WO2021190551A1 - 结合cd19的嵌合抗原受体及其用途 - Google Patents

结合cd19的嵌合抗原受体及其用途 Download PDF

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WO2021190551A1
WO2021190551A1 PCT/CN2021/082683 CN2021082683W WO2021190551A1 WO 2021190551 A1 WO2021190551 A1 WO 2021190551A1 CN 2021082683 W CN2021082683 W CN 2021082683W WO 2021190551 A1 WO2021190551 A1 WO 2021190551A1
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amino acid
car
cell
acid sequence
seq
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PCT/CN2021/082683
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French (fr)
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丁艳萍
鲁薪安
何霆
齐菲菲
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上海先博生物科技有限公司
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Publication of WO2021190551A1 publication Critical patent/WO2021190551A1/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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the present invention relates to the fields of immunology and molecular biology, especially chimeric antigen receptor (Chimeric Antigen Receptor, CAR) modified immune cells and uses thereof.
  • CAR Chimeric Antigen Receptor
  • Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) technology combines the specificity of antibodies and the killing effect of T cells to form an effective way of adoptive immunity.
  • the original CAR usually consists of an extracellular antigen binding domain, a hinge region, a transmembrane region, and an intracellular signal domain.
  • the second-generation CAR adds intracellular costimulatory domains, such as CD28, CD137, OX40, etc., which are usually located between the transmembrane region and the intracellular signal domain.
  • the third-generation CAR uses two costimulatory domains to enhance CAR-T activation.
  • CAR-T technology has achieved great success in the treatment of hematomas, especially CAR-T targeting CD19, but there are still some patients who fail to respond or relapse after responding, and there are still insufficient effects in durability and toxic and side effects.
  • Two important issues regarding the efficacy and safety of CAR-T are: 1) CAR-T expansion and continuous deficiency in the body; 2) CAR-T can cause cytokine storm, neurotoxicity and off-target effects and other toxic and side effects.
  • the killing ability, in vivo expansion ability and safety of CAR-T cells to target cells are affected by many factors, including CAR design, antigen density on the surface of target cells, CAR-T preparation process, etc., of which CAR design is the key It is necessary to consider the affinity of the antigen-binding domain of the CAR and the target, the composition of the costimulatory signal domain, and the composition of the CD3 ⁇ signal domain.
  • the design of each domain of the CAR molecule depends on the specificity of the target, expression abundance, structural characteristics on the cell membrane surface, as well as the pathological characteristics of the indications and treatment strategies.
  • the present invention discloses an isolated antigen binding domain, which comprises:
  • a single chain variable region fragment capable of binding to CD19 which includes a light chain variable region and a heavy chain variable region;
  • the protective peptide comprises a polypeptide with a length of 8-15 amino acids rich in polar amino acids, and the protective peptide is operably linked to the light chain variable Region or the N-terminal, C-terminal of the variable region of the heavy chain, or between the variable region of the light chain and the variable region of the heavy chain;
  • the protective peptide and the variable region of the light chain or the variable region of the heavy chain can be selectively connected via a flexible linker peptide rich in glycine and serine.
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO:1
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:2.
  • the protective peptide comprises an amino acid sequence selected from SEQ ID NOs: 6, 7, 8, 9 or 10 or at least 75% or more, 80% or more, 85% or more of the amino acid sequence thereof. %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the protective peptide is operably linked to the N-terminus or C-terminus of the light chain variable region; in one embodiment, the protective peptide is operably linked to the heavy chain The N-terminus or C-terminus of the variable region; in one embodiment, the protective peptide is operably linked between the light chain variable region and the heavy chain variable region.
  • the protective peptide and the light chain variable region or the heavy chain variable region can be selectively connected by a flexible linker peptide such as (G 4 S) 2 rich in glycine and serine, for example, in the light chain variable region
  • a flexible linker peptide such as (G 4 S) 2 rich in glycine and serine, for example, in the light chain variable region
  • the N-terminal can be operably connected to the protective peptide and the flexible linker peptide.
  • the antigen-binding domain comprises SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39.
  • the amino acid sequence of or with it has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or more , 98% or more, 99% or more identical amino acid sequences.
  • the present invention discloses an isolated chimeric antigen receptor, which includes the antigen binding domain as described above.
  • the chimeric antigen receptor further includes a costimulatory domain and/or an intracellular signal transduction domain.
  • the costimulatory domain is selected from CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C , B7-H3 or any combination thereof.
  • the intracellular signal transduction domain is selected from CD3 ⁇ .
  • the costimulatory domain is selected from 4-1BB.
  • the costimulatory domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof.
  • 4-1BB variants include one or more of Q20F, T21P, T22E, Q23E, F32Q, P33T, E34T, E35Q amino acid substitutions in SEQ ID NO: 41 or the variant is described in SEQ ID NO: 41
  • the N-terminal or C-terminal of the amino acid sequence is connected with an amino acid sequence as described in SEQ ID NO: 53, 54, 55.
  • the 4-1BB costimulatory domain comprises a variant of the amino acid sequence shown in SEQ ID NO: 41.
  • the 4-1BB variant comprises at least the Q20F amino acid substitution, and further comprises T21P And Q23E; in a specific embodiment, the variant includes the Q20F amino acid substitution in SEQ ID NO: 41; in a specific embodiment, the variant includes Q20F, T21P and Q23E in SEQ ID NO: 41 Amino acid substitutions; in a specific embodiment, the variants include Q20F, T21P, T22E, Q23E, F32Q, P33T, E34T, E35Q amino acid substitutions in SEQ ID NO: 41.
  • the costimulatory domain is selected from 4-1BB, preferably, the 4-1BB costimulatory domain comprises the amino acid shown in SEQ ID NOs: 43, 45, 47, 49 or 51 The sequence or it has at least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98 % And 99% or more identical amino acid sequences.
  • the CD3 ⁇ intracellular signal transduction domain comprises the amino acid sequence set forth in SEQ ID NO: 56 or a variant thereof, and the variants include V2L, D9E, Q14K, Q15K, Y90F, L104Y, One or more of H105R and M106H.
  • the CD3 ⁇ variant comprises a Q14K amino acid substitution.
  • the CD3 ⁇ variant includes Q15K amino acid substitutions, and may further include V2L and Y90F amino acid substitutions, and may further include D9E.
  • the CD3 ⁇ variants include V2L and Q15K.
  • the CD3 ⁇ variant comprises V2L, D9E, Q15K and Y90F amino acid substitutions; in one embodiment, the CD3 ⁇ variant comprises V2L, D9E, Q15K, Y90F, L104Y, H105R and M106H amino acid substitutions.
  • the CD3 ⁇ intracellular signal transduction domain comprises an amino acid sequence as shown in SEQ ID NOs: 58, 60, 62, 64 or 66 or has at least 75%, 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical amino acid sequences.
  • the chimeric antigen receptor may further include a hinge region and a transmembrane domain.
  • the hinge region and the transmembrane domain are selected from IgG1, IgG4, CD8 ⁇ , CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1 or CD34 The hinge region and transmembrane domain.
  • the chimeric antigen receptor may further include a signal peptide.
  • the chimeric antigen receptor includes sequentially linked:
  • Antigen-binding domain comprising or having the amino acid sequence shown in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39 At least 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99 Amino acid sequences with more than% identity;
  • 4-1BB costimulatory domain comprising the amino acid sequence shown in SEQ ID NOs: 41, 43, 45, 47, 49 or 51 or at least 75% or more, 80% or more, 85% or more or 90% therewith More than, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical amino acid sequence; and
  • CD3 ⁇ intracellular signal transduction domain which contains the amino acid sequence shown in SEQ ID NOs: 58, 60, 62, 64 or 66 or has at least 75%, 80%, 85%, 90% with it , 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identical amino acid sequence.
  • the chimeric antigen receptor includes SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, or 124 amino acid sequence or at least 75%, 80%, 85%, 90% More than, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical amino acid sequence.
  • the present invention further discloses an isolated nucleic acid molecule, which encodes the antigen binding domain or the chimeric antigen receptor as described in the present invention.
  • the present invention discloses a vector comprising the isolated nucleic acid molecule as described above; preferably, wherein the vector is selected from one of DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector kind or more.
  • the vector is an expression vector or a cloning vector.
  • the present invention discloses a cell comprising the isolated nucleic acid molecule as described above or the vector as described above.
  • the cells are T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, cytotoxic T cells, tumor infiltrating T cells, or regulatory T cells.
  • the cells are selected from human peripheral blood T lymphocytes.
  • the present invention discloses a pharmaceutical composition, which comprises one or more selected from the following:
  • a pharmaceutically acceptable carrier diluent or excipient.
  • the present invention discloses a method for preparing a cell as described above, which comprises: introducing a nucleic acid encoding a chimeric antigen receptor as described above into the cell.
  • the present invention also discloses the use of the aforementioned antigen-binding domain, chimeric antigen receptor, nucleic acid molecule, carrier, cell or pharmaceutical composition in the preparation of medicines; the aforementioned antigen-binding structure Use of domains, chimeric antigen receptors, nucleic acid molecules, vectors, cells or pharmaceutical compositions in the preparation of drugs for the treatment of diseases, wherein the diseases are preferably selected from tumors, autoimmune diseases, or infections caused by viruses or bacteria sexual disease.
  • the present invention discloses a method for carrying out cellular immunotherapy in a subject suffering from a disease, which comprises administering to the subject the pharmaceutical composition as described above or the cell as described above, wherein the disease is preferably selected from Tumors, autoimmune diseases, or infectious diseases caused by viruses or bacteria.
  • the tumor is a solid tumor or hematological cancer mediated by tumor-specific molecules
  • the solid tumor is preferably breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, Colorectal cancer, kidney cancer, liver cancer, brain cancer, gastric cancer, gastrointestinal stromal tumor, lung cancer and thyroid cancer
  • said hematological cancer is preferably selected from acute leukemia, which includes acute lymphocytic leukemia, acute myeloid leukemia , Acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia; chronic leukemia, which includes chronic myeloid (granulocyte) leukemia, chronic myelogenous leukemia And chronic lymphocytic leukemia and refractory CD19+ leukemia and lymphoma; polycythemia vera; lymphoma; mantle cell lymphoma; diffuse large B-cell lympho
  • the tumor is a recurring tumor or cancer.
  • FIG. 1 shows the transduction efficiency of different CAR molecules in T cells.
  • Figure 2 shows the comparison of biacore affinity data between FMC63 scFv and scFv after modification.
  • Figure 3 shows the binding of CD19 CAR-T to K562-CD19 cells (* represents P ⁇ 0.05, ** represents P ⁇ 0.01).
  • Figure 4 shows the killing efficiency of CD19 CAR-T on K562-CD19 cells.
  • Figure 5 shows the proliferation of CD19 CAR-T in K562-CD19 cells stimulated for 3 days (compared with FMC63 CAR-T, * means P ⁇ 0.05, ** means P ⁇ 0.01).
  • Figure 6 shows the cytokine expression of CD19 CAR-T after K562-CD19 cell stimulation for 12 hours (compared with FMC63 CAR-T, * means P ⁇ 0.05, ** means P ⁇ 0.01).
