CN111499766A - Immune effector cell aiming at chronic lymphocytic leukemia, preparation method and application thereof - Google Patents

Abstract

The invention discloses a Chimeric Antigen Receptor (CAR) targeting CD32b, an immune effector cell modified by the CAR, a preparation method thereof and application thereof in inhibiting chronic lymphocytic leukemia. The invention provides a new treatment scheme for refractory chronic lymphocytic leukemia.

Description

Immune effector cell aiming at chronic lymphocytic leukemia, preparation method and application thereof

Technical Field

The invention belongs to the field of medical biology, and particularly relates to an immune effector cell aiming at chronic lymphocytic leukemia, and a preparation method and application thereof.

Background

Adoptive immunotherapy based on immune effector cells in the art achieves a certain effect in some tumors and this immunotherapy approach can overcome the above-mentioned drawbacks of antibody therapy, but the efficacy in most tumors is still unsatisfactory [ Grupp SA, et al, adoptive cellular therapy.curr Top micro antibody, 2011: 344:149-72] in recent years, based on the discovery that recognition of target cells by cytotoxic T lymphocytes (CT L) is specifically dependent on T lymphocyte receptors (T Cell receptors TCR), fusion of scFv of antibodies against tumor Cell-associated antigens with intracellular signal activation motifs such as CD3 ζ or FcRI γ of T lymphocyte receptors into Chimeric Antigen Receptors (CAR) and its genetic modification on T lymphocyte surface by means of e.g. lentiviral infection, this CAR can be treated with major histocompatibility CAR (MHC-specific), and has a good prospect of tumor Cell-specific immune therapy.

Chimeric Antigen Receptors (CARs) consist of an antigen recognition region, a transmembrane region, and an intracellular signaling region, and CAR molecules are expressed on the surface of T cells, typically by viral transduction or electroporation, to form CAR-T cells. The CAR-T cell recognizes tumor antigens through an extracellular antigen recognition region, recruits T cells to the surface of tumor cells, and activates the T cells through an intracellular signal region, so that the T cells can perform a killing effect to kill the tumor cells, thereby achieving the purpose of treating tumors.

The present invention relates to a method for treating malignant tumor of lymphatic system, and more particularly to a method for treating malignant tumor of lymphatic system, which comprises the steps of treating malignant tumor of lymphatic system with a plurality of clinical multiple lines, wherein the malignant tumor of lymphatic system is difficult to treat, and particularly, the clinical common diseases are recurrent and refractory, chronic lymphocytic leukemia (C LL) is an incurable clinical multiple line therapy, and neither traditional drug chemotherapy nor emerging small molecule inhibitors can completely eliminate tumor, therefore, more advanced treatment methods need to be explored for patients at high risk of C LL and multiple relapses, such as CAR-T cell therapy, CD19 is one of cluster differentiation antigens, is an important membrane antigen related to B cell proliferation, differentiation, activation and antibody production, and is specifically expressed on the surfaces of B lymphocytes at different differentiation stages, more than 95% of B cell lymphoma and B lymphocytic leukemia express CD19 antigen, 865865, CD19CAR-T has achieved ideal treatment effect on acute lymphocytic leukemia of children, can induce more than 90% of acute lymphocytic leukemia (A LL) patients, but, although CD19 on tumor cells of C LL is expressed, the clinical multiple cell CAR-T-25 shows that the clinical multiple cell therapy is different from CD-T-3625, and the clinical multiple cell therapy of patients only shows that the CD-366323 is capable of inducing high clinical effect.

In developing CAR-modified immune effector cells, the antigen targeted is actually a more critical choice. However, given the complexity of gene expression in vivo and various uncontrollable factors, it is very difficult to successfully select an appropriate gene for CAR-based therapy, and in the extensive research work in this field, many tumor-specific antigens were found to be unsuitable for CAR-modification based targeted therapy. Furthermore, CAR-T therapy, although successful in some cases, is challenged by the complexity of its production and adverse events associated with cellular activity, such as cytokine storm (CRS), among others. There are currently no reports of CAR T cell therapy for many tumors

In conclusion, the present invention is to find a more ideal antigenic molecule against the disease C LL to achieve better therapeutic effect.

