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

Immune effector cells and their applications Technical Field

[0001] 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

[0002] Despite the remarkable efficacy of anti-CD19 CAR-T cells, CD19 chimeric antigen receptor (CAR) T cell therapy also has many challenges. Some patients still experience poor responses and are prone to relapse. Therefore, there is an urgent need to develop effective methods to treat tumors and prevent antigen escape.

[0003] 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] In a first aspect, the present invention provides a multifunctional immune effector cell, comprising a chimeric receptor that binds to CD19 and CD20, or comprising 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 that binds to CD19 and the second chimeric receptor that binds 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 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 the light chain of CD19 antibody; L2 V is the variable region of the light chain of CD20 antibody; H2 is the variable region of the CD20 antibody heavy chain; “-” is a connecting peptide or peptide bond.

[0017] In one embodiment, 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.

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

[0020] In one embodiment, the transmembrane domain is selected from the group consisting of CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 transmembrane domains. Preferably, the transmembrane domain is selected from the group consisting of CD8 and 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 set forth 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 the signal peptide of natural IgG, IgM, IgD, IgA or IgE.

[0026] In one example, the chimeric receptor comprises an antigen binding domain comprising 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 further includes a costimulatory signaling domain.

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

[0031] In one embodiment, 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 comprises 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 comprises 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 the group consisting of: CD19, CD20, BCMA, GPRC5D, GPC3, Claudin18.2, CD22, FAP, Mesothelin, NKG2D, NKG2A, CD94, and 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 modules.

[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 embodiment, the polynucleic acid comprises, from N-terminus to C-terminus:

[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 natural IgG, IgM, IgD, IgA or IgE light chain is operably linked to the light chain variable region of the first antigen-binding domain; the signal peptide of the natural IgG, IgM, IgD, IgA or IgE heavy chain 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 embodiment, 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 according to the claims above.

[0050] In one embodiment, the engineered cells are immune cells; preferably, the immune cells are selected from the group consisting of: 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 expression of TCR, B2M, HLA-II and / or NKG2A.

[0053] In a sixth aspect, the present invention provides a polynucleotide encoding a chimeric receptor that binds to CD19 and CD20, or encoding 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 that binds CD19 and the second chimeric receptor that binds 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 signaling molecule nucleic acid module; CD3ζ is a cytoplasmic signaling 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 the light chain of CD19 antibody; L2 V is the variable region of the light chain of CD20 antibody; H2 is the variable region of the 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 embodiment, the nucleotide sequences encoding the same component are codon-divergent.

[0068] In one embodiment, 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 costimulatory signal molecule component is selected from: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS intracellular domain or a combination thereof; preferably, the costimulatory signal molecule component is selected from: CD28, 4-1BB; more preferably, the costimulatory 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 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, or a fragment thereof.

[0074] The seventh aspect of the present invention provides a vector, which 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, which 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 expression of TCR, B2M, HLA-II and / or NKG2A.

[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] In an eleventh aspect, 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 shows the structure of a dual-target chimeric antigen receptor that binds to CD19 and CD20: including a signal peptide sequence (L), antigen recognition sequences that recognize CD19 and CD20, a hinge region (Hinge), a transmembrane region (TM), a costimulatory signaling domain (Co-stimulator), and a CD3ζ signaling domain (CD3ζ). Expression vectors CAR6, 8, and 9 are shown in Formula I; expression vector CAR10 is shown in Formula II; expression vector CAR7 is shown in Formula III; and expression vector CAR11 is shown in Formula IX.

[0083] FIG2 shows that Daudi expresses CD19 and CD20; and Nalm6 expresses CD19.

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

[0085] Figures 4A-4B show 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 can secrete high levels of IFN-γ, IL-2, and TNF-α.

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

[0087] Figure 6 shows that on the 7th and 14th days after 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 shows the treatment of orthotopic lymphoma in NPG mice by UTD, CD19-CAR-T, and dual-target CAR-T4, 9, 10, and 12 cells.

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

[0090] Figure 9 shows that UTD (TKO), CD19-CAR-T (DKO), CD20-CAR-T (DKO), CAR-T9 (DKO), CAR-T12 (DKO), and CAR-T9 (TKO) cells were used to treat in situ lymphoma in NPG mice.

[0091] Figure 10 shows 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 in situ lymphoma in NPG mice, D131 days after tumor cell infusion.

[0092] Figure 11 shows the tumor fluorescence intensity IVIS imaging of UTD (TKO), CD19-CAR-T (DKO), CD20-CAR-T (DKO), CAR-T9 (DKO), CAR-T12 (DKO), and CAR-T9 (TKO) cells in the treatment of orthotopic lymphoma in NPG mice.

[0093] Figure 12 shows that after co-incubation with tumor cells Daudi or Nalm6, CAR9 / NKG2A(1)-tko and CAR9 / NKG2A(2)-tko cells can significantly kill tumor cells.

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

[0095] Figure 14 shows that after incubation with primary human NK cells for 24 or 72 hours, CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells were able to significantly kill NK cells.

[0096] Figure 15 shows that CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells can 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 inventors unexpectedly discovered that immune effector cells expressing different forms of CD19 / CD20 chimeric receptors have different tumor-killing abilities. Based on this finding, the present invention was completed.

[0098] the term

[0099] Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly 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 examples of the present invention are merely illustrative and are not intended to be limiting. Based on the present disclosure, it will be understood by those skilled in the art that many changes or modifications can be made in the disclosed specific embodiments and still obtain the same or similar results 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 alterations 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 and are fully explained in the literature.

[0101] The term "about" refers to the usual error range for each value readily known to those skilled in the art. Reference herein to an "about" value or parameter includes embodiments referring to the value or parameter itself. For example, a description of "about X" includes a description of "X." As used herein, "about" can refer to an acceptable error range within the technical field. For example, a value or parameter can refer to a value or parameter within a range of ±10% of an "about" value or parameter, e.g., about 5 uM can include any number between 4.5 uM and 5.5 uM.

