Immune effector cell and application thereof

Abstract

The invention provides an immune effector cell targeting CD19 and CD20 and application of the immune effector cell. Comprising scFv targeting CD19 and CD20, a hinge region, a transmembrane region and an intracellular signal structural domain. The invention also provides a polynucleotide targeting CD19 and CD20, a corresponding expression vector, a CAR-T cell and an application of the CAR-T cell.

Description

[0001] This application is a divisional application of the invention application with an international application date of May 20, 2024, a date of entry into the Chinese national phase of December 31, 2024, a national application number of 202480003276.7, and an invention name of “Immune Effector Cells and Their Applications”. Technical Field

[0002] The present invention relates to the field of biomedicine, and more specifically to immune effector cells targeting CD19 and CD20 and applications thereof. Background Art

[0003] Although anti-CD19 CAR-T has outstanding efficacy, CD19 chimeric antigen receptor (CAR) T cell therapy also has many problems, and some patients still have poor efficacy and are prone to relapse. Therefore, there is an urgent need to develop methods to effectively treat tumors and prevent antigen escape in this field. Summary of the invention

[0004] The object of the present invention is to provide an immune effector cell that can recognize CD19 and CD20.

[0005] The first aspect of the present invention provides a multifunctional immune effector cell, wherein the immune effector cell comprises a chimeric receptor that binds to CD19 and CD20, or comprises a first chimeric receptor that binds to CD19 and a second chimeric receptor that binds to CD20, wherein the structure of the chimeric receptor that binds to CD19 and CD20 is shown in the following formula:

[0006] (1)LV L1 -V H2 -IV L2 -V H1 -H-TM-C-CD3ζ

[0007] (2)LV L2 -V H1 -IV L1 -V H2 -H-TM-C-CD3ζ

[0008] (3)LV H1 -V L2 -IV H2 -V L1 -H-TM-C-CD3ζ

[0009] (4)LV H2 -V L2 -IV L1 -V H1 -H-TM-C-CD3ζ

[0010] The structures of the first chimeric receptor binding to CD19 and the second chimeric receptor binding to CD20 are shown in the following formula:

[0011] (5)LV L1 -V H1 -H-TM-C-CD3ζ and LV H2 -V L2 -H-TM-C-CD3ζ

[0012] (6)LV L1 -V H1 -H-TM-C-CD3ζ and LV L2 -V H2 -H-TM-C-CD3ζ

[0013] (7)LV H2 -V L2 -H-TM-C-CD3ζ and LV H1 -V L1 -H-TM-C

[0014] (8)LV H1 -V L1 -H-TM-C-CD3ζ and LV H2 -V L2 -H-TM-C

[0015] (9)LV H1 -V L1 -H-TM-C-CD3ζ and LV H2 -V L2 -H-TM-C-CD3ζ

[0016] In the formula, each "-" is independently a connecting peptide or a peptide bond; L is an optional signal peptide sequence; I is a flexible linker; H is an optional hinge region; TM is a transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is an intracellular signal transduction sequence derived from CD3ζ; V H1 V is the variable region of the heavy chain of CD19 antibody; L1 V is the variable region of CD19 antibody light chain; L2 V is the variable region of CD20 antibody light chain; H2 It is the variable region of CD20 antibody heavy chain; “-” is a connecting peptide or peptide bond.

[0017] In one example, the V H1 comprising the amino acid sequence shown in SEQ ID NO: 21; V L1 comprising the amino acid sequence shown in SEQ ID NO: 22; said V H2 comprising the amino acid sequence shown in SEQ ID NO: 23; said V L2Comprising the amino acid sequence shown in SEQ ID NO:24.

[0018] In one example, the signal peptide is selected from: CD8α, GMCSF, GMCSFRa, IgGsL1, IgGsL2, IgGsL3, IgGsL4, IgGsH1, IgGsH2, IgGsH3 or IgGsH4 signal peptide; preferably, the signal peptide is selected from CD8α, GMCSFRa signal peptide; more preferably, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0019] In one example, the co-stimulatory signal molecule is selected from: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS intracellular domain or a combination thereof; preferably, the co-stimulatory signal molecule is selected from: CD28, 4-1BB; more preferably, the co-stimulatory signal molecule comprises the amino acid sequence shown in SEQ ID NO: 10 or 11.

[0020] In one example, the transmembrane domain is selected from: CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 transmembrane domain. Preferably, the transmembrane domain is selected from: CD8, CD28; more preferably, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 7, 8 or 9.

[0021] In one example, the flexible peptide linker is selected from: newlinker or (G4S)n, wherein n is 1, 2, 3, 4 or 5; preferably, the flexible peptide linker comprises the amino acid sequence shown in SEQ ID NO:14, 15 or 16.

[0022] In one example, the chimeric receptor comprises an amino acid sequence that is at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical to an amino acid sequence as shown in SEQ ID NO: 17, 29, 30, 31, 32, 33, 34 or 54, or a fragment thereof.

[0023] The second aspect of the present invention provides a polynucleotide, wherein the polynucleotide encodes the above-mentioned chimeric receptor.

[0024] The third aspect of the present invention provides a chimeric receptor, wherein the chimeric receptor is a polypeptide chain, the amino terminus of which is operably linked to a signal peptide that promotes antibody secretion.

[0025] In one example, the signal peptide is selected from: a signal peptide of natural IgG, IgM, IgD, IgA or IgE.

[0026] In one example, the chimeric receptor includes an antigen binding domain, and the antigen binding domain includes an antibody heavy chain variable region and / or an antibody light chain variable region.

[0027] In one example, (i) the amino terminus of the chimeric receptor is a heavy chain variable region, and the amino terminus of the chimeric receptor is operably linked to the signal peptide of the heavy chain of natural IgG, IgM, IgD, IgA or IgE; or (ii) the amino terminus of the chimeric receptor is a light chain variable region, and the amino terminus of the chimeric receptor is operably linked to the signal peptide of the light chain of natural IgG, IgM, IgD, IgA or IgE.

[0028] In one example, the antigen binding domain can bind to one antigen, two or more epitopes of one antigen, or two or more different antigens.

[0029] In one example, the chimeric receptor includes a transmembrane domain and an intracellular domain; preferably, the intracellular domain includes an intracellular signaling domain; more preferably, the intracellular domain also includes a co-stimulatory signaling domain.

[0030] In one example, the chimeric receptor further comprises a spacer region between the antigen binding domain and the transmembrane binding domain.

[0031] In one example, the spacer region comprises a hinge region.

[0032] In one example, the hinge region comprises an IgG1, IgG2, IgG4 hinge region or a fragment thereof, a CD8 hinge region or a fragment thereof, or a CD28 hinge region or a fragment thereof.

[0033] In one example, the hinge region includes the amino acid sequence encoded by the nucleic acid shown in SEQ ID NO: 3, 4, 5 or 6.

[0034] In one example, the chimeric receptor includes a chimeric antigen receptor (CAR) and a recombinant TCR receptor.

[0035] In one example, the antigen includes a tumor antigen and / or a pathogen antigen; preferably, the tumor antigen is selected from: CD19, CD20, BCMA, GPRC5D, GPC3, Claudin18.2, CD22, FAP, Mesothelin, NKG2D, NKG2A, CD94, CD38.

[0036] The fourth aspect of the present invention provides a polynucleotide, wherein the polynucleotide encodes the chimeric receptor described in the third aspect.

[0037] In one example, the chimeric receptor is a first chimeric receptor, and the polynucleotide further comprises a nucleotide sequence encoding a second chimeric receptor; and the first chimeric receptor comprises a first antigen binding domain, and the second chimeric receptor comprises a second antigen binding domain.

[0038] In one example, the first chimeric receptor and the second chimeric receptor are separated by one or more polycistronic components.

[0039] In one example, the signal peptide A linked to the first chimeric receptor is the same as or different from the signal peptide B linked to the second chimeric receptor.

[0040] In one example, the polynucleic acid, from N-terminus to C-terminus, comprises:

[0041] (i) a signal peptide of a natural IgG, IgM, IgD, IgA or IgE heavy chain is operably linked to the heavy chain variable region of the first antigen-binding domain; a signal peptide of a natural IgG, IgM, IgD, IgA or IgE heavy chain is operably linked to the heavy chain variable region of the second antigen-binding domain;

[0042] (ii) a signal peptide of a natural IgG, IgM, IgD, IgA or IgE heavy chain is operably linked to the heavy chain variable region of the first antigen-binding domain; a signal peptide of a natural IgG, IgM, IgD, IgA or IgE light chain is operably linked to the light chain variable region of the second antigen-binding domain;

[0043] (iii) the signal peptide of the light chain of natural IgG, IgM, IgD, IgA or IgE is operably linked to the light chain variable region of the first antigen-binding domain; the signal peptide of the heavy chain of natural IgG, IgM, IgD, IgA or IgE is operably linked to the heavy chain variable region of the second antigen-binding domain; or

[0044] (iv) the signal peptide of the light chain of natural IgG, IgM, IgD, IgA or IgE is operably linked to the light chain variable region of the first antigen-binding domain; the signal peptide of the light chain of natural IgG, IgM, IgD, IgA or IgE is operably linked to the light chain variable region of the second antigen-binding domain.

[0045] In one example, in the polynucleotide, the first and second antigen binding domains can bind to different epitopes of the same antigen, or the first and second antigen binding domains bind to different antigens.

[0046] In one example, the polynucleotide, wherein the nucleic acid sequence of one or more domains among the spacer region, transmembrane domain, and intracellular domain included in the second chimeric receptor is different from the nucleic acid sequence encoding the corresponding domain of the first chimeric receptor; and the amino acid sequence transcribed from the nucleic acid sequence encoding one or more domains among the spacer region, transmembrane domain, and intracellular domain included in the second chimeric receptor is the same as the amino acid sequence of the corresponding domain of the first chimeric receptor.

[0047] In one example, the polynucleotide is codon-optimized for expression in human cells.

[0048] In one example, the polynucleotide, wherein the second antigen includes a tumor antigen and / or a pathogen antigen; preferably, the tumor antigen is selected from: CD19, CD20, BCMA, GPRC5D, GPC3, Claudin18.2, CD22, FAP, Mesothelin, NKG2D, NKG2A, CD94, CD3.

[0049] The fifth aspect of the present invention provides an engineered cell comprising the nucleic acid molecule, vector, or chimeric receptor as claimed in claim 1.

[0050] In one example, the engineered cells are immune cells; preferably, the immune cells are selected from: T cells, NK cells, cytotoxic T cells, NKT cells, dendritic cells, macrophages, CIK cells, and stem cell-derived immune cells or a combination thereof;

[0051] In one embodiment, the engineered cells are autologous or allogeneic cells.

[0052] In one example, the engineered cells have low or no endogenous TCR, B2M, HLA-II and / or NKG2A expression.

[0053] The sixth aspect of the present invention provides a polynucleotide, which encodes a chimeric receptor that binds to CD19 and CD20, or encodes a first chimeric receptor that binds to CD19 and a second chimeric receptor that binds to CD20; wherein the polynucleotide component encoding the chimeric receptor that binds to CD19 and CD20 is as follows:

[0054] (1)LV L1 -V H2 -IV L2 -V H1 -H-TM-C-CD3ζ

[0055] (2)LV L2 -V H1 -IV L1 -V H2-H-TM-C-CD3ζ

[0056] (3)LV H1 -V L2 -IV H2 -V L1 -H-TM-C-CD3ζ

[0057] (4)LV H2 -V L2 -IV L1 -V H1 -H-TM-C-CD3ζ

[0058] The polynucleotide components encoding the first chimeric receptor binding to CD19 and the second chimeric receptor binding to CD20 are as follows:

[0059] (5)LV L1 -V H1 -H-TM-C-CD3ζ-2A-LV H2 -V L2 -H-TM-C-CD3ζ

[0060] (6)LV L1 -V H1 -H-TM-C-CD3ζ-2A-LV L2 -V H2 -H-TM-C-CD3ζ

[0061] (7)LV H2 -V L2 -H-TM-C-CD3ζ-2A-LV H1 -V L1 -H-TM-C

[0062] (8)LV H1 -V L1 -H-TM-C-CD3ζ-2A-LV H2 -V L2 -H-TM-C

[0063] (9)LV H1 -V L1 -H-TM-C-CD3ζ-2A-LV H2 -V L2 -H-TM-C-CD3ζ

[0064] In the formula, each "-" is independently a connecting peptide or peptide bond component; L is an optional nucleic acid component encoding a signal peptide;

[0065] I is a nucleic acid module encoding a flexible linker; H is an optional hinge region nucleic acid module; TM is a transmembrane domain nucleic acid module; C is a co-stimulatory signal molecule nucleic acid module; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ; 2A is an optional 2A self-cleaving peptide nucleic acid module; V H1 V is the variable region of the heavy chain of CD19 antibody; L1 V is the variable region of CD19 antibody light chain; L2 V is the variable region of CD20 antibody light chain; H2 It is the variable region of CD20 antibody heavy chain; “-” is a connecting peptide or peptide bond.

[0066] In one example, the polynucleotide is codon optimized for expression in human cells.

[0067] In one example, the nucleotide sequences encoding the same component are codon divergent.

[0068] In one example, the V H1 comprising the amino acid sequence shown in SEQ ID NO: 21; V L1 comprising the amino acid sequence shown in SEQ ID NO: 22; said V H2 comprising the amino acid sequence shown in SEQ ID NO: 23; said V L2 Comprising the amino acid sequence shown in SEQ ID NO:24.

[0069] In one example, the signal peptide component is selected from: CD8α, GMCSF, GMCSFRas, IgGsL1, IgGsL2, IgGsL3, IgGsL4, IgGsH1, IgGsH2, IgGsH3 or IgGsH4 signal peptide; preferably, the signal peptide component is selected from CD8α, GMCSF signal peptide; more preferably, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:1 or 2.

[0070] In one example, the co-stimulatory signal molecule component is selected from: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS intracellular domain or a combination thereof; preferably, the co-stimulatory signal molecule component is selected from: CD28, 4-1BB; more preferably, the co-stimulatory signal molecule comprises the amino acid sequence shown in SEQ ID NO: 10 or 11.

