WO2022083590A1

WO2022083590A1 - Chimeric receptor containing dap 12 and co-stimulatory signal molecule signal domain, and method for using same

Info

Publication number: WO2022083590A1
Application number: PCT/CN2021/124728
Authority: WO
Inventors: 王恩秀; 汪晨; 孙明
Original assignee: 南京卡提医学科技有限公司
Priority date: 2020-10-19
Filing date: 2021-10-19
Publication date: 2022-04-28

Abstract

Provided are a chimeric receptor containing a DAP 12 and a co-stimulatory signal molecule signal domain, and a method for using same. The chimeric antigen receptor comprises a first fusion peptide and a second fusion peptide. The first fusion peptide comprises an antigen-binding domain and a transmembrane domain. The second fusion peptide comprises a transmembrane domain, a cytoplasmic domain, and a co-stimulatory domain.

Description

Chimeric receptors comprising DAP12 and co-stimulatory signaling molecule signaling domains and methods of using the same

technical field

The present disclosure relates to the field of tumor cell therapy, and more particularly, to expressing a chimeric receptor comprising DAP12 and a signaling domain of a costimulatory signaling molecule, immune effector cells thereof, compositions comprising the same, and methods of making and using the same.

Background technique

CD19 is the Cluster of Differentiation 19 protein (CD19), which is an epitope detectable on leukemic precursor cells. CD19 is expressed on most B-lineage cancers, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. CD19 is also an early marker of B cell ancestry.

Mesothelin (MSLN), a glycoprotein linked to the cell membrane through the glycophosphatidylinositol domain (GPI domain), was initially identified as tumor-associated due to its limited expression in normal tissues and overexpression in tumors antigen. The mesothelin gene encodes the precursor 71-kDa protein, and after the N-terminal is excised by furin, the 40-kDa C-terminal (mesothelin) remains on the cell membrane, and the soluble 31-kDa N-terminal fragment MPF (megakaryotic potentiating factor, megakaryocyte enhancing factor) is released. Mesothelin is overexpressed in the vast majority of primary pancreatic adenomas, whereas rare and weak expression is seen in benign pancreatic tissue. Epithelial malignant pleural mesothelioma (MPM) ubiquitously expresses mesothelin whereas sarcomatoid MPM does not. Most serous epithelial ovarian tumors and related primary peritonomas express mesothelin. In ovarian cancer, mesothelin is a target of the innate immune response and has been proposed as a target for cancer immunotherapy. In patients with pancreatic cancer, the presence of mesothelin-specific CTLs is associated with overall survival. Soluble antibody fragments of anti-mesothelin antibodies conjugated to immunotoxins have been used to treat cancer patients with mesothelin-positive tumors. This approach has demonstrated adequate safety and some clinical activity in pancreatic cancer. In ovarian cancer, this treatment strategy produced a minimal response and stable disease in a second patient who had completely resolved their ascites according to RECIST criteria.

Disialoganglioside (GD2, pubchem: 6450346) is a sialic acid-containing glycosphingolipid, mainly expressed on the cell surface, which is found in tumors of neuroectodermal origin, including neuroblastoma and melanoma ), whereas expression in normal tissues is highly restricted. GD2 is densely, homogeneously and almost ubiquitously expressed on neuroblastomas. In normal tissues, GD2 expression is largely restricted to skin melanocytes, and peripheral pain fibrous myelin sheaths. In the CNS, GD2 appears to be an embryonic antigen, but is found dimly expressed in scattered oligodendrocytes and in the posterior pituitary. This makes GD2 very suitable for targeted antitumor therapy.

In recent years, although conventional tumor treatment methods such as radiotherapy, chemotherapy and surgery can play a certain therapeutic effect, the problems of tumor metastasis, recurrence and low survival rate of patients have not been properly solved. With the development of tumor immunology theory and technology, the role of immune cells in tumor therapy has been paid more and more attention. Among them, chimeric antigen receptor T cell (CAR-T) technology is a cell therapy technology that has developed very rapidly in recent years.

The chimeric antigen receptor (CAR) is the core component of CAR-T. Using the ligand-binding domain properties, CAR can redirect its specificity and reactivity against selected immune cells, thus giving T cells an HLA-independent manner. The ability to recognize tumor antigens allows CAR-engineered T cells to recognize a wider range of targets than the native T-cell surface receptor TCR. The basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFv segment of the antigen-binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region and an intracellular signal. Area. CAR-T therapy has shown promise in some hematological cancer trials. Clinical trials have shown that CAR-T cell therapy has great potential in controlling advanced acute lymphoblastic leukemia (ALL) and lymphoma.

However, some patients receiving CAR T-cell therapy and other immunotherapies experience dangerous, even life-threatening side effects known as cytokine release syndrome (CRS), in which the infused CAR-T cells produce a systemic inflammatory response, Among them, cytokines are rapidly released into the blood in large quantities, causing dangerously low blood pressure, high fever and chills.

In severe cases of CRS, patients experience a cytokine storm (also known as cytokine cascade or hypercytokineemia) in which there is a positive correlation between the presence of cytokines and leukocytes with highly elevated levels of cytokines. This can lead to potentially life-threatening complications, including cardiac dysfunction, respiratory distress syndrome, neurological toxicity, renal and/or hepatic failure, pulmonary edema, and disseminated intravascular coagulation.

CN107580628A discloses targeted cytotoxic cells with chimeric receptors for adoptive immunotherapy, in which DAP12/KIRS2 is used as the intracellular signaling domain of the CAR structure. However, due to the lack of costimulatory signaling molecules, it has technical problems of poor sustainability. When co-cultured with tumor cells in the experiment, the amount of IL-2 secretion of DAP12/KIRS2 CAR-T is very low, and in clinical treatment lead to ineffective treatment.

Therefore, new strategies are needed to enhance clinical efficacy and control cytokine release syndrome, especially cytokine storm, to maximize the potential of CAR-T cell therapy.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the present disclosure introduces costimulatory signal molecules and anti-tumor antigen single-chain antibodies into DAP12/KIRS2 to form a CAR structure of anti-tumor antigen antibodies (anti-tumor antigen single-chain antibody/ DAP12-4-1BB), and validated the in vitro pharmacodynamics (killing, cytokine secretion, proliferation, etc.) based on this CAR structure, demonstrating that the CAR structure is effective against tumor antigens (such as CD19 antigen, mesothelin antigen and GD2 antigen). ) expression-positive solid tumor cells have effective killing effect.

In one aspect, the present disclosure provides a chimeric antigen receptor comprising a first fusion peptide and a second fusion peptide, wherein:

the first fusion peptide comprises an antigen binding domain and a transmembrane domain;

The second fusion peptide comprises a transmembrane domain, a cytoplasmic domain and a costimulatory domain.

In some embodiments, the transmembrane domain of the second fusion peptide interacts with the transmembrane domain of the first fusion peptide through charge interactions, or the second fusion peptide interacts with phosphorylated ITAM within the cytoplasmic domain The sequence interacts with the signaling molecule.

In some embodiments, the transmembrane domain of the first fusion peptide is a KIR transmembrane domain; preferably, the KIR transmembrane domain is selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1. In some preferred embodiments, the KIR transmembrane domain is KIRS2 or KIR2DS2.

In some embodiments, the transmembrane domain of the second fusion peptide is the DAP12 transmembrane domain.

In some embodiments, the cytoplasmic domain is selected from the cytoplasmic domains of DAP12 and KIR.

In some embodiments, the antigen binding domain comprises an antibody or antigen binding fragment thereof, preferably the antigen binding domain comprises the heavy chain CDR1, CDR2 and CDR3 of the antibody and the CDR1, CDR2 and CDR3 of the light chain; preferably Preferably, the antigen binding domain comprises a heavy chain variable region and a light chain variable region of an antibody; preferably, the antigen binding domain comprises Fab, Fab', F(ab') ₂ , single-chain Fv (scFv ), Fv, dsFv, diabodies, Fd and Fd' fragments

In some embodiments, the antigen is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from the group consisting of: CD19, mesothelin, GD-2, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CEA, CTLA4, BCMA, CS1, c-Met, EPCAM, EGFR/EGFRvIII, gp100, GPC3, IGF1R, IGF-I receptor, MAGE A3, B7-H3, MUC1, NY-ESO-1, HER2, PD1, PSMA, ROR1, WT1, glycolipid F77 or any other tumor antigen or other modifier types and any combination thereof. In some preferred embodiments, the tumor-associated antigen is selected from the group consisting of: CD19, mesothelin, and GD-2.

In some embodiments, the costimulatory domain is selected from the group consisting of 4-1BB, CD28, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3. In some preferred embodiments, the costimulatory domain is 4-1BB.

In one aspect, the present disclosure provides nucleic acids encoding chimeric antigen receptors as previously described.

In one aspect, the present disclosure provides vectors comprising the aforementioned nucleic acids.

In one aspect, the present disclosure provides cells comprising the aforementioned vectors.

In one aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned chimeric antigen receptor or nucleic acid encoding the same and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a cell preparation method by introducing the aforementioned nucleic acid or vector into immune effector cells.

In one aspect, the present disclosure provides use of the aforementioned chimeric antigen receptor, nucleic acid, vector, cell and/or pharmaceutical composition in the manufacture of a medicament for the treatment and/or prevention of a disease or disorder.

In one aspect, the present disclosure provides a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a chimeric antigen receptor, nucleic acid, vector and/or cell comprising the foregoing.

In one aspect, the present disclosure provides a method of treating a mammal having a disease or disorder, comprising administering to the mammal an effective amount of the aforementioned chimeric antigen receptors, nucleic acids, vectors, and/or cells.

Description of drawings

Figure 1 shows the structural design of a DPK CAR targeting CD19.

Figure 2 shows the expansion and volume changes of four CAR-T cells targeting CD19.

Figure 3 shows the positive rates of four CAR-Ts targeting CD19.

Figure 4 shows the detection results of four CAR-T cell differentiation subtypes targeting CD19.

Figure 5 shows the lysis rate of target cells by CAR-T cells targeting CD19 and non-transduced T cells (NTD).

Figure 6 shows the results of IL-2 and IFN-γ assays for four CAR-T cells targeting CD19.

Figure 7 shows the results of CAR-T proliferation flow assay targeting CD19.

Figure 8 shows the trend of tumor changes in mice in different CAR-T administration groups targeting CD19.

Figure 9 shows the secretion of IL-6 and IL-10 in the blood of mice 14 days after injection of CAR-T targeting CD19.

Figure 10 shows CD19 CAR-T occupancy in blood T lymphocytes.

Figure 11 shows the content of PD-1 in CAR-T lymphocytes targeting CD19.

Figure 12 shows a flow chart of the clinical treatment regimen of CAR-T targeting CD19. In the figure, M1, M3, and M6 represent 1 month, 3 months, and 6 months; ** represents an important time node for evaluating the efficacy.

Figure 13 shows the structural design of mesothelin-DAP12/KIRS2 CAR targeting mesothelin.

Figure 14 shows expansion and volume changes of two CAR-T cells targeting mesothelin.

Figure 15 shows the positive rate of two CAR-T cells and NTD cells targeting mesothelin.

Figure 16 shows the results of two CAR-T cell and NTD cell differentiation subtype assays targeting mesothelin.

Figure 17 shows the lysis rate of target cells by two CAR-T cells targeting mesothelin and NTD cells.

Figure 18 shows the results of IL-2 and IFN-γ assays for two CAR-T cells targeting mesothelin and NTD cells.

Figure 19 shows the results of flow cytometry of the proliferation of two CAR-T cells and NTD cells targeting mesothelin.

Figure 20 shows the trend of tumor changes in mice in different CAR-T administration groups targeting mesothelin.

Figure 21 shows a flow chart of the clinical treatment protocol of CAR-T cells targeting mesothelin.

Figure 22 shows solid tumor survival statistics for mesothelin-targeted SS1-CAR-T therapy.

Figure 23 shows solid tumor progression-free survival statistics for mesothelin-targeted SS1-CAR-T therapy.

Figure 24 shows the structural design of a GD2-DAP12/KIRS2 CAR targeting GD2.

Figure 25 shows the expansion and volume changes of two CAR-T cells targeting GD2.

Figure 26 shows the positive rate of two CAR-T targeting GD2.

Figure 27 shows the results of two CAR-T cell differentiation subtype assays targeting GD2.

Figure 28 shows the lysis rate of target cells by CAR-T cells targeting GD2 and NTDs.

Figure 29 shows the results of IL-2 and IFN-γ assays for two CAR-T cells targeting GD2.

Figure 30 shows the results of GD2-targeted CAR-T proliferation flow assay.

Detailed ways

I. Definitions

In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are the terms and routine procedures widely used in the corresponding fields. Meanwhile, for a better understanding of the present disclosure, definitions and explanations of related terms are provided below.

As used herein and unless otherwise stated, the term "about" or "approximately" means within plus or minus 10% of the given value or range. Where a whole number is required, the term refers to within plus or minus 10% of the given value or range, rounded up or down to the nearest whole number.

"Chimeric Antigen Receptor" or "CAR," as the term is used herein, refers to a chimeric polypeptide that shares structural and functional properties with cellular immune function receptors or adaptor molecules from, eg, T cells or NK cells. In embodiments, the CAR comprises an antigen binding domain that binds to a cognate antigen (eg, a tumor antigen described herein). After binding to the cognate antigen, CAR can activate or inactivate the cytotoxic cells in which it is located, or modulate the antitumor activity of cells or modulate the immune response of cells.

The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to a target antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and can be derived from natural sources or from recombinant sources. Antibodies are usually tetramers of immunoglobulin molecules. Antibody molecules described herein can exist in a variety of forms in which the antigen-binding portion of the antibody is expressed as part of a contiguous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), and humanized or human antibodies , for example, as described herein.

With respect to antibody chain polypeptide sequences, the phrase "substantially identical" can be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains of 97%, 98%, 99% or more sequence identity. With respect to nucleic acid sequences, the term is to be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 A nucleotide sequence of %, 99% or greater sequence identity.

Sequence "identity" or "identity" has an art-recognized meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along regions of the molecule. While many methods exist for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).

A "substitutional" variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its same position. The substitutions can be single, wherein only one amino acid is substituted in the molecule, or multiple, wherein the same molecule has two or more amino acids substituted. Multiple substitutions can be made at consecutive sites. Likewise, one amino acid may be substituted by multiple residues, wherein such variants include both substitutions and insertions. An "insertional" variant is one in which one or more amino acids are inserted into an amino acid immediately adjacent to a specific position in a native sequence. Immediately adjacent to an amino acid means attachment to the alpha-carboxyl or alpha-amino functional group of the amino acid. A "deletion" variant is one in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of their molecule.

