WO2021190550A1

WO2021190550A1 - Chimeric antigen receptor containing protective peptide, and use thereof

Info

Publication number: WO2021190550A1
Application number: PCT/CN2021/082682
Authority: WO
Inventors: 丁艳萍; 鲁薪安; 何霆; 齐菲菲
Original assignee: 上海先博生物科技有限公司
Priority date: 2020-03-25
Filing date: 2021-03-24
Publication date: 2021-09-30
Also published as: WO2021190551A1; CN115867581A; CN115335407A

Abstract

Disclosed is an isolated antigen-binding domain, comprising a single-chain variable region fragment capable of binding to an antigen, which comprises a light-chain variable region and a heavy-chain variable region; and one or more protective peptides. The present invention further discloses the hinge region, transmembrane region, co-stimulatory signal domain and intracellular signal transduction domain of an isolated chimeric antigen receptor. The present invention further discloses the isolated chimeric antigen receptor, which comprises the antigen-binding domain, a nucleic acid molecule encoding the antigen-binding domain or the chimeric antigen receptor, a vector, a host cell, a composition and a preparation method. The host cell or the composition is used to prepare a drug for treating diseases such as a cancer or viral infection.

Description

Chimeric antigen receptor containing protective peptide and its use

Technical field

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.

Background technique

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 , Need to consider the affinity of the antigen binding domain of the CAR to the target, the length of the hinge region, the composition of the costimulatory signal domain, and the composition of the CD3ξ 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.

Summary of the invention

In one aspect, the present invention discloses a method for preparing a targeted antigen product through a protective peptide or the use of a protective peptide in preparing a product capable of binding antigen, wherein

The protective peptide is a peptide containing polar amino acids and a length of 8-15 amino acids;

The antigen is a tumor-specific antigen or a tumor-related antigen;

The product is selected from a protein comprising an antigen-binding domain, a chimeric antigen receptor comprising the antigen-binding domain, a nucleic acid encoding the antigen-binding domain or the chimeric antigen receptor, and a nucleic acid comprising the nucleic acid A vector, a cell containing the nucleic acid;

The antigen binding domain includes: a ligand or receptor protein fragment capable of binding to the antigen; a single chain variable region fragment capable of binding to the antigen, which includes the light chain variable region and the heavy chain variable region At least one; wherein the protective peptide is located at the N-terminal, C-terminal, or between the variable region of the light chain and the variable region of the heavy chain of the antigen binding domain;

The protective peptide has at least one of the following characteristics: the protective peptide can change the affinity of the antigen binding domain or the chimeric antigen receptor to the antigen; the protective peptide can change the antigen binding structure Interaction between cells containing the domain and target cells expressing the antigen; improving the killing efficiency of the cells containing the antigen-binding domain against the target cells expressing the antigen; improving the expansion of the cells containing the antigen-binding domain under antigen stimulation ability.

In one embodiment, the number of the polar amino acids in the protective peptide accounts for more than 60%, such as 65% or more, 70% or more, 80% or more, 90% or more, 95% or more. The number is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In a specific embodiment, at least 5 or more polar amino acids are present in the peptide of 8 amino acids in length, or at least 6 polar amino acids are present in the peptide of 9 amino acids in length, or There are at least 9 or more polar amino acids in the 11-amino acid-length peptide, or there are 11 or more polar amino acids in the 15-amino-acid-length peptide. The polar amino acids are selected from threonine (Thr), serine (Ser), cysteine (Cys), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), glycine ( Gly), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp) or glutamic acid (Glu).

In one embodiment, the protective peptide is selected from the amino acid sequence shown in SEQ ID NOs: 6, 7, 8, 9 or 10 or has at least 75%, 80%, 85%, 90%, Amino acid sequences that are 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.

In one embodiment, the protective peptide and the antigen binding domain, the light chain variable region, the heavy chain variable region, the ligand, or the receptor protein can be selectively connected via a flexible linker peptide rich in glycine and serine Preferably, the flexible linker peptide is (G ₄ S) _n , where n is 1, 2, 3, or 4.

In one embodiment, the tumor-specific antigen or tumor-associated antigen is selected from CD19, CD20, CD22, CD23, CD371, CD123, CD33, CD117, CS-1, ROR1, mesothelin, C-Met, PSMA , IL13Ra2, BCMA, Glypican-3, Claudin 18.2, Her2, GD-2, DOG-1, Trop2, GUYC2C, MAGE A3 TCR or a combination thereof.

In one embodiment, the chimeric antigen receptor further includes a costimulatory domain and/or an intracellular signal transduction domain; preferably, the costimulatory domain is selected from CD27, CD28, 4-1BB, OX -40, CD30, CD40, PD-1, CTLA-4, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, NKG2D, B7-H3 or any combination thereof; preferably, the intracellular signal transduction The guide domain is selected from CD3ζ.

In one embodiment, the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, and the variants include Q20F, T21P, T22E, Q23E, F32Q, P33T, E34T One or more of the E35Q amino acid substitutions, or the variant is the amino acid sequence described in SEQ ID NO: 41 connected to the N-terminus or C-terminus with an amino acid sequence described in SEQ ID NO: 53, 54 or 55.

In one embodiment, the 4-1BB costimulatory domain comprises an amino acid sequence as shown in SEQ ID NOs: 43, 45, 47, 49 or 51.

In one embodiment, the CD3ζ 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 amino acid substitutions.

In one embodiment, the CD3ζ intracellular signal transduction domain comprises an amino acid sequence as shown in SEQ ID NOs: 58, 60, 62, 64 or 66.

In one embodiment, the chimeric antigen receptor further includes a hinge region and a transmembrane domain. Preferably, the hinge region and the transmembrane domain are selected from IgG1, IgG4, CD8α, CD28, IL-2 receptor, The hinge region and transmembrane domain of IL-7 receptor, IL-11 receptor, PD-1 or CD34.