[0071] In one example, the transmembrane domain component is selected from: CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 transmembrane domain; preferably, the transmembrane domain component is selected from: CD8, CD28; more preferably, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:7, 8 or 9.

[0072] In one example, the flexible peptide linker is selected from: newlinker, (G4S)n, wherein n is 1, 2, 3, 4 or 5; preferably, the flexible peptide linker comprises the amino acid sequence shown in SEQ ID NO:14, 15 or 16.

[0073] In one example, the polynucleotide comprises an amino acid sequence or a fragment thereof that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least about 100% homologous or identical to SEQ ID NO: 17, 29, 30, 31, 32, 33, 34 or 54.

[0074] The seventh aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the sixth aspect.

[0075] The eighth aspect of the present invention provides a chimeric receptor comprising amino acids encoded by the polynucleotide according to the sixth aspect of claim; or amino acids encoded by the vector according to the seventh aspect.

[0076] The ninth aspect of the present invention provides an engineered cell, wherein the engineered cell comprises the vector of the seventh aspect, or the polynucleotide of the sixth aspect, or expresses the chimeric receptor of the eighth aspect.

[0077] In one example, the engineered cells are immune cells; preferably, the immune cells are selected from: T cells, NK cells, cytotoxic T cells, NKT cells, dendritic cells, macrophages, CIK cells, and stem cell-derived immune cells or a combination thereof; preferably, the engineered cells are autologous or allogeneic cells.

[0078] In one example, the engineered cells have low or no endogenous TCR, B2M, HLA-II and / or NKG2A expression.

[0079] The tenth aspect of the present invention provides a method for preparing engineered cells, wherein the engineered cells express the chimeric antigen receptor described above, comprising the following steps: (i) adding an input composition comprising cells to be transduced, a stimulator for cells to be transduced, and incubating for no more than 24 hours; (ii) adding viral vector particles containing recombinant nucleic acid and incubating for no more than 24 hours to obtain the engineered cells.

[0080] In one example, the total incubation time of steps (i) and (ii) is no more than 48 hours, no more than 32 hours, no more than 28 hours, or no more than 24 hours.

[0081] The eleventh aspect of the present invention provides a drug for preventing or treating tumors, comprising the polynucleotide, vector, chimeric receptor, or engineered cell described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The structure of the dual-target chimeric antigen receptor binding to CD19 and CD20 is shown: including a signal peptide sequence (L), an antigen recognition sequence that recognizes CD19 and CD20, a hinge region (Hinge), a transmembrane region (TM), a co-stimulatory factor signaling domain (Co-stimulator) and a CD3ζ signaling region (CD3ζ). Expression vectors CAR6, 8, 9 as shown in formula I; expression vector CAR10 as shown in formula II; expression vector CAR7 as shown in formula III; expression vector CAR11 as shown in formula IX.

[0083] Figure 2 It showed that Daudi expressed CD19 and CD20, and Nalm6 expressed CD19.

[0084] Figure 3A-3B It showed that CD19 / CD20 dual-target CAR-T1, 2, 4, 5, 6, 7, 8, 9, and 10 cells could significantly kill tumor cells Daudi and Nalm6.

[0085] Figure 4A-4B It was shown that after co-incubation with tumor cells Daudi or Nalm6, CD19 / CD20 dual-target CAR-T1, 2, 4, 5, 6, 7, 8, 9, and 10 cells could secrete higher levels of IFN-γ, IL-2, and TNF-α.

[0086] Figure 5 The inhibition rate of dual-target CAR-T1 and CAR-T11 on subcutaneously transplanted lymphoma (Daudi) in NPG mice is shown.

[0087] Figure 6 It showed that on the 7th and 14th days after the dual-target CAR-T cell infusion to treat mice with subcutaneously transplanted lymphoma (Daudi), surviving human T cells were detected in the peripheral blood of mice in the CAR-T1 and CAR-T11 groups.

[0088] Figure 7 UTD, CD19-CAR-T, and dual-target CAR-T4, 9, 10, and 12 cells were shown to treat in situ lymphoma in NPG mice.

[0089] Figure 8 Tumor fluorescence intensity IVIS imaging is shown for the treatment of orthotopic lymphoma in NPG mice with UTD, CD19-CAR-T, and dual-target CAR-T4, 9, 10, and 12 cells.

[0090] Fig. 9 UTD(TKO), CD19-CAR-T(DKO), CD20-CAR-T(DKO), CAR-T9(DKO), CAR-T12(DKO), and CAR-T9(TKO) cells were shown to treat orthotopic lymphoma in NPG mice.

[0091] Fig.10 The results show the survival of mice treated with UTD (TKO), CD19-CAR-T (DKO), CD20-CAR-T (DKO), CAR-T9 (DKO), CAR-T12 (DKO), and CAR-T9 (TKO) cells for orthotopic lymphoma in NPG mice, and the survival of mice on D131 days after tumor cell infusion.

[0092] Fig.11 Tumor fluorescence intensity IVIS imaging is shown for orthotopic lymphoma in NPG mice treated with UTD (TKO), CD19-CAR-T (DKO), CD20-CAR-T (DKO), CAR-T9 (DKO), CAR-T12 (DKO), and CAR-T9 (TKO) cells.

[0093] Fig.12 The results showed that after co-incubation with tumor cells Daudi or Nalm6, CAR9 / NKG2A(1)-tko and CAR9 / NKG2A(2)-tko cells could significantly kill tumor cells.

[0094] Fig.13 The results showed that CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells could secrete high levels of IL-2, TNF-α, and IFN-γ cytokines after co-incubation with tumor cells Daudi and Nalm6.

[0095] Fig.14 The results showed that after 24 or 72 hours of co-incubation with primary human NK cells, CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells could significantly kill NK cells.

[0096] Fig.15 The results showed that CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells could secrete high levels of IL-2, TNF-α, and IFN-γ cytokines after co-incubation with NK cells. DETAILED DESCRIPTION

[0097] After extensive and in-depth research, the inventor unexpectedly found that immune effector cells expressing different forms of CD19 / CD20 chimeric receptors have different tumor-killing abilities. Based on this, the present invention was completed.

[0098] the term

[0099] Unless specifically defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, wherein suitable methods and materials are described herein. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification shall prevail. In addition, unless otherwise specified, the materials, methods and embodiments of the present invention are merely illustrative and are not intended to be limiting. According to the present disclosure, it should be understood by those skilled in the art that many changes or modifications can be made in the disclosed specific embodiments, and the same or similar results are still obtained without departing from the spirit and scope of the present invention. The present invention is not limited in scope to the specific embodiments described herein (which are only intended to be illustrative of various aspects of the present invention), and functionally equivalent methods and components are within the scope of the present invention. The present invention includes modifications and modifications to the subject matter of the present invention for various uses and conditions.

[0100] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. These techniques are fully explained in the literature.

[0101] The term "about" refers to the usual error range of each value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include embodiments directed to the value or parameter itself. For example, a description of "about X" includes a description of "X". In this article, "about" can be an acceptable error range in the technical field. For example, it can refer to a value or parameter within the range of ±10% of an "about" value or parameter, for example, about 5uM can include any number between 4.5uM and 5.5uM.

[0102] Term "CD19": CD19 (Gene ID: 930) This gene encodes a member of the immunoglobulin gene superfamily. CD19 expression is restricted to B cell lymphocytes. The protein is a reliable marker for pre-B cells and in antibody-secreting plasma cells, where its expression decreases during terminal B cell differentiation.

[0103] Term "CD20": CD20 (Gene ID: 931) This gene encodes a B lymphocyte surface molecule that plays a role in the development and differentiation of B cells into plasma cells.

[0104] The term "FasL (Fas ligand)" is (Gene ID: 356), a member of the tumor necrosis superfamily, which activates the apoptosis signaling pathway of target cells by binding to Fas.

[0105] The term "NKG2A": NKG2A (Gene ID: 3821). NKG2A and CD94 form dimers on NK cells and act as inhibitory receptors of NK, and its ligand is HLA-E. NKG2A is generally expressed on NK cells and a small number of CD8 T cells.

[0106] Term "CD38": CD38 (Gene ID: 952). It is expressed on the surface of many immune cells, including CD4 T, CD8 T, B lymphocytes and NK cells. CD38 plays a role in cell adhesion, signal transduction and calcium signaling.

[0107] The term "T cell receptor (TCR)" mediates T cells to recognize specific major histocompatibility complex (MHC)-restricted peptide antigens, including classical TCR receptors and optimized TCR receptors. The classical TCR receptor is composed of two peptide chains, α and β, each of which can be divided into a variable region (V region), a constant region (C region), a transmembrane region and a cytoplasmic region. Its antigen specificity exists in the V region, and the V region (Vα, Vβ) each has three hypervariable regions CDR1, CDR2, and CDR3.

[0108] The term "recombinant T cell receptor (recombinant TCR)" includes chimeric receptors derived from one or more TCR subunits. For example, a recombinant TCR includes an extracellular domain, a transmembrane domain, and a TCR intracellular domain of at least part of a TCR subunit, and the TCR subunit portion is effectively connected to an antigen binding domain. For example, the TCR subunits in the recombinant TCR are derived from CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRγ and / or TCRδ subunits. For example, the recombinant TCR can be integrated into a TCR / CD3 complex expressed on a T cell. For example, a recombinant TCR includes constant regions and intracellular domains of TCRα and TCRβ subunits, and the subunit constant regions are effectively connected to an antigen binding domain. For example, a recombinant TCR includes constant regions and intracellular domains of TCRγ and TCRδ subunits, and the subunit constant regions are effectively connected to an antigen binding domain. For example, the recombinant TCR comprises a CD3ζ, CD3ε, CD3γ or CD3δ subunit, and the extracellular domain of the subunit is operably linked to an antigen binding domain.

[0109] The term "T cell antigen coupler (TAC)" includes three functional domains: 1. Antigen binding domain, including single-chain antibody, designed ankyrin repeat protein (DARPin) or other targeting groups; 2. Extracellular domain, single-chain antibody that binds to CD3, thereby bringing the TAC receptor into close proximity with the TCR receptor; 3. Transmembrane region and intracellular region of CD4 co-receptor, wherein the intracellular region is connected to protein kinase LCK, catalyzing the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as the initial step of T cell activation.

[0110] The term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides constituting TCR, which are capable of binding to surface antigens on target cells and interacting with other polypeptides of the complete TCR complex, usually co-localized on the surface of T cells. The chimeric T cell receptor is composed of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least a portion of the TCR extracellular domain, a transmembrane domain, and a stimulatory domain of the intracellular signaling domain of the TCR intracellular domain; the TCR subunit and the antibody domain are operatively connected, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε or CD3γ, and the chimeric T cell receptor is integrated into the TCR expressed on the T cell.

[0111] The term "chimeric antigen receptor" (CAR) includes at least one extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain includes a functional signaling domain of a stimulatory molecule and / or a co-stimulatory molecule. For example, the stimulatory molecule is from a ζ chain (such as CD3ζ) that binds to a T cell receptor complex. For example, the intracellular signaling domain further includes a functional signaling domain of one or more co-stimulatory molecules, such as 4-1BB (CD137), CD27, and / or CD28. For example, the polypeptide groups are connected to each other. For example, the intracellular signaling domain (or structural region) can be selected from the intracellular co-stimulatory domain of any one or more of the following polypeptides: CD27, CD28, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, CD83.

[0112] The term "primary signal domain" or "primary signal domain" regulates the initial activation of the TCR complex in a stimulating manner. On the one hand, the primary signal domain is triggered by the combination of, for example, the TCR / CD3 complex with the MHC molecule loaded with the peptide, thereby mediating T cell responses (including but not limited to, proliferation, activation, differentiation, etc.). The primary signal domain that acts in a stimulating manner may include a signal transduction motif of an immunoreceptor tyrosine activation motif or an ITAM. For example, a fragment of the primary signal domain containing ITAM includes, but is not limited to, an intracellular signal transduction domain derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD79a, CD79b, CD278 and CD66d.

[0113] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate the activity of the cell via a defined signaling pathway by producing a second messenger or by acting as an effector in response to such a messenger. An intracellular signaling domain may include the entire intracellular portion of a molecule, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0114] The term "costimulatory signal domain" or "costimulatory molecule": generally refers to the intracellular domain of a costimulatory molecule that can bind to a cell stimulatory signal molecule, such as TCR / CD3, and the combination leads to T cell proliferation and / or upregulation or downregulation of key molecules. Costimulatory molecules are usually associated binding partners on T cells, which specifically bind to costimulatory ligands and mediate co-stimulatory responses of T cells, including but not limited to proliferation. Costimulatory molecules are cell surface molecules or their ligands that are non-antigen receptors required for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18) and 4-1BB (CD137).

[0115] The term "CD3ζ (also known as CD3 Zeta)" includes the protein provided by GenBank Accession No. BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. "CD3ζ" is used interchangeably with "CD3z" and "CD3Z" in this application.

[0116] The term "newlinker" refers to a linker having a sequence as shown in GSTSGSGKPGSGEGSTKG (see Marc Whitlow et al., "An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability", Protein Engineering Vol. 6 No. 8 pp. 989-995, 1993).

[0117] The term "cell" refers to a cell of human or non-human, or animal origin.

[0118] The term "host" or "subject" refers to a recipient of a transplant, for example, an individual, such as a human, into which exogenous cells are implanted. For example, a "subject" may be a clinical patient, a clinical trial volunteer, an experimental animal, or the like. The subject may be suspected of having a disease characterized by cell proliferation or may be diagnosed with a disease characterized by cell proliferation. For example, the subject may be suffering from or may be suffering from an immune disease such as an autoimmune disease, or may be suffering from a tumor, or may be suffering from an inflammatory disease.