With respect to variable domains of antibodies, the term "variable" refers to certain portions of related molecules that differ widely in sequence between antibodies and are used for the specific recognition and binding of a particular antibody against its specific target. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called complementarity determining regions (CDRs; ie CDR1, CDR2 and CDR3) or hypervariable regions, all located within the variable domains of light and heavy chains. The more conserved portions of the variable domains are referred to as framework (FR) regions or framework sequences. Each variable domain of native heavy and light chains includes four FR regions, predominantly in a beta-sheet configuration, linked by three CDRs that form loops that connect the beta-sheet structure and Partial β-sheet structures are formed in some cases. The CDRs of each chain are usually linked in proximity by FR regions and, with the aid of CDRs from other chains, contribute to the formation of antibody target binding sites (epitopes or determinants). As used herein, the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. A CDR can have the ability to specifically bind to the cognate epitope.

The term "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length, but which comprises at least a portion of the variable region (e.g., one or more CDRs and/or the variable region of said antibody that binds an antigen) or one or more antibody binding sites), and thus retain the binding specificity and at least part of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, eg, recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into the antibody framework (eg, by substituting the corresponding region), results in an antibody that immunospecifically binds (ie, exhibits a Ka of at least or at least about ^107-108 M- ¹ ) to an antigen . A "functional fragment" is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, functional fragments generally have the same meaning as "antibody fragments" and, in the case of antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, eg, Fv, Fab , F(ab') ₂ , and so on. "Fv" fragments consist of a dimer ( _VH - _VL dimer) formed by non-covalent association of the variable domains of a heavy chain and the variable domains of a light chain. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the _VH - _VL dimer, as is the case with intact antibodies. The six CDRs collectively confer the target-binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind targets.

The term "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, eg, by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

The term full-length antibody is an antibody having two full-length heavy chains (eg VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, For example, antibodies produced naturally by antibody secreting B cells as well as synthetically produced antibodies having the same domains.

The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.

"Humanized" antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') ₂ or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.

In addition, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody . For example, PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, there are usually no more than one or two mutations within a CDR. Accordingly, the humanized antibodies described in the present disclosure also encompass antibodies comprising 1 or 2 two amino acid mutations within the CDRs.

The term "CDR" refers to complementarity-determining regions, and each of the heavy and light chains of antibody molecules is known to have 3 CDRs. The CDRs are also referred to as hypervariable regions, and are present in the variable regions of each heavy and light chain of antibodies, with sites of very high variability in the primary structure of the CDRs. In the present specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

The term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants typically comprise chemically active surface profiles of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.

The terms "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to an antibody or antigen-binding fragment through non-co-coding between the antibody-binding sites of the antibody and the antigen. The ability to form one or more non-covalent bonds with alloantigens. The antigen may be an isolated antigen or present in tumor cells. Typically, an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka of about or ^1x107 M ^-1 or ^1x108 M ^-1 or greater (or ^1x10-7 M or 1x A dissociation constant (Kd) of 10 ⁻⁸ M or lower binds the antigen. Affinity constants can be determined by standard kinetic methods of antibody responses, eg, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also US Pat. No. 7,229,619 describing exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27 :335).

The terms "polynucleotide" and "nucleic acid molecule" refer to oligomers or polymers comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid ( DNA) and ribonucleic acid (RNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

As used herein, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.

As used herein, "operably linked" in reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.

Also provided are "conservative sequence modifications" of the sequences described in the Sequence Listing described herein, ie, nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody to the antigen encoded by the nucleotide sequence or containing the amino acid sequence. These conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the Sequence Listing described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), amino acids with uncharged polar side chains amino acids (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g. alanine, valine) acid, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta branched side chains (e.g. threonine, valine, isoleucine) and Amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Methods for identifying conservative substitutions of nucleotides and amino acids that do not abolish antigen binding are well known in the art (for example, see Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12 ( 10): 879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

The term "expression" refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.

The term "host cell" is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

The term "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when transformed into an appropriate host cell. References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. References to vectors also include those that contain nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.

As used herein, a vector also includes a "viral vector" or "viral vector." A viral vector is an engineered virus that is operably linked to a foreign gene to transfer (either as a vehicle or shuttle) the foreign gene into a cell.

The term "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.

The term "stimulation" refers to mediating a primary response to a signaling event by stimulating the binding of a molecule (eg, the TCR/CD3 complex) to its cognate ligand, such as, but not limited to, via the TCR/CD3 complex signal transduction. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, among others.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates, in a stimulatory manner, the primary level of the TCR complex for at least some aspects of the T cell signaling pathway activation. In one aspect, the primary signal is initiated by, eg, binding of the TCR/CD3 complex to the peptide-loaded MHC molecule, and it results in the mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences particularly useful in the present disclosure include, but are not limited to, those derived from TCRδ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10 and DAP12. In a specific CAR of the present disclosure, the intracellular signaling domain in any one or more of the CARs of the present disclosure includes an intracellular signaling sequence.

The term "antigen presenting cell" or "APC" refers to an immune system cell, such as a helper cell (eg, B-cells, dendritic cells), that presents on its surface a foreign antigen complexed with the major histocompatibility complex (MHC). Wait). T-cells can recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

The term "intracellular signaling domain" refers to the intracellular portion of a molecule. Intracellular signaling domains can generate signals that promote the immune effector function of CAR-containing cells (eg, CAR-T cells or CAR-expressing NK cells). Examples of immune effector functions (eg, in CAR-T cells or CAR-expressing NK cells) include cytolytic activity and helper activity, including secretion of cytokines. In embodiments, intracellular signaling domains transduce effector function signals and direct cells to perform specialized functions. While the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain. In the case of using truncated portions of intracellular signaling domains, such truncated portions can be used in place of the full chain, so long as it transduces effector function signals. The term intracellular signaling domain is thus intended to include any truncated portion of the intracellular signaling domain sufficient to signal effector function.

In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signaling or antigen-independent stimulation. For example, in the case of CAR-expressing immune effector cells, such as CAR-T cells or CAR-expressing NK cells, the primary intracellular signaling domain may comprise the cytoplasmic sequence of the T cell receptor and co-stimulate intracellular signaling Domains may comprise cytoplasmic sequences from co-receptors or co-stimulatory molecules.

The primary intracellular signaling domain may include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3δ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, DAP10 and DAP12.

The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand to mediate a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response. Costimulatory molecules include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA , Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM -1, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1 , CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, LAT, GADS, SLP-76, PAG/ Cbp, CD19a and ligands that bind specifically to CD83.

A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain may include the entire intracellular portion or the entire native intracellular signaling domain of the molecule, or a functional fragment thereof.

The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence as provided in GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. and "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc.No..AAA62478.2, or equivalents from non-human species such as mouse, rodent, monkey, ape, etc. Residues. In one aspect, a "4-1BB costimulatory domain" is a sequence as provided in SEQ ID NO. 5 or equivalent residues from a non-human species such as mouse, rodent, monkey, ape, etc.

The term "immune effector cell" refers to a cell that is involved in an immune response, eg, promotes an immune effector response. Examples of immune effector cells include T cells, eg, alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

The term "immune effector function or immune effector response" refers to an immune effector cell, eg, a function or response that enhances or facilitates the immune attack of a target cell. For example, immune effector function or response refers to the properties of T cells or NK cells that promote killing of target cells or inhibit growth or proliferation. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

In some embodiments, a population of cells (eg, a harvested population of cells) comprises, eg, T cells or populations of T cells at various stages of differentiation. Stages of T cell differentiation ranging from least to most differentiated include naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and terminal effector T cells. Following antigen exposure, naive T cells proliferate and differentiate into memory T cells, such as stem central memory T cells and central memory T cells, and then differentiate into effector memory T cells. Memory T cells further differentiate into terminal effector T cells after receiving appropriate T cell receptors, co-stimulatory and inflammatory signals.

Naive T cells (TN) are characterized by the expression pattern of the following cell surface markers: CCR7+, CD62L+, CD45RO-, CD95-. Stem cell central memory T cells (TSCM) are characterized by the expression pattern of the following cell surface markers: CCR7+, CD62L+, CD45RO-, CD95+. Central memory T cells (TCM) are characterized by the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO+, CD95+. Effector memory T cells (TEM) were characterized by the expression pattern of the following cell surface markers: CCR7-, CD62L-, CD45RO+, CD95+. Terminal effector T cells (TEW) are characterized by the following expression pattern of cell surface markers: CCR7-, CD62L-, CD45RO-, CD95+.

Fresh T cells from healthy donors are typically divided into four subgroups based on CD45RA and CD62L expression: 1) naive T cells (CD45RA+CD62L+, termed Tn), 2) central memory T cells (CD45RA-CD62L+, termed Tcm) ), 3) effector memory T cells (CD45RA-CD62L-, referred to as Tem) and 4) CD45RA positive effector T cells (CD45RA+CD62L-, referred to as Temra). The expression of CCR7, CD27, CD28 and CD95 in each subgroup was then further assessed. CD95 expression was significantly upregulated after lentiviral transduction. The latter three T cell subsets were positive for CD95, while only a small fraction of Tn expressed CD95 (3.6±1.4% in CD4+ and 3.7±1.3% in CD8+ T cells). This small population also co-expresses CD27, CD28 and CCR7 and is considered memory stem cells (Tscm).

The term "effector function" refers to a specialized function of a cell. For example, the effector function of T cells can be cytolytic activity or helper activity, including secretion of cytokines.

The term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard bibliography in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition to any conventional media or agent incompatibility with the chimeric antigen receptor, its use in the composition is envisaged.

The term "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged following treatment. Thus, treatment includes prevention, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease. Treatment also includes any of the chimeric antigen receptors provided and any pharmaceutical uses of the compositions provided herein.

The term "efficacy" refers to an effect resulting from a treatment in an individual that alters, generally ameliorates or ameliorates the symptoms of a disease or condition, or cures the disease or condition.

The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of the disease or disorder.

The term "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, eg, preventing or delaying the onset of a disease or symptom or Relapse, reducing the likelihood of disease or symptoms developing or recurring. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

The term "patient" refers to a mammal, such as a human.

II. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In some embodiments, the transmembrane domain of the first fusion peptide is a KIR transmembrane domain; preferably, the KIR transmembrane domain is selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1; more preferably, the KIR transmembrane domain is KIRS2 or KIR2DS2; preferably, the KIRS2 comprises the An amino acid sequence showing that the amino acid sequence has 80% or more identity, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% More preferably, the amino acid sequence of the KIRS2 is as shown in SEQ ID NO.8; preferably, the KIR2DS2 contains 80% or more identity with the amino acid sequence shown in SEQ ID NO.9 amino acid sequence, preferably an amino acid sequence with more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more preferably an amino acid sequence with 98% or more identity; more Preferably, the amino acid sequence of the KIR2DS2 is shown in SEQ ID NO.9.

In some embodiments, the transmembrane domain of the second fusion peptide is a DAP12 transmembrane domain; preferably, the transmembrane domain of DAP12 comprises 80% or more of the amino acid sequence shown in SEQ ID NO. Amino acid sequences with the above identity, preferably amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more identity ; More preferably, the amino acid sequence of the transmembrane domain of the DAP12 is shown in SEQ ID NO.14.

In some embodiments, the first fusion peptide comprises a signal peptide, and the signal peptide is a CD8α signal peptide; preferably, the CD8α signal peptide comprises 80% or more identical to the amino acid sequence shown in SEQ ID NO. 4 amino acid sequence, preferably an amino acid sequence with more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more preferably an amino acid sequence with 98% or more identity; more Preferably, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.4.

In some embodiments, the second fusion peptide comprises a signal peptide, and the signal peptide is a DAP12 signal peptide or a CD8α signal peptide; preferably, the DAP12 signal peptide comprises an amino acid sequence having 80°C with the amino acid sequence shown in SEQ ID NO.1 % or more identical amino acid sequences, preferably amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identities, more preferably 98% or more. amino acid sequence; more preferably, the amino acid sequence of the DAP12 signal peptide is shown in SEQ ID NO.1; preferably, the CD8α signal peptide comprises 80% or more identity with the amino acid sequence shown in SEQ ID NO.4 amino acid sequence, preferably amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequence with 98% or more identity; more preferably Typically, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.4.

In some embodiments, the costimulatory domain is selected from the group consisting of 4-1BB, CD28, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3.

In some embodiments, the costimulatory domain is 4-1BB; preferably, the 4-1BB comprises an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO. 5, preferably 85 %, 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the 4-1BB The amino acid sequence of is shown in SEQ ID NO.5.

In some embodiments, the cytoplasmic domain is the cytoplasmic domain of DAP12; preferably, the cytoplasmic domain of DAP12 comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.15 Amino acid sequence, preferably amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequence with 98% or more identity; more preferably , the amino acid sequence of the cytoplasmic domain of the DAP12 is shown in SEQ ID NO.15.

In some embodiments, the antigen binding domain comprises an antibody or antigen binding fragment thereof, preferably the antigen binding domain comprises the heavy chain CDR1, CDR2 and CDR3 of the antibody and the CDR1, CDR2 and CDR3 of the light chain; preferably Preferably, the antigen binding domain comprises a heavy chain variable region and a light chain variable region of an antibody; preferably, the antigen binding domain comprises Fab, Fab', F(ab') ₂ , single-chain Fv (scFv ), Fv, dsFv, diabodies, Fd and Fd' fragments.

In some embodiments, the antigen is a tumor-associated antigen.

In some embodiments, the tumor-associated antigen is selected from the group consisting of: CD19, mesothelin, GD-2, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CEA, CTLA4, BCMA, CS1, c-Met, EPCAM, EGFR/EGFRvIII, gp100, GPC3, IGF1R, IGF-I receptor, MAGE A3, B7-H3, MUC1, NY-ESO-1, HER2, PD1, PSMA, ROR1, WT1, glycolipid F77 or any other tumor antigen or other modifier types and any combination thereof.

In some embodiments, the tumor-associated antigen is selected from the group consisting of CD19, mesothelin, and GD-2, and the antigen binding domain is selected from the group consisting of CD19 antibody or antigen-binding fragment thereof, mesothelin antibody or antigen-binding fragment thereof, and GD2 antibody or antigen-binding fragment thereof.

In some embodiments, the CD19 antibody or antigen-binding fragment thereof comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 7, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of the CD19 scFv is shown in SEQ ID NO.7 .

In some embodiments, the mesothelin antibody or antigen-binding fragment thereof comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 19 or SEQ ID NO. 20, preferably 85%, An amino acid sequence of 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence of 98% or more identity; more preferably, the amino acid sequence of the SS1 scFv As shown in SEQ ID NO.19 or SEQ ID NO.20.

In some embodiments, the GD2 antibody or antigen-binding fragment thereof comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 25, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; more preferably, the amino acid sequence of the GD2 scFv is shown in SEQ ID NO.25 .