Disclosure of Invention

The invention aims to provide a CAR-T therapeutic vector for chronic lymphocytic leukemia or Burkitt's lymphoma, a construction method and application thereof.

In a first aspect of the invention there is provided a Chimeric Antigen Receptor (CAR) comprising, connected in sequence: an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes CD32 b.

In a preferred embodiment, the protein specifically recognizing CD32b is an antibody; preferably, the antibody is a single chain antibody or a domain antibody; more preferably, the antibody is a single chain antibody, the heavy chain of which has the amino acid sequence shown in SEQ ID NO. 1 and the light chain of which has the amino acid sequence shown in SEQ ID NO. 2, or the heavy chain of which has the amino acid sequence shown in SEQ ID NO. 3 and the light chain of which has the amino acid sequence shown in SEQ ID NO. 4.

In another preferred embodiment, the transmembrane region is a sequence comprising the hinge region and the transmembrane region of CD8 or CD 28; preferably, the transmembrane region is a sequence comprising the hinge region and the transmembrane region of CD 8; more preferably, the hinge region has an amino acid sequence shown in SEQ ID NO. 5, and the transmembrane region has an amino acid sequence shown in SEQ ID NO. 6.

In another preferred embodiment, the intracellular signaling region comprises an intracellular signaling region sequence selected from 4-1BB, CD3 ζ, FcRI γ, CD27, CD28, CD134, ICOS, GITR, or a combination thereof; preferably, the intracellular signaling region comprises 4-1BB and CD3 ζ; more preferably, the 4-1BB has the amino acid sequence shown in SEQ ID NO. 7, and the CD3 zeta has the amino acid sequence shown in SEQ ID NO. 8.

In another preferred embodiment, the chimeric antigen receptor comprises an extracellular binding domain, a transmembrane domain and an intracellular signaling domain connected in this order as follows: a single chain antibody that specifically recognizes CD32b, a CD8hinge region, a CD8 transmembrane region, 4-1BB, and CD3 ζ.

In another preferred embodiment, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10.

In another preferred embodiment, the immune effector cells comprise cells selected from the group consisting of: t lymphocytes, NK cells or NKT cells.

In another aspect of the invention, there is provided a nucleic acid encoding the chimeric antigen receptor.

In a preferred embodiment, the nucleic acid has the nucleotide sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12.

In another aspect of the invention, there is provided an expression vector comprising said nucleic acid.

In a preferred embodiment, the expression vector takes pCDH as a skeleton plasmid.

In another aspect of the invention, there is provided a virus, such as a lentivirus, comprising said expression vector.

In another aspect of the invention, there is provided the use of said chimeric antigen receptor, said nucleic acid, said expression vector or said virus for the preparation of a genetically modified immune effector cell targeted to CD32 b.

In another aspect of the invention, there is provided a genetically modified immune effector cell transduced with said nucleic acid, said expression vector or said virus; or, the surface thereof expresses said chimeric antigen receptor.

In a preferred embodiment, the immune effector cells comprise cells selected from the group consisting of: t lymphocytes, NK cells or NKT cells.

In another preferred embodiment, the cell further expresses cytokines having characteristics including those selected from the group consisting of immunomodulatory activity or anti-tumor activity and enhanced immune effector cell function, preferably, the cytokines include, but are not limited to, I L-12, I L-15, I L-21, I L-2, I L-4, I L-7, I L-9, I L-17, I L-18, I L-23.

In another preferred embodiment, the cell also expresses a chemokine receptor (which blocks metastasis of a tumor); preferably, the chemokine receptors comprise: CCR2 or CCR 7.

In another preferred embodiment, the cell also expresses a modulator (e.g., siRNA) that reduces PD-1 expression or a protein that blocks PD-L1.

In another preferred embodiment, the cell further expresses a safety switch; preferably, the safety switch comprises: iCaspase-9, Truanated EGFR or RQR 8.

In another aspect of the invention there is provided the use of a genetically modified immune effector cell as described in any one of the preceding paragraphs for the manufacture of a medicament for the inhibition of chronic lymphocytic leukaemia, Burkitt's lymphoma.