[0119] The term "engineering" or "engineering" refers to the application of the principles and methods of cell biology and molecular biology, through some engineering means, at the level of the whole cell or the organelle level, to change the genetic material in the cell or obtain cell products according to people's wishes. For example, "engineering" refers to one or more changes in nucleic acids (such as nucleic acids in the genome of an organism). "Engineering" can refer to the change, addition and / or deletion of genes. "Engineered cells" can also refer to cells with added, deleted and / or changed genes.

[0120] The term "immune cell" refers to a cell that participates in an immune response and produces an immune effect, such as a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a dendritic cell, a CIK cell, a macrophage, a mast cell, etc. For example, the immune cell is a T cell, an NK cell, or a NKT cell. For example, the T cell can be an autologous T cell, a heterologous T cell, or an allogeneic T cell. For example, the NK cell can be an autologous NK cell or an allogeneic NK cell. For example, the immune cell is obtained by sorting a donor's peripheral blood mononuclear cells (PBMC).

[0121] The term "T cell" can be PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue and natural T cells obtained from infection sites, ascites, pleural effusion, spleen tissue, tumor tissue, or a cell population with specific phenotypic characteristics obtained through sorting, or a mixed cell population with different phenotypic characteristics, such as "T cells" can be cells comprising at least one T cell subset: memory stem cell-like T cells (stem cell-like memory T cells, Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (Tregs) and / or effector memory T cells (Tem). In some cases, "T cells" can be T cells of a certain specific subtype, such as αβT cells, γδT cells. In some cases, any number of techniques known to those skilled in the art, such as FicollTM separation and / or apheresis, can be used to obtain T cells from blood collected from an individual. For example, T cells are derived from induced pluripotent stem cells. For example, cells from circulating blood of an individual are obtained by a single blood draw. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. For example, the cells collected by apheresis can be washed to remove plasma molecules and the cells can be placed in a suitable buffer or culture medium for subsequent processing steps. The T cells can be derived from healthy donors or from individuals diagnosed with cancer. The T cells can be autologous T cells or allogeneic T cells. The T cells can be primary T cells.

[0122] The term "cell composition" generally refers to a combination of at least two types of cells, wherein the first type of cells can at least bind to CD19 and CD20; the second type of cells bind to NK cell markers. For example, each type of cell can be present in different containers, and can also be formulated into a desired preparation with a suitable adjuvant simultaneously or separately when necessary; each type of cell can be from different sources (e.g., prepared, produced or sold by different manufacturers; for example, naturally occurring T cells separated from donors and T cells derived from stem cells); each type of cell can be prepared into an independent preparation (solid, liquid, gel, etc.); each type of cell can be present in a mixed form. The cell composition can also include an effective amount of antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells, and can also include a pharmaceutically acceptable carrier.

[0123] The term "MHC" stands for histocompatibility complex. In human cells, MHC is called HLA antigen and plays an important role in transplantation reactions, with rejection mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.

[0124] The term "human leukocyte antigen" (HLA) is the encoding gene of the human major histocompatibility complex and is closely related to the human immune system function. HLA includes class I, class II and class III gene parts. HLA class I is a heterodimer composed of a heavy chain (α chain) and a light chain β2 microglobulin (B2M). The term "B2M" is β-2 microglobulin, also known as B2M, which is the light chain of the MHC class I molecule. HLA-II class genes include the HLA-D family, mainly HLA-DP, HLA-DQ and HLA-DR, etc., which are mainly distributed on the surface of professional antigen presenting cells such as B lymphocytes, macrophages and dendritic cells.

[0125] The term "exogenous" refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or the expression level is insufficient to achieve the function it has when overexpressed.

[0126] The term "endogenous" refers to a nucleic acid molecule, polypeptide, etc. that originates from the organism itself.

[0127] The terms "activation" and "activation" are used interchangeably and refer to the process by which cells change from a quiescent state to an active state. The process may include responses to phenotypic or genetic changes in antigens, migration and / or functional activity states. For example, the term "activation" may refer to the process by which T cells are gradually activated. The activation process is regulated by the first stimulation signal and the co-stimulation signal. The activation of T cells is a dynamically changing process, and its duration and degree of activation are affected by external stimulation. "T cell activation" or "T cell activation" refers to the state of T cells that are stimulated to induce detectable cell proliferation, cytokine production and / or detectable effector function. Using CD3 / CD28 magnetic beads, in vitro antigen stimulation or in vivo antigen stimulation will affect the degree and duration of T cell activation. For example, the engineered T cells are co-incubated with tumor cells containing specific target antigens or activated after viral infection.

[0128] The term "Genome editing, Gene editing" refers to a genetic engineering technology that uses site-specific nucleases to insert, knock out, modify or replace DNA at a specific location in the genome of an organism to change the DNA sequence. Gene editing can be used to achieve precise and efficient gene knockout or gene knock-in. Gene knockout technology using nucleases includes CRISPR / Cas technology, ZFN technology, TALEN technology and TALEN-CRISPR / Cas technology, single base editing (Base Editor) technology, guide editing (Prime Editor) technology homing nuclease (Meganuclease) technology. The guide sequence (gRNA) is a polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence, and the gRNA can guide the sequence-specific binding of the CRISPR complex to the target sequence. Whenever the sequence of the gRNA is involved in this application, it can be a targeted DNA sequence, or it can be a complete Cas9 guide sequence formed by the ribonucleotides corresponding to the DNA and crRNA and TracrRNA. gRNA is used to guide, bind or recognize the Cas enzyme. For example, endogenous TCR / B2M / FAS knockout or endogenous TCR / B2M knockout engineered cells are constructed using CRISPR technology. The gRNA sequences targeting TCR, B2M, FAS, and NKG2A are shown in SEQ ID NOs: 35, 36, 37, and 38, respectively.

[0129] "Low expression" described in the present application is that the protein and / or RNA level of target gene expression in engineered cells is lower than the expression level before cell engineering treatment. For example, low expression of B2M, TCR, NKG2A or FAS refers to that the expression of B2M, TCR, NKG2A or FAS in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. Expression or content of specific protein in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting or flow cytometry using specific antibodies.

[0130] The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.

[0131] The term "nucleic acid" or "nucleic acid molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form, and includes any nucleic acid molecule encoding a polypeptide of interest or a fragment thereof.

[0132] The terms "nucleic acid molecule encoding", "coding DNA sequence" and "coding DNA" refer to the sequence or order of deoxyribonucleotides along a deoxyribonucleic acid strand. For example, a nucleic acid sequence encodes an amino acid sequence. When referring to a nucleotide sequence, the "sequence" may include DNA or RNA, and may be single-stranded or double-stranded.

[0133] The term "homology" or "identity" refers to the subunit sequence identity between two polymer molecules, for example, between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. The term "substantial identity" or "substantial homology" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity with a reference amino acid sequence or nucleic acid sequence. Sequence identity can be measured using sequence analysis software (e.g., BLAST, BESTFIT, GAP or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and / or other modifications.

[0134] The present invention provides amino acid sequences or nucleic acid sequences that are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% homologous or identical to the amino acid sequences or nucleic acid sequences shown in the sequence table. The present invention provides amino acid sequences or nucleic acid sequences that are modified based on the amino acid sequences or nucleic acid sequences shown in the sequence table, and / or one or more amino acid substitutions, and / or deletions and / or additions of one or more amino acids and that have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homology or identity with the sequences in the sequence table.

[0135] The term "subject" refers to any animal, such as a mammal or marsupial. The subject of the present invention includes, but is not limited to, humans, non-human primates (such as rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

[0136] The term "peripheral blood mononuclear cell" (PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc. Density-based cell separation methods, for example, by lysing red blood cells or not lysing red blood cells and preparing PBMCs by Percoll Ficoll gradient centrifugation of peripheral blood or a single sample or leukapheresis sample.

[0137] The term "effective amount" or "therapeutically effective amount" refers to a dose sufficient to prevent or treat an individual disease (cancer). The effective dose for therapeutic or preventive use depends on the stage and severity of the disease being treated, the age, weight and general health of the subject, and the judgment of the prescribing physician. The size of the dose also depends on the selected active substance, the method of administration, the time and frequency of administration, the presence, nature and extent of adverse side effects that may accompany the administration of a specific active substance, and the desired physiological effect. According to the judgment of the prescribing physician or a person skilled in the art, one or more rounds, or multiple administrations of the engineered cells of the present application may be required.

[0138] The term "expression vector" refers to a vector containing a recombinant polynucleotide, which contains an expression control sequence operably linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by a host cell or an in vitro expression system. Expression vectors include plasmids, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0139] The term "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. It includes, but is not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. For example, it includes autonomously replicating plasmids or viruses. It also includes non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc.

[0140] The term "treatment" refers to intervention measures that attempt to change the course of a disease, which can be either preventive or intervention in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, improving or alleviating the condition, and alleviating or improving prognosis.

[0141] The term "prevention" refers to interventions that attempt to occur before disease (eg, tumor metastasis, recurrence) occurs.

[0142] The term "transplant rejection" refers to the process in which the host's immune system recognizes the foreign transplant as a "foreign component" after the host receives an allogeneic tissue, organ, or cell transplant, and initiates an immunological response to attack, destroy, and eliminate the transplant.

[0143] The term "graft" refers to a biological material or preparation derived from an individual other than a host and used to be implanted into a host. The graft may be from any animal source, such as a mammalian source, preferably from a human. The graft may be from a host, such as cells from a host that are cultured in vitro or transformed and then implanted into a host. The graft may be from another individual of the same species, such as cells from another person that are cultured in vitro or transformed and then implanted into a host.

[0144] The term "autologous" refers to originating from the same organism. For example, a sample can be removed from a subject (e.g., a cell), processed, and returned to the subject at a later time. Allogeneic refers to the donor and recipient being different individuals.

[0145] The term "scFv" refers to a fusion protein comprising an antibody fragment of the variable region of at least one light chain and an antibody fragment of the variable region of at least one heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., connected by a synthetic linker, such as a short flexible polypeptide linker) and can be expressed in the form of a single-chain polypeptide. The scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv may include VL-linker-VH or may include VH-linker-VL. The antigen binding function of an antibody may be performed by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen binding units". The term "antigen binding unit" also includes any molecular structure containing a polypeptide chain having a specific shape suitable for and recognizing an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.

[0146] The term "single domain antibody (sdAb)" may also be referred to as "VHH", or "VHH polypeptide", which contains a variable VHH domain responsible for antigen recognition. The antigen binding of the VHH domain is mediated by three CDRs, which are flanked by four relatively constant framework regions (FRs). It refers to a type of antibody that lacks the antibody light chain and only has the heavy chain variable region. Because of its small molecular weight, it is also called a nanobody. VHH can be truncated at the N-terminus or C-terminus so that it contains only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as the VHH basically maintains antigen binding and specificity.

[0147] The terms "recognize", "bind", and "target" are used interchangeably and refer to selective binding to a target antigen. For example, binding to a target cell refers to binding to a target antigen (eg, a target molecule) on a target cell.

[0148] The term "DDpp" refers to a target binding D domain (DD) polypeptide based on a non-traditional antibody structural scaffold. D domain polypeptides (DDpp) are characterized by high target binding affinity and a non-antibody structural scaffold. DDpp can be monovalent or multivalent. In some embodiments, DDpp is monospecific or multispecific. In other embodiments, monospecific and multivalent. In other embodiments, DDpp is multispecific and multivalent. For more information, see CN111727250A.

[0149] The term "variable region or variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in antibody antigen binding. The heavy chain variable domain (VH) and light chain variable domain (VL) of a natural antibody generally have similar structures, wherein each domain comprises four conserved FRs and three CDRs. A single VH or VL domain can confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains using a VH or VL domain from an antibody that binds to the antigen, respectively.

[0150] The term "hypervariable region" or "complementarity determining region" or "CDR" refers to each region of an antibody variable domain whose sequence is hypervariable, and / or forms structurally defined loops ("hypervariable loops"), and / or contains residues that contact the antigen ("antigen contacts"). Typically, an antibody comprises six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3).

[0151] The term "Fc region" or "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0152] "Framework (FR)" refers to variable domain residues other than hypervariable region (CDR) residues. The FR of a variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. In VH (or VL), CDR and FR sequences usually appear in the following order:

[0153] FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0154] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0155] The term "loop structure" generally refers to a curved, non-closed peptide segment, which can be a loop structure formed by a CD19 antigen binding domain and a CD20 antigen binding domain by a suitable connection method. For example, the CD19 antibody heavy chain variable region (VH1) and the CD19 antibody light chain variable region (VL1) are connected to form an antibody scFv1, and the CD20 antibody heavy chain variable region (VH2) and the CD20 antibody light chain variable region (VL2) are respectively connected to the two ends of the antibody scFv1. For example, the CD20 antibody heavy chain variable region (VH2) and the CD20 antibody light chain variable region (VL2) are connected to form an antibody scFv2, and the CD19 antibody heavy chain variable region (VH1) and the CD19 antibody light chain variable region (VL1) are respectively connected to the two ends of the antibody scFv2.

[0156] The terms "whole antibody", "full length antibody" and "intact antibody" are used interchangeably and refer to a complete full-length antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein or including an antigen-binding region.

[0157] The term "fully human antibody (or fully human antibody)" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequence.

[0158] The term "variant" refers to a different protein or polypeptide with respect to a protein or polypeptide having a specific sequence feature ("control protein" or "reference polypeptide"), having one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions and / or additions compared to the control protein or reference polypeptide. The change in the amino acid sequence can be an amino acid substitution. The change in the amino acid sequence can be a conservative amino acid substitution. The functional fragment or functional variant of a protein or polypeptide maintains the basic structure and functional properties of the control protein or polypeptide.

[0159] The term "cell marker" is also called a cell surface molecule or a cell surface protein, and preferably is a molecule present on the surface of an immune cell membrane. For example, "markers of T cells and / or NK cells" refer to markers present in T cells or NK cells, respectively, or in both T cells and NK cells, including but not limited to: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, SLAMF7, and TIGIT. For example, NK cell markers are selected from: NKG2 receptor family, killer immunoglobulin-like receptor (KIR) family, natural cytotoxicity receptor (NCR), and / or other NK cell-specifically expressed antigens. The NKG2 receptor family includes NKG2A, NKG2D, and NKG2C. The KIR family includes KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, and KIR3DS1. NCR includes NKP30, NKP44, NKP46, and NKp80. Other NK cell-specifically expressed antigens include CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, TIGIT, CS1, and IL-15R.