In one embodiment, the cells are lymphocytes, preferably, the lymphocytes are selected from T cells, B cells, natural killer cells or dendritic cells; more preferably, the cells are selected from cytotoxic T cells , Tumor infiltrating T cells or regulatory T cells.

In one embodiment, the target cell is a cell expressing the antigen.

In another aspect, the present invention discloses an isolated antigen binding domain, which includes:

a) A single chain variable region fragment capable of binding antigen, which includes a light chain variable region and a heavy chain variable region; or a ligand or receptor protein fragment capable of binding antigen;

b) one or more protective peptides, wherein the protective peptide comprises a polypeptide of 8-15 amino acids in length rich in polar amino acids, and the protective peptide is operably linked to the N-terminus of the antigen binding domain or C-terminal, or between the variable region of the light chain and the variable region of the heavy chain;

The antigen is a tumor-specific antigen or a tumor-related antigen;

Preferably, the protective peptide and the antigen binding domain, light chain variable region, heavy chain variable region, ligand, or receptor protein can be selectively connected via a flexible linker peptide rich in glycine and serine, preferably , The flexible linker peptide is (G ₄ S) _n , where n is 1, 2, 3, or 4.

In a specific embodiment, the tumor-specific antigen or tumor-associated antigen is CD19, the light chain variable region comprises the amino acid sequence shown in SEQ ID NOs:1, and the heavy chain variable region comprises The amino acid sequence shown in SEQ ID NOs: 2.

In a specific embodiment, the protective peptide comprises an amino acid sequence selected from SEQ ID NOs: 6, 7, 8, 9 or 10, or has 75% or more, 80% or more, 85% or more, or 90% or more of the amino acid sequence shown in SEQ ID NOs: 6, 7, 8, 9 or 10. , 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 sequences.

In one embodiment, the protective peptide is operably connected to the N-terminus or C-terminus of the variable region of the light chain; in one embodiment, the protective peptide is operably connected to the variable region of the heavy chain. The N-terminus or C-terminus of the region; in one embodiment, the protective peptide is operably linked between the variable region of the light chain and the variable region of the heavy chain. In a specific embodiment, the amino acid sequence of the flexible linker peptide rich in glycine and serine may be (G ₄ S) _n , where n is 1, 2, 3 or 4, preferably the flexible peptide may be (G ₄ S) ₂ , namely GGGGSGGGGS. The protective peptide and the variable region of the light chain or the variable region of the heavy chain can be selectively connected by a flexible linker peptide rich in glycine and serine such as (G ₄ S) ₂ , for example in the N of the light chain variable region. The end is operably connected to the protective peptide and the flexible linker peptide.

In one embodiment, 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.

In another aspect, the present invention discloses an isolated chimeric antigen receptor, which includes the antigen binding domain as described above.

In one embodiment, the chimeric antigen receptor further includes a costimulatory domain and/or an intracellular signal transduction domain.

In a specific embodiment, the costimulatory domain is selected from CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, CTLA-4, ICOS, LFA-1, CD-2, CD7 , LIGHT, NKG2C, NKG2D, B7-H3 or any combination thereof. In a specific embodiment, the intracellular signal transduction domain is selected from CD3ζ.

In a specific embodiment, the costimulatory domain is selected from 4-1BB, and the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, and the 4-1BB The variant includes one or more of Q20F, T21P, T22E, Q23E, F32Q, P33T, E34T, E35Q amino acid substitutions in SEQ ID NO: 41 or the variant is the amino acid sequence N described in SEQ ID NO: 41 The amino acid sequence described in SEQ ID NO: 53, 54, 55 is connected to the end or the C end.

In a specific embodiment, the 4-1BB costimulatory domain comprises a variant of the amino acid sequence shown in SEQ ID NO: 41. Preferably, the 4-1BB variant includes at least the Q20F amino acid substitution, and further includes T21P and Q23E; In one embodiment, the variant includes the Q20F amino acid substitution in SEQ ID NO: 41; in one embodiment, the variant includes the Q20F, T21P, and Q23E amino acid substitutions in SEQ ID NO: 41; In one embodiment, the variant includes amino acid substitutions of Q20F, T21P, T22E, Q23E, F32Q, P33T, E34T, E35Q in SEQ ID NO: 41.

In a specific embodiment, the costimulatory domain is selected from 4-1BB, and the 4-1BB costimulatory domain comprises an amino acid sequence as shown in SEQ ID NOs: 43, 45, 47, 49, or 51 or a combination thereof 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, Amino acid sequences with more than 99% identity.

In one embodiment, the CD3ζ 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. In a specific embodiment, the CD3ζ variant comprises a Q14K amino acid substitution. In a specific embodiment, the CD3ζ variant includes Q15K amino acid substitutions, and may further include V2L and Y90F amino acid substitutions, and may further include D9E. For example, in a specific embodiment, the CD3ζ variants include V2L and Q15K. And Y90F amino acid substitutions; in a specific embodiment, the CD3ζ variant comprises V2L, D9E, Q15K and Y90F amino acid substitutions; in one embodiment, the CD3ζ variant comprises V2L, D9E, Q15K, Y90F, L104Y, H105R and M106H amino acid substitutions.

In a specific embodiment, the CD3ζ 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.

In one embodiment, the chimeric antigen receptor may further include a hinge region and a transmembrane domain. In a specific embodiment, the hinge region and the transmembrane domain are selected from IgG1, IgG4, CD8α, 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.

In a specific embodiment, the chimeric antigen receptor includes sequentially linked:

a) Signal peptide;

b) 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;

c) Such as the hinge region of CD8α and the transmembrane domain of CD8α;

d) 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

e) CD3ζ 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.

In a specific embodiment, 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.

In another aspect, 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 step discloses a vector, which contains the isolated nucleic acid molecule as described above; preferably, wherein the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector One 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.