In some embodiments, the first fusion peptide comprises CD19 scFv and KIRS2, or comprises CD19 scFv and KIR2DS2.

In some embodiments, the CD19 scFv comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 7, preferably 85%, 90%, 95%, 96%, 97%, 98 %, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of the CD19 scFv is shown in SEQ ID NO.7.

In some embodiments, the amino acid sequence of the KIRS2 is shown in SEQ ID NO.8; preferably, the amino acid sequence of the KIR2DS2 is shown in SEQ ID NO.9.

In some embodiments, the second fusion peptide comprises a DAP12 transmembrane domain, a DAP12 cytoplasmic domain, and a costimulatory domain 4-1BB, or comprises a truncated DAP12 transmembrane domain, a DAP12 cytoplasmic domain, and Costimulatory domain 4-1BB; preferably, the DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.2, preferably 85% , 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the DAP12 transmembrane structure The amino acid sequence of the domain and the DAP12 cytoplasmic domain is shown in SEQ ID NO.2; preferably, the truncated DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise the amino acid sequence shown in SEQ ID NO.3. 80% or more identical amino acid sequences, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical The amino acid sequence of ; more preferably, the amino acid sequence of the truncated DAP12 transmembrane domain and DAP12 cytoplasmic domain is shown in SEQ ID NO.3.

In some embodiments, the chimeric antigen receptor is a CD19-KIRS2/Dap12-BB chimeric antigen receptor, and the CD19-KIRS2/Dap12-BB chimeric antigen receptor is formed by cleavage of a fusion protein by a T2A peptide , the fusion protein comprises DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4-1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIRS2; preferably, the fusion protein comprises An amino acid sequence with 80% or more identity with the amino acid sequence shown in SEQ ID NO.10, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, More preferably, the amino acid sequence with 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.10.

In some embodiments, the chimeric antigen receptor is a CD19-KIRS2/tDap12-BB chimeric antigen receptor, and the CD19-KIRS2/tDap12-BB chimeric antigen receptor is formed by cleavage of a fusion protein by a T2A peptide , the fusion protein consists of CD8α signal peptide, truncated DAP12 (tDap-12, including truncated transmembrane and cytoplasmic domains), 4-1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIRS2; preferably, the fusion protein comprises an amino acid sequence with 80% or more identity with the amino acid sequence shown in SEQ ID NO. 11, preferably 85%, 90%, 95%, 96%, 97%, 98% , an amino acid sequence of more than 99% identity, more preferably an amino acid sequence of 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.11.

In some embodiments, the chimeric antigen receptor is a CD19-KIR2DS2/Dap12-BB chimeric antigen receptor, and the CD19-KIR2DS2/Dap12-BB chimeric antigen receptor is formed by cleavage of a fusion protein with a T2A peptide , the fusion protein consists of DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4-1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIR2DS2; preferably, the fusion protein comprises An amino acid sequence with 80% or more identity with the amino acid sequence shown in SEQ ID NO.12, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, More preferably, the amino acid sequence with 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.12.

In some embodiments, the T2A cleavage site comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 6, preferably 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of the T2A cleavage site is shown in SEQ ID NO.6.

In some embodiments, the first fusion peptide comprises SS1 scFv and KIRS2, or comprises SS1 scFv and KIR2DS2.

In some embodiments, the SS1 scFv comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 19 or SEQ ID NO. 20, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences; more preferably, the amino acid sequence of the SS1 scFv is as shown in SEQ ID NO.19 or shown in SEQ ID NO.20.

In some embodiments, the chimeric antigen receptor is an SS1 scFv1-KIRS2/Dap12-BB chimeric antigen receptor, and the SS1 scFv1-KIRS2/Dap12-BB chimeric antigen receptor is cleaved by a fusion protein via a T2A peptide After formation, the fusion protein comprises DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4-1BB, T2A cleavage site, CD8α signal peptide, SS1 scFv1 and KIRS2; preferably, the fusion The protein comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 21, preferably amino acids with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity sequence, more preferably an amino acid sequence with 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.21.

In some embodiments, the chimeric antigen receptor is an SS1 scFv2-KIRS2/Dap12-BB chimeric antigen receptor, and the SS1 scFv2-KIRS2/Dap12-BB chimeric antigen receptor is cleaved from a fusion protein by a T2A peptide Formed, the fusion protein comprises CD8α signal peptide, truncated DAP12 (tDap-12, including truncated transmembrane and cytoplasmic domains), 4-1BB, T2A cleavage site, CD8α signal peptide, SS1 scFv2 and KIRS2; preferably, the fusion protein comprises an amino acid sequence with 80% or more identity with the amino acid sequence shown in SEQ ID NO.23, preferably 85%, 90%, 95%, 96%, 97%, 98 %, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.23.

In some embodiments, the first fusion peptide comprises GD2 scFv and KIRS2, or comprises GD2 scFv and KIR2DS2.

In some embodiments, the GD2 scFv comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 25, preferably 85%, 90%, 95%, 96%, 97%, 98% %, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of the GD2 scFv is shown in SEQ ID NO.25.

In some embodiments, the chimeric antigen receptor is a GD2 scFv-KIRS2/Dap12-BB chimeric antigen receptor, and the GD2 scFv-KIRS2/Dap12-BB chimeric antigen receptor is cleaved from a fusion protein by a T2A peptide Formed, the fusion protein comprises DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4-1BB, T2A cleavage site, CD8α signal peptide, GD2 scFv1 and KIRS2; preferably, the fusion protein Contains an amino acid sequence with 80% or more identity with the amino acid sequence shown in SEQ ID NO. , more preferably an amino acid sequence with 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.26.

In one aspect, the present disclosure provides a nucleic acid encoding the aforementioned chimeric antigen receptor; preferably, the nucleic acid comprises SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.22, The nucleotide sequences shown in SEQ ID NO.24, SEQ ID NO.27 and SEQ ID NO.29 have 80% or more identity, preferably 85%, 90%, 95%, 96%, A nucleotide sequence of 97%, 98%, 99% or more identity, more preferably a nucleotide sequence of 98% or more identity; preferably, the nucleic acid is selected from: SEQ ID NO.16, SEQ ID NO.16, SEQ ID NO.16, SEQ ID NO. The nucleic acid shown in ID NO.17, SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.24, SEQ ID NO.27; more preferably, the encoding nucleic acid is SEQ ID NO.16, SEQ ID NO.27 The nucleic acid shown in NO.22 or SEQ ID NO.27.

In one aspect, the present disclosure provides cells comprising the aforementioned nucleic acids or vectors.

In one aspect, the present disclosure provides a composition comprising the aforementioned chimeric antigen receptor, nucleic acid, vector and/or cell and a pharmaceutically acceptable carrier.

In one aspect, the present disclosure provides a method for preparing cells, the method comprising introducing the aforementioned nucleic acid and vector into immune effector cells.

In one aspect, the present disclosure provides use of the aforementioned chimeric antigen receptors, nucleic acids, vectors and/or cells in the manufacture of a medicament for the treatment and/or prevention of a disease or disorder.

In some embodiments, the use is in the manufacture of a medicament for the treatment and/or prevention of a disease or disorder associated with aberrant CD19 expression, activity and/or signaling. In some embodiments, the disease or disorder comprises a tumor, preferably, the tumor is a cancer disease; preferably, the cancer disease is selected from the group consisting of: brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, Liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thymoma, sarcoma, non- Hodgkin lymphoma, Hodgkin lymphoma or uterine cancer and any combination thereof.

In some embodiments, the chimeric antigen receptor can be used as a therapeutic agent. Such agents will typically be used to treat, alleviate and/or prevent a disease or pathology associated with aberrant mesothelin (MSLN) expression, activity and/or signaling in a subject. Treatment can be carried out using standard methods by identifying a subject, such as a human patient having (or at risk or developing) a disease or disorder associated with aberrant MSLN expression, activity and/or signaling, such as cancer or other neoplastic disorders Program. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect due to its binding to the target. The administered chimeric antigen receptor can abrogate or inhibit or interfere with the expression, activity and/or signaling function of the target (eg, MSLN). The administered chimeric antigen receptor can eliminate or inhibit or prevent the target (eg, MSLN) from binding to the endogenous ligand to which it naturally binds. For example, a chimeric antigen receptor binds to a target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with MSLN expression, activity, and/or signaling. In some embodiments, to treat a disease or disorder associated with aberrant MSLN expression, a chimeric antigen receptor having heavy and light chain CDRs can be administered to a subject. In one embodiment, the disease or disorder associated with aberrant MSLN expression may be cancer.

As a non-limiting example, diseases or disorders associated with aberrant MSLN expression, activity and/or signaling include solid tumors. Preferably, the mesothelin expression-related disease is selected from the group consisting of mesothelioma, epithelial-like malignant pleural mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, biliary tract cancer, endometrial cancer, thymic cancer, colon cancer cancer, breast cancer.

In some embodiments, the chimeric antigen receptor can be used as a therapeutic agent. Such agents will typically be used to treat, alleviate and/or prevent a GD2-related disease or pathology in a subject. Treatment regimens can be implemented using standard methods by identifying a subject, eg, a human patient having (or at risk or developing) a disease or disorder associated with GD2, eg, cancer or other neoplastic disorder. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect due to its binding to the target. The administered chimeric antigen receptor can abrogate or inhibit or interfere with the expression, activity and/or signaling function of the target (eg, GD2). The administered chimeric antigen receptor can eliminate or inhibit or prevent the target (eg, GD2) from binding to the endogenous ligand to which it naturally binds. For example, a chimeric antigen receptor binds to a target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with GD2 expression, activity, and/or signaling. In some embodiments, to treat a disease or disorder associated with GD2, a chimeric antigen receptor having heavy and light chain CDRs can be administered to a subject. In one embodiment, the disease or disorder associated with abnormal GD2 may be cancer.

As a non-limiting example, a GD2-related disease or disorder is a GD2-positive tumor. A GD2-positive tumor is one that is associated with elevated levels of GD2 expression, including, for example, neuroblastoma, glioblastoma, medulloblastoma, astrocytoma, melanoma, small cell lung cancer , desmoplastic small round cell tumor, osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma or liposarcoma, fibrosarcoma, leiomyosarcoma and other soft tissue sarcomas in adults. In one embodiment, the patient has primary refractory or recurrent high-risk neuroblastoma, or minimal residual disease with high-risk neuroblastoma. The patient may have been previously treated or concurrently treated with another therapy such as surgery, chemotherapy, radiation therapy, stem cell transplantation, cytokine therapy (eg, with IL-2 and/or GM-CSF), and/or tretinoin ( For example, the use of isotretinoin) to treat.

Symptoms associated with cancer and other neoplastic disorders include, for example, inflammation, fever, general malaise, fever, pain, often localized inflammation, loss of appetite, weight loss, swelling, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue and abdominal Symptoms such as abdominal pain, diarrhea or constipation.

In one aspect, the present disclosure provides a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a chimeric antigen receptor, nucleic acid, vector, cell and/or composition comprising the foregoing .

In one aspect, the present disclosure provides a method of treating a mammal having a disease or disorder, comprising administering to the mammal an effective amount of the aforementioned chimeric antigen receptor, nucleic acid, vector, cell, and/or composition .

The pharmaceutical compositions of the embodiments are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile injectable diluents such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate Or phosphate, and agents to adjust osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin to maintain the desired particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferred to include isotonic agents such as sugars, polyols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the chimeric antigen receptor in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the chimeric antigen receptor into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying to obtain a powder containing the active ingredient and any additional desired ingredient from a sterile-filtered solution of those previously enumerated. .

For administration by inhalation, the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a gas of a suitable propellant, such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the permeation barrier are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, one or more of the chimeric antigen receptors can be formulated into an ointment, ointment, gel, or cream as generally known in the art.

The compounds can also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.

In one embodiment, the chimeric antigen receptor can be prepared with a carrier that will prevent it from being rapidly eliminated by the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier one or more of the chimeric antigen receptors. The specifications for the dosage unit forms of the embodiments are indicated by and are directly dependent on the unique characteristics of the chimeric antigen receptor and the particular therapeutic effect to be achieved, and the field of formulation of such chimeric antigen receptors used to treat the individual. inherent limitations.

The pharmaceutical composition can be placed in a container, pack, or dispenser with instructions for administration.

The formulations described herein may also contain more than one such chimeric antigen receptor, preferably those that have complementary activities but do not negatively affect each other, depending on the particular condition to be treated. Alternatively or additionally, the composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably combined in amounts effective for the intended purpose. For example, it can be combined in a kit, and can also be combined in use.

In one embodiment, the production of the cytokine IL-6 is reduced or inhibited. In another embodiment, the production of the cytokine IL-10 is reduced or inhibited.

In one embodiment, cytokine release syndrome is graded. In another embodiment, Level 1 describes a cytokine release syndrome wherein symptoms are not life threatening and only symptomatic treatment is required, eg, fever, nausea, fatigue, headache, myalgia, malaise. In another embodiment, Grade 2 symptoms require and respond to moderate intervention, such as oxygen, fluids, or vasopressors for hypotension. In another embodiment, grade 3 symptoms require and respond to aggressive intervention. In another embodiment, the grade 4 symptoms are life-threatening symptoms requiring a ventilator and the patient exhibits organ toxicity.

In one embodiment, one or more of the chimeric antigen receptors may be administered in combination therapy, ie, with other agents such as therapeutic agents (which are useful in the treatment of pathological conditions or disorders, such as various forms of cancer, autoimmune disorders and inflammatory diseases). The term "combination" as used herein refers to the administration of the agents substantially simultaneously, simultaneously or sequentially. If administered sequentially, at the start of administration of the second compound, the first of the two compounds is still preferably detected at an effective concentration at the treatment site. In one instance, "combination" can also be the simultaneous inclusion of a chimeric antigen receptor of the present disclosure and other therapeutic agents in a kit.

For example, a combination therapy can comprise one or more chimeric antigen receptors described herein with one or more additional therapeutic agents (eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents) , metabolic inhibitors, enzyme inhibitors, and/or cytotoxins or cytostatics, as described in more detail below) are co-formulated and/or co-administered. Such combination therapy may advantageously utilize lower doses of the administered therapeutic agent, thus avoiding possible toxicity or complications associated with various monotherapies.

The present disclosure uses DAP12 in series with 4-1BB to increase the costimulatory signal molecular elements, and designs a chimeric antigen receptor with better anti-tumor effect. Its anti-tumor beneficial effect has the following four aspects: 1. (2) lower secretion of immunosuppressive factors in vivo (reduced secretion of IL-10); (3) good clinical safety and good efficacy (low-grade CRS, durable remission).

