CN114369168B

CN114369168B - Chimeric receptors comprising DAP12 and co-stimulatory signaling molecule signaling domains and methods of use thereof

Info

Publication number: CN114369168B
Application number: CN202011122767.3A
Authority: CN
Inventors: 王恩秀; 汪晨
Original assignee: Nanjing Cart Medical Technologies Ltd
Current assignee: Nanjing Cart Medical Technologies Ltd
Priority date: 2020-10-19
Filing date: 2020-10-19
Publication date: 2024-08-30
Anticipated expiration: 2040-10-19
Also published as: CN114369168A

Abstract

The present disclosure relates to chimeric receptors comprising DAP12 and a costimulatory signaling molecule signaling domain, and methods of use thereof. The chimeric antigen receptor comprises 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 disclosed by the invention not only improves the curative effect and sustainability, but also has better safety and good curative effect.

Description

Chimeric receptors comprising DAP12 and co-stimulatory signaling molecule signaling domains and methods of use thereof

Technical Field

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

Background

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

Chimeric Antigen Receptors (CARs) are the core component of CAR-T, which, by virtue of their ligand binding domain properties, are able to redirect their specificity and reactivity towards selected immune cells, thus conferring to T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR engineered T cells to recognize a wider range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a Tumor Associated Antigen (TAA) binding region (typically an scFv fragment derived from a monoclonal antibody antigen binding region), an extracellular hinge region, a transmembrane region and an intracellular signaling region. CAR-T therapy has shown promise in some hematologic cancer trials. Clinical experiments show that the CAR-T cell therapy has great potential in controlling advanced Acute Lymphoblastic Leukemia (ALL), lymphoma and the like.

However, some patients receiving CAR T cell therapy and other immunotherapy experience dangerous, even life threatening, side effects called Cytokine Release Syndrome (CRS), in which infused CAR-T cells produce systemic inflammatory responses in which cytokines are rapidly released in large amounts into the blood, resulting in dangerous hypotension, hyperthermia, and chills.

In severe CRS cases, patients experience a cytokine storm (also known as cytokine cascade or hypercytokinemia) in which cytokines are present and white blood cells with highly elevated cytokine levels are positively correlated. This can lead to potentially life-threatening complications including cardiac dysfunction, respiratory distress syndrome, nervous system toxicity, renal and/or liver failure, pulmonary edema and disseminated intravascular coagulation.

CN107580628a discloses targeted cytotoxic cells with chimeric receptors for adoptive immunotherapy, wherein DAP12/KIRS2 is employed as intracellular signaling domain of CAR structure. However, due to the lack of co-stimulatory signaling molecules, there are technical problems with poor sustainability, low IL-2 secretion of DAP12/KIRS2 CAR-T when co-cultured with tumor cells in experiments, and ineffective treatment in clinical treatment.

Thus, new strategies are needed to enhance clinical efficacy and control cytokine release syndromes, particularly cytokine storms, to maximize the potential of CAR-T cell therapy.

Disclosure of Invention

Aiming at the problems in the prior art, the co-stimulatory signal molecules are introduced into the DAP12/KIRS2, and different pre-clinical results (killing, cytokine secretion, proliferation, in-vivo efficacy and the like) are brought from different introduction sites of the co-stimulatory molecules, and the therapeutic effects of curing patients can be brought clinically.

Thus, in one aspect, the present disclosure provides chimeric antigen receptors.

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

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

In one aspect, the present disclosure provides a cell comprising the aforementioned vector.

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

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

In one aspect, the present disclosure provides the 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 foregoing chimeric antigen receptor, nucleic acid, vector, and/or cell.

Drawings

Figure 1 shows a DPK CAR structural design.

Figure 2 shows expansion and volume change of four CAR-T cells.

Figure 3 shows the positive rate of four CAR-ts.

Figure 4 shows the results of four CAR-T cell differentiation subtype assays.

Figure 5 shows CAR-T cell and NTD target cell lysis rates.

FIG. 6 shows the results of four CAR-T cell IL-2 and IFN-gamma assays.

Figure 7 shows CAR-T proliferation flow assay results.

Figure 8 trend of tumor change in mice of different CAR-T dosing groups.

FIG. 9 shows IL-6 and IL-10 secretion in blood of mice 14 days after CAR-T injection.

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

FIG. 11 shows the PD-1 content in T lymphocytes.

Fig. 12 shows a clinical study flow chart. In the figure, M1, M3 and M6 represent 1 month, 3 months and 6 months; * Represents a significant temporal node for assessing efficacy.

Detailed Description

I. Definition of the definition

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where integers are required, the term refers to rounding up or down to the nearest integer within plus or minus 10% of a given value or range.

"Chimeric antigen receptor" or "CAR", as that term is used herein, refers to a chimeric polypeptide that shares structural and functional properties with a cellular immune function receptor or adapter molecule from, for example, a T cell or NK cell. In embodiments, the CAR comprises an antigen binding domain that binds to a cognate antigen (e.g., a tumor antigen as described herein). Upon binding to the cognate antigen, the CAR can activate or inactivate the cytotoxic cells in which it resides, or modulate the antitumor activity of the cells or modulate the immune response of the 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. The antibody may be an intact immunoglobulin derived from natural sources or from recombinant sources, and may be an immunoreactive portion of an intact immunoglobulin. Antibodies may be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies are typically tetramers of immunoglobulin molecules. The antibody molecules described herein can exist in a variety of forms, wherein the antigen-binding portion of the antibody is expressed as part of a continuous polypeptide chain, including, for example, single domain antibody fragments (sdabs), single chain antibodies (scFv), and humanized or human antibodies, e.g., as described herein.

The phrase "substantially identical" in reference to an antibody chain polypeptide sequence is understood to mean an antibody chain that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. In terms of nucleic acid sequences, the term is understood to mean nucleotide sequences which exhibit at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

Sequence "identity" or "identity" has art-recognized meanings and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., ：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Pres,New York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987;and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991). although there are many methods of measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (carrilo, H. & Lipman, d., SIAM J APPLIED MATH: 1073 (1988)).

"Substitution type" variants are those in which at least one amino acid residue in the natural sequence has been removed and a different amino acid has been inserted at the same position. The substitution may be single, wherein only one amino acid in the molecule is substituted; or may be plural, in which the same molecule has two or more amino acids substituted. Multiple substitutions may be located at successive positions. Also, an amino acid may be substituted with multiple residues, where such variants include both substitutions and insertions. An "insertion" variant is a variant in which one or more amino acids are inserted at a particular position immediately adjacent to a native sequence. By immediately adjacent amino acid is meant a linkage to the alpha-carboxyl or alpha-amino functionality of the amino acid. A "deleted" variant is a variant in which one or more amino acids in the natural amino acid sequence have been removed. Typically, a deletion variant has one or two amino acids deleted in a particular region of its molecule.

With respect to the variable domains of antibodies, the term "variable" refers to portions of the relevant molecules that have wide sequence differences between antibodies and are used for specific recognition and binding of a particular antibody to its specific target. But the variability is not evenly distributed throughout the variable domains of the antibodies. Variability is focused on three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2 and CDR 3) or hypervariable regions, all located within the variable domains of the 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 the natural heavy and light chains comprises four FR regions, which adopt predominantly a β -sheet configuration, joined by three CDRs, which form a loop that connects the β -sheet structure and in some cases forms part of the β -sheet structure. The CDRs of each chain are typically joined in proximity by FR regions and aid in the formation of antibody target binding sites (epitopes or determinants) by means of CDRs from other chains (see Kabat et al Sequences of Proteins of Immunological Interest, nationalInstitute of Health, bethesda, MD (1987)). As used herein, numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al, unless otherwise indicated. One CDR may have the ability to specifically bind to a 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 that comprises at least a portion of the variable region (e.g., one or more CDRs and/or one or more antibody binding sites) of the antibody that binds an antigen, and thus retains binding specificity as well as at least a portion of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that comprises an antigen-binding portion that binds the same antigen as an antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, fab ', F (ab ') ₂, single chain Fv (scFv), fv, dsFv, diabodies, fd, and Fd ' fragments, and other fragments, including modified fragments (see, e.g., ,Methods in Molecular Biology,Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003);Chapter 1;p 3-25,Kipriyanov)., which fragments may include multiple chains linked together, e.g., 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 an antibody framework (e.g., by replacement of the corresponding region), obtains antibodies that immunospecifically bind (i.e., exhibit a Ka of at least or at least about 10 ⁷-10⁸ 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, a functional fragment is generally synonymous with an "antibody fragment" and, in the case of an antibody, may refer to a fragment, such as Fv, fab, F (ab') ₂, and the like, that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. The "Fv" fragment consists of a dimer (V _H-V_L dimer) of one heavy chain variable domain and one light chain variable domain formed by non-covalent binding. In this configuration, the three CDRs of each variable domain interact to determine the target binding site on the V _H-V_L dimer surface, as is the case with intact antibodies. The six CDRs together confer target binding specificity to the intact antibody. but even a single variable domain (or half of an Fv comprising only 3 target-specific CDRs) can have the ability to recognize and bind a target.

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 the other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies comprising antibodies having a plurality 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, e.g., by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to produce antibodies from clonal populations of host cells in vitro by transforming the host cells with plasmids carrying artificial sequences of nucleotides encoding the antibodies.

The term full length antibody is an antibody having two full length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH 4) and two full length light chains (VL-CL) and a hinge region, e.g., an antibody naturally produced by B cells by antibody secretion and an antibody synthetically produced with 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 an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

By "humanized" antibody is meant a form of non-human (e.g., mouse) antibody that is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (e.g., fv, fab, fab ', F (ab') ₂ or other antigen-binding subsequence of the antibody) that contains minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the Complementarity Determining Regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.