In one embodiment, the host cells are T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, cytotoxic T cells, tumor infiltrating T cells, or regulatory T cells. In a specific embodiment, the host cell is selected from human peripheral blood T lymphocytes.

The present invention discloses a pharmaceutical composition, which comprises one or more selected from the following:

i) The isolated antigen binding domain as described above;

ii) The isolated chimeric antigen receptor as described above;

iii) the isolated nucleic acid molecule as described above,

iv) the carrier as previously described, and

v) The host cell as described above;

And, a pharmaceutically acceptable carrier, diluent or excipient.

The present invention discloses a method for preparing a host cell as described above, which comprises: introducing a nucleic acid encoding a chimeric antigen receptor as described above into the host cell.

In another aspect, 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.

In one embodiment, the tumor disease is a solid tumor or hematological cancer mediated by a tumor-specific antigen or a tumor-associated antigen, wherein 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; wherein the 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 lymphoma; Hodgkin's disease; non-Hodgkin Multiple myeloma; Waldenstrom's macroglobulinemia; heavy chain disease; myelodysplastic syndrome; hairy cell leukemia; and myelodysplasia; wherein the hematological cancer is most preferably Acute lymphocytic leukemia or chronic lymphocytic leukemia. In a specific embodiment, the tumor disease is a relapsed or refractory tumor or cancer. Wherein the tumor-specific antigen or tumor-associated antigen is selected from CD19, CD20, CD22, CD23, CD371, CD123, CD33, CD117, CS-1, ROR1, mesothelin, C-Met, PSMA, IL13Ra2, BCMA, Glypican- 3. Claudin 18.2, Her2, GD-2, DOG-1, Trop2, GUYC2C, MAGE A3 TCR or a combination thereof.

Description of the drawings

Figure 1 shows the transduction efficiency of different CAR molecules in T cells.

Figure 2 shows the transduction efficiency of different CAR molecules in T cells.

Figure 3 shows the comparison of biacore affinity data between FMC63 scFv and scFv after modification.

Figure 4 shows the binding of CD19 CAR-T to K562-CD19 cells (* represents P<0.05, ** represents P<0.01).

Figure 5 shows the binding of BCMA CAR-T to H929 cells.

Figure 6 shows the binding of Glypican-3 CAR-T to Huh7 cells.

Figure 7 shows the killing efficiency of CD19 CAR-T on K562-CD19 cells.

Figure 8 shows the killing efficiency of CD19 CAR-T on K562-CD19 cells.

Figure 9 shows the killing efficiency of Glypican-3 CAR-T on Huh7 cells.

Figure 10 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 11 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).

Figure 12 shows the anti-tumor ability and the ability of inhibiting tumor recurrence of CD19 CAR-T cells

Figure 13 shows the tumor suppressive effect of CD19 CAR-T on tumor-bearing mice.

Figure 14 shows the amount of CD19 CAR-T in the peripheral blood of tumor-bearing mice.

Figure 15 shows a schematic diagram of the metabolic curve of CD19 CAR-T in vivo.

Detailed ways

The following will further illustrate the present invention through specific descriptions.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

In this application, the singular forms "a" and "the" include plural objects, unless the context clearly dictates otherwise.

The term "antigen" as used herein refers to a molecule that causes an immune response, which may involve the production of antibodies, or the activation of specific immunocompetent cells. Those skilled in the art can understand that any macromolecule, including all proteins or peptides, can be used as an antigen. In addition, the antigen can be derived from recombinant or genomic DNA. Those skilled in the art can understand any 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. In addition, those skilled in the art can understand that the antigen need not be encoded by the full-length nucleotide sequence of the gene alone. It is easily obvious that 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. In addition, those skilled in the art can understand that antigens need not be encoded by "genes" at all, and antigens can be produced, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

The term "chimeric antigen receptor" or "CAR" as used herein 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. In some aspects, 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). In some embodiments, the CAR can target cancer by redirecting the specificity of T cells expressing CARs specific for tumor-associated antigens. Herein, "CAR-n" and "CAR-T-n" are interchangeable and represent the CAR-T label of a specific sequence, where n=1 to any integer from 1 to 39.

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. Preferably the domain is capable of interacting with cell surface molecules. For example, 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. In the present invention, 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). 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). The CDRs of the antibodies and antigen binding domains disclosed in the present invention are defined or recognized by Kabat numbering.

The term "single chain variable region fragment", "single chain antibody" or "scFv" as used herein 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) 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. The 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.

As used herein, the term "protective peptide" refers to an oligopeptide operably linked to the N-terminal, C-terminal or any position in the middle of the scFv. This peptide can change the affinity of the scFv to the target such as CD19, but compared to the non-linked protection The interaction of the peptide CAR, the CAR cell connected to the protective peptide and the target protein is changed. 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.

As used herein, 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. In the present invention, 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. Those skilled in the art should understand that 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.

The terms "peptide", "polypeptide" and "protein" as used herein 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. As used herein, 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 term "vector" as used herein 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. Thus, the term "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. Examples of 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" as used herein 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" as used herein 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. For example, a 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.

As used herein, "isolated" refers to a change or removal from a natural state. For example, 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, such as, for example, a host cell.

Unless otherwise specified, the 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.

The term "operably linked" as used herein 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. For example, when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence, the first nucleic acid sequence and the second nucleic acid sequence are operably linked. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably linked to the coding sequence. Generally, DNA sequences that are operably linked are adjacent and, if necessary, join two protein coding regions in the same reading frame.

The term "variant" as used herein 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.

The term "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.

As used herein, the term "subject" includes any human or non-human animal. The term "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. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably. In the present invention, the preferred subject is a human.

As used herein, the term "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. For the purposes of this disclosure, 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. Therefore, those skilled in the art realize that treatment can improve the condition of the disease, but may not be a complete cure for the disease. As used herein, the term "treatment" includes prevention. Alternatively, 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.