[0160] The term "NK inhibitory receptor (NK inhibitory receptor, NKIR)" refers to a class of receptors on NK cells that can transduce killing inhibitory signals and inhibit the killing function of NK cells. Including HLA-specific and non-HLA-specific inhibitory receptors. Among them, HLA-specific inhibitory receptors include CD94, NKG2A and KIR. Non-HLA-specific NK inhibitory receptors include PD-1, Siglec7, LAIR1 and CD300A. NKIR includes, but is not limited to, having an immunoreceptor tyrosine-based inhibitory motif (ITIM). For example, NKIR includes: NKG2 / CD94 components, KIR family members, LIR family members, NKR-P1 family members, immune checkpoint receptors, immune checkpoint inhibitors, SIGLEC family members, Ly49 family members, or combinations thereof. For example, the NKG2 / CD94 component is selected from NKG2A, NKG2C and CD94. For example, the KIR family member is selected from KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2 and KIR3DL3. For example, the LIR family member is selected from LIR1, LIR2, LIR3, LIR5 and LIR8. For example, the NKR-P1 family member is selected from NKR-P1B and NKR-P1D. For example, immune checkpoint inhibitors include: (a) one or more antagonists of checkpoint molecules, which include PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, M ICA / B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha), TLR3, VISTA, NKG2A / HLA-E or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab and derivatives or functional equivalents thereof; or (c) at least one of atezolizumab, nivolumab and pembrolizumab, or one or more of venetoclax, azacitidine, pomalidomide. For example, the immune checkpoint receptor is selected from PD-1, TIGIT, CD96, TIM3 and LAG3. For example, the SIGLEC family member is selected from SIGLEC7 and SIGLEC9.For example, the Ly49 family member is selected from Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4.

[0161] The term "pathological cells" generally refers to any type of cell present in a patient that is believed to contribute to a worsening health condition, or to a malignant or infected cell that needs to be reduced or eliminated to obtain patient remission. The present invention relates to methods of novel adoptive immunotherapy strategies for treating diseases associated with the development of pathological cells, such as cancer (tumors), infections, and autoimmune diseases.

[0162] The term "tumor antigen" refers to an antigen that emerges or is overexpressed during the development and progression of a hyperproliferative disease.

[0163] The term "2A peptide" includes sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin). As used herein, "self-cleaving peptides" refer to oligopeptides that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins after translation. Including but not limited to "F2A", "E2A", "P2A" and "T2A".

[0164] The term "signal peptide (L or SP)", or leader sequence, is a short peptide chain (about 5-30 amino acids in length) that guides the transfer of newly synthesized proteins or polypeptides to the secretory pathway. The signal peptide is a short peptide located at the N-terminus (amino terminus) of the protein. For example, SP is a CD8 signal peptide or a GMCSFRa signal peptide. For example, SP is a natural signal peptide from a wild-type immunoglobulin superfamily (IgSF) member. For example, SP is modified from the natural signal peptide of IgSF.

[0165] For example, the signal peptide is a natural IgG, IgM, IgD, IgA or IgE heavy chain signal peptide. For example, the signal peptide is a natural κ or λ light chain signal peptide. For example, the SP connected to the antibody VL is a natural κ light chain signal peptide, such as IgGsL1, IgGsL2, IgGsL3. For example, the signal peptide connected to the antibody VL is a natural λ light chain signal peptide, such as IgGsL4.

[0166] The term "chimeric receptor" refers to a fusion molecule formed by connecting DNA fragments or protein corresponding cDNAs from different sources using genetic recombination technology, including an extracellular domain, a transmembrane domain, and an intracellular domain. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CAR), recombinant TCR receptors, and synthetic polypeptide receptors (synNotch receptors). Recombinant TCR receptors include TACs and chimeric T cell receptors. In one example, the extracellular domain includes an antigen binding domain. In one example, the extracellular domain also includes a hinge region.

[0167] The present invention discloses a bispecific CAR targeting CD19 and CD20. When tumor cells escape CD19 antigen or CD19 is not expressed in tumor cells, bispecific CAR eliminates tumor cells by recognizing CD20. Most leukemia tumor cells express CD20, including some CD19-negative patients after anti-CD19 CAR T therapy. CD20 is encoded by the MS4A gene, is a glycosylated protein, and is a B cell membrane marker. It is specifically expressed in more than 95% of normal and cancerous B cells. These cells are in the pre-B cell stage and subsequent developmental stages. CD20 does not stop expressing until the cells differentiate into plasma cells. The present invention uses CD20 as another target for immunotherapy of B cell malignancies. The present invention discloses a rationally optimized single-chain design and system, namely a bispecific CAR, which can be effectively integrated into primary human T cells and can simultaneously target CD19 and CD20 when T cells are activated. The CAR T cells of the present invention can recognize two antigens (CD19 and CD20). The present invention provides a very effective method for potentially preventing antigen escape.

[0168] The present invention uses CARs that simultaneously target CD19 and CD20. Compared with CARs that target a single antigen, affinity is enhanced, T cell activity is improved, and these targets have an additive or synergistic effect. CAR Ts that simultaneously target CD19 and CD20 on the surface of tumor cells can reduce the possibility of antigen escape caused by downregulation or loss of a single surface antigen. The antibodies of CD19 and CD20 are connected in a LOOP form. For example, the CD19 antibody heavy chain variable region (VH1) and the CD19 antibody light chain variable region (VL1) are connected as antibody scFv1, and the CD20 antibody heavy chain variable region (VH2) and the CD20 antibody light chain variable region (VL2) are respectively connected to the two ends of the antibody scFv1. For example, the CD20 antibody heavy chain variable region (VH2) and the CD20 antibody light chain variable region (VL2) are connected as antibody scFv2, and the CD19 antibody heavy chain variable region (VH1) and the CD19 antibody light chain variable region (VL1) are respectively connected to the two ends of the antibody scFv2.

[0169] The bispecific CAR in which the CD19 and CD20 antibody sequences are connected in a LOOP form disclosed in the present invention has enhanced affinity, improved T cell activity, and stronger ability to kill and inhibit CD19 and / or CD20 positive cells (e.g., tumor cells) in vivo and in vitro compared with the bispecific CAR in which the CD19 and CD20 antibody sequences are combined in a tandem form in the prior art.

[0170] In one example, the chimeric receptor includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain from the N-terminus to the C-terminus, and the extracellular region includes a domain that binds to CD19 and / or CD20. The domain that binds to CD19 includes an anti-CD19 antibody or a fragment thereof, or a synthetic binding domain. The domain that binds to CD20 includes an anti-CD20 antibody or a fragment thereof, or a synthetic binding domain. The domain that binds to CD19 and CD20 includes an anti-CD19 and CD20 antibody or a fragment thereof, or a synthetic binding domain. Among them, the domain that targets CD19 and / or CD20 includes an antigen binding unit that binds to CD19 and / or CD20. For example, the chimeric receptors of the present invention include the chimeric receptors shown in Tables 1, 2, 3 or 4. For example, the chimeric receptors of the present invention include the chimeric receptors shown in Tables 1, 2, 3 or 4. For example, the chimeric receptors of the present invention include the chimeric receptors shown in Tables 1, 2, 3 or 4. Figure 1 Chimeric receptors shown.

[0171] In one example, the anti-CD19 and / or CD20 antibody is selected from: a whole antibody, a scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody. In one example, the anti-CD19 and / or CD20 antibody is a scFv. In one example, the anti-CD19 and / or CD20 antibody is a single domain antibody. In one example, the anti-CD19 and / or CD20 antibody is a DDPP antibody.

[0172] In one example, the anti-CD19 antibody or fragment thereof comprises a heavy chain variable region (VH1) and a light chain variable region (VL1). In one embodiment, the VH1 comprises VH CDR1, VH CDR2, and VH CDR3, and the VL1 comprises VL CDR1, VL CDR2, and VLCDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are from VH having an amino acid sequence as shown in SEQ ID NO: 21, and VLCDR1, VL CDR2, and VL CDR3 are from VL having an amino acid sequence as shown in SEQ ID NO: 22.

[0173] In one example, the anti-CD20 antibody or fragment thereof comprises a heavy chain variable region (VH2) and a light chain variable region (VL2). In one embodiment, the VH2 comprises VH CDR1, VH CDR2, and VH CDR3, and the VL2 comprises VL CDR1, VL CDR2, and VLCDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are from VH2 having an amino acid sequence as shown in SEQ ID NO: 23, and VL CDR1, VL CDR2, and VL CDR3 are from VL2 having an amino acid sequence as shown in SEQ ID NO: 24.

[0174] In one embodiment, the anti-CD19 antibody or fragment thereof comprises VH1 and VL1. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 21 and 22, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto.

[0175] In one embodiment, the anti-CD20 antibody or fragment thereof comprises VH2 and VL2. In one embodiment, the VH1 and VL1 have the amino acid sequences shown in SEQ ID NOs: 23 and 24, respectively, or have at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity thereto.

[0176] It is well known in the art that some amino acid substitutions can occur in the VH and VL region sequences while the CDR sequence remains unchanged and retains its affinity for the target; and some amino acid substitutions can occur in the CDR sequence while the amino acids in contact with the target remain unchanged and retain its affinity for the target.

[0177] In one example, the anti-CD19 or CD20 antibody or fragment thereof is a scFv. The VH (heavy chain variable region) and VL (light chain variable region) in the scFv can be connected by a connecting peptide chain (linker), and the positions can be interchanged. In one example, the anti-CD19 or CD20 antibody includes, from the N-terminus to the C-terminus, VH, a connecting peptide chain, and VL. In one example, the anti-CD19 or CD20 antibody includes, from the N-terminus to the C-terminus, VL, a connecting peptide chain, and VH. Any connecting peptide chain that connects VH and VL together to form a fully functional single-chain antibody is applicable. In one example, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS, (GGGGS)3, or (GGGGS)4. In one example, the connecting peptide chain is selected from: SEQ ID NO:14, 15 or 16.

[0178] The chimeric receptor of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain from the N-terminus to the C-terminus, and the intracellular signal transduction domain can directly promote cellular responses when it contains one or more signal transduction domains or motifs, such as immunoreceptor tyrosine-based activation motifs (ITAMs), kinase domains, costimulatory domains, etc. The intracellular signal transduction domain will indirectly promote cellular responses by associating with one or more other proteins, which in turn directly promote cellular responses. The intracellular signaling domain or its functional fragment can be from CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD40, CD47, CD79A, CD79B, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD278 (ICOS), CD357 (GITR), CARD11, DAP10, DAP12, FcRα, FcRβ, FcRγ, Fyn, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, ROR2, Ryk, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2 or any combination thereof. The chimeric receptor provided by the present invention includes an intracellular signaling domain. In one example, the intracellular signal transduction domain includes an immunoreceptor tyrosine-based activation motif or an ITAM signaling motif.

[0179] In one example, the intracellular signal transduction domain includes an intracellular signal transduction domain selected from: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD79a, CD79b, CD278, or CD66d, or a combination thereof. In one example, the intracellular signal transduction domain includes a CD3ζ intracellular signaling domain (SEQ ID NO: 12 or 13).

[0180] In one example, the intracellular signal transduction domain included in the chimeric receptor provided by the present invention further includes a co-stimulatory domain. In one example, the co-stimulatory domain is selected from the intracellular signal transduction domain of CD137, CD28, CD27, TNFRSF9, TNFRSF4, TNFRSF8, TNFRSF14, TNFRSF18, CD40LG, ICOS, ITGB2, CD2, CD7, KLRC2, HAVCR1, LGALS9, or CD83, or a combination thereof. In one example, the co-stimulatory structure is the intracellular signal transduction domain of CD137 (SEQ ID NO: 11). In one example, the co-stimulatory structure is the intracellular signal transduction domain of CD28 (SEQ ID NO: 10).

[0181] The chimeric receptor of the present invention includes a transmembrane domain. In one embodiment, the transmembrane domain is selected from the group consisting of CD2, CD3ε, CD3δ, CD3ζ, CD8, CD25, CD27, CD28, CD40, CD79A, CD79B, CD80, CD86, CD95 (Fas), CD134 (OX40), CD137, CD150 (SLAMF1), CD152 (CTLA4), CD200R, CD223 (LAG3), CD270 (HVEM), CD272 (BTLA), CD273 (PD-L2), CD274 (PD -L1), CD278 (ICOS), CD279 (PD-1), CD300, CD357 (GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 or Zap70 transmembrane domain. In one example, the transmembrane domain is the transmembrane domain of CD28 (SEQ ID NO: 8 or 9). In one example, the transmembrane domain is a variant of the transmembrane domain of CD28 (SEQ ID NO: 8 or 9) with no more than three amino acid mutations. In one example, the transmembrane domain is the CD8 transmembrane domain (SEQ ID NO: 7). In one example, the transmembrane domain is a variant of the CD8 transmembrane domain (SEQ ID NO: 7) with no more than three amino acid mutations.

[0182] In one example, in the chimeric receptor provided by the present invention, the extracellular domain and the transmembrane domain are connected by a spacer region (also called a hinge region, Linker).

[0183] In one example, the hinge region is selected from: CD28 hinge region, CD8 hinge region, IgG spacer region or fragment thereof, or a combination thereof. In one example, the hinge region can be IgG1 spacer region or fragment thereof, IgG2 spacer region or fragment thereof, IgG3 spacer region or fragment thereof, or IgG4 spacer region or fragment thereof.

[0184] In one example, the hinge region is a CD8 hinge region (SEQ ID NO: 3), an IgG1 spacer region (SEQ ID NO: 4), or an IgG4 spacer region (SEQ ID NO: 5 or 6).

