Chimeric antigen receptor targeting CD19 and its use

Abstract

Provided are chimeric antigen receptors targeting CD19 and their use, and also provided are chimeric antigen receptors targeting CD19, corresponding nucleic acid molecules, vectors, immune effector cells, preparation methods and their products, pharmaceutical compositions, therapeutic uses, pharmaceutical uses, and methods for treating tumors or cancers.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to Chinese Patent Application No. 202210468137.4, filed on April 29, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] This application relates generally to the fields of biotechnology and cell therapy, and specifically to chimeric antigen receptors that target CD19 and uses thereof. [Background technology]

[0003] CD19 is a 95-kDa type I transmembrane glycoprotein belonging to the immunoglobulin superfamily, consisting of a single transmembrane domain, an N-terminal extracellular domain, and a C-terminal intracellular domain. The N-terminal extracellular domain is composed of two C2-type Ig-like domains, and the C-terminal intracellular domain is highly conserved and consists of 242 amino acids and nine tyrosine residues near the C-terminus. CD19 plays an important regulatory role in the activation and proliferation of B lymphocytes and is widely expressed on the surface of B cell malignant cells, mainly in chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), and non-Hodgkin's lymphoma (diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), and follicular lymphoma (FL).

[0004] In recent years, cellular immunotherapy technologies, particularly those based on chimeric antigen receptors (CARs), have made great strides. CD19 has become a popular target for CAR technology, and CAR-T products specifically targeting CD19 have already been approved by the U.S. FDA and are commercially available. However, conventional CD19 CART products use the immunogenic murine antibody FMC63, which can lead to the human body producing anti-drug antibodies (ADAs) or killer T lymphocytes (CTLs) against the xenoantibody used in CAR-T, resulting in rapid elimination of CAR-T cells and disease relapse in some patients.

[0005] Although CAR-modified T cells have antitumor activity, they have certain limitations in terms of logistics and clinical application. CAR T cells are prepared for each patient, which makes the preparation process complicated and expensive. Some patients experience significant toxic effects after receiving CAR T cell therapy, including cytokine release syndrome and neurotoxicity, requiring treatment at specialized medical institutions. Effective and safer allogeneic products could overcome these limitations. Summary of the Invention

[0006] In a first aspect, the present application provides a nucleic acid molecule encoding a chimeric antigen receptor that targets CD19, the chimeric antigen receptor comprising an extracellular domain comprising an antigen-binding region that specifically binds to CD19, a transmembrane domain linked to the extracellular domain, and an intracellular domain linked to the transmembrane domain, the antigen-binding region comprising a VH comprising HCDRs 1 to 3 and a VL comprising LCDRs 1 to 3, wherein: (1) the HCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 9 to 11, respectively, and the LCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 12 to 14, respectively; or (2) the HCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 15 to 17, respectively, and the LCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 18 to 20, respectively; or (3) the HCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 21 to 23, respectively, and the LCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 24 to 26, respectively; or (4) the HCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 27 to 29, respectively, and the LCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 30 to 32, respectively; or (5) The above HCDRs 1 to 3 each have at most 0 to 3 mutations compared to the corresponding CDR in any one of groups (1) to (4), and the above LCDRs 1 to 3 each have at most 0 to 3 mutations compared to the corresponding CDR in any one of groups (1) to (4).

[0007] In a second aspect, the present application provides a chimeric antigen receptor that targets CD19 encoded by a nucleic acid molecule according to the first aspect.

[0008] In a third aspect, the present application provides a vector comprising the nucleic acid molecule according to the first aspect.

[0009] In a fourth aspect, the present application provides an immune effector cell, wherein said immune effector cell is transfected with a nucleic acid molecule according to the first aspect, or transfected with a vector according to the third aspect, or expresses a chimeric antigen receptor according to the second aspect.

[0010] In a fifth aspect, the present application provides a method of preparing immune effector cells, said method comprising the steps of providing immune effector cells and introducing a nucleic acid molecule according to the first aspect or a vector according to the third aspect into said immune effector cells, optionally further comprising culturing said immune effector cells after introduction of said nucleic acid molecule or vector.

[0011] In a sixth aspect, the present application provides a product prepared according to the method of the fifth aspect.

[0012] In a seventh aspect, the present application provides a pharmaceutical composition, said pharmaceutical composition comprising a nucleic acid molecule according to the first aspect, or a vector according to the third aspect, or an immune effector cell according to the fourth aspect, or a product according to the sixth aspect, and a pharmaceutically acceptable vector.

[0013] In an eighth aspect, the present application provides the use of a nucleic acid molecule according to the first aspect, or a vector according to the third aspect, or an immune effector cell according to the fourth aspect, or a product according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect, in the preparation of a medicament for treating a tumor or cancer, wherein said tumor or cancer is preferably a CD19-associated tumor or cancer, more preferably a B-cell malignancy, such as acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, thyroid cancer ... Monoclonal gammopathy of undetermined significance, Waldenström's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, dual spectrum leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B-acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma.

[0014] In a ninth aspect, the present application provides a method of treating cancer or tumor, comprising administering to a subject in need thereof an effective amount of the nucleic acid molecule of the first aspect, or the vector according to the third aspect, or the immune effector cell according to the fourth aspect, or the product according to the sixth aspect, or the pharmaceutical composition according to the seventh aspect, wherein said tumor or cancer is preferably a CD19-associated tumor or cancer, more preferably a B-cell malignancy, such as acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, thyroid cancer ... Plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenström's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, dual spectrum leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B-acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma. [Brief explanation of the drawings]

[0015] [Figure 1] Schematic diagram of the structure of a chimeric antigen receptor that targets CD19. [Figure 2] Figure 1 shows the in vitro killing effect of different CD19 CAR NK on Nalm6 cells, of which the three columns in each group show the results from left to right at effector cell to target cell ratios (E:T) of 0.5:1, 1:1, and 2:1, and NT represents non-transfected CAR NK cells. [Figure 3] Figure 1 shows the in vitro killing effect of different CD19 CAR NK on Raji cells, of which the three columns in each group show the results from left to right at effector cell to target cell ratios (E:T) of 0.5:1, 1:1, and 2:1, and NT represents non-transfected CAR NK cells. [Figure 4]Figure 1 shows the in vitro killing effect of different CD19 CAR NK on Molm13 cells, of which the three columns in each group show the results from left to right at effector cell to target cell ratios (E:T) of 0.5:1, 1:1 and 2:1, and NT represents non-transfected CAR NK cells. [Figure 5] Figure 1 shows tumor imaging results of the Nalm6 xenograft model in animals after receiving different CD19 CAR NK treatments, of which the NT group corresponds to blank NK in Table 5, the Mab01-CAR1 group corresponds to Mab01-CAR in Table 5, the Mab03-CAR group corresponds to Mab03-CAR3 in Table 5, and the FMC63-CAR group corresponds to FMC63-CAR4 in Table 5. [Figure 6A] The expansion status of different CD19 CAR NKs is shown, of which UT represents untreated. [Figure 6B] Shows the CAR cell positivity rates of different CD19 CAR NKs during the expansion culture process. [Figure 7A] The killing effect of different CD19 CAR NKs on Raji cells is shown, with the results at 0 hours of resuscitation shown. [Figure 7B] The killing effect of different CD19 CAR NKs on Raji cells is shown, with results shown 24 hours after resuscitation. [Figure 8A] Tumor imaging results of the Nalm6 xenograft model in animals after receiving different CD19 CAR NK treatments, with results shown from days 5 to 26. [Figure 8B] Tumor imaging results of the Nalm6 xenograft model in animals after receiving different CD19 CAR NK treatments, with results shown from days 30 to 33. [Figure 8C] Photon dose results of Nalm6 xenograft model tumor imaging in animals after different CD19 CAR NK treatments are shown (starting from day 19; photon dose data was only collected up to day 19 due to deaths in the negative control group). [Figure 8D]Figure 1 shows the results of body weight detection of Nalm6 xenograft model animals after receiving different CD19 CAR NK treatments (starting on day 19; due to deaths in the negative control group, body weight data was only collected up to day 19). [Figure 8E] 1 shows survival curves of Nalm6 xenograft model animals after receiving different CD19 CAR NK treatments. [Figure 9A] Shows the percentage of CD56+CAR19+CD3− cells in peripheral blood samples obtained from Nalm6 xenograft model animals receiving different CD19 CAR NK treatments. [Figure 9B] Shows the percentage of CD56+CAR19-CD3- cells in peripheral blood samples obtained from Nalm6 xenograft model animals receiving different CD19 CAR NK treatments. [Figure 10] A summary of efficacy studies in the Nalm6 xenograft model for different CD19 CAR NK treatment arms is shown. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description of the Invention Definitions and explanations of terms Unless otherwise defined in this application, scientific and technical terms associated with this application shall have meanings that are understood by those skilled in the art.

