Chimeric antigen receptors that bind to CD19 and uses thereof

Abstract

Provided is a separated antigen binding domain comprising a single-chain variable region fragment capable of binding to an antigen, the single-chain variable region fragment comprising a light chain variable region and a heavy chain variable region; and one or more protective peptides. The invention also provides the chimeric antigen receptor which comprises a hinge region, a transmembrane region, a costimulatory signal structural domain and an intracellular signal transduction structural domain, and corresponding nucleic acid molecules, carriers, cells, compositions, preparation methods and medicines for treating diseases such as cancer or virus infection.

Description

[0001] This application is a divisional application of the invention patent application with application date of March 24, 2021, application number 202180019189.7 (international application number PCT / CN2021 / 082683), and invention name “Chimeric antigen receptor binding to CD19 and its use”. Technical Field

[0002] The present invention relates to the fields of immunology and molecular biology, in particular to immune cells modified with chimeric antigen receptors (CAR) and uses thereof. Background Art

[0003] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) technology combines the specificity of antibodies and the killing effect of T cells, thus forming 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 signaling domain. The second-generation CAR adds intracellular co-stimulatory domains, such as CD28, CD137, OX40, etc., which are usually located between the transmembrane region and the intracellular signaling domain. The third-generation CAR uses two co-stimulatory domains to enhance the activation of CAR-T. CAR-T technology has achieved great success in the treatment of hematological tumors, especially CAR-T targeting CD19, but some patients still do not respond or relapse after response, and there are still deficiencies in the durability of efficacy and toxicity. Two important issues in the efficacy and safety of CAR-T are: 1) the proliferation and persistence of CAR-T in the body is insufficient; 2) CAR-T can cause toxicity and side effects such as cytokine storm, neurotoxicity and off-target effects.

[0004] The killing ability of CAR-T cells on target cells, the ability to proliferate in vivo, and their safety are affected by many factors, including CAR design, antigen density on the surface of target cells, CAR-T preparation process, etc. Among them, CAR design is the key, and it is necessary to consider the affinity between the antigen binding domain of CAR and the target, the composition of the co-stimulatory signal domain, and the composition of the CD3ξ signal domain, etc. 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 indication and treatment strategy. Summary of the invention

[0005] In one aspect, the present invention discloses an isolated antigen binding domain comprising:

[0006] a) a single-chain variable region fragment capable of binding to CD19, comprising a light chain variable region and a heavy chain variable region;

[0007] b) one or more protective peptides, wherein the protective peptide segment comprises a polypeptide of 8-15 amino acids in length that is rich in polar amino acids, and the protective peptide is operably linked to the N-terminus, C-terminus, or between the light chain variable region and the heavy chain variable region of the light chain variable region or the heavy chain variable region;

[0008] The protective peptide and the light chain variable region or the heavy chain variable region can be selectively connected via a flexible linker peptide rich in glycine and serine.

[0009] In a specific embodiment, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, and the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:2.

[0010] In a specific embodiment, the protective peptide comprises an amino acid sequence selected from SEQ ID NOs: 6, 7, 8, 9 or 10, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0011] In one embodiment, the protective peptide is operably connected to the N-terminus or C-terminus of the light chain variable region; in one embodiment, the protective peptide is operably connected to the N-terminus or C-terminus of the heavy chain variable region; in one embodiment, the protective peptide is operably connected between the light chain variable region and the heavy chain variable region. In a specific embodiment, the amino acid sequence of the glycine- and serine-rich flexible linker peptide can be (G 4 S) n , wherein n=1-4, preferably the flexible peptide can be (G 4 S) 2 , i.e. 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 rich in glycine and serine, such as (G 4 S) 2 The protective peptide segment and the flexible linker peptide are operably connected, for example, at the N-terminus of the light chain variable region.

[0012] In one embodiment, the antigen binding domain comprises an amino acid sequence selected from SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0013] In another aspect, the present invention discloses an isolated chimeric antigen receptor comprising the antigen binding domain as described above.

[0014] In one embodiment, the chimeric antigen receptor further comprises a co-stimulatory domain and / or an intracellular signaling domain.

[0015] In a specific embodiment, 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. In a specific embodiment, the intracellular signal transduction domain is selected from CD3ζ.

[0016] In a specific embodiment, the costimulatory domain is selected from 4-1BB. Preferably, the 4-1BB costimulatory domain comprises an amino acid sequence as described in SEQ ID NO:41 or a variant thereof, wherein the 4-1BB variant includes one or more of the amino acid substitutions of Q20F, T21P, T22E, Q23E, F32Q, P33T, E34T, and E35Q in SEQ ID NO:41, or the variant is an amino acid sequence as described in SEQ ID NO:41 connected to the N-terminus or C-terminus of a segment of an amino acid sequence as described in SEQ ID NO:53, 54, or 55.

[0017] In a specific embodiment, the 4-1BB co-stimulatory 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 a specific embodiment, the variant includes the Q20F amino acid substitution in SEQ ID NO:41; in a specific embodiment, the variant includes the Q20F, T21P and Q23E amino acid substitutions in SEQ ID NO:41; in a specific embodiment, the variant includes the Q20F, T21P, T22E, Q23E, F32Q, P33T, E34T, E35Q amino acid substitutions in SEQ ID NO:41.