  • Fig. 7 shows the anti-tumor ability and the ability of inhibiting tumor recurrence of CD19 CAR-T cells.
  • Figure 8 shows the tumor suppressive effect of CD19 CAR-T on tumor-bearing mice.
  • Figure 9 shows the amount of CD19 CAR-T in the peripheral blood of tumor-bearing mice.
  • Figure 10 shows a schematic diagram of the metabolic curve of CD19 CAR-T in vivo.
  • antigen refers to a molecule that causes an immune response, which may involve the production of antibodies, or the activation of specific immunocompetent cells.
  • any macromolecule including all proteins or peptides, can be used as an antigen.
  • the antigen can be derived from recombinant or genomic DNA.
  • DNA that includes a nucleotide sequence or part of a nucleotide sequence that encodes a protein that causes an immune response, and encodes the term "antigen” as used herein.
  • the antigen need not be encoded by the full-length nucleotide sequence of the gene alone.
  • the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to elicit a desired immune response.
  • antigens do not need to be encoded by "genes" at all, and antigens can be produced, synthesized, or derived from biological samples.
  • biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.
  • chimeric antigen receptor refers to an artificial receptor that is engineered to express on immune effector cells and specifically bind to an antigen, such as an artificial T cell receptor.
  • CAR can be used as a therapy using adoptive cell transfer. Lymphocytes, such as T cells, are removed from the patient and modified so that they express receptors specific to the specific form of antigen.
  • CAR can also include an intracellular activation domain, a transmembrane domain, and an extracellular domain. The extracellular domain includes a tumor-associated antigen binding region.
  • the CAR includes a single-chain variable region fragment (scFv), which is fused to a transmembrane domain and an intracellular domain.
  • the specificity of CAR design can be derived from receptor ligands (e.g., peptides).
  • the CAR can target cancer by redirecting the specificity of T cells expressing CARs specific for tumor-associated antigens.
  • the term "antigen binding domain” as used herein is also called “ligand binding domain” and refers to an oligopeptide or polypeptide capable of binding a ligand.
  • the domain is capable of interacting with cell surface molecules.
  • the extracellular ligand binding domain can be selected to identify ligands that act as cell surface markers on target cells associated with a particular disease state. Therefore, examples of cell surface markers that can serve as ligands include those cell surface markers that are associated with viral, bacterial and parasitic infections, autoimmune diseases, and cancer cells.
  • the antigen-binding domain may include a single-chain variable region fragment (single-chain antibody). The antigen binding domain may only bind a part of the antigen.
  • the part of the antigen molecule responsible for the specific interaction with the antigen binding domain is called the "epitope" or "antigenic determinant”.
  • the antigen binding domain usually includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDR), interspersed with more conservative regions called framework regions (FR).
  • CDRs of the antibodies and antigen binding domains disclosed in the present invention are defined or recognized by Kabat numbering.
  • single chain variable region fragment refers to an antibody formed by recombinant DNA technology, in which the variable region of the immunoglobulin heavy chain and the variable region of the light chain are passed through amino acid peptides. Linker is connected.
  • a variety of methods for generating single-chain antibodies are known, including U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883 ; Ward et al. (1989) Nature 334: 54454; Skerra et al. (1988) Science 242: 1038-1041.
  • variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the CDR in the heavy chain is abbreviated as VH-CDR, such as VH-CDR1, VH-CDR2, VH-CDR3, and the CDR in the light chain is abbreviated as VL-CDR, such as VL-CDR1, VL-CDR2, VL-CDR3.
  • the CDRs of the antibodies and antigen binding domains disclosed in the present invention are defined or recognized by Kabat numbering.
  • peptide As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds.
  • the protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of the protein or peptide.
  • a polypeptide includes any peptide or protein that includes two or more amino acids joined to each other by peptide bonds.
  • the term refers to short chains, which are also commonly referred to in the art as peptides, oligopeptides, and oligomers; and both longer chains, which are commonly referred to in the art as proteins , Which has many types.
  • Polypeptide includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides or a combination thereof.
  • the terms "protective peptide” and “protective peptide” are used interchangeably, and refer to an oligopeptide operably linked to the N-terminal, C-terminal or any position in the middle of the scFv, and the peptide can reduce the scFv and the target.
  • the protective peptide may be connected between the N-terminus or C-terminus of the light chain variable region, the N-terminus or C-terminus of the heavy chain variable region, or between the light chain variable region and the heavy chain variable region.
  • the terms "flexible linker peptide” and “flexible peptide” are used interchangeably and refer to an amino acid sequence rich in glycine and serine, which can be (G 4 S) n , where n is an integer greater than or equal to 1, such as 1 Any integer from to 4 can be, for example, (G 4 S) 2 , that is, GGGGSGGGGS.
  • the protective peptide and the light chain variable region or the heavy chain variable region can be selectively connected by a flexible linker peptide such as (G 4 S) 2 rich in glycine and serine, for example, in the light chain variable region.
  • the N-terminal can be operably connected to the protective peptide and the flexible linker peptide.
  • any glycine and serine-rich amino acid sequence capable of attaching a protective peptide to the variable region of the light chain or the variable region of the heavy chain is within the scope of the flexible linker peptide.
  • vector refers to a molecular tool that transports, transduces, and expresses contained exogenous genes of interest (such as the polynucleotides of the present invention) in target cells, and the tools provide suitable molecular tools in target cells.
  • the nucleotide sequence that initiates transcription that is, the promoter.
  • the vector includes the isolated nucleic acid, and it can be used to deliver the isolated nucleic acid to the inside of the cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • the term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • Expression vector refers to a vector that includes a recombinant polynucleotide including an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • the expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art that incorporate recombinant polynucleotides, such as plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and Adeno-associated virus).
  • Identity refers to the sequence identity between two nucleic acid molecules or polypeptides. The identity can be determined by comparing the positions in each sequence aligned for comparison purposes. When a position in the compared sequence is occupied by the same base, then the molecules are the same at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences.
  • Various alignment algorithms and/or programs can be used to calculate the consistency between two sequences, including those available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, for example, default settings FASTA or BLAST.
  • polypeptide that has at least 70%, 85%, 90%, 95%, 98%, or 99% identity with a specific polypeptide described herein and preferably exhibits substantially the same function is envisaged, and encodes the above-mentioned polypeptide Of polynucleotides.
  • isolated refers to a change or removal from a natural state.
  • a nucleic acid or peptide naturally present in a living animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from its coexisting substance in its natural state is “isolated.”
  • the isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment, for example, in a host cell.
  • nucleotide sequence of a nucleic acid molecule that "encodes" a certain protein or an amino acid sequence of a certain protein used herein includes all nucleotide sequences that encode the same amino acid sequence in a degeneracy form.
  • the nucleotide sequence may also include one or more introns.
  • operably linked refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, which results in the expression of the heterologous nucleic acid sequence.
  • first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence
  • second nucleic acid sequence the first nucleic acid sequence and the second nucleic acid sequence are operably linked.
  • the promoter affects the transcription or expression of the coding sequence
  • the promoter is operably linked to the coding sequence.
  • the operably linked DNA sequences are adjacent and join two protein coding regions in the same reading frame when necessary.
  • variant means a polypeptide variant obtained by substituting at least one residue in the amino acid sequence of the parent molecule or adding at least one amino acid residue at the N-terminus or C-terminus.
  • anti-tumor ability or "tumor inhibition” as used herein is a biological effect, which can be caused by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or it is related to cancerous conditions The improvement of various physiological symptoms of the patient is clearly indicated.
  • Anti-tumor ability or “tumor inhibition” can also be expressed by the ability of the peptides, polynucleotides, cells and antibodies of the present disclosure to prevent tumors.
  • the term “subject” includes any human or non-human animal.
  • non-human animals includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, reptiles Wait.
  • the terms “patient” or “subject” are used interchangeably.
  • the preferred subject is a human.
  • treatment refers to administering to a subject an effective amount of a cell having a polynucleotide sequence of a target gene altered ex vivo according to the method described herein, so that the subject has the disease.
  • At least one symptom reduction or improvement of the disease for example, a beneficial or desired clinical outcome.
  • beneficial or desired clinical results include, but are not limited to, reduction of one or more symptoms, reduction of disease severity, stabilization of disease state (ie, no deterioration), delay or reduction of disease progression Slowness, improvement or remission of the disease state, and remission (whether partial or full remission), whether detectable or undetectable.
  • Treatment can refer to prolonging survival compared to expected survival in the absence of treatment.
  • treatment can improve the condition of the disease, but may not be a complete cure for the disease.
  • treatment includes prevention.
  • the treatment is “effective” if the progression of the disease is reduced or stopped.
  • Treatment can also mean prolonging survival compared to expected survival in the absence of treatment.
  • Patients in need of treatment include those who have been diagnosed with a condition related to the expression of the polynucleotide sequence, and may develop such a condition due to genetic susceptibility or other factors.
  • autoimmune disease as used herein is defined as a disorder caused by an autoimmune response. Autoimmune diseases are the result of inappropriate and excessive responses to self-antigens. Examples of autoimmune diseases include but are not limited to Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes (type 1), dystrophic bullous epidermis Lysis, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, spondyloarthropathy, thyroiditis, va
  • Example 1 CAR molecule construction and CAR-T preparation
  • This example takes the design of the preparation of CD19 CAR molecules and CAR-T cells with different antigen binding domains, costimulatory signal domains and CD3 ⁇ signal domains as an example.
  • Antigen-binding domain construction Without changing the nature and function of the scFv, replace the scFv antibody framework sequence with human germline antibody framework sequence; or at the N-terminal, C-terminal, or heavy chain variable region of the scFv A protective peptide with a length of 8-15 amino acids rich in polar amino acids is added between the variable regions of the light chain. Taking FMC63 scFv as an example below, the sequences of different antigen binding domains are shown in Table 1. The protective peptide sequence is selected from the amino acid sequence shown in SEQ ID NOs: 6, 7, 8, 9 or 10.
  • the protective peptide sequence is added to the N-terminal, C-terminal, or between the variable region of the heavy chain and the variable region of the light chain of the scFv to obtain the scFv with the protective peptide sequence added, such as SEQ ID NOs: 11, 13, 15, The amino acid sequence shown in 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39.