In another aspect of the invention, there is provided a pharmaceutical composition for inhibiting chronic lymphocytic leukemia or Burkitt's lymphoma comprising said genetically modified immune effector cells, and a pharmaceutically acceptable carrier or excipient.

In another aspect of the present invention, there is provided a kit for inhibiting chronic lymphocytic leukemia comprising: a container, and said pharmaceutical composition located in the container.

In another aspect of the present invention, there is provided a kit for inhibiting chronic lymphocytic leukemia comprising: a container, and the genetically modified immune effector cell located in the container.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

Fig. 1, a schematic molecular structure diagram of two CD32 b-specific Chimeric Antigen Receptors (CARs) in an embodiment of the invention.

FIG. 2 is a schematic diagram showing the structure of a lentiviral vector pCDH.

FIG. 3, CD32b CAR molecular enzyme cutting identification electrophoresis result.

Figure 4, results of flow cytometry to examine the efficiency of CAR-infected T cells.

Figure 5, CD32b expression levels in different tumor cell lines.

Figure 6, CD32b CAR-T release cytokine secretion.

Figure 7, results of CD32b CAR-T cells killing CD32b target cells in vitro.

FIG. 8A-B, CD32b CAR-T cell mice kill CD32b positive transplanted tumors in vivo.

FIGS. 9A-B, CD32B shows the expression positive rate (A) and expression density (B) in tumor cells of patients with chronic lymphocytic leukemia.

FIG. 10 ability of CD32b CAR-T cell body to kill tumor cells derived from C LL patient in animal body, wherein BM bone marrow, RB peripheral blood, SP spleen, L driver liver.

Detailed Description

The inventor of the invention, after extensive and intensive research, firstly discloses a Chimeric Antigen Receptor (CAR) targeting CD32b, an immune effector cell modified by the CAR, a preparation method thereof and application thereof in inhibiting chronic lymphocytic leukemia.

Term(s) for

As used herein, "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising an extracellular domain capable of binding an antigen, a transmembrane domain, and a cytoplasmic-to-cytoplasmic signaling domain (also referred to as an "intracellular signaling region"), wherein the intracellular signaling region comprises a functional signaling domain derived from a stimulatory molecule and/or a costimulatory molecule, e.g., a stimulatory molecule can be the ξ chain associated with a T cell receptor complex, and a costimulatory molecule can be 4-1BB (CD137) and/or CD 18.

As used herein, the term "immune cell" is used interchangeably with "immune effector cell" and includes: t lymphocytes, NK cells or NKT cells, etc.

As used herein, "single chain antibody (scFv) fragment" refers to an antibody fragment comprising the heavy chain variable region (VH) and the light chain variable region (V L) linked by a linker (linker) that associates the two domains to ultimately form an antigen binding site.

As used herein, "intracellular signaling domain" or "intracellular signaling region" or "intracellular signaling activation region" refers to the intracellular portion of a CAR molecule. The intracellular signaling domain produces a signal that can promote the immune effector function of CAR immune effector cells (e.g., CART cells). Examples of immune effector functions, such as in CART cells, include cytolytic activity and helper activity, including cytokine secretion.

As used herein, "specifically recognizes" means that the extracellular binding region of the invention (e.g., a single chain antibody) does not cross-react or substantially does not cross-react with any polypeptide other than the antigen of interest. The degree of specificity can be determined by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like. In the present invention, the specific recognition is preferably determined by flow cytometry, and the standard of specific recognition in a specific case can be judged by a person of ordinary skill in the art based on his or her knowledge in the art.

As used herein, the term "operably linked" or "operably linked" refers to a functional spatial arrangement of two or more nucleic acid/protein regions or nucleic acid/protein sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the term "construct" refers to a single-or double-stranded DNA molecule that has been artificially manipulated to contain DNA segments combined and arranged according to sequences not found in nature. The "construct" includes an expression vector; alternatively, the "construct" is included in an expression vector as part of the expression vector.

CD32b gene

Aiming at the refractory tumor of chronic lymphocytic leukemia, the inventor researches various tumor related genes in the early stage, finds that a large part of the genes are also expressed in normal cells of partial tissues and is difficult to be applied to CAR modified immune effector cell technology; some tumor specific genes have better tumor specific expression characteristics, but the CAR modified immune effector cells designed based on the tumor specific genes have no tumor cell killing activity or low activity, which is probably because the target can trigger tumor cells to secrete factors which have inhibitory effect on the immune effector cells. Through repeated examination and screening of a wide variety of genes, the inventors identified the CD32b gene as the target gene for designing CARs. The amino acid sequence of CD32b, disclosed in GenBank accession No. AF 543826.1.