[0185] In one example, the chimeric receptor includes a signal peptide. In one example, the signal peptide is a CD8 signal peptide (SEQ ID NO: 1) or a GMCSFRα signal peptide (SEQ ID NO: 2).

[0186] In one example, the chimeric receptor provided by the present invention includes (1) a domain of an anti-CD19 antibody (VH / VL is selected from SEQ ID NOs: 21, 22), and / or a domain of an anti-CD20 antibody (VH / VL is selected from SEQ ID NOs: 23, 24), (2) a CD8 hinge region (SEQ ID NO: 3), an IgG1 spacer region (SEQ ID NO: 4) or an IgG4 spacer region fragment (SEQ ID NO: 5 or 6), (3) a CD28 transmembrane domain (SEQ ID NO: 8 or 9) or a CD8 transmembrane domain (SEQ ID NO: 7), (4) a CD28 intracellular signaling domain (SEQ ID NO: 10) or a CD137 intracellular signaling domain (SEQ ID NO: 11); optionally, further includes (5) a CD3 zeta intracellular signaling domain (SEQ ID NO: 12 or 13).

[0187] In one example, the chimeric receptor provided by the present invention includes an anti-CD19 antibody or a fragment thereof, and an anti-CD20 antibody or a fragment thereof; wherein the anti-CD19 antibody or a fragment thereof includes a heavy chain variable region (VH1) and a light chain variable region (VL1), and the anti-CD20 antibody or a fragment thereof includes a heavy chain variable region (VH2) and a light chain variable region (VL2). Different variable regions are connected by a connecting peptide chain. In one example, the connecting peptide chain includes: the amino acid sequence shown in SEQ ID NO: 14, 15 and / or 16.

[0188] The antigen binding domain of the chimeric receptor can have different conformations, e.g. Figure 1Some of the ones shown. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VH1-VL1-VH2-VL2. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VL1-VH1-VL2-VH2. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VH1-VL1-VL2-VH2. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VL1-VH1-VH2-VL2. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VH1-VH2-VL2-VL1. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VL1-VH2-VL2-VH1. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VH1-VL2-VH2-VL1. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VL1-VL2-VH2-VH1. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VH2-VH1-VL1-VL2. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VL2-VH1-VL1-VH2. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VH2-VL1-VH1-VL2. In one example, the antigen binding domain includes, from the N-terminus to the C-terminus, VL2-VL1-VH1-VH2.

[0189] The chimeric receptors provided herein include an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 17, 29, 30, 31, 32, 33, 34, 55. The chimeric receptors provided herein include an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 39, 40, 41 or 42.

[0190] In one example, the immune cell marker is NKG2A, CD38, CD7, TIGIT, FasL or a combination thereof. In one example, the chimeric receptor includes an antibody or fragment against NKG2A. In one example, the antibody or fragment is selected from: a whole antibody, scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab')2 fragment, a Fd fragment, a sdAb, a multifunctional antibody, a DDPP antibody, a scFv-Fc antibody or an IgG4 antibody. In one example, the chimeric receptor that binds to an immune cell marker includes an IgG4 hinge region. In one example, the chimeric receptor that binds to an immune cell marker includes amino acids with at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 39, 40, 41 or 42. For example, the chimeric receptor comprises amino acids having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% homology or identity to the amino acid sequences set forth in SEQ ID NOs:25 and 39, SEQ ID NOs:25 and 40, SEQ ID NOs:25 and 41, SEQ ID NOs:25 and 42, SEQ ID NOs:26 and 39, SEQ ID NOs:26 and 40, SEQ ID NOs:26 and 41, SEQ ID NOs:26 and 42, SEQ ID NOs:27 and 39, SEQ ID NOs:27 and 40, SEQ ID NOs:27 and 41, SEQ ID NOs:27 and 42, SEQ ID NOs:28 and 39, SEQ ID NOs:28 and 40, SEQ ID NOs:28 and 41, or SEQ ID NOs:28 and 42.

[0191] In one example, the pathological cells are selected from malignant cells or infected cells. In one example, the pathological cells are selected from tumor cells or infected cells that are positive for CD19 and / or CD20. In one example, the pathological cells are selected from blood tumor cells and pathological cells of autoimmune diseases. In one example, the blood tumor is selected from: leukemia, lymphoma, myeloma; the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (systemic lupus erythematosus, SLE), rheumatoid arthritis (rheumatoid arthritis, RA), ankylosing spondylitis (AS), Sjogren syndrome (Sjogrensyndrome, SS), polymyositis / dermatomyositis.

[0192] The sequences provided in the present application are not limited to Bite binding to CD19 and CD20, and the antibody sequences of Bite binding to CD19 and CD20 are connected in a LOOP form.

[0193] For example, the chimeric receptor further includes a spacer (also referred to as a hinge region, Linker) located between the antigen binding domain and the transmembrane binding domain. For example, the spacer comprises a portion of an immunoglobulin. For example, the spacer comprises a sequence of a hinge region, a CH2 region, and / or a CH3 region. For example, the spacer comprises all or part of an IgG4 hinge region and / or an IgG2 hinge region, wherein the IgG4 hinge region is optionally a human IgG4 hinge region and the IgG2 hinge region is optionally a human IgG2 hinge region; the CH2 region contains all or part of IgG4 CH2 and / or IgG2 CH2, wherein IgG4 CH2 is optionally a human IgG4 CH2 and IgG2CH2 is optionally a human IgG2 CH2; and / or the CH3 region contains all or part of IgG4 CH3 and / or IgG2 CH3, wherein IgG4 CH3 is optionally a human IgG4 CH3 and the IgG2 CH3 is optionally a human IgG2 CH3. For example, the hinge region, CH2, and CH3 contain all or a portion of the hinge from human IgG4, all or a portion of the CH2, and all or a portion of the CH3. For example, one or more of the hinge region, CH2, and CH3 are chimeric and contain the hinge, CH2, and CH3 from human IgG4 and human IgG2. For example, the spacer comprises an IgG4 / 2 chimeric hinge region, or a modified IgG4 hinge region containing at least one amino acid substitution compared to the human IgG4 hinge; an IgG2 / 4 chimeric CH2 region; and an IgG4 CH3 region.

[0194] The present invention provides chimeric receptors that bind to CD19 as shown in Table 1.

[0195] Table 1. CD19-CAR structure

[0196]

[0197]

[0198] The present invention provides chimeric receptors that bind to CD20 as shown in Table 2.

[0199] Table 2. CD20-CAR structure

[0200] Carrier structure CAR20.4 <![CDATA[L-CD20V H -CD20V L -CD8(H)-CD28(TM)-CD28(C)-CD3ζ]]> CAR20.5 <![CDATA[L-CD20V H -CD20V L -CD8(H)-CD8(TM)-CD137(C)]]> CAR20.6 <![CDATA[L-CD20V H -CD20V L -CD8(H)-CD28(TM)-CD28(C)-CD137(C)]]> CAR20.7 <![CDATA[L-CD20V H -CD20V L -IgG4(H)-CD28(TM)-CD137(C)-CD3ζ]]> CAR20.8 <![CDATA[L-CD20V L -CD20V H -IgG4(H)-CD28(TM)-CD137(C)-CD3ζ]]> CD20-BBZ L-CD20VL-CD20VH-CD8(H)-CD8(TM)-CD137(C)

[0201] The present invention provides chimeric receptors that bind CD19 and CD20 as shown in Table 3.

[0202] Table 3. CD19-CD20-CAR structure

[0203]

[0204] The present invention provides chimeric receptors that bind to immune cell markers as shown in Table 4.

[0205] Table 4. CAR structures targeting immune cell markers

[0206] CAR vector structure NKG2A-s28z L-NKG2A(scFv)-IgG4(H)-CD28(TM)-CD28(C)-CD3ζ NKG2A-28z L-NKG2A(scFv)-CD8(H)-CD28(TM)-CD28(C)-CD3ζ CD38-S28Z L-CD38(scFv)-IgG4(H)-CD28(TM)-CD28(C)-CD3ζ CD38-28z L-CD38(scFv)-CD8(H)-CD28(TM)-CD28(C)-CD3ζ FasL-s28z L-FasL(scFv)-IgG4(H)-CD28(TM)-CD28(C)-CD3ζ FasL-28z L-FasL(scFv)-CD8(H)-CD28(TM)-CD28(C)-CD3ζ

[0207] In the above Tables 1, 2, 3, and 4, L: CD8 signal peptide (SEQ ID NO: 1), GMCSFRα signal peptide (SEQ ID NO: 2); CD8 (H): CD8α hinge (SEQ ID NO: 3); IgG4 (H): IgG4 short hinge (SEQ ID NO: 6); I1 (SEQ ID NO: 14), I2 (SEQ ID NO: 15), I3 (SEQ ID NO: 16); CD8TM (SEQ ID NO: 7), CD28TM (SEQ ID NO: 8 or 9); CD137 (C): CD137 costimulatory signaling domain (SEQ ID NO: 11); CD28 (C): CD28 costimulatory signaling domain (SEQ ID NO: 10); CD3ζ: CD3ζ signaling domain (SEQ ID NO: 12 or 13); CD19V H :CD19 antibody VH (SEQ ID NO: 21); CD19V L :CD19 antibody VL (SEQ ID NO: 22); CD20 V H :CD20 antibody VH (SEQ ID NO: 23); CD20V L :CD20 antibody VL (SEQ ID NO: 24); NKG2A (scFv): NKG2A antibody scFv (SEQ ID NO: 25); CD38 (scFv): CD38 antibody scFv (SEQ ID NO: 28), TIGIT (scFv): TIGIT antibody scFv (SEQ ID NO: 27); FasL (scFv): FasL antibody scFv (SEQ ID NO: 26). The corresponding lentivirus of the above expression vector was prepared using conventional molecular biological techniques.

[0208] The CAR-T1 cells in the embodiments disclosed in the present invention include CAR19.1 and CAR20.4 in Tables 1 and 2; CAR-T2 cells include CAR19.2 and CAR20.5 in Tables 1 and 2; CAR-T3 cells include CAR19.2 and CAR20.6 in Tables 1 and 2; CAR-T4 cells include CAR19.3 and CAR20.7 in Tables 1 and 2; CAR-T5 cells include CAR19.3 and CAR20.8 in Tables 1 and 2; CAR-T6 cells include CAR6 in Table 3; CAR-T7 cells include CAR7 in Table 3; CAR-T8 cells include CAR8 in Table 3; CAR-T9 cells include CAR9 in Table 3; CAR-T10 cells include CAR10 in Table 3; CAR-T11 cells include CAR11 in Table 3. The control group CAR-12 includes CAR12 in Table 3. CAR9 / NKG2A(1) T cells include CAR9 and NKG2A-28z in Tables 3 and 4. CAR9 / NKG2A(2) T cells contain CAR9 and NKG2A-s28z in Tables 3 and 4.

[0209] Carrier

[0210] Genetic modification of immune cells (e.g., T cells or NKT cells) in the composition can be accomplished by transducing a substantially homogeneous cell population with a recombinant nucleic acid molecule. In one example, a retroviral vector (gamma-retrovirus or slow virus) is used to introduce nucleic acid molecules into cells. For example, a polynucleotide encoding an exogenous receptor (e.g., CAR) can be cloned into a retroviral vector. Non-viral vectors can also be used. Transduction can use any suitable viral vector or non-viral delivery system. CAR can be constructed with auxiliary molecules (e.g., cytokines) in a single polycistronic expression cassette, multiple expression cassettes of a single vector, or multiple vectors. Examples of elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Baculovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis Virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides).

[0211] Other viral vectors that can be used include, for example, adenoviral, lentiviral and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus.

[0212] Non-viral methods can also be used for genetic modification of immune cells. For example, nucleic acid molecules can be introduced into immune cells by microinjection under lipid transfection, desialomycin-polylysine coupling, or surgical conditions. Other non-viral gene transfer methods include in vitro transfection using liposomes, calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. It is also possible to first transfer nucleic acid molecules to cell types that can be cultured in vitro (e.g., autologous or allogeneic primary cells or their progeny), and then inject the cells (or their progeny) modified by the nucleic acid molecules into the subject's target tissue or inject them systemically.

[0213] The present invention provides nucleic acid molecules encoding chimeric receptors as shown in Table 1, 2, 3 or 4.

[0214] The present invention provides that the nucleic acid molecule encodes any one of the CD19-CAR molecules shown in Table 1 and any one of the CD20-CAR molecules shown in Table 2. In one example, the polypeptide chain encoded by the nucleic acid molecule includes CD19-CAR and CD20-CAR from the amino terminus (N terminus) to the carboxyl terminus (C terminus); or includes CD20-CAR, CD19-CAR in sequence. In one example, the CAR binding to CD19 and the CAR binding to CD20 are connected by a hydrolyzable connecting peptide. In one example, the hydrolyzable connecting peptide is P2A (SEQ ID NO: 18), E2A (SEQ ID NO: 20), or F2A (SEQ ID NO: 19).

[0215] The present invention provides nucleic acid molecules encoding chimeric receptors binding to CD19 and CD20 as shown in Table 3, and chimeric receptors binding to immune cell markers as shown in Table 4. In one example, the polypeptide chain encoded by the nucleic acid molecule includes CAR binding to CD19 and CD20, and chimeric receptors binding to immune cell markers from the amino terminus (N terminus) to the carboxyl terminus (C terminus); or includes chimeric receptors binding to immune cell markers, and CAR binding to CD19 and CD20. In one example, the polypeptide chain encoded by the nucleic acid molecule includes CAR binding to CD19 and CD20, and chimeric receptors binding to NKG2A from the amino terminus (N terminus) to the carboxyl terminus (C terminus); or includes chimeric receptors binding to NKG2A, and CAR binding to CD19 and CD20. In one example, the polypeptide chain encoded by the nucleic acid molecule includes CARs that bind CD19 and CD20, and chimeric receptors that bind TIGIT from the amino terminus (N terminus) to the carboxyl terminus (C terminus); or includes chimeric receptors that bind TIGIT, and CARs that bind CD19 and CD20. In one example, the polypeptide chain encoded by the nucleic acid molecule includes CARs that bind CD19 and CD20, and chimeric receptors that bind CD38 from the amino terminus (N terminus) to the carboxyl terminus (C terminus); or includes chimeric receptors that bind CD38, and CARs that bind CD19 and CD20. In one example, the polypeptide chain encoded by the nucleic acid molecule includes CARs that bind CD19 and CD20, and chimeric receptors that bind FasL from the amino terminus (N terminus) to the carboxyl terminus (C terminus); or includes chimeric receptors that bind FasL, and CARs that bind CD19 and CD20.