The term "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, vasculitis, vitiligo, mucinous edema, malignancy Anemia, ulcerative colitis, etc.

Where numerical limits or ranges are stated herein, the endpoints are included. In addition, all values and sub-ranges within the numerical limit or range are specifically included.

Example

The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The present invention will be further understood with reference to the following non-limiting experimental examples.

Example 1: CAR molecule construction and CAR-T preparation

In this example, CAR molecules with different antigen binding domains, costimulatory signal domains and CD3ξ signal domains are designed. The antigens are tumor-specific antigens or tumor-related antigens, and the tumor-specific antigens or tumor-related antigens are selected from CD19 , CD20, CD22, CD33, CD123, CD371, CD117, CS-1, ROR1, Mesothelin, C-Met, PSMA, IL13Ra2, BCMA, Glypican-3, Claudin 18.2, Her2, GD-2, DOG-1, Trop2, GUYC2C, MAGEA3TCR, etc. and their combinations. The following takes the preparation of CAR-T cells as an example.

Construction of antigen binding domain modified by protective peptide: Without changing the nature and function of scFv, replace the scFv antibody framework sequence with human germline antibody framework sequence; or at the N-terminus, C-terminus, or heavy chain of scFv A protective peptide rich in polar amino acids with an amino acid length of 8-15 is added between the variable region and the variable region of the light chain. Taking FMC63 scFv, 11D5-3 scFv and GC33 scFv as examples 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. Add the protective peptide sequence 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, 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 amino acid sequence of CAR (CAR-4) with the protective peptide added to the C-terminus of FMC63 scFv is shown in SEQ ID NO: 74, and the nucleotide sequence is shown in SEQ ID NO: 75.

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 sequence is shown in SEQ ID NO: 80, and the nucleotide sequence is shown in SEQ ID NO: 81; the 4-1BB costimulatory domain The amino acid sequence of CAR (CAR-8) with an additional RFPEEEEGGCE sequence added to the C-terminus of the natural sequence is shown in SEQ ID NO: 82, and the nucleotide sequence is shown in SEQ ID NO: 83; the 4-1BB costimulatory domain is native The amino acid sequence of CAR (CAR-9) with the DLAMADLEQKV sequence added to the C-terminus of the sequence is shown in SEQ ID NO: 84, and the nucleotide sequence is shown in SEQ ID NO: 85.

The amino acid sequence of the initial CD3ζ 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 CD3ζ 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 CD3ζ 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 CD3ζ 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.

CAR (CAR-13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, and 29) amino acid sequences are as SEQ ID NO: 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, and 124, and the nucleotide sequences are as shown in SEQ ID NO: 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 and 125 are shown.

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).

The amino acid sequence of CAR (CAR-31 or CAR-34) with a protective peptide added to the N-terminus of FMC63 scFv is as SEQ ID NO: 130 or 133. Specifically, the FMC63 scFv encoding the protective peptide was synthesized by gene synthesis (Beijing Bomed Gene Technology Co., Ltd.), using the existing CAR plasmid targeting CD19 as a template (CAR nucleotide sequence targeting CD19) Refer to SEQ ID NO: 13 in the patent CN 105177031 B), using PCR to clone the CAR molecule containing the CD8α hinge region, CD8α transmembrane region, the intracellular region of 4-1BB (corresponding to NP_001552.2), and CD3ζ (corresponding to NP_000725.1) Nucleic acid fragment of the intracellular region. Using the synthesized FMC63 scFv gene and the obtained nucleic acid fragment as a template, the complete nucleic acid fragment of the CAR gene with the protective peptide was cloned by PCR. Through restriction enzyme digestion and ligation methods, the obtained complete nucleic acid fragment was inserted into the lentiviral vector pLenti6.3/V5 (Thermo Fisher, Waltham, MA, USA) to obtain a lentiviral transfer plasmid carrying the CAR gene. The lentiviral packaging plasmids pLP/VSVG, pLP1/MDK, pLP2/RSK (Thermo Fisher, Waltham, MA, USA) and the transfer plasmid were transfected into HEK293T cells with Lipofectamine 3000 (Thermo Fisher, Waltham, MA, USA), 48 After hours, the medium was collected and centrifuged at 300 g to remove cell debris, and then centrifuged at 25,000 rpm in an ultracentrifuge for 3 hours. Dissolve the precipitate with 1 mL of normal saline, which is the required lentiviral vector.

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 the ratio of the number of T cells to CD3/CD28 Dynabeads (Thermo Fisher) was 1:1 After adding Dynabeads to the culture system and adding IL-2 (Shandong Jintai Bioengineering Co., Ltd., China) (500IU/mL) for 48 hours, CAR was transduced into T cells via lentivirus. 24 hours after the virus infects the cells, 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 and Figure 2 show that when the MOI of lentivirus infection is 0.5, the transduction efficiency of each CAR gene is between 15-40%.

Table 1. Sequence information

Example 2: Comparison of affinity between FMC63 scFv and FMC63 scFv modified by protective peptides and CD19 protein

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 variable For example, the affinity of scFv (CAR-3 scFv) with protective peptide added between regions, and scFv (CAR-4 scFv) with protective peptide added to C-terminal of FMC63 scFv and CD19 protein.

A solution of human CD19-Fc protein (Acro BIOSYSTEMS, Newark, DE, USA) at a concentration of 50 μg/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 50μg/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. After blocking the chip with ethanolamine, it was incubated with CAR-1 scFv, CAR-2 scFv, CAR-3 scFv, CAR-4 scFv, and then with glycine at a suitable pH. The chip was washed with hydrochloric acid buffer, ^{and the affinity of each scFv to CD19 was detected and analyzed with Biacore TM} T200 equipment (GE healthcare, Chicago, IL, USA), including the binding rate constant Ka, the dissociation constant Kd, and the equilibrium dissociation constant KD.

