Chimeric antigen receptor targeting GPRC5D and / or BCMA and application thereof

Abstract

The invention provides a chimeric antigen receptor targeting GPRC5D and BCMA and application of the chimeric antigen receptor, and particularly provides nucleic acid for coding the chimeric antigen receptor targeting GPRC5D and BCMA, the corresponding chimeric antigen receptor, a carrier, an immune effector cell, a preparation method of the chimeric antigen receptor, a product of the chimeric antigen receptor, a pharmaceutical composition, pharmaceutical application and a tumor or cancer treatment method.

Description

Chimeric antigen receptors targeting GPRC5D and / or BCMA and their applications

[0001] Cross-references to Related Patent Applications

[0002] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office of China on July 22, 2022, with patent application number 202210871138.3 and invention name “Chimeric Antigen Receptors Targeting GPRC5D and / or BCMA and Applications Thereof”; and claims priority to the Chinese patent application filed with the State Intellectual Property Office of China on July 30, 2022, with patent application number 202210912614.1 and invention name “Chimeric Antigen Receptors Targeting GPRC5D and / or BCMA and Applications Thereof”. The present invention claims priority to the Chinese patent application number 202211580119.1 filed with the State Intellectual Property Office of China on December 9, 2022, entitled “Chimeric Antigen Receptor Targeting GPRC5D and / or BCMA and Its Applications”; and claims priority to the Chinese patent application number 202310302730.6 filed with the State Intellectual Property Office of China on March 24, 2023, entitled “Chimeric Antigen Receptor Targeting GPRC5D and / or BCMA and Its Applications”. The entire contents of the above-mentioned prior applications are incorporated into this application by reference. Technical Field

[0003] The present invention relates to the field of cell therapy, and in particular to a chimeric antigen receptor targeting GPRC5D and / or BCMA and applications thereof. Background Art

[0004] GPRC5D is a G protein-coupled receptor, class C, group 5, member D, and an orphan receptor. Studies have shown that GPRC5D is associated with cancer, with high expression specifically in myeloma and little expression in normal tissues. GPRC5D is a potential target for cancer therapy.

[0005] BCMA is a B-cell maturation antigen and a member of the tumor necrosis receptor (TNFR) family expressed by cells of the B-cell lineage. Upon binding to its ligands (such as APRIL or BAFF), BCMA activates B-cell proliferation and survival. BCMA is highly expressed on the surface of plasma cells and multiple myeloma cells. Anti-BCMA CAR T cells are currently used to treat multiple myeloma. However, due to tumor heterogeneity and downregulation of BCMA expression during treatment, some patients may still experience relapse after receiving anti-BCMA CAR T cell therapy.

[0006] Summary of the Invention

[0007] The present disclosure provides a chimeric antigen receptor targeting GPRC5D or targeting GPRC5D and BCMA, its encoding nucleic acid, and engineered immune effector cells, which have good binding effects on GPRC5D and / or BCMA antigens, obvious killing effects on tumor cells, and have broad application prospects.

[0008] In a first aspect, the present disclosure provides a chimeric antigen receptor (CAR), comprising a first polypeptide, wherein from N-terminus to C-terminus the first polypeptide comprises: an extracellular antigen binding domain, a hinge region, a transmembrane domain and at least one signal transduction domain, wherein the extracellular antigen binding region comprises a first antigen binding domain that specifically binds to GPRC5D.

[0009] In some embodiments, the extracellular antigen-binding domain of the first polypeptide further includes a second antigen-binding domain that specifically binds to BCMA; optionally, from N-terminus to C-terminus, the extracellular antigen-binding domain includes a first antigen-binding domain-linker-second antigen-binding domain, or a second antigen-binding domain-linker-first antigen-binding domain.

[0010] In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS.

[0011] In some embodiments, the chimeric antigen receptor further comprises a second polypeptide, which comprises, from N-terminus to C-terminus, an extracellular antigen binding domain, a hinge region, a transmembrane domain, and at least one signal transduction domain, wherein the extracellular antigen binding domain comprises a second antigen binding domain that specifically binds to BCMA.

[0012] In some embodiments, the first polypeptide and the second polypeptide are operably linked; optionally, the second polypeptide is operably linked to the N-terminus of the first polypeptide, or the first polypeptide is operably linked to the N-terminus of the second polypeptide; preferably, the first polypeptide and the second polypeptide are operably linked via a self-cleaving peptide; more preferably, after cleavage of the self-cleaving peptide, the first polypeptide and the second polypeptide form a dimer.

[0013] In some embodiments, in the first polypeptide and / or the second polypeptide:

[0014] (a) The hinge region contains the CD8 hinge region;

[0015] (b) The transmembrane domain contains the NKG2D transmembrane region;

[0016] (c) at least one signaling domain comprises a 2B4 co-stimulatory domain (natural killer cell receptor 2B4 co-stimulatory domain) and / or CD3ζ.

[0017] In some embodiments, the hinge region comprises the sequence shown in SEQ ID NO:53.

[0018] In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO:64.

[0019] In some embodiments, the signaling domain comprises the 2B4 costimulatory domain of SEQ ID NO:65 and / or the CD3 zeta of SEQ ID NO:56.

[0020] In some embodiments, in the first polypeptide and / or the second polypeptide:

[0021] (a) The hinge region contains the CD8 hinge region;

[0022] (b) the transmembrane domain contains the CD8 transmembrane region;

[0023] (c) at least one signaling domain comprises a CD137 costimulatory domain and / or CD3ζ.

[0024] In some embodiments, the hinge region comprises the sequence shown in SEQ ID NO:53.

[0025] In some embodiments, the transmembrane domain comprises the sequence shown in SEQ ID NO:54.

[0026] In some embodiments, the signaling domain comprises the CD137 costimulatory domain of SEQ ID NO:55 and / or the CD3 zeta of SEQ ID NO:56.

[0027] In some embodiments, the first antigen binding domain comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region (VL) and HCDR1, HCDR2 and HCDR3 of the heavy chain variable region (VH), wherein the light chain variable region comprises the LCDRs of the light chain variable region as shown in SEQ ID NO:9, 11, 13 or 15; and the heavy chain variable region comprises the HCDRs of the heavy chain variable region as shown in SEQ ID NO:10, 12, 14 or 16.

[0028] In some specific embodiments, the first antigen binding domain comprises the following combinations of LCDRs and HCDRs defined by the Kabat numbering system:

[0029] (1) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NOs: 17, 18 and 19, and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NOs: 20, 21 and 22;

[0030] (2) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NOs: 17, 18 and 19, and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NOs: 23, 24 and 25;

[0031] (3) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NOs: 26, 27 and 28 and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NOs: 29, 30 and 31; or

[0032] (4) A combination of LCDRs and HCDRs having 5, 4, 3, 2 or 1 amino acid substitutions, deletions or mutations compared to LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3 in any one of (1) to (3).

[0033] In some embodiments, the first antigen binding domain comprises a VL as shown in SEQ ID NO: 9, 11, 13 or 15 and a VH as shown in SEQ ID NO: 10, 12, 14 or 16;

[0034] Preferably, the following VL and VH combinations are present:

[0035] (1) VL shown in SEQ ID NO: 9 and VH shown in SEQ ID NO: 10;

[0036] (2) VL shown in SEQ ID NO: 11 and VH shown in SEQ ID NO: 12;

[0037] (3) VL shown in SEQ ID NO: 13 and VH shown in SEQ ID NO: 14;

[0038] (4) VL shown in SEQ ID NO: 15 and VH shown in SEQ ID NO: 16; or

[0039] (5) A sequence having 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to any one of (1) to (4).

[0040] In some embodiments, the VL and VH are connected by a glycine- and serine-rich linker peptide, which is GGGGSGGGGSGGGGS.

[0041] In some embodiments, the first antigen binding domain comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise the HCDR1, HCDR2, and HCDR3 of the VHH sequence of any one of SEQ ID NOs: 32-39;

[0042] Preferably, the HCDR1, HCDR2 and HCDR3 are selected from the following combinations:

[0043] (a) the sequences shown in SEQ ID NOs: 40, 41 and 42 as defined by the Kabat numbering system;

[0044] (b) the sequences set forth in SEQ ID NOs: 43, 44, and 45 as defined by the IMGT numbering system;

[0045] (c) the sequences represented by SEQ ID NOs: 46, 47, and 48 as defined by the IMGT numbering system;

[0046] (d) the sequences represented by SEQ ID NOs: 49, 50 and 51 as defined by the Kabat numbering system; or

[0047] (e) an amino acid sequence having one, two or three mutations in HCDR1, HCDR2 and / or HCDR3 as described in any one of (a) to (d); the mutations are selected from insertions, deletions and / or substitutions, and the substitutions are preferably substitutions of conservative amino acids.

[0048] In some embodiments, the first antigen binding domain comprises a sequence as shown in any one of SEQ ID NOs: 32-39, or a sequence that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to any one of SEQ ID NOs: 32-39.

[0049] In some embodiments, the second antigen binding domain comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise the HCDR1, HCDR2, and HCDR3 of the VHH sequence shown in any one of SEQ ID NOs: 1-2;

[0050] Preferably, the HCDR1, HCDR2 and HCDR3 are selected from the following combinations:

[0051] (a) the sequences shown in SEQ ID NOs: 3, 4 and 5 as defined by the Kabat numbering system;

[0052] (b) the sequences shown in SEQ ID NOs: 6, 7 and 8 as defined by the Kabat numbering system;

[0053] or (c) an amino acid sequence having one, two or three mutations in HCDR1, HCDR2 and / or HCDR3 as described in any one of (a) to (b); wherein the mutation is selected from insertion, deletion and / or substitution, and the substitution is preferably a conservative amino acid substitution.

[0054] In some embodiments, the second antigen binding domain comprises a sequence as shown in any one of SEQ ID NOs: 1-2, or a sequence having 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to any one of SEQ ID NOs: 1-2.

[0055] In some embodiments, the first and second antigen binding domains are humanized antibodies.

[0056] In some embodiments, the C-terminus of the chimeric antigen receptor further comprises an operably linked exogenous cytokine, wherein the exogenous cytokine is selected from IL2, IL4, IL6, IL7, IL10, IL11, IL12, IL15, IL18 and IL21; preferably, the cytokine is selected from IL-15, and more preferably, the IL15 has the sequence shown in SEQ ID NO:58.

[0057] In some embodiments, the C-terminus of the chimeric antigen receptor is operably linked to the cytokine via a self-cleaving peptide, and the self-cleaving peptide comprises a 2A peptide; more preferably, the self-cleaving peptide comprises a T2A peptide or a P2A peptide.

[0058] In some embodiments, the chimeric antigen receptor has a sequence as shown in any one of SEQ ID NOs: 59-63 and 66-76, or a sequence that is 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to a sequence as shown in any one of SEQ ID NOs: 59-63, 66-76.

[0059] In a second aspect, the present disclosure provides an isolated nucleic acid construct encoding the aforementioned chimeric antigen receptor.

[0060] In a third aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid construct.

[0061] In a fourth aspect, the present disclosure provides an immune effector cell comprising the expressed chimeric antigen receptor.

[0062] In some embodiments, the immune effector cells are selected from T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells; preferably, the immune effector cells of the present disclosure are NK cells.

[0063] In a fifth aspect, the present disclosure provides a method for preparing the immune effector cells expressing the chimeric antigen receptor, comprising introducing the nucleic acid construct or vector encoding the chimeric antigen receptor into the immune effector cells; further, the method may also include the step of inducing the expression of the chimeric antigen receptor.

[0064] In a sixth aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned chimeric antigen receptor, nucleic acid construct, vector and immune effector cells; further comprising a pharmaceutically acceptable carrier.

[0065] In a seventh aspect, the present disclosure provides a method for treating a tumor or cancer, comprising administering to a patient an effective amount of the aforementioned chimeric antigen receptor, nucleic acid construct, vector, immune effector cell and pharmaceutical composition; preferably, the tumor or cancer is selected from B-cell lymphoma; more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.

[0066] In an eighth aspect, the present disclosure provides a use of the aforementioned chimeric antigen receptor, nucleic acid construct, vector, immune effector cell and pharmaceutical composition in the preparation of a medicament for treating a tumor or cancer, wherein the tumor or cancer is selected from B-cell lymphoma; preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.

