WO2023193662A1

WO2023193662A1 - Chimeric antigen receptor targeting bcma, and application thereof

Info

Publication number: WO2023193662A1
Application number: PCT/CN2023/085504
Authority: WO
Inventors: 杨翠青; 王义芳; 姜福伟; 王超; 王庆杨; 曹卓晓
Original assignee: 上海先博生物科技有限公司
Priority date: 2022-04-03
Filing date: 2023-03-31
Publication date: 2023-10-12

Abstract

The present invention relates to an enhanced chimeric antigen receptor targeting BCMA, and to an application thereof, and comprises: a nucleic acid molecule for encoding a chimeric antigen receptor containing an NKG2D transmembrane domain, a corresponding chimeric antigen receptor, a vector, an immune effector cell, a preparation method and a product, a pharmaceutical composition, a pharmaceutical application and a tumor or cancer treatment method.

Description

Chimeric antigen receptor targeting BCMA and its application

Cross-references to related patent applications

This application is required to be submitted to the State Intellectual Property Office of China on April 3, 2022. The patent application number is 202210347351.4, the invention is titled "Enhanced Chimeric Antigen Receptor and its Application", and it is submitted to the Chinese National Intellectual Property Office on March 24, 2023. The patent application number submitted by the Intellectual Property Office is 202310296244.8, and the invention title is "Enhanced Chimeric Antigen Receptor and its Application". The entire contents of the above-mentioned prior applications are incorporated into this application by reference.

Technical field

The present disclosure belongs to the field of biotechnology, and specifically relates to chimeric antigen receptors targeting BCMA and their applications in treating cancer.

Background technique

B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily and is mainly expressed on the surface of terminally differentiated B cells. B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) are the main ligands of BCMA. They interact with BCMA to transmit cell stimulation signals, activate the TRAF-dependent NF-κB and JNK pathways, and increase the proliferation and survival of B cells. Rate. BCMA expression has been associated with a variety of cancers, autoimmune disorders, and infectious diseases. Cancers with increased BCMA expression include blood cancers such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. Anti-BCMA CAR T has been used to treat multiple myeloma. However, due to the heterogeneity of tumors and the downregulation of BCMA expression during treatment, some patients still have the possibility of recurrence after receiving Anti-BCMA CAR T cell therapy.

Natural killer cells (NK cells) are a type of lymphocytes that have a powerful killing effect on tumor cells and are MHC-independent. Their recognition of tumor cells mainly relies on the cross-regulation of their surface activating receptors and inhibitory receptors. After recognizing tumor cells, NK cells kill tumor cells through multiple channels such as releasing the killing mediators perforin and granzymes to cause target cell apoptosis, expressing membrane TNF family molecules to induce target cell apoptosis, and antibody-dependent cytotoxicity. Allogeneic NK cell transplantation will hardly induce graft-versus-host disease (GVHD), will not lead to CRS, and can be used as a completely "off-the-shelf" product. However, due to the decline in the number and quality of NK cells in tumor patients and the existence of tumor escape mechanisms, their anti-tumor functions in the body have not been fully exerted. Modifying NK cells with CAR is expected to enhance their ability to target and kill tumor cells and develop effector cells with powerful anti-tumor effects.

The structure of the functional chimeric antigen receptor (CAR) expressed by NK cells mainly includes: extracellular domain, transmembrane region and intracellular signaling domain. The selection of corresponding elements in the CAR structure, such as the transmembrane region and intracellular activation signal domain, affects the activity of CAR-NK cells. At present, most CAR-NK products still use the CAR structure commonly used in traditional CAR-T cell therapy. The intracellular co-activation signaling domain is CD28 or 4-1BB. Since the signaling pathways of NK cell activation and T cell activation are different, traditional Commonly used CARs for CAR-T cell therapy The structure may not necessarily function optimally in CAR-NK.

Therefore, in view of the problems of long preparation time, large patient burden and large number of tumor recurrences in the clinical application of Anti-BCMA CAR T, the effectiveness of Anti-BCMA CAR NK cell therapy on tumors can be improved by screening Anti-BCMA antibodies and optimizing the CAR structure. Overall therapeutic effect.

Contents of the invention

B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily and is mainly expressed on the surface of terminally differentiated B cells. B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) are the main ligands of BCMA. They interact with BCMA to transmit cell stimulation signals, activate the TRAF-dependent NF-κB and JNK pathways, and increase the proliferation and survival of B cells. Rate. BCMA expression has been associated with a variety of cancers, autoimmune disorders, and infectious diseases. Cancers with increased BCMA expression include blood cancers such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. BCMA is an ideal therapeutic target, and it is particularly important to develop corresponding chimeric antigen receptors for this target, especially chimeric antigen receptors suitable for NK cells. At the same time, this disclosure designs a CAR structure that is most suitable for CAR-NK cells to exert activity based on the unique signaling pathways activated by NK cells.

In view of this, the present disclosure provides nucleic acid molecules encoding chimeric antigen receptors targeting BCMA, corresponding chimeric antigen receptors, vectors, immune effector cells, preparation methods and products, pharmaceutical compositions, pharmaceutical uses and tumors or In a cancer treatment method, the chimeric antigen receptor targeting BCMA includes an anti-BCMA VHH antibody that binds two different epitopes, thereby enhancing the targeting of BCMA-positive tumor cells.

In a first aspect, the present disclosure provides a nucleic acid molecule, wherein the nucleic acid molecule comprises a first nucleic acid sequence encoding a chimeric anti-receptor targeting BCMA, the chimeric antigen receptor comprising an extracellular region comprising an antigen-binding region , a transmembrane region connected to the extracellular region and an intracellular domain connected to the transmembrane region, and the antigen-binding region includes:

(1)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; or,

Sequences as shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:13; or,

Sequences as shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18;

(2) VHH, including a sequence with at least 80% identity or up to 25 mutations to SEQ ID NO: 1 or 3;

(3)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21; or,

Sequences as shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:21; or,

Sequences as shown in SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26; or,

Sequences as shown in SEQ ID NO:19, SEQ ID NO:27 and SEQ ID NO:21;

(4) A sequence that is at least 80% identical or has at most 25 mutations to SEQ ID NO: 2 or 5; or,

(5)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30; or,

Sequences as shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:30; or,

Sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35;

(6) A sequence with at least 80% identity or up to 25 mutations to SEQ ID NO:3; or

(7) VHH1-linking peptide-VHH2, the VHH1 has the sequence shown in the group (1) or the group (2), and the VHH2 has the sequence shown in the group (3) or the group (4); or,

(8) VHH1-linking peptide-VHH2, said VHH1 has a sequence shown in the group (3) or group (4) and said VHH2 has a sequence shown in the group (1) or group (2); or,

(9) VHH1-linking peptide-VHH2, the VHH1 has a sequence shown in the group (1) or the group (2) and the VHH2 has a sequence shown in the group (5) or the group (6).

In some specific embodiments, the chimeric antigen receptor includes: CD8α hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3ζ intracellular signaling domain. For example, the chimeric antigen receptor has A sequence that is at least 80% identical or has at most 50 mutations compared to SEQ ID NO:52.