The inventor simultaneously finds that the relatively high antigen density (diversity) of CD32b on C LL can enhance the tumor killing capacity of CAR-T, release more anti-tumor cytokines and improve the activation degree of CAR-T.

Currently, although CAR T cells have become a potential therapeutic approach, the selection of therapeutic targets has been difficult, and there are no reports on CAR T cell therapy for many tumors. In the early stage of the present inventors' study, it was not known whether CD32b could be successfully used against chronic lymphocytic leukemia. Since the spatial structure of proteins appears to be that they move the whole body in a single turn, many monoclonal antibodies evolve into single-chain antibodies, often losing antigen-binding activity or specificity. After intensive research, the inventors have determined that a single-chain antibody is relatively suitable. The inventors' studies indicate that CAR T cells composed of two single chain antibodies of the invention retain a highly efficient selective killing effect on CD32b positive cells.

The present examples demonstrate that CD32 b-targeted CAR-modified T cells can selectively clear CD32 b-positive tumor cells without toxicity to other cells (e.g., non-tumor cells). The CAR T cell aiming at the CD32b is an effective new means applied to the treatment of chronic lymphocytic leukemia.

Chimeric antigen receptors and nucleic acids encoding same

The invention provides a CAR expressed on the surface of an immune effector cell, said CAR comprising, linked in sequence: an extracellular binding domain, a transmembrane domain and an intracellular signaling domain. The CARs of the invention combine specific extracellular binding domains with intracellular signaling domains. Expression of the CAR on the surface of immune effector cells can result in immune effector cells having highly specific cytotoxic effects against tumors expressing CD32 b.

The extracellular binding region comprises a protein that specifically recognizes CD32 b. Expression of the CAR on the surface of immune effector cells can result in immune effector cells with highly specific cytotoxic effects on tumor cells that highly express CD32 b.

As a more preferred aspect of the invention, the antibody to human CD32b is the single chain antibody scFv1 (including SEQ ID NO:1(VH), SEQ ID NO:2(V L)) or scFv2 (including SEQ ID NO:3(VH), SEQ ID NO:4(V L)). preferably, the VH and V L are linked by a flexible linker, for example, a linker having the amino acid sequence GGGGSGGGGSGGS, forming an scFv sequence.

In the chimeric antigen receptor of the present invention, the single-chain antibody may be operatively linked to an intracellular signal region by operatively linking to a hinge region sequence and a transmembrane region sequence. In a preferred form of the invention, the hinge region is a CD8hinge region.

In a preferred embodiment of the invention, the transmembrane region is selected from the transmembrane region of CD8(CD8a) or CD28 the CD8hinge region (hinge) is a flexible region, and thus CD8 or CD28 and the transmembrane region plus the hinge region can be used to link the target recognition domain of the CAR to the scFv signaling region.

CD3 molecule is composed of five subunits, wherein CD3 zeta subunit (also called CD3 zeta, abbreviated Z) contains 3 ITAM motifs which are important signal transduction regions in TCR-CD3 complex. FcRI gamma is mainly distributed on the surface of mast cells and basophils and contains an ITAM motif which is similar in structure, distribution and function to CD3 zeta. furthermore, CD28, 4-1BB, CD134 are costimulatory signaling molecules whose intracellular signaling segments produce a costimulatory effect after binding to the respective ligands which leads to the sustained proliferation of immune effector cells (mainly T lymphocytes) and can increase the level of cytokines such as I L-2 and IFN-gamma secreted by immune effector cells, while increasing the CAR's survival cycle and antitumor effect in vivo.