[0216] The present invention provides a CAR encoding binding to CD19 and CD20, and a CAR binding to an immune cell marker. For example, the nucleic acid molecule encodes any one of the CAR molecules binding to CD19 and CD20 as shown in Table 3, and any one of the CAR molecules binding to an immune cell marker as shown in Table 4. In one example, the CAR binding to CD19 and CD20 and the CAR binding to an immune cell marker are connected by a hydrolyzable connecting peptide. In one example, immune cell markers include: NKG2A, CD38, TIGIT and / or FasL.

[0217] The present invention provides a nucleic acid molecule 3 encoding a CAR that binds CD19 and CD20, and a nucleic acid molecule 4 encoding a CAR that binds an immune cell marker. In one example, the nucleic acid molecule 3 and the nucleic acid molecule 4 are constructed on a vector. In one example, the hydrolyzable connecting peptide is P2A (SEQ ID NO: 18), E2A (SEQ ID NO: 20), or F2A (SEQ ID NO: 19). In one example, the CAR that binds CD19 and CD20 and the CAR that binds an immune cell marker are connected by a hydrolyzable connecting peptide. In one example, the nucleic acid molecule 3 and the nucleic acid molecule 4 are constructed on different vectors. In one example, immune cell markers include: NKG2A, CD38, TIGIT and / or FasL.

[0218] Engineered cells

[0219] The present invention provides an engineered cell, which comprises the chimeric receptor disclosed in the present invention.

[0220] In one example, the engineered cells include a chimeric receptor that binds to CD19 (as shown in Table 1) and a chimeric receptor that binds to CD20 (as shown in Table 2).

[0221] In one example, the engineered cells include a chimeric receptor that binds to CD19 (as shown in Table 1), a chimeric receptor that binds to CD20 (as shown in Table 2), and a chimeric receptor that binds to an immune cell marker (as shown in Table 4).

[0222] In one example, the engineered cells include a chimeric receptor that binds CD19 and CD20 (as shown in Table 3).

[0223] In one example, the engineered cells include chimeric receptors that bind to CD19 and CD20 (as shown in Table 3), and also include chimeric receptors that bind to immune cell markers (as shown in Table 4).

[0224] In one example, the pathological cells are selected from malignant cells or infected cells. In one example, the pathological cells are selected from blood tumor cells and pathological cells of autoimmune diseases. In one example, the blood tumor cells are CD19 positive and / or CD20 positive tumor cells.

[0225] In one example, the blood tumor is selected from: leukemia, lymphoma, myeloma; the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjogren syndrome (SS), polymyositis / dermatomyositis.

[0226] In one embodiment, the present invention provides an engineered cell, which includes a polynucleotide encoding the present invention. In one embodiment, the present invention provides an engineered cell, which includes a vector encoding the present invention. In one embodiment, the present invention provides an engineered cell, which includes a virus encoding the present invention.

[0227] In one example, the engineering cell is an immune cell, a neuron, an epithelial cell, an endothelial cell or a stem cell. Stem cells include human pluripotent stem cells (including human induced pluripotent stem cells (iPSC) and human embryonic stem cells). For example, the engineering cell is an immune cell. For example, the engineering cell is a primary cell. For example, the engineering cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a T cell, a NKT cell or a combination thereof. The engineering cell can be a cell of autologous or allogeneic origin. For example, the engineering cell is derived from human PBMC cells.

[0228] In one example, the immune cells are selected from: B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, T cells, NKT cells, stem cell-derived immune effector cells, or a combination thereof.

[0229] In one example, the engineered cell is a T cell. In one example, the engineered cell is an allogeneic T cell. In one example, the engineered cell is a stem cell derived T cell. T cells can be cytotoxic T cells, helper T cells, αβT, or γδT, CD4+ / CD8+ double positive T cells, CD4+T cells, CD8+T cells, CD4 / CD8 double negative T cells, CD3+T cells, initial T cells, effector T cells, cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, Th0 cells, Th1 cells, Th2 cells, Th3 (Treg) cells, Th9 cells, Th17 cells, Thαβ helper cells, Tfh cells, stem cell-like central memory TSCM cells, central memory TCM cells, effector memory TEM cells or effector memory TEMRA cells. In one example, T cells are cytotoxic T cells. In some embodiments, the genetically engineered T cells provided by the present invention are separated. In one example, the genetically engineered T cells provided by the present invention are substantially purified.

[0230] In one example, the engineered cells provided by the present invention are derived from cells isolated from a subject. As used in the present invention, genetically engineered cells derived from source cells refer to genetically engineered cells obtained by obtaining source cells and genetically manipulating the source cells. The source cells may be from natural sources. For example, the source cells may be primary cells isolated from a subject. The source cells may also be cells that have been passaged or genetically manipulated in vitro.

[0231] In one example, the genetically engineered cells provided by the invention are derived from cells separated from human body.Immune effector cells (e.g., T cells) can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue of infection site, ascites, pleural effusion, spleen tissue and tumor. In one example, T cell lines available in the art can be used. In one example, the genetically engineered cells provided by the invention are derived from cells separated from peripheral blood. In one example, the genetically engineered cells provided by the invention are derived from cells separated from bone marrow. In one example, the genetically engineered cells provided by the invention are derived from cells separated from peripheral blood mononuclear cells (PBMC).

[0232] In one example, the engineered cells provided by the present invention are derived from cells differentiated in vitro from stem cells or progenitor cells. In one example, stem cells or progenitor cells are selected from the group consisting of T cell progenitor cells, hematopoietic stem / progenitor cells, hematopoietic multipotent progenitor cells, embryonic stem cells and induced pluripotent cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from T cell progenitor cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from hematopoietic stem / progenitor cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from hematopoietic multipotent progenitor cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from embryonic stem cells. In one example, the genetically engineered cells provided by the present invention are derived from cells differentiated in vitro from induced pluripotent cells.

[0233] In one example, the engineering cell also includes: low expression or non-expression of endogenous TCR, B2M, HLA-I, HLA-II, NKG2A, FAS and / or CD58. In one example, the engineering cell also includes low expression or non-expression of endogenous TCR and B2M. In one example, the engineering cell also includes low expression or non-expression of endogenous FAS. In one example, the engineering cell also includes: low expression or non-expression of endogenous CD58. In one example, the engineering cell also includes: low expression or non-expression of endogenous NKG2A. In one example, the engineering cell also includes: non-expression of endogenous TCR and B2M. In one example, the engineering cell also includes: non-expression of endogenous TCR / B2M / NKG2A. In one example, the engineering cell also includes: non-expression of endogenous TCR / B2M / FAS. In one example, the engineered cell further comprises: no expression of endogenous TCR / B2M / CD58.

[0234] In one example, the present invention provides a method for genetic engineering by transferring the polynucleotide provided by the present invention into a cell using gene editing. If necessary, techniques such as nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), regularly clustered short palindromic repeats (CRISPRs), homologous recombination, nonhomologous end joining, microhomology-mediated end joining, homology-mediated end joining, etc. can be used to achieve site-specific integration.

[0235] In one example, endogenous TCR / B2M, TCR / B2M / NKG2A, TCR / B2M / FAS, or TCR / B2M / CD58 are knocked out by CRISPR / Cas9 technology. In one example, the sgRNA sequences targeting TRAC, B2M, FAS, and NKG2A are shown in SEQ ID NOs: 35, 36, 37, and 38, respectively.

[0236] For example, in some embodiments, the present invention provides an engineered T cell expressing a chimeric receptor, the engineered T cell including the non-expression of endogenous TCR / B2M, and optionally, the non-expression of NKG2A, FAS and / or CD58; the chimeric receptor includes, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular signal transduction domain; wherein the extracellular region includes an antibody that binds to CD19 and CD20 disclosed herein, the transmembrane domain includes, for example, a transmembrane domain of CD28 or CD8, wherein the intracellular signal transduction domain includes, for example, a CD137 intracellular signaling domain, a CD28 intracellular signaling domain, and a CD3ζ intracellular signaling domain. Its extracellular domain and transmembrane domain may be connected by, for example, a CD8 hinge region, a CD28 hinge region, or an IgG spacer region or a fragment thereof. The engineered cell may also include a chimeric receptor that binds an immune cell marker.

[0237] In one example, the engineered cells expressing chimeric receptors of the present application can bind to target cells expressing CD19 and / or target cells expressing CD20. For example, the engineered cells of the present application can bind to NK cells. In one example, the engineered cells of the present application can bind to CD19 and / or CD20 positive cells, and can also bind to NK cells. For example, the engineered cells of the present application can bind to target cells expressing NK cell markers (e.g., CD7, NKG2A, CD38, CD94, CS1, TIGIT, NKG2DL). For example, the engineered cells expressing chimeric receptors targeting CD19 and CD20 disclosed in the present invention also express chimeric receptors targeting NKG2A and / or TIGIT. For example, the engineered cells expressing chimeric receptors targeting CD19 and CD20 disclosed in the present invention also express chimeric receptors targeting NKG2A. For example, the engineered cells expressing chimeric receptors targeting CD19 and CD20 disclosed in the present invention also express chimeric receptors targeting CD38. For example, the engineered cells expressing chimeric receptors targeting CD19 and CD20 disclosed in the present invention also express chimeric receptors targeting CD7. For example, the engineered cells expressing chimeric receptors targeting CD19 and CD20 disclosed in the present invention also express chimeric receptors targeting NKG2A and FasL. For example, the engineered cells expressing chimeric receptors targeting CD19 and CD20 disclosed in the present invention also express chimeric receptors targeting CD38 and FasL.

[0238] In one example, the engineered cells expressing chimeric receptors of the present application do not cause host rejection of the graft reaction. In one example, in the presence of host immune cells (e.g., NK cells), the engineered cells expressing chimeric receptors of the present application have a longer survival time and / or amplification capacity. For example, the engineered cells can kill the host's immune cells. For example, the engineered cells can kill the host's NK cells. For example, the engineered cells can kill allogeneic NK cells. For example, the engineered cells can resist the killing of host NK cells. For example, the engineered cells can resist the killing of allogeneic NK cells. For example, relative to cells that do not express the chimeric receptors of the present application, the engineered cells expressing the chimeric receptors of the present application have significantly improved ability to kill pathological cells.

[0239] In one example, the engineered cells have a prolonged survival time or enhanced proliferation ability in the presence of host immune cells. In one example, the engineered cells have an inhibitory or killing function on the host's immune cells. In one example, the engineered cells have an inhibitory or killing function on the host's T cells and NK cells. In one example, the engineered cells have an inhibitory or killing function on the host's NK cells. In one example, the engineered cells can enhance the survival, proliferation, and killing of pathological cells of another engineered cell that is introduced into the subject previously, simultaneously, or later.

[0240] In one example, in the presence of host immune cells (e.g., NK cells), the engineered cells of the present application that express in combination with CD19, CD20, and NK cell markers (e.g., NKG2A, CD38, CD94, CS1, TIGIT, NKG2DL) have longer survival time and / or amplification capacity. For example, the engineered cells have a stronger ability to kill host NK cells. For example, the engineered cells have a stronger ability to kill allogeneic NK cells. For example, the engineered cells have a stronger ability to resist the killing of host NK cells. For example, the engineered cells have a stronger ability to resist the killing of allogeneic NK cells.

[0241] In one example, the engineered cells expressing the chimeric receptor also express immune checkpoint inhibitors. Immune checkpoint inhibitors include any agent that blocks, inhibits or reduces the activity or function of the inhibitory pathway of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, ligands and / or receptor-ligand interactions. Exemplary immune checkpoint molecules targeted for blocking, inhibiting, regulating, enhancing and / or stimulating include, but are not limited to: PD-1 (CD279), PD-L1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40 (CD134, TNFRSF4), CXCR2, tumor-associated antigens (TAA), B7-H3, B7-H4, BTLA, HVE M, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and expressed on all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also known as BY55), CGEN-15049, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and transforming growth factor receptor (TGFR; e.g., TGFRβ). Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof or other binding proteins that bind to and block or inhibit and / or enhance or stimulate the activity of one or more of any of the molecules described.

[0242] Transduction

[0243] The present invention provides a method for transducing a viral vector into a cell (e.g., an immune effector cell), the method involving activation and transduction of the cell to be transduced, the activation and transduction of the cell can be performed simultaneously, that is, the input composition containing the cell to be transduced, the cell stimulator to be transduced, and the viral vector particles carrying the recombinant nucleic acid encoding the chimeric receptor of the present invention are incubated together, or the activation can be performed first and then the transduction can be performed, such as the input composition containing the cell to be transduced and the cell stimulator to be transduced are incubated together for activation, and then the viral vector particles carrying the recombinant nucleic acid encoding the chimeric receptor of the present invention are added for incubation, and the total time for transduction activation and transduction of the recombinant nucleic acid is controlled to be completed within 72 hours, preferably, within 48 hours, or within 36 hours, or within 24 hours. In some embodiments, the method provided involves incubating and / or contacting a retroviral vector particle (e.g., a lentiviral vector) with a cell (e.g., an immune cell, such as a T cell) group, and before and / or simultaneously and / or after contacting or incubating the cell with the viral particle, using an ex vivo cell activation reagent (e.g., an anti-CD3 / anti-CD28 reagent) to activate and / or activate the T cell. Preferably, the cells are activated first and then the virus is transduced. In one example, when the input composition containing the cells to be transduced, the stimulator of the cells to be transduced, and the viral vector particles carrying the recombinant nucleic acid encoding the chimeric receptor of the present invention are incubated together, the incubation time does not exceed 72 hours for harvesting to obtain an output composition, and the output composition contains cells transduced with the recombinant nucleic acid; preferably, it can be 1 hour to 72 hours; more preferably, the incubation time is 2 hours to 48 hours; more preferably, the incubation time is 2 hours to 36 hours; more preferably, the incubation time is 12 hours to 36 hours; more preferably, the incubation time is 12 hours to 24 hours; more preferably, the incubation time is 15 hours to 24 hours. In one example, after the output composition is purified by washing, centrifugation, etc., a pharmaceutical preparation can be prepared without further in vitro amplification culture, that is, the drug prepared using the output composition does not need to be amplified in vitro before being used in a subject (or patient).