In some specific embodiments, the nucleic acid molecule further includes a second nucleic acid sequence encoding IL15. Alternatively, the IL15 is selected from the group consisting of soluble IL15, membrane-bound IL15, or a complex of IL15 and its receptor or receptor fragment. , optionally, the IL15 has a sequence with at least 80% identity or at most 35 mutations compared with SEQ ID NO:51;

Optionally, the first nucleic acid sequence and the second nucleic acid sequence are connected by an IRES or a sequence encoding a self-cleaving peptide selected from 2A peptides, such as P2A, T2A, F2A or E2A, optionally , the self-cleaving peptide is P2A, for example, having a sequence with at least 80% identity or at most 5 mutations compared to SEQ ID NO:50;

Optionally, the core encodes a nucleic acid sequence having at least 85% identity or at most 100 mutations to a sequence compared to SEQ ID NO: 53.

In some specific embodiments, it includes a nucleic acid sequence encoding a sequence having at least 80% identity or up to 150 mutations compared to SEQ ID NO: 57 or SEQ ID NO: 63.

In a second aspect, the present disclosure provides a nucleic acid molecule comprising a first nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: an extracellular region comprising an antigen-binding region, An NKG2D transmembrane region connecting the extracellular region and an intracellular domain connecting the transmembrane region, the NKG2D transmembrane region having at least 80% identity or at most 9 mutations compared to SEQ ID NO: 41 sequence.

In some specific embodiments, the antigen-binding region has at least one of the properties of (1)-(4):

(1) The antigen-binding region is selected from an antibody or a fragment thereof or a ligand that binds the antigen, such as scFv, VHH, Fab, F(ab)' ₂ or ligand;

(2) The antigen-binding region binds or does not bind to NKG2DL;

(3) The antigen is selected from one or more of the following group: BCMA, GPRC5D, CLDN18.2, ROR1, CD19, CD33, CD5, CD70, HER2, IL13Rα2, GCC or GPC3;

(4) The antigen-binding region binds at least two different antigens, at least two different epitopes of the same antigen, or the number of the same epitope is more than two.

In some specific embodiments, the antigen-binding region specifically binds BCMA, including:

(1)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; or,

Sequences as shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:13; or,

Sequences as shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18;

(3)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21; or,

Sequences as shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:21; or,

Sequences as shown in SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26; or,

Sequences as shown in SEQ ID NO:19, SEQ ID NO:27 and SEQ ID NO:21;

(4) A sequence with at least 80% identity or at most 25 mutations with SEQ ID NO: 2 or 5;

(5)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30; or,

Sequences as shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:30; or,

Sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35;

In some specific embodiments, the extracellular region further includes a hinge region selected from one or more of the following group: CD8α hinge region, 2B4 hinge region, CD28 hinge region, IgG1 hinge region, IgD Hinge, IgG4 hinge region, GS hinge, KIR2DS2 hinge, KIR hinge, NCR hinge, SLAMF hinge, CD16 hinge, CD64 hinge or LY49 hinge; optionally, the hinge region is selected from the CD8α hinge region, for example, the hinge region Sequences with at least 80% identity or up to 10 mutations compared to SEQ ID NO:38.

In some specific embodiments, the extracellular region further includes a signal peptide selected from one or more of the following group: CD8α signal peptide, IgG1 heavy chain signal peptide or GM-CSFR2 signal peptide; Optionally, the signal peptide is a CD8α signal peptide, for example, the signal peptide has a sequence of at least 80% identity or at most 5 mutations compared to SEQ ID NO:36.

In some specific embodiments, the intracellular domain includes an intracellular signaling domain and/or a costimulatory domain;

Alternatively, the intracellular signaling domain is CD3ζ, for example, the intracellular signaling domain has a sequence of at least 80% identity or at most 35 mutations compared to SEQ ID NO: 49;

Optionally, the costimulatory domain is selected from one or more of the following group: CD27 costimulatory domain, 4-1BB costimulatory domain, OX40 costimulatory domain, 2B4 costimulatory domain, CD28 costimulatory domain Structural domain, ICOS costimulatory node domain, a DAP10 costimulatory domain or a DAP12 costimulatory domain; optionally, the costimulatory domain is a 2B4 costimulatory domain, for example, the costimulatory domain has at least 80% identity or up to 25 mutated sequences.

In some specific embodiments, the chimeric antigen receptor includes: CD8α hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3ζ intracellular signaling domain, for example, having the same structure as SEQ ID NO:52 Than sequences with at least 80% identity or at most 50 mutations.

Optionally, the nucleic acid encodes a nucleic acid sequence having at least 85% identity or at most 100 mutations compared to SEQ ID NO: 53.

In some specific embodiments, the nucleic acid is DNA or RNA, and the RNA is preferably mRNA.

In a third aspect, the present disclosure provides chimeric antigen receptors encoded according to the aforementioned nucleic acids.

In a fourth aspect, the present disclosure provides a vector, wherein the vector includes the aforementioned nucleic acid molecule.

In a fifth aspect, the present disclosure provides immune effector cells, wherein the immune effector cells include the aforementioned nucleic acid molecules or chimeric antigen receptors or vectors.

In some specific embodiments, the immune effector cells are NK cells, which are differentiated from iPSCs or derived from peripheral blood or umbilical cord blood.

In a sixth aspect, the present disclosure provides a method for preparing the aforementioned immune effector cells, which includes the steps of providing immune effector cells and transferring the nucleic acid molecules into the immune effector cells.

In a seventh aspect, the present disclosure provides products prepared according to the aforementioned method.

In an eighth aspect, the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition includes the aforementioned nucleic acid molecule or chimeric antigen receptor or carrier, immune effector cell or product, and a pharmaceutically acceptable carrier.

In a ninth aspect, the present disclosure provides the use of the aforementioned nucleic acid molecules or chimeric antigen receptors or vectors, immune effector cells, products or pharmaceutical compositions in the preparation of medicaments for the treatment of cancer or tumors selected from Hematological tumor or solid tumor; optionally, the hematological tumor is selected from myeloma, lymphoma or leukemia, such as multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma , follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or hairy cell leukemia (HCL); Alternatively, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer or adrenal cancer.

In the tenth aspect, the present disclosure provides the aforementioned nucleic acid molecules or chimeric antigen receptors or vectors, immune effector cells, products or Pharmaceutical composition for use in the treatment of cancer or tumors selected from hematological tumors or solid tumors; optionally, the hematological tumors are selected from myeloma, lymphoma or leukemia, such as multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), Acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer , skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer or adrenal cancer.

In an eleventh aspect, the present disclosure provides a method of treating cancer or tumors, the method comprising administering an effective amount of the aforementioned nucleic acid molecule or chimeric antigen receptor or vector, immune effector cell, product or drug to a subject in need thereof The composition, the cancer or tumor is selected from a hematological tumor or a solid tumor; optionally, the hematological tumor is selected from myeloma, lymphoma or leukemia, such as multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma Lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia Cellular leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, Uterine, prostate, or adrenal gland cancer.

Beneficial effects: The present disclosure provides optimized chimeric antigen receptors targeting BCMA, which have at least one of the following advantages: (1) higher CAR expression rate and CAR-NK proliferation rate; (2) higher sensitivity to BCMA-positive tumors. Strong killing effect.

Definitions and explanations of terms

Unless otherwise defined in this disclosure, scientific and technical terms related to this disclosure shall have the meanings understood by those of ordinary skill in the art.