  • the initial FMC63 scFv amino acid sequence is shown in SEQ ID NO: 3; the CAR (CAR-1) amino acid sequence after humanization of FMC63 scFv amino acids is shown in SEQ ID NO: 68, and the nucleotide sequence is shown in SEQ ID NO: 69;
  • the amino acid sequence of CAR (CAR-2) with a protective peptide added to the N-terminus of FMC63 scFv is shown in SEQ ID NO: 70, and the nucleotide sequence is shown in SEQ ID NO: 71;
  • FMC63 scFv The amino acid sequence of CAR (CAR-3) with a protective peptide added between the variable region of the heavy chain and the variable region of the light chain is shown in SEQ ID NO: 72, and the nucleotide sequence is shown in SEQ ID NO: 73;
  • the original 4-1BB amino acid sequence is shown in SEQ ID NO: 41, and the nucleotide sequence is shown in SEQ ID NO: 42; the PEEE motif in the natural sequence of the costimulatory domain of 4-1BB is replaced with the CAR of PEQE (CAR -5)
  • the amino acid sequence is shown in SEQ ID NO: 76, and the nucleotide sequence is shown in SEQ ID NO: 77; the TTQE motif in the natural sequence of the 4-1BB costimulatory domain is replaced with a PTEE motif, and the PEEE motif is replaced
  • the amino acid sequence of CAR (CAR-6) with the PEQE motif replaced by the sequence is shown in SEQ ID NO: 78, and the nucleotide sequence is shown in SEQ ID NO: 79; the QTTQE sequence in the natural sequence of the costimulatory domain of 4-1BB Substituting the FPEEE sequence, the CAR (CAR-7) amino acid sequence of the QTTQE sequence replaced by the FPEEE
  • the amino acid sequence of the initial CD3 ⁇ signal domain is shown in SEQ ID NO:56, and the nucleotide sequence is shown in SEQ ID NO:57; the amino acid sequence of CAR (CAR-10) after Q15K substitution of the CD3 ⁇ signal domain is shown in SEQ ID
  • the nucleotide sequence is shown in SEQ ID NO: 87; the amino acid sequence of CAR (CAR-11) after the CD3 ⁇ signal domain is replaced by V2L, Q15K and Y90F is shown in SEQ ID NO: 88.
  • the nucleotide sequence is shown in SEQ ID NO: 89; the CAR (CAR-12) amino acid sequence after the CD3 ⁇ signal domain is replaced by V2L, D9E, Q15K and Y90F is shown in SEQ ID NO: 90, and the nucleotide sequence is shown in SEQ ID NO: 91.
  • the nucleotide sequences of each of the above schemes are synthesized by gene synthesis, and then connected with other sequences by PCR or homologous recombination to finally form a gene molecule encoding CAR.
  • the gene molecule encoding CAR was inserted into the lentiviral vector pLenti6.3/V5 (Thermo Fisher, Waltham, MA, USA).
  • T cells were isolated from peripheral blood mononuclear cells (Miaotong (Shanghai) Biotechnology Co., Ltd., China) of healthy volunteers. The separated and purified T cells were inoculated and cultured into X-VIVO 15 medium (Lonza, Switzerland) at 1.0 ⁇ 10 6 cells/mL, and added according to the ratio of the number of T cells to CD3/CD28 Dynabeads (Thermo Fisher) at 1:1 Dynabeads was added to the culture system and IL-2 (Shandong Jintai Bioengineering Co., Ltd., China) (500IU/mL) was cultured for 48 hours, and then CAR was transduced into T cells via lentivirus.
  • X-VIVO 15 medium Lionza, Switzerland
  • IL-2 Handong Jintai Bioengineering Co., Ltd., China
  • the cells are centrifuged to change the medium, and fresh X-VIVO 15 containing IL-2 (500IU/mL) is added to continue the culture. After the cells were cultured for 4 days, all cells in the culture system were collected, and the Dynabeads in the culture system were removed with a magnetic stand. The T cells were centrifuged and counted. Group cell CAR content.
  • Figure 1 shows that when the MOI of lentivirus infection is 0.5, the transduction efficiency of each CAR gene is between 15-40%.
  • This example compares the initial FMC63 scFv, humanized FMC63 scFv (CAR-1 scFv), FMC63 scFv N-terminal scFv (CAR-2 scFv), FMC63 scFv heavy chain variable region and light chain.
  • CAR-1 scFv humanized FMC63 scFv
  • FMC63 scFv N-terminal scFv CAR-2 scFv
  • FMC63 scFv heavy chain variable region and light chain For example, the affinity of scFv (CAR-3 scFv) with protective peptide added between the variable regions and FMC63 scFv with protective peptide added to the C-terminal (CAR-4 scFv) and CD19 protein.
  • a solution of human CD19-Fc protein (Acro BIOSYSTEMS, Newark, DE, USA) at a concentration of 50 ⁇ g/mL was prepared using HBS-EP+ buffer (10 mM HEPES; 150 mM NaCl; 3 mM EDTA; 0.5% Tween20; pH 7.4).
  • CAR-1 scFv, CAR-2 scFv, CAR-3 scFv, CAR-4 scFv conjugated to Fc fragment were prepared by Beijing Tongli Haiyuan Biotechnology Co., Ltd.
  • the CAR with a concentration of 50 ⁇ g/mL was configured with HBS-EP+buffer -1 scFv, CAR-2 scFv, CAR-3 scFv and CAR-4 scFv solutions.
  • the Biacore CM5 chip (GE healthcare, Chicago, IL, USA) was pretreated with a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide After activation, it was incubated and coupled with human CD19-Fc protein solution.
  • Figure 2 shows that the initial KD value of FMC63 scFv combined with CD19 is 3.27E-10, and the KD value of CAR-1 scFv, CAR-2 scFv, CAR-3 scFv, CAR-4 scFv combined with CD19 are 7.48E- 8. 1.12E-8, 6.73E-8 and 2.95E-9, indicating that compared with the original FMC63 scFv, the modified CAR scFv has a significantly lower affinity with CD19.
  • Example 3 The binding efficiency of CD19 CAR-T containing the modified antigen binding domain from FMC63 scFv to target cells
  • the binding efficiency of the initial FMC63 scFv, FMC63 scFv with a protective peptide added to the N-terminal, and FMC63 scFv with a protective peptide added to the C-terminal to compare the binding efficiency of CD19-expressing target cells is taken as an example.
  • K562 and K562-CD19 cells that have not been incubated with T cells or CAR-T cells as a control. If CAR-T cells bind to target cells through CD19, the APC fluorescent antibody cannot bind to the surface of CD19 molecules. The flow cytometry result is APC The ratio of fluorescence signal decreases, and the ratio of FITC fluorescence signal increases.
  • FIG. 3 shows that T cells can hardly bind to K562-CD19, neither T cells nor CAR-T cells can bind to normal K562 cells, but CAR-T cells can bind to K562-CD19, indicating the antigen in these CAR molecules Both binding domains can specifically recognize CD19 target protein.
  • Example 4 The efficiency of CD19 CAR-T in killing target cells
  • This example takes as an example the detection of the killing efficiency of CAR-T containing modified antigen binding domains, different costimulatory signal domains, and different CD3 ⁇ signal domains on target cells expressing CD19.
  • Figure 4 shows that CAR-Ts designed with different antigen binding domains, costimulatory signal domains and CD3 ⁇ signal domains can effectively kill K562-CD19 cells by recognizing the CD19 target protein.
  • This example takes as an example the detection of the proliferation efficiency of CAR-T containing modified antigen binding domains, different costimulatory signal domains, and different CD3 ⁇ signal domains under the stimulation of target cells expressing CD19.
  • Figure 5 shows that, compared with FMC63 CAR-T, CAR-Ts containing different antigen binding domains, costimulatory signal domains and CD3 ⁇ signal domain modifications, especially CAR-Ts containing different costimulatory signal domains and CD3 ⁇ signal domain modifications.
  • the proliferation efficiency under the stimulation of K562-CD19 target cells is significantly improved, indicating that the modification of these components can enhance the expansion efficiency and duration of CAR-T.
  • This embodiment takes as an example the detection of the cytokine expression level of CAR-T containing modified antigen binding domains, different costimulatory signal domains, and different CD3 ⁇ signal domains under the stimulation of target cells expressing CD19.
  • Figure 6 shows that, compared with T cells, the expression levels of cytokines such as IL-6, IL-10, TNF, and IFN- ⁇ are significantly increased for various CAR-Ts stimulated by K562-CD19 target cells, and contain
  • the cytokine expression level of CAR-T with CD28 costimulatory signal domain is significantly higher than that of FMC63 CAR-T with 4-1BB costimulatory signal domain; compared with FMC63 CAR-T, it contains modified antigen binding domain and modified 4 -1BB costimulatory signal domain and modified CD3 ⁇ signal domain CAR-T, especially CAR-T containing different costimulatory signal domain and CD3 ⁇ signal domain modification, under the stimulation of K562-CD19 target cells, IL-6, IL-
  • the expression level of cytokines such as 10 and TNF was significantly reduced, indicating that the modification of these components can reduce the expression level of cytokines and reduce the cytokine storm caused by CAR-T in vivo while ensuring the killing efficiency of C
  • Example 7 Anti-tumor ability and anti-tumor recurrence ability of CD19 CAR-T cells in tumor-bearing mice
  • This example takes as an example the detection of the anti-tumor ability and the ability of inhibiting tumor recurrence of CAR-T containing the modified antigen-binding domain in tumor-bearing mice.
  • the isolated and purified T cells were inoculated and cultured at 1.5 ⁇ 10 6 cells/mL into fresh X-VIVO medium containing IL-2 (500IU/mL), and added according to the number of T cells and CD3/CD28 Dynabeads at a ratio of 1:1 After Dynabeads cultured the cells for 48 hours, the T cells were respectively infected with the corresponding CAR-containing lentivirus to prepare the corresponding CAR-T cells (at the same time, the T cells not infected with the lentivirus were cultured for control experiments).
  • the cells were centrifuged to change the medium, counted, inoculated and cultured at 0.8 ⁇ 10 6 cells/mL into fresh X-VIVO containing IL-2 (500IU/mL), and continued to maintain the original Dynabeads stimulated culture.
  • the cells were centrifuged every 48 hours to change the medium, and inoculated and cultured at 0.8 ⁇ 10 6 cells/mL into fresh X-VIVO containing IL-2 (500IU/mL).
  • the cells were cultured to the 11th day, the cells were harvested and counted, and kept at the same time.
  • mice There are 15 NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China) aged 6-8 weeks, divided into 5 mice/groups, 3 groups in total. After each mouse was injected with 1.0 ⁇ 10 6 Nalm-6-LAE cells (ATCC, USA) from the tail vein for 5 days (D5), the mice were subjected to luciferase in vivo imaging (Lumina II small animal in vivo imaging system, PerkinElmer) , USA) analysis to verify the success of the mouse leukemia model.
  • luciferase in vivo imaging Lumina II small animal in vivo imaging system, PerkinElmer
  • mice After successful creation of the mouse leukemia model, each group of mice was injected with CD19 CAR-T cells (2 ⁇ 10 6 cells/mouse) from the tail vein, and the other two groups of mice were injected with the corresponding number of T cells and corresponding cells. A volume of normal saline served as a control. Mice were subjected to mouse in vivo imaging analysis on the 2nd (D7), 8 (D13), and 15 (D20) days after CAR-T cell injection. On the 16th day after CAR-T cell injection, the mouse was taken from the tail vein of each mouse. 1.0 ⁇ 10 5 Nalm-6-LAE cells were re-injected, and mouse live imaging analysis was performed on the 27th day (D32) after the first CAR-T cell injection.
  • Figure 7 shows that the tumor-bearing mice in the T-cell injection group all died before 20 days after tumor inoculation, and the CAR-T-injected mice all survived 20 days after tumor inoculation, and CAR-T-2 and CAR- T-30 (the costimulatory signal domain is CD28) can significantly inhibit tumor growth; however, when the tumor is re-inoculated to simulate tumor recurrence, CAR-T-2 can still significantly inhibit tumor growth, indicating that CAR-T-2 It can inhibit tumor recurrence, but CAR-T-30 cannot inhibit tumor recurrence.