The experimental results show that the chimeric antigen receptor of the present disclosure not only improves the efficacy and sustainability, but also, in terms of safety, significantly reduces the secretion of IL-6 in vivo, which is a clinical side effect (cytokine release synthesis). Symptoms), the most critical cytokine, and decreased secretion of IL-10, indicating decreased immunosuppressive effect, IL-10 is an immunosuppressive factor that plays an immunosuppressive role in anti-tumor therapy, in addition to the cytokine results in vivo, In the clinical study, 4 patients were all low-grade CRS, indicating a good clinical safety.

The present disclosure increases costimulatory signaling molecular elements through DAP12 in series with 4-1BB, links different anti-CD19, anti-mesothelin or anti-GD2 scFV antibodies, and designs anti-CD19, anti-mesothelin or anti-GD2 expression-positive solid tumors. Chimeric antigen receptors, forming a chimeric antigen receptor structure in parallel with a fusion peptide comprising an antigen binding domain and a transmembrane domain and another fusion peptide comprising a transmembrane domain, a cytoplasmic domain and a costimulatory domain. The chimeric antigen receptor has good killing activity on CD19, mesothelin or GD2 positive tumor cells, and can promote the secretion of cytokines (including IL-2 and INFγ). The CAR structure disclosed in the present disclosure is expected to greatly improve the therapeutic effect of CAR-T cells in the clinical application of MSLN-positive solid tumors in terms of the low efficacy of CAR-T in the clinical treatment of solid tumors. For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments, however, it is to be understood that the scope of the present disclosure may include combinations of all or some of the described features some embodiments.

Example 1: Structural design of DPK CAR targeting CD19

The following four chimeric antigen receptors were designed in this example (Fig. 1):

(1) CD19-KIRS2/Dap12-BB chimeric antigen receptor

As shown in Figure 1(A), the CD19-KIRS2/Dap12-BB chimeric antigen receptor comprises a first fusion peptide CD19-KIRS2 and a second fusion peptide Dap12-BB, wherein:

The first fusion peptide CD19-KIRS2 comprises an antigen binding domain and a transmembrane domain, the antigen binding domain is a CD19 scFv, and the transmembrane domain is a KIRS2 transmembrane domain;

The second fusion peptide Dap12-BB comprises a DAP12 transmembrane domain, a DAP12 cytoplasmic domain and a costimulatory domain 4-1BB.

The CD19-KIRS2/Dap12-BB chimeric antigen receptor is formed by the cleavage of DPK01 protein by T2A peptide, and the DPK01 fusion protein comprises DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4- 1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIRS2, the amino acid sequence of the DPK01 fusion protein is shown in SEQ ID NO.10.

DPK01: DAP12 signal peptide+DAP12+4-1BB+T2A+CD8α signal peptide+CD19 scFv+KIRS2

(2) CD19-KIRS2/tDap12-BB chimeric antigen receptor

As shown in Figure 1(B), the CD19-KIRS2/tDap12-BB chimeric antigen receptor comprises a first fusion peptide CD19-KIRS2 and a second fusion peptide tDap12-BB, wherein:

The second fusion peptide tDap12-BB comprises a truncated DAP12 transmembrane domain, a DAP12 cytoplasmic domain and a costimulatory domain 4-1BB.

The CD19-KIRS2/tDap12-BB chimeric antigen receptor is formed by the cleavage of the DPK02 protein by the T2A peptide, and the DPK02 fusion protein comprises the CD8α signal peptide, the truncated DAP12 (tDap12, including the transmembrane domain and the cytoplasmic structure). domain), 4-1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIRS2, the amino acid sequence of the DPK02 fusion protein is shown in SEQ ID NO.11.

DPK02: CD8α signal peptide+tDap12+4-1BB+T2A+CD8α signal peptide+CD19 scFv+KIRS2

(3) CD19-KIR2DS2/Dap12-BB Chimeric Antigen Receptor

As shown in Figure 1(C), the CD19-KIR2DS2/Dap12-BB chimeric antigen receptor comprises a first fusion peptide CD19-KIR2DS2 and a second fusion peptide Dap12-BB, wherein:

The first fusion peptide CD19-KIR2DS2 comprises an antigen binding domain and a transmembrane domain, the antigen binding domain is a CD19 scFv, and the transmembrane domain is a KIR2DS2 transmembrane domain;

The CD19-KIR2DS2/Dap12-BB chimeric antigen receptor is formed by the cleavage of DPK03 protein by T2A peptide, and the DPK03 fusion protein comprises DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4- 1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIR2DS2, the amino acid sequence of the DPK03 fusion protein is shown in SEQ ID NO.12.

DPK03: DAP12 signal peptide+DAP12+4-1BB+T2A+CD8α signal peptide+CD19 scFv+KIR2DS2

(4) CD19-KIRS2/Dap12 Chimeric Antigen Receptor

As shown in Figure 1(D), the CD19-KIRS2/Dap12 chimeric antigen receptor comprises a first fusion peptide CD19-KIRS2 and a second fusion peptide Dap12, wherein:

The second fusion peptide Dap12 comprises a DAP12 transmembrane domain and a DAP12 cytoplasmic domain.

The CD19-KIRS2/Dap12 chimeric antigen receptor is formed by the cleavage of the pKT011 protein by the T2A peptide, and the pKT011 fusion protein comprises a DAP12 signal peptide, DAP12 (including a transmembrane domain and a cytoplasmic domain), and a T2A cleavage site. , CD8α signal peptide, CD19 scFv and KIRS2, the amino acid sequence of the pKT011 fusion protein is shown in SEQ ID NO.13.

pKT011: DAP12 signal peptide+DAP12+T2A+CD8α signal peptide+CD19 scFv+KIRS2

in:

The amino acid sequence of the DAP12 signal peptide is as follows:

The amino acid sequence of DAP12 includes the DAP12 transmembrane domain and the DAP12 cytoplasmic domain, and the specific sequence is as follows:

The truncated amino acid sequence of DAP12 includes a truncated DAP12 transmembrane domain and a DAP12 cytoplasmic domain, and the specific sequence is as follows:

The amino acid sequence of the CD8α signal peptide is as follows:

The amino acid sequence of 4-1BB is as follows:

The amino acid sequence of the T2A cleavage site is as follows:

The amino acid sequence of CD19 scFv is as follows:

The amino acid sequence of KIRS2 is as follows:

The amino acid sequence of KIR2DS2 is as follows:

The amino acid sequence of DPK01 is as follows:

The amino acid sequence of DPK02 is as follows:

The amino acid sequence of DPK03 is as follows:

The amino acid sequence of pKT011 is as follows:

The amino acid sequence of the transmembrane domain of DAP12 is as follows:

The amino acid sequence of the cytoplasmic domain of DAP12 is as follows:

The nucleic acid sequence of DPK01 is as follows:

The nucleic acid sequence of DPK02 is as follows:

The nucleic acid sequence of DPK03 is as follows:

Example 2: Preparation of lentivirus

(1) 293T cells were passaged every other day

Seed ⁵ x 106 cells per T150 cell flask. After 48 hours, the number of cells should reach 20-25 million/vial.

(2) 293T cell plating

a) Taking 1 T150 cell culture flask as an example, gently wash the cells twice with about 15 ml of 1×PBS.

b) Add 3ml of 0.25% trypsin-2.21mM EDTA

c) Wait until the cells are detached, add 12 ml of DMEM medium (purchased from corning) with 10% (wt) FBS (purchased from Gibico) to the detached cells.

d) Collect and transfer cells to sterile centrifuge tubes, centrifuge at 1000 rpm for 10 minutes.

e) Aspirate the supernatant and resuspend the pellet in 10 ml of 10% (wt) FBS in DMEM.

f) Cell count, the volume required for ¹² x 106 cells was calculated based on the cell concentration.

g) Combine the cells and 25ml of 10% (wt) FBS in DMEM medium, put them into a T150 cell flask, shake gently to make the cells evenly distributed to the bottom of the cell flask, and culture overnight in a 37°C, 5% CO ₂ incubator .

(3) Cell transfection

The cells were observed, and the cell density reached approximately 80%-90%, at which point the transfection started.

a) 30-60 minutes before transfection, gently aspirate the culture medium.

b) Mix plasmid DNA and calcium chloride solution, take a T150 bottle as an example, 28μg pRSV.rev (purchased from Invitrogen Company), 28 μg pGAG-Pol (purchased from Invitrogen Company), 11 μg pVSVG (purchased from Invitrogen Company), 23 μg of lentiviral expression plasmids (plasmids DPK01, DPK02, DPK03, pKT011) were added to 1.5 ml of calcium chloride solution and mixed.

c) Add 1.5ml of borate buffer solution into a 15ml sterile centrifuge tube, mix the DNA-calcium chloride solution with a 1ml pipette tip, and then add dropwise to the borate buffer solution, and mix quickly for 15- 20 at room temperature for 25-30 minutes.

d) The DNA-calcium chloride-borate buffer mixture (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) was uniformly added dropwise to the T150 bottle with a 5ml pipette. Incubate in a 37°C cell incubator with 5% carbon dioxide, and change the medium for 6h.

e) Change the medium after 6h. Gently shake the culture plate several times to fully suspend some calcium phosphate precipitates, aspirate the medium containing calcium phosphate precipitates, add 20 ml of fresh DMEM medium of 5% (wt) FBS, and continue the culture.

(4) Initial collection of virus supernatant

a) The culture supernatant of 293T cells transfected the previous day was collected into a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, labeled, and temporarily stored in a 4°C refrigerator.

b) Add 20 ml of pre-warmed DMEM medium of 5% (wt) FBS into the cell flask, and continue to cultivate overnight in a 37°C cell incubator.

(5) The virus supernatant was collected for the second time (48h/fourth day).

(6) Filter the supernatant

The supernatants from the two collections were pooled and filtered through a 0.45 μm filter to remove cell debris.

(7) Virus concentration

Centrifuge overnight at 12000-24000 rpm at 4°C.

(8) Virus storage

After centrifugation, the supernatant was poured out, fresh 5% (wt) FBS DMEM medium was added to resuspend, and the virus was subpackaged (denoted as DPK01, DPK02, DPK03, pKT011), and quickly stored in a -80°C refrigerator for later use.

Example 3: In vitro functional test of CD19 CAR-T

(1) Cell Preparation

Plasma was separated from fresh blood samples (for use after inactivation), PBMCs were separated from the blood cell suspension with lymphocyte separation medium, and the obtained PBMCs were subjected to T cell sorting with a T cell sorting kit. T cells were activated by adding Dynabeads CD3/CD28 (added by cell: Dynabeads = 1:1), using activation medium (X-VIVO15, 5% plasma, 300 IU/ml IL-2) to adjust the final cell concentration to 1 × 10 ⁶ /ml at 37°C, 5% CO ₂ . 24h later, the CAR lentivirus (DPK01, DPK02, DPK03, pKT011) prepared in Example 2 was added to transfect T cells, and the lentivirus was removed after 48h of infection. On day 4 (D4), the cells were observed in supplemental culture every 1-2 days, and the cell density was maintained at 0.8×10 ⁶ cells/mL. Activation medium was used on days 4-5 (D4-D5), and expansion medium (X-VIVO15, 300 IU/ml IL-2) was used after day 5 (D5). Four kinds of CAR-T cells (DPK01, DPK02, DPK03, pKT011) were obtained by continuous culture until the 11th day (D11).

There was no significant difference in the activation and expansion of T cells among the four different CAR structures (Fig. 2A); the four CAR-T cells were in a state of expansion during the culture, and the volume gradually increased in the first 4 days, and then gradually decreased ( Figure 2B).

(2) Detection of positive rate of CAR-T cells

Four CAR-T cells of DPK01, DPK02, DPK03, and pKT011 on the 8th day (D8) were taken and incubated with anti-human CD19 antibody (PE) primary antibody and Streptavidin-PE secondary antibody, without transfection Transduced T cells (NTD) were incubated in the same way as a control, and the positive rate of CAR-T cells was detected by flow cytometry. The positive rates of the four types of CD19 CAR-T cells were detected, and there was little difference in the expression of the positive rates of the four CAR-T cells on day 8 (D8) (Figure 3).

(3) Detection of T cell differentiation subsets

CAR-T cell subset detection antibodies: anti-human CD3 (APC-Cy7), anti-human CCR7 antibody (BV421), anti-human CD45RO antibody (APC), CD4-BB515, CD8-BV510, CD45RA (PE- Cy7), CD62L-PE.

On the 11th day (D11) of the four CAR-T cell cultures of DPK01, DPK02, DPK03, and pKT011, samples were collected and the CAR-T cell subset detection antibody was used for flow detection. Detect CAR-T cell typing, including Tn, Tscm, Tcm, Tem, and TemRA in CD4+ and CD8+ T cells. The T cell differentiation subtypes of the four CAR-Ts (DPK01, DPK02, DPK03, pKT011) showed that compared with pKT011, DPK01, DPK02, and DPK03 had more memory cells and could achieve a lower differentiation state of CAR-T, It is more persistent and has memory activity in vivo (Figure 4).

(4) target cell culture

293T, MCF-7 and MC-7-CD19 were cultured in DMEM medium (DMEM+10% FBS+1% Penicillin/Streptomycine); Nalm6 and L428 were cultured in 1640 medium (1640+10 %FBS+1% penicillin/streptomycin).

(5) CAR-T specific killing

a. The target cells were digested and counted, and the target cell suspension with a density of 2×10 ⁵ cells/ml was adjusted with culture medium (DMEM+10% FBS);

b. Add 50 μl of cell culture medium to the Eplate plate, put the Eplate into the DP monitoring tank, and prepare to measure the baseline.

c. After the baseline measurement, take out the Eplate, add 1×10 ⁴ (50 μl) of target cells to each well, and place at room temperature for 30 minutes.

d. Put the E-plate with the cell suspension back into the corresponding monitoring tank to start monitoring.

e. After 18h, count the CAR-T and NTD cells, and adjust the effector cells to the corresponding density according to the effector-target ratio (E:T) in the following table.

E:TE:T	靶细胞数目number of target cells	细胞密度及每孔加入体积Cell density and volume added per well
0:10:1	00	50μl培养液50μl culture medium
1:11:1	1×10 ⁴ 1×10 ⁴	2×10 ⁵细胞/ml,50μl 2×10 ⁵ cells/ml, 50 μl
5:15:1	5×10 ⁴ 5×10 ⁴	1×10 ⁶细胞/ml,50μl 1×10 ⁶ cells/ml, 50 μl
10:110:1	1×10 ⁵ 1×10 ⁵	2×10 ⁶细胞/ml,50μl 2×10 ⁶ cells/ml, 50 μl

f. Suspend the data collection of all detection slots;

g. Take out the overnight E-plate, and add 50 μl of corresponding effector cells to each well;

h. Put the E-plate back into the corresponding monitoring tank, and continue the data collection of the real-time label-free dynamic cell analysis technology (RTCA) system;

i. After the effector cells are added, the monitoring time shall not exceed 24h.