[0244] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0245] The biological materials used in the examples of this application are shown in Table 5.

[0246] Table 5

[0247]

[0248] Example 1. Vector construction

[0249] The vector expressing the chimeric receptor disclosed in the present application was constructed using conventional molecular biology techniques. Full-length DNA was synthesized to construct the expression vectors CAR6-12 of the bispecific CAR shown in Table 3, which were respectively integrated into the pRRLSIN vector (Addgene). The corresponding lentivirus of the above expression vector was prepared using conventional molecular biology techniques.

[0250] Full-length DNA was synthesized to construct expression vectors CAR19.1, 19.2, 19.3, and / or CAR20.4, 20.5, 20.6, 20.7, 20.8 binding to CD19 and CD20 as shown in Tables 1 and 2, and integrated into pRRLSIN vector (Addgene). The corresponding lentivirus of the above expression vectors was prepared using conventional molecular biological techniques.

[0251] Example 2. Construction of CAR-T cells

[0252] The conventional method for preparing CAR-T in the art was adopted: donor PBMC cells were collected, activated by magnetic beads (Life Technologies, 40203D) containing anti-CD3 and CD28 antibodies, and T cells were infected with lentivirus containing the above-mentioned vector to prepare CAR-T1-12 cells expressing the corresponding chimeric receptor. T cells not transfected with the virus were regarded as UTD.

[0253] 1×10 were collected for each CAR-T sample 5 The cells were incubated with 5 μg / ml biotinylated anti-FMC63 antibody (Acrobiosystems, FM3-BY54) and anti-Fab antibody (Jackson, 109-066-006) at room temperature for 1 hour, and then Streptavidin BV421 (Biolegend, 405225, 1:200 dilution) antibody was added and incubated at room temperature for 30 minutes. The CAR positivity rate was detected by FACS.

[0254] Example 3. Detection of the expression of CD19 and CD20 in human B lymphoid leukemia cells Nalm6 and human Burkitt's lymphoma cells Daudi

[0255] Nalm6 cells (Nalm6-Luci) and Daudi cells (Daudi-Luci) expressing exogenous luciferase were constructed using conventional molecular biological techniques, and anti-CD19 APC-H7 (BD, 560727) and anti-CD20 APC antibodies (Invitrogen, 17-0209-4) were used to detect the expression of CD19 and CD20 in different cells by flow cytometry (FACS). Figure 2 The results showed that Daudi expressed CD19 and CD20, while Nalm6 expressed CD19.

[0256] Example 4. In vitro cytotoxic effects of dual-target CAR-T cells on lymphoma cells

[0257] Daudi and Nalm6 cells were inoculated in 96-well plates at 10,000 cells / well. After 18 hours of co-incubation with Daudi and Nalm6 cells at an effector-target ratio of 3:1, 1:1 or 1:3, the killing effect of CAR T cells on lymphoma cells was detected by FACS. Figure 3A ,3B showed that CD19 / CD20 dual-target CAR-T1, 2, 4, 5, 6, 7, 8, 9, and 10 cells could significantly kill tumor cells Daudi and Nalm6.

[0258] Example 5. Cytokine secretion after co-incubation of dual-target CAR-T cells with tumor cells

[0259] Daudi and Nalm6 lymphoma cells were inoculated in 96-well plates at 40,000 cells / well. CD19 / CD20 dual-target CAR-T cells were co-incubated with Daudi and Nalm6 cells at a target ratio of 1:1 for 18 hours, and the secretion levels of interferon-γ (IFN-γ), IL-2, and TNF-α in the supernatant were detected using the CBA method (BD, 558264). Figure 4A ,B shows that CD19 / CD20 dual-target CAR-T1, 2, 4, 5, 6, 7, 8, 9, and 10 cells can secrete high levels of IFN-γ, IL-2, and TNF-α.

[0260] Example 6. Dual-target CAR-T for the treatment of subcutaneous transplanted lymphoma in NPG mice

[0261] On D0, each NPG mouse was subcutaneously inoculated with 3×10 6 Daudi cells, 5 in each group. On D12, the average tumor volume was about 130 mm 3 On D13, each mouse was injected intravenously with 3×10 6 CD19 / 20 dual-target CART cells.

[0262] Figure 5 The results showed that on D34, compared with the UTD group, the tumor volume inhibition rates of the CAR-T1 and CAR-T11 groups were 100%; among them, the tumors of all 5 mice in the CAR-T1 group regressed, and the tumors of 2 mice in the CAR-T11 group regressed.

[0263] Example 7. Detection of the survival of human T cells in mouse peripheral blood

[0264] Peripheral blood was randomly collected from 4 mice in each group after CAR-T treatment as described in Example 6. 50 μL of each blood sample was used for detection using antibodies anti-CD45 APC-H7 (1 ul / test, BD, 560178) and CD3 percp-cy5.5 / CD4 FITC / CD8PE (10 ul / test, BD, 340298) using BD absolute counting tubes (BD, Cat: 340334, Lot: 21258, Beads Number: 49450 / tube). Figure 6 It shows that on the 7th and 14th days after CAR-T cell infusion, surviving human T cells were detected in the peripheral blood of mice in the CAR-T1 and CAR-T11 groups.

[0265] Example 8. Dual-target CAR-T for the treatment of in situ lymphoma in NPG mice

[0266] On D0, 3×10^6 Daudi-Luci cells were injected into the tail vein of each NPG mouse, with 5 mice in each group. Fluorescence imaging (IVIS LUMINA III SYSTEM) was performed at different time points to observe the photon intensity of the tumor. On D9, the photon intensity reached an average of 2.47×10 4 At p / s / cm2 / sr, each mouse was injected with 1×10 6 CAR-T cells. Figure 7 , 8 The results showed that when the mice were observed until D80, three mice in the CD19-CAR-T group died and the tumor fluorescence value of one mouse continued to rebound from D31; the tumor fluorescence value of one mouse in the CAR-T12 group continued to rebound from D45; and the CAR-T4, CAR-T9, and CAR-T10 groups had the best tumor inhibition effects: there was no tumor rebound, and all mice survived and were in good condition.

[0267] Example 9. UCAR-T cell preparation

[0268] In vitro synthesis of sgRNA sequences targeting TRAC, B2M, FAS, and NKG2A (shown in SEQ ID NOs: 35, 36, 37, and 38, respectively) (Kaixing Diagnostics), knocking out the endogenous TCR / B2M or TCR / B2M / NKG2A of the CAR-T9 and CAR-T12 cells constructed in Example 2 by CRISPR / Cas9 technology (Cas9 protein, Kaixia Biotechnology (Shanghai) Co., Ltd., CAS-EE109), and obtaining CAR-T9 (DKO), CAR-T9 (TKO), and CAR-T12 (DKO) by magnetic bead sorting (Miltenyi Biopharmaceuticals, 130-048-801). Untransduced UTD cells with TCR / B2M / NKG2A knockout were used as controls. 1×10 5 The cells were incubated with 5 μg / ml biotinylated anti-FMC63 antibody (Acrobiosystems, FM3-BY54) or anti-Fab antibody (Jackson, 109-066-006) or NKG2A protein (Kaixing Diagnostics, KD1140) at room temperature for 1 hour, and then Streptavidin BV421 (Biolegend, 405225, 1:200 dilution) or Streptavidin PE antibody (Biolegend, 405225) were added and incubated at room temperature for 30 minutes, and the CAR positivity rate was detected by FACS.

[0269] Example 10. Dual-target CAR-T for the treatment of in situ lymphoma in NPG mice

[0270] On D0, each NPG mouse was injected with 3×10^6 Daudi-Luci cells through the tail vein, with 5 mice in each group. Fluorescence imaging (IVIS LUMINA III SYSTEM) was performed at different time points to observe the photon intensity of the tumor. On D10 (when the photon intensity reached an average of 6.4×10 4 Each mouse was injected intravenously with 1×10 6 UCAR-T cells. Fig. 9 , 10 , 11 showed that when the mice were observed until D131, all mice in the CD19-CAR-T (DKO) group died; 3 mice in the CD20-CAR-T (DKO) group died; and 2 mice in the CAR-T12 (DKO) group died. The CAR-T9 (DKO) and CAR-T9 (TKO) groups had the best tumor inhibition effects: the tumors did not rebound, and all mice survived and were in good condition.

[0271] Example 11. Vectors and cell construction targeting immune cells

[0272] Conventional molecular biology techniques were used to construct vectors expressing chimeric receptors targeting immune cell markers. Full-length DNA was synthesized, and expression vectors of NKG2A-28z and NKG2A-s28z as shown in Table 4 were constructed and integrated into the pRRLSIN vector (Addgene).

[0273] CAR9 / NKG2A(1) T cells and CAR9 / NKG2A(2) T cells were constructed with reference to Example 2.

[0274] Example 12. Preparation of CD19 / CD20-NKG2A UCAR-T cells

[0275] Referring to Example 9, CAR9 / NKG2A(1)-tko cells and CAR9 / NKG2A(2)-tko cells were constructed by knocking out TCR / B2M / NKG2A in CAR9 / NKG2A(1) T cells or CAR9 / NKG2A(2) T cells. Untransduced UTD cells with TCR / B2M / NKG2A knockout were used as controls. 1×10 5 The cells were incubated with 5 μg / ml biotinylated anti-FMC63 antibody (Acrobiosystems, FM3-BY54) or anti-Fab antibody (Jackson, 109-066-006) or NKG2A protein (Kaixing Diagnostics, HD1140) at room temperature for 1 hour, and then Streptavidin BV421 (Biolegend, 405225, 1:200 dilution) or Streptavidin PE antibody (eBioscience, 12-4317-87) were added and incubated at room temperature for 30 minutes, and the CAR positivity rate was detected by FACS.

[0276] Example 13. In vitro cytotoxic effects of CD19 / CD20-NKG2A UCAR-T cells on lymphoma cells

[0277] 30,000 Daudi and Nalm6 cells were inoculated in each well of a 96-well plate. CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells were taken and incubated for 18 h at an effector-target ratio of 1:1 or 1:3. Then, 7-AAD (BD, 559925) and CountingBeads (Invitrogen, C36995) were added to quantify the two types of cells. After detection by flow cytometry, the remaining numbers of the two types of cells were counted and calculated as follows: % cytotoxicity = (1-experimental group / control group)*100. Fig.12The results showed that both CAR9 / NKG2A(1)-tko and CAR9 / NKG2A(2)-tko cells could significantly kill lymphoma cells.

[0278] Example 14. Cytokine secretion after co-incubation of CD19 / CD20-NKG2A UCAR-T cells with tumor cells

[0279] The supernatant of CD19 / CD20-NKG2A UCAR-T cells co-cultured with Daudi and Nalm6 for 18 hours was obtained, and the secretion of IL-2, TNF-α, and IFN-γ cytokines was detected using CBA-Human Soluble Protein Master Buffer Kit (Cat: 558265). Fig.13 The results showed that CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells could secrete high levels of IL-2, TNF-α, and IFN-γ cytokines after co-incubation with tumor cells.

[0280] Example 15. In vitro killing of human primary NK cells by CD19 / CD20-NKG2A UCAR-T cells

[0281] Peripheral blood PBMC cells from donors were collected and NK cells were screened using CD56 magnetic beads (Miltenyi Biotec, 130-050-401) according to the manufacturer's instructions.

[0282] Collect 3×10 4 CD19 / CD20-NKG2A UCAR-T cells were co-cultured with the above-collected primary human NK cells at a ratio of 1:1 and 1:2 for 24 h and 72 h, and B2M-KO T cells were labeled with HLA-ABC (Thermo Fisher, 17-9983-42) and NK cells were labeled with CD56 (BD 555516). 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells. CountingBeads (Invitrogen, C36995) were added, and the number of aNK cells was detected by flow cytometry, and the lysis rate of NK cells was calculated. Fig.14 The results showed that after 24 or 72 hours of co-incubation with NK cells, CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells could significantly kill NK cells.

[0283] Example 16. Secretion of cytokines IL-2, TNF-α, and IFN-γ after co-incubation of CD19 / CD20-NKG2A UCAR-T cells with NK cells

[0284] The supernatant was obtained after co-culture of CD19 / CD20-NKG2A UCAR-T cells and human primary NK cells for 24 hours, and the secretion of IL-2, TNF-α, and IFN-γ cytokines was detected using CBA-Human Soluble Protein Master Buffer Kit (Cat: 558265). Fig.15 The results showed that CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells could secrete high levels of IL-2, TNF-α, and IFN-γ cytokines after co-incubation with NK cells.

[0285] Example 17. Rapid preparation process of CAR-T cells targeting CD19 and CD20

[0286] T cells were activated with magnetic beads (Miltenyi Biotec, 170-076-156) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 h, and then transduced with a virus containing CAR targeting CD19 and CD20 (prepared by conventional molecular biology techniques) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 h. The transduced T cells were collected and cryopreserved, or the T cell density was adjusted to approximately 1 × 10 6 cells / ml were inoculated into 24-well plates, cultured for more than 24 h, and cryopreserved.