[0102] Term "CD19": CD19 (Gene ID: 930) encodes a member of the immunoglobulin gene superfamily. CD19 expression is restricted to B-cell lymphocytes. This protein is a reliable marker for pre-B cells and 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 a member of the tumor necrosis superfamily and activates the apoptosis signaling pathway of target cells by binding to Fas.

[0105] The term "NKG2A" refers to NKG2A (Gene ID: 3821). NKG2A dimers with CD94 on NK cells and acts as an inhibitory receptor for NK cells. Its ligand is HLA-E. NKG2A is generally expressed on NK cells and a small percentage of CD8 T cells.

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

[0107] The term "T cell receptor (TCR)" refers to the T cell receptor that mediates T cell recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens, encompassing both classical and optimized TCRs. Classical TCRs are composed of two peptide chains, α and β. Each peptide chain is subdivided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region. Antigen specificity resides in the V region, which (Vα and Vβ) each contain 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 comprises an extracellular domain, a transmembrane domain, and a TCR intracellular domain of at least a portion of a TCR subunit, wherein 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 comprises the 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 comprises the 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. The antigen-binding domain, which can include a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting groups; 2. The extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; and 3. The transmembrane region and the intracellular region of the CD4 co-receptor, which is connected to the protein kinase LCK and catalyzes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex as the initial step in T cell activation.

[0110] The term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides that constitute the 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. A 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 linked, 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 the 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 signaling domain" or "primary signaling domain" regulates the initial activation of the TCR complex in a stimulating manner. On the one hand, the primary signaling domain is triggered by the binding of, for example, the TCR / CD3 complex to the MHC molecule loaded with the peptide, thereby mediating T cell responses (including but not limited to, proliferation, activation, differentiation, etc.). The primary signaling domain that acts in a stimulating manner can include a signaling motif of an immunoreceptor tyrosine activation motif or an ITAM. For example, a fragment of the primary signaling domain comprising an ITAM includes but is not limited to, an intracellular signaling 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 generating a second messenger or by acting as an effector in response to such a messenger. The intracellular signaling domain can 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 signaling domain" or "costimulatory molecule" generally refers to an intracellular domain of a costimulatory molecule that can bind to a cell stimulatory signaling molecule, such as TCR / CD3, and the combination leads to T cell proliferation and / or upregulation or downregulation of key molecules. Costimulatory molecules are typically cognate binding partners on T cells that 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 mouse, rodent, monkey, ape, etc. "CD3ζ" is used interchangeably with "CD3z" and "CD3Z" in this application.

[0116] The term "newlinker" refers to a linker with a sequence such as 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 into which a transplant is transplanted, for example, an individual into which exogenous cells are implanted, such as a human. For example, a "subject" can 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 certain engineering means, to change the genetic material in a cell or obtain a cell product according to people's wishes at the level of the whole cell or the organelle level. 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 altered genes.

[0120] The term "immune cell" refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, etc. For example, the immune cells are T cells, NK cells, and NKT cells. For example, the T cells can be autologous T cells, xenogeneic T cells, or allogeneic T cells. For example, the NK cells can be autologous NK cells or allogeneic NK cells. For example, the immune cells are obtained by sorting donor peripheral blood mononuclear cells (PBMC).

[0121] The term "T cell" can be a natural T cell obtained from PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and from an infection site, ascites, pleural effusion, spleen tissue, or tumor tissue. It can also be a cell population with a specific phenotypic characteristic 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 and 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 the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. For example, cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or culture medium for subsequent processing steps. The T cells can be derived from a healthy donor or from an individual 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 bind to at least CD19 and CD20; the second type of cells binds to NK cell markers. For example, each type of cell can be present in a different container and can be formulated into a desired preparation with a suitable adjuvant simultaneously or separately when needed; each type of cell can be from a different source (e.g., prepared, produced, or sold by different manufacturers; for example, naturally occurring T cells isolated from a donor 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 referred to as HLA antigens and plays an important role in transplantation reactions, with rejection mediated by T cells that respond to histocompatibility antigens on the surface of the transplanted tissue.

[0124] The term "human leukocyte antigen" (HLA) refers to the gene encoding the human major histocompatibility complex and is closely related to the function of the human immune system. 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, 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, polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or is expressed at a level insufficient to achieve the function it would have 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 transition from a resting state to an active state. This process may include responses to antigens, migration and / or phenotypic or genetic changes in functional activity state. For example, the term "activation" may refer to the process by which T cells are gradually activated. This activation process is jointly regulated by a first stimulation signal and a co-stimulation signal. The activation of T cells is a dynamic process, and its duration and degree of activation are affected by external stimulation conditions. "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. The use of 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" 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 and homing nuclease (Meganuclease) technology. The guide sequence (gRNA) is a polynucleotide sequence that has sufficient complementarity with the target polynucleotide sequence so as 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 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 to or recognize the Cas enzyme. For example, CRISPR technology is used to construct endogenous TCR / B2M / FAS knockout or endogenous TCR / B2M knockout engineered cells. The gRNA sequences targeting TCR, B2M, FAS, and NKG2A are shown in SEQ ID NOs: 35, 36, 37, and 38, respectively.

[0129] "Low expression" as described herein means that the protein and / or RNA level of the target gene expressed in the engineered cell is lower than the expression level before the 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 the cell 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%. The expression or content of a specific protein in the cell 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 that encodes 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, "sequence" can include DNA or RNA, and can 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 to 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 listing. 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 listing, and / or have one or more amino acid substitutions, and / or deletions and / or additions of one or more amino acids and have 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more homology or identity to the sequences in the sequence listing.

[0135] The term "subject" refers to any animal, such as a mammal or marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (e.g., 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, can be used to obtain PBMCs by centrifugation of peripheral blood or apheresis samples or leukapheresis samples with or without lysis of red blood cells using a Percoll-Ficoll gradient.

[0137] The term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to prevent or treat an individual disease (cancer). The effective dose for therapeutic or prophylactic 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 active substance selected, 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. One or more rounds, or multiple administrations of the engineered cells of the present application may be necessary at the discretion of the prescribing physician or a person skilled in the art.