[0017] Also, unless otherwise indicated herein, singular terms herein shall include the plural and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "one," "an," and "such" include plural referents unless the context clearly dictates otherwise.

[0018] In this specification, the terms "comprise," "include," and "have" are used interchangeably and are intended to indicate the inclusive nature of a solution, meaning that other elements than the listed elements may be present in the solution. At the same time, it should be understood that the terms "comprise," "include," and "have" used in this specification also provide a solution "consisting of." Illustratively, "a composition comprising A and B" means A composition consisting of A and B , and Compositions containing other ingredients in addition to A and B All of these should be understood as technical solutions that fall within the scope of the aforementioned "composition."

[0019] The term "and / or" as used herein includes the meaning of "and", "or" and "all or any other combination of the elements linked by the term to which it belongs".

[0020] Notwithstanding the numerical ranges and approximations of parameters set forth in the broad scope of this application, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical values necessarily contain certain errors resulting from the standard deviations inherent in their respective measurements. Also, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, a recited range of "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (inclusive), i.e., all subranges beginning with a minimum value of 1 or greater, e.g., 1 to 6.1, and subranges ending with a maximum value of 10 or less, e.g., 5.5 to 10. Also, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0021] As used herein, "chimeric antigen receptor (CAR)" refers to an artificial immune effector cell surface receptor engineered to express on immune effector cells and specifically bind to an antigen, which comprises at least (1) an extracellular antigen-binding domain, such as the variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR to the immune effector cell, and (3) an intracellular signaling domain. CARs can use the extracellular antigen-binding domain to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in an MHC-unrestricted manner. The extracellular domain of a chimeric antigen receptor can further comprise a signal peptide and / or a hinge region. The intracellular domain of a chimeric antigen receptor can further comprise a costimulatory domain.

[0022] As used herein, the term "signal peptide" in the context of a chimeric antigen receptor refers to a fragment of a protein or polypeptide that directs the protein or polypeptide into the secretory pathway and translocates it to the cell membrane and / or cell surface. A non-limiting example of a signal peptide is the CD8α signal peptide.

[0023] As used herein, the term "hinge region" in the context of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that functions to link the transmembrane region and the antigen-binding region. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antigen-binding region. The hinge region may be derived in whole or in part from a natural molecule, for example, from the extracellular region of CD8, CD4, or CD28, or from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence.

[0024] As used herein, the term "transmembrane (TM) region" in the context of a chimeric antigen receptor refers to a polypeptide structure that allows the chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, an NK cell, or an NKT cell) and can induce a cellular response of the immune cell against a target cell. The transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein. When the chimeric antigen receptor binds to a target antigen, the transmembrane domain can transduce signals. A non-limiting example of a hinge region is the CD8 transmembrane region.

[0025] As used herein, the term "intracellular signaling domain" in the context of a chimeric antigen receptor refers to a protein portion that transmits an effector function signal and instructs a cell to perform a specific function. The intracellular signaling domain is involved in the primary intracellular signal transduction after the antigen-binding domain binds to an antigen, thereby resulting in the activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. An exemplary intracellular signaling domain includes CD3ζ.

[0026] As used herein, the term "costimulatory domain" in the context of a chimeric antigen receptor refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that provides a second signal necessary for efficient activation and function of T lymphocytes after binding to an antigen. A non-limiting example of a costimulatory domain is the CD28 costimulatory domain.

[0027] As used herein, the term "immune effector cell" or "effector cell" refers to a cell that is involved in an immune response, e.g., a cell that promotes an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0028] As used herein, the term "specifically binds" refers to an antigen-binding molecule (e.g., an antibody or a ligand of an antigen) that typically specifically binds to an antigen and a substantially identical antigen with high affinity, but does not bind to unrelated antigens with high affinity. Affinity is typically reflected by the equilibrium dissociation constant (KD), where a relatively low KD indicates a relatively high affinity. For example, in the case of an antibody, high affinity is typically about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, approximately 1 x 10 -9 M or less, approximately 1 x 10 -10 M or less, 1 x 10 -11 M or less, or 1 x 10 -12 This refers to a KD of M or less. The KD is calculated as follows: KD = Kd / Ka, where Kd represents the dissociation rate and Ka represents the binding rate. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis measurement. For example, see the methods for obtaining KD values shown in the Examples of this specification.

[0029] As used herein, the term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region such that it can specifically bind to an antigen. As used herein, "antibody" encompasses various forms and structures, including intact antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.

[0030] As used herein, the term "antibody" includes typical "four-chain antibodies" belonging to the immunoglobulin class consisting of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting, from N- to C-terminus, of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. If the full-length antibody is of the IgE isotype, it optionally further contains a heavy chain constant region CH4 domain. The light chain refers to a polypeptide chain consisting, from N- to C-terminus, of a light chain variable region (VL) and a light chain constant region (CL), with the heavy and light chains linked by disulfide bonds to form a "Y" structure. Immunoglobulin heavy chain constant regions differ in amino acid composition and sequence, and therefore their antigenicity also differs. Thus, "immunoglobulins" as used herein can be divided into five types, or immunoglobulin isotypes, namely, IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε chains, respectively. Ig types of the same type can be further divided into subclasses, such as IgG1, IgG2, IgG3, and IgG4, and IgA IgA1 and IgA2, depending on the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. Light chains are divided into κ chains and λ chains depending on the constant region. Each of the five types of Ig may have either a κ chain or a λ chain.

[0031] As used herein, the terms "antigen-binding fragment" and "antibody fragment" can be used interchangeably and include only local or local variants of an intact antibody that do not have the entire structure of an intact antibody and have the ability to bind to an antigen. As used herein, "antigen-binding fragment" or "antibody fragment" includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, scFv, and VHH.

[0032] As used herein, the term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH domains are linked by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Hustone et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers include GSTSGSGKPGSGEGSTKG and consist of a repeated GGGGS amino acid sequence or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, although variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers for use in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may be present between the VH and VL of an scFv, forming a disulfide-linked Fv (dsFv).

[0033] As used herein, an "antibody" may be derived from any animal, including, but not limited to, humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or chondrichthyes (e.g., sharks).