Furthermore, unless otherwise indicated herein, singular terms herein shall include the plural form and plural terms shall include the singular form. More specifically, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

The terms "comprises,""comprises," and "having" are used interchangeably herein and are intended to indicate an inclusive nature, meaning that there may be elements other than those listed. At the same time, it should be understood that the descriptions of "including", "including" and "having" are used in this article, and the solution of "consisting of" is also provided. For example, "a composition including A and B" should be understood as the following technical solution: a composition composed of A and B , as well as a composition containing other components in addition to A and B, all fall into the category Within the scope of the aforementioned "a composition".

The term "and/or" when used herein includes the meaning of "and", "or" and "all or any other combination of elements linked by the applicable term".

As used herein, "antigen chimeric receptor (CAR)" refers to an artificial immune effector cell surface receptor engineered to be expressed on immune effector cells and specifically bind an antigen, which contains at least (1) an extracellular antigen-binding structure Domains, such as the variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into immune effector cells, and (3) an intracellular signaling domain. CARs are able to utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.

As used herein, the "transmembrane (TM) region" of a chimeric antigen receptor refers to the ability of the chimeric antigen receptor to be expressed on the surface of immune cells (such as lymphocytes, NK cells, or NKT cells) and to direct immune cells to target target cell cellular response Polypeptide structure. The transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain enables signaling when the chimeric antigen receptor binds to the target antigen.

As used herein, the "hinge region" of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that serves to connect the transmembrane region and the antigen-binding region. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antigen-binding region. The hinge region may be derived in whole or in part from a natural molecule, 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 may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence.

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

As used herein, the term "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for effective activation and function of T lymphocytes upon binding to an antigen.

The term "immune effector cell" or "effector cell" as used herein refers to cells that participate in an immune response, such as promoting an immune effector response. Examples of immune effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

The term "specifically binds" herein means that an antigen-binding molecule (eg, an antibody) specifically binds an antigen and substantially the same antigen, typically with high affinity, but does not bind with high affinity to an unrelated antigen. Affinity is usually reflected in terms of equilibrium dissociation constant (KD), where a lower KD indicates higher affinity. Taking antibodies as an example, high affinity generally refers to having about 10 ^-6 M or less, about 10 ^-7 M or less, about 10 ^-8 M or less, about 1×10 ^-9 M or less, about 1× KD of 10 ^-10 M or less, 1×10 ^-11 M or less, or 1×10 ^-12 M or less. KD is calculated as follows: KD=Kd/Ka, where Kd represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (such as Biacore) or equilibrium dialysis measurement. For example, see the method for obtaining the KD value shown in the embodiments herein.

The term "antibody" is used herein in its broadest sense and refers to a polypeptide 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 capable of specifically binding to an antigen. or peptide combinations. "Antibody" herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity. "Antibody" herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds, which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds, which contain, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

"Antibody" herein includes a typical "quadruple chain antibody", which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to such a polypeptide chain, which The direction from N-terminus to C-terminus consists of heavy chain variable region (VH), heavy chain Chain constant region CH1 domain, hinge region (HR), heavy chain constant region CH2 domain, heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally also includes Heavy chain constant region CH4 domain; light chain is a polypeptide chain composed of light chain variable region (VL) and light chain constant region (CL) in the N-terminal to C-terminal direction; between heavy chain and heavy chain, heavy chain The chain and light chain are connected through disulfide bonds to form a "Y"-shaped structure. Because the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, their antigenicity is also different. Based on this, the "immunoglobulins" in this article can be divided into five categories, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain and δ chain respectively. , γ chain, α chain and ε chain. The same type of Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. IgA can be divided into IgA1 and IgA2. Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.

"Antibodies" herein also include antibodies that do not contain light chains, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Alpaca ( Heavy-chain antibodies (HCAbs) produced by Vicugna pacos, etc., and immunoglobulin neoantigen receptors (Ig new antigen receptor, IgNAR) discovered in sharks and other cartilaginous fishes.

The "antibodies" herein can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camelids and llamas. , ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (such as shark).

"Antigen-binding fragment" and "antibody fragment" are used interchangeably herein. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have the ability to bind Antigen capabilities. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , scFv, and VHH.

Papain digestion of intact antibodies generates two identical antigen-binding fragments, termed "Fab" fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ). As such, the term "Fab fragment" herein refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residues of the constant domain carry free thiol groups. Pepsin treatment produces an F(ab') ₂ fragment with two antigen binding sites (two Fab fragments) and part of the Fc region.

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

The term "nanobody" in this article refers to the natural heavy chain antibody lacking the light chain that exists in camels and other bodies, and its variable region is cloned Single domain antibodies consisting only of heavy chain variable regions can be obtained, also called VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.

In this article, the terms "VHH domain" and "single domain antibody" (single domain antibody, sdAb) have the same meaning and can be used interchangeably. They refer to the variable region of a cloned heavy chain antibody, which is constructed from only one heavy chain variable region. A single-domain antibody is the smallest fully functional antigen-binding fragment. Usually, after obtaining a heavy chain antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

The term "variable region" herein refers to the region of the heavy or light chain of an antibody involved in enabling the antibody to bind to the antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR", and "light chain variable region" is used interchangeably. ” can be used interchangeably with “VL” and “LCVR”. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably in this article, and usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region. Among them, the heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The term "framework region" or "FR region" is used interchangeably and refers to those amino acid residues other than CDRs in the heavy or light chain variable region of an antibody. Usually, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

For further description of CDR, refer to Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. . Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003) ; and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDR" herein can be annotated and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but are not limited to AbRSA website (http://cao.labshare. cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. cgi#results). CDRs herein include overlaps and subsets of differently defined amino acid residues.

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).

The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol .196:901-917; Chothia et al. (1989) Nature 342:878-883).

The term "IMGT numbering system" in this article generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol. 27:55-77, 2003.

As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the alignment of sequences and the introduction of gaps, if necessary, to achieve maximum percent sequence identity ( For example, for the best Alignment, after which gaps can be introduced in one or both of the candidate and reference sequences, and non-homologous sequences can be ignored for comparison purposes), the amino acid (or nucleotide) residues of the candidate sequence are consistent with those of the reference sequence. The percentage of amino acid (or nucleotide) residues in a sequence that are identical. For the purpose of determining percent sequence identity, alignment can be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% decrease in to 100% sequence identity. The length of the candidate sequences aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. . When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

As used herein, "at least 80% identity" is preferably 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

As used herein, "mutation" includes insertion mutations, deletion mutations and substitution mutations. Preferably, the substitution mutations are conservative amino acid substitutions. As used herein, "conserved amino acids" generally refer to amino acids that are in the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). For example, the amino acids in each of the following groups belong to each other's conserved amino acid residues, and the substitution of amino acid residues within the group belongs to the substitution of conservative amino acids:

Illustratively, the following six groups are examples of amino acids that are considered conservative substitutions for each other:

1)Alanine (A), serine (S), threonine (T);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), lysine (K), histidine (H);

5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

As used herein, "up to 5 mutations" is preferably up to 4, 3, 2, 1 or 0 mutations.

As used herein, "up to 9 mutations" is preferably up to 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

As used herein, "up to 10 mutations" is preferably at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

As used herein, "up to 25 mutations" is preferably at most 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

As used herein, "up to 30 mutations" is preferably at most 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

As used herein, "up to 50 mutations" is preferably at most 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 20, 29, 28, 27 , 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 1, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations.