[0067] In a ninth aspect, the present disclosure provides use of immune effector cells and immune checkpoint inhibitors, or immune effector cells and CBL-b inhibitors, or immune effector cells and immune checkpoint inhibitors and CBL-b inhibitors, in the preparation of a medicament for treating cancer or tumors.

[0068] In some embodiments, the immune effector cells are selected from T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells; preferably, the immune effector cells of the present disclosure are NK cells.

[0069] In some embodiments, the immune effector cells express a chimeric antigen receptor, preferably a chimeric antigen receptor targeting BCMA or GPRC5D, more preferably a chimeric antigen receptor targeting BCMA and GPRC5D; more preferably, the immune effector cells express the chimeric antigen receptor described in the first aspect of the present disclosure.

[0070] In some embodiments, the immune checkpoint inhibitor is soluble PD-L1.

[0071] In some embodiments, the immune checkpoint inhibitor is further fused to a cytokine.

[0072] In some embodiments, the cytokine is IL15.

[0073] In some embodiments, the immune checkpoint inhibitor comprises a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:77.

[0074] In some embodiments, the CBL-b inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt of the compound of formula (I):

[0075] In some embodiments, the cancer or tumor is selected from a solid tumor or a hematological tumor.

[0076] In some embodiments, the hematological neoplasm is selected from B-cell lymphoma.

[0077] In some embodiments, the B-cell lymphoma is multiple myeloma.

[0078] In some embodiments, the multiple myeloma is refractory or relapsed multiple myeloma.

[0079] In a tenth aspect, the present disclosure provides a method for treating cancer or tumors, comprising administering to a subject an effective amount of immune effector cells, the method further comprising: (1) stimulating the immune effector cells with a CBL-b inhibitor before administering the immune effector cells, and / or, (2) administering to the subject an effective amount of an immune checkpoint inhibitor.

[0080] In some embodiments, the immune effector cells express a chimeric antigen receptor, preferably a chimeric antigen receptor targeting BCMA or GPRC5D, more preferably a chimeric antigen receptor targeting BCMA and GPRC5D; more preferably, the immune effector cells express the chimeric antigen receptor described in the first aspect of the present disclosure.

[0081] In some embodiments, the immune checkpoint inhibitor is soluble PD-L1.

[0082] In some embodiments, the immune checkpoint inhibitor is further fused to a cytokine.

[0083] In some embodiments, the cytokine is IL15.

[0084] In some embodiments, the immune checkpoint inhibitor comprises a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:77.

[0085] In some embodiments, the CBL-b inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt of the compound of formula (I):

[0086] In some embodiments, the cancer or tumor is selected from a solid tumor or a hematological tumor.

[0087] In some embodiments, the hematological neoplasm is selected from B-cell lymphoma.

[0088] In some embodiments, the B-cell lymphoma is multiple myeloma.

[0089] In some embodiments, the multiple myeloma is refractory or relapsed multiple myeloma.

[0090] In a tenth aspect, the present disclosure provides a method for enhancing the expression of a chimeric antigen receptor by an immune effector cell and / or enhancing the killing ability of an immune effector cell, the method comprising contacting the immune effector cell with a CBL-b inhibitor under conditions that activate the immune effector cell and / or transfect the chimeric antigen receptor.

[0091] In some embodiments, the CBL-b inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt of the compound of formula (I):

[0092] In some embodiments, the immune effector cells express a chimeric antigen receptor, preferably a chimeric antigen receptor targeting BCMA or GPRC5D, more preferably a chimeric antigen receptor targeting BCMA and GPRC5D; more preferably, the immune effector cells express the chimeric antigen receptor described in the first aspect of the present disclosure.

[0093] In some embodiments, the immune effector cells are selected from T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells; preferably, the immune effector cells of the present disclosure are NK cells. Beneficial effects:

[0094] The present disclosure has one or more of the following effective effects:

[0095] 1. The present disclosure provides a chimeric antigen receptor targeting GPRC5D, which can be used to kill tumor cells expressing GPRC5D antigen and has broad application prospects.

[0096] 2. The present disclosure provides a chimeric antigen receptor targeting GPRC5D and BCMA, enhances the killing ability of the chimeric antigen receptor, and improves the treatment failure or recurrence of BCMA CART due to tumor heterogeneity or BCMA antigen escape.

[0097] 3. The present disclosure compares and screens the antibody sequences, CAR elements and / or CAR structures of the aforementioned chimeric antigen receptors to improve the performance of the chimeric antigen receptors in at least one aspect of binding to the target antigen, CAR expression, proliferation or killing effect (especially long-term killing effect).

[0098] 4. The chimeric antigen receptor disclosed herein is particularly suitable for use in NK cells and is suitable for providing "off-the-shelf" GPRC5D CAR or "off-the-shelf" GPRC5D×BCMA CAR, which can overcome the defects of existing autologous CAR T therapy, such as high production cost, complex preparation process, and potential toxic reactions or adverse events.

[0099] 5. The present disclosure combines immune effector cells expressing chimeric antigen receptors with CBL-b inhibitors and / or PDL1-IL15 fusion proteins to enhance the killing effect of the immune effector cells on tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1A-1D are schematic diagrams of the structures of dual-target chimeric antigen receptors (BI-CARs) targeting BCMA and GPRC5D;

[0101] Figure 2 Schematic diagram of the structure of the NKG2D_TM dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D;

[0102] Figure 3. Statistics of BI-CAR-NK cell proliferation fold;

[0103] Figures 4A and 4B show the results of multiple rounds of killing experiments of MOLP8 cells and RPMI 8226 cells by BI-CAR-NK;

[0104] 5A-5B of FIG. 5 are schematic diagrams of the structure of a dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D;

[0105] Figure 6. Statistics of BI-CAR-NK cell proliferation fold;

[0106] Figures 7A to 7F show the results of multiple rounds of killing experiments of BI-CAR-NK cells against NCI H929 cells, RPMI 8226 cells, MOLP8 cells and tumor heterogeneous cells;

[0107] Figure 8: Positive rate of BI-CAR-NK;

[0108] Figure 9 shows the results of multiple rounds of killing experiments on NCI H929+10% NCI H929-hBCMA-KO cells using BI-CAR-NK in combination with CBL-b inhibitor and / or PDL1-IL15.

[0109] 10A to 10D show the positive rate of BI-CAR-NK and the effect of cryopreservation on the positive rate and viability of BI-CAR-NK.

[0110] 11A to 11C show the killing activity of BI-CAR-NK and the effect of cryopreservation on the killing activity of BI-CAR-NK.

[0111] 12A to 12D show the anti-tumor efficacy of BI-CAR-NK in the NCI H929 tumor model.

[0112] 13A to 13D show the anti-tumor efficacy of BI-CAR-NK in the MOLP8 tumor model.

[0113] Figure 14 is a schematic diagram of the structure of BCMA and GPRC5D single-targeting CAR;

[0114] 15A to 15D show the anti-tumor efficacy of BI-CAR-NK in the BCMA tumor escape model.

[0115] 16A to 16D show the anti-tumor efficacy test of BI-CAR-NK combined with CBL-b inhibitor and / or PDL1-IL15 in the NCI H929 tumor model.

[0116] Definitions and Explanations of Terms

[0117] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0118] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0119] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0120] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the subject term."

[0121] The term "BCMA" in this article stands for B cell maturation antigen, a member of the tumor necrosis factor receptor family. BCMA is primarily expressed on the surface of late-stage B cells, short-lived proliferating plasmablasts, and long-lived plasma cells, but is not expressed on naive B cells, CD34-positive hematopoietic stem cells, and other normal tissue cells. However, it is highly expressed in MM cells and plays a key role in the survival, proliferation, metastasis, and drug resistance of MM cells by mediating downstream signaling pathways. Therefore, BCMA is an ideal antigen target for the treatment of MM. An exemplary human BCMA sequence can be found in GenBank Protein Accession No: NP_001183.2.

[0122] The term GPRC5D herein refers to the G protein-coupled receptor C5 family subtype D, an orphan receptor, a seven-transmembrane protein with no known ligand. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while expression in normal tissue is restricted to the hair follicle region. Studies have shown that 65% of multiple myeloma patients have GPRC5D expression exceeding the 50% threshold. This characteristic makes GPRC5D a potential target for the treatment of multiple myeloma. An exemplary human GPRC5D sequence can be found in GenBank Protein Accession No: NP_061124.1.

[0123] The term "chimeric antigen receptor (CAR)" herein refers to an artificial cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen, which comprises at least (1) an extracellular antigen binding domain, such as an antibody heavy chain variable region and / or light chain variable region, (2) a transmembrane domain that anchors CAR into immune effector cells, and (3) an intracellular signaling domain. CAR is able to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding domain.

[0124] The term "signal peptide" herein refers to a fragment of a protein or polypeptide that is used to guide the protein or polypeptide into the secretory pathway and transferred to the cell membrane and / or cell surface. In some embodiments, the signal peptide is a CD8α signal peptide, optionally having at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) to the following sequence: MALPVTALLLPLALLLHAARP.

[0125] As used herein, the "hinge region" of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane region and the antigen binding region. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antigen binding region. The hinge region can be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a fully synthetic hinge sequence.

[0126] As used herein, the "transmembrane (TM) region" of a chimeric antigen receptor refers to a polypeptide structure that enables the chimeric antigen receptor to be expressed on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and guides the immune cell to respond to the target cell. The transmembrane domain can be natural or synthetic, or derived from any membrane-bound protein or transmembrane protein. When the chimeric antigen receptor binds to the target antigen, the transmembrane domain can conduct signal transduction. Exemplarily, the hinge region can be a CD8 transmembrane region or a NKG2D transmembrane region.

[0127] As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs cells to perform designated functions. The intracellular signaling domain is responsible for primary signaling within the cell after the antigen binding domain binds to the antigen, leading to activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed. Exemplary intracellular signaling domains include CD3ζ.

[0128] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0129] The term "immune effector cell" herein refers to a cell that is involved in the performance of effector functions during an immune response. "Immune effector cell" is preferably capable of binding to an antigen, such as an antigen expressed on the cell surface or a cell characterized by presenting an antigen or an antigenic peptide derived from an antigen, and mediating an immune response. For example, such cells secrete cytokines and / or chemokines, kill microorganisms, secrete antibodies, identify infected or cancerous cells and optionally eliminate these cells. For example, immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells. Preferably, in the context of the present disclosure, "immune response cells" are NK cells.

[0130] The terms "T lymphocyte" and "T cell" are used interchangeably herein and refer to the main type of white blood cell that completes maturation in the thymus and has multiple roles in the immune system, including recognizing specific foreign antigens in the body and activating and inactivating other immune cells in an MHC class I restricted manner. T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from mammals. T cells can be CD3+ cells. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulatory T cells, γδT cells (gamma delta T cell / γδT cell), etc. Other types of helper T cells include cells such as Th3 (Treg), Th17, Th9 or Tfh cells. Other types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically engineered T cells, such as T cells modified to express T cell receptors (TCR) or chimeric antigen receptors (CAR). T cells or T cell maintenance effector cells can also be differentiated by stem cells or progenitor cells. T cell-like derived effector cells can have T cell lineages in some aspects, but at the same time have one or more functional characteristics not present in primary T cells.

[0131] The term "NK cell" or "natural killer cell" herein refers to a subpopulation of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As used herein, the terms "adaptive NK cell" and "memory NK cell" are interchangeable and refer to a subpopulation of NK cells whose phenotype is CD3 - and CD56 + , expresses at least one of NKG2C and CD57 and optionally CD16, but lacks expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some embodiments, the isolated CD56 + NK cell subsets include expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and / or DNAM-1. +It can be weaker or stronger expression. NK cells or NK cell-like effector cells can be differentiated from stem cells or progenitor cells. NK cell-like derived effector cells can have NK cell lineage in some aspects, but at the same time have one or more functional characteristics not present in primary T cells.

[0132] As used herein, the term "NKT cell" or "natural killer T cell" refers to a T cell restricted to CD1d that expresses a T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d (a non-classical MHC molecule). Two types of NKT cells are identified. Constant or type I NKT cells express a very limited TCR library: a combination of a typical α chain (Vα24-Jα18 in humans) and a limited spectrum of β chains (Vβ11 in humans). A second NKT cell population (called non-classical or non-constant type II NKT cells) shows a more uneven TCR α β utilization rate. Type I NKT cells are considered suitable for immunotherapy. Adaptive or invariant (type I) NKT cells can be identified by expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.