[0034] The term "humanized antibody" refers to an antibody obtained by grafting CDR sequences derived from another mammalian species, such as the germline of a mouse, onto human framework sequences. Some residues in the framework segment, called FR, can be modified to retain binding affinity. Humanized antibodies or fragments thereof according to the present application can be prepared by techniques known to those skilled in the art.

[0035] As used herein, the term "variable region" refers to the region of an antibody heavy or light chain involved in binding the antibody to an antigen; "heavy chain variable region" may be used interchangeably with "VH" and "HCVR," and "light chain variable region" may be used interchangeably with "VL" and "LCVR." The heavy and light chain variable domains of a natural antibody (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. As used herein, the terms "complementarity-determining region" and "CDR" are used interchangeably and generally refer to the hypervariable region (HVR) of a heavy chain variable region (VH) or a light chain variable region (VL). Because these regions can form precise complementarity with antigen epitopes in their spatial conformation, they are also called complementarity-determining regions. The CDR of a heavy chain variable region can be abbreviated as HCDR, and the CDR of a light chain variable region can be abbreviated as LCDR. The terms "framework region" or "FR region" are also used interchangeably and refer to those amino acid residues other than CDRs in an antibody heavy chain variable region or light chain variable region. A typical antibody variable region consists of four FR regions and three CDR regions, in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0036] For further descriptions of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol., 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein may be represented and defined in any manner known in the art, including but not limited to the Kabat numbering system, and may be used in tools such as the abYsis site (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0037] As used herein, the term "Kabat numbering system" generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0038] As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the percentage of amino acid (or nucleotide) residues in a candidate sequence that are identical to the amino acid (or nucleotide) residues in a reference sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced in one or both of the candidate and reference sequences, and non-homologous sequences can be ignored for comparison purposes). For purposes of determining percent sequence identity, alignment can be achieved in a variety of ways known to those of skill in the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. Those of skill in the art can determine appropriate parameters to use for measuring alignment, including any algorithms required to achieve maximum alignment within the full length of the sequences being compared. For example, a reference sequence aligned for comparison to a candidate sequence can show that the candidate sequence exhibits 50% to 100% sequence identity over the entire length of the candidate sequence or over a selected portion of consecutive amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, the molecules are identical at that position.

[0039] As used herein, "mutation" includes insertion mutations, deletion mutations, and substitution mutations, and in some embodiments, the substitution mutations are preferably conservative amino acid substitutions.

[0040] As used herein, "conservative amino acids" generally refer to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation and rigidity). Illustratively, amino acids within each of the following groups are conserved amino acid residues, and substitutions of amino acid residues within the groups are conserved amino acid substitutions.

[0041] By way of example, the following six groups are illustrative of amino acids that are considered conservative substitutions for one another: 1) Alanine (A), serine (S), threonine (T), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), Histidine (H), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0042] As used herein, "at most X mutations" refers to the number of mutations that can be selected from any natural number in the range of 0 to X.

[0043] As used herein, "at most 0 to 3 mutations" may mean 3 mutations, 2 mutations, 1 mutation, or 0 mutations. As used herein, "each" in "the above-mentioned HCDRs 1 to 3 each have at most 0 to 3 mutations" means that the number of mutations in each CDR in HCDRs 1 to 3 is independent of each other and may each be independently selected from 0 to 3. Similarly, as used herein, "each" in "the above-mentioned LCDRs 1 to 3 each have at most 0 to 3 mutations" means that the number of mutations in each CDR in LCDRs 1 to 3 is independent of each other.

[0044] As used herein, a "vector" may comprise an isolated nucleic acid and may be comprised of a substance used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be interpreted to include non-plasmid and non-viral compounds that contribute to the introduction of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, etc.

[0045] As used herein, the terms "subject," "subject," and "patient" refer to a living organism undergoing treatment for a particular disease or condition (e.g., cancer or an infectious disease) described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the bovine family (e.g., domestic cattle, bison, buffalo, elephants, yaks), cows, sheep, horses, and bison, that are undergoing treatment for a disease or condition (e.g., a cell proliferative disorder such as cancer or an infectious disease).

[0046] As used herein, the term "treatment" refers to surgical or therapeutic treatment aimed at preventing or slowing (reducing) the progression of an undesirable physiological change or pathology in a subject, such as a cell proliferative disorder (such as cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, a decrease in the extent of disease, a stable disease state (i.e., not worsening), a delay or slowing of disease progression, an improvement or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Subjects in need of treatment include those already suffering from a disease or disorder, as well as those susceptible to a disease or disorder, or those in whom a disease or disorder is to be prevented. References to terms such as slowing, alleviating, reducing, mitigating, and remission also include conditions such as resolution, elimination, and non-occurrence.

[0047] As used herein, the term "effective amount" refers to the amount of a therapeutic agent that, when administered to a cell, tissue, or subject alone or in combination with another therapeutic agent, is effective to prevent or ameliorate the symptoms of a disease or the progression of that disease. "Effective amount" also refers to the amount of a compound sufficient to ameliorate a symptom, e.g., treat, cure, prevent, or ameliorate an associated medical condition, or treat, cure, prevent, or ameliorate an increased rate of such a condition. When an active ingredient is administered alone to an individual, the therapeutically effective dose is the amount of that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined dose of the active ingredients that produces the therapeutic effect, regardless of combined, sequential, or simultaneous administration.

[0048] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes benign and malignant cancers. As used herein, the term "tumor" or "mass" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

[0049] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug and, optionally, a pharmaceutically acceptable vector or additive, combined together to achieve a specific purpose. In certain embodiments, a pharmaceutical composition includes a combination that is temporally and / or spatially separated, as long as they can act together to achieve the purpose of the present application. For example, the components included in the pharmaceutical composition (e.g., CAR-NK cells according to the present application) may be administered to an individual together or separately. When the components included in the pharmaceutical composition are administered to an individual separately, the components may be administered to the individual simultaneously or sequentially. A pharmaceutical composition according to the present application may contain conventional cell culture components to maintain the activity of CAR-NK cells. The pharmaceutically acceptable vector may further include water, a buffered aqueous solution, an isotonic salt solution such as PBS (phosphate buffer solution), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or a polyalkylene glycol such as polypropylene glycol or a triglyceride. The pharmaceutical composition or pharmaceutical formulation according to the present application can be administered by any suitable route, for example, intravenous administration, intradermal, subcutaneous, intramuscular injection, etc. The composition according to the present application may contain, as additives, wetting agents, emulsifying agents, or buffer substances.

[0050] After extensive research, development, and testing, the inventors of the present application have developed a novel chimeric antigen receptor that targets CD19 and uses thereof, and provide a chimeric antigen receptor that targets CD19, as well as corresponding nucleic acid molecules, vectors, immune effector cells, preparation methods and products thereof, pharmaceutical compositions, therapeutic uses, pharmaceutical uses, and methods for treating tumors or cancer. The inventions in each section of the present application achieve at least one of the following beneficial effects: (1) specific killing of CD19-positive tumor cells and / or inhibitory effects on the growth of CD19-associated tumors; (2) the chimeric antigen receptor that targets CD19 is humanized to reduce immunogenicity; and (3) taking NK cells as an exemplary effector cell, it has the potential for use in "shelf" therapeutic products.