Furthermore, 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 (e.g., affinity) of the antibody. Mutations, such as PCR-mediated mutations, can be introduced, and their effect on antibody binding or other functional properties can be assessed using in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations may be amino acid substitutions, additions or deletions. In addition, mutations within the CDRs typically do not exceed one or two. Thus, the humanized antibodies of the present invention also encompass antibodies comprising 1 or 2 amino acid mutations within the CDRs.

The term "CDR" refers to the complementarity determining region (complementarity-DETERMINING REGION), known as an antibody molecule, having 3 CDRs per heavy and light chain. CDRs are also known as hypervariable regions and are found in the variable regions of each of the heavy and light chains of antibodies with very high variability sites in the primary structure of the CDRs. In the present specification, CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino terminal sequence of the heavy chain, and CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino terminal sequence of the light chain. These sites are adjacent to each other in 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. Epitope determinants generally comprise chemically active surface groupings of molecules such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics.

The terms "specific binding" or "immunospecifically binding" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with an alloantigen through non-covalent interactions between the antibody and the antibody binding site of the antigen. The antigen may be an isolated antigen or present in a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) an antigen binds the antigen with an affinity constant Ka (or dissociation constant (Kd) of 1x10 ^-7 M or 1x10 ^-8 M or less) of about or 1x10 ⁷M^-1 or 1x10 ⁸M^-1 or more. Affinity constants can be determined by standard kinetic methods of antibody reaction, e.g., immunoassays, surface Plasmon Resonance (SPR) (Rich and Myszka (2000) curr. Opin. Biotechnol 11:54; englebiene (1998) analysis 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 U.S. Pat. No. 7,229,619) describing exemplary SPR and ITC methods for calculating binding affinity of antibodies. Instruments and methods for detecting and monitoring binding rates in real time 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 an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) that are typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules as well as RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, may 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" with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter may 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 herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of antibodies encoded by or containing the nucleotide sequence to an antigen. 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 an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Methods for identifying nucleotide and amino acid conservative substitutions that do not eliminate antigen binding are well known in the art (see, e.g.) 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 of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods of determining 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 after gel electrophoresis, lowry protein assay, and Bradford protein assay.

The term "host cell" is a cell that is used to receive, hold, replicate, and amplify a vector. Host cells may also be used to express the polypeptides encoded by the vectors. When the host cell is divided, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell may be a eukaryotic cell or a prokaryotic cell. 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 may be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those comprising nucleic acids encoding polypeptides. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell, for amplifying the nucleic acid or for expressing/displaying the polypeptide encoded by the nucleic acid. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. Vectors for artificial chromosomes are also contemplated, 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, vectors also include "viral vectors" or "viral vectors". The vector of the virus is an engineered virus operably linked to a foreign gene to transfer (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 may include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that when introduced into an appropriate host cell results in expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of a host cell.

The term "stimulation" refers to the binding of a stimulatory molecule (e.g., a TCR/CD3 complex) to its cognate ligand, thereby mediating a primary response to a signaling event, such as, but not limited to, signaling via the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as down-regulation of TGF- β, and/or recombination 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 activation of the TCR complex for at least some aspects of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for example, binding of the TCR/CD3 complex to an MHC molecule loaded with a peptide, and which results in mediating T cell responses including, but not limited to, proliferation, activation, differentiation, and the like. The primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of first order cytoplasmic signaling sequences containing ITAM which are particularly useful in the present invention include, but are not limited to, those derived from TCR ζ, fcRγ, fcRβ, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), fcεRI, CD66d, DAP10, and DAP12. In the specific CARs of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence.

The term "antigen presenting cell" or "APC" refers to an immune system cell, such as a helper cell (e.g., B-cell, dendritic cell, etc.), that presents foreign antigens complexed with a Major Histocompatibility Complex (MHC) on its surface. T-cells can recognize these complexes using their T-cell receptor (TCR). APCs process antigens and present them to T-cells.

The term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes immune effector function of a CAR-containing cell (e.g., CAR-T cell or CAR-expressing NK cell). Examples of immune effector functions (e.g., in CAR-T cells or CAR-expressing NK cells) include cytolytic activity and helper activity, including secretion of cytokines. In embodiments, the intracellular signaling domain transduces effector function signals and directs the cell to perform a specialized function. Although the entire intracellular signaling domain may be used, in many cases the entire strand need not be used. In the case of using a truncated portion of an intracellular signaling domain, such truncated portion may be used in place of the complete strand, so long as it transduces an effector function signal. The term intracellular signaling domain is thus intended to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

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 co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory 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 a cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

The primary intracellular signaling domain may include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of first order cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from CD3 ζ, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), fc εRI, 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 activating molecules (SLAM proteins), activating NK cell receptors, BTLA, toll ligand receptors ,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 CD 83.

Costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain may include all intracellular portions of the molecule or all native intracellular signaling domains, or functional fragments thereof.

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

The term "immune effector cell" refers to a cell that is involved in an immune response, e.g., that promotes an immune effector response. Examples of immune effector cells include T cells, e.g., α/β T cells and γ/δ T cells, B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

The term "immune effector function or immune effector response" refers to an immune effector cell, e.g., a function or response that enhances or promotes immune attack by a target cell. For example, immune effector function or response refers to the property 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 co-stimulation are examples of immune effector functions or responses.

In some embodiments, the cell population (e.g., harvested cell population) comprises, for example, T cells or T cell populations at different differentiation stages. T cell differentiation stages range from least to most differentiated including naive T cells, stem central memory T cells, effector memory T cells and terminal effector T cells. After 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, upon receiving appropriate T cell receptors, co-stimulation and inflammatory signals, differentiate further into terminal effector T cells.

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 following expression patterns of cell surface markers: CCR7+, CD62L+, CD45RO-, CD95+. Central memory T Cells (TCM) are characterized by the following expression patterns of cell surface markers: CCR7+; CD62L+: CD45RO + cd95+. Effector memory T cells (TEM) are characterized by the following expression patterns of cell surface markers: CCR7-, CD62L-, CD45ro+, cd95+. Terminal effector T cells (TEff) are characterized by the following expression patterns 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+, referred to as Tn), 2) central memory T cells (CD 45RA-CD62L+, referred to as Tcm), 3) effector memory T cells (CD 45RA-CD62L-, referred to as Tem) and 4) CD45RA positive effector T cells (CD45RA+CD62L-, abbreviated Temra). The expression of each subgroup CCR7, CD27, CD28 and CD95 was then further assessed. CD95 expression was significantly up-regulated 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-expressed CD27, CD28 and CCR7, and was considered memory stem cells (Tscm).

The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may 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 version of Remington's Pharmaceutical Sciences, a standard reference in the art, 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 carriers, 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. Except insofar as any conventional medium or agent is incompatible with the chimeric antigen receptor, its use in the composition is contemplated.

The term "treating" an individual with a disease or condition means that the symptoms of the individual are partially or fully alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or disease progression. Treatment also includes any chimeric antigen receptor provided and any pharmaceutical use of the compositions provided herein.

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

The term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition of matter comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block or partially block the symptoms of a 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, e.g., prevent or delay the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom. The fully prophylactically effective dose need not occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

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

Detailed description of the preferred embodiments

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.

According to the chimeric antigen receptor of the previous aspect, the transmembrane domain of the second fusion peptide interacts with the transmembrane domain of the first fusion peptide by charge interactions, or the second fusion peptide interacts with the signal molecule by phosphorylated ITAM sequences within the cytoplasmic domain.

In some embodiments, 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, KIRS2 comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO 8, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of KIRS2 is shown as SEQ ID NO 8; preferably, the KIR2DS2 comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO 9, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of KIR2DS2 is shown in SEQ ID NO 9.

In some embodiments, the transmembrane domain of the second fusion peptide is the DAP12 transmembrane domain; preferably, the transmembrane domain of DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence depicted in SEQ ID NO 14, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably an amino acid sequence that is 98% or more identical; more preferably, the amino acid sequence of the transmembrane domain of DAP12 is set forth in SEQ ID NO 14.

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, B, 7-H3.

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

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

In some embodiments, the cytoplasmic domain is the cytoplasmic domain of DAP 12; preferably, the cytoplasmic domain of DAP12 comprises an amino acid sequence that is 80% or more identical to the amino acid sequence depicted in SEQ ID NO 15, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably an amino acid sequence that is 98% or more identical; more preferably, the cytoplasmic domain of DAP12 has the amino acid sequence depicted in SEQ ID NO 15.

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

In some embodiments, the tumor-associated antigen is selected from ：CD19、CD20、CD22、CD30、CD33、CD38、CD123、CD138、CEA、CTLA4、BCMA、CS1、c-Met、EPCAM、EGFR/EGFRvIII、gp100、GPC3、GD-2、IGF1R、IGF-I receptor、MAGE A3、mesothelin、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.