[0018] In a specific embodiment, the costimulatory domain is selected from 4-1BB. Preferably, the 4-1BB costimulatory domain comprises an amino acid sequence as shown in SEQ ID NOs: 43, 45, 47, 49 or 51, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0019] In one embodiment, the CD3ζ intracellular signal transduction domain comprises an amino acid sequence as described in SEQ ID NO: 56 or a variant thereof, wherein the variant comprises one or more of V2L, D9E, Q14K, Q15K, Y90F, L104Y, H105R, and M106H. In a specific embodiment, the CD3ζ variant comprises a Q14K amino acid substitution. In a specific embodiment, the CD3ζ variant comprises a Q15K amino acid substitution, and may further comprise V2L and Y90F amino acid substitutions, and may further comprise D9E, for example, in a specific embodiment, the CD3ζ variant comprises V2L, Q15K, and Y90F amino acid substitutions; in a specific embodiment, the CD3ζ variant comprises V2L, D9E, Q15K, and Y90F amino acid substitutions; in an embodiment, the CD3ζ variant comprises V2L, D9E, Q15K, Y90F, L104Y, H105R, and M106H amino acid substitutions.

[0020] 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 an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0021] 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 the hinge region and the transmembrane domain of IgG1, IgG4, CD8α, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1 or CD34. The chimeric antigen receptor may further include a signal peptide.

[0022] In a specific embodiment, the chimeric antigen receptor comprises, linked in sequence:

[0023] a) signal peptide;

[0024] b) an antigen binding domain comprising an amino acid sequence as shown in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto;

[0025] c) such as the hinge region of CD8α and the transmembrane domain of CD8α;

[0026] d) a 4-1BB costimulatory domain comprising an amino acid sequence as shown in SEQ ID NOs: 41, 43, 45, 47, 49 or 51, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and

[0027] e) a CD3ζ intracellular signal transduction domain comprising an amino acid sequence as shown in SEQ ID NOs: 58, 60, 62, 64 or 66, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0028] In a specific embodiment, the chimeric antigen receptor comprises an amino acid sequence as shown in 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, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0029] In yet another aspect, the present invention further discloses an isolated nucleic acid molecule encoding the antigen binding domain or the chimeric antigen receptor as described in the present invention.

[0030] The present invention discloses a vector, which comprises the isolated nucleic acid molecule as described above; preferably, the vector is selected from one or more of DNA, RNA, plasmid, lentiviral vector, adenoviral vector and retroviral vector. The vector is an expression vector or a cloning vector.

[0031] The present invention discloses a cell, which comprises the isolated nucleic acid molecule or the vector as described above.

[0032] In one embodiment, the cell is a T lymphocyte, a B lymphocyte, a natural killer cell, a dendritic cell, a cytotoxic T cell, a tumor-infiltrating T cell or a regulatory T cell. In a specific embodiment, the cell is selected from human peripheral blood T lymphocytes.

[0033] The present invention discloses a pharmaceutical composition comprising one or more selected from the following:

[0034] i) an isolated antigen-binding domain as described above;

[0035] ii) an isolated chimeric antigen receptor as described above;

[0036] iii) an isolated nucleic acid molecule as described above;

[0037] iv) a vector as described above; and

[0038] v) cells as described above;

[0039] and pharmaceutically acceptable carriers, diluents or excipients.

[0040] The present invention discloses a method for preparing the cell as described above, comprising: introducing a nucleic acid encoding the chimeric antigen receptor as described above into the cell.

[0041] In another aspect, the present invention also discloses the use of the aforementioned antigen binding domain, chimeric antigen receptor, nucleic acid molecule, vector, cell or pharmaceutical composition in the preparation of a drug; the use of the aforementioned antigen binding domain, chimeric antigen receptor, nucleic acid molecule, vector, cell or pharmaceutical composition in the preparation of a drug for treating a disease, wherein the disease is preferably selected from tumors, autoimmune diseases, or infectious diseases caused by viruses or bacteria.

[0042] The present invention discloses a method for performing cellular immunotherapy in a subject suffering from a disease, comprising administering the pharmaceutical composition or the cell as described above to the subject, wherein the disease is preferably selected from tumors, autoimmune diseases, or infectious diseases caused by viruses or bacteria.

[0043] In one embodiment, the tumor is a solid tumor or hematological cancer mediated by tumor-specific molecules, 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, including acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, granulocytic-monocytic, monocytic and erythroleukemia; chronic leukemia Diseases, which include chronic myeloid (granulocytic) leukemia, chronic myeloid 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's lymphoma; 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. Wherein the tumor-specific molecule is CD19.

[0044] In a specific embodiment, the tumor is a recurrent tumor or cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The transduction efficiency of different CAR molecules in T cells is shown.

[0046] Figure 2 The biacore affinity data of FMC63 scFv and the engineered scFv are compared.

[0047] Figure 3 The binding of CD19 CAR-T to K562-CD19 cells is shown (* represents P < 0.05, ** represents P < 0.01).

[0048] Figure 4 The killing efficiency of CD19 CAR-T on K562-CD19 cells is shown.

[0049] Figure 5 The figure shows the proliferation of CD19 CAR-T cells after 3-day stimulation of K562-CD19 cells (compared with FMC63CAR-T, * represents P < 0.05, ** represents P < 0.01).

[0050] Figure 6 The cytokine expression of CD19 CAR-T after 12 hours of stimulation of K562-CD19 cells is shown (compared with FMC63CAR-T, * represents P < 0.05, ** represents P < 0.01).

[0051] Figure 7 The results show that CD19 CAR-T cells have anti-tumor ability and the ability to inhibit tumor recurrence.

[0052] Figure 8 The figure shows the tumor inhibitory effect of CD19 CAR-T on tumor-bearing mice.

[0053] Fig. 9 The figure shows the changes in the number of CD19 CAR-T in the peripheral blood of tumor-bearing mice.