The killing effect of DPK01, DPK02, DPK03, pKT011 CAR-T four CD19 CAR-T cells and non-transduced T cells (NTD) on tumor cells MCF7-CD19 was detected, and the killing ability of various CAR-T cells was compared (Figure 5 ). The results showed that NTD had no killing effect on MCF-7-CD19; when the effector-target ratio (E:T) was 0:1, 1:1, 5:1, 10:1, all four CAR-T cells were It showed obvious killing effect on MCF7-CD19 at two effector-target ratios of 5:1 and 10:1. There was no significant difference in the lysis rate of target cells by the three CAR-T cells DPK01, DPK02 and DPK03; the anti-tumor effect of pKT011 was relatively weak, and the killing efficiency on target cells was significantly lower than that of DPK01, DPK02 and DPK03 CAR-T.

(6) Detection of cytokine secretion

According to E:T=2:1, DPK01, DPK02, DPK03, pKT011 CAR-T were co-cultured with Nalm6 (positive target cells) and L428 (negative target cells) for 24 hours, respectively; IFN-γ.

Analysis of the 4 kinds of CAR-T cells and NTDs co-cultured with the positive target cell Nalm6 for 24 hours, the supernatant was taken, and the cytokines were detected by ELISA. And the secretion of IFN-γ of DPK01 CAR-T cells was significantly similar to that of DPK02 and DPK03, and the secretion of IFN-γ of pKT011 was significantly lower than that of other CAR-T groups (Figure 6). The four CAR-T cells secreted IL-2 after co-culture with the positive target cell Nalm6, and the IFN-γ secretion of DPK01 CAR-T cells was significantly similar to that of DPK02 and DPK03, while pKT011 hardly secreted IL-2. Taken together, IFN-γ and IL-2 secreted by DPK01 CAR-T cells were optimal under the stimulation of tumor antigens.

(7) Antigen stimulates proliferation

a. CAR-T was cultured to the 8th day (D8) to demagnetize the beads, and continued to culture for 2 days with IL-2-free medium;

b. On day 10 (D10), count effector cells (CAR-T) and NTDs;

c. Take the required CAR-T (5×10 ⁶ /ml), wash 2 times with PBS, and finally add 500 μl PBS to resuspend the cells to make the concentration 1×10 ⁷ /ml;

d. Add the fluorescent dye CFSE (purchased from Sigma) (the original concentration is 5 mM) to make the final concentration 5 μM, put it into the incubator and incubate for 10 min.

e. Add 9 times the incubation volume of PBS and centrifuge;

f. After discarding the supernatant, add 10 ml of medium containing 10% FBS to resuspend, centrifuge, and discard the supernatant.

g. Resuspend to 1×10 ⁶ /ml with 5 ml of medium (10% FBS, 300 UI/ml IL-2). stand-by.

h. Count the target cells (Nalm-6 and 293T), and adjust the cell suspension with a density of 1×10 ⁶ /ml with a culture medium (1640+10% FBS+300UI/ml IL-2) for use;

i. According to the corresponding effector-target ratio (E:T), add 1 ml of CAR-T cells to each well, place them in a 37°C, 5% CO ₂ incubator, and change the medium by centrifugation for 1-2 days.

j. On the fifth day (D5), flow detection was performed.

The CFSE fluorescence intensity flow detection results of the four CAR-Ts (DPK01, DPK02, DPK03, pKT011) showed that (Figure 7), compared with the single CAR-T control group, after stimulation of MCF7-CD19 positive target cells, the four The left shift of the fluorescence intensity of CFSE of CAR-T was weakened (green column in the figure), confirming that the positive target cells can stimulate the proliferation of these four CAR-Ts.

Example 4: In vivo functional test of CD19 CAR-T

Tumor cells: Nalm6 (1×10 ⁷ cells)

Tumor formation method: subcutaneous tumor formation

Administration group: NTD, DPK01, DPK02, DPK03

Dosage: 1×10 ⁶ CAR-T

(1) In vivo efficacy

The antitumor activities of NTD, DPK01, DPK02, and DPK03 in animals were further validated (Figure 8). The experimental results showed that the tumor volume of the mice in the NTD group increased rapidly and reached the end point of the experiment soon; DPK01, DPK02, DPK03, and pKT011 had similar tumor clearance efficiencies in mice. Long lasting effect.

(2) Cytokine secretion in vivo

Quantification of soluble cytokines was performed using Luminex bead array technology and kits purchased from Life technologies (Invitrogen). Assays were performed using an 8-point standard curve generated by 3-fold serial dilutions according to the manufacturer's protocol. Each standard spot and sample was evaluated in duplicate at a 1:3 dilution; the calculated %CV was less than 15% for both measurements. The standard curve quantification range was determined by the 80-120% (observed/expected) range. Individual analyte quantification ranges are reported in the figure captions. DPK01, DPK02 and DPK03 of the present disclosure significantly decreased the levels of endocrine IL-6 in mice (Fig. 9), indicating that the present disclosure can achieve better clinical safety, and IL-10 was also significantly decreased, indicating that the production of lower immunosuppressive factors, Promote clinical efficacy.

(3) CAR-T pharmacokinetics in vivo

The occupancy rates of DPK01, DPK02, DPK03, and pKT011 four CAR-T cells detected in the peripheral blood of mice 28 days after administration were taken (Figure 10), and anti-human CD19 antibody (PE) primary antibody and streptavidin ( Streptavidin)-PE secondary antibody incubation, the positive rate of CAR-T cells was detected by flow cytometry, the positive rate of DPK01, DPK02, DPK03 was not significantly different, and the CD19 CAR-T occupancy rate of pKT011 was significantly lower, indicating that DPK01, DPK02, DPK03 Better anti-tumor effect.

(4) In vivo CAR-T depletion marker expression

The PD-1 expression of four CAR-T cells, DPK01, DPK02, DPK03, and pKT011 detected in the peripheral blood of mice 28 days after administration (Figure 11), the positive rate of CAR-T cells was detected by flow cytometry, and the PD-1 expression of pKT011 1 was significantly up-regulated, indicating that DPK01, DPK02, and DPK03 were better able to resist depletion.

Example 5: CD19 CAR-T clinical study

(1) Cell Preparation

On the 14th day (D-14) before the reinfusion of cells, the patient's blood was drawn, and the plasma was separated from the fresh blood sample (for inactivation), the blood cell suspension was separated from PBMC with lymphocyte separation medium, and the obtained PBMC was separated by T cell sorting kit. T cell sorting was performed. T cells were activated by adding Dynabeads CD3/CD28 (added by cell: Dynabeads = 1:1), using activation medium (X-VIVO15, 5% plasma, 300 IU/ml IL-2) to adjust the final cell concentration to 1 × 10 ⁶ /ml at 37°C, 5% CO ₂ . T cells were transfected with DPK01 lentivirus after 24 h, and the lentivirus was removed after 48 h of infection. On day 4 (D4), the cells were observed in supplemental culture every 1-2 days, and the cell density was maintained at 0.8×10 ⁶ cells/mL. Activation medium was used on days 4-5 (D4-D5), and expansion medium (X-VIVO15, 300 IU/ml IL-2) was used after day 5 (D5). Culture was continued until days 7-12 (D7-D12).

The CAR clinical-grade vector was manufactured in Nanjing Aide Immunotherapy Research Institute Co., Ltd. At the end of the CAR-T cell culture, the cells are cryopreserved in an injectable freezing medium. Administer CAR-T cells in accordance with the dose administered. Each bag contains an aliquot of freezing medium (volume depends on dose), and cryopreservation solution is CS5. Bags (10-100ml) containing CD19 CAR-T cells were stored in a -135°C liquid nitrogen box under test. Cryopreservation bags are stored in the freezer until needed. To increase safety, the dose of the first reinfused cells was given in divided doses on Day 0 (D0) and Day 1 (Dl), with approximately 30% cells on

Day

0 and 70% cells on Day 1.

(2) Cell thawing

Frozen cells are shipped to the laboratory or patient on dry ice. Using a water bath maintained at 37°C, cells were thawed and massaged gently until cells were just thawed. There should be no frozen pieces left in the container. If the CD19 CAR-T cell product appears to be in a damaged or leaking pocket, it should not be reinfused.

(3) Preoperative medication

The CD19 CAR-T preparation should not be left at room temperature for too long after warming, so the warming time needs to be determined after all preparations for the subject's treatment are completed. Prepare necessary first aid equipment and medicines before reinfusion. 30-60 minutes before reinfusion, subjects were premedicated with acetaminophen, depakine, and diphenhydramine or other H1 antihistamines. Since CD19 CAR-T is an autologous T cell therapy, the packaging bag is dedicated to the patient, and the subject information on the bag should be confirmed before reinfusion. Check the package for damage before returning it. If it is damaged, do not return it.

(4) Return input

Confirm the subject information on the packaging bag again; flush the tubing for injection with normal saline; flush the tubing with 50ml of normal saline before and after CAR-T cell infusion; complete the intravenous infusion within 30 minutes.

(5) Entry criteria

(1) CD19-positive relapsed and refractory B-cell hematological malignancies; (2) under 70 years of age; (3) KPS score ≥ 60, expected survival period ≥ 3 months; (4) absolute platelet count ≥ 30×10 ⁹ /L; (5) the absolute number of lymphocytes ≥ 0.15×10 ⁹ /L; (6) serum ALT≤100U/L, AST≤100U/L; (7) total bilirubin≤30μmol; (8) creatinine≤ 200 μmol/L; (9) Women of childbearing age who had a negative urine pregnancy test before the start of administration, and agreed to take effective contraceptive measures during the trial period until the last follow-up; (10) Voluntary enrollment, good compliance, and able to cooperate with the trial observed, and signed written informed consent.

(6) Exclusion criteria

Patients were not eligible for this study if they met any of the following criteria:

(1) Clinical diagnosis (symptoms, signs, imaging, cerebrospinal fluid) of central nervous system leukemia; (2) accompanied by hyperleukemia (white blood cell count ≥ 50×10 ⁹ /L) or the subjects were enrolled according to the The investigator judged that the disease progressed rapidly and could not ensure the completion of a complete treatment cycle; (3) patients with infections including fungal, bacterial, viral or other uncontrollable infections or infections that required four-level isolation treatment; (4) HIV , HBV, HCV positive test; (5) patients with central nervous system diseases including stroke, epilepsy, dementia and other central nervous system diseases or autoimmune central nervous system diseases; (6) within 12 months before enrollment Infection, cardiac angiography or stent, active angina pectoris or other obvious clinical symptoms, or associated with cardiac asthma or cardiovascular lymphocyte infiltration; (7) Those who are receiving anticoagulation therapy or have severe coagulation dysfunction (APTT>70 ); (8) Research in which the drug treatment the patient is receiving will affect the safety and efficacy of this project according to the investigator's judgment; (9) Patients with allergies or a history of allergies to the biological agents used in this project; (10) Pregnancy or Lactating women; (11) Those who have systematically used systemic and systemic steroids within 2 weeks before participating in treatment (except those who are using inhaled steroids recently or currently); (12) Have other uncontrolled diseases, according to the investigators Those who are not suitable to join; (13) any situation that the investigator believes may increase the risk of patients or interfere with the results of the trial; (14) patients who participate in other clinical studies at the same time.

(7) Treatment plan

Figure 9 shows the flow chart of the clinical treatment protocol of CD19 CAR-T cells.

(8) Preprocessing scheme

On the 5th, 4th, 3rd, 2nd and 1st days before the infusion of cells: intravenous infusion of fludarabine for 5 consecutive days, the dose is 30 mg/m ² / day;

On the 5th and 4th days before the reinfusion of cells: intravenous infusion of cyclophosphamide for 2 consecutive days at a dose of 500 mg/m ² /day.

(9) Dosing schedule

(1) Reinfusion dose: according to the existing international counterpart dose

The experimental drug DPK01 was administered according to the clinical situation, 1.2×10 ⁶ CAR-T cells/kg intravenous drip was administered in two doses, 30% was administered on the 0th day (D0), and 70% was administered on the 1st day (D1). %. The above dosing regimen was taken as a course of treatment.

(10) Treatment results

NO.NO.	适应症Indications	肿瘤负荷tumor burden	剂量dose	CRSCRS	疗效curative effect	随访结果(天)Follow-up results (days)
11	B-ALLB-ALL	74.5(BM)74.5 (BM)	1.2×10 ⁶ 1.2×10 ⁶	11	完全缓解complete relief	复发(154)Relapse (154)
22	B-ALLB-ALL	96(BM)96 (BM)	1.2×10 ⁶ 1.2×10 ⁶	22	完全缓解complete relief	持续缓解(380)Sustained Relief (380)
33	B-ALLB-ALL	7.5(BM)7.5 (BM)	1.2×10 ⁶ 1.2×10 ⁶	11	完全缓解complete relief	持续缓解(137)Sustained Relief (137)
44	B-ALLB-ALL	56.5(BM)56.5 (BM)	1.2×10 ⁶ 1.2×10 ⁶	11	完全缓解complete relief	持续缓解(368)Sustained Relief (368)

CD19-CAR-T therapy in 4 adult patients with acute B lymphocytic leukemia (B-ALL) had a 1-month complete remission rate of 100% and a 6-month complete remission rate of 50%. All 4 patients developed CRS, with grade 1 CRS occurring in 75% and grade 2 CRS in 25%, and no patient had central system toxicity. The results of clinical studies show that DPK01 produces controllable low-grade CRS in clinical treatment, exhibits better safety, and has a high complete remission rate and strong lasting effect in vivo.