Figure 3 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 is 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.

The above results show that adding a protective peptide at the N-terminus, C-terminus, or between the variable region of the light chain and the variable region of the heavy chain of the scFv can change and reduce the affinity of the scFv to the target.

Example 3: The binding efficiency of CD19 CAR-T containing the antigen-binding domain of FMC63 scFv modified with protective peptides and target cells

In this embodiment, the binding efficiency of the initial FMC63 scFv, FMC63 scFv with a protective peptide added to the N-terminus, and FMC63 scFv with a protective peptide added to the C-terminus of the scFv and CD19-expressing target cells is used as an example to compare the binding efficiency.

^{Add 6×10 5} CD19-overexpressing K562 cells (K562-CD19) or normal K562 cells to each well of a 24-well cell culture plate (Corning Incorporated, Corning, NY, USA) (National Laboratory Cell Resource Sharing Platform, China ), add T cells or CAR-T cells according to E:T=3:1 (effective target ratio), mix gently, transfer the above cell plate to 37℃, 5% CO ₂ cell incubator (Thermo Fisher) After incubating for 2 hours, incubate with homemade CD19 CAR antibody coupled with FITC fluorescent molecule and CD19 antibody coupled with APC fluorescent molecule (Biolegend, San Diego, CA, USA) for 20 minutes, and then use a flow cytometer (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA) detects the expression of CAR and CD19. Use 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.

Figure 4 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. When CAR-T is incubated with K562-CD19 target cells for 1 hour, CAR-T containing the original FMC63 scFv (FMC63 CAR-T, its signal peptide, transmembrane region and intracellular region sequence are the same as CAR-T-1, CAR- T-2, CAR-T-3, CAR-T-4 are the same, only the extracellular region antigen binding domain sequence uses the original FMC63 scFv, does not contain protective peptides and flexible linker peptides) The binding rate to target cells Significantly higher than CAR-T (CAR-T-2 or CAR-T-4) containing FMC63 scFv with protective peptide added to the N-terminus or C-terminus; when CAR-T is incubated with K562-CD19 target cells for 2 hours, almost all FMC63 CAR-T, CAR-T-2 and CAR-T-4 all effectively bind to target cells. This shows that the original FMC63 scFv can bind to the target protein on the cell surface faster than the modified antigen binding domain, that is, when the target cell exists, the activation speed of FMC63 CAR-T is faster, while CAR-T-2 and CAR-T-2 CAR-T-4 can be activated more gently.

Example 4: Binding efficiency of BCMA CAR-T containing the antigen binding domain of 11D5-3 scFv modified by protective peptide to target cells

This embodiment takes as an example the comparison of the binding efficiency of CAR-T containing the initial 11D5-3 scFv, 11D5-3 scFv, N-terminal or C-terminal scFv with a protective peptide added to the BCMA-expressing target cell.

^{Add 6×10 5} U266 cells expressing BCMA (National Laboratory Cell Resource Sharing Platform, China) to each well of a 24-well cell culture plate (Corning Incorporated, Corning, NY, USA), press E:T=3:1 to add T cells or CAR-T cells, mix gently, transfer the above cell plate to 37°C, 5% CO ₂ cell incubator (Thermo Fisher) and incubate for 1 hour, then mix with homemade BCMA CAR coupled with FITC fluorescent molecules After incubating the antibody and BCMA antibody (Biolegend, San Diego, CA, USA) coupled with APC fluorescent molecule for 20 minutes, the expression of CAR and BCMA was detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA) . If CAR-T cells bind to the target cell through BCMA, the antibody cannot bind to the surface of the BCMA molecule, and the result of flow cytometry is that the expression of the BCMA molecule changes from positive to negative.

Figure 5 shows that T cells can hardly bind to H929 cells effectively, and CAR-T cells can bind to H929, indicating that the antigen binding domains in these CAR molecules can specifically recognize the BCMA target protein. When CAR-T is incubated with H929 target cells for 1 hour, CAR-T (11D5-3 CAR-T, CAR-38, SEQ ID NO: 137) containing the initial 11D5-3 scFv has a significantly higher binding rate to target cells CAR-T (CAR-T-32 or CAR-T-35) with 11D5-3 scFv added to the N-terminus or C-terminus of the protective peptide. This shows that the original 11D5-3 scFv can bind to the target protein on the cell surface faster than the modified antigen binding domain, that is, when the target cell exists, the 11D5-3 CAR-T activates faster, while the CAR- T-32 and CAR-T-35 can be activated more gently.

Example 5: The binding efficiency of Glypican-3 CAR-T containing the antigen binding domain of GC33 scFv modified with protective peptides and target cells

This embodiment takes as an example the comparison of the binding efficiency of CAR-T containing the initial GC33 scFv and GC33 scFv with a protective peptide added to the N-terminus of the CAR-T and the target cell expressing Glypican-3 (GPC3).

^{Add 6×10 5} Huh7 cells expressing Glypican-3 (National Experimental Cell Resource Sharing Platform, China) into each well of a 24-well cell culture plate (Corning Incorporated, Corning, NY, USA), press E:T=5: 1 Add T cells or CAR-T cells, mix gently, transfer the above cell plate to 37℃, 5% CO ₂ cell incubator (Thermo Fisher) and incubate for 1 hour. Glypican-3 CAR antibody and Glypican-3 antibody coupled with APC fluorescent molecule (Sino Biological, Bei Jing, China) were incubated for 20 minutes and then detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA) Expression of CAR and Glypican-3. If CAR-T cells bind to target cells through Glypican-3, the antibody cannot bind to the surface of Glypican-3 molecules, and the result of flow cytometry is that the expression of Glypican-3 molecules changes from positive to negative.

