - 62

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