[0054] Fig.10 The figure shows the metabolic curve pattern of CD19 CAR-T in vivo. DETAILED DESCRIPTION

[0055] The present invention will be further illustrated by detailed description below.

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0057] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0058] The term "antigen" or "Ag" as used herein refers to a molecule that causes an immune response, which may involve antibody production, or the activation of specific immunocompetent cells. It will be appreciated by those skilled in the art that any macromolecule, including all proteins or peptides, can be used as an antigen. In addition, antigens may be derived from recombinant or genomic DNA. It will be appreciated by those skilled in the art that any DNA includes a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response, encoding the term "antigen" as used herein. In addition, it will be appreciated by those skilled in the art that antigens do not necessarily have to be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in different combinations to cause a desired immune response. In addition, it will be appreciated by those skilled in the art that antigens do not necessarily have to be encoded by "genes" at all, and that antigens may be produced, synthesized, or may be derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0059] The term "chimeric antigen receptor" or "CAR" as used herein refers to an artificial receptor, such as an artificial T cell receptor, that is engineered to express and specifically bind to an antigen on an immune effector cell. 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 a specific form of antigen. CAR can also include an intracellular activation domain, a transmembrane domain, and an extracellular domain, and the extracellular domain includes a tumor-associated antigen binding region. In some aspects, 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 a ligand (e.g., a peptide) of a receptor. In some embodiments, CAR can target cancer by redirecting the specificity of T cells expressing CAR specific to tumor-associated antigens.

[0060] The term "antigen binding domain" as used herein is also referred to as "ligand binding domain", which refers to an oligopeptide or polypeptide capable of binding a ligand. Preferably, the domain is capable of interacting with a cell surface molecule. For example, an extracellular ligand binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a specific disease state. Therefore, examples of cell surface markers that can be used 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 comprise a single-chain variable region fragment (single-chain antibody). The antigen binding domain may only bind to a portion of the antigen. The portion of the antigen molecule that is responsible for specific interaction with the antigen binding domain is referred to as an "epitope" or "antigenic determinant". The antigen binding domain typically 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 complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). The CDRs of the antibodies and antigen binding domains disclosed herein are defined or identified by Kabat numbering.

[0061] 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 immunoglobulin heavy chain variable region and the light chain variable region are connected by an amino acid peptide segment (linker). Various methods for generating single-chain antibodies are known, including those described in 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 a binding domain that interacts with an antigen. The CDRs in the heavy chain are abbreviated as VH-CDR, such as VH-CDR1, VH-CDR2, VH-CDR3, and the CDRs in the light chain are 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 identified by Kabat numbering.

[0062] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A 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 a protein or peptide. Polypeptides include 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 both short chains, which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers; and longer chains, which are commonly referred to in the art as proteins, which have many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0063] The terms "protective peptide" and "protective peptide" used herein are used interchangeably and refer to an oligopeptide that is operably connected to the N-terminus, C-terminus or any position in the middle of the scFv, which can reduce the affinity of the scFv to the target, such as CD19, but compared to the CAR without the protective peptide, the CAR cell connected to the protective peptide can bind to the target protein more slowly. The protective peptide can be connected to 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.

[0064] 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 (G4 S) n , wherein n is an integer greater than or equal to 1, such as any integer from 1 to 4, for example, it can be (G 4 S) 2 , i.e. 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 rich in glycine and serine, such as (G 4 S) 2 The protective peptide segment and the flexible linker peptide are operably connected, for example, at the N-terminus of the light chain variable region. It should be understood by those skilled in the art that any amino acid sequence rich in glycine and serine that can connect the protective peptide segment to the light chain variable region or the heavy chain variable region is within the scope of the flexible linker peptide.

[0065] The term "vector" as used herein refers to a molecular tool for transporting, transducing and expressing the contained exogenous target gene (e.g., the polynucleotide of the present invention) in a target cell, and the tool provides a suitable nucleotide sequence for initiating transcription in the target cell, i.e., a promoter. The vector includes an isolated nucleic acid, and it can be used to deliver the isolated nucleic acid to the interior of the cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphipathic compounds, plasmids and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmids and non-viral compounds that facilitate the transfer of nucleic acids into cells, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, etc.

[0066] As used herein, "expression vector" refers to a vector comprising a recombinant polynucleotide comprising 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).

[0067] "Identity" as used herein refers to the sequence identity between two nucleic acid molecules or polypeptides.Identity can be determined by comparing the position in each sequence aligned for comparison purposes.When the position in the sequence compared is occupied by the same base, the molecules are identical 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 the position shared by the nucleic acid sequence.The consistency between two sequences can be calculated using various alignment algorithms and / or programs, including FASTA or BLAST that can be obtained as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, for example, default settings.For example, it is envisioned that there is at least 70%, 85%, 90%, 95%, 98% or 99% consistency with the specific polypeptide described in this article and preferably a polypeptide that exhibits substantially the same function, and a polynucleotide encoding the above-mentioned polypeptide.

[0068] As used herein, "isolated" means changed or removed from its 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 the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment, for example, a host cell.

[0069] Unless otherwise specified, the nucleotide sequence "encoding" a nucleic acid molecule of a protein or an amino acid sequence of a protein used herein includes all nucleotide sequences that are degenerate and encode the same amino acid sequence. The nucleotide sequence may also include one or more introns.

[0070] The term "operably connected" as used herein refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, when a first nucleic acid sequence is in a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably connected to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably connected to the coding sequence. Typically, operably connected DNA sequences are adjacent and, if necessary, join two protein coding regions in the same reading frame.