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

[0139] The term "vector" refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into cells. This includes, but is not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. For example, these include autonomously replicating plasmids or viruses. Also included are non-plasmid and non-viral compounds that facilitate nucleic acid transfer into cells, such as polylysine compounds and liposomes.

[0140] The term "treatment" refers to interventions that attempt to alter the course of a disease, including both preventative and clinical pathological interventions. 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 the disease, preventing metastasis, slowing the progression of a disease, improving or relieving symptoms, and alleviating or improving prognosis.

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

[0142] The term "transplant rejection" refers to the situation in which, after a host receives an allogeneic tissue, organ, or cell transplant, the host's immune system recognizes the foreign transplant as a "foreign component" 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 the host, intended for implantation into the host. The graft may be derived from any animal, such as a mammal, preferably from a human. The graft may be derived from the host, such as cells cultured or modified in vitro from the host and then implanted into the host. The graft may also be derived from another allogeneic individual, such as cells cultured or modified in vitro from another human and then implanted into the host.

[0144] The term "autologous" refers to originating from the same organism. For example, a sample can be removed from a subject (e.g., cells), processed, and returned to the subject at a later time. Allogeneic refers to when the donor and recipient are 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 can 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 can include VL-linker-VH or can include VH-linker-VL. The antigen binding function of an antibody can 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 with a specific shape that is suitable for and recognizes 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)", also known as "VHH" or "VHH polypeptide", contains a variable VHH domain responsible for antigen recognition. Antigen binding of the VHH domain is mediated by three CDRs, which are flanked by four relatively constant framework regions (FRs). SdAbs are a type of antibody that lacks the light chain and only has the variable region of the heavy chain. Due to their small molecular weight, they are also called nanobodies. VHHs can be truncated at the N-terminus or C-terminus, so that they only contain part of FR1 and / or FR4, or lack one or both of those framework regions, as long as the VHH maintains substantial 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 (e.g., a target molecule) on the 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. DDpps can be monovalent or multivalent. In some embodiments, DDpps are monospecific or multispecific. In other embodiments, they are monospecific and multivalent. In other embodiments, DDpps are 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 participates in antibody antigen binding. The heavy chain variable domain (VH) and light chain variable domain (VL) of a natural antibody typically 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, respectively, using a VH or VL domain from an antibody that binds to the antigen.

[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 the hypervariable region (CDR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. In VH (or VL), CDR and FR sequences typically 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 to refer to a full-length antibody having a structure substantially similar to a native antibody structure or having heavy chains containing an Fc region as defined herein or including an intact antigen-binding region.

[0157] The term "fully human antibody (or fully human antibody)" is an antibody having an amino acid sequence that corresponds 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 characteristic ("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. A 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 refers to 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 the group consisting of: 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. NCRs include 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. It includes 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, immunoreceptor tyrosine-based inhibitory motifs (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, a KIR family member is selected from KIR2DL1, KIR2DL2 / 3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3. For example, a LIR family member is selected from LIR1, LIR2, LIR3, LIR5, and LIR8. For example, a 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, including 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, and 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 cell" generally refers to any type of cell present in a patient that is believed to contribute to a worsening health condition, or refers to a malignant or infected cell that needs to be reduced or eliminated to achieve patient remission. The present invention relates to methods for 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 (approximately 5-30 amino acids in length) that directs the transfer of newly synthesized proteins or polypeptides into the secretory pathway. A signal peptide is a short peptide located at the N-terminus (amino terminus) of a protein. For example, the SP is a CD8 signal peptide or a GMCSFRa signal peptide. For example, the SP is derived from the natural signal peptide of a wild-type immunoglobulin superfamily (IgSF) member. For example, the SP is modified from the natural signal peptide of IgSF.

[0165] For example, the signal peptide is a natural heavy chain signal peptide of IgG, IgM, IgD, IgA, or IgE. For example, the signal peptide is a natural κ or λ light chain signal peptide. For example, the SP linked to the antibody VL is a natural κ light chain signal peptide, such as IgGsL1, IgGsL2, or IgGsL3. For example, the signal peptide linked 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 (CARs), 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] This invention discloses bispecific CARs targeting CD19 and CD20. When tumor cells escape CD19 antigens or lack CD19 expression, bispecific CARs can eliminate them by recognizing CD20. Most leukemia tumor cells express CD20, including some CD19-negative patients after anti-CD19 CAR T therapy. CD20, encoded by the MS4A gene, is a glycosylated protein and a B cell membrane marker. It is specifically expressed in over 95% of normal and cancerous B cells during the pre-B cell stage and subsequent developmental stages, with CD20 expression not fading until the cells differentiate into plasma cells. This invention utilizes CD20 as another target for immunotherapy of B cell malignancies. This 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 simultaneously targets CD19 and CD20 upon T cell activation. The CAR T cells of this invention are capable of recognizing two antigens (CD19 and CD20). This invention provides a highly effective method for potentially preventing antigen escape.

[0168] The present invention uses CARs that simultaneously target CD19 and CD20. Compared with CARs targeting 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 deletion 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 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.

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

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

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

[0172] In one embodiment, an 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 a VH CDR1, a VH CDR2, and a VH CDR3, and the VL1 comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1, VH CDR2, and VH CDR3 are derived from a VH having an amino acid sequence as set forth in SEQ ID NO: 21, and the VL CDR1, VL CDR2, and VL CDR3 are derived from a VL having an amino acid sequence as set forth in SEQ ID NO: 22.

[0173] In one embodiment, an 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 VL CDR3, wherein VH CDR1, VH CDR2, and VH CDR3 are derived from VH2 having the amino acid sequence set forth in SEQ ID NO: 23, and VL CDR1, VL CDR2, and VL CDR3 are derived from VL2 having the amino acid sequence set forth 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 set forth in SEQ ID NOs: 21 and 22, respectively, or are at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical 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 of SEQ ID NOs: 23 and 24, respectively, or are at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical thereto.