In some embodiments, the first fusion peptide comprises a CD19 scFv and KIRS2, or comprises a CD19 scFv and KIR2DS2; preferably, the amino acid sequence of KIRS is shown in SEQ ID NO 8; preferably, the amino acid sequence of 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 a costimulatory domain, 4-1BB; preferably, the DAP12 transmembrane domain and DAP12 cytoplasmic domain comprise an amino acid sequence that is 80% or more identical to the amino acid sequence depicted in SEQ ID NO 2, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably an amino acid sequence that is 98% or 99% or more identical; more preferably, the amino acid sequences of the DAP12 transmembrane domain and the DAP12 cytoplasmic domain are shown in SEQ ID NO 2; preferably, the truncated DAP12 transmembrane domain and DAP12 cytoplasmic domain comprise an amino acid sequence that is 80% or more identical to the amino acid sequence depicted in SEQ ID NO 3, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably an amino acid sequence that is 98% or more identical; more preferably, the amino acid sequences of the truncated DAP12 transmembrane domain and DAP12 cytoplasmic domain are shown in SEQ ID NO 3.

In some embodiments, the first fusion peptide comprises a signal peptide that is a CD8 a signal peptide; preferably, the CD8 a signal peptide comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO 4, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of the CD8 a signal peptide is shown in SEQ ID NO 4.

Preferably, the second fusion peptide comprises a signal peptide which is a DAP12 signal peptide or a CD 8a signal peptide; preferably, the DAP12 signal peptide comprises an amino acid sequence that is 80% or more identical to the amino acid sequence depicted in SEQ ID NO 1, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably an amino acid sequence that is 98% or more identical; more preferably, the amino acid sequence of the DAP12 signal peptide is shown in SEQ ID NO 1; preferably, the CD 8a signal peptide comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO 4, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of the CD 8a signal peptide is shown in SEQ ID NO 4.

In some embodiments, the chimeric antigen receptor is a CD19-KIRS2/Dap12-BB chimeric antigen receptor, which CD19-KIRS2/Dap12-BB chimeric antigen receptor is formed from a DPK01 protein upon cleavage of a T2A peptide, which DPK01 fusion protein consists of a Dap12 signal peptide, dap12 (including transmembrane and cytoplasmic domains), 4-1BB, a T2A cleavage site, a CD8 a signal peptide, CD19 scFv, and KIRS2, preferably the DPK01 fusion protein comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity to the amino acid sequence set forth in SEQ ID NO 10; more preferably, the amino acid sequence of the DPK01 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, which CD19-KIRS 2/tDap-BB chimeric antigen receptor is formed from a DPK02 protein upon cleavage of a T2A peptide, which DPK02 fusion protein consists of a CD8 a signal peptide, truncated DAP12 (tDap-12, including a transmembrane domain and cytoplasmic domain), 4-1BB, a T2A cleavage site, a CD8 a signal peptide, a CD19 scFv, and KIRS2, preferably the DPK02 fusion protein comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity to the amino acid sequence set forth in SEQ ID NO 11; more preferably, the amino acid sequence of the DPK02 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, which is formed from a DPK03 protein after cleavage of a T2A peptide, which DPK03 fusion protein consists of a Dap12 signal peptide, dap12 (including a transmembrane domain and a cytoplasmic domain), 4-1BB, a T2A cleavage site, a CD 8a signal peptide, a CD19 scFv, and KIR2DS2, preferably, the DPK03 fusion protein comprises an amino acid sequence having 80% or more identity, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity to the amino acid sequence set forth in SEQ ID NO 12; more preferably, the amino acid sequence of the DPK03 fusion protein is shown in SEQ ID NO 12.

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

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

In one aspect, the present disclosure provides nucleic acids encoding the foregoing chimeric antigen receptors; preferably, the nucleic acid comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO 16, SEQ ID NO 17 or SEQ ID NO 18, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; preferably, the nucleic acid is selected from: nucleic acids shown in SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18; more preferably, the coding nucleic acid is the nucleic acid shown in SEQ ID NO 16.

In one aspect, the present disclosure provides a cell comprising the foregoing nucleic acid or vector.

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 of preparing a cell, the method comprising introducing the aforementioned nucleic acid, vector, into an immune effector cell.

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

In some embodiments, the disease or disorder comprises a tumor, preferably, the tumor is a cancer disease; preferably, the cancer disease is selected from: 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, renal cancer, skin cancer, thymoma, sarcoma, non-hodgkin's lymphoma, or uterine cancer.

The pharmaceutical compositions of the embodiments are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline, 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 ethylenediamine tetraacetic acid (EDTA); buffers, such as acetates, citrates or phosphates, and agents for regulating the osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may 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 ^TM (BASF, parsippany, N.J.), or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and must be able to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required 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 preferable to include isotonic agents, for example, sugars, polyalcohols (such as mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition agents delaying absorption, for example, aluminum monostearate and gelatin.

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

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

Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated 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 may be formulated as a cream, ointment, gel, or ointment as is commonly known in the art.

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

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

It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a dosage unit form refers to a physically separable unit suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of one or more of the chimeric antigen receptors calculated to produce the desired therapeutic effect in combination with the desired drug carrier. The specifications for the dosage unit forms of the embodiments are indicated below and directly depend on: unique characteristics of chimeric antigen receptors and the particular therapeutic effect to be achieved, and limitations inherent in the field of formulation of such chimeric antigen receptors for treating individuals.

The pharmaceutical composition may be placed in a container, package, or dispenser together with instructions for administration.

The formulations described herein may also comprise more than one of the chimeric antigen receptors, preferably those having complementary activity without adversely affecting each other, depending on the particular situation to be treated. Alternatively or in addition, 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 present in combination in an amount effective for the intended purpose. For example, they may be present in combination in a kit, or may be present in combination in use.

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

In one embodiment, the cytokine release syndrome is graded. In another embodiment, class 1 describes a cytokine release syndrome wherein the symptoms are not life threatening and only require symptomatic treatment, e.g., fever, nausea, weakness, headache, myalgia, discomfort. In another embodiment, the symptoms of grade 2 require and respond to a moderate intervention, such as oxygen supply, fluid or vasopressors for hypotension. In another embodiment, the symptoms of level 3 require and respond to positive intervention. In another embodiment, the grade 4 symptom is a life threatening symptom, 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, i.e., in combination with other agents such as therapeutic agents (which may be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders, and inflammatory diseases). The term "in combination" herein means that the agents are administered substantially simultaneously, simultaneously or sequentially. If administered sequentially, the first of the two compounds is still preferably detected at the treatment site at an effective concentration at the time of initiation of administration of the second compound. In one instance, "in combination" may also be in a kit comprising both the chimeric antigen receptor of the present disclosure and the other therapeutic agent.

For example, the combination therapy may comprise the co-formulation and/or co-administration of one or more chimeric antigen receptors described herein with one or more additional therapeutic agents (e.g., one or more cytokines and growth factor inhibitors, immunosuppressives, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxins or cytostatics, as described in more detail below). Such combination therapy may advantageously utilize lower doses of the therapeutic agent administered, thus avoiding the potential toxicity or complications associated with various monotherapy regimens.

The chimeric antigen receptor with better anti-tumor effect is designed by adding the co-stimulatory signal molecule element through DAP12 serial connection 4-1BB, and the anti-tumor beneficial effect has the following four aspects: ① Better in vivo safety (reduced IL-6 secretion); ② Lower secretion of immunosuppressive factors in vivo (reduced secretion of IL-10); ③ Has good clinical safety and good curative effect (low-grade CRS, persistent remission).

Experimental results show that the chimeric antigen receptor not only improves the curative effect and sustainability, but also has significantly reduced in vivo IL-6 secretion from safety, IL-6 is the most critical cytokine for clinical side effects (cytokine release syndrome), and IL-10 secretion is reduced, which indicates that the immunosuppression is reduced, IL-10 is an immunosuppression factor, and plays an immunosuppressive role in anti-tumor treatment, and 4 patients are low-grade CRS in clinical study except for in vivo cytokine results, which indicates that the clinical safety is good.

For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the invention may include some embodiments with a combination of all or some of the features described.

Example 1: DPK CAR structure design

This example designed four chimeric antigen receptors (fig. 1):

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

As shown in FIG. 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-KIRS comprises an antigen-binding domain that is a CD19 scFv and a transmembrane domain that 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 cutting a DPK01 protein by a T2A peptide, the DPK01 fusion protein consists of a DAP12 signal peptide, DAP12 (comprising a transmembrane domain and a cytoplasmic domain), 4-1BB, a T2A cutting site, a CD8 alpha signal peptide, CD19 scFv and KIRS, and the amino acid sequence of the DPK01 fusion protein is shown as SEQ ID NO 10.

DPK01: DAP12 Signal peptide+DAP12+4-1BB+T2A+CD8 alpha Signal peptide+CD 19 scFv+ KIRS2

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

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

the second fusion peptide tDap-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 cutting a DPK02 protein by a T2A peptide, the DPK02 fusion protein consists of a CD8 alpha signal peptide, truncated DAP12 (tDap comprising a transmembrane domain and a cytoplasmic domain), 4-1BB, a T2A cutting site, the CD8 alpha signal peptide, CD19 scFv and KIRS2, and the amino acid sequence of the DPK02 fusion protein is shown as SEQ ID NO 11.

DPK02: CD8α signal peptide +383012+4-1BB+T2A+CD8 alpha Signal peptide+CD 19 scFv+ KIRS2

(3) CD19-KIR2DS2/Dap12-BB chimeric antigen receptor

As shown in FIG. 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, wherein the antigen binding domain is CD19 scFv, and the transmembrane domain is KIR2DS2 transmembrane domain;

The CD19-KIR2DS2/Dap12-BB chimeric antigen receptor is formed by cutting a DPK03 protein by a T2A peptide, the DPK03 fusion protein consists of a DAP12 signal peptide, DAP12 (comprising a transmembrane domain and a cytoplasmic domain), 4-1BB, a T2A cutting site, a CD8 alpha signal peptide, CD19 scFv and KIR2DS2, and the amino acid sequence of the DPK03 fusion protein is shown as SEQ ID NO 12.