[0133] As used herein, the term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into that individual. "Allogeneic" refers to a transplant derived from a different individual of the same species.

[0134] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an antibody with a specific affinity of about 10 -6 M or lower, 10 -7 M or lower, about 10 -8 M or lower, about 10 -9 The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0135] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0136] The term "antibody" is used herein in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibodies" generally comprise a glycoprotein, or antigen-binding portion thereof, of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further divided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that can interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Due to the different amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain, their antigenicity also varies. Accordingly, "immunoglobulins" herein can be divided into five categories, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. Igs of the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG is divided into IgG1, IgG2, IgG3, and IgG4, and IgA is divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0137] "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds including, for example, biocompatible polymers. See, for example, Korndorfer, IP, Beste, G. & Skerra, A. (2003). Proteins, 53, 121–129.; Roque, ACA, Lowe, CR & Taipa, MA Antibodies and genetically engineered related molecules: production and purification. Biotechnol. Prog. 20, 639-654 (2004). Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for transplanting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, etc.

[0138] The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0139] The term "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0140] The term "heavy chain antibody" herein refers to an antibody lacking the light chains of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.

[0141] The term "nanoantibody" in this article refers to heavy chain antibodies that are naturally missing light chains and exist in camels and other organisms. Cloning their variable regions can produce single-domain antibodies consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

[0142] The terms "VHH domain," "nanobody," and "single domain antibody" (sdAb) are synonymous and used interchangeably herein. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy chain variable region, obtained by cloning the variable region of a heavy chain antibody. This single-domain antibody (sdAb) is the smallest fully functional antigen-binding fragment. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting solely of a single heavy chain variable region.

[0143] For further description of “heavy chain antibodies” and “single domain antibodies”, “VHH domains” and “nanoantibodies”, see: Hamers-Casterman et al., Nature. 363, 446-8 (1993). Naturally occurring antibodies devoid of light chains.; a review article by Muyldermans (J Biotechnol. 2001 Jun; 74(4): 277-302. Single domain camel antibodies: current status); and the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193.

[0144] The term "multi-specificity" herein refers to the ability of an antibody or antigen-binding fragment to bind to, for example, different antigens or at least two different epitopes on the same antigen. Therefore, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes that an antibody can bind to. For example, conventional monospecific IgG antibodies have two identical antigen-binding sites (paratopes) and therefore can only bind to the same epitope (rather than binding to different epitopes). In contrast, multispecific antibodies have at least two different types of paratopes / binding sites and therefore can bind to at least two different epitopes. As described herein, "complementary determining region" refers to the antigen-binding site of an antibody. In addition, a single "specificity" can refer to one, two, three, or more than three identical complementary determining regions (the actual number of complementary determining regions / binding sites in a single antibody molecule is referred to as "valence") in a single antibody. For example, a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes. Accordingly, a multispecific antibody comprises at least two (different) complementary determining regions / binding sites. Therefore, the term "multispecific antibody" refers to an antibody having more than one paratope and having the ability to bind to two or more different epitopes. The term "multispecific antibody" particularly includes bispecific antibodies as defined above, but generally also includes proteins, e.g. antibodies that specifically bind three or more different epitopes, scaffolds, i.e. antibodies with three or more paratopes / binding sites.

[0145] The term "valent" herein refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0146]

[0014] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of a native antibody.

[0147] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. Exemplarily, "antigen-binding fragment" or "antibody fragment" herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0148] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune response, etc.

[0149] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0150] The term "variable region" herein refers to the region of an antibody heavy or light chain involved in antigen binding. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0151] The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) found in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT), but considered to be outside the hypervariable region under a different set of criteria (such as KABAT or IMGT). One or more of these positions can also be found in extended hypervariable regions. The present invention includes antibodies comprising modifications at these hybrid hypervariable positions. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region can be abbreviated as LCDRs. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3), which form loops connecting the sheet structure and, in some cases, form part of the sheet structure. The CDRs in each chain are held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and contribute to the formation of the antibody antigen-binding site with the CDRs from other antibody chains.

[0152] "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0153] The term "IMGT numbering system" herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc, MP, Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., ... & Lefranc, G. (2003). IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Developmental & Comparative Immunology, 27(1), 55-77.

[0154] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0155] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the positions of structural loop regions (see, e.g., Chothia, C., & Lesk, AM (1987). Canonical structures for the hypervariable regions of immunoglobulins. Journal of molecular biology, 196(4), 901-917.).

[0156] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0157] 1) Alanine (A), serine (S), threonine (T);

[0158] 2) Aspartic acid (D), glutamic acid (E);

[0159] 3) Asparagine (N), glutamine (Q);

[0160] 4) Arginine (R), Lysine (K), Histidine (H);

[0161] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0162] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0163] The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences varies as the number of identical positions shared by the sequences changes, taking into account the number of spaces that need to be introduced and the length of each space for optimal comparison of the two sequences.

[0164] Mathematical algorithms can be used to compare sequences between two sequences and calculate percent identity. For example, the Needlema and Wunsch algorithms (available at www.gcg.com) that have been integrated into the GAP program of the GCG software package are used, using a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program (available at www.gcg.com) in the GCG software package is used, using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0165] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for directly expressing the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into a subject to produce antibodies in vivo (see, e.g., Stadler, CR, et al., 2001). H., Celik, L., Hebich, B., Roth, AS, Roth, RP, ... & Sahin, U. (2017). Elimination of large tumors in mice by mRNA-encoded bispecific antibodies. Nature medicine, 23(7), 815-817. or EP2101823B1).

[0166] The term "control sequence" herein refers to DNA sequences required for expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0167] A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0168] As used herein, the term "operably linked" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest in a relationship that allows them to act in a desired manner, regardless of the presence of spacers or linkers. When applied to polypeptides, the term is intended to indicate that the polypeptide sequences are linked in a manner that allows the linked product to have the desired biological function. Illustratively, two or more polypeptide sequences can be linked using a self-cleaving peptide that causes the polypeptide sequences to separate from each other after expression, rather than fusing together, so that each performs the function of the polypeptide sequence. The term can also be applied to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), the term is intended to indicate that the polynucleotide sequences are linked in a manner that allows the expression of the polypeptide to be regulated by the polynucleotide.

[0169] As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0170] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0171] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0172] The term "pharmaceutical composition" herein refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0173] The term "pharmaceutically acceptable carrier" herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and the like, and combinations thereof, which are known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th edition. Mack Printing Company, 1990, pp. 1289-1329). Except in cases where it is incompatible with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.

[0174] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as cancer and tumors. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0175] The term "subject" herein refers to an organism that is being treated for a particular disease or condition as described herein. Exemplarily, a "subject" includes a mammal, such as a human, primate (e.g., monkey), or non-primate mammal, being treated for a disease or condition.

[0176] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0177] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0178] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.

[0179] The term "CBL-b" herein refers to Casitas B-lineage lymphoma proto-oncogene-b, an E3 ubiquitin ligase. The term "CBL-b inhibitor" herein refers to a compound that inhibits the activity of the CBL-b protein.

[0180] The term "NCI H929 + 20% H929-hBCMA-KO heterogeneous tumor cells" herein refers to a mixture of NCI H929 cells and H929-hBCMA-KO cells, in which NCI H929 cells comprise 80% and NCI H929-hBCMA-KO cells comprise 20%. The proportions of each type of cell in heterogeneous tumor cells for NCI H929 + 10% H929-hBCMA-KO and NCI H929 + 20% H929-hBCMA-KO are similarly calculated. DETAILED DESCRIPTION

[0181] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0182] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0183] General Methods

[0184] Biacore (SPR method) detection of antibody affinity

[0185] Recombinant candidate antibodies were captured using a Protein A chip (GE Healthcare; 29-127-558). Sample and running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69). The flow-through pool was set to 25°C. The sample block was set to 16°C. Both were pretreated with running buffer. In each cycle, the antibody to be tested was first captured using a Protein A chip, then a single concentration of antigen protein was injected, the binding and dissociation process of the antibody and antigen protein was recorded, and finally the chip was regenerated using Glycine pH 1.5 (GE Healthcare; BR-1003-54). Binding was measured by injecting different concentrations of human antigen protein in solution for 240 seconds, with a flow rate of 30 μL / min, starting from 200 nM (see detailed results for the actual concentration tested), diluted 1:1, and a total of 5 concentrations. The dissociation phase was monitored for up to 600 seconds and triggered by switching from the sample solution to the running buffer. The surface was regenerated by washing with a 10 mM glycine solution (pH 1.5) for 30 seconds at a flow rate of 30 μL / min. Bulk refractive index differences were corrected by subtracting the response obtained from the goat anti-human Fc surface. A blank injection (= double reference) was also subtracted. To calculate the apparent KD and other kinetic parameters, a Langmuir 1:1 model was used. The association rate (Ka), dissociation rate (Kdis) and binding affinity (KD) were calculated.

[0186] Example 1. Preparation of antibodies

[0187] 1.1 Preparation of anti-BCMA nanobodies

[0188] Anti-BCMA nanobodies were prepared by immunizing alpacas with human BCMA (Met1-Ala54)-hFc protein, followed by booster immunizations with a mixture of human BCMA (Met1-Ala54)-hFc and cynomolgus macaque BCMA (Met1-Ala53)-hFc proteins. Blood was collected one week after each booster immunization, and serum antibody titer and specificity were assessed by ELISA. After verifying that the serum bound to both human and cynomolgus macaque BCMA-hFc proteins to varying degrees, phage library construction and panning were performed.

[0189] Peripheral blood was collected, PBMCs were isolated, and total RNA was extracted. cDNA was prepared by reverse transcription and nested amplification to obtain VHH fragments. After both the VHH fragments and vector were digested and ligated, the ligated products were transformed by electroporation and cultured on plates. After overnight growth, the E. coli library was expanded. Helper phage M13K07 was added, cultured, and titered. Clones with a high binding rate to human and monkey BCMA proteins were identified by ELISA and panned over multiple rounds. Positive clones were sequenced and analyzed by cluster analysis.

[0190] Using ELISA and Biacore methods familiar to those skilled in the art, VHH antibodies with excellent binding to human and monkey BCMA proteins were screened and identified for humanized design. By comparing the IMGT (http: / / imgt.cines.fr) human antibody heavy and light chain variable region germline gene database, heavy chain variable region germline genes with high homology to the VHH nanobody were selected as templates. The CDRs were then transplanted into the corresponding humanized templates. Key amino acids in the framework sequence were backmutated to those corresponding to the VHH nanobody to maintain the original affinity. Furthermore, if the antibody contained sites susceptible to chemical modification, point mutations were performed to eliminate the risk of modification, resulting in humanized monoclonal antibodies. The CDR amino acid residues were assigned and annotated using the Kabat numbering system. These antibodies were then validated by Biacore. The resulting alpaca-derived VHH antibody was designated BCMA-Lab02, and its humanized anti-BCMA antibody was designated BCMA-Hab02-VHH7. Biacore affinity data are shown in Table 1. The antibody sequences are shown in Table 2, and the Kabat numbered CDR sequences are shown in Table 3.

[0191] Table 1 Affinity of BCMA-Lab02 and its humanized antibodies to human BCMA protein

[0192] Table 2 Anti-BCMA Nanobody Sequence List

[0193] Table 3 Anti-BCMA Nanobody Kabat Numbering CDR Sequence List

[0194] 1.2 Preparation of anti-GPRC5D scFv

[0195] Anti-human GPRC5D monoclonal antibodies were produced by immunizing mice using a conventional hybridoma method. Alternate immunizations were performed using a human GPRC5D-overexpressing cell line (HEK293T-hGPRC5D, constructed in-house) and a monkey GPRC5D-overexpressing cell line (HEK293T-cynoGPRC5D, constructed in-house). Blood was collected and serum was separated. Serum specific antibody titers were determined using a cell-based enzyme-linked immunosorbent assay (ELISA). Mice with high titers were selected, and their spleen cells were fused with SP2 / 0 myeloma cells. Positive clones that bound to both human and monkey GPRC5D-overexpressing cells and NCI-H929 cells were screened using cell-based ELISA and FACS.