Figure 6 shows that T cells can hardly bind to Huh7 cells effectively, and CAR-T cells can bind to Huh7, indicating that the antigen binding domains in these CAR molecules can specifically recognize the Glypican-3 target protein. When CAR-T is incubated with Huh7 target cells for 1 hour, CAR-T containing the initial GC33 scFv (GC33 CAR-T, CAR-39, SEQ ID NO: 138) has a significantly higher binding rate to target cells than that containing GC33 scFv CAR-T (CAR-T-33 or CAR-T-36) with a protective peptide added to the N-terminus. This shows that the original GC33 scFv can bind to the target protein on the cell surface faster than the modified antigen binding domain, that is, when the target cell exists, the activation speed of GC33 CAR-T is faster, and CAR-T-33 and CAR-T-33 CAR-T-36 can be activated more gently.

Example 6: 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 CD3ξ signal domains on target cells expressing CD19.

Resuspend K562-CD19 target cells in 1 mL of normal saline, add 5 μL Calcein-AM (concentration 1 μg/μL, ThermoFisher, USA), mix gently, and then incubate in a 37°C water bath for 5 minutes to label target cells. ^{Add 1×10 5} K562-CD19 cells labeled above to each well of a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA), press E:T=5:1 to add various CAR-T cells, set Incubate in a 37°C, 5% CO ₂ cell incubator for 6 hours, and detect the fluorescence value of the cell supernatant (excitation wavelength: 495 nm, emission wavelength: 515 nm) with a fluorescence microplate reader (Varioscan Lux, ThermoFisher).

Figure 7 shows that CAR-Ts designed with different antigen binding domains, costimulatory signal domains and CD3ξ signal domains can all effectively kill K562-CD19 cells by recognizing the CD19 target protein.

Example 7: Killing efficiency of CAR-T containing antigen-binding domain with or without protective peptide modification on target cells expressing CD19

This example takes as an example the detection of the killing efficiency of CAR-T containing the antigen-binding domain with or without protective peptide modification on target cells expressing CD19.

Resuspend K562-CD19 target cells in 1 mL of normal saline, add 5 μL Calcein-AM (concentration 1 μg/μL, ThermoFisher, USA), mix gently, and then incubate in a 37°C water bath for 5 minutes to label target cells. ^{Add 1×10 5} K562-CD19 cells labeled above to each well of a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA), press E:T for 1:1, 5:1 or 10:1 to add Incubate various CAR-T cells in a 37°C, 5% CO ₂ cell incubator for 6 hours. Use a fluorescence microplate reader (Varioscan Lux, ThermoFisher) to detect the fluorescence value of the cell supernatant (excitation wavelength: 495nm, emission wavelength) : 515nm).

Figure 8 shows that CAR-T can effectively kill K562-CD19 cells by recognizing the CD19 target protein, and the killing efficiency of CAR-T-31 and CAR-T-34 is better than FMC63 CAR-T (CAR-37, SEQ ID NO: 136), indicating that these CAR-T cells can be activated and lysed to the target cells within a short period of incubation with the target cells, and the CAR-T modified by the protective peptide has a higher killing efficiency.

Example 8: The efficiency of Glypican-3 CAR-T in killing target cells

This example takes as an example the detection of the killing efficiency of CAR-T containing the antigen-binding domain with or without protective peptide modification on target cells expressing Glypican-3.

Resuspend Huh7 target cells in 1 mL of normal saline, add 5 μL Calcein-AM (concentration 1 μg/μL, ThermoFisher, USA), mix gently, and then incubate in a 37°C water bath for 5 minutes to label target cells. ^{Add 1×10 5} of the above-labeled Huh7 cells to each well of a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA), press E:T for 1:1, 5:1 or 10:1 to add various CAR-T cells were incubated in a 37°C, 5% CO ₂ cell incubator for 6 hours, and the fluorescence value of the cell supernatant (excitation wavelength: 495nm, emission wavelength: 515nm) was detected with a fluorescent microplate reader (Varioscan Lux, ThermoFisher) ).

Figure 9 shows that CAR-T can effectively kill Huh7 cells by recognizing the Glypican-3 target protein, and the killing efficiency of CAR-T-33 is better than that of CAR-T-36 and GC33 CAR-T, indicating that the short period of incubation with target cells Within a period of time, these CAR-T cells can all be activated and lyse the target cells, and the CAR-T modified by the protective peptide has a higher killing efficiency.

It can be seen from the above experimental results that the inventors' design of the protective peptide for the antigen binding domain of the CAR molecule is beneficial to the function of CAR-T to recognize the target and kill tumor cells.

Example 9: Proliferation of CD19 CAR-T stimulated by target cells

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 CD3ξ signal domains under the stimulation of target cells expressing CD19.

^{Add 1×10 5} K562-CD19 cells to each well of a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA), press E:T=5:1 to add various CAR-T cells, and place at 37°C , 5% CO ₂ cell culture incubator for 3 days, stain the cells with trypan blue to count the total number of viable cells, and label the cells with a self-made CD19 CAR antibody coupled to FITC fluorescent molecules, and use a flow cytometer (NovoCyte 2060R , ACEA Biosciences, San Diego, CA, USA) detected the proportion of CAR-T in each group of cells, and calculated the proliferation fold of CAR-T in each group.

Figure 10 shows that compared with FMC63 CAR-T, CAR-Ts containing different antigen binding domains, costimulatory signal domains and CD3ξ signal domain modifications, especially CAR-Ts containing different costimulatory signal domains and CD3ξ 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.

Example 10: Cytokine expression of CD19 CAR-T stimulated by target cells

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 CD3ξ signal domains under the stimulation of target cells expressing CD19.

^{Add 1×10 5} K562-CD19 cells to each well of a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA), press E:T=5:1 to add various CAR-T cells, and place at 37℃ Incubate for 12 hours in a 5% CO ₂ cell incubator, and use a cytometric bead array (BD Biosciences, San Jose, CA, USA) on the cell supernatant with a flow cytometer (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA) Detect the expression level of various cytokines.