DPK03: DAP12 Signal peptide+DAP12+4-1BB+T2A+CD8 alpha Signal peptide+CD 19 scFv+KIR2DS2

(4) CD19-KIRS2/Dap12 chimeric antigen receptor

As shown in FIG. 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 cutting a pKT011 protein through a T2A peptide, the pKT011 fusion protein consists of a DAP12 signal peptide, DAP12 (comprising a transmembrane domain and a cytoplasmic domain), a T2A cutting site, a CD8 alpha signal peptide, CD19 scFv and KIRS2, and the amino acid sequence of the pKT011 fusion protein is shown as SEQ ID NO 13.

PKT011: DAP12 Signal peptide +: DAP12+T2A+CD8 alpha Signal peptide +: CD19scFv+ KIRS2

Wherein:

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

MGGLEPCSRFLLLPLLLAVSG(SEQ ID NO 1)

The amino acid sequence of DAP12 comprises a DAP12 transmembrane domain and a DAP12 cytoplasmic domain, and is specifically as follows:

LRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK(SEQ ID NO 2)

The truncated amino acid sequence of DAP12 comprises a truncated DAP12 transmembrane domain and DAP12 cytoplasmic domain, and is specifically the following:

CSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK(SEQ ID NO 3)

the amino acid sequence of the CD8 a signal peptide is as follows:

MALPVTALLLPLALLLHAARP(SEQ ID NO 4)

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

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO 5)

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

EGRGSLLTCGDVEENPG(SEQ ID NO 6)

the amino acid sequence of CD19 scFv is as follows:

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO 7)

KIRS2 has the following amino acid sequence:

SKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA(SEQ ID NO 8)

the amino acid sequence of KIR2DS2 is as follows:

HEGVHRKPSLLAHPGPLVKSEETVILQCWSDVRFEHFLLHREGKYKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRFSAGPKVNGTFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA(SEQ ID NO 9)

the amino acid sequence of DPK01 is as follows:

MGGLEPCSRFLLLPLLLAVSGLRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKDIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRVEGGGEGRGSLLTCGDVEENPGPRMALPVTALLLPLALLLHAARPGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSASGGGGSGGGGSSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA(SEQ ID NO 10)

the amino acid sequence of DPK02 is as follows:

MALPVTALLLPLALLLHAARPCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKDIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRVEGGGEGRGSLLTCGDVEENPGPRMALPVTALLLPLALLLHAARPGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSASGGGGSGGGGSSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA(SEQ ID NO 11)

the amino acid sequence of DPK03 is as follows:

MGGLEPCSRFLLLPLLLAVSGLRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKDIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSRVEGGGEGRGSLLTCGDVEENPGPRMALPVTALLLPLALLLHAARPGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSASGGGGSGGGGSHEGVHRKPSLLAHPGPLVKSEETVILQCWSDVRFEHFLLHREGKYKDTLHLIGEHHDGVSKANFSIGPMMQDLAGTYRCYGSVTHSPYQLSAPSDPLDIVITGLYEKPSLSAQPGPTVLAGESVTLSCSSRSSYDMYHLSREGEAHERRFSAGPKVNGTFQADFPLGPATHGGTYRCFGSFRDSPYEWSNSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA(SEQ ID NO 12)

The amino acid sequence of pKT011 is as follows:

MGGLEPCSRFLLLPLLLAVSGLRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKVEGGGEGRGSLLTCGDVEENPGPRMALPVTALLLPLALLLHAARPGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSASGGGGSGGGGSSPTEPSSKTGNPRHLHVLIGTSVVKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA(SEQ ID NO 13)

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

GVLAGIVMGDLVLTVLIALAV(SEQ ID NO 14)

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

YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK(SEQ ID NO 15)

the nucleic acid sequence of DPK01 is as follows:

ATGGGGGGACTTGAACCCTGCAGCAGGTTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAGTGGTCTCCGTCCTGTCCAGGTCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAGATATCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGTCTAGAGTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGGATCCGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGCTAGCGGTGGCGGAGGTTCTGGAGGTGGGGGTTCCTCACCCACTGAACCAAGCTCCAAAACCGGTAACCCCAGACACCTGCATGTTCTGATTGGGACCTCAGTGGTCAAAATCCCTTTCACCATCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCTGCTGTAATGGACCAAGAGCCTGCAGGGAACAGAACAGTGAACAGCGAGGATTCTGATGAACAAGACCATCAGGAGGTGTCATACGCA(SEQ ID NO 16)

The nucleic acid sequence of DPK02 is as follows:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAGATATCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGTCTAGAGTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGGATCCGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGCTAGCGGTGGCGGAGGTTCTGGAGGTGGGGGTTCCTCACCCACTGAACCAAGCTCCAAAACCGGTAACCCCAGACACCTGCATGTTCTGATTGGGACCTCAGTGGTCAAAATCCCTTTCACCATCCTCCTCTTCTTTCTCCTTCATCGCTGGTGCTCCAACAAAAAAAATGCTGCTGTAATGGACCAAGAGCCTGCAGGGAACAGAACAGTGAACAGCGAGGATTCTGATGAACAAGACCATCAGGAGGTGTCATACGCA(SEQ ID NO 17)

The nucleic acid sequence of DPK03 is as follows:

ATGGGGGGACTTGAACCCTGCAGCAGGTTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAGTGGTCTCCGTCCTGTCCAGGTCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTCAACACACAGAGGCCGTATTACAAAGATATCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGTCTAGAGTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGGATCCGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGCTAGCGGTGGCGGAGGTTCTGGAGGTGGGGGTTCCCATGAAGGTGTGCACAGAAAGCCTTCACTGCTTGCCCATCCAGGTCCGCTGGTTAAGTCCGAGGAAACCGTCATTCTTCAATGTTGGTCCGATGTCCGGTTCGAACACTTCCTGCTGCACCGGGAAGGGAAGTACAAAGATACCTTGCATTTGATTGGCGAACACCATGATGGAGTAAGTAAAGCTAACTTCAGTATCGGACCCATGATGCAGGACCTTGCAGGAACATACAGATGTTATGGCAGCGTTACCCATAGTCCTTACCAGCTGAGCGCACCCAGCGATCCGCTCGATATTGTAATCACGGGTCTCTATGAAAAACCTAGTCTCTCTGCCCAACCTGGCCCAACAGTGCTGGCAGGTGAAAGCGTAACCTTGTCTTGCAGTAGCAGGTCTAGCTACGATATGTACCACCTCTCCAGAGAGGGTGAAGCACATGAGAGGAGATTCTCTGCCGGACCTAAGGTTAACGGGACATTTCAGGCAGACTTTCCATTGGGACCGGCTACCCATGGTGGCACATACCGGTGTTTCGGCAGCTTCCGCGACTCCCCTTATGAGTGGTCTAATAGTTCAGACCCTCTGCTCGTGTCCGTGACTGGCAATCCCTCTAACTCATGGCCGTCTCCAACCGAGCCAAGTTCAAAAACAGGCAATCCACGGCATCTGCATGTGCTGATTGGCACTTCTGTAGTAAAAATCCCCTTTACGATCTTGCTGTTTTTTCTGCTGCATCGGTGGTGCAGCAACAAGAAGAATGCTGCCGTGATGGATCAGGAACCCGCTGGCAACAGAACCGTTAACAGTGAGGATTCCGATGAGCAAGACCACCAGGAGGTGAGCTACGCC(SEQ ID NO 18)

Example 2: preparation of lentiviruses

(1) Passage of 293T cells on alternate days

Each T150 cell flask was seeded with 5X 10 ⁶ cells. After 48 hours, the cell number should reach 20-25 million/bottle.

(2) 293T cell laying bottle

A) Taking 1T 150 cell flask as an example, cells were gently washed twice with about 15ml of 1 XPBS.

B) 3ml of 0.25% pancreatin-2.21 mM EDTA was added

C) Until the cells had fallen off, 12ml of 10% (wt) FBS (purchased from Gibico) DMEM medium (purchased from burning) was added to the cells that had fallen off.

D) The cells were collected and transferred to a sterile centrifuge tube, 1000rpm, and centrifuged for 10 minutes.

E) The supernatant was aspirated off and the pellet was resuspended in 10ml of 10% (wt) FBS in DMEM medium.

F) Cell counts, the volume required for 12×10 ⁶ cells was calculated from the cell concentration.

G) The cells were combined with 25ml of DMEM medium containing 10% (wt) FBS, placed in T150 cell flasks, and gently shaken to allow uniform distribution of the cells to the bottom of the flasks, and cultured overnight at 37 ℃ in a 5% co ₂ incubator.

(3) Cell transfection

Cells were observed to reach approximately 80% -90% cell density, at which point transfection could begin.

A) 30-60 minutes before transfection, the medium was gently aspirated.

B) The plasmid DNA and the calcium chloride solution were mixed, taking one T150 bottle as an example, 28. Mu.g pRSV. Rev (purchased from Invitrogen corporation), 28. Mu.g pGAG-Pol (purchased from Invitrogen corporation), 11. Mu. g pVSVG (purchased from Invitrogen corporation), 23. Mu.g lentiviral expression plasmid (plasmids DPK01, DPK02, DPK03, pKT 011), and each of the lentiviral expression plasmids was added to 1.5ml of the calcium chloride solution and mixed.

C) 1.5ml of borate buffer solution is added into a 15ml sterile centrifuge tube, the DNA-calcium chloride solution is evenly mixed by a 1ml gun head and then is dripped into the borate buffer solution, and the mixture is quickly evenly mixed for 15-20 minutes at room temperature for incubation for 25-30 minutes.