In an embodiment of the invention, we provide a CAR (scFv1-CD8-4-1BB-CD3 ζ, scFv2-CD8-4-1BB-CD3 ζ) preferred by the inventors. It will also be understood that CARs with some alterations or modifications based on the optimized CARs of the invention may also be encompassed by the invention, e.g., a CAR of the invention may be selected from, but not limited to, being linked sequentially in the following specific manner:

scFv1 or 2-CD8-CD3 ζ,

scFv1 or 2-CD28a-CD28b-CD3 ζ,

scFv1 or 2-CD28a-CD28b-4-1BB-CD3 ζ.

Wherein, in a related CAR, CD8 comprises a CD8hinge region and a transmembrane region; CD28a represents the transmembrane region of the CD28 molecule, and CD28b represents the intracellular signaling region of the CD28 molecule.

As a preferred mode of the present invention, the sequence of the hinge region of CD8 is shown as SEQ ID NO. 5, the sequence of the transmembrane region is shown as SEQ ID NO. 6, the sequence of the intracellular signal region 4-1BB is shown as SEQ ID NO. 7, and the sequence of CD3 zeta is shown as SEQ ID NO. 8.

As one mode of the invention, the CAR further comprises a leader sequence N-terminal to the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

The invention also includes nucleic acids encoding the CARs. The nucleic acid sequences of the invention may be in the form of DNA or in the form of RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The nucleic acid codons encoding the CAR protein amino acid sequences of the invention can be degenerate, i.e., multiple degenerate nucleic acid sequences encoding the same amino acid sequence are included within the scope of the invention. Degenerate nucleic acid codons encoding the corresponding amino acids are well known in the art.

The invention also includes variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

Expression constructs and immune effector cells

The invention also provides genetically modified immune effector cells transduced with a nucleic acid of the invention or transduced with a recombinant plasmid of the invention comprising the nucleic acid, or a virus comprising the plasmid. The cell is a cell or an intercellular cell containing the cell, preferably a T cell or a cell group containing the T cell.

Nucleic acid transduction methods conventional in the art, including non-viral and viral transduction methods, can be used in the present invention. Non-viral based transduction methods include electroporation and transposon methods.

The invention also provides expression constructs (vectors), including viral vectors or non-viral vectors, comprising the above-described nucleic acids encoding chimeric antigen receptor proteins expressed on the surface of immune effector cells.

The transduction efficiency OF the non-viral vector system such as a Sleeping Beauty transposon (Sleeping Beauty system) or PiggyBac transposon system is greatly improved compared with that OF common electroporation, and the combination application OF a nuclear inductor transfectator and the Sleeping Beauty transposon system has been reported [ Davies JK., et al. combining CD19redirection and allo-orientation to generation child-specific human T cells for inductive cell therapy OF B-cell mallicines. cancer Res, 2010, 70(10): OF1-10 ], so that the method not only has higher transduction efficiency, but also can realize the targeted integration OF target genes. In addition, mRNA transfection techniques may be used.

In one particular embodiment of the invention, the backbone vector used in the invention is a lentiviral plasmid vector pCDH (more specifically pCDH-CMV-MCS-EF 1-Puro). in a pCDH vector, expression of the CAR molecule is driven by the EF1 promoter for transcriptional expression, and the CAR lentiviral vector plasmid is co-transfected with HEK293T cells in the presence of helper packaging plasmids Rev, VSV-G and pMD L, i.e., can be packaged as a lentivirus with the CAR molecule.

The invention also includes a virus packaged with the viral vector. The virus can be lentivirus, adenovirus, adeno-associated virus and the like, and also comprises viruses formed by further modifying the viruses. The virus of the present invention includes a packaged virus having infectivity, and also includes a virus to be packaged which contains components necessary for packaging the virus having infectivity. It is to be understood that although lentiviruses are preferred for use in the present invention, other viruses and their corresponding plasmid vectors known in the art for use in the transfer of foreign genes into immune effector cells may also be used in the present invention.

In one embodiment of the invention, the method of transduction of immune effector cells to achieve genetic modification of the chimeric antigen receptor is based on transduction of viruses such as lentiviruses. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, shortened time for in vitro culture of immune effector cells to reach clinical level, etc. On the surface of the transgenic immune effector cell, the transduced nucleic acid is expressed on its surface by transcription and translation. In vitro cytotoxicity experiments on various cultured tumor cells prove that the anti-CD 32b chimeric antigen receptor gene modified immune effector cells have high specific tumor cell killing effect (also called cytotoxicity). Thus, the nucleic acid encoding the chimeric antigen receptor protein, the plasmid containing the nucleic acid, the virus containing the plasmid and the transgenic immune effector cell transduced with the nucleic acid, the plasmid or the virus of the present invention can be effectively used for immunotherapy of tumors, particularly chronic lymphocytic leukemia.