[0071] The term "variant" used herein means a polypeptide variant obtained by replacing 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.

[0072] The term "anti-tumor ability" or "tumor inhibition" as used herein is a biological effect that can be clearly represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancerous conditions. "Anti-tumor ability" or "tumor inhibition" can also be represented by the ability of the peptides, polynucleotides, cells and antibodies disclosed herein to prevent tumors.

[0073] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably. In the present invention, the preferred subject is a human.

[0074] As used herein, the term "treatment" refers to administering to a subject an effective amount of cells having a polynucleotide sequence of a target gene altered in vitro according to the methods described herein, so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, a beneficial or desired clinical outcome. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or total relief), whether detectable or undetectable. Treatment may refer to prolonging survival compared to the expected survival without treatment. Therefore, those skilled in the art recognize that treatment may improve the disease condition, but may not be a complete cure for the disease. As used herein, the term "treatment" includes prevention. Alternatively, treatment is "effective" in the case of a reduction or cessation of disease progression. "Treatment" may also mean prolonging survival compared to the expected survival without treatment. Patients in need of treatment include those who have been diagnosed with a condition associated with the expression of a polynucleotide sequence, and those who may develop such a condition due to genetic susceptibility or other factors.

[0075] As used herein, the term "autoimmune disease" is defined as a disorder resulting from 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 parotitis, Crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, 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, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and the like.

[0076] Where numerical limits or ranges are stated herein, the endpoints are included. In addition, all values ​​and sub-ranges within the numerical limits or ranges are specifically included.

[0077] Example

[0078] The experimental methods in the following examples are conventional methods unless otherwise specified. The present invention will be further understood with reference to the following non-limiting experimental examples.

[0079] Example 1: CAR molecule construction and CAR-T preparation

[0080] This example takes the design of CD19CAR molecules with different antigen binding domains, co-stimulatory signal domains and CD3ξ signal domains and the preparation of CAR-T cells as an example.

[0081] Antigen binding domain construction: without changing the properties and functions of scFv, the antibody framework sequence of scFv is replaced with the human germline antibody framework sequence; or a protective peptide segment rich in polar amino acids with an amino acid length of 8-15 is added to the N-terminus, C-terminus, or between the heavy chain variable region and the light chain variable region of scFv. Taking FMC63 scFv as an example, 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-terminus, C-terminus, or between the heavy chain variable region and the light chain variable region of scFv to obtain an scFv with an added protective peptide sequence, such as 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.

[0082] The initial FMC63 scFv amino acid sequence is shown in SEQ ID NO: 3; the amino acid sequence of CAR (CAR-1) after humanization of the FMC63 scFv amino acid 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 the FMC63 scFv is shown in SEQ ID NO: 70, and the nucleotide sequence is shown in SEQ ID NO: 71; the amino acid sequence of CAR (CAR-3) with a protective peptide added between the heavy chain variable region and the light chain variable region of the FMC63 scFv 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 a protective peptide added to the C-terminus of the FMC63 scFv is shown in SEQ ID NO: 74, and the nucleotide sequence is shown in SEQ ID NO: 75.

[0083] The initial 4-1BB amino acid sequence is shown in SEQ ID NO:41, and the nucleotide sequence is shown in SEQ ID NO:42; the CAR (CAR-5) amino acid sequence in which the PEEE motif in the natural sequence of the 4-1BB costimulatory domain is replaced with PEQE 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 with a PEQE motif. The CAR (CAR-6) amino acid 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 4-1BB costimulatory domain is replaced with an FPEEE sequence, and 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 CAR (CAR-8) amino acid sequence in which an additional RFPEEEEGGCE sequence is added to the C-terminus of the natural sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO:82, and the nucleotide sequence is shown in SEQ ID NO:83; the amino acid sequence of CAR (CAR-9) with the DLAMADLEQKV sequence added to the C-terminus of the natural sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO:84, and the nucleotide sequence is shown in SEQ ID NO:85.

[0084] 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 the CD3ζ signal domain is replaced by Q15K is shown in SEQ ID NO:86, and 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, and the nucleotide sequence is shown in SEQ ID NO:89; the amino acid sequence of CAR (CAR-12) 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.

[0085] The amino acid sequences of CARs (CAR-13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29) after different combinations of different antigen binding domains, costimulatory signal domains and CD3ζ signal domains mentioned in Table 1 are shown in SEQ ID NOs: 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122 and 124, respectively, and the nucleotide sequences are shown in SEQ ID NOs: 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 and 125, respectively.

[0086] The nucleotide sequences 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 is inserted into the lentiviral vector pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA).

[0087] T cells were isolated from peripheral blood mononuclear cells of healthy volunteers (Miaotong (Shanghai) Biotechnology Co., Ltd., China). The isolated and purified T cells were concentrated at 1.0×10 6Cells / mL were inoculated and cultured in X-VIVO 15 medium (Lonza, Switzerland). Dynabeads were added to the culture system at a ratio of 1:1 between the number of T cells and CD3 / CD28 Dynabeads (Thermo Fisher), and IL-2 (Shandong Jintai Bioengineering Co., Ltd., China) (500 IU / mL) was added. After 48 hours of culture, CAR was transduced into T cells by lentivirus. 24 hours after the virus infected the cells, the cells were centrifuged and the medium was changed, and fresh X-VIVO 15 containing IL-2 (500 IU / mL) was added to continue the culture. After 4 days of cell culture, all cells in the culture system were collected, and the Dynabeads in the culture system were removed by magnetic stand. T cells were centrifuged and counted, and the CAR content of each group of cells was detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA). Figure 1 It was shown that when the lentiviral infection MOI = 0.5, the transduction efficiency of each CAR gene was between 15-40%.