[0051] In a first aspect, the present application provides a nucleic acid molecule encoding a chimeric antigen receptor that targets CD19, the chimeric antigen receptor comprising an extracellular domain comprising an antigen-binding region that specifically binds to CD19, a transmembrane domain linked to the extracellular domain, and an intracellular domain linked to the transmembrane domain, the antigen-binding region comprising a VH comprising HCDRs 1 to 3 and a VL comprising LCDRs 1 to 3, wherein: (1) the HCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 9 to 11, respectively, and the LCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 12 to 14, respectively; or (2) the HCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 15 to 17, respectively, and the LCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 18 to 20, respectively; or (3) the HCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 21 to 23, respectively, and the LCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 24 to 26, respectively; or (4) the HCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 27 to 29, respectively, and the LCDRs 1 to 3 have the sequences shown in SEQ ID NOs: 30 to 32, respectively; or (5) The HCDRs 1 to 3 each have at most 0 to 3 mutations (e.g., 0, 1, 2, or 3 mutations) compared to the corresponding CDR in any one of groups (1) to (4), and the LCDRs 1 to 3 each have at most 0 to 3 mutations (e.g., 0, 1, 2, or 3 mutations) compared to the corresponding CDR in any one of groups (1) to (4).

[0052] In some embodiments, (1) the VH has the sequence set forth in SEQ ID NO: 1, and the VL has the sequence set forth in SEQ ID NO: 2, or (2) the VH has the sequence set forth in SEQ ID NO: 40, and the VL has the sequence set forth in SEQ ID NO: 39, or (3) the VH has the sequence set forth in SEQ ID NO: 41, and the VL has the sequence set forth in SEQ ID NO: 38, or (4) the VH has the sequence set forth in SEQ ID NO: 3, and the VL has the sequence set forth in SEQ ID NO: 4, (5) the VH has the sequence set forth in SEQ ID NO: 5, and the VL has the sequence set forth in SEQ ID NO: 6, (6) the VH has the sequence set forth in SEQ ID NO: 7 or 43, and the VL has the sequence set forth in SEQ ID NO: 8 or 42, or (7) the VH has at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, The VL has a sequence with at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, or at most 25 mutations (e.g., at most 20 mutations, at most 15 mutations, at most 10 mutations, at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation), and the VL has a sequence with at least 80% of the VL corresponding to any one of groups (1) to (6). or at most 20 mutations (e.g., at most 15 mutations, at most 10 mutations, at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation).

[0053] In some embodiments, the antigen-binding region is in the form of an scFv.

[0054] In some embodiments, the antigen binding region in scFv form is: (1) the amino acid sequence set forth in SEQ ID NO: 51, 55, or 56; or (2) the amino acid sequence shown in SEQ ID NO: 52, or (3) the amino acid sequence shown in SEQ ID NO: 53, or (4) the amino acid sequence shown in SEQ ID NO: 54 or 57, or (5) An amino acid sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), or at most 50 mutations (e.g., at most 45 mutations, at most 40 mutations, at most 35 mutations, at most 30 mutations, at most 25 mutations, at most 20 mutations, at most 15 mutations, at most 10 mutations, at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation) compared to the amino acid sequence set forth in any one of SEQ ID NOs: 51 to 57.

[0055] EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKFLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQASSIPRMFTFGSGTKLEIKGSTSGSGKPGSGEGSTKGQVQLQQPGAELVVPGTSVKLSCKASGYTFTNYWMHWVKQRPGQGLEWIGEIDPSDNYANYNQEFQGKATLTVDKSSSTAYMQLSSLTSDDSAVYYCARHDGYFGALDYWGQGTSVTVSS (SEQ ID NO: 51, contained in Mab01-CAR) DVVLTQTPLSLPVSLGDQASISCRSSQSLENSNGNSYLNWYLQKPGQSPQLLIYRVSNRFSGVLDRFSGSGSGTDFTLKISRVEAEDLGVYFCLQITHVPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGEVQLQQSGPELVKPGASVKMSCKASGYTFTDYVMHWVRQTPGQGLEWIGYFNPYNDGTNYNEKFKVKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGVYYYGRDFDYWGQGTTLTVSS (SEQ ID NO: 52, contained in Mab02-CAR) DVVMTQTPLTLSVTIGQSASISCKSSQSLLESDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSASGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQVQLQQSGAELVRPGASVTLSCKASGYTFPDYEIHWVKQTPVHGLEWIGAIDPETGGIGYNQKFTGKAMLTADKSSSTAYMELRSLTSEDSAVYFCTRNYGSRWGQGSTLTVSS (SEQ ID NO: 53, contained in Mab03-CAR) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 54, contained in FMC63-CAR) EIVLTQSPATLSLSPGERATLSCSASSSISSNYLHWYQQKPGQAPRFLIYRTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQASSIPRMFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLQQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQRPGQGLEWMGEIDPSDNYANYNQEFQGRVTITVDKSASTAYMELSSLRSEDTAVYYCARHDGYFGALDYWGQGTTVTVSS (SEQ ID NO: 55, contained in Hab01-4-CAR) EIVLTQSPDFQSVTPKEKVTITCSASSSISSNYLHWYQQKPDQSPKFLIYRTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQASSIPRMFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQRPGQGLEWMGEIDPSDNYANYNQEFQGRVTITVDKSASTAYMELSSLRSEDTAVYYCARHDGYFGALDYWGQGTTVTVSS (SEQ ID NO: 56, Hab01-11-CAR) DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGGAVKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPYTFGGGTKVEIKGSTSGSGKPGSGEGSTKGEVTLKESGPVLVKPTETLTLTCTVSGFSLSDYGVSWIRQPPGKALEWLAVIWGSETTYYNSALKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCAKHYYYGGSYAMDYWGQGTTVTVSS (SEQ ID NO: 57, contained in hFMC63-CAR)

[0056] In some embodiments, the chimeric antigen receptor comprises a CD8α signal peptide, an antigen-binding region that specifically binds to CD19, a CD8 hinge region, a CD8 transmembrane region, a CD28 costimulatory domain, and CD3ζ.

[0057] In some embodiments, the CD8α signal peptide has the amino acid sequence set forth in SEQ ID NO: 33 or at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 33 or at most 10 mutations (e.g., at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation).

[0058] MALPVTALLLPLALLLHAARP (SEQ ID NO: 33)

[0059] In some embodiments, the CD8α hinge region has the amino acid sequence set forth in SEQ ID NO: 47, or at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 47, or at most 10 mutations (e.g., at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation).

[0060] FVPVFLPAKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 47)

[0061] In some embodiments, the CD8 transmembrane domain has the amino acid sequence set forth in SEQ ID NO:48, or at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO:48, or at most 10 mutations (e.g., at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation).

[0062] IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 48)

[0063] In some embodiments, the CD3ζ has the amino acid sequence set forth in SEQ ID NO:49, or at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO:49, or at most 10 mutations (e.g., at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation).

[0064] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 49)

[0065] In some embodiments, the CD28 costimulatory domain has an amino acid sequence set forth in SEQ ID NO: 50, or has at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 50, or at most 10 mutations (e.g., at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation).

[0066] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 50)

[0067] In some embodiments, the chimeric antigen receptor further comprises a cytokine linked by a self-cleaving peptide. In some embodiments, the cytokine is IL15. In some embodiments, the self-cleaving peptide is selected from F2A, T2A, P2A, and E2A.

[0068] In some embodiments, the chimeric antigen receptor has a sequence set forth in any one of SEQ ID NOs: 34 to 37 or 44 to 46, or has at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) or at most 120 amino acid mutations compared to any one of SEQ ID NOs: 34 to 37 or 44 to 46. (e.g., at most 110 mutations, at most 100 mutations, at most 90 mutations, at most 80 mutations, at most 70 mutations, at most 60 mutations, at most 50 mutations, at most 45 mutations, at most 40 mutations, at most 35 mutations, at most 30 mutations, at most 25 mutations, at most 20 mutations, at most 15 mutations, at most 10 mutations, at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations, or 1 mutation).