As used herein, "up to 100 mutations is preferably up to 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87 , 86, 85, 84, 83, 82, 81, 70, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70 , 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 or 50 mutations, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37 , 36, 35, 34, 33, 32, 31, 20, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1 or 0 mutations.

As used herein, "up to 150 mutations is preferably at most 149, 148, 147, 146, 145, 144, 143, 142, 141, 140, 139, 138, 137 , 136, 135, 134, 133, 132, 131, 120, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87 , 86, 85, 84, 83, 82, 81, 70, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70 , 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 or 50 mutations, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37 , 36, 35, 34, 33, 32, 31, 20, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1 or 0 mutations.

As used herein, a "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, etc.

As used herein, the terms "subject," "subject," and "patient" refer to an organism undergoing treatment for a particular disease or condition (eg, cancer or infectious disease) as described herein. Examples of subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, members of the Bovidae family (such as domestic cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.

As used herein, the term "treatment" refers to surgical or therapeutic treatment, The purpose is to prevent, slow down (reduce) the progression of undesirable physiological changes or pathologies in the treatment subject, such as cell proliferative disorders (such as cancer or infectious diseases). Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable. Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented. When referring to terms such as slow down, alleviation, weakening, alleviation, alleviation, their meanings also include elimination, disappearance, non-occurrence, etc.

As used herein, the term "effective amount" refers to an amount of a therapeutic agent that is effective when administered alone or in combination with another therapeutic agent to a cell, tissue or subject to prevent or alleviate the symptoms of a disease or the progression of the disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions. When the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

Description of the drawings

Figure 1. Schematic diagram of the CAR-NK structure containing different NKG2D TM region elements.

Figure 2. shows the CAR expression rate in BCMA-CAR NK cells.

Figure 3. Statistics of proliferation fold of -BCMA-CAR NK.

Figure 4. shows the cell flow cytometric detection results of MOLP8 and NCI H929-hBCMA-KO.

Figure 5. Multiple rounds of killing effects of BCMA-CAR NK on MOLP8 cells.

Figure 6 is a schematic structural diagram of BCMA-CAR-NK-6 (BCMA-CAR6) and BCMA-CAR-NK-7 (BCMA-CAR7).

Figure 7. shows the CAR expression rate in the prepared dual-epitope BCMA-CAR-NK cells in the form of NKG2D-TM1;

Figure 8. BCMA-CAR-NK cell proliferation fold statistics;

Figure 9A. Results of the 4-hour short-term killing experiment of BCMA-CAR-NK on NCI H929 cells; Figure 9B. Results of the 4-hour short-term killing experiment of BCMA-CAR-NK on NCI H929-hBCMA-KO cells;

Figure 10A. Results of multiple rounds of killing experiments by BCMA-CAR-NK on NCI H929 cells; Figure 10B. Results of multiple rounds of killing experiments by BCMA-CAR-NK on MOLP8 cells;

Figure 11. shows the CAR expression rate in BCMA-CAR NK cells used in animal experiments.

Figure 12A ~ Figure 12E. Anti-tumor efficacy experiments of different forms of BCMA-CAR-NK on NCI H929-luc multiple myeloma animal model. Figure 12A shows the detection results of animal tumor bio-luminescence signal; Figure 12B shows the animal body weight change rate %; Figure 12C shows the animal tumor biosignal photon quantity; Figure 12D shows the animal tumor growth inhibition rate %; Figure 12E shows the animal survival rate %.

Detailed ways

The present disclosure will be further described below in conjunction with specific embodiments, and the advantages and features of the present disclosure will become clearer with the description. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

The embodiments of the disclosure are only exemplary and do not limit the scope of the disclosure in any way. Those skilled in the art should understand that the details and forms of the technical solution of the present disclosure can be modified or replaced without departing from the spirit and scope of the present disclosure, but these modifications and substitutions all fall within the protection scope of the present disclosure.

Unless otherwise specified, the target cells mentioned in the following examples were all transformed into luciferase genes and expressed luciferase. The fluorescence intensity is detected by luciferase reporter gene detection reagent, which reflects the cell viability and the killing effect of NK cells. The formula for calculating the kill rate is as follows:
Killing rate = (target cell well reading value - test well reading value)/target cell well reading value × 100%.

Unless otherwise specified, the target cells used in the following examples are A549 cells, 786-O cells, RPMI 8226 cells, MOLP8 and H929-hBCMA-KO. All cells contain luciferase reporter genes through conventional gene manipulation methods. Among them, H929-hBCMA-KO is an H929 cell line that uses conventional gene manipulation methods to knock out BCMA.

Example 1 Screening and Preparation of Antibodies

The protein containing the extracellular domain of human BCMA was used as an antigen to immunize alpacas. Peripheral blood was collected after the fourth and fifth immunizations (the antibody titer and specificity had been verified by ELISA), PBMCs were isolated, total RNA was extracted, and reverse transcription and Nested PCR amplified the VHH fragment, constructed a phage library, and panned and identified clones that were cross-positive with human and monkey BCMA. The variable region sequences of the positive clones were obtained by sequencing, named VHH1, VHH2 and VHH3, and were identified through the Kabat numbering system and Chothia numbering system (http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT Numbering system (https://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi) determines its CDR region sequence.

By comparing the IMGT (http://imgt.cines.fr) human antibody heavy and light chain variable region germline gene database, we selected the heavy chain variable region germline gene (IGHV3-64* with high homology to VHH1 antibody 04 and IGHJ3*01) or the heavy chain variable region germline gene with high homology to the VHH2 antibody (IGHV3-7*01 and IGHJ6*01) as a template, and the CDR of VHH1 or VHH2 (determined by the IMGT numbering system) were transplanted into corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. If necessary, backmute the key amino acids in the backbone sequence to the amino acids corresponding to the VHH nanobody to ensure the original affinity; if the antibody has sites prone to chemical modification, point mutations at these sites will eliminate the risk of modification. Humanized antibodies hu-VHH1 and hu-VHH2 were obtained. The sequence information and CDR information of VHH antibodies and their humanized antibodies are shown in Tables 1-2.

Recombine the VHH1, VHH2, VHH3, hu-VHH1, and hu-VHH2 sequences into the expression vector of human IgG1Fc, and obtain chimeric antibodies and humanized antibodies VHH1-hFc, VHH2-hFc, VHH3-hFc, hu- VHH1-hFc and hu-VHH2-hFc. After verification by ELISA and FACS, the aforementioned antibodies all have good binding activity to human BCMA protein and monkey BCMA protein, and they all bind well to H929 and U266 cells that endogenously express BCMA. The affinity test results further verified that VHH1-hFc , VHH2-hFc, VHH3-hFc, hu-VHH1-hFc and hu-VHH2-hFc have high affinity to human BCMA protein, with KD values of 5.27E-10M (VHH1-hFc) and 7.16E-11M (VHH2-hFc) respectively. ), 2.97E-11M (VHH3-hFc), 6.13E-10M (hu-VHH1-hFc) and 8.82E-11M (hu-VHH2-hFc).