[0196] Positive clones that meet the criteria are selected and subcloned using semi-solid culture medium. The culture is gradually expanded and evaluated for binding activity to human and monkey GPRC5D-overexpressing cells and endogenous cells (NCI-H929). The optimal clone is selected and expanded. RNA from the hybridoma cells is collected, and the amino acid sequences of the light and heavy chain variable regions of the positive clones are determined and clustered. Chimeric antibodies containing human IgG1 constant regions are constructed based on the sequenced light and heavy chain variable region sequences. Cell-based ELISA and FACS screening, familiar to those skilled in the art, are used to obtain monoclonal antibodies that bind to both human and monkey GPRC5D-overexpressing cells and NCI-H929 cells for subsequent humanization design.

[0197] By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes, we selected heavy and light chain variable region germline genes with high homology to mouse antibodies as templates. The CDRs of the mouse antibodies were then transplanted into the corresponding humanized templates. Key amino acids in the FR regions were backmutated to their mouse counterparts as needed to maintain the original affinity. Furthermore, if the antibody contained sites susceptible to chemical modification, point mutations were performed to eliminate the risk of modification. Humanized monoclonal antibodies were generated. The CDR amino acid residues were assigned and annotated using the Kabat numbering system. Subsequently, validation was performed using cell-based ELISA. The resulting mouse antibodies were designated GPRC5D-mab03 and GPRC5D-mab06, and their humanized counterparts were designated GPRC5D-Hab03 and GPRC5D-Hab06. The sequences of the light and heavy chain variable regions are shown in Table 4, and the CDR sequences are shown in Table 5.

[0198] Table 4 VL and VH sequences of anti-GPRC5D mouse and humanized antibodies

[0199] Table 5 Kabat numbering of LCDRs and HCDRs sequences of anti-GPRC5D antibodies

[0200] 1.3 Preparation of anti-GPRC5D nanobodies

[0201] Alpaca-derived VHH antibodies were prepared and screened as described in Example 1.1, using CHO-K1 cells overexpressing human GPRC5D as the immunogen. The CDR amino acid residues of the antibodies were identified and annotated using the IMGT or Kabat numbering system. This yielded two alpaca sequences, designated GPRC5D-Lab05 and GPRC5D-Lab06, and two humanized sequences, designated GPRC5D-Hab05-H2 and GPRC5D-Hab06-H1. The amino acid sequences are shown in Table 6, and the numbered CDR sequences are shown in Table 7.

[0202] Preparation of camel-derived VHH antibodies: Camels approximately three weeks old were selected, and 5 ml of blood was collected before immunization to serve as a negative control serum. The first three immunizations used human GPRC5D protein, followed by four immunizations using a CHO-K1 cell line overexpressing human GPRC5D. After verifying that the serum binds to both human and monkey GPRC5D proteins, a phage library was constructed and screened. PBMCs were isolated from peripheral blood, and total RNA was extracted and reverse transcribed to obtain cDNA. Nested PCR was used to amplify the nanobody (VHH) fragments. The vector and target product were digested and ligated. The ligated product was then electroporated into TG1 competent cells, diluted and plated for culture. After overnight expansion, the culture was supplemented with helper phage M13K07, cultured, and titered.

[0203] Phage library panning was performed using cells overexpressing human GPRC5D as an antigen. After multiple rounds of panning, positive phage were continuously enriched during the panning process, and FACS was used to screen for nanobodies with good specificity and high affinity for overexpressing cells and cells endogenously expressing human GPRC5D. After multiple rounds of optimization and screening, several positive clones capable of simultaneously recognizing human and monkey GPRC5D were obtained. Sequencing was performed, and humanized antibodies were constructed as described in Example 1.1. The CDRs of the antibodies were annotated by IMGT or Kabat. After FACS verification, several strains of humanized antibodies with high affinity were obtained. The camel antibodies were named GPRC5D-Lab03 and GPRC5D-Lab04, and their humanized antibodies were named GPRC5D-Hab03-H10 and GPRC5D-Hab04-H5. The nanobody sequences are shown in Table 6, and the CDRs sequences are shown in Table 7.

[0204] Table 6 Anti-GPRC5D Nanobody Amino Acid Sequences

[0205] Table 7 Anti-GPRC5D Nanobody CDRs Sequences

[0206] Example 2. Preparation of PBMC-derived CAR-NK

[0207] 2.1 Construction of target plasmid

[0208] Conventional molecular biology methods in the art were used to load the nucleic acid sequence of the chimeric antigen receptor into a retroviral vector to construct a target plasmid. The specific steps are as follows: amplify and obtain the CAR nucleotide sequence by PCR, perform a double enzyme digestion reaction on the target gene fragment and the retroviral vector plasmid, and incubate at 37°C for 60 minutes. After the enzyme digestion reaction, the nucleic acid fragment was purified by agarose gel (using a Taraka gel purification kit). The specific operation steps were carried out according to the kit instructions. The target nucleic acid molecule after gel purification was incubated at 16°C for 10-12 hours under the action of T4 ligase. The obtained ligation product was transformed into DH5α competent cells by heat shock. The competent cells were transferred into liquid LB medium without antibiotics, shaken at 37°C at 200rpm, cultured for 2 hours, then centrifuged at 4000rpm for 2 minutes, and then resuspended in fresh LB medium by centrifugation. The bacterial solution was evenly applied to solid LB medium containing antibiotics and cultured in a 37°C incubator overnight. Positive clones were picked and identified, and the target plasmid of the positive clones was extracted for standby use.

[0209] 2.2 Virus packaging

[0210] One day before virus packaging, 293T cells (purchased from ATCC) were trypsinized and cultured at a rate of 1×10 7 When transfecting cells, mix the packaging plasmid and the target plasmid and add them to the α-MEM culture medium. In another centrifuge tube containing α-MEM culture medium, add Transfection reagent (Promega, E2311). Add the diluted transfection reagent dropwise to the diluted plasmid, mix thoroughly, and let stand at room temperature for 15 minutes. Finally, add the plasmid and transfection reagent mixture to a 10 cm culture dish, gently shake 10 times to mix thoroughly, and place in the incubator. Three days after cell transfection, harvest the virus and transfer 10 ml of the virus-containing culture supernatant to a 50 ml centrifuge tube. Incubate at 4°C, 1250 rpm, for 5 minutes to remove dead 293T cells. Filter the virus-containing supernatant and concentrate using Retro-X Concentrator (Clontech, 631455). Aliquot and store at -80°C until needed.

[0211] 2.3 Methods for inducing PBMC into NK cells and NK cell activation

[0212] Fresh PBMCs were centrifuged at 500 g for 7 minutes at room temperature. The supernatant was discarded, and NK cells were isolated using the Human NK Cell Isolation Kit (Stemcell, 17955). The isolated NK cells were activated with K562 cells using the following protocol: On Day 0, cells were counted using AO / PI, mixed at a 1:2 ratio of NK:K562, and added to a non-treated 6-well plate in NK cell culture medium (Miltenyi Biotec, 130-114-429) supplemented with 200 IU / ml human IL-2. The plate was then incubated in an incubator (37°C, 5% CO2). On Day 4, 3 mL of culture medium was added to each well. On Day 6, cell activation was complete and ready for transfection.

[0213] 2.4 Methods for constructing CAR-NK cells through viral infection.

[0214] On Day 1, coat a 24-well plate with 500 μL of RetroNectin reagent (Takara, T202) at a concentration of 7 μg / mL, and incubate at 4°C overnight. On Day 2, discard the upper RetroNectin layer and wash once with PBS. Infect NK cells at an MOI of 5, calculate the amount of virus based on the virus titer, and add the virus to the 24-well plate. Centrifuge the 24-well plate with the virus at 2000g, 4-8°C for 60 minutes. Discard the upper viral liquid. Count the NK cells and calculate the number of cells per well. 5 Add 100 cells / well to a 24-well plate and centrifuge at 400g for 5 minutes at room temperature. Place the 24-well plate in an incubator (37°C, 5% CO2). On Day 3, transfer the transfected NK cells to a non-treated 6-well plate. On Day 6, assay CAR expression and CAR NK cell proliferation.

[0215] Example 3. Construction of 4-1BB chimeric antigen receptor and screening of extracellular antibody combinations

[0216] 3.1 Construction of 4-1BB CAR-NK

[0217] Using the BCMA-Hab02-VHH7 antibody and GPRC5D-scFv (GPRC5D-Hab06) antibody obtained by screening in Example 1, a dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D as shown in 1A-1D of Figure 1 was designed and constructed, respectively, and named BI-CAR01 to BI-CAR04; BCMA-Hab02-VHH7 and GPRC5D-scFv (GPRC5D-Hab03) antibodies were used to design and construct BI-CAR05 with a structure as shown in 1A of Figure 1; the scFv structures were all designed with the N-terminal-C-terminal VL-VH structure. The molecular biology method shown in Example 2 was used to load the nucleic acid sequence of BI-CAR into a retroviral vector, construct the target plasmid and viral vector, induce NK cells, and prepare CAR-NK cells. The amino acid sequences of each domain of BI-CAR and exemplary BI-CAR01 to 05 sequences are shown in Table 8.

[0218] Table 8 BI-CAR domains and exemplary BI-CAR amino acid sequence

[0219] 3.2 Detection of chimeric antigen receptor expression rate

[0220] On the 6th day after retroviral infection of NK cells, the expression rate of CAR on the surface of NK cell membrane was detected by flow cytometry. The method for detecting the expression of BCMA-CAR NK surface molecules in BI-CAR is as follows: 2×10 5 Cells were transferred to a 96-well U-shaped plate and centrifuged. The supernatant was discarded and the cells were washed with buffer. 100 μl of FITC-hBCMA-Fc (ACRO Biosystems, BCA-HF254) with a final concentration of 2 μg / ml was added and incubated at 4°C in the dark for 1 hour. After the incubation, the cells were centrifuged, the supernatant was discarded, and the cells were washed with buffer and resuspended. The BCMA-CAR expression rate was detected by BD FACS Canto II flow cytometer.

[0221] The method for detecting the expression of GPRC5D-CAR NK cell surface molecules in BI-CAR is as follows: 2×10 5Cells were transferred to a 96-well U-shaped plate, centrifuged, the supernatant discarded, washed with buffer, and then 100 μl of human GPRC5D-his (ACROBiosystems, GPD-H52D4) with a final concentration of 10 μg / ml was added. The cells were incubated at 4°C in the dark for 1 hour. After the incubation, the cells were centrifuged, the supernatant discarded, washed with buffer, and then 100 μl of THETM His Tag Antibody [iFluor 647] (GenScript, A01802) with a final concentration of 1 μg / ml was added. The cells were incubated at 4°C in the dark for 1 hour. After the incubation, the cells were centrifuged, the supernatant discarded, and the cells were resuspended after washing with buffer. The expression rate of GPRC5D scFv in BI-CAR was detected using a BD FACS Canto II flow cytometer.

[0222] The FACS expression detection results of BI-CAR are shown in Table 9. The results showed that in the formats of Figure 1 1A and Figure 1 1B, the expression of anti-BCMA and anti-GPRC5D was generally high and had no position dependence; while in the formats of Figure 1 1C and Figure 1 1D, the expression of BCMA-CAR or GPRC5D-CAR was affected by its location.

[0223] Table 9 BI-CAR expression rate detection

[0224] 3.3 Detection of BI-CAR-NK’s rapid killing rate of target cells within 4 hours in vitro

[0225] Six days after retroviral infection of NK cells, a 4-hour in vitro killing experiment was performed: the target cells selected were NCI H929, RPMI8226, MOLP8, and NCI H929-hBCMA-KO. All cells were genetically manipulated to contain a luciferase reporter gene. NCI H929-hBCMA-KO is an NCI H929 cell line in which BCMA was knocked out using conventional genetic manipulation methods. Target cells diluted in 1640 medium were incubated at 2×10 4 50 μl / well of the solution was added to a white opaque 96-well plate, and NK cells were added to the target cells at effector-target ratios of 5:1, 2.5:1, and 1.25:1. The 96-well plate was cultured in a 37°C, 5% CO2 incubator. After 4 hours, 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added. After incubation at room temperature in the dark for 10 minutes, the killing rate was measured using a microplate reader and calculated.