[0069] In some embodiments, the nucleic acid molecule is DNA or RNA.

[0070] In some embodiments, the RNA is mRNA.

[0071] In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule. In some embodiments, the nucleic acid molecule is operably linked to a regulatory sequence that can be recognized by a host cell transformed with the vector.

[0072] In a second aspect, the present application provides a chimeric antigen receptor that targets CD19 encoded by a nucleic acid molecule according to the first aspect.

[0073] In a third aspect, the present application provides a vector (eg, an expression vector) comprising the nucleic acid molecule according to the first aspect.

[0074] In a fourth aspect, the present application provides an immune effector cell, wherein said immune effector cell is transfected with a nucleic acid molecule according to the first aspect, or transfected with a vector according to the third aspect, or expresses a chimeric antigen receptor according to the second aspect.

[0075] In some embodiments, the immune effector cells are NK cells, T cells, or NKT cells. In some embodiments, the immune effector cells are NK cells. In some embodiments, the NK cells are differentiated from iPSCs or derived from peripheral blood or umbilical cord blood.

[0076] Techniques for culturing, expanding, and obtaining NK cells in vitro are numerous, and the general principles and methodologies are known to those skilled in the art.

[0077] In some embodiments, NK cells are obtained by in vitro culture and expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells. PBMCs are one of the main sources of NK cells, and have advantages such as relatively easy collection, easy in vitro expansion, and no toxic side effects. However, the percentage of NK cells in PBMCs is generally 10% to 15%. Methods for expanding PBMC-derived NK cells include stimulating in vitro proliferation of NK cells using a combination of cytokines, feeder cells, or membrane particles.

[0078] In some embodiments, NK cells are obtained by in vitro culture and expansion of umbilical cord blood-derived NK cells. Generally, there are two different methods by which large amounts of NK cells can be obtained from umbilical cord blood: one is to expand NK cells in umbilical cord blood, and the other is to induce the differentiation of umbilical cord blood CD34+ hematopoietic stem / progenitor cells into NK cells, followed by expansion.

[0079] In some embodiments, the NK cells are obtained from the in vitro culture and expansion of an NK cell line. Illustratively, NK-92 is a homogenous immortalized NK lymphoma cell line, the first NK cell-based immunotherapy approved by the FDA for clinical trials.

[0080] In some embodiments, the NK cells are obtained from induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs) that have been induced, cultured, and expanded in vitro.

[0081] In a fifth aspect, the present application provides a method of preparing immune effector cells, the method comprising the steps of providing immune effector cells and introducing into the immune effector cells a nucleic acid molecule according to the first aspect or a vector according to the third aspect. In some embodiments, the method further comprises culturing the immune effector cells after introducing the nucleic acid molecule or vector.

[0082] In a sixth aspect, the present application provides a product prepared according to the method of the fifth aspect.

[0083] In a seventh aspect, the present application provides a pharmaceutical composition, said pharmaceutical composition comprising a nucleic acid molecule according to the first aspect, or a vector according to the third aspect, or an immune effector cell according to the fourth aspect, or a product according to the sixth aspect, and a pharmaceutically acceptable vector.

[0084] In some embodiments, the pharmaceutical composition is used to treat a tumor or cancer. In some embodiments, the tumor or cancer is a CD19-associated tumor or cancer. In some embodiments, the tumor or cancer is a B-cell malignancy. In some embodiments, the tumor or cancer is acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, dual spectrum leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma.

[0085] In some embodiments, a CD19-associated tumor or cancer refers to the expression of CD19 on the surface of tumor or cancer cells. In some embodiments, the tumor is a tumor that highly expresses CD19 (CD19+). In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 60% of tumor cells in a tumor cell population expressing CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 70% of tumor cells in a tumor cell population expressing CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 80% of tumor cells in a tumor cell population expressing CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 90% of tumor cells in a tumor cell population expressing CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 95% of tumor cells in a tumor cell population expressing CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 98% of tumor cells in a tumor cell population expressing CD19. In some embodiments, a tumor that highly expresses CD19 (CD19+) refers to at least 99% of tumor cells in a tumor cell population expressing CD19.

[0086] In an eighth aspect, the present application provides use of a nucleic acid molecule according to the first aspect, or a vector according to the third aspect, or an immune effector cell according to the fourth aspect, or a product according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect, in the preparation of a medicament for treating a tumor or cancer. In some embodiments, the tumor or cancer is a CD19-associated tumor or cancer. In some embodiments, the tumor or cancer is a B-cell malignancy. In some embodiments, the tumor or cancer is acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, dual spectrum leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma.

[0087] In a ninth aspect, the present application provides a method of treating cancer or tumor, comprising administering to a subject in need thereof an effective amount of a nucleic acid molecule according to the first aspect, or a vector according to the third aspect, or an immune effector cell according to the fourth aspect, or a product according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect. In some embodiments, the tumor or cancer is a CD19-associated tumor or cancer. In some embodiments, the tumor or cancer is a B-cell malignancy. In some embodiments, the tumor or cancer is acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin's lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt's lymphoma, dual spectrum leukemia, biphenotypic leukemia, hairy cell leukemia, precursor B acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma, or marginal zone B-cell non-Hodgkin's lymphoma.

[0088] It should be understood that the above detailed description is merely intended to enable those skilled in the art to more clearly understand the contents of the present application, and is not intended to be limiting in any way. Those skilled in the art may make various modifications and changes to the described embodiments. [Example]

[0089] The present application will be further described below with reference to specific examples, and the advantages and features of the present application will become more apparent with the following description. Unless specific conditions are specified in the examples, general conditions or conditions recommended by the manufacturer are used. Unless the manufacturer of the reagents or equipment used is specified, they are all common products available on the market.

[0090] The examples of the present application are merely illustrative and do not limit the scope of the present application. Those skilled in the art may modify or replace the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and it should be understood that all such modifications and replacements fall within the scope of protection of the present application.

[0091] Materials and Methods

[0092] SPR detection method

[0093] Anti-human CD19 antibodies were captured using a Protein A chip (GE Healthcare; 29-127-558). The sample and running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare, BR-1006-69). The flow cell was set to 25°C. The sample block was set to 16°C. Both were pretreated with running buffer. In each cycle, the antibody to be measured was first captured on the Protein A chip. Next, a single concentration of CD19 antigen protein was injected, and the binding and dissociation processes between the antibody and antigen protein were recorded. Finally, the chip was regenerated using Glycine pH 1.5 (GE Healthcare, BR-1003-54). Binding was measured by continuous injection of different concentrations of recombinant human CD19-His in solution for 240 seconds, with a flow rate of 30 μL / min, starting at 200 nM (see detailed results for actual concentrations tested), for a total of five concentrations at 1:1 dilutions. The dissociation phase was monitored for up to 600 seconds and triggered by switching from the sample solution to the running buffer. The surface was regenerated by washing with 10 mM glycine solution (pH 1.5) for 30 seconds at a flow rate of 30 μL / min. Differences in bulk refractive index were corrected by subtracting the response obtained from the goat anti-human Fc surface. A blank injection was also subtracted (= double referencing). The apparent KD and other kinetic parameters were calculated using the Langmuir 1:1 model.