Example 6: Structural design of a chimeric antigen receptor targeting mesothelin

In this example, the following two mesothelin-targeting chimeric antigen receptors (MSLN CARs) were designed (Figure 13):

(1) MSLN SS1 scFv1-KIRS2/Dap12-BB Chimeric Antigen Receptor

As shown in Figure 13(A), the MSLN SS1 scFv1-KIRS2/Dap12-BB chimeric antigen receptor comprises the first fusion peptide MSLN SS1 scFv1-KIRS2 and the second fusion peptide Dap12-BB, wherein:

The first fusion peptide MSLN SS1 scFv1-KIRS2 comprises an antigen binding domain and a transmembrane domain, the antigen binding domain is MSLN SS1 scFv1, and the transmembrane domain is a KIRS2 transmembrane domain;

The MSLN SS1 scFv1-KIRS2/Dap12-BB chimeric antigen receptor is formed by the cleavage of the pKT032 fusion protein by the T2A peptide, and the pKT032 fusion protein comprises the DAP12 signal peptide, DAP12 (including the transmembrane domain and the cytoplasmic domain) , 4-1BB, T2A cleavage site, CD8α signal peptide, MSLN SS1 scFv1 and KIRS2, the amino acid sequence of the pKT032 fusion protein is shown in SEQ ID NO.21, and its encoding nucleic acid is shown in SEQ ID NO.22.

pKT032: DAP12 signal peptide+DAP12+4-1BB+T2A+CD8α signal peptide+SS1scFv1+KIRS2

(2) MSLN SS1 scFv2-KIRS2/Dap12-BB Chimeric Antigen Receptor

As shown in Figure 13(B), the MSLN SS1 scFv2-KIRS2/Dap12-BB chimeric antigen receptor comprises the first fusion peptide MSLN SS1 scFv2-KIRS2 and the second fusion peptide Dap12-BB, wherein:

The first fusion peptide MSLN SS1 scFv2-KIRS2 comprises an antigen binding domain and a transmembrane domain, the antigen binding domain is MSLN SS1 scFv2, and the transmembrane domain is a KIRS2 transmembrane domain;

The MSLN SS1 scFv2-KIRS2/Dap12-BB chimeric antigen receptor is formed by the cleavage of the pKT108 fusion protein by the T2A peptide, and the pKT0108 fusion protein comprises the DAP12 signal peptide, DAP12 (including the transmembrane domain and the cytoplasmic domain) , 4-1BB, T2A cleavage site, CD8α signal peptide, MSLN SS1 scFv2 and KIRS2, the amino acid sequence of the pKT108 fusion protein is shown in SEQ ID NO.23, and its encoding nucleic acid is shown in SEQ ID NO.24.

pKT108: DAP12 signal peptide+DAP12+41BB+T2A+CD8α signal peptide+SS1 scFv2+KIRS2

in:

The amino acid sequence of SS1 scFv1 is as follows:

The amino acid sequence of SS1 scFv2 is as follows:

The amino acid sequence of pKT032 is as follows:

The nucleotide sequence of pKT032 is as follows:

The amino acid sequence of pKT108 is as follows:

The nucleotide sequence of pKT108 is as follows:

Example 7: Preparation of lentivirus

(1) 293T cells were passaged every other day

(2) 293T cell plating

b) Add 3ml of 0.25% trypsin-2.21mM EDTA

(3) Cell transfection

a) 30-60 minutes before transfection, gently aspirate the culture medium.

b) Mix plasmid DNA and calcium chloride solution, take a T150 bottle as an example, 28μg pRSV.rev (purchased from Invitrogen Company), 28 μg pGAG-Pol (purchased from Invitrogen Company), 11 μg pVSVG (purchased from Invitrogen Company), 23 μg of lentiviral expression plasmids (plasmids pKT032, pKT108, synthesized by Shanghai Sangong) were added to 1.5 ml of calcium chloride solution and mixed.

d) Using a 5ml pipette, the DNA-calcium chloride-borate buffer mixture (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) was evenly added dropwise to the T150 bottle. Incubate in a 37°C cell incubator with 5% carbon dioxide, and change the medium for 6h.

(4) Initial collection of virus supernatant

(5) The virus supernatant was collected for the second time (48h/fourth day).

(6) Filter the supernatant

(7) Virus concentration

Centrifuge overnight at 12000-24000 rpm at 4°C.

(8) Virus storage

After centrifugation, the whole supernatant was poured, and fresh 5% (wt) FBS DMEM medium was added to resuspend, and the virus was divided into packaging (referred to as lentivirus pKT032, pKT108), and quickly stored in a -80°C refrigerator for later use.

Example 8: In vitro functional test of MSLN CAR-T cells

(1) Cell Preparation

Plasma was separated from fresh blood samples (for use after inactivation), PBMCs were separated from the blood cell suspension with lymphocyte separation medium, and the obtained PBMCs were subjected to T cell sorting with a T cell sorting kit. T cells were activated by adding Dynabeads CD3/CD28 (added by cell: Dynabeads = 1:1), using activation medium (X-VIVO15, 5% plasma, 300 IU/ml IL-2) to adjust the final cell concentration to 1 × 10 ⁶ /ml at 37°C, 5% CO ₂ . 24h later, the CAR lentivirus (virus pKT032, pKT108) prepared in Example 2 was added to transfect T cells, and the lentivirus was removed after 48h of infection. On day 4 (D4), the cells were observed in supplemental culture every 1-2 days, and the cell density was maintained at 0.8×10 ⁶ cells/mL. Activation medium was used on days 4-5 (D4-D5), and expansion medium (X-VIVO15, 300 IU/ml IL-2) was used after day 5 (D5). Two kinds of CAR-T cells targeting mesothelin (CAR-T cells pKT032, pKT108) were obtained by continuous culture until the 11th day (D11).

There was no significant difference in the activation and expansion of T cells between the two different CAR structures (Fig. 14A); the two CAR-T cells were in a state of expansion during the culture process, and the volume gradually increased in the first 4 days, and then gradually decreased ( Figure 14B).

(2) Detection of positive rate of CAR-T cells

Take two CAR-T cells of pKT032 and pKT108 on the 8th day (D8), add anti-mouse Fab antibody (PE) primary antibody and streptavidin (Streptavidin)-PE secondary antibody to incubate, and incubate untransduced T cells in the same way Incubation was used as a control, and the positive rate of CAR-T cells was detected by flow cytometry. The positive rates of the two SS1 CAR-T cells were detected, and there was little difference in the expression of the positive rates of the two CAR-T cells on day 8 (Figure 15).

(3) Detection of T cell differentiation subsets

On the 12th day of culture of two CAR-T cells, pKT032 and pKT108, samples were added to detect antibody for CAR-T cell subsets for flow detection. Detection of CAR-T cell typing, including the proportion of Tn, Teff, Tcm, and Tem in CD4+ and CD8+ T cells. The T cell differentiation subtypes of the two CAR-Ts (pKT032, pKT108) showed that pKT032 (73.67%), pKT108 (74.21%) were significantly more efficient than untransduced T cells (NTD) (Tcm ratio was 29.28%). There were more cells in the memory population (Figure 16). The present disclosure can achieve a lower differentiated state of CAR-T, which will be more durable and have memory activity in vivo.

(4) target cell culture

SK-OV-3 cells were cultured using Myco5A medium (Myco5A+10%FBS+1% Penicillin/Streptomycine); OVCAR-3 cells were cultured using 1640 medium (1640+10%FBS+1 % penicillin/streptomycin).

(5) CAR-T specific killing

a. Digestion and counting of target cells, adjust the target cell suspension with a density of 2×10 ⁵ cells/ml with culture medium (Myco5A or 1640+10% FBS);

f. Suspend the data collection of all detection slots;

i. After the effector cells are added, the monitoring time should not exceed 48h.

The killing effects of two SS1 CAR-T cells, pKT032 and pKT108, and non-transduced T cells (NTD) on tumor cells SK-OV-3 and OVCAR-3 were detected, and the CAR-T cells containing different scFVs against mesothelin were compared. lethality (Figure 17). The results showed that NTD had no killing effect on SK-OV-3 and OVCAR-3 cells; when the effector-target ratio (E:T) was 0:1, 1:1, 5:1, 10:1, the three Both CAR-T cells showed significant killing effects on SK-OV-3 and OVCAR-3 at two effector-target ratios of 5:1 and 10:1. There was no significant difference in the lysis rate of target cells between pKT032 and pKT108 CAR-T cells.

(6) Detection of cytokine secretion

According to E:T=2:1, pKT032 and pKT108 CAR-T were co-cultured with SK-OV-3 and OVCAR-3 cells for 24h, respectively; the cell supernatants were collected, and IL-2 and IFN-γ were detected by ELISA.

Analysis of two kinds of CAR-T cells and NTD co-cultured with SK-OV-3 and OVCAR-3 cells for 24 hours, the supernatant was taken, and cytokines were detected by ELISA. Both secreted IFN-γ and IL2, and pKT032 CAR-T cells secreted IFN-γ (respectively: 2997.99 pg/ml and 4622.92 pg/ml), IL2 (respectively: 1214.24 pg/ml and 4769.07 pg/ml) The amount was significantly higher than that in the pKT108 CAR-T group (Figure 18). Taken together, pKT032 CAR-T cells secreted IFN-γ and IL-2 optimally under the stimulation of tumor antigens.

(7) Antigen stimulates proliferation

a. CAR-T cells were cultured to the 8th day (D8) with demagnetized beads, and continued to culture for 2 days with IL-2-free medium;

b. On day 10 (D10), count effector cells (CAR-T) and NTDs;

c. Take the required CAR-T cells (5×10 ⁶ /ml), wash twice with PBS, and finally add 500 μl PBS to resuspend the cells to make the concentration 1×10 ⁷ /ml;

d. Add the fluorescent dye CFSE (purchased from BD Bioscience, the original concentration is 5mM) to make the final concentration of CFSE 5μM, put it into the incubator and incubate for 10min;

e. Add 9 times the incubation volume of PBS and centrifuge;

f. After discarding the supernatant, add 10 ml of medium containing 10% FBS to resuspend, centrifuge, and discard the supernatant;

g. Resuspend to 1×10 ⁶ /ml with 5ml medium (10%FBS, 300UI/ml IL-2), set aside;

h. Count the target cells (SK-OV-3 and OVCAR-3), and adjust the cell suspension to a density of 1×10 ⁶ /ml with culture medium (Myco5A or 1640+10%FBS+300UI/ml IL-2). ,spare;

j. On the fifth day (D5), flow detection was performed.

Flow detection results of CFSE fluorescence intensity of two CAR-T cells (pKT032, pKT108) showed (Fig. 19), compared with NTD control group, after stimulation of mesothelin-positive target cells, the fluorescence intensity of CFSE of pKT032 CAR-T The left shift is weakened, confirming that the positive target cells can stimulate the proliferation of pKT032 CAR-T; while the fluorescence intensity of CFSE of pKT108 CAR-T has little left shift, confirming that the proliferation of pKT108 CAR-T stimulated by positive target cells is weak, not as good as that of pKT032 CAR-T. The proliferative capacity of T.

Example 9: In vivo functional test of MSLN CAR-T cells

Tumor cells: SKOV-3 (5×10 ⁶ cells)

Tumor formation method: subcutaneous tumor formation

Administration group: NTD, pKT032, pKT108

Dosage: 5×10 ⁶ CAR-T

In vivo efficacy:

The antitumor activity of three CAR-T cells (NTD, pKT032 and pKT108) in animals was further validated (Figure 20). The results showed that the tumor volume of the mice in the NTD group increased rapidly and reached the end point of the experiment; pKT032 and pKT108 had similar tumor clearance efficiencies, and pKT032 made the tumor regress more rapidly and brought a more durable curative effect.

Example 10: Clinical study of MSLN CAR-T cells

(1) Cell Preparation

Plasma was separated from fresh blood samples (for use after inactivation), PBMCs were separated from the blood cell suspension with lymphocyte separation medium, and the obtained PBMCs were subjected to T cell sorting with a T cell sorting kit. T cells were activated by adding Dynabeads CD3/CD28 (added by cell: Dynabeads = 1:1), using activation medium (X-VIVO15, 5% plasma, 300 IU/ml IL-2) to adjust the final cell concentration to 1 × 10 ⁶ /ml at 37°C, 5% CO ₂ . T cells were transfected with pKT032 lentivirus after 24 hours, and the lentivirus was removed after 48 hours of infection. On the 4th day (D4), the cell culture was observed every 1-2 days, and the cell density was maintained at 0.8×10 ⁶ cells/mL. Activation medium was used on days 4-5 (D4-D5), and expansion medium (X-VIVO15, 300 IU/ml IL-2) was used after day 5 (D5). Culture was continued until days 7-12 (D7-D12).

The CAR clinical-grade vector was manufactured in Nanjing Aide Immunotherapy Research Institute Co., Ltd. At the end of the CAR-T cell culture, the cells are cryopreserved in an injectable freezing medium. Administer CAR-T cells in accordance with the dose administered. Each bag contains an aliquot of freezing medium (volume depends on dose), and cryopreservation solution is CS5. Bags (10-100 ml) containing mesothelin CAR-T cells were stored in a -135°C liquid nitrogen box under test. Cryopreservation bags are stored in the freezer until needed. For added safety, the first dose was given in divided doses on

days

0 and 1, with approximately 30% cells on

day

0 and 70% cells on day 1.

(2) Cell thawing

Frozen cells are shipped to the laboratory or patient on dry ice. Using a water bath maintained at 37°C, cells were thawed and massaged gently until cells were just thawed. There should be no frozen pieces left in the container. If the mesothelin CAR-T cell product appears to be in a damaged or leaking pocket, it should not be reinfused.

(3) Preoperative medication

The mesothelin CAR-T preparation should not be left at room temperature for too long after warming, so the warming time needs to be determined after the subject's treatment preparations are completed. Prepare necessary first aid equipment and medicines before reinfusion. 30-60 minutes before reinfusion, subjects were premedicated with acetaminophen, depakine, and diphenhydramine or other H1 antihistamines. Since mesothelin CAR-T is an autologous T cell therapy, the packaging bag is dedicated to the patient, and the subject information on the bag should be confirmed before reinfusion. Check the package for damage before returning it. If it is damaged, do not return it.

(4) Return input

(5) Inlet and discharge standard

(1) Mesothelin-positive recurrent and refractory ovarian cancer and pancreatic cancer; (2) Age 18-70 years old, no gender restriction; (3) Relapse after receiving chemotherapy or targeted drugs and other second-line or above treatments; ( 4) The immunohistochemical results of tumor tissue are positive for MSLN antigen expression, and the antigen expression rate is ≥15%; (5) According to RECIST1.1 criteria, the patient has at least one evaluable tumor lesion, which can be accurately measured at baseline; ( 6) The ECOG score is 0-2, and the expected survival time is more than 12 weeks; (7) There is venous access for venous blood collection or apheresis; (8) The patients themselves participate voluntarily and sign the informed consent in writing.

(6) Exclusion criteria

(1) Pregnant or breastfeeding women; (2) Those who used chemotherapy or radiotherapy within 3 days before the blood collection period; (3) Those who used systemic steroids within 5 days before the blood collection period (except those who were recently or currently using inhaled steroids); (4) Those who have used drugs to stimulate bone marrow hematopoietic cell production within 5 days before the blood collection period; (5) Those who have used any gene or cell therapy products; (6) Those who have a history of epilepsy or other central nervous system diseases; (7) Active type B Subjects with hepatitis or hepatitis C (defined as: hepatitis B surface antigen HBsAg or hepatitis B core antibody HBcAb positive and peripheral blood HBV DNA titers above the upper limit of detection); hepatitis C disease HCV antibody positive and peripheral blood HCV RNA positive HIV, syphilis infection; (8) other tumors in the past 5 years; (9) severe chest and ascites; (10) active infection requiring systemic treatment or uncontrollable within 14 days before enrollment Infection; (11) Other anti-tumor treatments (except pretreatment chemotherapy) were performed within two weeks before the start of the study; (12) The investigator assessed that the patient was unable or unwilling to comply with the requirements of the study protocol.