Table 1 Antibody sequence information

Table 2 CDR information of VHH1 and VHH2

Example 2 Design of different forms of chimeric antigen receptors

Design NKG2D TM1 and load it into the chimeric antigen receptor as a transmembrane region element to construct a new chimeric antigen receptor. At the same time, chimeric antigen receptors loaded with other NKG2D TM sequences were constructed, as well as chimeric antigen receptors using 2B4 or CD28 in the hinge region-transmembrane region and costimulatory domain, in which NKG2D-TM4 was derived from WO2021071962A1, (see for details Sequence 57) disclosed in WO2021071962A1. In subsequent examples, the functional differences (CAR positive rate, NK cell proliferation and killing) between CAR NK cells loaded with the NKG2D transmembrane region and other CARNK cells loaded with different transmembrane regions are evaluated, as well as the evaluation of CAR loaded with the NKG2D TM1 element. Functional differences between NK cells and CARNK cells loaded with other NKG2D TM sequences. The molecular structure of the chimeric antigen receptor is shown in Figure 1 and Figure 6 for details, the element sequence information is shown in Table 3, and the CAR sequence information is shown in Table 4. In Figures 1 and 6, BCMA-CAR-NK-1 is referred to as BCMA-CAR1, BCMA-CAR-NK-2 is referred to as BCMA-CAR2, BCMA-CAR-NK-3 is referred to as BCMA-CAR3, and BCMA-CAR- NK-4 will be referred to as BCMA-CAR4, BCMA-CAR-NK-5 will be referred to as BCMA-CAR5, BCMA-CAR-NK-6 will be referred to as BCMA-CAR6, and BCMA-CAR-NK-7 will be referred to as BCMA-CAR7.

Table 3 CAR element sequence information

Table 4 CAR sequence information

Example 3 Preparation of Chimeric Antigen Receptor Retrovirus

Using conventional molecular biology methods in this field, the nucleic acid sequence encoding the CAR described in Example 2 (Table 4) is loaded into a retroviral vector to construct a target plasmid. The day before virus packaging, 293T cells (purchased from ATCC) were trypsinized and inoculated into a culture dish at 1E7 cells/10cm. When transfecting cells, mix the packaging plasmid and the target plasmid, add them to α-MEM medium, and add them to another centrifuge tube containing α-MEM medium. HD transfection reagent (Promega, E2311). Add the diluted transfection reagent drop by drop on top of the diluted plasmid, mix well, and let stand at room temperature for 15 minutes. Finally, add the mixture of plasmid and transfection reagent into a 10cm petri dish, shake gently 10 times, mix well, and place in the incubator box. Three days after cell transfection, harvest the virus. Transfer 10 ml of the virus-containing culture supernatant into a 50 ml centrifuge tube at 4°C, 1250 rpm for 5 minutes. Remove the dead 293T cells, filter the virus-containing supernatant, and use Retro-X Concentrator concentration reagent ( Clontech, 631455), aliquot and store at -80°C for later use.

Example 4 Preparation of CAR-NK

1. Isolation and activation of NK lymphocytes

Fresh PBMC were centrifuged at 500 g for 7 min at room temperature, the upper culture medium was discarded, and NK cells were isolated according to the Human NK Cell isolation kit (Stemcell, 17955). The isolated NK cells were activated with K562 cells. The activation method was: Day0, count with AO/PI, mix the cells according to NK:K562=1:2, and add the mixed cells to the Non-Treated 6-well plate at 2ml/well (culture The base is NK cell culture medium (Miltenyi Biotec, 130-114-429) containing 200 IU/ml human IL2, and cultured in an incubator (37°C, 5% CO ₂ ); on Day 4, add 3 mL of culture medium to each well. Base; Day 6, cell activation is complete and can be transfected.

2. Retrovirus infection of NK cells

Day1, coat a 24-well plate with RetroNectin reagent (Takara, T202) at a concentration of 7 μg/mL, 500 μL per well. Leave overnight at 4°C. On Day 2, discard the upper layer of RetroNectin and wash once with PBS. Use MOI=5 to infect NK cells, calculate the amount of virus based on the virus titer, and add the virus to a 24-well plate. Centrifuge the 24-well plate after adding the virus at 2000g and 4-8°C for 60 minutes. Discard the upper layer of virus liquid. Count NK cells, add 3E5 cells/well to a 24-well plate, and centrifuge at 400g for 5 minutes at room temperature. Place the 24-well plate into an incubator (37°C, 5% CO ₂ ). On Day 3, replace the transfected NK into the Non-Treated 6-well plate. Day6, detect CAR expression and CAR NK cell proliferation.

Example 5 Detection of expression efficiency and proliferation efficiency of different forms of chimeric antigen receptors

On the 6th day after retrovirus infection of NK cells, flow cytometry was used to detect the expression rate of CAR on the NK cell membrane surface and CAR NK cell proliferation was detected by a cell counter.

1. Detection of CAR expression rate in NK cells

Take 2E5 cells into a 96-well U-shaped plate, centrifuge, discard the supernatant, wash with buffer, add 100 μl of FITC-hBCMA-his (ACROBiosystems, BCA-HF254) with a final concentration of 2 μg/ml, and incubate at 4°C in the dark. 1 hour; after incubation, centrifuge, discard the supernatant, wash with buffer and resuspend the cells; BD FACS Canto II flow cytometer detects BCMA-CAR expression rate. The flow cytometric detection results of BCMA-CAR NK cell surface molecule expression are shown in Figure 2. The results show that the expression efficiency of BCMA-CAR in NK cells is about 70-90%, and the expression efficiency from high to low is BCMA-CAR4 (89.7%) > BCMA-CAR5 (85.9%) > BCMA-CAR1 (73.8%) )>BCMA-CAR2 (73%)>BCMA-CAR3 (65.1%).

2. Detect the proliferation rate of CAR NK cells

The proliferation of BCMA-CAR NK cells was detected by AO/PI counting, and the results are shown in Figure 3. BCMA-CAR NK cells proliferate about 40 to 70 times 6 days after transfection. The proliferation rates from high to low are: BCMA-CAR1 (73 times) > BCMA-CAR2 (68 times) > BCMA-CAR4 (66 times) > BCMA -CAR3 (63 times)>parental NK (53 times)>BCMA-CAR5 (44 times).

Description of results:

According to the above results, in BCMA-CAR-NK, in terms of CAR expression rate, those loaded with traditional CD28-TM elements have a higher CAR expression rate; the overall expression rate of CARs loaded with NKG2D TM elements is low, among which The expression rate of the NKG2D-TM1 component is the highest. In terms of CAR-NK cell proliferation rate, the proliferation rate of CAR NK cells loaded with NKG2D TM elements is generally higher than that of CAR NK cells using other transmembrane elements. Among them, CAR NK cells loaded with NKG2D-TM1 elements have the fastest proliferation rate.

Overall, different forms of BCMA-CAR-NK have certain expression and proliferation, among which the NKG2D-TM1 form has the best overall performance.

Example 6 4h in vitro killing functional evaluation of different forms of CAR NK

Flow cytometry was used to detect the expression of BCMA in multiple myeloma MOLP8 cells and H929-hBCMA-KO cells. See Figure 4 for the results.

On the 6th day after retrovirus infection of NK cells, a 4-hour in vitro killing experiment was performed: target cells MOLP8 or H929-hBCMA-KO diluted in 1640 culture medium were added to a white opaque 96-well plate at 2E4 cells/50 μl/well, and the effect-to-target ratio was 10 :1, 5:1, 2.5:1 Add NK cells to the above target cells, and place the 96-well plate in a 37°C, 5% CO ₂ incubator; after 4 hours, add 30 μl FIREFLYGLO luciferase reporter gene detection reagent (Meilun Bio, MA0519-1), incubate at room temperature in the dark for 10 minutes, then measure with a microplate reader and calculate the killing rate.