[0088] Table 1. Sequence information

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] Example 2: Comparison of affinity between modified CD19 CAR antigen binding domain and FMC63 scFv and CD19 protein

[0111] This example compares the affinity of the initial FMC63 scFv, humanized FMC63 scFv (CAR-1scFv), scFv with a protective peptide added to the N-terminus of FMC63 scFv (CAR-2scFv), scFv with a protective peptide added between the heavy chain variable region and the light chain variable region of FMC63scFv (CAR-3scFv), and scFv with a protective peptide added to the C-terminus of FMC63 scFv (CAR-4scFv) to CD19 protein as an example.

[0112] A human CD19-Fc protein (Acro BIOSYSTEMS, Newark, DE, USA) solution with 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-1scFv, CAR-2scFv, CAR-3scFv, and CAR-4scFv coupled to the Fc fragment were prepared by Beijing Tongli Haiyuan Biotechnology Co., Ltd., and CAR-1scFv, CAR-2scFv, CAR-3scFv, and CAR-4scFv solutions with a concentration of 50 μg / mL were prepared using HBS-EP+ buffer. The Biacore CM5 chip (GE healthcare, Chicago, IL, USA) was pretreated with a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide for activation, and then incubated with a human CD19-Fc protein solution for coupling. After the chip was blocked with ethanolamine, it was incubated with CAR-1scFv, CAR-2scFv, CAR-3scFv, and CAR-4scFv, respectively. The chip was then rinsed with a glycine-hydrochloric acid buffer of appropriate pH and the chip was analyzed by Biacore.TM The T200 instrument (GE healthcare, Chicago, IL, USA) was used to detect and analyze the affinity of each scFv to CD19, including the binding rate constant Ka, dissociation constant Kd, and equilibrium dissociation constant KD.

[0113] Figure 2 The results showed that the KD value of the initial FMC63 scFv binding to CD19 was 3.27E-10, and the KD values ​​of CAR-1scFv, CAR-2scFv, CAR-3scFv, and CAR-4scFv binding to CD19 were 7.48E-8, 1.12E-8, 6.73E-8, ​​and 2.95E-9, respectively, indicating that compared with the initial FMC63 scFv, the affinity of the modified CAR scFv to CD19 was significantly reduced.

[0114] The above results show that adding a protective peptide to the N-terminus, C-terminus, or between the light chain variable region and the heavy chain variable region of scFv can reduce the affinity of scFv to targets such as CD19.

[0115] Example 3: Binding efficiency of CD19 CAR-T containing modified antigen binding domain from FMC63 scFv to target cells

[0116] This example takes the comparison of the binding efficiency of the original FMC63 scFv, the scFv with a protective peptide added to the N-terminus of the FMC63 scFv, and the scFv with a protective peptide added to the C-terminus of the FMC63 scFv with target cells expressing CD19 as an example.

[0117] 6 × 10 cells were added to each well of a 24-well cell culture plate (Corning Incorporated, Corning, NY, USA). 5 K562 cells overexpressing CD19 (K562-CD19) or normal K562 cells (National Laboratory Cell Resource Sharing Platform, China) were added with T cells or CAR-T cells at an E:T (effector-target ratio) of 3:1, and the cells were gently mixed. The cell plates were transferred to 37°C, 5% CO 2After incubation in a cell culture incubator (Thermo Fisher) for 2 hours, the cells were incubated with homemade CD19CAR antibodies coupled to FITC fluorescent molecules and CD19 antibodies coupled to APC fluorescent molecules (Biolegend, San Diego, CA, USA) for 20 minutes, and then the expression of CAR and CD19 was detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA). K562 and K562-CD19 cells that were not incubated with T cells or CAR-T cells were used as controls. If CAR-T cells bind to target cells through CD19, the APC fluorescent antibody cannot bind to the surface of the CD19 molecule. The flow cytometry results show that the proportion of APC fluorescent signals decreases and the proportion of FITC fluorescent signals increases.

[0118] Figure 3 The results showed that T cells could hardly bind to K562-CD19, and both T cells and CAR-T cells could not bind to normal K562 cells, but CAR-T cells could bind to K562-CD19, indicating that the antigen binding domains in these CAR molecules could specifically recognize the CD19 target protein. When CAR-T was incubated with K562-CD19 target cells for 1 hour, the binding rate of CAR-T containing the initial FMC63 scFv (FMC63 CAR-T, whose signal peptide, transmembrane region and intracellular region sequences are consistent with those in CAR-T-1, CAR-T-2, CAR-T-3, and CAR-T-4, only the extracellular antigen binding domain sequence uses the initial FMC63 scFv, and does not contain protective peptides and flexible linker peptides) to target cells was significantly higher than that of CAR-T containing FMC63 scFv with protective peptides added to the N-terminus or C-terminus (CAR-T-2 or CAR-T-4); when CAR-T was incubated with K562-CD19 target cells for 2 hours, almost all FMC63CAR-T, CAR-T-2, and CAR-T-4 effectively bound to target cells. This indicates that the initial FMC63 scFv can bind to the target protein on the cell surface faster than the modified antigen binding domain, that is, when target cells are present, FMC63 CAR-T is activated faster, while CAR-T-2 and CAR-T-4 can be activated more slowly.