D) The DNA-calcium chloride-borate buffer mixture (available from Shanghai Biyun biotechnology Co., ltd.) was added dropwise to the T150 flask with a 5ml pipette. Culturing in a cell culture box containing 5% carbon dioxide at 37 ℃ for 6h, and changing the liquid.

E) After 6h, the liquid is changed. The plate was gently shaken several times to suspend some of the calcium phosphate precipitate thoroughly, the culture solution containing the calcium phosphate precipitate was aspirated, and 20ml of fresh 5% (wt) FBS DMEM medium was added to continue the culture.

(4) Primary collection of viral supernatants

A) The supernatant of 293T cell culture transfected the previous day was collected into a centrifuge tube, centrifuged at 1000rpm for 5 minutes, labeled, and buffered in a refrigerator at 4 ℃.

B) 20ml of 5% (wt) FBS DMEM medium, preheated beforehand, was added to the cell flask, and the cell incubator was continued to culture overnight at 37 ℃.

(5) Viral supernatant was collected a second time (48 h/fourth day).

(6) Filtering the supernatant

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

(7) Virus concentration

Centrifuge overnight at 12000-24000rpm at 4 ℃.

(8) Virus storage

After centrifugation, the whole supernatant was poured, resuspended in fresh 5% (wt) FBS-containing DMEM medium, and virus split-packed (designated DPK01, DPK02, DPK03, pKT 011) and rapidly stored in a-80℃refrigerator for use.

Example 3: CD19 CAR-T in vitro functional test

(1) Cell preparation

Plasma was isolated from fresh blood samples (inactivated ready for use), PBMCs were isolated from the blood cell suspensions with lymphocyte separation fluid and the obtained PBMCs were T-cell sorted with T-cell sorting kit. T cells were activated by adding Dynabeads CD3/CD28 (Dynabeads=1:1 addition) and cultured in an activation medium (X-VIVO 15,5% plasma, 300IU/ml IL-2) at a final cell concentration of 1X10 ⁶/ml at 37℃and 5% CO ₂. T cells were transfected 24h later with CAR lentiviruses (DPK 01, DPK02, DPK03, pKT 011) prepared in example 2, and lentiviruses were removed 48h later after infection. Cell culture was observed every 1-2 days starting on day 4 (D4) maintaining cell densities at 0.8X10 ⁶ cells/mL. The activation medium was used on days 4-5 (D4-D5), and the amplification medium (X-VIVO 15, 300IU/ml IL-2) was used after day 5 (D5). Four CAR-T cells (DPK 01, DPK02, DPK03, pKT 011) were obtained by continuous culture until day 11 (D11).

There was no significant difference in T cell activation expansion for four different CAR structures (fig. 2A); four CAR-T cells were in an expanded state throughout the culture, gradually increasing in volume for the first 4 days, and gradually decreasing thereafter (fig. 2B).

(2) CAR-T cell positive rate detection

Taking four CAR-T cells of DPK01, DPK02, DPK03 and pKT011 on day 8 (D8), adding an anti-human CD19 antibody (PE) primary antibody and a Streptavidin-PE secondary antibody for incubation, and incubating non-transduced T cells (NTD) in the same way as a control, so as to detect the positive rate of the CAR-T cells in a flow mode. The positive rates of four CD19 CAR-T cells were detected with little difference in expression of the positive rates of four CAR-T cells on day 8 (D8) (fig. 3).

(3) T cell differentiation subpopulation assay

CAR-T cell subpopulation detection antibodies: anti-human CD3 (APC-Cy 7), anti-human CCR7 antibody (BV 421), anti-human CD45 RO Antibody (APC), CD4-BB515, CD8-BV510, CD45RA (PE-Cy 7), CD62L-PE.

Samples were taken on day 11 (D11) of four CAR-T cell cultures, DPK01, DPK02, DPK03, pKT011, and a CAR-T cell subset detection antibody was added for flow detection. CAR-T cell typing was detected, including Tn, tscm, tcm, tem, temRA in cd4+ and cd8+ T cells. The T cell differentiation subtypes of the four CAR-T (DPK 01, DPK02, DPK03, pKT 011) showed that DPK01, DPK02, DPK03 had more memory cells than pKT011, and the present invention enabled a lower differentiation state of CAR-T, which was more durable in vivo and possessed memory activity (fig. 4).

(4) Target cell culture

DMEM medium (DMEM+10% FBS+1% Penicillin/streptomycin (Penicilliin/Streptomycine)) was used for both 293T, MCF-7 and MC-7-CD19 cultures; nalm6 and L428 cultures were performed using 1640 medium (1640+10% FBS+1% penicillin/streptomycin).

(5) CAR-T specific killing

A. Target cell digestion counts, conditioned with culture medium (dmem+10% fbs) to a target cell suspension density of 2×10 ⁵ cells/ml;

b. Add 50. Mu.l of cell culture medium to Eplate plates and place Eplate in the DP monitoring tank in preparation for measuring baseline.

C. At the end of the baseline measurement, eplate were removed, 1X 10 ⁴ (50. Mu.l) target cells were added to each well and left at room temperature for 30min.

D. the E-plate with the cell suspension was returned to the corresponding monitoring well and monitoring was started.

E. after 18h, CAR-T and NTD cells were counted and effector cells were adjusted to the corresponding densities according to the effective target ratio (E: T) of the table.

E:T	Number of target cells	Cell density and volume added per well
			0:1	0	50 Μl of culture medium
1:1	1×10⁴	2X 10 ⁵ cells/ml, 50. Mu.l
			5:1	5×10⁴	1X 10 ⁶ cells/ml, 50. Mu.l
10:1	1×10⁵	2X 10 ⁶ cells/ml, 50. Mu.l

F. Suspending data acquisition of all detection grooves;

g. the E-plate placed overnight was removed and 50. Mu.l of corresponding effector cells were added to each well;

h. the E-plate is put back into the corresponding monitoring groove, and the data acquisition of a real-time label-free dynamic cell analysis technology (RTCA) system is continued;

i. after effector cell addition, the monitoring time does not exceed 24 hours.

The killing effect of four CD19 CAR-T cells including DPK01, DPK02, DPK03 and pKT011 CAR-T and non-transduced T cells (NTD) on tumor cells MCF7-CD19 was examined, and the killing ability of various CAR-T cells was compared (FIG. 5). The result shows that NTD has no killing effect on MCF-7-CD 19; at an effective target ratio (E: T) of 0:1,1:1,5:1, 10:1, all four CAR-T cells showed significant killing of MCF7-CD19 at both 5:1 and 10:1 effective target ratios. There was no significant difference in the lysis rates of the three CAR-T cells DPK01, DPK02 and DPK03 to the target cells; the anti-tumor effect of pKT011 is relatively weak, and the killing efficiency on target cells is obviously lower than that of DPK01, DPK02 and DPK03 CAR-T.

(6) Cytokine secretion assay

According to E, t=2: 1, co-culturing DPK01, DPK02, DPK03 and pKT011 CAR-T with Nalm6 (positive target cells) and L428 (negative target cells) for 24h respectively; cell supernatants were collected and ELISA was used to detect IL-2 and IFN-gamma.

Analysis of 4 CAR-T cells and NTD were co-cultured with positive target cells Nalm6 for 24 hours, and the supernatant was taken, cytokine was detected by ELISA, all four CAR-T cells were secreting IFN- γ after co-culture with positive target cells Nalm6, and the amount of IFN- γ secretion by DPK01 CAR-T cells was significantly similar to DPK02, DPK03, pKT011 secreting IFN- γ significantly lower than other CAR-T groups (fig. 6). IL-2 is secreted by four CAR-T cells after co-culture with positive target cell Nalm6, and IFN-gamma secretion of DPK01 CAR-T cells is obviously similar to DPK02 and DPK03, and pKT011 hardly secretes IL-2. Taken together, IFN-gamma and IL-2 secretion by DPK01 CAR-T cells under stimulation of tumor antigens is optimal.

(7) Antigen-stimulated proliferation

A. CAR-T was incubated to day 8 (D8) demagnetized beads and continued for 2 days using IL-2 free medium;

b. Day 10 (D10), effector cells (CAR-T) and NTD were counted;

c. The desired CAR-T (5X 10 ⁶/ml) was taken, washed 2 times with PBS and finally the cells were resuspended to a concentration of 1X 10 ⁷/ml by adding 500. Mu.l PBS;

d. fluorescent dye CFSE (purchased from Sigma) (original concentration 5 mM) was added to give a final concentration of 5 μm, and incubated in an incubator for 10min.

E. Adding 9 times of incubation volume of PBS, and centrifuging;

f. after discarding the supernatant, 10ml of medium containing 10% FBS was added for resuspension, and the supernatant was discarded.

G. The suspension was resuspended to 1X 10 ⁶/ml with 5ml of medium (10% FBS,300UI/ml IL-2). And (5) standby application.

H. Counting target cells (Nalm-6 and 293T), and preparing a cell suspension with density of 1×10 ⁶/ml with culture solution (1640+10% FBS+300UI/ml IL-2) for use;

i. according to the corresponding effective target ratio (E: T), 1ml of CAR-T cells are added into each hole, and the mixture is placed in a 37 ℃ and 5% CO ₂ incubator for culture, and the solution is centrifugally changed for 1-2 days.