The 4h in vitro cell killing effect on MOLP8 cells is detailed in Table 5. Compared with other CAR structures, the CAR structure loaded with NKG2D TM region showed stronger tumor cell killing function at the effect-to-target ratio of 10:1, 5:1, and 2.5:1. At the same time, the CAR loaded with NKG2D TM1 ( BCMA-CAR1) has the strongest killing function and is superior to other NKG2D TM forms. In addition, the killing of H929-hBCMA-KO cells by NK cells transfected with BCMA-CAR1~5 and partial NK was basically equivalent, indicating that BCMA-CAR has no non-specific killing of H929-hBCMA-KO cells.

Table 5 BCMA-CAR NK 4h in vitro killing rate of MOLP8 cells (unit, %)

Note: ND stands for "no kill"

Example 7 Evaluation of multiple rounds of killing functions of different forms of CAR NK

On the 6th day after retrovirus infection of NK cells, the in vitro multi-round killing effect of BCMA-CAR NK cells on MOLP8 cells was detected: (1) First round of killing: 2.5E5 cells/500μl/well were diluted with 1640 medium Target cells MOLP8 were placed in 12-well plates. Add NK cells to a 12-well plate at an effective-to-target ratio of 1:1 and culture them in a 37°C, 5% _CO2 incubator for 24 hours. After culturing for 24 hours, add 100 μl of the above-mentioned mixed cells into a 96-well plate with a white bottom and opaque bottom, add 30 μl FIREFLYGLO luciferase reporter gene detection reagent (Meilun Biotech, MA0519-1), incubate at room temperature in the dark for 10 minutes, and then use A microplate reader measures the fluorescence intensity and calculates the NK cell killing efficiency. After that, directly enter the next round of killing test, or measure the NK cell killing rate again after 24 hours of culture and then enter the next round of killing test. (2) Next round of killing: Take the cells from the previous round of 12-well plate, count the NK cells, and add the previous round of NK cells to the 12-well plate seeded with new target cells at an effective-to-target ratio of 1:1 , repeat step (1), measure the NK cell killing rate and continue the next round of killing test.

The results of multiple rounds of BCMA-CAR NK cell killing tests are shown in Figure 5. As shown in Figure 5, the killing effect of BCMA-CAR loaded with CD28TM elements is weak, and other forms of BCMA-CAR can maintain the killing effect for 3 to 5 rounds. In addition, BCMA-CAR1 loaded with NKG2D-TM1 had the best killing effect and could still maintain a cell killing rate of 98% in the third round of killing (R3-2d).

Example 8 Evaluation of cytokine release function of different forms of CAR NK

BCMA-CAR NK was cultured for 14 days after transfection, and incubated with MOLP8 cells for 24 hours at an effective-to-target ratio of 10:1 and 2.5:1. The supernatant was collected and detected according to the instructions of the human IFN-γ quantitative ELISA kit (BD, 555142). Medium IFN-γ content. Calculate the IFN-γ content in the supernatant of the sample to be tested based on the standard curve of the standard product. The results are shown in Table 6.

The results in Table 6 show that, except for BCMA-CAR3 and BCMA-CAR4, all can release higher concentrations of IFN-γ under high-efficiency target ratios, and have potentially strong tumor killing effects. Among them, BCMA-CAR1 can upregulate the expression of the tumor-killing cytokine IFN-γ at different effect-to-target ratios and on different target cells, and is overall better than other forms of CAR-NK.

Table 6 Cytokine IFN-γ content detection results

Note: ND means "not detected"

Example 9. Construction and screening of dual BCMA chimeric antigen receptors in the form of NKG2D-TM1

1.Construction of double BCMA chimeric antigen receptor in the form of NKG2D-TM1

The HuVHH1 and VHH3 antibodies were designed to construct a CAR as shown in Figure 6. The dual-epitope chimeric antigen receptor targeting BCMA includes CD8α signal peptide (SP), anti-BCMA-VHH and anti-BCMA-VHH, CD8α hinge region, and NKG2D. -TM1 transmembrane domain, 2B4 costimulatory domain and CD3ζ, and IL15 linked via the self-cleaving peptide P2A. Named BCMA-CAR7 (for the sequence, see SEQ ID NO: 63 recorded in Table 4 of Example 2). Referring to the method in Example 4, BCMA-CAR1 and BCMA-CAR7 were prepared.

2. Expression detection of BCMA-CAR-NK dual epitope in the form of NKG2D-TM1

The BCMA-CAR-NK expression detection method is the same as in Example 5. The flow cytometry expression detection results of BCMA-CAR-NK are shown in Figure 7. The results showed that BCMA CAR expression could be detected after infecting NK cells at MOI=5, BCMA-CAR1 expression could reach 95.3%, and BCMA-CAR7 expression could reach 96.0%.

3. Determination of proliferation rate of NKG2D-TM1 dual epitope BCMA-CAR-NK cells

The proliferation of BI-CAR cells was detected by AO/PI counting, and the results are shown in Figure 8. Thirteen days after NK activation, the proliferation rates of BCMA-CAR-NK cells from high to low were: BCMA-CAR1 (2100 times) > NK (1400 times) > BCMA-CAR7 (1100 times).

4. 4h rapid killing rate detection of NKG2D-TM1 dual epitope BCMA-CAR-NK cells

On the 6th day after retrovirus infection of NK cells, a 4-h in vitro killing experiment was performed on the prepared BCMA-CAR-NK cells: target cells NCI H929 and NCI H929-hBCMA-KO diluted in 1640 culture medium were used at 2 × 10 ⁴ cells/ Add 50 μl/well to a white opaque 96-well plate, and add NK cells to the above at an effective-to-target ratio of 10:1, 3:1, 1:1, 0.3:1, 0.1:1, 0.03:1. In the target cells, the killing rate was measured and calculated with reference to the method of Example 6.

The 4-hour in vitro cell killing effect of BCMA-CAR-NK on the above target cells is detailed in Figure 9A-Figure 9B and Table 7 and Table 8. According to Figure 9A-Figure 9B and Table 7 and Table 8, it can be seen that BCMA-CAR1 and BCMA-CAR7 both have good specific killing effect on NCI H929 tumor cells, among which BCMA-CAR1 has a better specific killing effect on NCI H929 than BCMA- The non-specific killing of NCI H929-hBCMA-KO by CAR7; BCMA-CAR1 and BCMA-CAR7 was both smaller and lower than that of blank control NK cells.

Table 7. 4h rapid killing of NCI H929-lu cells by BI-CAR

Note: "-" means no killing detected.

Table 8. Non-specific killing of NCI H929-hBCMA-KO cells by BI-CAR

Note: "-" means no killing detected.

5. NKG2D-TM1 dual-epitope BCMA-CAR-NK multi-round killing effect detection

On the 6th day after the retrovirus infects NK cells, the in vitro multiple rounds of killing effects of BCMA-CAR1 and BCMA-CAR7 on NCI H929 and MOLP8 tumor cells were detected. For specific experimental methods, refer to Example 7.