Figure 11 shows that, compared with T cells, the expression levels of cytokines such as IL-6, IL-10, TNF, and IFN-γ are significantly increased under the stimulation of K562-CD19 target cells for various CAR-Ts, 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 CD3ξ signal domain CAR-T, especially CAR-T containing different costimulatory signal domain and CD3ξ 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 CAR-T on target cells. The risk of improving safety.

Example 11: 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). 24 hours after the virus infection, 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). When the cells were cultured to the 11th day, the cells were harvested and counted, and kept at the same time. 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. 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. 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 12 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 12: 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 CD3ξ 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). 24 hours after the virus infection, 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). When the cells were cultured to the 11th day, the cells were harvested and counted, and kept at the same time. 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. A total of 36 NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., China) aged 6-8 weeks were divided into 6 mice/groups for a total of 6 groups. After each mouse was injected with 1.0×10 ⁶ Nalm-6-LAE cells (ATCC, USA) from the tail vein for 5 days, 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. 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 13 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 14 shows that compared with FMC63 CAR-T, CAR-T-13, CAR-T-15 and CAR-T-17 have significantly improved persistence in the body. 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. Higher levels of CAR-T cells can still be detected in the blood, indicating that compared with FMC63 CAR-T, CAR-T cells containing modified antigen binding domains, modified costimulatory signal domains, and modified CD3ξ signal domains The sustained performance in the body has been significantly improved.

From the above in vitro and in vivo experimental results, it can be seen that the inventors have been very successful in designing the antigen binding domain, costimulatory signal domain and CD3ξ signal domain of the CD19 CAR molecule, especially for the design of the costimulatory signal domain and CD3ξ signal domain. Significantly improve the amplification efficiency and duration of CD19 CAR molecules in vivo, as shown in Figure 15 for the in vivo amplification curve model.

As mentioned above, the inventor has described the embodiments based on the present invention, but the present invention is not limited to this. It should be understood by those skilled in the art that modifications and changes can be implemented within the scope of the present invention. The manner of deformation and modification should belong to the protection scope of the present invention.

Claims

The use of a protective peptide in the preparation of a product targeting an antigen, wherein

The protective peptide is a peptide containing polar amino acids and a length of 8-15 amino acids;

The antigen is a tumor-specific antigen or a tumor-related antigen;

The product is selected from a protein comprising an antigen-binding domain, a chimeric antigen receptor comprising the antigen-binding domain, a nucleic acid encoding the antigen-binding domain or the chimeric antigen receptor, and a nucleic acid comprising the nucleic acid A vector, a cell containing the nucleic acid;

The antigen binding domain includes: a ligand or receptor protein fragment capable of binding to the antigen; a single chain variable region fragment capable of binding to the antigen, which includes the light chain variable region and the heavy chain variable region At least one; wherein the protective peptide is located at the N-terminal, C-terminal, or between the variable region of the light chain and the variable region of the heavy chain of the antigen binding domain;

The protective peptide has at least one of the following characteristics: the protective peptide can change the affinity of the antigen binding domain or the chimeric antigen receptor to the antigen; the protective peptide can change the antigen binding structure Interaction between cells containing the domain and target cells expressing the antigen; improving the killing efficiency of the cells containing the antigen-binding domain against the target cells expressing the antigen; improving the expansion of the cells containing the antigen-binding domain under antigen stimulation ability.
The use according to claim 1, wherein the number of the polar amino acids in the protective peptide accounts for more than 60%, and the number of the polar amino acids is 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, or 15.
The use according to claim 1, wherein the protective peptide is selected from the amino acid sequence shown in SEQ ID NOs: 6, 7, 8, 9 or 10 or has at least 75%, 80%, 85%, An amino acid sequence that is 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, or 99% or more identical.
The use according to claim 1, wherein the protective peptide and the antigen binding domain, light chain variable region, heavy chain variable region, ligand, or receptor protein can selectively pass through a glycine- and serine-rich The flexible linker peptide is connected. Preferably, the flexible linker peptide is (G 4 S) n , where n is 1, 2, 3, or 4.
The use according to claim 1, wherein the tumor-specific antigen or tumor-associated antigen is selected from the group consisting of CD19, CD20, CD22, CD23, CD371, CD123, CD33, CD117, CS-1, ROR1, mesothelin, C -Met, PSMA, IL13Ra2, BCMA, Glypican-3, Claudin 18.2, Her2, GD-2, DOG-1, Trop2, GUYC2C, MAGE A3 TCR or a combination thereof.
The use according to claim 1, wherein the chimeric antigen receptor further comprises a costimulatory domain and/or an intracellular signal transduction domain; preferably, the costimulatory domain is selected from CD27, CD28, 4 -1BB, OX-40, CD30, CD40, PD-1, CTLA-4, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3 or any combination thereof; preferably, the intracellular The signal transduction domain is selected from CD3ζ.
The use according to claim 6, wherein the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof, and the variants include Q20F, T21P, T22E, Q23E, F32Q, One or more of the amino acid substitutions of P33T, E34T, E35Q, or the variant is the amino acid sequence described in SEQ ID NO: 41. The N-terminus or C-terminus of the amino acid sequence described in SEQ ID NO: 41 is connected with a section as described in SEQ ID NO: 53, 54 or 55. Amino acid sequence.
The use according to claim 6, wherein the 4-1BB costimulatory domain comprises an amino acid sequence as shown in SEQ ID NOs: 43, 45, 47, 49 or 51.
The use according to claim 6, wherein the CD3ζ 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, One or more of Y90F, L104Y, H105R, M106H amino acid substitutions.
The use according to claim 6, wherein the intracellular signal transduction domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NOs: 58, 60, 62, 64 or 66.
The use according to claim 6, wherein the chimeric antigen receptor further comprises a hinge region and a transmembrane domain, preferably the hinge region and a transmembrane domain are selected from IgG1, IgG4, CD8α, CD28, IL- 2 Hinge region and transmembrane domain of receptor, IL-7 receptor, IL-11 receptor, PD-1 or CD34.
The use according to claim 1, wherein the cells are lymphocytes, preferably, the lymphocytes are selected from T cells, B cells, natural killer cells or dendritic cells; more preferably, the cells are selected from Cytotoxic T cells, tumor-infiltrating T cells, or regulatory T cells.
The use according to claim 1, wherein the target cell is a cell expressing the antigen.
An isolated antigen binding domain, which comprises:

a) A single chain variable region fragment capable of binding antigen, which includes at least one of a light chain variable region and a heavy chain variable region; or a ligand or receptor protein fragment capable of binding antigen;

b) One or more protective peptides, wherein the protective peptide comprises a polypeptide of 8-15 amino acids in length rich in polar amino acids, and the protective peptide is operably linked to the variable region of the light chain or the heavy chain N-terminal, C-terminal of the variable region, or between the variable region of the light chain and the variable region of the heavy chain;

The antigen is a tumor-specific antigen or a tumor-related antigen;

Preferably, the protective peptide and the antigen binding domain, light chain variable region, heavy chain variable region, ligand, or receptor protein can be selectively connected via a flexible linker peptide rich in glycine and serine.
The antigen binding domain of claim 14, wherein the tumor-specific antigen or tumor-associated antigen is selected from the group consisting of CD19, CD20, CD22, CD23, CD371, CD123, CD33, CD117, CS-1, ROR1, mesothelin, C-Met, PSMA, IL13Ra2, BCMA, Glypican-3, Claudin 18.2, Her2, GD-2, DOG-1, Trop2, GUYC2C, MAGE A3 TCR or a combination thereof.
An isolated chimeric antigen receptor comprising the antigen binding domain of claim 14 or 15.
The chimeric antigen receptor according to claim 16, which further comprises a costimulatory domain and/or an intracellular signal transduction domain, preferably the costimulatory domain is selected from CD27, CD28, 4-1BB, OX- 40, CD30, CD40, PD-1, CTLA-4, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, NKG2D, B7-H3 or any combination thereof, preferably the intracellular signal transduction domain Selected from CD3ζ.
The chimeric antigen receptor according to claim 17, wherein the costimulatory domain is selected from 4-1BB, and the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 41 or a variant thereof The variant includes one or more amino acid substitutions of Q20F, T21P, T22E, Q23E, F32Q, P33T, E34T, E35Q, or the variant is the N-terminal or C of the amino acid sequence described in SEQ ID NO: 41 The end is connected with an amino acid sequence as described in SEQ ID NO: 53, 54 or 55.
The chimeric antigen receptor according to claim 18, wherein the costimulatory domain is selected from 4-1BB, and the 4-1BB costimulatory domain comprises as shown in SEQ ID NOs: 43, 45, 47, 49 or 51 The amino acid sequence shown.
The chimeric antigen receptor according to claim 17, wherein the CD3ζ 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, H105R, M106H amino acid substitutions one or more.
The chimeric antigen receptor according to claim 20, wherein the intracellular signal transduction domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NOs: 58, 60, 62, 64 or 66.
The chimeric antigen receptor according to claim 17, which further comprises a hinge region and a transmembrane domain, preferably the hinge region and the transmembrane domain are selected from IgG1, IgG4, CD8α, CD28, IL-2 receptor , IL-7 receptor, IL-11 receptor, PD-1 or CD34 hinge region and transmembrane domain.
An isolated nucleic acid molecule encoding the antigen binding domain of claim 14 or 15 or the chimeric antigen receptor of any one of 16-22.
A vector comprising the isolated nucleic acid molecule of claim 23; preferably, wherein the vector is selected from one or more of DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector kind.
A cell comprising the isolated nucleic acid molecule according to claim 23 or the vector according to claim 24.
The cell according to claim 25, wherein the cell is selected from T lymphocytes, B lymphocytes, natural killer cells or dendritic cells, preferably, the cells are selected from cytotoxic T cells, tumor infiltrating T cells or Regulatory T cells.
A pharmaceutical composition comprising one or more selected from the following:

i) The isolated antigen binding domain of claim 14 or 15;

ii) The isolated chimeric antigen receptor of any one of claims 16 to 22;

iii) the isolated nucleic acid molecule of claim 23,

iv) the vector of claim 24, and

v) The cell of claim 25 or 26;

And, a pharmaceutically acceptable carrier, diluent or excipient.
A method for preparing the cell of claim 25 or 26, which comprises:

The nucleic acid encoding the chimeric antigen receptor of any one of claims 16 to 22 is introduced into the cell.
Use of the pharmaceutical composition according to claim 27 or the cell according to claim 25 or 26 in the preparation of a medicament for the treatment of tumor diseases, autoimmune diseases, infectious diseases caused by viruses or bacteria.
A method for cellular immunotherapy in subjects suffering from tumor diseases, autoimmune diseases, infectious diseases caused by viruses or bacteria, which comprises administering to the subject the pharmaceutical composition according to claim 27 or The cell of 25 or 26 is required.
The use of claim 29 or the method of claim 30, wherein the tumor disease is a solid tumor or hematological cancer mediated by a tumor-specific antigen or a tumor-associated antigen, wherein 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, etc., wherein the hematology The cancer is selected from: acute leukemia, including acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia; chronic leukemia , Including chronic myeloid (granulocyte) leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and refractory CD19+ leukemia and lymphoma; polycythemia vera, lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma Tumor, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplastic ; Preferably, the tumor disease is a relapsed or refractory tumor or cancer.