  • Example 8 Anti-tumor ability, expansion ability and sustained performance of CD19 CAR-T cells in tumor-bearing mice
  • This example takes as an example the detection of the anti-tumor ability, in vivo amplification ability and persistence ability of CAR-T containing the modified antigen binding domain, the modified costimulatory signal domain, and the modified CD3 ⁇ signal domain in tumor-bearing mice. .
  • the isolated and purified T cells were inoculated and cultured at 1.5 ⁇ 10 6 cells/mL into fresh X-VIVO medium containing IL-2 (500IU/mL), and added according to the number of T cells and CD3/CD28 Dynabeads at a ratio of 1:1 After Dynabeads cultured the cells for 48 hours, the T cells were respectively infected with the corresponding CAR-containing lentivirus to prepare the corresponding CAR-T cells (at the same time, the T cells not infected with the lentivirus were cultured for control experiments).
  • the cells were centrifuged to change the medium, counted, inoculated and cultured at 0.8 ⁇ 10 6 cells/mL into fresh X-VIVO containing IL-2 (500IU/mL), and continued to maintain the original Dynabeads stimulated culture.
  • the cells were centrifuged every 48 hours to change the medium, and inoculated and cultured at 0.8 ⁇ 10 6 cells/mL into fresh X-VIVO containing IL-2 (500IU/mL).
  • the cells were cultured to the 11th day, the cells were harvested and counted, and kept at the same time.
  • mice The corresponding cell samples were subjected to flow cytometry to analyze the CAR expression rate, and the cells were resuspended in cryopreservation solution and stored in liquid nitrogen for later use.
  • Each mouse was injected with 1.0 ⁇ 10 6 Nalm-6-LAE cells (ATCC, USA) from the tail vein for 5 days, and the mice were subjected to luciferase in vivo imaging (Lumina II small animal in vivo imaging system, PerkinElmer, USA) analysis To verify the success of the mouse leukemia model.
  • luciferase in vivo imaging Lumina II small animal in vivo imaging system, PerkinElmer, USA
  • mice After successful creation of the mouse leukemia model, each group of mice was injected with CD19 CAR-T cells (2 ⁇ 10 6 cells/mouse) from the tail vein, and the other two groups of mice were injected with the corresponding number of T cells and corresponding cells. A volume of normal saline served as a control.
  • Mouse peripheral blood CAR-T test was performed on days 2, 4, 8, 12, 21, and 28 after CAR-T cell injection. The day before or after blood collection (3rd, 7th day after CAR-T cell injection) , 13, 20, 27 days) for mouse live imaging analysis.
  • Figure 8 shows that the tumor-bearing mice in the T cell group and the saline group had all died before 28 days after the injection, and all the CAR-T-injected mice had survived, and FMC63 CAR-T, CAR-T-13, CAR -Both T-15 and CAR-T-17 can significantly inhibit tumor growth.
  • Figure 9 shows that, compared with FMC63 CAR-T, the persistence of CAR-T-13, CAR-T-15 and CAR-T-17 in the body is significantly improved. Specifically, after Nalm-6 cells were injected into NCG mice to create a B-ALL leukemia model, the mice were injected with anti-CD19 CAR-T cells into the tail vein to detect CAR-T cells in the peripheral blood of the mice. The peripheral blood of the FMC63 CAR-T group mice has been unable to detect CAR-T cells after 21 days, while the CAR-T-13, CAR-T-15 and CAR-T-17 mice have been peripherally detected 28 days after the CAR-T injection.
  • the inventors designed the antigen binding domain, costimulatory signal domain and CD3 ⁇ signal domain of the CD19 CAR molecule, especially the design of the costimulatory signal domain and CD3 ⁇ signal domain, which can significantly improve CD19 CAR.
  • the amplification efficiency and duration of the molecule in vivo are shown in the model of the in vivo amplification curve as shown in FIG. 10.

Abstract

提供了一种分离的抗原结合结构域，其包括能够结合抗原的单链可变区片段，其包括轻链可变区和重链可变区；和一个或多个保护性肽。还提供了嵌合抗原受体，包括铰链区、穿膜区、共刺激信号结构域以及胞内信号转导结构域，以及相应的的核酸分子、载体、细胞、组合物、制备方法和用于制备治疗癌症或病毒感染等疾病的药物。

Description

结合CD19的嵌合抗原受体及其用途 技术领域
本发明涉及免疫学和分子生物学领域，特别是嵌合抗原受体(Chimeric Antigen Receptor,CAR)修饰的免疫细胞及其用途。
背景技术
嵌合抗原受体T细胞免疫疗法(Chimeric Antigen Receptor T-Cell Immunotherapy，简称CAR-T)技术结合了抗体的特异性及T细胞的杀伤效应，从而形成了一种过继性免疫的有效途径。最初的CAR通常由胞外抗原结合域、铰链区、跨膜区和胞内信号域构成。第二代CAR增加了胞内共刺激域，如CD28,CD137,OX40等，通常位于跨膜区与胞内信号域之间。第三代CAR使用两个共刺激域，以增强CAR-T的激活。CAR-T技术在血液瘤治疗方面取得了巨大成功，尤其是靶向CD19的CAR-T，但是仍有一些患者无响应或响应后复发，而且在疗效持久性及毒副反应上仍有不足。CAR-T疗效和安全性的两个重要问题是：1)CAR-T在体内的扩增和持续不足；2)CAR-T会引起细胞因子风暴、神经毒性和脱靶效应等毒副反应。
CAR-T细胞对靶细胞的杀伤能力、体内扩增能力和其安全性受多种因素影响，包括CAR的设计、靶细胞表面的抗原密度、CAR-T制备工艺等，其中CAR的设计是关键，需要考虑CAR的抗原结合结构域与靶点的亲和力、共刺激信号域的组成、以及CD3ξ信号域的组成等。CAR分子各结构域的设计取决于靶点的特异性、表达丰度、在细胞膜表面的结构特征，以及适应症的病理特征和治疗策略等。
发明内容
一方面，本发明公开了一种分离的抗原结合结构域，其包括：
a)能够结合CD19的单链可变区片段，其包括轻链可变区和重链可变 区；
b)一个或多个保护性肽，其中所述保护性肽段包含富含极性氨基酸的长度为8-15个氨基酸的多肽，所述保护性肽可操作性连接在所述轻链可变区或重链可变区的N端、C端、或轻链可变区和重链可变区之间；
所述保护性肽与轻链可变区或重链可变区之间可选择性地通过富含甘氨酸和丝氨酸的柔性接头肽连接。
在一个具体实施方案中，所述轻链可变区包含如SEQ ID NO:1所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:2所示的氨基酸序列。
在一个具体实施方案中，所述保护性肽包含选自SEQ ID NOs:6、7、8、9或10所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
在一个实施方案中，所述保护性肽可操作性连接在所述轻链可变区的N端或C端；在一个实施方案中，所述保护性肽可操作性连接在所述重链可变区的N端或C端；在一个实施方案中，所述保护性肽可操作性连接在轻链可变区和重链可变区之间。在一个具体实施例中，所述富含甘氨酸和丝氨酸的柔性接头肽的氨基酸序列可以为(G 4S) n，其中n＝1-4，优选所述柔性肽可以为(G 4S) 2，即GGGGSGGGGS。所述保护性肽与轻链可变区或重链可变区之间可选择性地通过富含甘氨酸和丝氨酸的柔性接头肽例如(G 4S) 2连接，例如在轻链可变区的N端可操作性连接保护性肽段和柔性接头肽。