[0226] The 4-hour in vitro cytotoxicity of BI-CAR against the above target cells is detailed in Table 10. At an effector-target ratio of 5:1, BI-CAR01 to BI-CAR05 all showed specific cytotoxicity against NCI H929-Lu and NCI H929-hBCMA-KO-Lu. BI-CAR01 to BI-CAR03 and BI-CAR05 all showed good specific cytotoxicity against the four tumor cell lines. Among them, BI-CAR01, BI-CAR-03, and BI-CAR05 showed stronger cytotoxicity against NCI H929-hBCMA-KO-Lu tumor cells, indicating that BI-CAR01, BI-CAR03, and BI-CAR05 have a better cytotoxic effect on tumor cells with downregulated or missing BCMA expression.

[0227] Table 10 Results of BI-CAR-NK rapid killing rate of target cells in vitro within 4 hours Note: “-” means no killing effect was detected.

[0228] Example 4. Design of NKG2D-F chimeric antigen receptor

[0229] 4.1 Construction of NKG2D-F chimeric antigen receptor.

[0230] To verify the effect of different transmembrane domains on the activity of chimeric antigen receptors, the antibodies BCMA-Hab02-VHH7 and GPRC5D-Hab06 corresponding to BI-CAR01 and BI-CAR03 obtained by screening in Example 3 were used to design and construct a dual-target chimeric antigen receptor targeting BCMA and GPRC5D as shown in Figure 2, named BI-CAR06. The dual-target chimeric antigen receptor targeting BCMA and GPRC5D includes CD8α signal peptide (SP), anti-GPRC5D-scFv and anti-BCMA-VHH, CD8 hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3ζ, and IL15 connected by self-cleaving peptide P2A. The specific sequence is shown in Table 11. The molecular biology method in Example 2 was used to load the nucleic acid sequence into the retroviral vector to construct the target plasmid.

[0231] Table 11 BI-CAR domains and exemplary BI-CAR amino acid sequence

[0232] 4.2 Determination of CAR expression rate in CAR-NK cells with different structures

[0233] Referring to the method shown in Example 2, NK cells were infected with the same MOI to prepare BI-CAR-NK cells with different transmembrane regions and intracellular stimulatory domain structures, and the expression of CAR was detected by the method in Reference Example 3.2. The results are shown in Table 12. The results show that the expression of the CAR shown in Figure 1A is better than the expression of the CAR shown in Figure 1C.

[0234] Table 12 CAR expression rate results in different structures of CAR-NK

[0235] 4.3 Determination of proliferation rate of CAR-NK cells with different structures

[0236] The proliferation of BI-CAR cells was detected by AO / PI counting, and the results are shown in Figure 3. After 14 days of NK activation, the proliferation rates of BI-CAR cells were as follows: BI-CAR06 (5566 times) > NK (3124 times) > BI-CAR03 (1705 times) > BI-CAR05 (1245.2 times) > BCMA-CAR01 (1012 times). According to the above results, the proliferation rate of BI-CAR cells loaded with NKG2D™ elements after optimization is significantly better than that of BI-CAR cells using other transmembrane elements.

[0237] 4.4 Determination of 4-hour rapid killing rate of CAR-NK cells with different structures

[0238] On the 6th day after retroviral infection of NK cells, a 4-hour in vitro rapid killing experiment was performed: target cells NCI H929, RPMI 8226, MOLP8 and NCI H929-hBCMA-KO diluted in 1640 medium were cultured at 2×10 4 50 μl / well of the solution was added to a white opaque 96-well plate, and NK cells were added to the target cells at an effector-target ratio of 9:1 and 3:1. The 96-well plate was cultured in a 37°C, 5% CO2 incubator. After 4 hours, 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added. After incubation at room temperature in the dark for 10 minutes, the killing rate was measured using a microplate reader and calculated.

[0239] The 4h in vitro cell killing effect of BI-CAR on the above target cells is detailed in Table 13. The results showed that BI-CAR05, BI-CAR01, BI-CAR-03, and BI-CAR-06 all showed strong tumor cell killing function.

[0240] Table 13 Results of 4h rapid killing rate of CAR-NK cells with different structures Note: “-” means no killing effect was detected.

[0241] 4.5 Detection of multiple rounds of killing effects of CAR-NK cells with different structures

[0242] Six days after retroviral infection of NK cells, the in vitro multiple-round killing effects of BI-CAR01, BI-CAR-03, BI-CAR-05, and BI-CAR-06 on RPMI 8226 cells and MOLP8 cells were detected: (1) First round of killing: 2.5×10 5 Target cells RPMI8226 or MOLP8 diluted with 1640 culture medium were placed in a 12-well plate at a concentration of 500 μl / well. NK cells were added to the 12-well plate at an effector-target ratio of 1:1 and cultured in a 37°C, 5% CO2 incubator for 24 hours. After culturing for 24 hours, 100 μl of the above mixed cells were added to a 96-well plate with an opaque white bottom, and 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added. After incubation at room temperature in the dark for 10 minutes, the fluorescence intensity was measured using a microplate reader to calculate the NK cell killing efficiency. After that, the next round of killing test was carried out. (2) Next round of killing: Take the cells in the 12-well plate in the previous round, count the NK cells, add the NK cells from the previous round to the 12-well plate inoculated with new target cells at an effector-target ratio of 1:1, repeat step (1), measure the NK cell killing rate and continue the next round of killing test.

[0243] The results of the multi-round killing test of BI-CAR NK cells are shown in Figure 4. As shown in Figure 4, the killing effect of BI-CAR06 loaded with NKG2D™ element is better than that of other forms of BI-CAR; BI-CAR06 can still maintain a 50% cell killing rate in the third round of killing (R3-1d) of MOLP8 cells and a 40% cell killing rate in the third round of killing (R3-1d) of RPMI 8226 cells.

[0244] Example 5 BCMA-VHH×GPRC5D-VHH Screening

[0245] 5.1 Screening of BCMA-VHH and GPRC5D-VHH Antibody Combinations

[0246] To verify the effects of different BCMA-VHH×GPRC5D-VHH combinations on chimeric antigen receptor expression and activity and to exclude BCMA-VHH×GPRC5D-VHH CARs with nonspecific killing, BCMA-Hab02-VHH7 and GPRC5D-VHH antibodies were used to construct the BI-CAR (Figure 1A). The dual-target chimeric antigen receptor targeting BCMA and GPRC5D includes the CD8α signal peptide (SP), anti-GPRC5D-VHH and anti-BCMA, CD8 hinge region, CD8 transmembrane region, CD137 costimulatory domain, and CD3ζ. After preparing BI-CAR T cells, CAR expression, specific killing function, and nonspecific killing function were tested. Antibody combinations with significant effects on CAR expression and killing, as well as those with strong nonspecific killing, were eliminated. BI-CARs with high BI-CAR expression, strong specific killing, and no nonspecific killing were selected for further construction into BI-CAR NK cells for validation.

[0247] 5.2 Design and Expression Detection of Dual VHH BI-CAR-NK

[0248] The BI-CAR obtained by screening in Example 5.1 was further constructed into a BI-CAR NK with a CD8 transmembrane region (the structure is shown in 1A of Figure 1) to verify its expression. The BI-CAR sequence is shown in Table 14. The BI-CAR NK expression detection method is the same as in Example 3.2, and the flow cytometry expression detection results of BI-CAR are shown in Table 15. The results showed that the expression of BCMA CAR and GPRC5D CAR could be detected after infection of NK cells at MOI = 5, with BCMA CAR expression reaching more than 80% and GPRC5D CAR expression reaching more than 50%.

[0249] Table 14 Dual VHH BI-CAR NK sequence information

[0250] Table 15 Dual VHH BI-CAR-NK structure expression detection

[0251] 5.3 Dual VHH BI-CAR-NK 4h Rapid Killing Rate Detection

[0252] On the 6th day after retroviral infection of NK cells, the BI-CAR NK cells prepared in Example 5.2 were subjected to a 4-h in vitro killing experiment: target cells NCI H929, RPMI 8226, MOLP8 and NCI H929-hBCMA-KO diluted in 1640 medium were inoculated at 2×10 450 μl / well of the solution was added to a white opaque 96-well plate, and NK cells were added to the target cells at effector-target ratios of 5:1, 2.5:1, and 1.25:1. The 96-well plate was cultured in a 37°C, 5% CO2 incubator. After 4 hours, 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added. After incubation at room temperature in the dark for 10 minutes, the killing rate was measured using a microplate reader and calculated.

[0253] The 4-hour in vitro cell killing effect of BI-CAR on the above target cells is detailed in Table 16. As shown in Table 16, all dual-VHH BI-CARs have good specific killing effects on tumor cells. Among them, BI-CAR07 showed a tumor cell killing effect comparable to BI-CAR01, and the tumor killing effects of humanized BI-CAR14 and BI-CAR15 were lower than those of non-humanized BI-CAR16 and BI-CAR17.

[0254] Table 16 Dual VHH BI-CAR-NK 4h rapid killing results Note: “-” means no killing effect was detected.

[0255] Example 6 NKG2D-F CAR constructed with humanized antibodies

[0256] 6.1 Construction of NKG2D-F CAR

[0257] The dual-target chimeric antigen receptor targeting BCMA and GPRC5D was designed and constructed, including the CD8α signal peptide (SP), anti-GPRC5D antibody and anti-BCMA, CD8 hinge region, NKG2D-F transmembrane region, 2B4 costimulatory domain and CD3ζ, and IL15 connected by the self-cleaving peptide P2A. The structures of BI-CAR18 and BI-CAR20 are shown in Figure 5A, and the structures of BI-CAR19 and BI-CAR21 are shown in Figure 5B. The sequences are shown in Table 17.

[0258] Table 17 NKG2D-F CAR amino acid sequence

[0259] 6.2 NKG2D-F CAR Expression Detection

[0260] Referring to Example 3.2, the expression of BI CARs 18 to 21 was detected. The results are shown in Table 18. The results show that the expression of BCMA CAR and GPRC5D CAR can be detected after infection of NK cells at an MOI of 5. The BCMA CAR positive rate is relatively high, reaching over 60%; the GPRC5D CAR positive rate is relatively low, with the expression rates from high to low being BI-CAR06 (69.60%) > BI-CAR21 (54.20%) > BI-CAR20 (54.00%) > BI-CAR18 (32.30%) > BI-CAR19 (13.80%).

[0261] Table 18 NKG2D-F CAR expression rate results

[0262] 6.3 NKG2D-F CAR-NK Cell Proliferation Assay

[0263] The proliferation of BI-CAR NK cells was detected by AO / PI counting, and the results are shown in Figure 6 and Table 19. On the 11th day after activation, BI-CAR NK cells proliferated by about 1473 to 2166 times, and the proliferation rates from high to low were: BI-CAR19 (2166 times) = BI-CAR20 (2166 times) > BI-CAR21 (1950 times) > Parental NK (1690 times) > BI-CAR18 (1538 times) > BI-CAR06 (1473 times). According to the above results, the proliferation rate of BI-CAR18, BI-CAR19, BI-CAR20 and BI-CAR21 cells constructed with humanized antibodies was better than that of BI-CAR06.

[0264] Table 19 NKG2D-F CAR-NK cell proliferation detection

[0265] 6.4 NKG2D-F CAR-NK cell killing effect detection in multiple rounds

[0266] Six days after retroviral infection of NK cells, the in vitro multi-round killing effects of BI-CAR18, BI-CAR19, BI-CAR20, BI-CAR21, and BI-CAR06 on heterogeneous tumor cells containing NCI H929, RPMI 8226, MOLP8, NCI H929-hBCMA-KO, and NCI H929+20% (or 10% or 5%) H929-hBCMA-KO were detected: (1) First round of killing: 2.5×10 5The target cells diluted with 1640 culture medium were placed in a 12-well plate at a concentration of 500 μl / well. NK cells were added to the 12-well plate at an effector-target ratio of 1:1 and cultured in a 37°C, 5% CO2 incubator for 24 hours. After culturing for 24 hours, 100 μl of the above mixed cells were added to a 96-well plate with an opaque white bottom, and 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added. After incubation at room temperature in the dark for 10 minutes, the fluorescence intensity was measured with a microplate reader to calculate the NK cell killing efficiency. After that, the next round of killing test was carried out. (2) Next round of killing: Take the cells in the 12-well plate in the previous round, count the NK cells, add the NK cells from the previous round to the 12-well plate inoculated with new target cells at an effector-target ratio of 1:1, repeat step (1), measure the NK cell killing rate and continue the next round of killing test.