[0094] Virus packaging

[0095] The day before virus packaging, 293T cells (purchased from ATCC) were digested with pancreatin and seeded in 10 cm dishes at a density of 1E7 cells per dish. To transfect the cells, the packaging plasmid and target plasmid were mixed and added to α-MEM medium. FuGENE® HD Transfection Reagent (Promega, E2311) was added to a separate α-MEM-containing centrifuge tube. The diluted transfection reagent was added dropwise on top of the diluted plasmid, mixed evenly, and allowed to stand at room temperature for 15 minutes. Finally, the mixture of the plasmid and transfection reagent was added to the 10 cm dish, gently shaken 10 times to mix evenly, and placed in an incubator. After transfection of the cells for 3 days, the virus was harvested, and 10 mL of the virus-containing culture supernatant was transferred to a 50 mL centrifuge tube and centrifuged at 1250 rpm at 4°C for 5 minutes to remove dead 293T cells. The virus-containing supernatant was filtered and concentrated using Retro-X Concentrator (Clontech, 631455). After aliquoting, it was stored at -80°C for further use.

[0096] NK cell purification and activation

[0097] NK cells are isolated from mononuclear cells and purified in two steps. The first step is CD3 + In a second step, the cells are removed and CD56 + Cells were enriched by washing, incubation with CD3 magnetic beads, and resuspension using Sepax, followed by CD3 enrichment using CliniMACS. + The cells were removed. Sepax was used again to remove CD3 + CD3 after cell removal - The cell product was washed, incubated with CD56 magnetic beads, and resuspended, followed by CD56 analysis using the CliniMACS. + The cells were enriched. After enrichment, CD56 +The cells were washed with Sepax, replaced with complete medium, and K562 feeder cells were added and transferred to a 37°C incubator for activation for 5 days.

[0098] Viral transduction

[0099] On day 1, RetroNectin reagent was added to a 24-well plate at 500 μL / well and coated overnight at 4°C. On day 2, the upper layer of RetroNectin was discarded, and the plate was washed with PBS. Afterwards, packaged retrovirus was added and centrifuged at 2000 g for 60 minutes at 4-8°C. The upper layer of virus was discarded. NK cells activated for 5 days were added to a 24-well plate at 3E5 cells / well, centrifuged at 400 g for 5 minutes at room temperature, and then cultured in a 37°C incubator (37°C, 5% CO2). On day 3, NK cells were transferred to a non-tissue culture-treated 6-well plate and cultured. On day 6, CAR expression was detected to ensure that the CAR positivity of the NK cells used for in vitro or in vivo killing was in the range of 60-80%.

[0100] Measurement of in vitro tumor cell killing rate

[0101] Unless otherwise specified, all target cells shown in the following examples were stably transfected with the luciferase gene and expressed luciferase. The fluorescence intensity detected by the luciferase reporter gene detection reagent reflected the cell activation rate and NK cell killing effect. The killing rate of the test well was calculated as follows: Killing rate = (target cell well read value - test well read value) / target cell well read value × 100%.

[0102] Example 1 Preparation of CD19 antibody

[0103] Mice were immunized with human CD19 protein (NCBI: NP_001761.3) as an immunogen. Mice with high serum antibody titers and platform-specific titers were selected, and their splenocytes were collected and fused with myeloma cells, SP2 / 0. After culture and screening, positive monoclonal hybridoma cells were obtained. The antibodies secreted by the positive monoclonal hybridoma cells were identified, and positive monoclonal hybridomas Mab01, Mab02, and Mab03 capable of secreting CD19 antibodies were obtained. RNA from the positive monoclonal hybridoma cells was extracted and reverse transcribed into cDNA. Sequences of the heavy and light chain variable regions of the hybridoma-positive clone antibodies were obtained by sequencing. The variable region sequences and their CDR (Kabat numbering system) sequence information are shown in Tables 1 and 2.

[0104] The heavy and light chain variable regions of Mab01, Mab02, Mab03, and the positive control FMC63 were cloned into an expression vector containing a signal peptide and a human IgG1 heavy chain constant region, and another expression vector containing a signal peptide and a human IgG1 light chain constant region, to express human-mouse chimeric antibodies. The binding ability of the chimeric antibodies to human CD19 protein was detected by SPR (Biacore). The SPR detection results are shown in Table 3. Mab01 to Mab03 all have relatively good affinity for human CD19 protein.

[0105] Table 1. Antibody sequence information [Table 1]

[0106] Table 2. CDRs corresponding to antibodies [Table 2]

[0107] Table 3. Detection of affinity of chimeric antibodies to human CD19 by SPR (Biacore) [Table 3]

[0108] Example 2 Screening for chimeric antigen receptors targeting CD19

[0109] The obtained anti-CD19 antibodies and the positive control FMC63 antibody were screened as described in Example 1, and a chimeric antigen receptor targeting CD19 was constructed as shown in Figure 1. Specifically, the chimeric antigen receptor targeting CD19 contains, in order, a CD8α signal peptide (SP), an anti-CD19 scFv, a CD8 hinge region, a CD8 transmembrane region, a CD28 costimulatory domain, and CD3ζ, as well as IL15 linked by the self-cleaving peptide P2A. The specific sequence is detailed in Table 4. The nucleic acid molecule encoding the chimeric antigen receptor was cloned into a retroviral vector, packaged, and then used to transfect NK cells.

[0110] Six days after retroviral infection of NK cells, we detected the specific killing of CD19-expressing target cells, Nalm6 and Raji, and the nonspecific killing of non-CD19-expressing target cells, Molm13, by CD19-CAR NK cells. Three gradients of CAR NK-CD19 cells:target cells (effector cell to target cell ratios) were set up: 0.5:1, 1:1, and 2:1, respectively. Target cells were seeded at 1E4 / well in a white opaque 96-well plate, and NK cells were added according to the effector cell to target cell ratio. The plates were cultured for 4 hours in a 37°C, 5% CO2 incubator. 30 μL of FIREFLYGLO luciferase reporter gene detection reagent (MeilunBio, MA0519-1) was added, and the plates were incubated at room temperature for 10 minutes in the dark. The killing rate was then measured using a microplate reader. The detection results are shown in Figures 2 to 4. The results showed that FMC63-CAR, Mab01-CAR, and Mab03-CAR all had relatively strong specific killing effects against CD19-positive cells (Nalm6 and Raji cells), and there was no significant difference in the killing effects. FMC63-CAR, Mab01-CAR, Mab02-CAR, and Mab03-CAR did not show nonspecific killing against CD19-negative cells.

[0111] The tumor-inhibitory effects of Mab01-CAR and Mab03-CAR were further compared using a mouse tumor model. Specifically, NSG mice were weighed and randomly divided into groups according to Table 5. Human acute lymphoblastic leukemia cells Nalm6 (harboring the luciferase gene) were inoculated at a cell dose of 0.25E6. CD19 CAR NK cells (3E6 CAR-NK cells per mouse) were intravenously injected in a single dose, and the inhibitory effect on tumor cell growth in mice was observed. The results are shown in Figure 5. Compared to NT (non-CD19-CAR transfected NK cells), Mab01-CAR, Mab03-CAR, and FMC63-CAR all exhibited tumor-inhibitory effects. Among them, Mab01-CAR's tumor-inhibitory effect was stronger and significantly superior to Mab03-CAR and FMC63-CAR. Mab01-CAR was selected for further development.