(7) Treatment plan

Figure 21 shows a flow chart of the MSLN CAR-T cell clinical treatment protocol.

(8) Preprocessing scheme

Day 3 before infusion: intravenous infusion of fludarabine at a dose of 75-100 mg/ ^m2 /day;

Day 3 before infusion: Intravenous infusion of cyclophosphamide at a dose of 750 mg/ ^m2 /day.

(9) Dosing schedule

(1) Reinfusion dose: according to the existing international counterpart dose

The experimental drug pKT032 was administered by intravenous infusion of 3×10 ⁶ CAR-T cells/kg once according to the clinical situation. The above dosing regimen was taken as a course of treatment.

(10) Treatment results

Remarks: ①+ represents the date of statistics; ② Caused by pretreatment; PR, partial remission; SD, stable disease.

The solid tumor patients treated with pKT032 CAR-T cells are all relapsed and refractory. They have been treated with more than 4 kinds of treatments before, but the disease cannot be controlled. For example, 2A patients were enrolled after 40 times of chemotherapy, chemotherapy and targeted drug resistance. CAR-T therapy. According to reports in the literature, the median survival time of patients with relapsed and refractory ovarian cancer was 4.8 months, and the median survival time of patients with solid tumors treated with pKT032 CAR-T cells was 11.6 months (Figure 22). The median progression-free survival in solid tumors treated with pKT032 CAR-T cells was 7 months (Figure 23), compared with 2.1 months in the first phase of Penn CAR-T cell therapy (Mol Ther. 2019 Nov 6;27(11):1919-1929) data have obvious benefits.

Example 11: Structural Design of a Chimeric Antigen Receptor Targeting GD2

This example designed the following two GD2-targeting chimeric antigen receptors (GD2 CARs) (Figure 24):

(1) GD2-KIRS2/Dap12-BB chimeric antigen receptor

As shown in Figure 24(A), the GD2-KIRS2/Dap12-BB chimeric antigen receptor comprises a first fusion peptide GD2-KIRS2 and a second fusion peptide Dap12-BB, wherein:

The first fusion peptide GD2-KIRS2 comprises an antigen binding domain and a transmembrane domain, the antigen binding domain is a GD2 scFv, and the transmembrane domain is a KIRS2 transmembrane domain;

Described GD2-KIRS2/Dap12-BB chimeric antigen receptor is formed by pKT081 fusion protein after T2A peptide cleavage, described pKT081 fusion protein comprises DAP12 signal peptide, DAP12 (comprising transmembrane domain and cytoplasmic domain), 4 -1BB, T2A cleavage site, CD8α signal peptide, GD2 scFv and KIRS2, the amino acid sequence of the pKT081 fusion protein is shown in SEQ ID NO.12, and its encoding nucleic acid sequence is shown in SEQ ID NO.13.

pKT081: DAP12 signal peptide+DAP12+4-1BB+T2A+CD8α signal peptide+GD2 scFv+KIRS2

(2) pKT082 chimeric antigen receptor

As shown in Figure 24(B), the GD2-41BB-CD3δ chimeric antigen receptor comprises a fusion peptide GD2-CD8-CD8Hinge-CD8TM-41BB-CD3δ, which comprises an antigen binding domain, a transmembrane domain, a costimulatory Domain 4-1BB and signaling domain CD3δ, wherein the antigen binding domain is GD2 scFv, and the transmembrane domain is CD8TM transmembrane domain.

The GD2-41BB-CD3delta chimeric antigen receptor is formed from a pKT082 fusion protein comprising CD8 (including a transmembrane domain and a cytoplasmic domain), 4-1BB, CD3delta and GD2, the pKT082 fusion The amino acid sequence of the protein is shown in SEQ ID NO.14, and its encoding nucleic acid sequence is shown in SEQ ID NO.15.

in:

The amino acid sequence of GD2 scFv is as follows:

The amino acid sequence of pKT081 is as follows:

The nucleic acid sequence of pKT081 is as follows:

The amino acid sequence of pKT082 is as follows:

The nucleic acid sequence of pKT082 is as follows:

Example 12: Preparation of lentivirus

(1) 293T cells were passaged every other day

(2) 293T cell plating

b) Add 3ml 0.25% trypsin-2.21mM EDTA

c) Wait until the cells are detached, add 12 ml of 10% (wt) FBS (purchased from Gibico) in DMEM medium (purchased from corning) to the detached cells.

(3) Cell transfection

The cells were observed, and the cell density reached approximately 80%-90%, at which point the transfection was started.

a) 30-60 minutes before transfection, gently aspirate the culture medium.

b) Mix plasmid DNA and calcium chloride solution, take a T150 bottle as an example, 28μg pRSV.rev (purchased from Invitrogen Company), 28 μg pGAG-Pol (purchased from Invitrogen Company), 11 μg pVSVG (purchased from Invitrogen Company), 23 μg of lentiviral expression plasmids (plasmids pKT075, pKT094, pKT095, synthesized by Shanghai Sangong) were added to 1.5 ml of calcium chloride solution and mixed.

e) Change the medium after 6h. Gently shake the culture plate several times to fully suspend some calcium phosphate precipitates, aspirate the culture medium containing calcium phosphate precipitates, add 20 ml of fresh 5% (wt) FBS DMEM medium, and continue culturing.

(4) Initial collection of virus supernatant

a) The culture supernatant of 293T cells transfected the previous day was collected into a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, labeled, and temporarily stored in a refrigerator at 4°C.

(5) The virus supernatant was collected for the second time (48h/fourth day).

(6) Filter the supernatant

(7) Virus concentration

Centrifuge overnight at 12000-24000 rpm at 4°C.

(8) Virus storage

After centrifugation, the whole supernatant was poured out, fresh 5% (wt) FBS DMEM medium was added to resuspend, and the virus was divided into packaging (referred to as lentivirus pKT081, pKT082), and quickly stored in a -80°C refrigerator for later use.

Example 13: In vitro functional test of GD2 CAR-T

(1) Cell Preparation

Plasma was separated from fresh blood samples (for use after inactivation), PBMCs were separated from the blood cell suspension with lymphocyte separation medium, and the obtained PBMCs were subjected to T cell sorting with a T cell sorting kit. T cells were activated by adding Dynabeads CD3/CD28 (added by cell: Dynabeads = 1:1), using activation medium (X-VIVO15, 5% plasma, 300 IU/ml IL-2) to adjust the final cell concentration to 1 × 10 ⁶ /ml at 37°C, 5% CO ₂ . 24h later, the CAR lentivirus (virus pKT081, pKT082) prepared in Example 2 was added to transfect T cells, and the lentivirus was removed after 48h of infection. On day 4 (D4), the cells were observed in supplemental culture every 1-2 days, and the cell density was maintained at 0.8×10 ⁶ cells/mL. Activation medium was used on days 4-5 (D4-D5), and expansion medium (X-VIVO15, 300 IU/ml IL-2) was used after day 5 (D5). Two kinds of GD2-targeting CAR-T cells (CAR-T cells pKT081, pKT082) were obtained by continuous culture until the 11th day (D11).

There was no significant difference in the activation and expansion of T cells between the two different CAR structures (Fig. 25A); the two CAR-T cells were in a state of expansion during the culture, and the volume gradually increased in the first 4 days, and then gradually decreased ( Figure 25B).

(2) Detection of positive rate of CAR-T cells

Take two CAR-T cells pKT081 and pKT082 on the 8th day, add anti-mouse Fab antibody (PE) primary antibody and streptavidin (Streptavidin)-PE secondary antibody to incubate, and incubate untransduced T cells in the same way as In contrast, the positive rate of CAR-T cells was detected by flow cytometry. The positive rate of two GD2 CAR-T cells was detected, and the positive rate of pKT081 was higher than that of pKT082 on day 8

Take two CAR-T cells of pKT081 and pKT082 on the 8th day (D8), add anti-mouse Fab antibody (PE) primary antibody and streptavidin (Streptavidin)-PE secondary antibody to incubate, untransduced T cells in the same way Incubation was used as a control, and the positive rate of CAR-T cells was detected by flow cytometry. The positive rates of the two GD2 CAR-T cells were detected, and the results of the positive rates of the two CAR-T cells on the 8th day showed that the CAR positive rate of pKT081 was 66.87%, which was higher than that of pKT082, which was 47.91% (Figure 26).

(3) Detection of T cell differentiation subsets

On the 12th day of the culture of pKT081 and pKT082 two CAR-T cells, samples were added to detect antibody of CAR-T cell subsets for flow detection. Detection of CAR-T cell typing, including the proportion of Tn, Teff, Tcm, Tem, and Teff in CD4+ and CD8+ T cells. The two T cell differentiation subtypes of pKT081 and pKT082 showed that the proportion of memory cells in pKT081 was 86.74% more, which was significantly higher than that of pKT082 (25.98%) (Fig. 27). The present disclosure can achieve a lower differentiated state of CAR-T, which will be more durable and have memory activity in vivo.

(4) target cell culture

LAN-1 culture uses DMEM medium (DMEM+10%FBS+1% Penicillin/Streptomycine); H1299 culture uses 1640 medium (1640+10%FBS+1% Penicillin/Streptomycine) .

(5) CAR-T specific killing

a. The target cells were digested and counted, and the target cell suspension was adjusted to a density of 2×10 ⁵ cells/ml with culture medium (DMEM or 1640+10% FBS);

f. Suspend the data collection of all detection slots;

The killing effects of two GD2 CAR-T cells, pKT081 and pKT082, and non-transduced T cells (NTD) on tumor cells H1299 and LAN-1 were detected, and the killing abilities of different CAR-T cells were compared (Figure 28). The results showed that the killing effect of NTD on H1299 and LAN-1 cells was very weak; in the case of effector-target ratio (E:T) of 0:1, 1:1, 5:1, 10:1, the two CAR-T Cells showed obvious killing effect on H1299 and LAN-1 at two effector-target ratios of 5:1 and 10:1. The lysis rate of pKT081 cells to target cells was higher than that of pKT082 cells.

(6) Detection of cytokine secretion

According to E:T=2:1, pKT081 and pKT082 were co-cultured with H1299 and LAN-1 for 24 hours, respectively; the cell supernatant was collected, and IL-2 and IFN-γ were detected by ELISA.

Analysis of 2 kinds of CAR-T cells and NTD co-cultured with H1299 and LAN-1 cells after 24 hours, the supernatant was taken, and cytokines were detected by ELISA. Both CAR-T and positively selected target cells secreted IFN- γ and IL2, the secretion of IFN-γ and IL2 of pKT081 CAR-T cells was slightly higher than that of pKT082 CAR-T group. Taken together, both CAR-T cells secreted IFN-γ and IL-2 upon stimulation by tumor antigens (Figure 29).

(7) Antigen stimulates proliferation

b. On day 10 (D10), count effector cells (CAR-T) and NTDs;

d. Add the fluorescent dye CFSE (purchased from BD Life Sciences, the original concentration is 5mM) to make the final concentration of CFSE 5μM, put it into the incubator and incubate for 10min;

e. Add 9 times the incubation volume of PBS and centrifuge;

h. Count the target cells (H1299 and LAN-1), and adjust the cell suspension to a density of 1×10 ⁶ /ml with culture medium (DMEM or 1640+10% FBS+300UI/ml IL-2), for use;

j. On the fifth day (D5), flow detection was performed.

The CFSE fluorescence intensities of the two CAR-Ts (pKT081, pKT082) were detected by flow cytometry (Fig. 30), compared with the NTD control group, after stimulation of GD2-positive target cells, the fluorescence intensities of the CFSEs of the two CAR-Ts shifted to the left Attenuated, it was confirmed that the positive target cells were able to stimulate the proliferation of these two CAR-Ts.

Claims

A chimeric antigen receptor comprising a first fusion peptide and a second fusion peptide, wherein:

the first fusion peptide comprises an antigen binding domain and a transmembrane domain;

The second fusion peptide comprises a transmembrane domain, a cytoplasmic domain and a costimulatory domain.
The chimeric antigen receptor of claim 1, wherein the transmembrane domain of the second fusion peptide interacts with the transmembrane domain of the first fusion peptide through charge interaction, or the second fusion peptide interacts with the transmembrane domain of the first fusion peptide through a charge interaction. Phosphorylated ITAM sequences within the cytoplasmic domain interact with signaling molecules;

Preferably, the transmembrane domain of the first fusion peptide is a KIR transmembrane domain; preferably, the KIR transmembrane domain is selected from KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3 , KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1; more preferably, the KIR transmembrane domain is KIRS2 or KIR2DS2; preferably, the KIRS2 comprises the amino acid sequence shown in SEQ ID NO.8 Amino acid sequences with 80% or more identity, preferably amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or more identity more preferably, the amino acid sequence of the KIRS2 is as shown in SEQ ID NO.8; preferably, the KIR2DS2 comprises an amino acid with 80% or more identity with the amino acid sequence shown in SEQ ID NO.9 sequence, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more identity; more preferably, The amino acid sequence of the KIR2DS2 is shown in SEQ ID NO.9;

Preferably, the transmembrane domain of the second fusion peptide is a DAP12 transmembrane domain; preferably, the transmembrane domain of DAP12 comprises 80% or more identity with the amino acid sequence shown in SEQ ID NO.14 amino acid sequence, preferably amino acid sequence with more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more preferably amino acid sequence with 98% or more identity; more preferably Ground, the amino acid sequence of the transmembrane domain of the DAP12 is shown in SEQ ID NO.14;

Preferably, the first fusion peptide comprises a signal peptide, and the signal peptide is a CD8α signal peptide; preferably, the CD8α signal peptide comprises an amino acid having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 4 sequence, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more identity; more preferably, The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.4;

Preferably, the second fusion peptide comprises a signal peptide, and the signal peptide is a DAP12 signal peptide or a CD8α signal peptide; preferably, the DAP12 signal peptide comprises 80% or more of the amino acid sequence shown in SEQ ID NO.1 An amino acid sequence of identity, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more identity; More preferably, the amino acid sequence of the DAP12 signal peptide is shown in SEQ ID NO.1; preferably, the CD8α signal peptide comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.4 , preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more identity; more preferably, the The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.4.
The chimeric antigen receptor of claim 1 or 2, wherein the costimulatory domain is selected from the group consisting of 4-1BB, CD28, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3;

Preferably, the costimulatory domain is 4-1BB; preferably, the 4-1BB comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.5, preferably 85%, 90% %, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of 4-1BB As shown in SEQ ID NO.5.
The chimeric antigen receptor of any one of claims 1-3, wherein the cytoplasmic domain is selected from DAP12 and KIR;