[0267] The results of the multi-round killing test of BI-CAR NK cells are shown in Figures 7A-7F. The results show that the multi-round killing effects of BI-CAR19, BI-CAR20, and BI-CAR21, which are simultaneously loaded with the NKG2D TM element and the 2B4 costimulatory signaling domain, are better than those of BI-CAR18 and BI-CAR06. BI-CAR21 had the best killing effect on NCI H929 cells, maintaining a cell killing rate of 30% in the fourth round of killing (R4-1d) experiments; BI-CAR19, BI-CAR20 and BI-CAR21 had the best killing effect on NCI H929+20% (or 10% or 5%) H929-hBCMA-KO heterogeneous tumor cells, RPMI 8226 cells and MOLP8 cells, maintaining a cell killing rate of more than 30% in the fourth round of killing (R4-1d) experiments on NCI H929+20% (or 10% or 5%) H929-hBCMA-KO heterogeneous tumor cells, and maintaining a cell killing rate of about 10% in the fifth round of killing (R5-1d) experiments on RPMI 8226 cells and MOLP8 cells.

[0268] Example 7 Combination of CAR-NK targeting GPRC5D and BCMA with CBL-b inhibitors and / or PDL1-IL15

[0269] 7.1 Synthesis of CBL-b Inhibitors

[0270] Referring to patent WO2020210508A1, the CBL-b inhibitor shown below was synthesized.

[0271] 7.2 CBL-b inhibitors increase CAR positivity

[0272] BI-CAR21 was prepared with reference to Example 2, NK cells were transfected, and the CBL-b inhibitor prepared by Example 7.1 (final concentration 1 μM) was added during the activation and transfection of NK cells. Six days after retroviral transfection of NK cells, the CAR positivity of NK cells was detected with reference to Example 3.2. The results showed that the CAR positivity of NK cells increased after the addition of the CBL-b inhibitor. Exemplarily, as shown in Figure 8, the expression rate of BI-CAR21 was detected by BCMA, wherein the expression rate of BI-CAR increased from 80% to 92.5% after the addition of the BL-b inhibitor, compared with the control to which the CBL-B inhibitor was not added.

[0273] 7.3 CBL-b inhibitors and / or PDL1-IL15 enhance the killing effect of CAR-NK cells

[0274] Referring to Example 2, NK cells were transfected with retrovirus and cultured for 6 days. Referring to Example 6.4, the multi-round killing effect of CAR-NK (BI-CAR21) targeting GPRC5D and BCMA in combination with CBL-b inhibitor and / or PDL1-IL15 on NCI H929 + 10% NCI H929-hBCMA-KO cells was detected. The experimental design is shown in Table 20, wherein the CBL-b inhibitor and its addition method are shown in 7.2, and the PDL1-IL15 is added during the incubation of target cells and NK cells. The results of multiple rounds of killing are shown in Figure 9. Using CBL-b inhibitors to stimulate NK cells during culture or adding PDL1-IL15 fusion protein during CAR-NK cell killing can enhance the killing effect of BCMA-GPRC5D CAR-NK. The combination of CBL-b inhibitors and PDL1-IL15 can further enhance the killing effect of BCMA-GPRC5D CAR-NK. The G5 group still maintained a high target cell killing rate at R5-1d, which was significantly higher than that of the G3 and G4 groups.

[0275] Table 20 Experimental design Note: “ / ” means not added.

[0276] The sequence information of PDL1-IL15 is as follows:

[0277] Example 8 Antitumor efficacy test of CAR-NK targeting GPRC5D and BCMA in a mouse myeloma tumor model

[0278] 8.1 Preparation and In Vitro Activity Verification of BI-CAR06, BI-CAR19, BI-CAR20, and BI-CAR21

[0279] BI-CAR06, BI-CAR19, BI-CAR20, and BI-CAR21 were prepared for in vivo efficacy verification with reference to Example 2 and transfected into NK cells. Six days after retroviral transfection of NK cells, the CAR positivity of NK cells was detected with reference to Example 3.2. As shown in Figure 10A, the CAR positivity of NK cells was greater than 80% by BCMA detection. The CAR positivity of NK cells was greater than 70% by GPRC5D detection. As shown in Figures 10B and 10C, the CAR positivity of NK cells decreased by approximately 10% at 0 hour after cryopreservation and resuscitation. The viability of BI-CAR cells after resuscitation was detected by AO / PI counting. As shown in Figure 10D, the viability of BI-CAR cells was lowest 24 hours after cryopreservation and resuscitation, and the viability reached greater than 80% after 72 hours of cryopreservation and resuscitation. The above results indicate that BI-CAR has good stability.

[0280] 4h in vitro killing experiment of BI-CAR on NCI H929 cells before freezing and 72 hours after thawing: NCI H929 cells were selected as target cells, and all cells were genetically manipulated by conventional methods to contain luciferase reporter genes. Target cells diluted in 1640 medium were incubated at 2×10 4 50 μl / well of the solution was added to a white opaque 96-well plate, and NK cells were added to the target cells at effector-target ratios of 5:1, 2.5:1, and 1.25:1. The 96-well plate was cultured in a 37°C, 5% CO2 incubator. After 4 hours, 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added. After incubation at room temperature in the dark for 10 minutes, the killing rate was measured using a microplate reader and calculated.

[0281] The 4-hour in vitro cytotoxicity of BI-CAR against the aforementioned target cells is detailed in Figures 11A and 11B. BI-CAR demonstrated strong specific cytotoxicity against NCI H929 cells before cryopreservation. BI-CAR19 and BI-CAR06 demonstrated enhanced cytotoxicity against NCI H929 cells 72 hours after resuscitation.

[0282] Long-term in vitro killing experiment of CHOK1-hBCMA and CHOK1-hGPRC5D cells by BI-CAR at 0 hours after recovery: The experiment was performed according to the cell killing experiment operation steps of the xCELLigence RTCA instrument operation manual with a target effector ratio of 1:1. The target cells were CHOK1-hBCMA and CHOK1-hGPRC5D cells, mixed at a ratio of 1:1, and diluted to 2×10 in DMEM / F-12 medium. 4 BI-CAR NK cells were diluted to 2×10 cells / 50 μl / well with DMEM / F-12 medium. 4Cells were plated in 50 μl / well. When the cell index approached 0, the next round of cytotoxicity testing was initiated, and the cytotoxicity of BI-CAR NK cells was monitored in real time using an instrument. The long-term in vitro cytotoxicity of BI-CAR against the aforementioned target cells is detailed in Figure 11C. As shown, at 0 hours post-resuscitation, BI-CAR19 and BI-CAR06 exhibited enhanced cytotoxicity against CHOK1-hBCMA and CHOK1-hGPRC5D cells.

[0283] 8.2 Antitumor Efficacy Tests of BI-CAR06, BI-CAR19, BI-CAR20, and BI-CAR21 in the NCI H929 (Human Myeloma Cell) Tumor Model

[0284] In order to evaluate the anti-tumor efficacy of GPRC5D and BCMA dual-targeting CAR-NK cells BI-CAR06, BI-CAR19, BI-CAR20, and BI-CAR21, a myeloma intravenous transplant tumor model was used for anti-tumor efficacy testing. The details are as follows:

[0285] NCI H929-Luc cells in the logarithmic growth phase and in good growth condition were collected and 2×10 cells were inoculated into the tail vein of each NPG mouse (combined immunodeficient mouse). 6 Cells. One day after tumor inoculation, the mice were weighed and randomly grouped based on the random number principle. Mice weighing approximately 19.76 to 25.52 g were selected, with an average weight of 21.66 g. On the day of grouping, frozen-thawed CAR-NK cells (8 × 10 6 cells / cell), CAR-T cells (3×10 6 The injection volume was 200 μl / mouse, and the CAR-NK / T cell injection date was Day 0. The mouse groups and CAR-NK cell injection conditions are shown in Table 21. Tumor growth (fluorescence signal ROI value) was monitored using an IVIS in vivo imager, and the mice were weighed twice a week.

[0286] BCMA-CAR-T preparation: The nucleic acid encoding the chimeric antigen receptor for T cells was cloned into a retroviral shuttle plasmid by conventional molecular cloning technology and viral packaging was performed by packaging plasmid. T cells were isolated from fresh PBMC using the Stemcell Easy Sep Kit (Stemcell, Cat#19055). The isolated T cells were transferred to culture dishes pre-coated with 1 μg / ml CD3 / CD28 antibodies (MY-ebioscience-16-0289-85, MY-ebioscience-16-0037-85) for culture. The culture medium components were X-VIVO15 (Lonza, Cat#BEBP02-054Q), 5% human AB serum (Gemini, Cat#100-512), 100 U / ml penicillin-streptomycin (Gibco, Cat#15140-122) and 200 IU / ml human IL2 (Beijing Shuanglu, Cat#S19991007). Cell counts were performed twice a week. When the cell density reached 2.5×10 6 / ml when passage and expansion.

[0287] Take the above T cells in good condition and calculate 1×10 6 Cells were seeded into a 12-well cell culture plate pre-coated with 5 μg / ml recombinant human fibrin (Takara, Cat#T100B), 50 μl of concentrated lentivirus was added, and the cells were centrifuged at 1000 g for 1 hour at 4°C. After centrifugation, the cells were cultured at 37°C and 5% CO2. The cells were counted twice a week and the cells were counted when the cell density reached 2.5×10 6 The prepared CAR-T cells were cryopreserved for future use.

[0288] The BCMA-CAR-T sequence information is as follows:

[0289] Table 21. Grouping of in vivo anti-tumor experiments

[0290] The results of the fluorescent signal detection of mouse tumor growth are shown in Figure 12A. The results show that on the 22nd day after injection of CAR-NK cells, the BI-CAR06, BI-CAR19 and BI-CAR20 groups showed significant inhibition of tumor burden and effective control of tumor progression. In the three groups, 4, 2 and 3 animals had complete tumor elimination, respectively. The BI-CAR21 group had the most recurrences as the detection time prolonged. The BI-CAR21 treatment group was able to slow tumor growth but could not eliminate the tumor. After administration, the photon value of the mouse tumor was continuously measured. There was a significant difference between the CAR-NK treatment groups and the Parental NK group (P < 0.001***). In particular, the BI-CAR06, BI-CAR19 and BI-CAR20 treatment groups all had extremely significant therapeutic effects (as shown in Figure 12B).

[0291] During the experiment, the survival rate was monitored until 29 days. The mortality rates of the BI-CAR06, BI-CAR19, and BI-CAR21 groups were 20%, 40%, and 40%, respectively. After analysis, all of them died due to the rapid proliferation of NK cells and the heavy burden on the major organs. No animal deaths occurred in the other treatment groups (as shown in Figure 12C). At the same time, the weight of mice in the three groups treated with BI-CAR06, BI-CAR19, and BI-CAR21 showed a downward trend in the experiment due to the high proliferation of NK cells in some animals in the group, resulting in poor condition. Although the weight of mice in the other CAR-NK treatment groups fluctuated after administration, they showed an overall growth trend (as shown in Figure 12D).

[0292] 8.3 Antitumor efficacy studies of BI-CAR06, BI-CAR19, BI-CAR20, and BI-CAR21 in the Molp8 (human multiple myeloma) tumor model

[0293] In order to evaluate the anti-tumor efficacy advantages of the structure-optimized GPRC5D and BCMA dual-targeting CAR-NK cell, the Molp8 myeloma intravenous transplant tumor model was used to conduct anti-tumor efficacy tests. The details are as follows:

[0294] Molp8-Luc cells in the logarithmic growth phase and in good growth condition were collected and 5×10 cells were inoculated into the tail vein of each NPG mouse (combined immunodeficient mouse). 5 One day after tumor inoculation, the weight and tumor fluorescence signal of mice were measured. Mice weighing 21.81 to 27.25 g were randomly selected according to the random number principle, with an average weight of 23.98 g for random grouping. On the day of grouping, frozen-thawed CAR-NK cells (8 × 10 6 cells / cell), CAR-T cells (3×10 6The injection volume was 200 μl per mouse, and the CAR-NK / T cell injection date was Day 0. The mouse grouping and CAR-NK cell injection status are shown in Table 22. Tumor growth (fluorescence signal ROI value) was monitored using an IVIS in vivo imager. Mouse body weight was measured and recorded twice a week, and mouse survival rate was observed.