[0112] Table 4. CD19 chimeric antigen receptor sequence information [Table 4-1] [Table 4-2]

[0113] Table 5. NSG mouse grouping status [Table 5]

[0114] Example 3 Humanization of Mouse Monoclonal Antibody Mab01

[0115] To reduce the immunogenicity of Mab01-CAR, Mab01 was humanized. Specifically, the heavy chain variable region germline genes IGHV1-3*01 and IGHJ6*01, and the light chain variable region germline genes IGKV6-21*01 / IGKV3-11*01 and IGKJ2*01, which are highly homologous to the mouse antibody Mab01, were selected as templates based on a comparison with the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database. The CDRs of the mouse antibody were then grafted onto the corresponding human templates to form a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Where necessary, key amino acids in the framework sequence were backmutated to their corresponding amino acids in the mouse antibody to ensure the original affinity. For example, sites prone to chemical modification in the antibody were mutated to eliminate the risk of modification. The CDR amino acid residues of the antibody were determined using the Kabat numbering system. The sequence information of the humanized VH and VL is shown in Table 6, and the combinations of humanized antibody variable regions formed are shown in Table 7.

[0116] The heavy chain variable regions and light chain variable regions of Hab01-4 and Hab01-11 were cloned into an expression vector containing a signal peptide and a mouse IgG1 heavy chain constant region, and an expression vector containing a signal peptide and a mouse IgG1 light chain constant region, respectively, to express humanized antibodies of the variable regions. The binding ability of the chimeric antibodies to human CD19 protein was detected by SPR (Biacore), as shown in Table 8. Both Hab01-4 and Hab01-11 have strong binding ability to human CD19 protein.

[0117] Using the same method, FMC63 was humanized, and the humanized antibody hFMC63 was constructed and expressed. The binding ability of the antibody to human CD19 protein was detected by SPR (Biacore). For detailed information, see Tables 6 and 9. Both hFMC63 and human CD19 have relatively strong binding ability.

[0118] Table 6. Humanized antibody sequence information for Mab01 and FMC63 [Table 6]

[0119] Table 7. Mab01 humanized antibody variable region combinations [Table 7]

[0120] Table 8. Detection of affinity of Mab01 humanized antibody to human CD19 by SPR (Biacore) [Table 8]

[0121] Table 9. Detection of affinity of FMC63 humanized antibody to human CD19 by SPR (Biacore) [Table 9]

[0122] Example 4 Screening of humanized chimeric antigen receptors targeting CD19

[0123] The humanized antibodies Hab01-4, Hab01-11, and hFMC63 prepared in Example 3 were loaded onto the chimeric antigen receptor (CAR) shown in Figure 1 to construct a humanized CAR targeting CD19 (see Table 10 for specific sequence information). Nucleic acid molecules corresponding to the CAR were cloned into retroviral vectors, packaged, and transfected into umbilical cord blood-derived NK cells. The NK cell proliferation and CAR positivity were monitored after transfection, and the results are shown in Figures 6A and 6B. As shown in Figure 6A, the NK cells transfected with the different CAR constructs had essentially equivalent proliferation capabilities. As shown in Figure 6B, Hab01-4-CAR and Hab01-11-CAR had the highest CAR positivity, while hFMC63-CAR had a slightly lower CAR positivity. FMC63-CAR had the lowest CAR positivity, but all were within the 60-80% range, meeting the requirements. Six days after infection of NK cells with the retrovirus, sufficient cells were collected and cryopreserved.

[0124] Prior to the experiment to detect target cell killing activity, cryopreserved CAR-NK cells were resuscitated. The same number of CD19-CAR NK cells was mixed with target cell Raji cells immediately after resuscitation (0 h) and 24 h after resuscitation. The in vitro killing effect of NK cells against target cells (Raji: 1E6 cells / mL) was measured at different effector cell-to-target cell ratios. The results are shown in Figures 7A-7B. Immediately after resuscitation (Figure 7A) and 24 h after resuscitation (Figure 7B), FMC63-CAR, hFMC63-CAR, Hab01-4-CAR, and Hab01-11-CAR all had relatively strong killing effects against Raji cells. In contrast, the killing effect of CAR-NK cells was superior after 24 h of resuscitation.

[0125] A Nalm6 xenograft mouse model was established to evaluate the tumor-suppressing effects of FMC63-CAR, hFMC63-CAR, Hab01-4-CAR, and Hab01-11-CAR. Specifically, NOG mice were selected, weighed, and randomly assigned to groups (the grouping scheme is detailed in Table 11). Human acute lymphoblastic leukemia Nalm6 (harboring the luciferase gene) was injected at a dose of 2.5E6 cells per mouse. Two days later, CD19 CAR NK cells (4E6 CAR-NK cells per mouse) were intravenously injected at a single dose. The injection of CD19 CAR NK cells was defined as day 0. The mice in G1, G3, G4, G6, G8, and G10 groups were continuously monitored for in vivo tumor progression (fluorescence imaging), mouse weight, and survival. Mice in groups G2, G5, G7, G9, and G11 were given peripheral blood samples on days 0, 7, 16, and 21 to inject CAR19. + CD56 + and CAR19 - CD56 + On days 0, 7, 14 and 28, peripheral blood was collected for routine blood tests and blood biochemistry indicators, and the results are shown in Figures 8A to 8E, 9A to 9B and 10.

[0126] As shown in FIGS. 8A to 8E, the tumor signals of the post-tumor negative control groups (G1 group and G3 group) continuously increased, and all four CD19 CAR NK treatment groups (G4, G6, G8, and G10) showed significant anti-tumor efficacy and could significantly extend the survival period of mice. Among the four CD19 CAR NK treatment groups, the treatment effect of the Hab01-4-CAR NK treatment group (G8 group) was optimal. The G8 group, like the other treatment groups, had a recurrence of tumor metastasis to the head and face on the 26th day. However, during the period from the 30th to the 33rd day, through the observation of dorsal imaging and ventral imaging, it was found that no recurrence occurred in other parts of the G8 group, while recurrence was also observed in other parts of the other treatment groups besides the head and face. At the same time, from the survival curve, the survival rate of the mice in the G8 group was 80% (on the 36th day), which was better than that of the other treatment groups. In terms of weight change, the four CD19 CAR-NK treatment groups had good mouse conditions and no treatment-related toxicity such as weight loss until the 19th day. From the 19th day, weight loss began to appear, and at the same time, a fluorescence signal was detected in the mandible, and weight loss of the mice due to tumor recurrence was observed. As shown in FIGS. 9A to 9B, CD19 CAR + CD56 + In terms of the ratio, G5 group > G9 group > G11 group > G7 group, and in terms of the ratio of CD19 CAR - CD56 + G11 group < G9 group = G7 group < G5 group. In the G5, G7, G9, and G11 groups using humanized antibodies, the relative number of CAR + NK cells in peripheral blood mononuclear cells of the G9 group was higher. Routine blood tests and blood biochemical indexes are shown in detail in FIG. 10. In the G9 group, the levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were lower compared with those of the other CD19 CAR NK treatment groups, indicating that it was safer overall.

[0127] Table 10. Sequence information of humanized chimeric antigen receptors targeting CD19

Table 10-1

Table 10-2

[0128] Table 11. NOG mouse grouping status [Table 11]

[0129] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0130] Although exemplary embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the embodiments contained in the appended claims.