The results of multiple rounds of BCMA-CAR-NK cell killing tests are shown in Figure 10A-Figure 10B. The results showed that BCMA-CAR1 and BCMA-CAR7 had a strong multi-round killing effect on H929 cells. Among them, BCMA-CAR1 had a slightly better multi-round killing effect on NCI H929 cells than BCMA-CAR7; BCMA-CAR1 had a multi-round killing effect on MOLP8 cells. The round killing effect is significantly better than BCMA-CAR7. BCMA-CAR1 and BCMA-CAR7 can still maintain a 50% cell killing rate on NCI H929 cells in the third round of killing (R3-2d) experiments. BCMA-CAR1 can still maintain a cell killing rate of 60% in the third round of killing (R3-2d) experiments on MOLP8 cells.

Example 10 In vivo efficacy evaluation of different forms of BCMA-BCMA CAR-NK

BCMA-CAR1 loaded with NKG2D TM1 element, BCMA-CAR2 loaded with NKG2D TM2 element, BCMA-CAR6 loaded with NKG2D TM4 element, and BCMA-CAR4 and BCMA-CAR5 not loaded with NKG2D TM element (for the CAR structure, see Figure 1 and Figure 6; sequence information See Table 4), and the efficacy was evaluated on the NCI H929-luc mouse model.

1. Preparation of BCMA CAR-NK cells

Referring to the preparation method of Example 4, BCMA CAR-NK was performed using NK cells from different sources than those in Example 4. Cell preparation, infect NK cells at MOI=5, calculate the amount of virus based on the virus titer, and add the virus to a 24-well plate. Centrifuge the 24-well plate after adding the virus at 2000g and 4-8°C for 60 minutes. Discard the upper layer of virus liquid. Count NK cells, add 3E5 cells/well to a 24-well plate, and centrifuge at 400g for 5 minutes at room temperature. Place the 24-well plate into an incubator (37°C, 5% CO ₂ ). On Day 3, replace the transfected NK into the Non-Treated 6-well plate. On Day9, CAR expression was detected as shown in Figure 11.

According to the results in Figure 11, in this experiment, in terms of CAR expression rate, except for BCMA-CAR2, the rest of the BCMA-CARs had an expression rate of about 70% or more.

2. Anti-tumor efficacy test of BCMA-BCMA CAR-NK loaded with NKG2D-TM element in NCI H929-luc mouse tumor model

In order to evaluate the anti-tumor efficacy of BCMA-BCMA CAR-NK loaded with NKG2D-TM elements, a mouse bone marrow cancer vein transplant tumor model was used to conduct an anti-tumor efficacy test. details as follows:

H929-Luc cells in the logarithmic growth phase and in good growth status were collected, and a total of 2 × 10 ⁶ cells were inoculated into the tail veins of NPG mice (combined immunodeficient mice). On the first day after tumor inoculation, the weight and inoculation status of the mice were measured, and mice with a weight of about 18.85-23.52g were selected based on the random number principle, with an average value of 21.92g for random grouping. On the 1st day after tumor inoculation, that is, on the day of grouping, freshly prepared CAR-NK cells (5×10 ⁶ cells/animal) were injected into the tail vein. The injection volume was 200 μl/animal. The CAR-NK cell injection diary was Day 0. The grouping of mice and the injection of CAR-NK cells are shown in Table 9. Continuously monitor the tumor growth fluorescence signal ROI value and body weight changes with the IVIS intravital imager. Measure and record twice a week and calculate the tumor inhibition rate. The calculation formula: tumor inhibition rate TGI (%) = (PBS group mouse tumor photon signal value - Photon signal value of mouse tumor in the experimental group)/photon signal value of mouse tumor in the PBS group × 100%.

Table 9. Grouping status of anti-tumor experiments in vivo

The fluorescence signal detection results of mouse tumor growth are shown in Figure 12A. The results show that on the 76th day after the injection of CAR-NK cells, the BCMA-CAR 1 treatment group was able to significantly inhibit the tumor growth burden of mice, and four of the animals had tumors. Cured, only 1 animal had tumor recurrence and growth; BCMA-CAR 6 treatment group could significantly inhibit tumor growth load, but could not control tumor progression in this group of animals. Among them, 2 animals had tumor recurrence and growth, 2 had tumors that completely disappeared, and 1 animal had tumor recurrence and growth. Death occurred; BCMA-CAR 2 treatment group was able to inhibit the growth rate of mouse tumors. One animal was cured of tumor, but 4 animals in the group had tumor recurrence and two died; BCMA-CAR 4 and BCMA-CAR 5 were administered It was able to inhibit the tumor growth rate of mice in the last 39 days. In the later stage of the experiment, the tumor control effect was poor as the tumors recurred, and all BCMA-CAR 4 died on the 76th day.

At the same time, although the body weight of mice in each group treated with CAR-NK fluctuated after administration, it showed an overall upward growth trend (as shown in Figure 12B).

After administration, the photon value of the mouse tumor was continuously measured. Except for the BCMA-CAR 5 treatment group, the other CARNK treatment groups showed a decreasing trend compared with the Parental NK group, P<0.05*, P<0.001***, especially In the BCMA-CAR1 and BCMA-CAR6 treatment groups, extremely significant therapeutic effects were demonstrated (as shown in Figure 12C);

The mouse tumor volume was measured on day 76, and the tumor inhibition rate was calculated. As shown in Figure 12D, the tumor inhibition rates of the three treatment groups BCMA-CAR1, BCMA-CAR6, and BCMA-CAR4 reached 93%, 94%, and 84% respectively, showing good inhibitory effects on tumor growth, while BCMA-CAR 5 treatment The tumor inhibition rate of the group was only 25%. Since all the animals in the BCMA-CAR 4 group died before 76 days, the tumor inhibition rate cannot be calculated;

During the experiment, the survival rate was continuously monitored until 130 days. As shown in Figure 12E, one animal died in the Parental NK treatment group starting from day 57, and the mortality rate was 100% by day 80; the BCMA-CAR 6 treatment group started from day 67 One animal died, and the mortality rate was 60% on day 130; one animal died in the BCMA-CAR 2 treatment group starting on day 39 (the mouse died on the same day after the tumor growth fluorescence signal was detected on day 39). The mortality rate at 130 days was 60%; in the BCMA-CAR4 treatment group, one animal died from day 48, and the mortality rate was 100% at day 76; in the BCMA-CAR 5 treatment group, one animal died from day 63, and by day 108 The daily mortality rate was 100%; only one animal died in the BCMA-CAR 1 treatment group on day 85. This group showed a durable therapeutic effect and good safety.