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

[0177] In one embodiment, 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 embodiment, the anti-CD19 or CD20 antibody comprises, from the N-terminus to the C-terminus, a VH, a connecting peptide chain, and a VL. In one embodiment, the anti-CD19 or CD20 antibody comprises, from the N-terminus to the C-terminus, a VL, a connecting peptide chain, and a VH. Any connecting peptide chain that connects the VH and VL together to form a functionally complete single-chain antibody is applicable. In one embodiment, the connecting peptide chain is a GS connecting peptide chain, for example, GGGGS, (GGGGS)3, or (GGGGS)4. In one embodiment, 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. When the intracellular signal transduction domain contains one or more signal transduction domains or motifs, such as immunoreceptor tyrosine-based activation motifs (ITAMs), kinase domains, costimulatory domains, etc., it can directly promote cellular responses. 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 comprises 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 comprises 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 costimulatory domain. In one example, the costimulatory 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 costimulatory structure is the intracellular signal transduction domain of CD137 (SEQ ID NO: 11). In one example, the costimulatory 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. 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 embodiment, 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 embodiment, the hinge region is selected from the group consisting of a CD28 hinge region, a CD8 hinge region, an IgG spacer region or a fragment thereof, or a combination thereof. In one embodiment, the hinge region can be an IgG1 spacer region or a fragment thereof, an IgG2 spacer region or a fragment thereof, an IgG3 spacer region or a fragment thereof, or an IgG4 spacer region or a 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 embodiment, the chimeric receptor comprises a signal peptide, and in one embodiment, 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 comprising an anti-CD19 antibody (VH / VL selected from SEQ ID NOs: 21, 22), and / or a domain comprising an anti-CD20 antibody (VH / VL 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 comprising (5) a CD3ζ intracellular signaling domain (SEQ ID NO: 12 or 13).

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

[0188] The antigen binding domain of the chimeric receptor can have different conformations, such as some of those shown in Figure 1. In one example, the antigen binding domain comprises, from N-terminus to C-terminus, VH1-VL1-VH2-VL2. In one example, the antigen binding domain comprises, from N-terminus to C-terminus, VL1-VH1-VL2-VH2. In one example, the antigen binding domain comprises, from N-terminus to C-terminus, VH1-VL1-VL2-VH2. In one example, the antigen binding domain comprises, from N-terminus to C-terminus, VL1-VH1-VH2-VL2. In one example, the antigen binding domain comprises, from N-terminus to C-terminus, VH1-VH2-VL2-VL1. In one example, the antigen binding domain comprises, from N-terminus to C-terminus, VL1-VH2-VL2-VH1. In one example, the antigen binding domain comprises, from the N-terminus to the C-terminus, VH1-VL2-VH2-VL1. In one example, the antigen binding domain comprises, from the N-terminus to the C-terminus, VL1-VL2-VH2-VH1. In one example, the antigen binding domain comprises, from the N-terminus to the C-terminus, VH2-VH1-VL1-VL2. In one example, the antigen binding domain comprises, from the N-terminus to the C-terminus, VL2-VH1-VL1-VH2. In one example, the antigen binding domain comprises, from the N-terminus to the C-terminus, VH2-VL1-VH1-VL2. In one example, the antigen binding domain comprises, from the N-terminus to the C-terminus, VL2-VL1-VH1-VH2.

[0189] The chimeric receptors provided herein comprise 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, or 55. The chimeric receptors provided herein comprise 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 comprises an anti-NKG2A antibody or fragment. In one example, the antibody or fragment is selected from: a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab')2 fragment, Fd fragment, sdAb, multifunctional antibody, DDPP antibody, scFv-Fc antibody, or IgG4 antibody. In one example, the chimeric receptor that binds to an immune cell marker comprises an IgG4 hinge region. In one example, the chimeric receptor that binds to an immune cell marker comprises amino acids that are 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. For example, the chimeric receptor comprises amino acids that are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% homologous or identical 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 embodiment, the pathological cells are selected from malignant cells or infected cells. In one embodiment, the pathological cells are selected from tumor cells or infected cells that are CD19 and / or CD20 positive. In one embodiment, the pathological cells are selected from blood tumor cells and pathological cells of autoimmune diseases. In one embodiment, the blood tumor is selected from leukemia, lymphoma, and myeloma; and the autoimmune disease is selected from multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), and polymyositis / dermatomyositis.

[0192] The sequences provided in this application are not limited to Bite that binds to CD19 and CD20. The antibody sequences of the Bite that binds to CD19 and CD20 are connected in a LOOP format.

[0193] For example, the chimeric receptor further comprises 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 a portion 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 comprises all or a portion of IgG4 CH2 and / or IgG2 CH2, wherein IgG4 CH2 is optionally a human IgG4 CH2 and IgG2 CH2 is optionally a human IgG2 CH2; and / or the CH3 region comprises all or a portion 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 both 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] The present invention provides chimeric receptors that bind to CD20 as shown in Table 2.

[0197] Table 2. CD20-CAR structure

[0198] The present invention provides chimeric receptors that bind to CD19 and CD20 as shown in Table 3.

[0199] Table 3. CD19-CD20-CAR structure

[0200] The present invention provides chimeric receptors that bind to immune cell markers as shown in Table 4.

[0201] Table 4. CAR structures targeting immune cell markers

[0202] In Tables 1, 2, 3, and 4 above, 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 vectors was prepared using conventional molecular biology techniques.

[0203] The CAR-T1 cells in the embodiments disclosed herein comprise CAR19.1 and CAR20.4 in Tables 1 and 2; CAR-T2 cells comprise CAR19.2 and CAR20.5 in Tables 1 and 2; CAR-T3 cells comprise CAR19.2 and CAR20.6 in Tables 1 and 2; CAR-T4 cells comprise CAR19.3 and CAR20.7 in Tables 1 and 2; CAR-T5 cells comprise CAR19.3 and CAR20.8 in Tables 1 and 2; CAR-T6 cells comprise CAR6 in Table 3; CAR-T7 cells comprise CAR7 in Table 3; CAR-T8 cells comprise CAR8 in Table 3; CAR-T9 cells comprise CAR9 in Table 3; CAR-T10 cells comprise CAR10 in Table 3; and CAR-T11 cells comprise CAR11 in Table 3. The control group CAR-12 comprises CAR12 in Table 3. CAR9 / NKG2A(1) T cells comprise CAR9 and NKG2A-28z in Tables 3 and 4. CAR9 / NKG2A(2) T cells contain CAR9 and NKG2A-s28z in Tables 3 and 4.