[0119] Example 4: Efficiency of CD19 CAR-T in killing target cells

[0120] This example takes the detection of the killing efficiency of CAR-T containing modified antigen binding domains, different co-stimulatory signal domains, and different CD3ξ signal domains on target cells expressing CD19 as an example.

[0121] K562-CD19 target cells were resuspended in 1 mL of saline, and 5 μL of Calcein-AM (concentration 1 μg / μL, ThermoFisher, USA) was added. The cells were gently mixed and then placed in a 37°C water bath for 5 minutes to label the target cells. 1×10 5 The K562-CD19 cells labeled as above were added with various CAR-T cells at an E:T ratio of 5:1 and placed at 37°C and 5% CO 2 The cells were incubated in a cell culture incubator for 6 hours, and the fluorescence value of the cell supernatant was detected using a fluorescence microplate reader (Varioscan Lux, ThermoFisher) (excitation wavelength: 495 nm, emission wavelength: 515 nm).

[0122] Figure 4 It was shown that CAR-T cells designed with different antigen binding domains, co-stimulatory signal domains and CD3ξ signal domains can effectively kill K562-CD19 cells by recognizing the CD19 target protein.

[0123] Example 5: Proliferation of CD19 CAR-T cells under stimulation of target cells

[0124] This example takes the detection of the proliferation efficiency of CAR-T containing a modified antigen binding domain, different co-stimulatory signal domains, and different CD3ξ signal domains under the stimulation of target cells expressing CD19 as an example.

[0125] 1×10 cells were added to each well of a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA). 5 K562-CD19 cells were added with various CAR-T cells at an E:T ratio of 5:1 and placed at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator for 3 days, and the total number of live cells was counted by staining with trypan blue. The cells were labeled with a homemade CD19 CAR antibody coupled to FITC fluorescent molecules, and the proportion of CAR-T in each group of cells was detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA), and the proliferation multiples of CAR-T in each group were calculated.

[0126] Figure 5The results showed that compared with FMC63 CAR-T, CAR-T containing different antigen binding domains, co-stimulatory signal domains and CD3ξ signal domain modifications, especially CAR-T containing different co-stimulatory signal domains and CD3ξ signal domain modifications, had significantly improved proliferation efficiency under stimulation of K562-CD19 target cells, indicating that the modification of these components can enhance the expansion efficiency and duration of CAR-T.

[0127] Example 6: Cytokine expression of CD19 CAR-T under target cell stimulation

[0128] This example takes the detection of cytokine expression levels of CAR-T containing modified antigen binding domains, different co-stimulatory signal domains, and different CD3ξ signal domains under the stimulation of target cells expressing CD19 as an example.

[0129] 1×10 50 μL of culture medium was added to each well of a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA). 5 K562-CD19 cells were added with various CAR-T cells at an E:T ratio of 5:1 and placed at 37°C and 5% CO 2 After incubation in a cell culture incubator for 12 h, the cell supernatant was analyzed by cytometric bead array (BD Biosciences, San Jose, CA, USA) and the expression levels of various cytokines were detected by flow cytometry (NovoCyte 2060R, ACEA Biosciences, San Diego, CA, USA).

[0130] Figure 6 The results showed that compared with T cells, the expression levels of cytokines such as IL-6, IL-10, TNF and IFN-γ of various CAR-T cells were significantly increased under the stimulation of K562-CD19 target cells, and the cytokine expression level of CAR-T containing CD28 co-stimulatory signal domain was significantly higher than that of FMC63 CAR-T containing 4-1BB co-stimulatory signal domain; compared with FMC63 CAR-T, CAR-T containing modified antigen binding domain, modified 4-1BB co-stimulatory signal domain and modified CD3ξ signal domain, especially CAR-T containing different co-stimulatory signal domains and CD3ξ signal domain, under the stimulation of K562-CD19 target cells, the expression levels of cytokines such as IL-6, IL-10 and TNF were significantly reduced, indicating that the modification of these components can reduce the cytokine expression level while ensuring the killing efficiency of CAR-T on target cells, reduce the risk of CAR-T inducing cytokine storm in vivo, and improve safety.

[0131] Example 7: Anti-tumor ability and tumor recurrence inhibition ability of CD19 CAR-T cells in tumor-bearing mice

[0132] This example takes the detection of the anti-tumor ability and tumor recurrence inhibition ability of CAR-T containing a modified antigen binding domain in tumor-bearing mice as an example.

[0133] The isolated and purified T cells were cultured at a volume of 1.5 × 10 6 Cells / mL were inoculated and cultured in fresh X-VIVO medium containing IL-2 (500 IU / mL). Dynabeads were added at a ratio of 1:1 between the number of T cells and CD3 / CD28 Dynabeads. After culturing the cells for 48 hours, the T cells were infected with lentivirus containing the corresponding CAR to prepare the corresponding CAR-T cells (while culturing T cells not infected with lentivirus for control experiments). 24 hours after virus infection, the cells were centrifuged and the medium was changed, and the cells were counted and counted at 0.8×10 6 Cells / mL were inoculated and cultured in fresh X-VIVO containing IL-2 (500 IU / mL), and the original Dynabeads stimulation culture was continued. The cells were centrifuged and the medium was changed every 48 hours at 0.8×10 6 Cells / mL were inoculated and cultured in fresh X-VIVO containing IL-2 (500 IU / mL). On the 11th day of culture, the cells were harvested and counted. At the same time, the corresponding cell samples were retained for flow cytometry analysis of CAR expression rate. The cells were resuspended in freezing solution and stored in liquid nitrogen for later use. A total of 15 6-8 week old NCG mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., China) were divided into 3 groups of 5 mice / group. Each mouse was injected with 1.0×10 6 After 5 days (D5) of inoculation with Nalm-6-LAE cells (ATCC, USA), the mice were analyzed by luciferase live imaging (Lumina II small animal live imaging system, PerkinElmer, USA) to verify whether the mouse leukemia model was successful. After the mouse leukemia model was successfully established, CD19 CAR-T cells (2×10 6 The other two groups of mice were injected with the corresponding number of T cells and the corresponding volume of saline as controls. The mice were subjected to in vivo imaging analysis on days 2 (D7), 8 (D13), and 15 (D20) after CAR-T cell injection. On day 16 after CAR-T cell injection, 1.0×10 5 Nalm-6-LAE cells were used for in vivo imaging analysis of mice on day 27 (D32) after the first CAR-T cell injection.