Claims

Nucleic acid molecule, wherein said nucleic acid molecule comprises a first nucleic acid sequence encoding a chimeric anti-receptor targeting BCMA, said chimeric antigen receptor comprising an extracellular region comprising an antigen-binding region, a region connecting said extracellular region A transmembrane region and an intracellular domain connecting the transmembrane region, and the antigen-binding region includes:

(1)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; or,

Sequences as shown in SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:13; or,

Sequences as shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18;

(2) VHH, including a sequence with at least 80% identity or up to 25 mutations to SEQ ID NO: 1 or 3;

(3)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21; or,

Sequences as shown in SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:21; or,

Sequences as shown in SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26; or,

Sequences as shown in SEQ ID NO:19, SEQ ID NO:27 and SEQ ID NO:21;

(4) A sequence with at least 80% identity or at most 25 mutations with SEQ ID NO: 2 or 5;

(5)VHH, its CDR1-3 include:

Sequences as shown in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30; or,

Sequences as shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:30; or,

Sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35;

(6) A sequence that is at least 80% identical or has at most 25 mutations to SEQ ID NO:3; or,

(7) VHH1-linking peptide-VHH2, the VHH1 has the sequence shown in the group (1) or the group (2), and the VHH2 has the sequence shown in the group (3) or the group (4); or,

(8) VHH1-linking peptide-VHH2, said VHH1 has a sequence shown in the group (3) or group (4) and said VHH2 has a sequence shown in the group (1) or group (2); or,

(9) VHH1-linking peptide-VHH2, the VHH1 has a sequence shown in the group (1) or the group (2) and the VHH2 has a sequence shown in the group (5) or the group (6).
The nucleic acid molecule according to claim 1, wherein the extracellular region further includes a hinge region, the hinge region is selected from one or more of the following group: CD8α hinge region, 2B4 hinge region, CD28 hinge region, IgG1 Hinge region, IgD hinge, IgG4 hinge region, GS hinge, KIR2DS2 hinge, KIR hinge, NCR hinge, SLAMF hinge, CD16 hinge, CD64 hinge or LY49 hinge; Optionally, the hinge region is selected from the CD8α hinge region, for example, The hinge region has a sequence that is at least 80% identical or at most 10 mutated compared to SEQ ID NO:38.
The nucleic acid molecule according to any one of claims 1-2, wherein the extracellular region further includes a signal peptide selected from one or more of the following group: CD8α signal peptide, IgG1 heavy chain signal peptide or GM-CSFR2 signal peptide; optionally, the signal peptide is a CD8α signal peptide, for example, the signal peptide has a sequence of at least 80% identity or at most 5 mutations compared to SEQ ID NO: 36.
The nucleic acid molecule according to any one of claims 1-3, wherein the intracellular domain includes intracellular signaling domains and/or costimulatory domains;

Alternatively, the intracellular signaling domain is CD3ζ, for example, the intracellular signaling domain has a sequence of at least 80% identity or at most 35 mutations compared to SEQ ID NO: 49;

Optionally, the costimulatory domain is selected from one or more of the following group: CD27 costimulatory domain, 4-1BB costimulatory domain, OX40 costimulatory domain, 2B4 costimulatory domain, CD28 costimulatory domain domain, ICOS co-stimulatory domain, DAP10 co-stimulatory domain or DAP12 co-stimulatory domain; optionally, the co-stimulatory domain is a 2B4 co-stimulatory domain, for example, the co-stimulatory domain has the same structure as SEQ ID NO:47 compared to sequences with at least 80% identity or at most 25 mutations.
The nucleic acid molecule according to any one of claims 1-4, wherein the chimeric antigen receptor includes: CD8α hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3ζ intracellular signaling domain, for example , the chimeric antigen receptor has a sequence that is at least 80% identical or has at most 50 mutations compared to SEQ ID NO: 52.
The nucleic acid molecule according to any one of claims 1 to 5, wherein the nucleic acid molecule further includes a second nucleic acid sequence encoding IL15. Alternatively, the IL15 is selected from the group consisting of soluble IL15, membrane-bound IL15 or IL15 and its A complex of receptors or receptor fragments, optionally, the IL15 has a sequence with at least 80% identity or at most 35 mutations compared to SEQ ID NO: 51;

Optionally, the first nucleic acid sequence and the second nucleic acid sequence are connected by an IRES or a sequence encoding a self-cleaving peptide selected from 2A peptides, such as P2A, T2A, F2A or E2A, optionally , the self-cleaving peptide is P2A, for example, having a sequence with at least 80% identity or at most 5 mutations compared to SEQ ID NO:50;

Optionally, the nucleic acid encodes a nucleic acid sequence having at least 85% identity or at most 100 mutations compared to SEQ ID NO: 53.
The nucleic acid molecule according to any one of claims 1-6, comprising a nucleic acid sequence encoding a sequence having at least 80% identity or at most 150 mutations compared to SEQ ID NO: 57 or SEQ ID NO: 63.
The nucleic acid molecule according to any one of claims 1 to 7, wherein the nucleic acid is DNA or RNA, and the RNA is preferably mRNA.
The nucleic acid molecule according to claims 1-8, wherein the chimeric antigen receptor includes: CD8α hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3ζ intracellular signaling domain, for example, A chimeric antigen receptor has a sequence that is at least 80% identical or has up to 50 mutations compared to SEQ ID NO:52.
The chimeric antigen receptor encoded by the nucleic acid molecule according to any one of claims 1-9.
A vector, wherein the vector includes the nucleic acid molecule of any one of claims 1-9.
Immune effector cells, wherein the immune effector cells include the nucleic acid molecule of any one of claims 1-9, the chimeric antigen receptor of claim 10, or the vector of claim 11.
The immune effector cell according to claim 12, wherein the immune effector cell is an NK cell, and the NK cell is selected from the group consisting of NK cells differentiated from iPSCs, peripheral blood or cord blood-derived NK cells, or NK92 cells.
The method for preparing immune effector cells according to any one of claims 12 to 13, which includes the steps of providing immune effector cells and transferring the nucleic acid molecules into the immune effector cells.
The product prepared according to the method of claim 14.
Pharmaceutical composition, wherein the pharmaceutical composition includes the nucleic acid molecule of any one of claims 1-9 or the chimeric antigen receptor of claim 10 or the vector of claim 11, any one of claims 12-13 The immune effector cells described in claim 15 or the product described in claim 15, and a pharmaceutically acceptable carrier.
The nucleic acid molecule described in any one of claims 1-9, the chimeric antigen receptor described in claim 10, the vector described in claim 11, the immune effector cell described in any one of claims 12-13, or the immune effector cell described in claim 15 The product or the use of the pharmaceutical composition according to claim 16 in the preparation of a medicament for the treatment of cancer or tumors, the cancer or tumor being selected from hematological tumors or solid tumors; optionally, the hematological tumors are selected from myeloma , lymphoma or leukemia, such as multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia ( AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal, stomach, pancreatic, liver, skin, bladder, ovarian, uterine, prostate or adrenal cancer.
The nucleic acid molecule described in any one of claims 1-9, the chimeric antigen receptor described in claim 10, the vector described in claim 11, the immune effector cell described in any one of claims 12-13, or the immune effector cell described in claim 15 The product or the pharmaceutical composition according to claim 16, for use in the treatment of cancer or tumors, the cancer or tumor being selected from hematoma or solid tumor; optionally, the hematoma is selected from myeloma, lymphoma or Leukemias, such as multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, acute myeloid leukemia (AML), chronic Lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) or hairy cell leukemia (HCL); optionally, the solid tumor is selected from lung cancer, breast cancer, colorectal cancer, stomach, pancreas, liver, skin, bladder, ovary, uterus, prostate or adrenal gland cancer.
A method for treating cancer or tumors, the method comprising administering an effective amount of the nucleic acid molecule of any one of claims 1-9 or the chimeric antigen receptor of claim 10 or the chimeric antigen receptor of claim 11 to a subject in need. The carrier, the immune effector cell of any one of claims 12-13, the product of claim 15 or the pharmaceutical composition of claim 16, the cancer or tumor is selected from hematological tumors or solid tumors; optionally , the hematological tumor is selected from myeloma, lymphoma or leukemia, such as multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell Lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), or hairy cell leukemia (HCL); optionally, The solid tumor is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, skin cancer, bladder cancer, ovarian cancer, uterine cancer, prostate cancer or adrenal cancer.