[0204] carrier

[0205] Genetic modification of immune cells (e.g., T cells or NKT cells) in the composition can be completed 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).

[0206] 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.

[0207] Non-viral methods can also be used for the genetic modification of immune cells. For example, nucleic acid molecules can be introduced into immune cells by lipofection, asialomucoid-polylysine coupling, or microinjection under 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 the nucleic acid molecules into a cell type 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 with the nucleic acid molecules into the subject's target tissue or inject them systemically.

[0208] The present invention provides nucleic acid molecules encoding chimeric receptors as shown in Table 1, 2, 3 or 4.

[0209] The present invention provides nucleic acid molecules encoding 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 that binds to CD19 and the CAR that binds 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).

[0210] The present invention provides nucleic acid molecules encoding chimeric receptors for binding to CD19 and CD20 as shown in Table 3 and chimeric receptors for binding to immune cell markers as shown in Table 4. In one example, the polypeptide chain encoded by the nucleic acid molecule includes, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), CARs that bind to CD19 and CD20 and chimeric receptors that bind to immune cell markers; or includes, in sequence, a chimeric receptor that binds to immune cell markers and CARs that bind to CD19 and CD20. In one example, the polypeptide chain encoded by the nucleic acid molecule includes, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), CARs that bind to CD19 and CD20 and chimeric receptors that bind to NKG2A; or includes, in sequence, a chimeric receptor that binds to NKG2A and CARs that bind to CD19 and CD20. In one example, the polypeptide chain encoded by the nucleic acid molecule includes, from the amino terminus (N terminus) to the carboxyl terminus (C terminus), CAR that binds to CD19 and CD20, and a chimeric receptor that binds to TIGIT; or includes, in sequence, a chimeric receptor that binds to TIGIT, and a CAR that binds to CD19 and CD20. In one example, the polypeptide chain encoded by the nucleic acid molecule includes, from the amino terminus (N terminus) to the carboxyl terminus (C terminus), CAR that binds to CD19 and CD20, and a chimeric receptor that binds to CD38; or includes, in sequence, a chimeric receptor that binds to CD38, and a CAR that binds to CD19 and CD20. In one example, the polypeptide chain encoded by the nucleic acid molecule includes, from the amino terminus (N terminus) to the carboxyl terminus (C terminus), CAR that binds to CD19 and CD20, and a chimeric receptor that binds to FasL; or includes, in sequence, a chimeric receptor that binds to FasL, and a CAR that binds to CD19 and CD20.

[0211] The present invention provides a CAR encoding a CAR that binds to CD19 and CD20, and a CAR that binds to an immune cell marker. For example, the nucleic acid molecule encodes any one of the CAR molecules that bind to CD19 and CD20 shown in Table 3, and any one of the CAR molecules that bind to an immune cell marker shown in Table 4. In one example, the CAR that binds to CD19 and CD20 and the CAR that binds to an immune cell marker are connected by a hydrolyzable connecting peptide. In one example, the immune cell markers include: NKG2A, CD38, TIGIT and / or FasL.

[0212] The present invention provides a nucleic acid molecule 3 encoding a CAR that binds to CD19 and CD20, and a nucleic acid molecule 4 encoding a CAR that binds to 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 to CD19 and CD20 and the CAR that binds to 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, the immune cell markers include: NKG2A, CD38, TIGIT and / or FasL.

[0213] Engineered cells

[0214] The present invention provides an engineered cell, comprising the chimeric receptor disclosed in the present invention.

[0215] 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).

[0216] 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).

[0217] In one embodiment, the engineered cells include chimeric receptors that bind CD19 and CD20 (as shown in Table 3).

[0218] 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).

[0219] In one embodiment, the pathological cells are selected from malignant cells or infected cells. In one embodiment, the pathological cells are selected from blood tumor cells and pathological cells of autoimmune diseases. In one embodiment, the blood tumor cells are CD19-positive and / or CD20-positive tumor cells.

[0220] In one embodiment, the hematological tumor is selected from the group consisting of leukemia, lymphoma, and myeloma; the autoimmune disease is selected from the group consisting of multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), and polymyositis / dermatomyositis.

[0221] In one embodiment, the present invention provides an engineered cell comprising a polynucleotide encoding the present invention. In another embodiment, the present invention provides an engineered cell comprising a vector encoding the present invention. In another embodiment, the present invention provides an engineered cell comprising a virus encoding the present invention.

[0222] 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.

[0223] 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.

[0224] In one example, the engineered cells are T cells. In one example, the engineered cells are allogeneic T cells. In one example, the engineered cells are stem cell-derived T cells. The 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, naive 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 TSC cells, central memory TSC cells, effector memory TEM cells, or effector memory TEMRA cells. In one example, the T cells are cytotoxic T cells. In some embodiments, the genetically engineered T cells provided herein are isolated. In one example, the genetically engineered T cells provided herein are substantially purified.

[0225] In one embodiment, the engineered cells provided herein are derived from cells isolated from a subject. As used herein, a genetically engineered cell derived from a source cell refers to a genetically engineered cell obtained by obtaining a source cell and genetically manipulating the source cell. The source cell can be derived from a natural source. For example, the source cell can be a primary cell isolated from a subject. The source cell can also be a cell that has been passaged or genetically manipulated in vitro.

[0226] In one example, the genetically engineered cells provided by the invention derive from cells separated from human body.Immune effector cells (for example, 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 derive from cells separated from peripheral blood. In one example, the genetically engineered cells provided by the invention derive from cells separated from bone marrow. In one example, the genetically engineered cells provided by the invention derive from cells separated from peripheral blood mononuclear cells (PBMC).

[0227] 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.