在一个实施方案中，所述抗原结合结构域包含选自SEQ ID NOs:11、13、15、17、19、21、23、25、27、29、31、33、35、37或39所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
另一方面，本发明公开了一种分离的嵌合抗原受体，其包括如前所述的抗原结合结构域。
在一个实施方案中，所述嵌合抗原受体进一步包括共刺激结构域和/或 胞内信号转导结构域。
在一个具体实施方案中，所述共刺激结构域选自CD27、CD28、4-1BB、OX-40、CD30、CD40、PD-1、ICOS、LFA-1、CD-2、CD7、LIGHT、NKG2C、B7-H3或其任意组合。在一个具体实施方案中，所述胞内信号转导结构域选自CD3ζ。
在一个具体实施方案中，所述共刺激结构域选自4-1BB，优选地，所述4-1BB共刺激结构域包含如SEQ ID NO:41所述的氨基酸序列或其变体，所述4-1BB变体包括SEQ ID NO:41中的Q20F、T21P、T22E、Q23E、F32Q、P33T、E34T、E35Q氨基酸替代的一种或多种或所述变体为SEQ ID NO:41所述的氨基酸序列N端或C端连接一段如SEQ ID NO:53、54、55所述的氨基酸序列。
在一个具体实施方案中，所述4-1BB共刺激结构域包含如SEQ ID NO:41所示氨基酸序列的变体，优选地，所述4-1BB变体至少包括Q20F氨基酸替代，进一步包括T21P和Q23E；在一个具体实施例中，所述变体包括SEQ ID NO:41中的Q20F氨基酸替代；在一个具体实施例中，所述变体包括SEQ ID NO:41中的Q20F、T21P和Q23E氨基酸替代；在一个具体实施例中，所述变体包括SEQ ID NO:41中的Q20F、T21P、T22E、Q23E、F32Q、P33T、E34T、E35Q氨基酸替代。
在一个具体实施方案中，所述共刺激结构域选自4-1BB，优选地，所述4-1BB共刺激结构域包含如SEQ ID NOs:43、45、47、49或51所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
在一个实施方案中，所述CD3ζ胞内信号转导结构域包含如SEQ ID NO:56所述的氨基酸序列或其变体，所述变体包括V2L、D9E、Q14K、Q15K、Y90F、L104Y、H105R、M106H中的一种或多种。在一个具体实施例中，所述CD3ζ变体包含Q14K氨基酸替代。在一个具体实施例中，所述CD3ζ变体包含Q15K氨基酸替代，进一步还可以包含V2L和Y90F氨基酸替代，进 一步还可以包含D9E，例如在一个具体实施例中，所述CD3ζ变体包含V2L、Q15K和Y90F氨基酸替代；在一个具体实施例中，所述CD3ζ变体包含V2L、D9E、Q15K和Y90F氨基酸替代；在一个实施例中，所述CD3ζ变体包含V2L、D9E、Q15K、Y90F、L104Y、H105R和M106H氨基酸替代。
在一个具体实施方案中，所述CD3ζ胞内信号转导结构域包含如SEQ ID NOs:58、60、62、64或66所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
在一个实施方案中，所述嵌合抗原受体还可以进一步包括铰链区和跨膜结构域。在一个具体实施例中，所述铰链区和和跨膜结构域选自IgG1、IgG4、CD8α、CD28、IL-2受体、IL-7受体、IL-11受体、PD-1或CD34的铰链区和跨膜结构域。所述嵌合抗原受体还可以进一步包括信号肽。
在一个具体实施方案中，所述嵌合抗原受体包括依次连接的：
a)信号肽；
b)抗原结合结构域，包含如SEQ ID NOs:11、13、15、17、19、21、23、25、27、29、31、33、35、37或39所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列；
c)诸如CD8α的铰链区和CD8α的跨膜结构域；
d)4-1BB共刺激结构域，包含如SEQ ID NOs:41、43、45、47、49或51所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列；和
e)CD3ζ胞内信号转导结构域，包含如SEQ ID NOs:58、60、62、64或66所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
在一个具体实施方案中，所述嵌合抗原受体包括如SEQ ID NO:68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122或124所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
又一方面，本发明进一步公开了一种分离的核酸分子，其编码如本发明中所述的抗原结合结构域或所述的嵌合抗原受体。
本发明公开了一种载体，其包含如前所述的分离的核酸分子；优选的，其中所述载体选自DNA、RNA、质粒、慢病毒载体、腺病毒载体和逆转录病毒载体中的一种或多种。所述载体是表达载体或克隆载体。
本发明公开了一种细胞，其包含如前所述的分离的核酸分子或如前所述的载体。
在一个实施方案中，所述细胞为T淋巴细胞、B淋巴细胞、自然杀伤细胞、树突状细胞、细胞毒性T细胞、肿瘤浸润T细胞或调节性T细胞。在一个具体实施例中，所述细胞选自人外周血T淋巴细胞。
本发明公开了一种药物组合物，其包括选自下述的一项或多项：
i)如前所述的分离的抗原结合结构域；
ii)如前所述的分离的嵌合抗原受体；
iii)如前所述的分离的核酸分子；
iv)如前所述的载体；和
v)如前所述的细胞；
以及，药学上可接受的载体、稀释剂或赋形剂。
本发明公开了用于制备如前所述的细胞的方法，其包括：将编码如前所述的嵌合抗原受体的核酸引入所述细胞中。
再一方面，本发明还公开了如前所述的抗原结合结构域、嵌合抗原受体、核酸分子、载体、细胞或药物组合物在制备药物中的用途；如前所述 的抗原结合结构域、嵌合抗原受体、核酸分子、载体、细胞或药物组合物在制备用于治疗疾病的药物中的用途，其中所述疾病优选选自肿瘤、自身免疫病、或病毒或细菌引起的感染性疾病。
本发明公开了在患有疾病的受试者中进行细胞免疫治疗的方法，其包括向受试者施用如前所述的药物组合物或如前所述的细胞，其中所述疾病优选选自肿瘤、自身免疫病、或病毒或细菌引起的感染性疾病。
在一个实施方案中，所述的肿瘤是肿瘤特异性分子介导的实体瘤或血液学癌症，其中所述实体瘤优选为乳腺癌、前列腺癌、卵巢癌、宫颈癌、皮肤癌、胰腺癌、结肠直肠癌、肾癌、肝癌、脑癌、胃癌、胃肠间质瘤、肺癌和甲状腺癌；其中所述的血液学癌症优选地选自急性白血病，其包括急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病；慢性白血病，其包括慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病和难治疗的CD19+白血病和淋巴瘤；真性红细胞增多症；淋巴瘤；套细胞淋巴瘤；扩散大B-细胞淋巴瘤；霍奇金氏疾病；非霍奇金氏淋巴瘤；多发性骨髓瘤；瓦尔登斯特伦氏巨球蛋白血症；重链疾病；骨髓增生异常综合征；多毛细胞白血病；和脊髓发育不良；其中所述的血液学癌症最优选为急性淋巴细胞白血病或慢性淋巴细胞白血病。其中肿瘤特异性分子为CD19。
在一个具体实施方案中，所述的肿瘤是复发的肿瘤或癌症。
附图说明
图1示出不同CAR分子在T细胞中的转导效率。
图2示出FMC63 scFv与改造后scFv的biacore亲和力数据对比。
图3示出CD19 CAR-T对K562-CD19细胞的结合(*代表P<0.05，**代表P<0.01)。
图4示出CD19 CAR-T对K562-CD19细胞的杀伤效率。
图5示出CD19 CAR-T在K562-CD19细胞刺激3天的增殖(与FMC63 CAR-T比，*代表P<0.05，**代表P<0.01)。
图6示出CD19 CAR-T在K562-CD19细胞刺激12小时后的细胞因子表达(与FMC63 CAR-T比，*代表P<0.05，**代表P<0.01)。
图7示出CD19 CAR-T细胞的抗肿瘤能力和抑制肿瘤复发能力。
图8示出CD19 CAR-T对荷瘤小鼠的肿瘤抑制效果。
图9示出CD19 CAR-T在荷瘤小鼠外周血中的数量变化。
图10示出CD19 CAR-T在体内的代谢曲线模式图。
具体实施方式
下面将通过具体描述，对本发明作进一步的说明。
除非另有限定，本文中所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解相同的含义。
本申请中，单数形式“一个”、“该”包括复数对象，除非上下文另外清楚规定。
本文所用的术语“抗原”或“Ag”是指引起免疫应答的分子，该免疫应答可涉及抗体产生，或特异性免疫活性细胞的活化。本领域技术人员均可理解任何大分子包括所有的蛋白质或肽，可用作抗原。此外，抗原可源自重组或基因组DNA。本领域技术人员均可理解任何DNA其包括编码引起免疫应答的蛋白质的核苷酸序列或部分核苷酸序列，编码如本文使用的术语“抗原”。此外，本领域技术人员均可理解抗原不必单独地由基因的全长核苷酸序列编码。容易显而易见的是本公开包括但不限于，多于一个的基因的部分核苷酸序列的用途，并且这些核苷酸序列以不同的组合进行布置，以引起期望的免疫应答。此外，本领域技术人员均可理解抗原根本不必由“基因”进行编码，抗原可被产生、合成或可源自生物学样本。这种生物学样本可包括但不限于组织样本、肿瘤样本、细胞或生物学流体。
本文使用的术语“嵌合抗原受体”或“CAR”是指被工程化以在免疫效应细胞上表达和特异性地结合抗原的人工受体，例如人工T细胞受体。CAR可以被用作使用过继细胞转移的疗法。淋巴细胞例如T细胞从患者移出并且进行修饰，使得它们表达特异于具体形式的抗原的受体。CAR还可以包括胞内活化结构域、跨膜结构域和胞外结构域，胞外结构域包括肿 瘤相关抗原结合区。在一些方面，CAR包括单链可变区片段(scFv)，其被融合至跨膜结构域和胞内结构域。CAR设计的特异性可以源自受体的配体(例如，肽)。在一些实施方式中，通过重定向表达特异于肿瘤相关抗原的CAR的T细胞的特异性，CAR可以靶向癌症。
本文所用的术语“抗原结合结构域”亦称为“配体结合结构域”，是指能够结合配体的寡肽或多肽。优选地该结构域能够与细胞表面分子相互作用。例如，可以选择胞外配体结合结构域以识别作用为在与特定疾病状态相关的靶细胞上的细胞表面标记物的配体。因此，可以作为配体的细胞表面标记物的实例包括那些细胞表面标记物，其与病毒、细菌和寄生虫感染、自身免疫病和癌细胞相关。本发明中，抗原结合结构域可以包含单链可变区片段(单链抗体)。抗原结合结构域可只结合抗原的一部分。抗原分子中负责与抗原结合结构域特异性相互作用的部分被称为“表位”或“抗原决定簇”。抗原结合结构域通常包括抗体轻链可变区(VL)和抗体重链可变区(VH)。VH和VL区可以进一步细分成高变区，称为互补决定区(CDR)，与更保守的称为构架区(FR)的区域散布。本发明公开的抗体和抗原结合结构域的CDR由Kabat编号所定义或识别。
本文使用的术语“单链可变区片段”、“单链抗体”或“scFv”是指通过重组DNA技术形成的抗体，其中免疫球蛋白重链可变区和轻链可变区通过氨基酸肽段(linker)连接而成。生成单链抗体的多种方法是已知的，包括在美国专利号4,694,778；Bird(1988)Science 242:423-442；Huston等(1988)Proc.Natl.Acad.Sci.USA 85:5879-5883；Ward等(1989)Nature 334:54454；Skerra等(1988)Science 242:1038-1041中描述的那些。重链和轻链的可变区包含与抗原相互作用的结合结构域。重链中的CDR缩写为VH-CDR，例如VH-CDR1、VH-CDR2、VH-CDR3，轻链中的CDR缩写为VL-CDR，例如VL-CDR1、VL-CDR2、VL-CDR3。本发明公开的抗体和抗原结合结构域的CDR由Kabat编号所定义或识别。