J. Fifth day (D5) the flow test was performed.

CFSE fluorescence intensity flow assay results for four CAR-ts (DPK 01, DPK02, DPK03, pKT 011) showed that the fluorescence intensities of CFSE for four CAR-ts were reduced to the left after MCF7-CD19 positive target cell stimulation (green bars shown) relative to CAR-T control alone, confirming that positive target cells were able to stimulate proliferation of these four CAR-ts.

Example 4: CD19 CAR-T in vivo functional test

Tumor cells: nalm6 (1X 10 ⁷ cells)

The tumor forming mode is as follows: subcutaneous neoplasia

Drug administration group: NTD, DPK01, DPK02, DPK03

Dosage of administration: 1X 10 ⁶ CAR-T

(1) In vivo efficacy

The antitumor activity of NTD, DPK01, DPK02, and DPK03 in animals was further verified (fig. 8). Experimental results show that the tumor volume of the NTD group mice increases faster and reaches the experimental endpoint soon; DPK01, DPK02, DPK03 and pKT011 have similar tumor eliminating efficiency to mice, and DPK01, DPK02 and DPK03 can make tumor subsidence faster and can bring longer lasting curative effect.

(2) In vivo cytokine secretion

Quantification of soluble cytokines was performed using Luminex bead array technology and kits purchased from Life technologies (Invitrogen). The assay was performed using an 8-point standard curve generated from a 3-fold serial dilution according to the manufacturer's protocol. Each standard point and sample was evaluated in duplicate at 1:3 dilutions; for both measurements, the calculated% CV is less than 15%. The standard curve quantization range is determined by a range of 80-120% (observed/expected). Individual analyte quantification ranges are reported in the figure illustrations. The invention DPK01, DPK02 and DPK03 have obviously reduced IL-6 secretion level in mice (figure 9), which shows that the invention can realize better clinical safety, and the same obviously reduced IL-10 shows that lower immunosuppression factors are produced, thereby promoting clinical curative effect.

(3) In vivo CAR-T pharmacokinetics

Taking four CAR-T cell occupancy rates (figure 10) of DPK01, DPK02, DPK03 and pKT011 detected by peripheral blood of mice 28 days after administration, adding an anti-human CD19 antibody (PE) primary antibody and a Streptavidin-PE secondary antibody for incubation, detecting the positive rate of the CAR-T cells in a flow mode, wherein the expression difference of the positive rates of DPK01, DPK02 and DPK03 is not large, the CD19 CAR-T occupancy rate of the pKT011 is remarkably lower, and the anti-tumor effect of DPK01, DPK02 and DPK03 is better.

(4) In vivo CAR-T depletion marker expression

Four CAR-T cell PD-1 expression conditions (figure 11) of DPK01, DPK02, DPK03 and pKT011 detected by peripheral blood of mice 28 days after administration are taken, and the positive rate of the CAR-T cells is detected in a flow mode, and the PD-1 of the pKT011 is obviously up-regulated, so that the DPK01, DPK02 and DPK03 can better resist the depletion effect.

Example 5: CD19 CAR-T clinical study

(1) Cell preparation

Patient blood was withdrawn on day 14 (D-14) prior to cell reinfusion, plasma was isolated from fresh blood samples (inactivated ready for use), PBMCs were isolated from the blood cell suspension with lymphocyte separation fluid, and the obtained PBMCs were T-cell sorted with T-cell sorting kit. T cells were activated by adding Dynabeads CD3/CD28 (Dynabeads=1:1 addition) and cultured in an activation medium (X-VIVO 15,5% plasma, 300IU/ml IL-2) at a final cell concentration of 1X 10 ⁶/ml at 37℃and 5% CO ₂. After 24h, DPK01 lentivirus was added to transfect T cells, and after 48h infection, lentivirus was removed. Cell culture was observed every 1-2 days starting on day 4 (D4) maintaining cell densities at 0.8X10 ⁶ cells/mL. The activation medium was used on days 4-5 (D4-D5), and the amplification medium (X-VIVO 15, 300IU/ml IL-2) was used after day 5 (D5). Cultivation was continued until day 7-12 (D7-D12).

The CAR clinical grade vector was manufactured by south kyi ald immunotherapy institute limited. At the end of CAR-T cell culture, the cells are cryopreserved in infusible frozen solution. CAR-T cells are administered in compliance with the dosing dose. Each bag contains an aliquot of frozen medium (volume dependent on dose) with CS5 as the frozen stock. Bags (10-100 ml) containing CD19 CAR-T cells were stored in a liquid nitrogen box at-135 ℃ under test. The freezing bag is stored in the freezer until needed. To increase safety, the dose of first reinfusion cells was given in divided doses on day 0 (D0) and day 1 (D1), with about 30% cells on day 0 and 70% cells on day 1.

(2) Cell thawing

The frozen cells are transported in dry ice to a laboratory or patient. The cells were thawed and gently massaged using a water bath maintained at 37 ℃ until the cells just thawed. No frozen mass should remain in the container. If the CD19 CAR-T cell product shows a damaged or leaky pocket, it should not be reinfused.

(3) Preoperative medication

The CD19 CAR-T formulation is not suitable for long periods of time at room temperature after warming, and therefore it is desirable to determine the warming time after the subject treatment preparation is fully completed. Necessary first-aid devices and medicines are prepared before reinfusion. The subjects were pre-operatively dosed with acetaminophen, debark, and diphenhydramine or other H1 antihistamines 30-60 minutes prior to return. Since CD19 CAR-T is an autologous T cell therapy, the packaging bag is patient specific and subject information on the bag should be confirmed prior to reinfusion. Before the return, whether the package is damaged or not is checked, and if the package is damaged, the return is not needed.

(4) Reinfusion

Again confirming the subject information on the packaging bag; flushing the injection pipeline with physiological saline; a 50ml physiological saline wash pipe is used before and after the infusion of the CAR-T cells; venous return was completed within 30 minutes.

(5) Group entry criteria

(1) Recurrence of CD19 positive, refractory B-cell hematological malignancy; (2) under 70 years old; (3) KPS score is not less than 60, expected lifetime is not less than 3 months; (4) The absolute number of the platelets is more than or equal to 30 multiplied by 10 ⁹/L; (5) The absolute number of lymphocytes is more than or equal to 0.15X10 ⁹/L; (6) serum ALT is less than or equal to 100U/L, AST is less than or equal to 100U/L; (7) total bilirubin is less than or equal to 30 mu mol; (8) creatinine is less than or equal to 200 mu mol/L; (9) Women of childbearing age were negative in the pregnancy test before dosing began and agreed to take effective contraceptive measures during the test until the last follow-up; (10) Voluntary group participation, compliance is good, can cooperate experimental observation, and signs written informed consent.

(6) Exclusion criteria

Patients were unable to enter the study if they met any of the following criteria:

(1) Clinical diagnosis is (symptoms, signs, imaging, cerebrospinal fluid) central nervous system leukemia; (2) Patients with hypercholesteremia (white blood cell count: 50 x 10 ⁹/L) or whose disease progression is rapidly judged by the investigator at the time of group entry cannot be assured of completing a complete treatment cycle; (3) Patients with infections including fungi, bacteria, viruses or other uncontrollable infections or requiring a quaternary isolation treatment; (4) HIV, HBV, HCV patients who tested positive; (5) Patients with central nervous system diseases or autoimmune central nervous system lesions including stroke, epilepsy, dementia, etc.; (6) Within the first 12 months of the group, with symptoms including myocardial infection, cardiac angiography or stenting, active angina or other obvious clinical symptoms, or with cardiac asthma or cardiovascular lymphocyte infiltration; (7) Patients undergoing anticoagulant therapy or having severe coagulation dysfunction (APTT > 70); (8) Studies that judge, according to the researchers, that the patient is receiving medication will affect the safety and effectiveness of the project; (9) Patients having allergies or a history of allergies to the biological agents used in the project; (10) pregnant or lactating women; (11) Systemic, systemic steroid medications (except for the most recent or current use of inhaled steroids) were administered systemically within 2 weeks prior to treatment; (12) With other uncontrolled diseases, researchers consider it unsuitable for the enrollees; (13) Any situation that the researcher believes may increase patient risk or interfere with the test results; (14) patients who are concurrently enrolled in other clinical studies.

(7) Treatment regimen

(8) Pretreatment protocol

Day 5,4,3,2,1 before reinfusion of cells: intravenous fludarabine was instilled for 5 consecutive days at a dose of 30mg/m ²/day;

Day 5,4 before reinfusion of cells: cyclophosphamide was instilled intravenously for 2 consecutive days at a dose of 500mg/m ²/day.

(9) Dosing regimen

(1) Reinfusion dose: according to internationally existing peer doses

Test drug DPK01, 1.2X10 ⁶ CAR-T cells/kg intravenous drip was administered in duplicate, 30% on day 0 (D0) and 70% on day 1 (D1) according to the clinical situation. The above dosage regimen was used as a course of treatment.

(10) Therapeutic results

NO.

Indication of disease

Tumor burden

Dosage of

CRS

Curative effect

Follow-up result (Tian)

1

B-ALL

74.5(BM)

1.2×10⁶

1

Complete alleviation of

Recurrence (154)

2

B-ALL

96(BM)

1.2×10⁶

2

Complete alleviation of

Sustained release (380)

3

B-ALL

7.5(BM)

1.2×10⁶

1

Complete alleviation of

Sustained release (137)

4

B-ALL

56.5(BM)

1.2×10⁶

1

Complete alleviation of

Sustained release (368)

The complete remission rate of CD19-CAR-T in 4 adult patients with acute B-lymphoblastic leukemia (B-ALL) is 100% in 1 month, and can reach 50% in 6 months. CRS was present in all 4 patients, 75% at grade 1 CRS, 25% at grade 2 CRS, and no central toxicity was present in the patients. Clinical research results show that DPK01 generates controllable low-level CRS in clinical treatment, shows better safety, and has high complete remission rate and strong in-vivo lasting effect.