[0228] In one example, the engineered cells further include: low expression or absence of endogenous TCR, B2M, HLA-I, HLA-II, NKG2A, FAS, and / or CD58. In one example, the engineered cells further include: low expression or absence of endogenous TCR and B2M. In one example, the engineered cells further include: low expression or absence of endogenous FAS. In one example, the engineered cells further include: low expression or absence of endogenous CD58. In one example, the engineered cells further include: low expression or absence of endogenous NKG2A. In one example, the engineered cells further include: absence of endogenous TCR and B2M. In one example, the engineered cells further include: absence of endogenous TCR / B2M / NKG2A. In one example, the engineered cells further include: absence of endogenous TCR / B2M / FAS. In one example, the engineered cells further comprise: no expression of endogenous TCR / B2M / CD58.

[0229] In one example, the present invention provides a method for genetically engineering a polynucleotide provided by the present invention by transferring it into a cell using gene editing. If necessary, techniques such as nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats (CRISPRs), homologous recombination, nonhomologous end joining, microhomology-mediated end joining, homology-mediated end joining, etc. can be used to achieve site-directed integration.

[0230] In one embodiment, endogenous TCR / B2M, TCR / B2M / NKG2A, TCR / B2M / FAS, or TCR / B2M / CD58 is knocked out using CRISPR / Cas9 technology. In one embodiment, the sgRNA sequences targeting TRAC, B2M, FAS, and NKG2A are shown in SEQ ID NOs: 35, 36, 37, and 38, respectively.

[0231] For example, in some embodiments, the present invention provides an engineered T cell expressing a chimeric receptor, the engineered T cell including the absence of endogenous TCR / B2M expression, and optionally, the absence of NKG2A, FAS and / or CD58 expression; 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.

[0232] In one example, the engineering cells expressing chimeric receptors of the present application can bind to target cells expressing CD19 and / or target cells expressing CD20. For example, the engineering cells of the present application can bind to NK cells. In one example, the engineering cells of the present application can bind to CD19 and / or CD20 positive cells, and can also bind to NK cells. For example, the engineering cells of the present application can bind to target cells expressing NK cell markers (such as CD7, NKG2A, CD38, CD94, CS1, TIGIT, NKG2DL). For example, the engineering 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 engineering cells expressing chimeric receptors targeting CD19 and CD20 disclosed in the present invention also express chimeric receptors targeting NKG2A. For example, the engineering 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.

[0233] 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 a significantly improved ability to kill pathological cells.

[0234] In one example, the engineered cells have a prolonged survival time or enhanced proliferation capacity 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 by another engineered cell that was previously, simultaneously, or subsequently introduced into the subject.

[0235] 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.

[0236] 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 an 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.

[0237] transduction

[0238] 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, an input composition comprising the cell to be transduced, a cell stimulator to be transduced, and a viral vector particle carrying a recombinant nucleic acid encoding a chimeric receptor of the present invention are co-incubated, or activation can be performed first and then transduced, such as incubating an input composition comprising the cell to be transduced and a cell stimulator to be transduced for activation, and then adding a viral vector particle carrying a recombinant nucleic acid encoding a chimeric receptor of the present invention for incubation, and performing transduction of the recombinant nucleic acid. The total time for activation and transduction 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 population (e.g., an immune cell, such as a T cell), and activating and / or activating the T cell using an ex vivo cell activation reagent (e.g., an anti-CD3 / anti-CD28 reagent) before and / or simultaneously and / or after contacting or incubating the cell with the viral particle. Preferably, the cells are activated first and then viral transduction is performed. In one example, when an input composition comprising cells to be transduced, a stimulator of cells to be transduced, and viral vector particles carrying a recombinant nucleic acid encoding a chimeric receptor of the present invention are incubated together, the incubation time does not exceed 72 hours for harvesting to obtain an output composition, wherein the output composition contains cells transduced with the recombinant nucleic acid; preferably, the incubation time 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 purification treatments such as washing and centrifugation, the output composition can be prepared into a pharmaceutical preparation without further in vitro amplification and culture, that is, the drug prepared using the output composition does not require in vitro amplification before use in a subject (or patient).

[0239] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning Laboratory Manual, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0240] The biological materials used in the examples of this application are shown in Table 5.

[0241] Table 5

[0242] Example 1. Vector construction

[0243] Conventional molecular biology techniques were used to construct vectors expressing the chimeric receptors disclosed herein. Full-length DNA was synthesized to construct expression vectors CAR6-12 for the bispecific CARs shown in Table 3, which were then integrated into the pRRLSIN vector (Addgene). Lentivirus encoding the expression vectors was prepared using conventional molecular biology techniques.

[0244] 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, and 20.8, binding to CD19 and CD20, as shown in Tables 1 and 2, and integrated into the pRRLSIN vector (Addgene). Lentivirus encoding the above expression vectors was prepared using conventional molecular biology techniques.

[0245] Example 2. Construction of CAR-T cells

[0246] Conventional CAR-T cell preparation methods in the field were used: donor PBMC cells were collected and activated with magnetic beads containing anti-CD3 and CD28 antibodies (Life Technologies, 40203D). T cells were then 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 considered UTD.

[0247] Each CAR-T sample collected 1×10 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.

[0248] Example 3. Detection of CD19 and CD20 Expression in Human B-lymphoid Leukemia Cells Nalm6 and Human Burkitt's Lymphoma Cells Daudi

[0249] Using conventional molecular biology techniques, Nalm6 cells (Nalm6-Luci) and Daudi cells (Daudi-Luci) expressing exogenous luciferase were constructed. Anti-CD19 APC-H7 (BD, 560727) and anti-CD20 APC antibodies (Invitrogen, 17-0209-4) were used to analyze CD19 and CD20 expression on different cell lines using flow cytometry (FACS). Figure 2 shows that Daudi cells express CD19 and CD20. Nalm6 cells express CD19.

[0250] Example 4. In vitro cytotoxic effects of dual-target CAR-T cells on lymphoma cells

[0251] Daudi and Nalm6 cells were seeded in 96-well plates at 10,000 cells / well. CAR-T cells were co-incubated with Daudi and Nalm6 cells at effector-target ratios of 3:1, 1:1, or 1:3 for 18 hours. The cytotoxicity of CAR T cells against lymphoma cells was assessed by FACS. Figures 3A and 3B show that CD19 / CD20 dual-target CAR-T cells 1, 2, 4, 5, 6, 7, 8, 9, and 10 were able to significantly kill both Daudi and Nalm6 tumor cells.