本文使用的术语“肽”、“多肽”和“蛋白质”可交换地使用，是指由肽键共价连接的氨基酸残基组成的化合物。蛋白或肽必须包含至少两个氨基酸，并且对可以构成蛋白质或肽的序列的氨基酸的最大数目没有限制。 多肽包括任何肽或蛋白质，所述肽或蛋白质包括通过肽键相互接合的两个或更多个氨基酸。如本文使用的，该术语指的是短链，其在本领域中也通常被称为例如肽、寡肽和寡聚物；和较长链二者，其在本领域中通常被称为蛋白质，其具有许多类型。“多肽”包括例如生物学活性片段、基本上同源的多肽、寡肽、同二聚体、异二聚体、多肽的变体、修饰的多肽、衍生物、类似物、融合蛋白等。多肽包括天然肽、重组肽、合成肽或其组合。
本文使用的术语“保护性肽”、“保护肽”可交换地使用，是指可操作性连接在scFv的N端、C端或中间任意位置的寡肽，该肽可降低scFv与靶点例如CD19的亲和力，但相比未连接保护肽的CAR，连接保护肽的CAR细胞可以较慢地与靶蛋白结合。所述保护肽可以连接在轻链可变区的N端或C端、所述重链可变区的N端或C端、或轻链可变区和重链可变区之间。
本文使用的术语“柔性接头肽”、“柔性肽”可交换地使用，是指富含甘氨酸和丝氨酸的氨基酸序列，可以为(G 4S) n，其中n为大于等于1的整数，例如1至4中的任一整数，例如可以为(G 4S) 2，即GGGGSGGGGS。本发明中保护性肽与轻链可变区或重链可变区之间可选择性地通过富含甘氨酸和丝氨酸的柔性接头肽例如(G 4S) 2连接，例如在轻链可变区的N端可操作性连接保护性肽段和柔性接头肽。本领域技术人员应理解，能够将保护性肽段连接在轻链可变区或重链可变区的任何富含甘氨酸和丝氨酸的氨基酸序列均在柔性接头肽的范围中。
本文所用的术语“载体”是指运输、转导和在靶细胞表达被包含的外源目的基因(例如本发明所述的多核苷酸)的分子工具，所述工具提供合适的在靶细胞中起始转录的核苷酸序列，即启动子。载体其包括分离的核酸，并且其可以用于递送分离的核酸至细胞内部。众多载体在本领域是已知的，包括但不限于线性多核苷酸、与离子或两亲性化合物相关联的多核苷酸、质粒和病毒。因而，术语“载体”包括自主复制的质粒或病毒。该术语也应当解释为包括便于将核酸转移入细胞的非质粒和非病毒化合物，例如，聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于仙台病毒载体、腺病毒载体、腺伴随病毒载体、逆转录病毒载体、慢病毒载体等。
本文使用的“表达载体”是指包括重组多核苷酸的载体，所述重组多核苷酸包括可操作地连接至待表达的核苷酸序列的表达控制序列。表达载体包括足够的用于表达的顺式作用元件；用于表达的其它元件可以由宿主细胞供应或在体外表达系统中供应。表达载体包括所有本领域已知的并入重组多核苷酸的那些，比如质粒(例如，裸露或包含在脂质体中)和病毒(例如，仙台病毒、慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。
本文使用的“同一性”是指在两种核酸分子或多肽之间的序列一致性。可以通过比较为比较目的而对齐的每个序列中位置来确定同一性。当比较的序列中的位置被相同碱基占据时，那么在该位置处分子是相同的。在核酸或氨基酸序列之间相似性或同一性的程度是在由核酸序列共享的位置处相同或匹配核苷酸的数目的函数。可使用各种比对算法和/或程序来计算两个序列之间的一致性，包括可获得为GCG序列分析包(University of Wisconsin，Madison，Wis.)的一部分，并且可以以例如默认设置使用的FASTA或BLAST。例如，设想了与在本文中描述的特定多肽具有至少70％、85％、90％、95％、98％或99％的一致性并且优选展现出基本上相同的功能的多肽，以及编码上述多肽的多核苷酸。
本文使用的“分离的”是指从自然状态改变或移出。例如，天然存在于活动物中的核酸或肽不是“分离的”，但是部分或完全与它的自然状态的共存物质分开的相同的核酸或肽是“分离的”。分离的核酸或蛋白质可以以基本上纯化的形式存在，或可以存在于非自然环境，例如，宿主细胞中。
除非另外规定，本文使用的“编码”某蛋白的核酸分子或某蛋白的氨基酸序列的核苷酸序列包括简并形式(Degeneracy)并且编码相同的氨基酸序列的所有的核苷酸序列。该核苷酸序列还可以包括一个或多个内含子。
本文使用的术语“可操作地连接”是指调控序列和异源核酸序列之间的功能连接，其导致异源核酸序列的表达。例如，当第一核酸序列处于与第二核酸序列的功能关系中时，第一核酸序列与第二核酸序列可操作地连接。例如，如果启动子影响编码序列的转录或表达，则启动子可操作地连接至编码序列。通常地，可操作地连接的DNA序列是邻近的，并且在必 要时在同一阅读框中接合两个蛋白编码区。
本文使用的术语“变体”意指通过在母体分子的氨基酸序列中替代至少一个残基或者在N端或C端添加至少一个氨基酸残基所获得的多肽变体。
本文所用的术语“抗肿瘤能力”或“抑制肿瘤”，是一种生物学效应，其可由肿瘤体积的减少、肿瘤细胞数的减少、转移数的减少、预期寿命的增加或与癌性病症相关的各种生理症状的改善清楚表示。“抗肿瘤能力”或“抑制肿瘤”也可由本公开的肽、多核苷酸、细胞和抗体在预防肿瘤方面能力表示。
如本文所用，术语“受试者”包括任何人或非人动物。术语“非人类动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、大鼠、小鼠、两栖动物、爬行动物等。除非另有说明，否则术语“患者”或“受试者”可互换使用。在本发明中，优选的受试者是人类。
如本文所用，术语“治疗”是指向受试者施用有效量的具有根据本文所述的方法离体改变的靶基因的多核苷酸序列的细胞，以使得所述受试者具有所述疾病的至少一种症状的减少或所述疾病的改善，例如，有益的或所需的临床结果。出于本公开的目的，有益的或所需的临床结果包括但不限于一种或多种症状的减轻、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和，以及缓解(无论是部分缓解还是全部缓解)，无论是可检测的或是不可检测的。治疗可指与未接受治疗情况下的预期存活期相比，延长存活期。因此，本领域的技术人员意识到治疗可改善疾病状况，但可能不是疾病的完全治愈。如本文所用，术语“治疗”包括预防。或者，治疗在疾病的进展减少或停止的情况下是“有效的”。“治疗”还可意指与在未接受治疗情况下的预期存活期相比，延长存活期。需要治疗的病人包括已经被诊断具有与多核苷酸序列的表达相关的病症，以及由于遗传易感性或其他因素可能发展这种病症。
如本文所用的术语“自身免疫病”被定义为由自身免疫应答产生的紊 乱。自体免疫疾病是对自身抗原的不适当和过度应答的结果。自身免疫疾病的例子包括但不限于阿狄森氏疾病、斑秃、强直性脊柱炎、自身免疫肝炎、自身免疫腮腺炎、克罗恩氏疾病、糖尿病(1型)、营养不良性大疱性表皮松解症、附睾炎、肾小球性肾炎、格雷夫斯氏疾病、吉兰-巴雷综合征、桥本氏疾病、溶血性贫血、系统性红斑狼疮、多发性硬化症、重症肌无力、寻常型天疱疮、牛皮癣、风湿热、类风湿性关节炎、结节病、硬皮病、斯耶格伦氏综合征、脊椎关节病变、甲状腺炎、血管炎、白癜风、粘液性水肿、恶性贫血、溃疡性结肠炎等等。
在本文中陈述数值极限或范围的情况下，包括端点。此外，具体包括在数值极限或范围内的所有值和子范围。
实施例
下述实施例中的实验方法，如无特殊说明，均为常规方法。将参照下述非限制性实验实施例进一步理解本发明。
实施例1：CAR分子构建和CAR-T制备
本实施例以设计具有不同抗原结合结构域、共刺激信号域和CD3ξ信号域的CD19 CAR分子和CAR-T细胞的制备为例。
抗原结合结构域构建：在不改变scFv性质和功能的同时，将scFv的抗体骨架区序列更换为人源胚系抗体骨架区序列；或在scFv的N末端、C末端、或重链可变区和轻链可变区之间添加氨基酸长度为8-15个的富含极性氨基酸的保护肽段。以下以FMC63 scFv为例，不同抗原结合结构域的序列如表1中所示。保护性肽序列选自SEQ ID NOs:6、7、8、9或10所示的氨基酸序列。将保护性肽序列添加在scFv的N末端、C末端、或重链可变区和轻链可变区之间，获得添加了保护肽序列的scFv，如SEQ ID NOs:11、13、15、17、19、21、23、25、27、29、31、33、35、37或39所示的氨基酸序列。
初始的FMC63 scFv氨基酸序列如SEQ ID NO:3所示；将FMC63 scFv氨基酸进行人源化改造后的CAR(CAR-1)氨基酸序列如SEQ ID NO:68所示，核苷酸序列如SEQ ID NO:69所示；将FMC63 scFv的N末端添加 保护肽段的CAR(CAR-2)氨基酸序列如SEQ ID NO:70所示，核苷酸序列如SEQ ID NO:71所示；将FMC63 scFv的重链可变区和轻链可变区之间添加保护肽段的CAR(CAR-3)氨基酸序列如SEQ ID NO:72所示，核苷酸序列如SEQ ID NO:73所示；将FMC63 scFv的C末端添加保护肽段的CAR(CAR-4)氨基酸序列如SEQ ID NO:74所示，核苷酸序列如SEQ ID NO:75所示。
初始的4-1BB氨基酸序列如SEQ ID NO:41所示，核苷酸序列如SEQ ID NO:42所示；将4-1BB共刺激域天然序列中的PEEE基序替代成PEQE的CAR(CAR-5)氨基酸序列如SEQ ID NO:76所示，核苷酸序列如SEQ ID NO:77所示；将4-1BB共刺激域天然序列中的TTQE基序替代成PTEE基序，将PEEE基序替代成PEQE基序的CAR(CAR-6)氨基酸序列如SEQ ID NO:78所示，核苷酸序列如SEQ ID NO:79所示；将4-1BB共刺激域天然序列中的QTTQE序列替代成FPEEE序列，将FPEEE序列替代的QTTQE序列的CAR(CAR-7)氨基酸序列如SEQ ID NO:80所示，核苷酸序列如SEQ ID NO:81所示；将4-1BB共刺激域天然序列中的C端添加额外的RFPEEEEGGCE序列的CAR(CAR-8)氨基酸序列如SEQ ID NO:82所示，核苷酸序列如SEQ ID NO:83所示；将4-1BB共刺激域天然序列中的C端添加DLAMADLEQKV序列的CAR(CAR-9)氨基酸序列如SEQ ID NO:84所示，核苷酸序列如SEQ ID NO:85所示。
初始的CD3ζ信号域的氨基酸序列如SEQ ID NO:56所示，核苷酸序列如SEQ ID NO:57所示；将CD3ζ信号域进行Q15K替代后的CAR(CAR-10)氨基酸序列如SEQ ID NO:86所示，核苷酸序列如SEQ ID NO:87所示；将CD3ζ信号域进行V2L、Q15K和Y90F替代后的CAR(CAR-11)氨基酸序列如SEQ ID NO:88所示，核苷酸序列如SEQ ID NO:89所示；将CD3ζ信号域进行V2L、D9E、Q15K和Y90F替代后的CAR(CAR-12)氨基酸序列如SEQ ID NO:90所示，核苷酸序列如SEQ ID NO:91所示。
将如表1中提及的不同抗原结合结构域、共刺激信号域和CD3ζ信号域进行不同组合后的CAR(CAR-13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28和29)的氨基酸序列分别如SEQ ID NO:92、 94、96、98、100、102、104、106、108、110、112、114、116、118、120、122和124所示，核苷酸序列分别如SEQ ID NO:93、95、97、99、101、103、105、107、109、111、113、115、117、119、121、123和125所示。
上述各方案核苷酸序列通过基因合成的方法合成，然后与其他序列通过PCR或同源重组连接，最终形成编码CAR的基因分子。将编码CAR的基因分子插入慢病毒载体pLenti6.3/V5(Thermo Fisher，Waltham，MA，USA)。