Sequence listing

<110> Nanjing card medical science and technology Co., ltd

<120> Chimeric receptors comprising DAP12 and a costimulatory signaling molecule signaling domain and methods of use thereof

<130> MTI20185

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly

20

<210> 2

<211> 92

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp Cys Ser Cys Ser Thr

1 5 10 15

Val Ser Pro Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu

20 25 30

Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu Gly Arg Leu Val Pro

35 40 45

Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr

50 55 60

Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val

65 70 75 80

Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys

85 90

<210> 3

<211> 81

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

1 5 10 15

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

20 25 30

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

35 40 45

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

50 55 60

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

65 70 75 80

Lys

<210> 4

<211> 21

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 5

<211> 42

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 6

<211> 17

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly

<210> 7

<211> 242

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 8

<211> 69

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

Ser Lys Thr Gly Asn Pro Arg His Leu His Val Leu Ile Gly Thr Ser

1 5 10 15

Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe Phe Leu Leu His Arg

20 25 30

Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala

35 40 45

Gly Asn Arg Thr Val Asn Ser Glu Asp Ser Asp Glu Gln Asp His Gln

50 55 60

Glu Val Ser Tyr Ala

65

<210> 9

<211> 283

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

His Glu Gly Val His Arg Lys Pro Ser Leu Leu Ala His Pro Gly Pro

1 5 10 15

Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln Cys Trp Ser Asp Val

20 25 30

Arg Phe Glu His Phe Leu Leu His Arg Glu Gly Lys Tyr Lys Asp Thr

35 40 45

Leu His Leu Ile Gly Glu His His Asp Gly Val Ser Lys Ala Asn Phe

50 55 60

Ser Ile Gly Pro Met Met Gln Asp Leu Ala Gly Thr Tyr Arg Cys Tyr

65 70 75 80

Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser Ala Pro Ser Asp Pro

85 90 95

Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys Pro Ser Leu Ser Ala

100 105 110

Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser Val Thr Leu Ser Cys

115 120 125

Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu Ser Arg Glu Gly Glu

130 135 140

Ala His Glu Arg Arg Phe Ser Ala Gly Pro Lys Val Asn Gly Thr Phe

145 150 155 160

Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly Gly Thr Tyr Arg

165 170 175

Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu Trp Ser Asn Ser Ser

180 185 190

Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro Ser Asn Ser Trp Pro

195 200 205

Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu His

210 215 220

Val Leu Ile Gly Thr Ser Val Val Lys Ile Pro Phe Thr Ile Leu Leu

225 230 235 240

Phe Phe Leu Leu His Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val

245 250 255

Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Val Asn Ser Glu Asp Ser

260 265 270

Asp Glu Gln Asp His Gln Glu Val Ser Tyr Ala

275 280

<210> 10

<211> 535

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys Asp Ile Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

115 120 125

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

130 135 140

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ser Arg Val

145 150 155 160

Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

165 170 175

Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala Leu Leu

180 185 190

Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser Asp Ile

195 200 205

Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg

210 215 220

Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

225 230 235 240

Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His

245 250 255

Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

260 265 270

Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp

275 280 285

Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe

290 295 300

Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly

325 330 335

Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val

340 345 350

Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro

355 360 365

Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr

370 375 380

Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp

385 390 395 400

Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp

405 410 415

Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser

420 425 430

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

435 440 445

Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro Thr Glu

450 455 460

Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu His Val Leu Ile Gly

465 470 475 480

Thr Ser Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe Phe Leu Leu

485 490 495

His Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu

500 505 510

Pro Ala Gly Asn Arg Thr Val Asn Ser Glu Asp Ser Asp Glu Gln Asp

515 520 525

His Gln Glu Val Ser Tyr Ala

530 535

<210> 11

<211> 524

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu

20 25 30

Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu

35 40 45

Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala

50 55 60

Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr

65 70 75 80

Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr

85 90 95

Gln Arg Pro Tyr Tyr Lys Asp Ile Lys Arg Gly Arg Lys Lys Leu Leu

100 105 110

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

115 120 125

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

130 135 140

Glu Leu Ser Arg Val Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu

145 150 155 160

Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro

165 170 175

Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg

180 185 190

Pro Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala

195 200 205

Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile

210 215 220

Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys

225 230 235 240

Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg

245 250 255

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn

260 265 270

Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr

275 280 285

Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly

290 295 300

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

305 310 315 320

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

325 330 335

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

340 345 350

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

355 360 365

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

370 375 380

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

385 390 395 400

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

405 410 415

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

420 425 430

Val Thr Val Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

435 440 445

Ser Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu

450 455 460

His Val Leu Ile Gly Thr Ser Val Val Lys Ile Pro Phe Thr Ile Leu

465 470 475 480

Leu Phe Phe Leu Leu His Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala

485 490 495

Val Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Val Asn Ser Glu Asp

500 505 510

Ser Asp Glu Gln Asp His Gln Glu Val Ser Tyr Ala

515 520

<210> 12

<211> 743

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys Asp Ile Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

115 120 125

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

130 135 140

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ser Arg Val

145 150 155 160

Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val

165 170 175

Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala Leu Leu

180 185 190

Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser Asp Ile

195 200 205

Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg

210 215 220

Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

225 230 235 240

Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His

245 250 255

Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

260 265 270

Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp

275 280 285

Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe

290 295 300

Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly

325 330 335

Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val

340 345 350

Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro

355 360 365

Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr

370 375 380

Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp

385 390 395 400

Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp

405 410 415

Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser

420 425 430

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

435 440 445

Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Glu Gly Val

450 455 460

His Arg Lys Pro Ser Leu Leu Ala His Pro Gly Pro Leu Val Lys Ser

465 470 475 480

Glu Glu Thr Val Ile Leu Gln Cys Trp Ser Asp Val Arg Phe Glu His

485 490 495

Phe Leu Leu His Arg Glu Gly Lys Tyr Lys Asp Thr Leu His Leu Ile

500 505 510

Gly Glu His His Asp Gly Val Ser Lys Ala Asn Phe Ser Ile Gly Pro

515 520 525

Met Met Gln Asp Leu Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Val Thr

530 535 540

His Ser Pro Tyr Gln Leu Ser Ala Pro Ser Asp Pro Leu Asp Ile Val

545 550 555 560

Ile Thr Gly Leu Tyr Glu Lys Pro Ser Leu Ser Ala Gln Pro Gly Pro

565 570 575

Thr Val Leu Ala Gly Glu Ser Val Thr Leu Ser Cys Ser Ser Arg Ser

580 585 590

Ser Tyr Asp Met Tyr His Leu Ser Arg Glu Gly Glu Ala His Glu Arg

595 600 605

Arg Phe Ser Ala Gly Pro Lys Val Asn Gly Thr Phe Gln Ala Asp Phe

610 615 620

Pro Leu Gly Pro Ala Thr His Gly Gly Thr Tyr Arg Cys Phe Gly Ser

625 630 635 640

Phe Arg Asp Ser Pro Tyr Glu Trp Ser Asn Ser Ser Asp Pro Leu Leu

645 650 655

Val Ser Val Thr Gly Asn Pro Ser Asn Ser Trp Pro Ser Pro Thr Glu

660 665 670

Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu His Val Leu Ile Gly

675 680 685

Thr Ser Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe Phe Leu Leu

690 695 700

His Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met Asp Gln Glu

705 710 715 720

Pro Ala Gly Asn Arg Thr Val Asn Ser Glu Asp Ser Asp Glu Gln Asp

725 730 735

His Gln Glu Val Ser Tyr Ala

740

<210> 13

<211> 489

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

Met Gly Gly Leu Glu Pro Cys Ser Arg Phe Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Val Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys Val Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

115 120 125

Asp Val Glu Glu Asn Pro Gly Pro Arg Met Ala Leu Pro Val Thr Ala

130 135 140

Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Gly Ser

145 150 155 160

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

165 170 175

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

180 185 190

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

195 200 205

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

210 215 220

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

225 230 235 240

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

245 250 255

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

275 280 285

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

290 295 300

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

305 310 315 320

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

325 330 335

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

340 345 350

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

355 360 365

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

370 375 380

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

385 390 395 400

Ser Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Pro

405 410 415

Thr Glu Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu His Val Leu

420 425 430

Ile Gly Thr Ser Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe Phe

435 440 445

Leu Leu His Arg Trp Cys Ser Asn Lys Lys Asn Ala Ala Val Met Asp

450 455 460

Gln Glu Pro Ala Gly Asn Arg Thr Val Asn Ser Glu Asp Ser Asp Glu

465 470 475 480

Gln Asp His Gln Glu Val Ser Tyr Ala

485

<210> 14

<211> 21

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 14

Gly Val Leu Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu

1 5 10 15

Ile Ala Leu Ala Val

20

<210> 15

<211> 52

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 15

Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala

1 5 10 15

Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu

20 25 30

Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg

35 40 45

Pro Tyr Tyr Lys

50

<210> 16

<211> 1605

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaag atatcaaacg gggcagaaag 360