Preferably, the cytoplasmic domain is the cytoplasmic domain of DAP12; preferably, the cytoplasmic domain of DAP12 comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO.15, Preferably, amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity are preferred, and amino acid sequences with 98% or more identity are more preferred; more preferably, the The amino acid sequence of the cytoplasmic domain of DAP12 is shown in SEQ ID NO.15.
The chimeric antigen receptor of any one of claims 1-4, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof, preferably, the antigen-binding domain comprises heavy chain CDR1, CDR2 of an antibody and CDR3 and CDR1, CDR2 and CDR3 of the light chain; preferably, the antigen binding domain comprises the heavy chain variable region and light chain variable region of an antibody; preferably, the antigen binding domain comprises Fab, Fab' , F(ab') 2 , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd'fragments;

Preferably, the antigen is a tumor-associated antigen;

Preferably, the tumor-associated antigen is selected from the group consisting of: CD19, mesothelin, GD-2, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CEA, CTLA4, BCMA, CS1, c-Met, EPCAM, EGFR /EGFRvIII, gp100, GPC3, IGF1R, IGF-I receptor, MAGE A3, B7-H3, MUC1, NY-ESO-1, HER2, PD1, PSMA, ROR1, WT1, glycolipid F77 or any other tumor antigen or other modification type and any combination thereof;

More preferably, the tumor-associated antigen is selected from: CD19, mesothelin and GD-2, and the antigen binding domain is selected from CD19 antibody or its antigen-binding fragment, mesothelin antibody or its antigen-binding fragment and GD2 antibody or an antigen-binding fragment thereof;

Preferably, the CD19 antibody or antigen-binding fragment thereof comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 7, preferably 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of the CD19 scFv is as shown in SEQ ID NO.7;

Preferably, the mesothelin antibody or antigen-binding fragment thereof comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.19 or SEQ ID NO.20, preferably 85%, 90%, An amino acid sequence of 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence of 98% or more identity; more preferably, the amino acid sequence of the SS1 scFv is as shown in SEQ ID NO.19 or SEQ ID NO.20;

Preferably, the GD2 antibody or antigen-binding fragment thereof comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 25, preferably 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of the GD2 scFv is shown in SEQ ID NO.25.
The chimeric antigen receptor of any one of claims 1-5, wherein the first fusion peptide comprises CD19 scFv and KIRS2, or comprises CD19 scFv and KIR2DS2;

Preferably, the CD19 scFv comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 7, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% The amino acid sequence with more than % identity, more preferably the amino acid sequence with more than 98% or 99% identity; more preferably, the amino acid sequence of the CD19 scFv is shown in SEQ ID NO.7;

Preferably, the amino acid sequence of the KIRS2 is shown in SEQ ID NO.8; preferably, the amino acid sequence of the KIR2DS2 is shown in SEQ ID NO.9;

Preferably, the second fusion peptide comprises DAP12 transmembrane domain, DAP12 cytoplasmic domain and costimulatory domain 4-1BB, or comprises a truncated DAP12 transmembrane domain, DAP12 cytoplasmic domain and costimulatory structure Domain 4-1BB; preferably, the DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.2, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the DAP12 transmembrane domain and DAP12 The amino acid sequence of the cytoplasmic domain is shown in SEQ ID NO.2; preferably, the truncated DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise 80% or more of the amino acid sequence shown in SEQ ID NO.3. Amino acid sequences with the above identity, preferably amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more identity More preferably, the amino acid sequence of the truncated DAP12 transmembrane domain and DAP12 cytoplasmic domain is as shown in SEQ ID NO.3;

Preferably, the chimeric antigen receptor is a CD19-KIRS2/Dap12-BB chimeric antigen receptor, and the CD19-KIRS2/Dap12-BB chimeric antigen receptor is formed by cleaving a fusion protein through a T2A peptide, and the The fusion protein comprises DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4-1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIRS2; preferably, the fusion protein comprises and SEQ ID The amino acid sequence shown in NO.10 has an amino acid sequence of 80% or more identity, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably has An amino acid sequence of 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.10;

Preferably, the chimeric antigen receptor is a CD19-KIRS2/tDap12-BB chimeric antigen receptor, and the CD19-KIRS2/tDap12-BB chimeric antigen receptor is formed by cleaving a fusion protein through a T2A peptide, and the Fusion protein consists of CD8α signal peptide, truncated DAP12 (tDap-12, including truncated transmembrane and cytoplasmic domains), 4-1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIRS2; preferably Preferably, the fusion protein comprises an amino acid sequence with 80% or more identity with the amino acid sequence shown in SEQ ID NO. 11, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% The amino acid sequence of the above identity, more preferably the amino acid sequence with 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.11;

Preferably, the chimeric antigen receptor is a CD19-KIR2DS2/Dap12-BB chimeric antigen receptor, and the CD19-KIR2DS2/Dap12-BB chimeric antigen receptor is formed by cleaving a fusion protein through a T2A peptide, and the The fusion protein is composed of DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4-1BB, T2A cleavage site, CD8α signal peptide, CD19 scFv and KIR2DS2; preferably, the fusion protein comprises and SEQ ID The amino acid sequence shown in NO.12 has an amino acid sequence of 80% or more identity, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably has An amino acid sequence of 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.12;

Preferably, the T2A cleavage site comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 6, preferably 85%, 90%, 95%, 96%, 97%, 98% , an amino acid sequence of more than 99% identity, more preferably an amino acid sequence of 98% or more identity; more preferably, the amino acid sequence of the T2A cleavage site is shown in SEQ ID NO.6.
The chimeric antigen receptor of any one of claims 1-5, wherein the first fusion peptide comprises SS1 scFv and KIRS2, or comprises SS1 scFv and KIR2DS2;

Preferably, the SS1 scFv comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 19 or SEQ ID NO. 20, preferably 85%, 90%, 95%, 96%, 97% %, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the amino acid sequence of the SS1 scFv is such as SEQ ID NO.19 or SEQ ID NO .20 shown;

Preferably, the amino acid sequence of the KIRS2 is shown in SEQ ID NO.8; preferably, the amino acid sequence of the KIR2DS2 is shown in SEQ ID NO.9;

Preferably, the second fusion peptide comprises DAP12 transmembrane domain, DAP12 cytoplasmic domain and costimulatory domain 4-1BB, or comprises a truncated DAP12 transmembrane domain, DAP12 cytoplasmic domain and costimulatory structure Domain 4-1BB; preferably, the DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.2, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the DAP12 transmembrane domain and DAP12 The amino acid sequence of the cytoplasmic domain is shown in SEQ ID NO.2; preferably, the truncated DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise 80% or more of the amino acid sequence shown in SEQ ID NO.3. Amino acid sequences with the above identity, preferably amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more identity ; More preferably, the amino acid sequence of the truncated DAP12 transmembrane domain and DAP12 cytoplasmic domain is as shown in SEQ ID NO.3;

Preferably, the chimeric antigen receptor is an SS1 scFv1-KIRS2/Dap12-BB chimeric antigen receptor, and the SS1 scFv1-KIRS2/Dap12-BB chimeric antigen receptor is formed by cleaving a fusion protein through a T2A peptide, The fusion protein comprises DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4-1BB, T2A cleavage site, CD8α signal peptide, SS1 scFv1 and KIRS2; preferably, the fusion protein comprises and The amino acid sequence shown in SEQ ID NO.21 has an amino acid sequence of 80% or more identity, preferably an amino acid sequence of 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more Preferably, the amino acid sequence has an identity of 98% or more than 99%; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.21;

Preferably, the chimeric antigen receptor is an SS1 scFv2-KIRS2/Dap12-BB chimeric antigen receptor, and the SS1 scFv2-KIRS2/Dap12-BB chimeric antigen receptor is formed by cleaving a fusion protein through a T2A peptide, The fusion protein comprises CD8α signal peptide, truncated DAP12 (tDap-12, including truncated transmembrane and cytoplasmic domains), 4-1BB, T2A cleavage site, CD8α signal peptide, SS1 scFv2 and KIRS2 Preferably, the fusion protein comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.23, preferably 85%, 90%, 95%, 96%, 97%, 98%, An amino acid sequence with more than 99% identity, more preferably an amino acid sequence with more than 98% or more identity; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.23;

Preferably, the T2A cleavage site comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 6, preferably 85%, 90%, 95%, 96%, 97%, 98% , an amino acid sequence of more than 99% identity, more preferably an amino acid sequence of 98% or more identity; more preferably, the amino acid sequence of the T2A cleavage site is shown in SEQ ID NO.6.
The chimeric antigen receptor of any one of claims 1-5, wherein the first fusion peptide comprises GD2 scFv and KIRS2, or comprises GD2 scFv and KIR2DS2;

Preferably, the GD2 scFv comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 25, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% The amino acid sequence with more than % identity, more preferably the amino acid sequence with more than 98% or 99% identity; more preferably, the amino acid sequence of the GD2 scFv is shown in SEQ ID NO.25;

Preferably, the amino acid sequence of the KIRS2 is shown in SEQ ID NO.8; preferably, the amino acid sequence of the KIR2DS2 is shown in SEQ ID NO.9;

Preferably, the second fusion peptide comprises DAP12 transmembrane domain, DAP12 cytoplasmic domain and costimulatory domain 4-1BB, or comprises a truncated DAP12 transmembrane domain, DAP12 cytoplasmic domain and costimulatory structure Domain 4-1BB; preferably, the DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO.2, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or more identical amino acid sequences; more preferably, the DAP12 transmembrane domain and DAP12 The amino acid sequence of the cytoplasmic domain is shown in SEQ ID NO.2; preferably, the truncated DAP12 transmembrane domain and the DAP12 cytoplasmic domain comprise 80% or more of the amino acid sequence shown in SEQ ID NO.3. Amino acid sequences with the above identity, preferably amino acid sequences with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more identity ; More preferably, the amino acid sequence of the truncated DAP12 transmembrane domain and DAP12 cytoplasmic domain is as shown in SEQ ID NO.3;

Preferably, the chimeric antigen receptor is a GD2 scFv-KIRS2/Dap12-BB chimeric antigen receptor, and the GD2 scFv-KIRS2/Dap12-BB chimeric antigen receptor is formed by cleaving a fusion protein through a T2A peptide, The fusion protein comprises DAP12 signal peptide, DAP12 (including transmembrane domain and cytoplasmic domain), 4-1BB, T2A cleavage site, CD8α signal peptide, GD2 scFv1 and KIRS2; preferably, the fusion protein comprises and The amino acid sequence shown in SEQ ID NO.26 has an amino acid sequence of 80% or more identity, preferably an amino acid sequence with more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more Preferably, the amino acid sequence has an identity of 98% or more than 99%; more preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO.26;

Preferably, the T2A cleavage site comprises an amino acid sequence with 80% or more identity to the amino acid sequence shown in SEQ ID NO. 6, preferably 85%, 90%, 95%, 96%, 97%, 98% , an amino acid sequence of more than 99% identity, more preferably an amino acid sequence of 98% or more identity; more preferably, the amino acid sequence of the T2A cleavage site is shown in SEQ ID NO.6.
Nucleic acid encoding the chimeric antigen receptor described in any one of claims 1-8; preferably, the nucleic acid comprises and SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO. 22. The nucleotide sequences shown in SEQ ID NO.24, SEQ ID NO.27, SEQ ID NO.29 have 80% or more identity, preferably 85%, 90%, 95%, 96 %, 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or more identical nucleotide sequences; preferably, the nucleic acid is selected from: SEQ ID NO.16 , SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.22, SEQ ID NO.24, the nucleic acid shown in SEQ ID NO.27; more preferably, the encoding nucleic acid is SEQ ID NO.16, The nucleic acid shown in SEQ ID NO.22 or SEQ ID NO.27.
A vector comprising the nucleic acid of claim 9.
A cell comprising the nucleic acid of claim 9 or the vector of claim 10.
A composition comprising the chimeric antigen receptor of any one of claims 1-8, the nucleic acid of claim 9, the carrier of claim 10 and/or the cell of claim 11 and A pharmaceutically acceptable carrier.
A method for preparing cells, the method comprising introducing the nucleic acid of claim 9 and the vector of claim 10 into immune effector cells.
The chimeric antigen receptor of any one of claims 1-8, the nucleic acid of claim 9, the vector of claim 10, the cell of claim 11 and/or the combination of claim 12 Use of a substance in the manufacture of a medicament for the treatment and/or prevention of a disease or disorder.
The use according to claim 14, which is the use in the preparation of a medicine for the treatment and/or prevention of a disease or disorder related to abnormal CD19 expression, activity and/or signal transduction;

Preferably, the disease or disorder includes a tumor, preferably, the tumor is a cancer disease; preferably, the cancer disease is selected from the group consisting of: brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, Lymphoma, Leukemia, Lung Cancer, Melanoma, Metastatic Melanoma, Mesothelioma, Neuroblastoma, Ovarian Cancer, Prostate Cancer, Pancreatic Cancer, Kidney Cancer, Skin Cancer, Thymoma, Sarcoma, Non-Hodgkin Lymphoma , Hodgkin lymphoma, uterine cancer, and any combination thereof.
The use according to claim 14, which is the use in the preparation of a medicine for the treatment and/or prevention of a disease or disorder associated with abnormal mesothelin expression, activity and/or signal transduction;

Preferably, the disease or disorder associated with aberrant mesothelin expression, activity and/or signaling comprises a tumor or cancer;

Preferably, the tumor is a solid tumor;

More preferably, the mesothelin expression-related disease is selected from the group consisting of mesothelioma, epithelial-like malignant pleural mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, biliary tract cancer, endometrial cancer, thymic cancer, Colon cancer, breast cancer.
The use described in claim 14, which is the use in the preparation of a medicine for the treatment and/or prevention of a disease or disorder related to abnormal GD2;

Preferably, the GD2-related disease or disorder is a GD2-positive tumor;

Preferably, the tumor is selected from neuroblastoma, glioblastoma, medulloblastoma, astrocytoma, melanoma, small cell lung cancer, desmoplastic small round cell tumor, osteosarcoma, rhabdomyoma Sarcoma, Ewing's sarcoma, or liposarcoma, fibrosarcoma, leiomyosarcoma, and other soft tissue sarcomas in adults.
A method for providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of the chimeric antigen receptor according to any one of claims 1-8, the nucleic acid according to claim 9, the The vector of claim 10, the cell of claim 11 and/or the composition of claim 12.
A method of treating a mammal suffering from a disease or disorder, comprising administering to the mammal an effective amount of a chimeric antigen receptor comprising the chimeric antigen receptor of any one of claims 1-8, the nucleic acid of claim 9, The vector of claim 10, the cell of claim 11 and/or the composition of claim 12.