从健康志愿者的外周血单个核细胞(妙通(上海)生物科技有限公司，中国)中分离T细胞。将分离纯化后的T细胞以1.0×10 6个细胞/mL接种培养至X-VIVO 15培养基(Lonza，Switzerland)中，按T细胞数与CD3/CD28Dynabeads(Thermo Fisher)比例为1:1加入Dynabeads于培养体系中，并加入IL-2(山东金泰生物工程有限公司，中国)(500IU/mL)培养48小时后，将CAR通过慢病毒转导至T细胞中。病毒感染细胞24小时后，细胞离心换液，并加入含IL-2(500IU/mL)的新鲜X-VIVO 15继续培养。细胞培养4天后，收集培养体系中的所有细胞，并用磁力架去除培养体系中的Dynabeads，T细胞离心并计数，用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测各组细胞CAR含量。图1显示，在慢病毒感染MOI＝0.5时，各CAR基因的转导效率均在15-40％之间。
表1.序列信息
Figure PCTCN2021082683-appb-000001
Figure PCTCN2021082683-appb-000002
Figure PCTCN2021082683-appb-000003
Figure PCTCN2021082683-appb-000004
Figure PCTCN2021082683-appb-000005
Figure PCTCN2021082683-appb-000006
Figure PCTCN2021082683-appb-000007
Figure PCTCN2021082683-appb-000008
Figure PCTCN2021082683-appb-000009
Figure PCTCN2021082683-appb-000010
Figure PCTCN2021082683-appb-000011
Figure PCTCN2021082683-appb-000012
Figure PCTCN2021082683-appb-000013
Figure PCTCN2021082683-appb-000014
Figure PCTCN2021082683-appb-000015
Figure PCTCN2021082683-appb-000016
Figure PCTCN2021082683-appb-000017
Figure PCTCN2021082683-appb-000018
Figure PCTCN2021082683-appb-000019
Figure PCTCN2021082683-appb-000020
Figure PCTCN2021082683-appb-000021
实施例2：改造的CD19 CAR抗原结合结构域和FMC63 scFv与CD19蛋白的亲和力比较
本实施例以比较初始的FMC63 scFv、人源化FMC63 scFv(CAR-1 scFv)、FMC63 scFv N末端添加保护肽段的scFv(CAR-2 scFv)、FMC63 scFv重链可变区和轻链可变区之间添加保护肽段的scFv(CAR-3 scFv)、FMC63 scFv C末端添加保护肽段的scFv(CAR-4 scFv)与CD19蛋白的亲和力为例。
利用HBS-EP+缓冲液(10mM HEPES；150mM NaCl；3mM EDTA；0.5％Tween20；pH 7.4)配置浓度为50μg/mL的人CD19-Fc蛋白(Acro  BIOSYSTEMS，Newark，DE，USA)溶液。偶联Fc片段的CAR-1 scFv、CAR-2 scFv、CAR-3 scFv、CAR-4 scFv由北京同立海源生物科技有限公司制备，利用HBS-EP+缓冲液配置浓度均为50μg/mL的CAR-1 scFv、CAR-2 scFv、CAR-3 scFv和CAR-4 scFv溶液。将Biacore CM5芯片(GE healthcare，Chicago，IL，USA)用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺的混合物预处理活化后，与人CD19-Fc蛋白溶液孵育偶联，用乙醇胺封闭芯片后，分别与CAR-1 scFv、CAR-2 scFv、CAR-3 scFv、CAR-4 scFv孵育，随后用适宜pH的甘氨酸-盐酸缓冲液冲洗芯片，用Biacore TMT200设备(GE healthcare，Chicago，IL，USA)检测和分析各scFv与CD19的亲和力，包括结合速率常数Ka、解离常数Kd和平衡解离常数KD。
图2显示，初始的FMC63 scFv与CD19结合的KD值为3.27E-10，CAR-1 scFv、CAR-2 scFv、CAR-3 scFv、CAR-4 scFv与CD19结合的KD值分别为7.48E-8、1.12E-8、6.73E-8和2.95E-9，说明与初始的FMC63 scFv相比，经过改造后的CAR scFv与CD19的亲和力均显著降低。
上述结果显示，在scFv的N端、C端、或者轻链可变区和重链可变区之间添加保护性肽，可降低scFv与靶点例如CD19的亲和力。
实施例3：包含改造的来自FMC63 scFv的抗原结合结构域的CD19 CAR-T与靶细胞的结合效率
本实施例以比较包含初始的FMC63 scFv、FMC63 scFv N末端添加保护肽段的scFv、FMC63 scFv C末端添加保护肽段的scFv与表达CD19的靶细胞的结合效率为例。
向24孔细胞培养板(Corning Incorporated，Corning，NY，USA)每孔中加入6×10 5个过表达CD19的K562细胞(K562-CD19)或正常的K562细胞(国家实验细胞资源共享平台，中国)，按E:T(效靶比)＝3:1加入T细胞或CAR-T细胞，轻轻混匀，将上述细胞板转移至37℃、5％CO 2细胞培养箱(Thermo Fisher)中孵育2小时后，与自制的偶联FITC荧光分子的CD19 CAR抗体和偶联APC荧光分子的CD19抗体(Biolegend,San  Diego,CA,USA)孵育20分钟后，用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测CAR和CD19的表达。用未与T细胞或CAR-T细胞孵育的K562和K562-CD19细胞做对照，如果CAR-T细胞通过CD19与靶细胞结合，APC荧光抗体则不能结合到CD19分子表面，流式检测结果为APC荧光信号比例降低，FITC荧光信号比例升高。
图3显示，T细胞几乎无法与K562-CD19结合，T细胞与CAR-T细胞均无法与正常的K562细胞结合，但CAR-T细胞均能与K562-CD19结合，说明这些CAR分子中的抗原结合结构域均能特异识别CD19靶蛋白。当CAR-T与K562-CD19靶细胞孵育1小时，包含初始的FMC63 scFv的CAR-T(FMC63 CAR-T，其信号肽、跨膜区和胞内区序列与CAR-T-1、CAR-T-2、CAR-T-3、CAR-T-4中的一致，仅胞外区抗原结合结构域序列使用了初始的FMC63 scFv，不含有保护肽和柔性接头肽)与靶细胞的结合率显著高于含有FMC63 scFv的N末端或C末端添加保护肽段的CAR-T(CAR-T-2或CAR-T-4)；当CAR-T与K562-CD19靶细胞孵育2小时，几乎所有的FMC63 CAR-T、CAR-T-2、CAR-T-4都与靶细胞有效结合。这说明初始的FMC63 scFv比改造后的抗原结合结构域能更快地与细胞表面的靶蛋白结合，即当靶细胞存在时，FMC63 CAR-T的活化速度更快，而CAR-T-2和CAR-T-4能较缓和地被激活。
实施例4：CD19 CAR-T杀伤靶细胞的效率
本实施例以检测包含改造的抗原结合结构域、不同共刺激信号域、和不同CD3ξ信号域的CAR-T对表达CD19的靶细胞的杀伤效率为例。
利用1mL生理盐水重悬K562-CD19靶细胞，加入5μL Calcein-AM(浓度1μg/μL，ThermoFisher，USA)，轻轻混匀，然后放入37℃水浴中孵育5分钟，以标记靶细胞。向48孔细胞培养板(Corning Incorporated,Corning,NY,USA)每孔中加入1×10 5个上述标记的K562-CD19细胞，按E:T＝5:1加入各种CAR-T细胞，置于37℃、5％CO 2细胞培养箱中孵育6小时，用荧光酶标仪(Varioscan Lux，ThermoFisher)检测细胞上清的荧光值(激发波长：495nm，发射波长：515nm)。
图4显示，具有不同抗原结合结构域、共刺激信号域和CD3ξ信号域设计的CAR-T均能通过识别CD19靶蛋白有效杀伤K562-CD19细胞。
实施例5：CD19 CAR-T在靶细胞刺激下的增殖
本实施例以检测包含改造的抗原结合结构域、不同共刺激信号域、和不同CD3ξ信号域的CAR-T在表达CD19的靶细胞刺激下的增殖效率为例。
向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)每孔中加入1×10 5个K562-CD19细胞，按E:T＝5:1加入各种CAR-T细胞，置于37℃、5％CO 2细胞培养箱中培养3天，用台盼蓝将细胞染色计活细胞总数，并将细胞用自制的偶联FITC荧光分子的CD19 CAR抗体标记，用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测各组细胞中CAR-T的比例，计算出各组中CAR-T的增殖倍数。
图5显示，与FMC63 CAR-T相比，包含不同抗原结合结构域、共刺激信号域和CD3ξ信号域改造的CAR-T，尤其是包含不同共刺激信号域和CD3ξ信号域改造的CAR-T，在K562-CD19靶细胞刺激下的增殖效率显著提高，说明这些构件的改造能增强CAR-T的扩增效率和持续时间。
实施例6：CD19 CAR-T在靶细胞刺激下的细胞因子表达
本实施例以检测包含改造的抗原结合结构域、不同共刺激信号域、和不同CD3ξ信号域的CAR-T在表达CD19的靶细胞刺激下的细胞因子表达水平为例。
向48孔细胞培养板(Corning Incorporated,Corning,NY,USA)每孔中加入1×10 5个K562-CD19细胞，按E:T＝5:1加入各种CAR-T细胞，置于37℃、5％CO 2细胞培养箱中孵育12小时，将细胞上清用cytometric bead array(BD Biosciences，San Jose，CA，USA)，用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测各种细胞因子的表达水平。
图6显示，与T细胞相比，各种CAR-T在K562-CD19靶细胞刺激下， IL-6、IL-10、TNF和IFN-γ等细胞因子的表达水平均显著升高，且含有CD28共刺激信号域的CAR-T的细胞因子表达水平显著高于含有4-1BB共刺激信号域的FMC63 CAR-T；与FMC63 CAR-T相比，包含改造的抗原结合结构域、改造的4-1BB共刺激信号域和改造的CD3ξ信号域的CAR-T，尤其是包含不同共刺激信号域和CD3ξ信号域改造的CAR-T，在K562-CD19靶细胞刺激下，IL-6、IL-10和TNF等细胞因子的表达水平均显著降低，说明这些构件的改造能在保证CAR-T对靶细胞的杀伤效率的前提下，降低细胞因子表达水平，减少CAR-T在体内引发细胞因子风暴的风险，提高安全性。
实施例7：CD19 CAR-T细胞在荷瘤小鼠中的抗瘤能力和抑制肿瘤复发能力
本实施例以检测包含改造的抗原结合结构域的CAR-T在对荷瘤小鼠的抗瘤能力及抑制肿瘤复发能力为例。