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 420

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gtctagagtc 480

gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 540

ggccctagga tggccttacc agtgaccgcc ttgctcctgc cgctggcctt gctgctccac 600

gccgccaggc cgggatccga catccagatg acacagacta catcctccct gtctgcctct 660

ctgggagaca gagtcaccat cagttgcagg gcaagtcagg acattagtaa atatttaaat 720

tggtatcagc agaaaccaga tggaactgtt aaactcctga tctaccatac atcaagatta 780

cactcaggag tcccatcaag gttcagtggc agtgggtctg gaacagatta ttctctcacc 840

attagcaacc tggagcaaga agatattgcc acttactttt gccaacaggg taatacgctt 900

ccgtacacgt tcggaggggg gaccaagctg gagatcacag gtggcggtgg ctcgggcggt 960

ggtgggtcgg gtggcggcgg atctgaggtg aaactgcagg agtcaggacc tggcctggtg 1020

gcgccctcac agagcctgtc cgtcacatgc actgtctcag gggtctcatt acccgactat 1080

ggtgtaagct ggattcgcca gcctccacga aagggtctgg agtggctggg agtaatatgg 1140

ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat catcaaggac 1200

aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga cacagccatt 1260

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggccaa 1320

ggaacctcag tcaccgtctc ctcagctagc ggtggcggag gttctggagg tgggggttcc 1380

tcacccactg aaccaagctc caaaaccggt aaccccagac acctgcatgt tctgattggg 1440

acctcagtgg tcaaaatccc tttcaccatc ctcctcttct ttctccttca tcgctggtgc 1500

tccaacaaaa aaaatgctgc tgtaatggac caagagcctg cagggaacag aacagtgaac 1560

agcgaggatt ctgatgaaca agaccatcag gaggtgtcat acgca 1605

<210> 17

<211> 1572

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgtgcagtt gctctacggt gagcccgggc gtgctggcag ggatcgtgat gggagacctg 120

gtgctgacag tgctcattgc cctggccgtg tacttcctgg gccggctggt ccctcggggg 180

cgaggggctg cggaggcagc gacccggaaa cagcgtatca ctgagaccga gtcgccttat 240

caggagctcc agggtcagag gtcggatgtc tacagcgacc tcaacacaca gaggccgtat 300

tacaaagata tcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 360

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 420

gaaggaggat gtgaactgtc tagagtcgag ggcggcggag agggcagagg aagtcttcta 480

acatgcggtg acgtggagga gaatcccggc cctaggatgg ccttaccagt gaccgccttg 540

ctcctgccgc tggccttgct gctccacgcc gccaggccgg gatccgacat ccagatgaca 600

cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 660

agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 720

ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 780

gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 840

tacttttgcc aacagggtaa tacgcttccg tacacgttcg gaggggggac caagctggag 900

atcacaggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc tgaggtgaaa 960

ctgcaggagt caggacctgg cctggtggcg ccctcacaga gcctgtccgt cacatgcact 1020

gtctcagggg tctcattacc cgactatggt gtaagctgga ttcgccagcc tccacgaaag 1080

ggtctggagt ggctgggagt aatatggggt agtgaaacca catactataa ttcagctctc 1140

aaatccagac tgaccatcat caaggacaac tccaagagcc aagttttctt aaaaatgaac 1200

agtctgcaaa ctgatgacac agccatttac tactgtgcca aacattatta ctacggtggt 1260

agctatgcta tggactactg gggccaagga acctcagtca ccgtctcctc agctagcggt 1320

ggcggaggtt ctggaggtgg gggttcctca cccactgaac caagctccaa aaccggtaac 1380

cccagacacc tgcatgttct gattgggacc tcagtggtca aaatcccttt caccatcctc 1440

ctcttctttc tccttcatcg ctggtgctcc aacaaaaaaa atgctgctgt aatggaccaa 1500

gagcctgcag ggaacagaac agtgaacagc gaggattctg atgaacaaga ccatcaggag 1560

gtgtcatacg ca 1572

<210> 18

<211> 2229

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

atggggggac ttgaaccctg cagcaggttc ctgctcctgc ctctcctgct ggctgtaagt 60

ggtctccgtc ctgtccaggt ccaggcccag agcgattgca gttgctctac ggtgagcccg 120

ggcgtgctgg cagggatcgt gatgggagac ctggtgctga cagtgctcat tgccctggcc 180

gtgtacttcc tgggccggct ggtccctcgg gggcgagggg ctgcggaggc agcgacccgg 240

aaacagcgta tcactgagac cgagtcgcct tatcaggagc tccagggtca gaggtcggat 300

gtctacagcg acctcaacac acagaggccg tattacaaag atatcaaacg gggcagaaag 360

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 420

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gtctagagtc 480

gagggcggcg gagagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc 540

ggccctagga tggccttacc agtgaccgcc ttgctcctgc cgctggcctt gctgctccac 600

gccgccaggc cgggatccga catccagatg acacagacta catcctccct gtctgcctct 660

ctgggagaca gagtcaccat cagttgcagg gcaagtcagg acattagtaa atatttaaat 720

tggtatcagc agaaaccaga tggaactgtt aaactcctga tctaccatac atcaagatta 780

cactcaggag tcccatcaag gttcagtggc agtgggtctg gaacagatta ttctctcacc 840

attagcaacc tggagcaaga agatattgcc acttactttt gccaacaggg taatacgctt 900

ccgtacacgt tcggaggggg gaccaagctg gagatcacag gtggcggtgg ctcgggcggt 960

ggtgggtcgg gtggcggcgg atctgaggtg aaactgcagg agtcaggacc tggcctggtg 1020

gcgccctcac agagcctgtc cgtcacatgc actgtctcag gggtctcatt acccgactat 1080

ggtgtaagct ggattcgcca gcctccacga aagggtctgg agtggctggg agtaatatgg 1140

ggtagtgaaa ccacatacta taattcagct ctcaaatcca gactgaccat catcaaggac 1200

aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga cacagccatt 1260

tactactgtg ccaaacatta ttactacggt ggtagctatg ctatggacta ctggggccaa 1320

ggaacctcag tcaccgtctc ctcagctagc ggtggcggag gttctggagg tgggggttcc 1380

catgaaggtg tgcacagaaa gccttcactg cttgcccatc caggtccgct ggttaagtcc 1440

gaggaaaccg tcattcttca atgttggtcc gatgtccggt tcgaacactt cctgctgcac 1500

cgggaaggga agtacaaaga taccttgcat ttgattggcg aacaccatga tggagtaagt 1560

aaagctaact tcagtatcgg acccatgatg caggaccttg caggaacata cagatgttat 1620

ggcagcgtta cccatagtcc ttaccagctg agcgcaccca gcgatccgct cgatattgta 1680

atcacgggtc tctatgaaaa acctagtctc tctgcccaac ctggcccaac agtgctggca 1740

ggtgaaagcg taaccttgtc ttgcagtagc aggtctagct acgatatgta ccacctctcc 1800

agagagggtg aagcacatga gaggagattc tctgccggac ctaaggttaa cgggacattt 1860

caggcagact ttccattggg accggctacc catggtggca cataccggtg tttcggcagc 1920

ttccgcgact ccccttatga gtggtctaat agttcagacc ctctgctcgt gtccgtgact 1980

ggcaatccct ctaactcatg gccgtctcca accgagccaa gttcaaaaac aggcaatcca 2040

cggcatctgc atgtgctgat tggcacttct gtagtaaaaa tcccctttac gatcttgctg 2100

ttttttctgc tgcatcggtg gtgcagcaac aagaagaatg ctgccgtgat ggatcaggaa 2160

cccgctggca acagaaccgt taacagtgag gattccgatg agcaagacca ccaggaggtg 2220

agctacgcc 2229

Claims

1. A chimeric antigen receptor selected from any one of the following: the chimeric antigen receptor is a CD19-KIRS2/Dap12-BB chimeric antigen receptor, the CD19-KIRS2/Dap12-BB chimeric antigen receptor is formed by cutting a DPK01 fusion protein through a T2A peptide, and the amino acid sequence of the DPK01 fusion protein is shown as SEQ ID NO. 10; or (b)

The chimeric antigen receptor is a CD19-KIRS2/tDap12-BB chimeric antigen receptor, the CD19-KIRS 2/tDap-BB chimeric antigen receptor is formed by cutting a DPK02 fusion protein by a T2A peptide, and the amino acid sequence of the DPK02 fusion protein is shown as SEQ ID NO. 11; or (b)

The chimeric antigen receptor is a CD19-KIR2DS2/Dap12-BB chimeric antigen receptor, the CD19-KIR2DS2/Dap12-BB chimeric antigen receptor is formed by cutting a DPK03 fusion protein through a T2A peptide, and the amino acid sequence of the DPK03 fusion protein is shown as SEQ ID NO. 12.

2. A nucleic acid encoding the chimeric antigen receptor of claim 1.

3. The nucleic acid of claim 2, wherein the encoding nucleic acid is the nucleic acid set forth in SEQ ID No. 16.

4. A vector comprising the nucleic acid of claim 2 or 3.

5. An immune effector cell comprising the nucleic acid of claim 2 or 3 or the vector of claim 4.

6. A composition comprising the chimeric antigen receptor of claim 1, the nucleic acid of claim 2 or 3, the vector of claim 4, and/or the immune effector cell of claim 5, and a pharmaceutically acceptable carrier.

7. A method of making a cell, the method comprising introducing the nucleic acid of claim 2 or 3, the vector of claim 4, into an immune effector cell.

8. Use of the chimeric antigen receptor of claim 1, the nucleic acid of claim 2 or 3, the vector of claim 4 and/or the immune effector cell of claim 5 in the manufacture of a medicament for the treatment and/or prevention of leukemia.

9. Use of the chimeric antigen receptor of claim 1, the nucleic acid of claim 2 or 3, the vector of claim 4 and/or the immune effector cell of claim 5 in the preparation of a medicament for treating mammalian leukemia.