Claims

Claim 1 A nucleic acid molecule encoding a chimeric antigen receptor targeting CD19, wherein the chimeric antigen receptor comprises an extracellular domain comprising an antigen-binding region that specifically binds to CD19, a transmembrane domain linked to the extracellular domain, and an intracellular domain linked to the transmembrane domain, and the antigen-binding region comprises a VH comprising HCDR1-3 and a VL comprising LCDR1-3, wherein (1) the HCDR1-3 each have the sequences shown in SEQ ID NOs: 9-11, and the LCDR1-3 each have the sequences shown in SEQ ID NOs: 12-14, or (2) the HCDR1-3 each have the sequences shown in SEQ ID NOs: 15-17, and the LCDR1-3 each have the sequences shown in SEQ ID NOs: 18-20, or (3) the HCDR1-3 each have the sequences shown in SEQ ID NOs: 21-23, and the LCDR1-3 each have the sequences shown in SEQ ID NOs: 24-26, or (4) the HCDR1-3 each have the sequences shown in SEQ ID NOs: 27-29, and the LCDR1-3 each have the sequences shown in SEQ ID NOs: 30-32, or (5) the HCDR1-3 each have 0 to 3 mutations compared to each corresponding CDR in any one of groups (1) to (4), and the LCDR1-3 each have 0 to 3 mutations compared to each corresponding CDR in any one of groups (1) to (4), Nucleic acid molecule. Claim 2 (1) the VH has the sequence shown in SEQ ID NO: 1, and the VL has the sequence shown in SEQ ID NO: 2, or (2) the VH has the sequence shown in SEQ ID NO: 40, and the VL has the sequence shown in SEQ ID NO: 39, or (3) the VH has the sequence shown in SEQ ID NO: 41, and the VL has the sequence shown in SEQ ID NO: 38, or (4) the VH has the sequence shown in SEQ ID NO: 3, and the VL has the sequence shown in SEQ ID NO: 4, (5) the VH has the sequence set forth in SEQ ID NO: 5, and the VL has the sequence shown in SEQ ID NO: 6, (6) the VH has the sequence set forth in SEQ ID NO: 7 or 43, and the VL has the sequence shown in SEQ ID NO: 8 or 42, or (7) The VH has a sequence having at least 80% identity or at most 25 mutations as compared to each corresponding VH in any one of groups (1) to (6), and the VL has a sequence having at least 80% identity or at most 20 mutations as compared to the VL corresponding to any one of groups (1) to (6). The nucleic acid molecule according to claim 1.

3. The antigen-binding region is in the form of a scFv, and the scFv (1) the amino acid sequence shown in SEQ ID NO: 51, 55, or 56, or (2) the amino acid sequence shown in SEQ ID NO: 52, or (3) the amino acid sequence shown in SEQ ID NO: 53, or (4) the amino acid sequence shown in SEQ ID NO: 54 or 57, or, (5) has an amino acid sequence having at least 80% identity or at most 50 mutations as compared to the amino acid sequence shown in any one of SEQ ID NOs: 51 to 57. The nucleic acid molecule according to any one of claims 1 to 2.

4. The chimeric antigen receptor comprises a CD8α signal peptide, an antigen-binding region that specifically binds to CD19, a CD8 hinge region, a CD8 transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ, and preferably, the CD8α signal peptide has the amino acid sequence shown in SEQ ID NO: 33, or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 33, or has at most 10 mutations, and / or the CD8α hinge region has the amino acid sequence shown in SEQ ID NO: 47, or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 47, or has at most 10 mutations, and / or the CD8 transmembrane region has the amino acid sequence shown in SEQ ID NO: 48, or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 48, or has at most 10 mutations, and / or the CD3ζ has the amino acid sequence shown in SEQ ID NO: 49, or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 49, or has at most 10 mutations, and / or the CD28 co-stimulatory domain has the amino acid sequence shown in SEQ ID NO: 50, or has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 50, or has at most 10 mutations. The nucleic acid molecule according to any one of claims 1 to 3.

5. The chimeric antigen receptor further comprises a cytokine linked by a self-cleaving peptide, and optionally, the cytokine is IL15, and / or the self-cleaving peptide is selected from F2A, T2A, P2A, and E2A. The nucleic acid molecule according to any one of claims 1 to 4.

6. The chimeric antigen receptor has a sequence shown in any one of SEQ ID NOs: 34 to 37 or 44 to 46, or has at least 80% identity compared to any one of SEQ ID NOs: 34 to 37 or 44 to 46, or has a sequence having at most 120 amino acid mutations. The nucleic acid molecule according to any one of claims 1 to 5.

7. The nucleic acid molecule is DNA or RNA, and preferably, the RNA is mRNA. The nucleic acid molecule according to any one of claims 1 to 6.

8. A chimeric antigen receptor targeting CD19, encoded by the nucleic acid molecule according to any one of claims 1 to 7. Chimeric antigen receptor.

9. A vector comprising the nucleic acid molecule according to any one of claims 1 to 7. Vector.

10. An immune effector cell, wherein the immune effector cell has introduced therein the nucleic acid molecule according to any one of claims 1 to 7, or has introduced therein the vector according to claim 9, or expresses the chimeric antigen receptor according to claim 8. Immune effector cell.

11. The immune effector cell is an NK cell, a T cell, or an NKT cell, and preferably, the NK cell. The immune effector cell according to claim 10.

12. The NK cell is differentiated from iPSC or is derived from peripheral blood or umbilical cord blood. The immune effector cell according to claim 11.

13. A method for preparing the immune effector cell according to any one of claims 10 to 12, the method comprising the steps of providing an immune effector cell and introducing into the immune effector cell the nucleic acid molecule according to any one of claims 1 to 7 or the vector according to claim 9, and optionally, the method further comprises culturing the immune effector cell after introduction of the nucleic acid molecule or the vector. Method.

14. A product prepared by the method according to claim 13. Product.

15. A pharmaceutical composition comprising the nucleic acid molecule according to any one of claims 1 to 7, the vector according to claim 9, the immune effector cell according to any one of claims 10 to 12 or the product according to claim 14, and a pharmaceutically acceptable vector. Pharmaceutical composition.

16. The pharmaceutical composition according to claim 15 for use in the treatment of a tumor or cancer, wherein the tumor or cancer is preferably a CD19-related tumor or cancer, more preferably a B-cell malignancy, such as acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenström macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt lymphoma, dual spectrum leukemia, dual phenotype leukemia, hairy cell leukemia, precursor B acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma or marginal zone B-cell non-Hodgkin lymphoma. The pharmaceutical composition according to claim 15.

17. Use of the nucleic acid molecule according to any one of claims 1 to 7, the vector according to claim 9, the immune effector cell according to any one of claims 10 to 12, the product according to claim 14, or the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating a tumor or cancer, wherein the tumor or cancer is preferably a CD19-related tumor or cancer, more preferably a B-cell malignancy, such as acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenström macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large cell lymphoma, Burkitt lymphoma, dual spectrum leukemia, dual phenotype leukemia, hairy cell leukemia, precursor B acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma or marginal zone B-cell non-Hodgkin lymphoma. Use

18. A method for treating cancer or a tumor, comprising administering to a subject in need thereof an effective amount of the nucleic acid molecule according to any one of claims 1 to 7, the vector according to claim 9, the immune effector cell according to any one of claims 10 to 12, the product according to claim 14, or the pharmaceutical composition according to claim 15, wherein the tumor or cancer is preferably a CD19-related tumor or cancer, more preferably a B-cell malignancy, such as acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, plasmacytoma, monoclonal gammopathy of undetermined significance, Waldenström macroglobulinemia (lymphoplasmacytic lymphoma), heavy chain disease, primary amyloidosis, post-transplant lymphoproliferative disorder, Hodgkin lymphoma, MALT lymphoma, B-cell lymphoma, mantle cell lymphoma, (germinal center-like) diffuse large B-cell lymphoma, Burkitt lymphoma, dual spectrum leukemia, dual phenotype leukemia, hairy cell leukemia, precursor B acute lymphoblastic leukemia / lymphoma, primary cutaneous follicle center lymphoma, follicular lymphoma or marginal zone B-cell non-Hodgkin lymphoma Method

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