将分离纯化的T细胞按1.5×10 6个细胞/mL接种培养至含IL-2(500IU/mL)的新鲜X-VIVO培养基中，按T细胞数与CD3/CD28 Dynabeads为1:1加入Dynabeads培养细胞48小时后，将T细胞分别感染含相应CAR的慢病毒以制备相应CAR-T细胞(同时培养不感染慢病毒的T细胞以供对照实验使用)。病毒感染24小时后，细胞离心换液，计数，按0.8×10 6个细胞/mL接种培养至含IL-2(500IU/mL)的新鲜X-VIVO中，继续维持原有的Dynabeads刺激培养，细胞每48小时离心换液，按0.8×10 6个细胞/mL接种培养至含IL-2(500IU/mL)的新鲜X-VIVO中，培养至第11天时，收获细胞并计数，同时留取相应细胞样品进行流式检测分析CAR表达率，细胞以冻存液重悬后保存至液氮中待用。6-8周龄NCG小鼠(江苏集萃药康生物科技有限公司，中国)共15只，分为5只/组，共3组。每只小鼠从尾静脉注射1.0×10 6个Nalm-6-LAE细胞(ATCC，USA)5天(D5)后，对小鼠进行荧光素酶活体成像(Lumina II小动物活体成像系统，PerkinElmer，USA)分析，以验证小鼠白血病模型是否成功。小鼠白血病模型制作成功后，每组小鼠分别从尾静脉注射CD19 CAR-T细胞(2×10 6 个细胞/只)，同时在另外两组小鼠分别注射相应细胞数的T细胞及相应体积的生理盐水作为对照。小鼠于CAR-T细胞注射后第2(D7)、8(D13)、15(D20)天进行小鼠活体成像分析，在CAR-T细胞注射后第16天，从每只小鼠尾静脉重新注射1.0×10 5个Nalm-6-LAE细胞，于首次CAR-T细胞注射后第27天(D32)进行小鼠活体成像分析。
图7显示，注射T细胞组的荷瘤小鼠已于肿瘤接种后20天前全部死亡，注射CAR-T的小鼠于肿瘤接种后20天前全部存活，且CAR-T-2和CAR-T-30(共刺激信号结构域为CD28)均能显著抑制肿瘤的生长；但是，当重新接种肿瘤模拟肿瘤复发后，CAR-T-2仍能显著抑制肿瘤的生长，说明CAR-T-2能抑制肿瘤复发，而CAR-T-30不能抑制肿瘤复发。
实施例8：CD19 CAR-T细胞在荷瘤小鼠中的抗瘤能力、扩增能力及持续性能
本实施例以检测包含改造的抗原结合结构域、改造的共刺激信号域、和改造的CD3ξ信号域的CAR-T在对荷瘤小鼠的抗瘤能力、体内扩增能力及持续能力为例。
将分离纯化的T细胞按1.5×10 6个细胞/mL接种培养至含IL-2(500IU/mL)的新鲜X-VIVO培养基中，按T细胞数与CD3/CD28 Dynabeads为1:1加入Dynabeads培养细胞48小时后，将T细胞分别感染含相应CAR的慢病毒以制备相应CAR-T细胞(同时培养不感染慢病毒的T细胞以供对照实验使用)。病毒感染24小时后，细胞离心换液，计数，按0.8×10 6个细胞/mL接种培养至含IL-2(500IU/mL)的新鲜X-VIVO中，继续维持原有的Dynabeads刺激培养，细胞每48小时离心换液，按0.8×10 6个细胞/mL接种培养至含IL-2(500IU/mL)的新鲜X-VIVO中，培养至第11天时，收获细胞并计数，同时留取相应细胞样品进行流式检测分析CAR表达率，细胞以冻存液重悬后保存至液氮中待用。6-8周龄NCG小鼠(江苏集萃药康生物科技有限公司，中国)共36只，分为6只/组，共6组。每只小鼠从尾静脉注射1.0×10 6个Nalm-6-LAE细胞(ATCC，USA)5天后，对小鼠进行荧光素酶活体成像(Lumina II小动物活体成像系统，PerkinElmer， USA)分析，以验证小鼠白血病模型是否成功。小鼠白血病模型制作成功后，每组小鼠分别从尾静脉注射CD19 CAR-T细胞(2×10 6个细胞/只)，同时在另外两组小鼠分别注射相应细胞数的T细胞及相应体积的生理盐水作为对照。小鼠于CAR-T细胞注射后第2、4、8、12、21、28天进行小鼠外周血CAR-T检测，于采血前一天或后一天(CAR-T细胞注射后第3、7、13、20、27天)进行小鼠活体成像分析。
图8显示，注射T细胞组及生理盐水组的荷瘤小鼠已于注射后28天前全部死亡，注射CAR-T的小鼠全部存活，且FMC63 CAR-T、CAR-T-13、CAR-T-15和CAR-T-17均能显著抑制肿瘤的生长。
图9显示，与FMC63 CAR-T相比，CAR-T-13、CAR-T-15和CAR-T-17在体内的持续性有显著提高。具体而言，Nalm-6细胞注射NCG小鼠制作B-ALL白血病模型成功后，小鼠尾静脉注射抗CD19 CAR-T细胞治疗后小鼠外周血中检测CAR-T细胞。FMC63 CAR-T组小鼠外周血在21天后就一直检测不到CAR-T细胞，而CAR-T-13、CAR-T-15和CAR-T-17小鼠在CAR-T注射28天后外周血中仍能检测到较高水平CAR-T细胞，说明与FMC63 CAR-T相比，包含改造的抗原结合结构域、改造的共刺激信号域、和改造的CD3ξ信号域的CAR-T细胞在体内的持续性能有显著提高。
从上述体外、体内实验结果可知，发明人对CD19 CAR分子的抗原结合结构域、共刺激信号域和CD3ξ信号域的设计，尤其是对共刺激信号域和CD3ξ信号域的设计能显著提高CD19 CAR分子在体内的扩增效率和持续时间，如图10所示体内扩增曲线模型。
以上，发明人对基于本发明的实施方式进行了说明，但本发明不限定于此，本领域的技术人员应该明白，在本发明的主旨范围内能够以进行变形和变更的方式实施，这样的变形和变更的方式，理应属于本发明的保护范围。

Claims (22)

  1. 一种分离的抗原结合结构域，其包括：
    a)能够结合CD19的单链可变区片段，其包括轻链可变区和重链可变区；
    b)一个或多个保护性肽，其中所述保护性肽包含富含极性氨基酸的长度为8-15个氨基酸的多肽，所述保护性肽可操作性连接在所述轻链可变区或重链可变区的N端、C端、或轻链可变区和重链可变区之间；
    所述保护性肽与轻链可变区或重链可变区之间可选择性地通过富含甘氨酸和丝氨酸的柔性接头肽连接。
  2. 如权利要求1所述的抗原结合结构域，所述轻链可变区包含如SEQ ID NO:1所示的氨基酸序列，所述重链可变区包含如SEQ ID NO:2所示的氨基酸序列。
  3. 如权利要求1或2所述的抗原结合结构域，其中，所述保护性肽包含选自SEQ ID NOs:6、7、8、9或10所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
  4. 如权利要求3所述的抗原结合结构域，其中所述抗原结合结构域包含选自SEQ ID NOs:11、13、15、17、19、21、23、25、27、29、31、33、35、37或39所示的氨基酸序列或与其具有至少80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
  5. 一种分离的嵌合抗原受体，其包括如权利要求1至4任一项所述的抗原结合结构域。
  6. 如权利要求5所述的嵌合抗原受体，其进一步包括共刺激结构域和/或胞内信号转导结构域；优选地，所述共刺激结构域选自CD27、CD28、4-1BB、OX-40、CD30、CD40、PD-1、ICOS、LFA-1、CD-2、CD7、LIGHT、 NKG2C、B7-H3或其任意组合；更优选地，所述胞内信号转导结构域选自CD3ζ胞内信号转导结构域。
  7. 如权利要求6所述嵌合抗原受体，其中所述共刺激结构域选自4-1BB；优选地，所述4-1BB共刺激结构域包含如SEQ ID NO:41所述的氨基酸序列或其变体；更优选地，所述变体包括Q20F、T21P、T22E、Q23E、F32Q、P33T、E34T、E35Q氨基酸替代的一种或多种，或所述变体为SEQ ID NO:41所述的氨基酸序列N端或C端连接一段如SEQ ID NO:53、54或55所述的氨基酸序列。
  8. 如权利要求7所述嵌合抗原受体，其中所述共刺激结构域选自4-1BB，所述4-1BB共刺激结构域包含如SEQ ID NOs:43、45、47、49或51所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
  9. 如权利要求6至8任一项所述嵌合抗原受体，其中所述CD3ζ胞内信号转导结构域包含如SEQ ID NO:56所述的氨基酸序列或其变体；优选地，所述变体包括V2L、D9E、Q14K、Q15K、Y90F、L104Y、H105R、M106H氨基酸替代的一种或多种。
  10. 如权利要求9所述嵌合抗原受体，其中所述CD3ζ胞内信号转导结构域包含如SEQ ID NOs:58、60、62、64或66所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
  11. 如权利要求5至10任一项所述的嵌合抗原受体，其进一步包括铰链区和跨膜结构域，优选所述铰链区和跨膜结构域选自IgG1、IgG4、CD8α、CD28、IL-2受体、IL-7受体、IL-11受体、PD-1或CD34的铰链区和跨膜结构域。
  12. 如权利要求5至11任一项所述嵌合抗原受体，其中所述嵌合抗原受体包括如SEQ ID NOs:68、70、72、74、76、78、80、82、84、86、88、 90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120、122或124所示的氨基酸序列或与其具有至少75％以上、80％以上、85％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、99％以上同一性的氨基酸序列。
  13. 一种分离的核酸分子，其编码如权利要求1至4中任一项所述的抗原结合结构域或5至12中任一项所述的嵌合抗原受体。
  14. 一种载体，其包含权利要求13所述的分离的核酸分子；优选的，其中所述载体选自DNA、RNA、质粒、慢病毒载体、腺病毒载体和逆转录病毒载体中的一种或多种。
  15. 一种细胞，其包含如权利要求13所述的分离的核酸分子或权利要求14所述的载体。
  16. 如权利要求15所述的细胞，其中所述细胞为T淋巴细胞、B淋巴细胞、自然杀伤细胞、树突状细胞、细胞毒性T细胞、肿瘤浸润T细胞或调节性T细胞。
  17. 一种药物组合物，其包括选自下述的一项或多项：
    i)如权利要求1至4中任一项所述的分离的抗原结合结构域；
    ii)如权利要求5至12中任一项所述的分离的嵌合抗原受体；
    iii)如权利要求13所述的分离的核酸分子；
    iv)如权利要求14所述的载体；和
    v)如权利要求15或16所述的细胞；
    以及，药学上可接受的载体、稀释剂或赋形剂。
  18. 用于制备如权利要求15或16所述的细胞的方法，其包括：
    将编码如权利要求5至12中任一项所述的嵌合抗原受体的核酸引入所述细胞中。
  19. 如权利要求1至4任一项所述的抗原结合结构域、如权利要求5至12中任一项所述的嵌合抗原受体、如权利要求13所述的核酸分子、如权利要求14所述的载体、如权利要求15或16所述的细胞或如权利要求17所述的药 物组合物在制备用于治疗疾病的药物中的用途，优选所述疾病选自肿瘤、自身免疫病、或病毒或细菌引起的感染性疾病。
  20. 在患有疾病的受试者中进行细胞免疫治疗的方法，其包括向受试者施用如权利要求17所述的药物组合物或如权利要求15或16所述的细胞，优选所述疾病选自肿瘤、自身免疫病、或病毒或细菌引起的感染性疾病。
  21. 如权利要求19所述的用途或权利要求20所述的方法，其中所述的肿瘤是肿瘤特异性分子介导的实体瘤或血液学癌症，其中所述实体瘤优选选自乳腺癌、前列腺癌、卵巢癌、宫颈癌、皮肤癌、胰腺癌、结肠直肠癌、肾癌、肝癌、脑癌、胃癌、胃肠间质瘤、肺癌和甲状腺癌；所述血液学癌症优选选自急性白血病，其包括急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病；慢性白血病，其包括慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病和难治疗的CD19+白血病和淋巴瘤；真性红细胞增多症；淋巴瘤；套细胞淋巴瘤；扩散大B-细胞淋巴瘤；霍奇金氏疾病；非霍奇金氏淋巴瘤；多发性骨髓瘤；瓦尔登斯特伦氏巨球蛋白血症；重链病；骨髓增生异常综合征；多毛细胞白血病；和脊髓发育不良；所述血液学癌症最优选为急性淋巴细胞白血病或慢性淋巴细胞白血病。
  22. 如权利要求19所述的用途或权利要求20或21所述的方法，其中所述的肿瘤是复发的肿瘤或癌症。
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