[0252] Example 5. Cytokine secretion after co-incubation of dual-target CAR-T cells with tumor cells

[0253] Daudi and Nalm6 lymphoma cells were seeded in 96-well plates at 40,000 cells / well. CD19 / CD20 dual-targeted CAR-T cells were co-incubated with Daudi and Nalm6 cells, respectively, at a 1:1 effector-target ratio for 18 hours. Interferon-γ (IFN-γ), IL-2, and TNF-α secretion levels in the supernatant were measured using a CBA assay (BD, 558264). Figures 4A and 4B show that CD19 / CD20 dual-targeted CAR-T cells 1, 2, 4, 5, 6, 7, 8, 9, and 10 all secreted high levels of IFN-γ, IL-2, and TNF-α.

[0254] Example 6. Dual-target CAR-T therapy for subcutaneously transplanted lymphoma in NPG mice

[0255] On D0, each NPG mouse was subcutaneously inoculated with 3×10 6 Daudi cells, 5 per group. On D12, the average tumor volume was approximately 130 mm 3 On D13, each mouse was injected with 3×10 6 CD19 / 20 dual-target CART cells.

[0256] Figure 5 shows that on D34, compared with the UTD group, the tumor volume inhibition rate of the CAR-T1 and CAR-T11 groups was 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.

[0257] Example 7. Detection of human T cell survival in mouse peripheral blood

[0258] Peripheral blood was randomly collected from 4 mice per group after CAR-T treatment as described in Example 6. 50 μL of blood was collected from each sample and detected using antibodies anti-CD45 APC-H7 (1 ul / test, BD, 560178) and CD3 percp-cy5.5 / CD4 FITC / CD8 PE (10 ul / test, BD, 340298) using BD absolute counting tubes (BD, Cat: 340334, Lot: 21258, Beads Number: 49450 / tube). Figure 6 shows that on days 7 and 14 after CAR-T cell infusion, viable human T cells were detected in the peripheral blood of mice in both the CAR-T1 and CAR-T11 groups.

[0259] Example 8. Dual-target CAR-T therapy for in situ lymphoma in NPG mice

[0260] 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 used at different time points to observe the photon intensity of the tumor. On D9, the photon intensity reached an average of 2.47×10^6. 4 Each mouse was injected with 1×10 6 Figures 7 and 8 show that, after observation for 80 days, three mice in the CD19-CAR-T group died, and one mouse experienced a continuous rebound in tumor fluorescence starting on D31. In the CAR-T12 group, one mouse experienced a continuous rebound in tumor fluorescence starting on D45. The CAR-T4, CAR-T9, and CAR-T10 groups showed the best tumor suppression effects: no tumors rebounded, and all mice survived and were in good condition.

[0261] Example 9. UCAR-T cell preparation

[0262] 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) was performed. The endogenous TCR / B2M or TCR / B2M / NKG2A of the CAR-T9 and CAR-T12 cells constructed in Example 2 was knocked out by CRISPR / Cas9 technology (Cas9 protein, Kaika Biotechnology (Shanghai) Co., Ltd., CAS-EE109), and CAR-T9 (DKO), CAR-T9 (TKO), and CAR-T12 (DKO) were obtained by magnetic bead sorting (Miltenyi Biopharmaceuticals, 130-048-801). Untransduced UTD cells with TCR / B2M / NKG2A knockout were used as controls. 1×10 cells were collected for each CAR-T sample. 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. The CAR positivity rate was detected by FACS.

[0263] Example 10. Dual-target CAR-T therapy for in situ lymphoma in NPG mice

[0264] 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 used at different time points to observe the photon intensity of the tumor. On D10 (when the photon intensity reached an average of 6.4×10^6), the tumor photon intensity was significantly increased. 4 Each mouse was injected with 1×10 6 Figures 9, 10, and 11 show that by day 131, all mice in the CD19-CAR-T (DKO) group had died; three mice in the CD20-CAR-T (DKO) group had died; and two mice in the CAR-T12 (DKO) group had died. The CAR-T9 (DKO) and CAR-T9 (TKO) groups had the best tumor suppression effects: no tumors rebounded, and all mice survived and remained in good condition.

[0265] Example 11. Vectors and cell construction targeting immune cells

[0266] Conventional molecular biology techniques were used to construct vectors expressing chimeric receptors targeting immune cell markers. Full-length DNA was synthesized, and expression vectors for NKG2A-28z and NKG2A-s28z, as shown in Table 4, were constructed and integrated into the pRRLSIN vector (Addgene).

[0267] CAR9 / NKG2A(1) T cells and CAR9 / NKG2A(2) T cells were constructed with reference to Example 2.

[0268] Example 12. Preparation of CD19 / CD20-NKG2A UCAR-T cells

[0269] Referring to Example 9, TCR / B2M / NKG2A in CAR9 / NKG2A(1) T cells or CAR9 / NKG2A(2) T cells were knocked out to construct CAR9 / NKG2A(1)-tko cells and CAR9 / NKG2A(2)-tko 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. The CAR positivity rate was detected by FACS.

[0270] Example 13. In vitro cytotoxicity of CD19 / CD20-NKG2A UCAR-T cells against lymphoma cells

[0271] 30,000 Daudi and Nalm6 cells were inoculated into each well of a 96-well plate, and CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells were taken and incubated for 18 hours at an effector-target ratio of 1:1 or 1:3. 7-AAD (BD, 559925) and Counting Beads (Invitrogen, C36995) were added to quantify the two types of cells. After flow cytometry detection, the remaining number of the two types of cells was counted and the calculation formula was: % cytotoxicity = (1-experimental group / control group) * 100. Figure 12 shows that both CAR9 / NKG2A(1)-tko and CAR9 / NKG2A(2)-tko cells can significantly kill lymphoma cells.