[0134] Figure 7The results showed that all the tumor-bearing mice in the T cell injection group had died 20 days after tumor inoculation, while all the mice injected with CAR-T survived 20 days after tumor inoculation, and both CAR-T-2 and CAR-T-30 (with the co-stimulatory signal domain being CD28) could significantly inhibit tumor growth; however, when the tumor was re-inoculated to simulate tumor recurrence, CAR-T-2 could still significantly inhibit tumor growth, indicating that CAR-T-2 could inhibit tumor recurrence, while CAR-T-30 could not.

[0135] Example 8: Anti-tumor ability, expansion ability and sustained performance of CD19 CAR-T cells in tumor-bearing mice

[0136] This example takes the detection of the anti-tumor ability, in vivo expansion ability and sustainability of CAR-T cells containing a modified antigen binding domain, a modified co-stimulatory signal domain, and a modified CD3ξ signal domain in tumor-bearing mice as an example.

[0137] The isolated and purified T cells were cultured at a volume of 1.5 × 10 6 Cells / mL were inoculated and cultured in fresh X-VIVO medium containing IL-2 (500 IU / mL). Dynabeads were added at a ratio of 1:1 between the number of T cells and CD3 / CD28 Dynabeads. After culturing the cells for 48 hours, the T cells were infected with lentivirus containing the corresponding CAR to prepare the corresponding CAR-T cells (while culturing T cells not infected with lentivirus for control experiments). 24 hours after virus infection, the cells were centrifuged and the medium was changed, and the cells were counted and counted at 0.8×10 6 Cells / mL were inoculated and cultured in fresh X-VIVO containing IL-2 (500 IU / mL), and the original Dynabeads stimulation culture was continued. The cells were centrifuged and the medium was changed every 48 hours at 0.8×10 6 Cells / mL were inoculated and cultured in fresh X-VIVO containing IL-2 (500 IU / mL). On the 11th day of culture, the cells were harvested and counted. At the same time, the corresponding cell samples were retained for flow cytometry analysis of CAR expression rate. The cells were resuspended in freezing solution and stored in liquid nitrogen for later use. A total of 36 6-8 week old NCG mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., China) were divided into 6 groups of 6 mice / group. Each mouse was injected with 1.0×10 6 After 5 days of inoculation with Nalm-6-LAE cells (ATCC, USA), the mice were analyzed by luciferase live imaging (Lumina II small animal live imaging system, PerkinElmer, USA) to verify whether the mouse leukemia model was successful. After the mouse leukemia model was successfully established, CD19 CAR-T cells (2×10 6The mice were injected with the corresponding number of T cells and the corresponding volume of saline as controls. The peripheral blood CAR-T test was performed on the 2nd, 4th, 8th, 12th, 21st and 28th day after CAR-T cell injection, and the in vivo imaging analysis of mice was performed on the day before or after blood collection (3rd, 7th, 13th, 20th and 27th day after CAR-T cell injection).

[0138] Figure 8 The results showed that all tumor-bearing mice in the T cell injection group and the saline group had died 28 days after injection, while all mice injected with CAR-T survived, and FMC63 CAR-T, CAR-T-13, CAR-T-15 and CAR-T-17 could significantly inhibit tumor growth.

[0139] Fig. 9 It was shown that compared with FMC63 CAR-T, the persistence of CAR-T-13, CAR-T-15 and CAR-T-17 in vivo was significantly improved. Specifically, after the successful establishment of B-ALL leukemia model by injecting Nalm-6 cells into NCG mice, CAR-T cells were detected in the peripheral blood of mice after the tail vein injection of anti-CD19 CAR-T cells. No CAR-T cells were detected in the peripheral blood of mice in the FMC63 CAR-T group after 21 days, while high levels of CAR-T cells were still detected in the peripheral blood of CAR-T-13, CAR-T-15 and CAR-T-17 mice 28 days after CAR-T injection, indicating that compared with FMC63 CAR-T, CAR-T cells containing modified antigen binding domains, modified costimulatory signaling domains, and modified CD3ξ signaling domains have significantly improved persistence in vivo.

[0140] From the above in vitro and in vivo experimental results, it can be seen that the inventors' design of the antigen binding domain, costimulatory signal domain and CD3ξ signal domain of the CD19 CAR molecule, especially the design of the costimulatory signal domain and CD3ξ signal domain, can significantly improve the expansion efficiency and duration of the CD19 CAR molecule in vivo. Fig.10 Model of in vivo amplification curves shown.

[0141] The inventors have described the embodiments based on the present invention above, but the present invention is not limited thereto. Those skilled in the art should understand that the present invention can be implemented in a modified and altered manner within the scope of the present invention, and such modified and altered manners should belong to the protection scope of the present invention.