[0287] The embodiments of the present invention include the embodiments as any single embodiment or in combination with any other embodiment or part thereof. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0288]

[0289]

[0290]

[0291]

[0292] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a bispecific chimeric receptor in the preparation of a drug for treating tumors, wherein: The chimeric receptor comprises an antigen binding domain, a transmembrane domain and an intracellular domain; the antigen binding domain comprises an anti-CD19 antibody heavy chain variable region (V H1 ) and antibody light chain variable region (V L1 ), anti-CD20 antibody heavy chain variable region (V H2 ) and antibody light chain variable region (V L2 ), the V H1 、V L1 、V H2 、V L2 Connect in a loop structure.

2. The use according to claim 1, wherein The antigen binding domain comprises: (1) Anti-CD20 antibody heavy chain variable region (V H2 ) and antibody light chain variable region (V L2 ) is connected to antibody 2, the variable region of the heavy chain of the anti-CD19 antibody (V H1 ) and antibody light chain variable region (V L1 ) are connected to both ends of the antibody 2; or (2) the variable region of the heavy chain of the anti-CD19 antibody (V H1 ) and antibody light chain variable region (V L1 ) is connected to antibody 1, the variable region of the heavy chain of the anti-CD20 antibody (V H2 ) and antibody light chain variable region (V L2 ) are respectively connected to both ends of the antibody 1; Preferably, from N-terminus to C-terminus, the antibody 2 comprises V H2、 V L2 or V L2 、V H2 ; or from N-terminus to C-terminus, the antibody 1 includes V H1、 V L1 or V L1 、V H1 .

3. The use according to claim 2, wherein From N-terminus to C-terminus, the antigen binding domain of the chimeric receptor 1 is shown in any of the following formulas: (1)V L1 -V H2 -IV L2 -V H1 、 (2)V H1 -V H2 -IV L2 -V L1 、 (3)V H1 -V L2 -IV H2 -V L1 、 (4)V L1 -V L2 -IV H2 -V H1 、 (5)V L2 -V H1 -IV L1 -V H2 、 (6)V H2 -V H1 -IV L1 -V L2 、 (7)V H2 -V L1 -IV H1 -V L2 、 (8)V L2 -V L1 -IV H1 -V H2 Each "-" is independently a connecting peptide or a peptide bond; I is a flexible linker.

4. The use according to any one of claims 1 to 3, wherein The V H1 comprising CDR1, CDR2 and CDR3 having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively, wherein the V L1 comprising CDR1, CDR2 and CDR3 having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48, respectively; and / or the V H2 comprising CDR1, CDR2 and CDR3 having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, respectively, wherein the V L2 comprising amino acid sequences CDR1, CDR2, and CDR3 having about 80% to about 100% identity to the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively; Preferably, the V H1 and V L1 An amino acid sequence having about 80% to about 100% identity with the amino acid sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; and / or said V H2 and V L2 An amino acid sequence having about 80% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

5. The use according to claim 3, wherein The flexible peptide linker is selected from: newlinker or (G4S)n, wherein n is 1, 2, 3, 4 or 5; preferably, the flexible peptide linker comprises the amino acid sequence shown in SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:

16.

6. The use according to any one of claims 1 to 5, wherein The co-stimulatory signal molecule included in the intracellular domain is selected from: CD27, CD28, CD137, OX40, CD30, CD40, PD-1, ICOS intracellular domain or a combination thereof; preferably, the co-stimulatory signal molecule is selected from: CD28 (SEQ ID NO: 10), CD137 (SEQ ID NO: 11); preferably, the intracellular domain includes the cytoplasmic signaling domain of CD3ζ; preferably, the cytoplasmic signaling domain of CD3ζ includes the amino acid sequence shown in SEQ ID NO: 12 or 13.

7. The use according to any one of claims 1 to 6, wherein The antigen binding domain is connected to the transmembrane region through a hinge region, and the hinge region is selected from: IgG1, IgG2, IgG4 hinge region or fragments thereof, CD8 hinge region or fragments thereof, CD28 hinge region or fragments thereof; preferably, the hinge region includes the amino acid sequence shown in SEQ ID NO: 3, 4, 5, 6.

8. The use according to any one of claims 1 to 7, wherein The transmembrane domain is selected from: CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 transmembrane domain; preferably, the transmembrane domain is selected from: CD8, CD28; more preferably, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:7, 8, 9.

9. The use according to any one of claims 1 to 8, wherein The chimeric receptor comprises an amino acid sequence having about 80% to 100% identity to the amino acid sequence shown in any one of SEQ ID NOs: 29, 30, 31, 32, 33, and 34.

10. Use of engineered cells in the preparation of drugs for treating tumors, wherein: The engineered cell comprises the chimeric receptor according to any one of claims 1-9.

11. Use of an engineered cell comprising a chimeric receptor that binds CD19 and CD20 in the preparation of a drug for treating a tumor, wherein: The engineered cell comprises a first chimeric receptor that binds to CD19 and a second chimeric receptor that binds to CD20, and the structures of the first chimeric receptor that binds to CD19 and the second chimeric receptor that binds to CD20 are shown in the following formula: (1)L-V L1 -V H1 -H-TM-C-CD3ζ and L-V H2 -V L2 -H-TM-C1-CD3ζ (2)L-V L1 -V H1 -H-TM-C-CD3ζ and L-V L2 -V H2 -H-TM-C1-CD3ζ (3)L-V H2 -V L2 -H-TM-C-CD3ζ and L-V H1 -V L1 -H-TM-C1 (4)L-V H1 -V L1 -H-TM-C-CD3ζ and L-V H2 -V L2 -H-TM-C1 (5)L-V H1 -V L1 -H-TM-C-CD3ζ and L-V H2 -V L2 -H-TM-C1-C2 In the formula, each "-" is independently a connecting peptide or a peptide bond; L is an optional signal peptide sequence; H is an optional hinge region; TM is a transmembrane domain; C1 is a co-stimulatory signal molecule 1, C2 is a co-stimulatory signal molecule 2; CD3ζ is an intracellular signal transduction sequence derived from CD3ζ; V H1 V is the variable region of the heavy chain of CD19 antibody; L1 V is the variable region of CD19 antibody light chain; L2 V is the variable region of CD20 antibody light chain; H2 is the variable region of the heavy chain of CD20 antibody; "-" is a connecting peptide or peptide bond.

12. The use according to claim 11, wherein The V H1 comprising CDR1, CDR2 and CDR3 having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45, respectively, wherein the V L1 comprising CDR1, CDR2 and CDR3 having an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence shown in SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:48; and / or said V H2 comprising CDR1, CDR2 and CDR3 having an amino acid sequence that is about 80% to about 100% identical to the amino acid sequence shown in SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51, wherein the V L2 comprising CDR1, CDR2, and CDR3 having amino acid sequences that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54, respectively; Preferably, the V H1 and V L1 An amino acid sequence having about 80% to about 100% identity with the amino acid sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; and / or said V H2 and V L2 An amino acid sequence having about 80% to about 100% identity with the amino acid sequence shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

13. The use according to claim 11 or 12, wherein The co-stimulatory signal molecule included in the intracellular domain is selected from: CD27, CD28, CD137, OX40, CD30, CD40, PD-1, ICOS intracellular domain or a combination thereof; preferably, the co-stimulatory signal molecule is selected from: CD28 (SEQ ID NO: 10) and / or CD137 (SEQ ID NO: 11).

14. The use according to any one of claims 11 to 13, wherein The antigen binding domain is connected to the transmembrane region through a hinge region, and the hinge region is selected from: IgG1, IgG2, IgG4 hinge region or fragments thereof, CD8 hinge region or fragments thereof, CD28 hinge region or fragments thereof; preferably, the hinge region includes the amino acid sequence shown in SEQ ID NO: 3, 4, 5, 6.

15. The use according to any one of claims 11 to 14, wherein The transmembrane domain is selected from: CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 transmembrane domain; preferably, the transmembrane domain is selected from: CD8 (SEQ ID NO: 7), CD28 (SEQ ID NO: 8, 9).

16. The use according to any one of claims 11 to 15, wherein The first chimeric receptor and the second chimeric receptor have amino acid sequences with about 80% to 100% identity to the amino acid sequences shown in SEQ ID NOs: 17 and 55, respectively.

17. The use according to any one of claims 10 to 16, wherein The engineered cell further comprises a third chimeric receptor, which binds to at least one or two or more immune cell markers; preferably, the immune cell marker is selected from: T cell markers and / or NK cell markers; preferably, the immune cell marker is a NK inhibitory receptor (NKIR); preferably, the immune cell marker is selected from: CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD138, CD160, CD161, CD178, CD218, CD 226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, TIGIT, TRAIL, SLAMF7, NKG2F, NKG2H, NKp30, NKp44, NKp46, NKp80, SLAM family members, L-selectin, natural cytotoxicity receptor NCR1, NCR2, NCR3, or a combination thereof.

18. The use according to any one of claims 10 to 17, wherein The third chimeric receptor comprises the amino acid sequence shown in SEQ ID NO: 25, 26, 27, 28 and / or 39, 40, 41, 42.

19. The use according to any one of claims 10 to 18, wherein The engineered cells also include: low expression or no expression of endogenous TCR, B2M, HLA-I, HLA-II, NKG2A, FAS and / or CD58; preferably, the engineered cells also include: no expression of endogenous TCR and B2M, or no expression of endogenous TCR / B2M / NKG2A.

20. The use according to any one of claims 10 to 19, wherein The engineered cells are immune cells, neurons, epithelial cells, endothelial cells, stem cells or a combination thereof; preferably, the engineered cells are immune cells selected from: T cells, NK cells, cytotoxic T cells, NKT cells, dendritic cells, macrophages, CIK cells, and stem cell-derived immune cells or a combination thereof; preferably, the engineered cells are autologous or allogeneic cells.

21. The use according to any one of claims 1 to 20, for treating, preventing or improving a tumor in a subject in need thereof; preferably, the tumor comprises: Leukemia, lymphoma, myeloma.

22. Use of multifunctional immune effector cells in the preparation of drugs for treating tumors, wherein: The immune effector cell comprises a chimeric receptor that binds CD19 and CD20, or comprises a first chimeric receptor that binds CD19 and a second chimeric receptor that binds CD20, The structure of the chimeric receptor that binds CD19 and CD20 is shown in any of the following formulas: (1)L-V L1 -V H2 -I-V L2 -V H1 -H-TM-C-CD3ζ (2)L-V L2 -V H1 -I-V L1 -V H2 -H-TM-C-CD3ζ (3)L-V H1 -V L2 -I-V H2 -V L1 -H-TM-C-CD3ζ (4)L-V H2 -V L2 -I-V L1 -V H1 -H-TM-C-CD3ζ The structures of the first chimeric receptor binding to CD19 and the second chimeric receptor binding to CD20 are shown in any of the following formulas: (5)L-V L1 -V H1 -H-TM-C-CD3ζ and L-V H2 -V L2 -H-TM-C-CD3ζ (6)L-V L1 -V H1 -H-TM-C-CD3ζ and L-V L2 -V H2 -H-TM-C-CD3ζ (7)L-V H2 -V L2 -H-TM-C-CD3ζ and L-V H1 -V L1 -H-TM-C (8)L-V H1 -V L1 -H-TM-C-CD3ζ and L-V H2 -V L2 -H-TM-C (9)L-V H1 -V L1 -H-TM-C-CD3ζ and L-V H2 -V L2 -H-TM-C-CD3ζ In the formula, Each "-" is independently a connecting peptide or a peptide bond; L is an optional signal peptide sequence; I is a flexible linker; H is an optional hinge region; TM is a transmembrane domain; C is a co-stimulatory signal molecule; CD3ζ is an intracellular signal transduction sequence derived from CD3ζ; V H1 V is the variable region of the heavy chain of CD19 antibody; L1 V is the variable region of CD19 antibody light chain; L2 V is the variable region of CD20 antibody light chain; H2 is the variable region of the heavy chain of CD20 antibody; "-" is a connecting peptide or peptide bond.

23. Use of a polynucleotide in the preparation of a drug for treating tumors, wherein: The polynucleotide encodes a chimeric receptor that co-binds CD19 and CD20, or encodes a first chimeric receptor that binds CD19 and a second chimeric receptor that binds CD20; The polynucleotide encoding the chimeric receptor binding CD19 and CD20 is shown in any of the following formulas: (1)L-V L1 -V H2 -I-V L2 -V H1 -H-TM-C-CD3ζ (2)L-V L2 -V H1 -I-V L1 -V H2 -H-TM-C-CD3ζ (3)L-V H1 -V L2 -I-V H2 -V L1 -H-TM-C-CD3ζ (4)L-V H2 -V L2 -I-V L1 -V H1 -H-TM-C-CD3ζ The polynucleotide encoding the first chimeric receptor binding to CD19 and the second chimeric receptor binding to CD20 is shown in any of the following formulas: (5)L-V L1 -V H1 -H-TM-C-CD3ζ-2A-L-V H2 -V L2 -H-TM-C-CD3ζ (6)L-V L1 -V H1 -H-TM-C-CD3ζ-2A-L-V L2 -V H2 -H-TM-C-CD3ζ (7)L-V H2 -V L2 -H-TM-C-CD3ζ-2A-L-V H1 -V L1 -H-TM-C (8)L-V H1 -V L1 -H-TM-C-CD3ζ-2A-L-V H2 -V L2 -H-TM-C (9)L-V H1 -V L1 -H-TM-C-CD3ζ-2A-L-V H2 -V L2 -H-TM-C-CD3ζ In the formula, Each "-" is independently a connecting peptide or peptide bond component; L is an optional nucleic acid component encoding a signal peptide; I is a nucleic acid module encoding a flexible linker; H is an optional hinge region nucleic acid module; TM is a transmembrane domain nucleic acid module; C is a co-stimulatory signal molecule nucleic acid module; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ; 2A is an optional 2A self-cleaving peptide nucleic acid module; V H1 V is the variable region of the heavy chain of CD19 antibody; L1 V is the variable region of CD19 antibody light chain; L2 V is the variable region of CD20 antibody light chain; H2 is the variable region of the heavy chain of CD20 antibody; "-" is a connecting peptide or peptide bond.

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