[0272] Example 14. Cytokine secretion after co-incubation of CD19 / CD20-NKG2A UCAR-T cells with tumor cells

[0273] The supernatant of CD19 / CD20-NKG2A UCAR-T cells co-cultured with Daudi and Nalm6 for 18 hours was collected and the secretion of IL-2, TNF-α, and IFN-γ cytokines was detected using CBA-Human Soluble Protein Master Buffer Kit (Cat: 558265). Figure 13 shows that CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells can secrete high levels of IL-2, TNF-α, and IFN-γ cytokines after co-incubation with tumor cells.

[0274] Example 15. In vitro killing of primary human NK cells by CD19 / CD20-NKG2A UCAR-T cells

[0275] Donor-derived peripheral blood PBMC cells were collected and NK cells were screened using CD56 magnetic beads (Miltenyi Biotec, 130-050-401) according to the manufacturer's instructions.

[0276] Collect 3×10 4 CD19 / CD20-NKG2A UCAR-T was co-cultured with the above-collected primary human NK cells at a ratio of 1:1 and 1:2 for 24 hours and 72 hours, respectively. HLA-ABC (Thermo Fisher, 17-9983-42) was used to label B2M-KO T cells, and CD56 (BD 555516) was used to label NK cells; 7-AAD fluorescent dye (BD, 559925) was used to distinguish dead cells from live cells. Counting Beads (Invitrogen, C36995) was added, and the number of aNK cells was detected by flow cytometry, and the lysis rate of NK cells was calculated. Figure 14 shows that after 24 or 72 hours of co-incubation with NK cells, CAR9 / NKG2A (1)-tko or CAR9 / NKG2A (2)-tko cells can significantly kill NK cells.

[0277] 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

[0278] The supernatant of CD19 / CD20-NKG2A UCAR-T cells co-cultured with human primary NK cells for 24 hours was collected and the secretion of IL-2, TNF-α, and IFN-γ cytokines was detected using the CBA-Human Soluble Protein Master Buffer Kit (Cat: 558265). Figure 15 shows that CAR9 / NKG2A(1)-tko or CAR9 / NKG2A(2)-tko cells can secrete high levels of IL-2, TNF-α, and IFN-γ cytokines after co-incubation with NK cells.

[0279] Example 17. Rapid preparation process for CAR-T cells targeting CD19 and CD20

[0280] 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 hours and then transduced with a virus containing CAR targeting CD19 and CD20 (prepared using conventional molecular biology techniques). The transduction time was 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 hours. 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 then cryopreserved.

[0281] The embodiments described herein include any embodiment as a single embodiment or in combination with any other embodiment or portion 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 appended hereto.

[0282] Sequence information

[0283] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also 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 that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A bispecific chimeric receptor, comprising 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 chimeric receptor 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 respectively connected to both ends of the antibody 2; or, (2) Anti-CD19 antibody heavy chain variable region (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 chimeric receptor according to claim 2, wherein From N-terminus to C-terminus, the antigen binding domain of the chimeric receptor 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 chimeric receptor 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 chimeric receptor 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 chimeric receptor 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 chimeric receptor 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 chimeric receptor 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 chimeric receptor 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. An engineered cell, wherein: The engineered cell comprises the chimeric receptor according to any one of claims 1-9.

11. An engineered cell comprising a chimeric receptor that binds CD19 and CD20, 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 engineered cell 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 engineered cell 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 engineered cell 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 engineered cell 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 engineered cell according to any one of claims 11 to 15, wherein: The first chimeric receptor and the second chimeric receptor comprise amino acid sequences having about 80% to 100% identity with the amino acid sequences shown in SEQ ID NOs: 17 and 55, respectively.

17. The engineered cell 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 engineered cell 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 engineered cell 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 engineered cell 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. A polynucleotide, wherein The polynucleotide encodes: the chimeric receptor described in any one of claims 1-9, the chimeric receptor described in any one of claims 1-9 and the third chimeric receptor described in any one of claims 17-20, the first and second chimeric receptors described in any one of claims 11-20, the first and second chimeric receptors described in any one of claims 11-20, and the third chimeric receptor described in any one of claims 17-20.

22. A vector, wherein The vector comprises the polynucleotide of claim 21; preferably, the chimeric receptor and the third chimeric receptor are located in the same expression vector; or the first and second chimeric receptors are located in the same expression vector.

23. A method for rapidly preparing engineered cells, wherein: The engineered cells contain the polynucleotide of claim 21, and include the following steps: (i) incubating an input composition containing cells to be transduced and a stimulator for cells to be transduced for no more than 24 hours, and (ii) adding viral vector particles containing the polynucleotide of claim 21 for incubation for no more than 24 hours to obtain the engineered cells; preferably, the vector is derived from a retroviral vector; preferably, the vector is a lentiviral vector; preferably, no in vitro amplification is required before administration to a subject in need.

24. A preparation, characterized in that The preparation comprises the polynucleotide of claim 21, the vector of claim 22, or the chimeric receptor of claims 1-9, or the engineered cell of any one of claims 10-20, and a pharmaceutically acceptable carrier, diluent or excipient.

25. A drug for preventing or treating a disease, wherein: The drug comprises the polynucleotide of claim 21, the vector of claim 22, or the chimeric receptor of claims 1-9, or the engineered cell of any one of claims 10-20.

26. The chimeric receptor according to any one of claims 1 to 9, the engineered cell according to any one of claims 10 to 20, which is used to kill or inhibit CD19 and / or CD20 positive pathological cells; preferably, it is used to prepare a drug for treating an autoimmune disease or an inflammatory disease; preferably, it is used to treat, prevent or improve an autoimmune disease or an inflammatory disease in a subject in need; preferably, the autoimmune disease is selected from: multiple sclerosis, autoimmune liver disease, type 1 diabetes, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), Sjögren's syndrome (SS), polymyositis / dermatomyositis; preferably, it is used to prepare a drug for treating a tumor; preferably, it is used to treat, prevent or improve a tumor in a subject in need; preferably, the tumor comprises: Leukemia, lymphoma, myeloma.

27. A multifunctional immune effector cell, 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.

28. A polynucleotide, 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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