Claims

1. Use of an isolated chimeric antigen receptor that binds to CD19, a nucleic acid molecule encoding the chimeric antigen receptor, a vector comprising the nucleic acid molecule, or a cell comprising the nucleic acid molecule or the vector in the preparation of a medicament for treating a tumor, an autoimmune disease, or an infectious disease caused by a virus or bacteria, wherein the chimeric antigen receptor comprises: an antigen binding domain, a costimulatory domain, and an intracellular signal transduction domain, wherein (1) The antigen-binding domain comprises, from the N-terminus to the C-terminus, a protective peptide, a light chain variable region and a heavy chain variable region; the protective peptide is selected from the amino acid sequence shown in SEQ ID NO: 7; the light chain variable region is the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 2; the protective peptide and the N-terminus of the light chain variable region can be selectively connected via a flexible linker peptide rich in glycine and serine; The costimulatory domain is selected from the 4-1BB costimulatory domain, and the 4-1BB costimulatory domain is selected from the amino acid sequence shown in SEQ ID NO: 41, 43, 47, 49 or 51; The intracellular signal transduction domain is selected from the CD3ζ intracellular signal transduction domain, and the CD3ζ intracellular signal transduction domain is selected from the amino acid sequence shown in SEQ ID NO: 64; or (2) The antigen-binding domain comprises, from the N-terminus to the C-terminus, a protective peptide, a light chain variable region and a heavy chain variable region; the protective peptide is selected from the amino acid sequence shown in SEQ ID NO: 7; the light chain variable region is the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 2; the protective peptide and the light chain variable region can be selectively connected by a flexible linker peptide rich in glycine and serine; The costimulatory domain is selected from the 4-1BB costimulatory domain, and the 4-1BB costimulatory domain is selected from the amino acid sequence shown in SEQ ID NO: 41; The intracellular signal transduction domain is selected from the CD3ζ intracellular signal transduction domain, and the CD3ζ intracellular signal transduction domain is selected from the amino acid sequence shown in SEQ ID NO: 58, 60 or 62; or (3) The antigen-binding domain comprises, from the N-terminus to the C-terminus, a protective peptide, a light chain variable region and a heavy chain variable region; the protective peptide is selected from the amino acid sequence shown in SEQ ID NO: 7; the light chain variable region is the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 2; the protective peptide and the N-terminus of the light chain variable region can be selectively connected via a flexible linker peptide rich in glycine and serine; The costimulatory domain is selected from the 4-1BB costimulatory domain, and the 4-1BB costimulatory domain is selected from the amino acid sequence shown in SEQ ID NO: 43, 45, 47, 49 or 51; The intracellular signal transduction domain is selected from the CD3ζ intracellular signal transduction domain, and the CD3ζ intracellular signal transduction domain is selected from the amino acid sequence shown in SEQ ID NO: 66; or (4) The antigen-binding domain comprises, from the N-terminus to the C-terminus, a light chain variable region, a protective peptide and a heavy chain variable region; the light chain variable region is the amino acid sequence shown in SEQ ID NO: 1, and the protective peptide is selected from the amino acid sequence shown in SEQ ID NO: 7; the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 2; the protective peptide can be selectively connected to the light chain variable region or the heavy chain variable region through a flexible linker peptide rich in glycine and serine; The costimulatory domain is selected from the 4-1BB costimulatory domain, and the 4-1BB costimulatory domain is selected from the amino acid sequence shown in SEQ ID NO: 41, 43, 45, 47, 49 or 51; The intracellular signal transduction domain is selected from the CD3ζ intracellular signal transduction domain, and the CD3ζ intracellular signal transduction domain is selected from the amino acid sequence shown in SEQ ID NO: 66; or (5) The antigen-binding domain comprises, from the N-terminus to the C-terminus, a light chain variable region, a heavy chain variable region and a protective peptide; the light chain variable region is the amino acid sequence shown in SEQ ID NO: 1, and the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 2; the protective peptide is selected from the amino acid sequence shown in SEQ ID NO: 7; the protective peptide and the C-terminus of the heavy chain variable region can be selectively connected through a flexible linker peptide rich in glycine and serine; The costimulatory domain is selected from the 4-1BB costimulatory domain, and the 4-1BB costimulatory domain is selected from the amino acid sequence shown in SEQ ID NO: 41, 43, 45, 47, 49 or 51; The intracellular signal transduction domain is selected from the CD3ζ intracellular signal transduction domain, and the CD3ζ intracellular signal transduction domain is selected from the amino acid sequence shown in SEQ ID NO:

66.

2. The use according to claim 1, wherein the antigen binding domain is selected from the amino acid sequence shown in SEQ ID NO: 17, 19 or 21.

3. The use according to claim 1, further comprising a hinge region and a transmembrane region, wherein the hinge region and the transmembrane domain are selected from the hinge region and the transmembrane domain of IgG1, IgG4, CD8α, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1 or CD34.

4. The use of any one of claims 1 to 3, wherein the chimeric antigen receptor comprises an amino acid sequence as shown in SEQ ID NO: 70, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122 or 124.

5. The use according to any one of claims 1 to 4, wherein the vector is selected from one or more of DNA, RNA, lentiviral vector, adenoviral vector and retroviral vector.

6. The use according to claim 5, wherein the DNA is a plasmid.

7. The use according to any one of claims 1 to 4, wherein the cells are T lymphocytes, B lymphocytes, natural killer cells, dendritic cells.

8. The use according to claim 7, wherein the cells are cytotoxic T cells, tumor infiltrating T cells or regulatory T cells.

Images