[0295] Table 22. Grouping of in vivo anti-tumor experiments

[0296] The results of the fluorescence signal detection of mouse tumor growth are shown in Figure 13A. The results show that on the 15th day after the injection of CAR-NK cells, the BI-CAR21+CBL / B inhibitor treatment group showed significant inhibition of tumor growth, and the tumors of 3 animals were completely cleared; on Day 15, the tumors of 2 animals in the BI-CAR06 group were completely cleared; the BI-CAR19, BI-CAR20 and BI-CAR21 treatment groups were able to significantly inhibit the growth rate of mouse tumors, but could not completely clear the tumors, as shown by the recurrence and growth of all animal tumors. After administration, the photon value of the mouse tumor was continuously measured. Compared with the Parental NK group, the photon value of each CAR NK treatment group showed a significant decrease (as shown in Figure 13B).

[0297] During the experiment, the survival rate was monitored until day 29. The mortality rates of the BI-CAR06, BI-CAR19, BI-CAR21, and Parental NK groups were 80%, 20%, 60%, and 100%, respectively. As mentioned above, these deaths were caused by the rapid proliferation and accumulation of NK cells in major organs, which led to heavy burdens. No animals died in the other treatment groups (as shown in Figure 13C). At the same time, the body weights of mice in the BI-CAR06, BI-CAR19, and BI-CAR21 treatment groups showed a downward trend during the experiment. This was due to the poor condition of some animals in the groups due to high NK cell proliferation, while the Parental NK group lost weight due to the onset of the model (as shown in Figure 13D).

[0298] 8.4 Antitumor Efficacy of BI-CAR06 in NCI H929 and NCI H929-hBCMA-KO Mixed Tumor Models

[0299] 8.4.1 Preparation and In Vitro Characterization of BCMA and GPRC5D Single-Targeting CAR-NK Cells

[0300] To compare the efficacy of single-targeting BCMA and GPRC5D versus simultaneous BCMA-GPRC5D targeting, a single-targeting CAR-NK cell, designated mono-B-CAR, was constructed using the BCMA-Hab02-VHH7 sequence. A single-targeting CAR-NK cell, designated mono-G-CAR, was constructed using the GPRC5D-Hab06 humanized antibody with an N-terminal-C-terminal VL-VH scFv. Both single-targeting CARs utilize the NKG2D transmembrane region and the 2B4 intracellular domain. The structures are shown in Figure 14 , and the sequences are listed in Table 23 .

[0301] Table 23. Single-target CAR sequences

[0302] Referring to the method shown in Example 2, NK cells were infected with the same MOI to prepare BCMA single-targeted mono-B-CAR and GPRC5D single-targeted mono-G-CAR and BCMA and GPRC5D dual-targeted BI-CAR06-NK cells, and the expression of CAR was detected by the method in Reference Example 3.2. The results are shown in Table 24. The results showed that after NK cells were infected with MOI = 5, BI-CAR06 could detect the expression of BCMA CAR (90.3%) and GPRC5D CAR (96.3%), and CAR expression was comparable to that of single-targeted mono-B-CAR and mono-G-CAR.

[0303] Table 24. CAR expression detection

[0304] On the 6th day after retroviral infection of NK cells, the prepared mono-B-CAR, mono-G-CAR and BI-CAR06 NK cells were subjected to a 4-h in vitro killing experiment: target cells NCI H929-Lu and NCI H929-hBCMA-KO-Lu carrying luciferase reporter genes were diluted in 1640 medium and incubated at 2×10 4 Add 50 μl of the solution per well to a white opaque 96-well plate, add NK cells to the target cells at effector-target ratios of 5:1, 2.5:1, and 1.25:1, and incubate the 96-well plate in a 37°C, 5% CO2 incubator. After 4 hours, add 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1), incubate at room temperature in the dark for 10 minutes, and then measure and calculate the killing rate using a microplate reader (the killing rate calculation formula is the same as in Section 4.4).

[0305] The 4h in vitro cell killing effects of mono-B-CAR, mono-G-CAR and BI-CAR06NK cells on the above target cells are shown in Table 25. As shown in Table 25, mono-B-CAR, mono-G-CAR and BI-CAR06NK cells all have good specific killing effects on NCI H929 tumor cells, mono-G-CAR and BI-CAR06NK cells all have good specific killing effects on NCI H929-hBCMA-KO-Lu tumor cells, and the killing effect of mono-B-CAR on NCI H929-hBCMA-KO-Lu tumor cells is only comparable to that of Parental NK.

[0306] Table 25. 4h rapid killing experiment

[0307] 8.4.2 Verification of the Antitumor Efficacy of CAR-NK Cells Simultaneously Targeting BCMA and GPRC5D in a Mix H929-lu Tumor Xenograft Model

[0308] In order to evaluate the anti-tumor efficacy of CAR-NK cells mono-B-CARNK, mono-G-CARNK and BI-CAR06, an anti-tumor efficacy test was conducted using a mouse myeloma intravenous transplantation model. The details are as follows:

[0309] 50% of H929-Luc cells and 50% of H929-Luc (BCMA-KO) cells in the logarithmic growth phase and in good growth condition were collected and mixed, and 2×10 cells were inoculated into the tail vein of each NPG mouse (combined immunodeficient mouse). 6 cells. One day after tumor inoculation, the mice were weighed and randomly grouped based on the random number principle. Mice weighing 21.57 to 27.47 g were selected, with an average weight of 24.37 g. On the day of grouping, freshly prepared CAR-NK cells (5 × 10 6 The injection volume was 200 μl / mouse. The CAR-NK cell injection date was Day 0. Mouse grouping and CAR-NK cell injection status are shown in Table 26. Tumor growth fluorescence signal ROI value was monitored using an IVIS in vivo imager. Mouse weight was weighed and recorded twice weekly.

[0310] Table 26. Grouping of in vivo anti-tumor experiments

[0311] Referring to the method shown in Example 2, NK cells were infected with the same MOI to prepare BCMA single-targeted mono-B-CAR and GPRC5D single-targeted mono-G-CAR and BCMA and GPRC5D dual-targeted BI-CAR06 cells, and the expression of CAR was detected by the method in Reference Example 3.2. The results are shown in Table 27. The results showed that after NK cells were infected with MOI = 5, BI-CAR06 could detect the expression of BCMA CAR (90.3%) and GPRC5D CAR (96.3%), and CAR expression was comparable to that of single-targeted mono-B-CAR and mono-G-CAR.

[0312] Table 27. CAR expression detection

[0313] On the 6th day after retroviral infection of NK cells, the prepared mono-B-CAR, mono-G-CAR and BI-CAR06 NK cells were subjected to a 4-h in vitro killing experiment: the target cells NCI H929 and NCI H929-hBCMA-KO diluted in 1640 medium were inoculated with 2×10 4 50 μl / well of the solution was added to a white opaque 96-well plate, and NK cells were added to the target cells at effector-target ratios of 10:1, 5:1, 2.5:1, and 1.25:1. The 96-well plate was cultured in a 37°C, 5% CO2 incubator. After 4 hours, 30 μl of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1) was added. After incubation at room temperature in the dark for 10 minutes, the killing rate was measured using a microplate reader and calculated.

[0314] The 4-hour in vitro cell killing effects of mono-B-CAR, mono-G-CAR and BI-CAR06 NK cells on the above target cells are detailed in Table 28. As shown in Table 28, mono-B-CAR, mono-G-CAR and BI-CAR06 NK cells all have good specific killing effects on NCI H929 tumor cells, with mono-B-CAR and mono-G-CAR slightly better than BI-CAR06; mono-G-CAR and BI-CAR06 NK cells all have good specific killing effects on NCI H929-hBCMA-KO tumor cells, and the killing effect of mono-G-CAR on NCI H929-hBCMA-KO tumor cells is only comparable to that of Parental NK.

[0315] Table 28.4hr rapid killing experiment

[0316] The results of the fluorescence signal detection of mouse tumor growth are shown in Figure 15A. The results show that on the 60th day after the injection of CAR-NK cells, the mono-B-CAR and mono-G-CAR treatment groups were able to inhibit the growth of mouse tumors, but neither group could eliminate the tumors, as shown by the recurrence of all animal tumors. Compared with the BI-CAR06 treatment group, the tumor growth was significantly inhibited and the progression of the tumor was effectively controlled. At 60 days, only 2 animals had recurrence of tumors, and the remaining 3 were completely eliminated. After administration, the photon quantity of mouse tumors was continuously measured. There was a significant difference between the CARNK treatment groups and the Parental NK group, P < 0.001***, especially the CAR NK_BI-CAR06 group, which was significantly better than the therapeutic effect of single-target CAR NK (as shown in Figure 15B).

[0317] During the experiment, the survival rate was continuously monitored until 96 days. In the PBS group, one animal died starting from day 53, and the mortality rate was 100% by day 96. In the mono-B-CAR group, one animal died on day 61, and the mortality rate was 40% by day 96. In the mono-G-CAR group, one animal died on day 69, and the mortality rate was 20% by day 96. No animal died in the BI-CAR06 group (as shown in Figure 15C). At the same time, none of the three groups of mice treated with CARNK showed any weight loss after administration, and the overall weight showed an upward trend. Only the PBS group continued to lose weight after 40 days due to the occurrence of the model disease (as shown in Figure 15D).

[0318] 8.5 Anti-tumor efficacy study of BI-CAR06 combined with CBL / B inhibitor and PD-L1 / IL-15 antibody in the NCI H929 tumor model

[0319] In order to verify the anti-tumor therapeutic advantages of the preferred dual-target CAR-NK cell BI-CAR06 combined with CBL / B inhibitors and PD-L1 / IL-15 antibody drugs, an anti-tumor efficacy test was conducted using a mouse myeloma intravenous transplant tumor model. The details are as follows:

[0320] H929-Luc cells in the logarithmic growth phase and in good growth condition were collected and 2×10 cells were inoculated into the tail vein of each NPG mouse (combined immunodeficient mouse). 6 Cells. One day after tumor inoculation, the mice were weighed and randomly grouped based on the random number principle. Mice weighing approximately 21.03 to 27.65 g were selected, with an average weight of 24.42 g. On the day of grouping, frozen-thawed CAR-NK cells (6×10 6 cells / cell), CAR-T cells (3×10 6The injection volume was 200 μl / mouse, and the CAR-NK / T cell injection date was Day 0. The mouse groups and CAR-NK / T cell injection conditions are shown in Table 29. Tumor growth (fluorescence signal ROI value) was monitored using an IVIS in vivo imager.

[0321] Table 29. Grouping of in vivo anti-tumor experiments

[0322] The results of fluorescent signal detection of mouse tumor growth are shown in Figure 16A. The results show that on the 27th day after the injection of CAR-NK cells, the BI-CAR06+PD-L1 / IL-15+CBL / B inhibitor combined treatment group showed a significant effect in controlling tumors. The tumors of all four animals in the experiment were cleared, and the effect of inhibiting tumor growth was better than that of the BI-CAR06, BI-CAR06+PDL1 / IL15 and BI-CAR06+CBL / B inhibitor treatment groups.

[0323] After drug administration, the photon value of mouse tumors was continuously measured. The CAR NK treatment group and the CAR T treatment group showed significant differences compared with the Parental NK group (P<0.001***). In particular, the CAR NK_BI-CAR06+PD-L1 / IL-15+CBL / B inhibitor combination group showed an extremely significant therapeutic effect (as shown in Figure 16B).

[0324] During the experiment, the survival rate was monitored until 42 days. The mortality rates of the BI-CAR06 and BI-CAR06+PD-L1 / IL-15+CBL / B inhibitor combination treatment groups were 75% and 25%, respectively. Analysis showed that this was due to the rapid proliferation and accumulation of NK cells in major organs, which led to high burden and death. The mortality rates of the Parental NK group, the PD-L1 / IL-15 group, and the CBL / B inhibitor group were 25%, 25%, and 75%, respectively. According to in vivo imaging and clinical observations, the deaths of animals in these three groups were caused by tumor recurrence. No animals died in the other treatment groups (as shown in Figure 16C). The weight of animals in the BI-CAR06 group continued to decrease due to the high proliferation of NK cells in the animals in this group, resulting in poor condition. Although there were fluctuations in the weight of mice in the other treatment groups after administration, they showed an overall growth trend (as shown in Figure 16D).

Claims

1. A chimeric antigen receptor (CAR), comprising a first polypeptide, wherein: The first polypeptide comprises, from N-terminus to C-terminus, an extracellular antigen binding domain, a hinge region, a transmembrane domain and at least one signal transduction domain, wherein the extracellular antigen binding region includes a first antigen binding domain that specifically binds to GPRC5D.

2. The chimeric antigen receptor according to claim 1, the extracellular antigen-binding domain of the first polypeptide further comprises a second antigen-binding domain that specifically binds to BCMA; optionally, the extracellular antigen-binding domain comprises, from N-terminus to C-terminus, a first antigen-binding domain-linker-second antigen-binding domain, or a second antigen-binding domain-linker-first antigen-binding domain; preferably, the linker is GGGGSGGGGSGGGGSGGGGS.

3. The chimeric antigen receptor according to claim 1, wherein It also includes a second polypeptide, which comprises, from N-terminus to C-terminus: an extracellular antigen binding domain, a hinge region, a transmembrane domain and at least one signal transduction domain, wherein the extracellular antigen binding domain includes a second antigen binding domain that specifically binds to BCMA.

4. The chimeric antigen receptor according to claim 3, wherein The first polypeptide and the second polypeptide are operably linked; optionally, the second polypeptide is operably linked to the N-terminus of the first polypeptide, or the first polypeptide is operably linked to the N-terminus of the second polypeptide; preferably, the first polypeptide and the second polypeptide are operably linked via a self-cleaving peptide; more preferably, after cleavage of the self-cleaving peptide, the first polypeptide and the second polypeptide form a dimer.

5. The chimeric antigen receptor according to any one of claims 1 to 4, wherein In the first polypeptide and / or the second polypeptide: (a) The hinge region contains the CD8 hinge region; (b) The transmembrane domain contains the NKG2D transmembrane region; (c) at least one signaling domain comprises a natural killer cell receptor 2B4 costimulatory domain and / or CD3ζ.

6. The chimeric antigen receptor according to claim 5, wherein The hinge region comprises the sequence shown in SEQ ID NO:

53.

7. The chimeric antigen receptor according to claim 5, wherein The transmembrane domain comprises the sequence shown in SEQ ID NO:

64.

8. The chimeric antigen receptor according to claim 5, wherein The signaling domain comprises the 2B4 costimulatory domain of SEQ ID NO: 65 and / or the CD3 zeta of SEQ ID NO:

56.

9. The chimeric antigen receptor according to any one of claims 1 to 4, wherein In the first polypeptide and / or the second polypeptide: (a) The hinge region contains the CD8 hinge region; (b) the transmembrane domain contains the CD8 transmembrane region; (c) at least one signaling domain comprises a CD137 costimulatory domain and / or CD3ζ.

10. The chimeric antigen receptor according to claim 9, wherein The hinge region comprises the sequence shown in SEQ ID NO:

53.

11. The chimeric antigen receptor according to claim 9, wherein The transmembrane domain comprises the sequence shown in SEQ ID NO:

54.

12. The chimeric antigen receptor according to claim 9, wherein The signaling domain comprises the CD137 costimulatory domain of SEQ ID NO: 55 and / or the CD3 zeta of SEQ ID NO:

56.

13. The chimeric antigen receptor according to any one of claims 1 to 12, wherein The first antigen binding domain comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region (VL) and HCDR1, HCDR2 and HCDR3 of the heavy chain variable region (VH), wherein the light chain variable region comprises the LCDRs of the light chain variable region as shown in SEQ ID NO: 9, 11, 13 or 15; and the heavy chain variable region comprises the HCDRs of the heavy chain variable region as shown in SEQ ID NO: 10, 12, 14 or 16.

14. The chimeric antigen receptor according to any one of claims 1 to 13, wherein The first antigen binding domain comprises the following combination of LCDRs and HCDRs defined by the Kabat numbering system: (1) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NOs: 17, 18 and 19, and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NOs: 20, 21 and 22; (2) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NOs: 17, 18 and 19, and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NOs: 23, 24 and 25; (3) LCDR1, LCDR2 and LCDR3 of the sequences shown in SEQ ID NOs: 26, 27 and 28 and HCDR1, HCDR2 and HCDR3 of the sequences shown in SEQ ID NOs: 29, 30 and 31; or (4) A combination of LCDRs and HCDRs having 5, 4, 3, 2 or 1 amino acid substitutions, deletions or mutations compared to LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3 in any one of (1) to (3).

15. The chimeric antigen receptor according to any one of claims 1 to 14, wherein the first antigen binding domain comprises a VL as shown in SEQ ID NO: 9, 11, 13 or 15 and a VH as shown in SEQ ID NO: 10, 12, 14 or 16; Preferably, the following VL and VH combinations are present: (1) VL shown in SEQ ID NO: 9 and VH shown in SEQ ID NO: 10; (2) VL shown in SEQ ID NO: 11 and VH shown in SEQ ID NO: 12; (3) VL shown in SEQ ID NO: 13 and VH shown in SEQ ID NO: 14; (4) VL shown in SEQ ID NO: 15 and VH shown in SEQ ID NO: 16; or (5) A sequence having 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to any one of (1) to (4).

16. The chimeric antigen receptor according to any one of claims 13 to 15, wherein The VL and VH are connected by a connecting peptide rich in glycine and serine, and the connecting peptide is GGGGSGGGGSGGGGS.

17. The chimeric antigen receptor according to any one of claims 1 to 12, wherein The first antigen-binding domain comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise the HCDR1, HCDR2, and HCDR3 of the VHH sequence shown in any one of SEQ ID NOs: 32 to 39; Preferably, the HCDR1, HCDR2 and HCDR3 are selected from the following combinations: (a) the sequences shown in SEQ ID NOs: 40, 41 and 42 as defined by the Kabat numbering system; (b) the sequences set forth in SEQ ID NOs: 43, 44, and 45 as defined by the IMGT numbering system; (c) the sequences represented by SEQ ID NOs: 46, 47, and 48 as defined by the IMGT numbering system; (d) the sequences represented by SEQ ID NOs: 49, 50 and 51 as defined by the Kabat numbering system; or (e) an amino acid sequence having one, two or three mutations in HCDR1, HCDR2 and / or HCDR3 as described in any one of (a) to (d); the mutations are selected from insertions, deletions and / or substitutions, and the substitutions are preferably substitutions of conservative amino acids.

18. The chimeric antigen receptor according to any one of claims 1 to 12 and claim 17, wherein The first antigen binding domain comprises a sequence as shown in any one of SEQ ID NOs: 32 to 39, or a sequence having 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to any one of SEQ ID NOs: 32 to 39.

19. The chimeric antigen receptor according to any one of claims 1 to 12, wherein The second antigen-binding domain comprises CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise the HCDR1, HCDR2, and HCDR3 of the VHH sequence shown in any one of SEQ ID NOs: 1-2; Preferably, the HCDR1, HCDR2 and HCDR3 are selected from the following combinations: (a) the sequences shown in SEQ ID NOs: 3, 4 and 5 as defined by the Kabat numbering system; (b) the sequences shown in SEQ ID NOs: 6, 7 and 8 as defined by the Kabat numbering system; or (c) an amino acid sequence having one, two or three mutations in HCDR1, HCDR2 and / or HCDR3 as described in any one of (a) to (b); wherein the mutation is selected from insertion, deletion and / or substitution, and the substitution is preferably a conservative amino acid substitution.

20. The chimeric antigen receptor according to any one of claims 1 to 12 and claim 19, wherein The second antigen binding domain comprises a sequence as shown in any one of SEQ ID NOs: 1 to 2, or a sequence having 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with any one of SEQ ID NOs: 1 to 2.

21. The chimeric antigen receptor according to any one of claims 1 to 20, wherein The first and second antigen binding domains are humanized antibodies.

22. The chimeric antigen receptor according to any one of claims 1 to 21, wherein the C-terminus of the chimeric antigen receptor further comprises an operably linked exogenous cytokine, wherein the exogenous cytokine is selected from IL2, IL4, IL6, IL7, IL10, IL11, IL12, IL15, IL18 and IL21; preferably, the cytokine is selected from IL-15.

23. The chimeric antigen receptor according to claim 22, wherein the C-terminus of the chimeric antigen receptor is operably linked to the cytokine via a self-cleaving peptide, and the self-cleaving peptide comprises a 2A peptide; more preferably, the self-cleaving peptide comprises a T2A peptide or a P2A peptide.

24. The chimeric antigen receptor of any one of claims 1 to 23, wherein the chimeric antigen receptor has a sequence as shown in any one of SEQ ID NOs: 59 to 63 and 66 to 77, or a sequence having 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to a sequence as shown in any one of SEQ ID NOs: 59 to 63 and 66 to 77.

25. An isolated nucleic acid construct encoding the chimeric antigen receptor of any one of claims 1 to 24. A vector comprising the nucleic acid construct according to claim 25 .

27. An immune effector cell comprising an expressed chimeric antigen receptor according to any one of claims 1 to 24.

28. The immune effector cell according to claim 27, wherein The immune effector cells are selected from T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells; preferably, the immune effector cells are NK cells.

29. A method for preparing the immune effector cell of claim 27 or 28, comprising introducing the nucleic acid construct of claim 25 or the vector of claim 26 into the immune effector cell; further, the method may further comprise the step of inducing expression of the chimeric antigen receptor.

30. A pharmaceutical composition comprising the chimeric antigen receptor of any one of claims 1 to 24, the nucleic acid construct of claim 25, the vector of claim 26, or the immune effector cell of claim 27 or 28; further comprising a pharmaceutically acceptable carrier.

31. A method for treating a tumor or cancer, wherein: The method comprises administering to a patient an effective amount of the chimeric antigen receptor according to any one of claims 1 to 24, the nucleic acid construct according to claim 25, the vector according to claim 26, the immune effector cell according to claim 27 or 28, or the pharmaceutical composition according to claim 30; preferably, the tumor or cancer is selected from B-cell lymphoma; more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.

32. Use of the chimeric antigen receptor according to any one of claims 1 to 24, the nucleic acid construct according to claim 25, the vector according to claim 26, the immune effector cell according to claim 27 or 28, or the pharmaceutical composition according to claim 30 in the preparation of a medicament for treating tumors or cancer, wherein: The tumor or cancer is selected from B cell lymphoma; preferably, the B cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.

33. Use of immune effector cells and CBL-b inhibitors, or immune effector cells and PDL1-IL15 fusion protein, or immune effector cells, CBL-b inhibitors and PDL1-IL15 fusion protein in the preparation of a drug for treating tumors or cancer, wherein: The immune effector cells express chimeric antigen receptors.

34. The use according to claim 33, wherein: The chimeric antigen receptor is the chimeric antigen receptor according to any one of claims 1 to 24.

35. The use according to any one of claims 33-34, wherein The immune effector cells are selected from T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells; preferably, the immune effector cells are NK cells.

36. The use according to any one of claims 33 to 35, wherein The CBL-b inhibitor is a chemical compound represented by formula (I) or a pharmaceutically acceptable salt of the compound represented by formula (I):

37. The use according to any one of claims 33 to 36, wherein The PDL1-IL15 fusion protein has the sequence shown in SEQ ID NO:

77.

38. The use according to any one of claims 33 to 37, wherein The tumor or cancer is selected from B-cell lymphoma; more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.

39. A method for treating cancer or tumor, wherein: The method comprises administering to a subject an effective amount of immune effector cells expressing a chimeric antigen receptor, and further comprises: (1) stimulating the immune effector cells with a CBL-b inhibitor prior to administration, and / or (2) administering to the subject an effective amount of a PDL1-IL15 fusion protein.

40. The method of claim 39, wherein The chimeric antigen receptor is the chimeric antigen receptor according to any one of claims 1 to 24.

41. The method according to any one of claims 39-40, wherein The immune effector cells are selected from T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages and dendritic cells; preferably, the immune effector cells are NK cells.

42. The method according to any one of claims 39 to 41, wherein The CBL-b inhibitor is a chemical compound represented by formula (I) or a pharmaceutically acceptable salt of the compound represented by formula (I):

43. The method according to any one of claims 39 to 42, wherein: The PDL1-IL15 fusion protein has the sequence shown in SEQ ID NO:

77.

44. The method according to any one of claims 39 to 43, wherein The tumor or cancer is selected from B-cell lymphoma; more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.

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