The immune cell of the present invention may also carry coding sequence of exogenous cell factor, including but not limited to I L-12, I L-15 or I L-21, etc. these cell factors have immunoregulation or antitumor activity, and can strengthen the function of effector T cell and activated NK cell or exert antitumor effect directly.

The immune cells of the present invention may also express a chimeric antigen receptor other than the chimeric antigen receptor described above, which may not contain CD3 ζ but may contain the intracellular signaling domain of CD28, the intracellular signaling domain of CD137, or a combination thereof.

The immune cells of the invention may also express chemokine receptors; such chemokine receptors include, but are not limited to, CCR 2. It will be appreciated by those skilled in the art that the CCR2 chemokine receptor allows CCR2 in vivo to compete for binding, and is advantageous for blocking tumor metastasis.

The immune cell can also express siRNA capable of reducing PD-1 expression or protein capable of blocking PD-L1, and the skilled in the art can understand that competitive blocking of the interaction between PD-L1 and the receptor PD-1 thereof is beneficial to restoring anti-tumor T cell reaction, thereby inhibiting tumor growth.

The immune cells of the invention may also express a safety switch; preferably, the safety switch comprises: iCaspase-9, Truanated EGFR or RQR 8. As previously mentioned, current CA therapies are also challenged by the complexity of their production and by adverse events associated with cellular activity, such as cytokine storm (CRS) and the like. Therefore, drugs or therapies that are effective in modulating CAR-T, such as setting a safety switch, are more preferred.

In one embodiment of the invention, the genetically modified immune effector cell of the invention expresses a CAR on the surface, wherein the amino acid sequence of the CAR is shown as SEQ ID No. 9 and SEQ ID No. 10; more preferably it is encoded and expressed by the nucleic acid shown in SEQ ID NO. 11 or SEQ ID NO. 12.

In a specific embodiment of the invention, there is provided a method of constructing the CAR molecule described above into a lentiviral vector pCDH; means are provided for packaging the CAR molecule as a lentivirus, infecting T cells, and determining the efficiency of infection; provides for the construction of CD32b target cells demonstrating the ability of CAR-T cells to kill CD32b target cells; and, demonstrating the ability of CAR-T cells to inhibit the growth of CD32b positive tumors in animals.

In one example, the inventors incubated CD32b CAR-T cells with the CD32b expressing tumor cell line Raji and the results showed that CD32b CAR-T cells were able to produce IFN- γ, I L-2 and TNF- α in large amounts under stimulation of target cells this example demonstrates the good specific targeting of CD32b CAR-T cells, being able to activate T cell killing function under CD32b stimulation.

In another example, the inventors demonstrated that CD32 bCR-T cells can effectively kill CD32b positive tumor cells at a certain effective target ratio (CAR-T cells: target cells). This example demonstrates that CD32b CAR-T is able to effectively lyse CD32b positive tumor cells in vitro.

In another example, the inventors constructed NSG (NOD-Prkdc) using the CD32b positive tumor cell line Raji^scidIL2rg^tm1/Bcgen) immunodeficient animals subcutaneous transplantation tumor model. The results of treating the tumor-bearing animals with the inventive CD32b CAR-T cell scFv1-BBZ demonstrated that CD32b CAR-T cells can effectively eliminate CD32b positive tumor cells, and that the treated group of animals survived all 30 days; however, tumors in the control pCDH group grew continuously and no animals survived at 30 days.

Pharmaceutical composition

The genetically modified immune effector cells of the invention may be used in the preparation of compositions, in particular pharmaceutical compositions. The composition may comprise, in addition to an effective amount of the immune effector cell, a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, e.g.

Wetting agents, such as sodium lauryl sulfate; a colorant; seasoningAn agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like.

The compositions of the present invention may be formulated into various dosage forms as desired, and may be administered by a physician in a dosage amount beneficial to the patient, depending on such factors as the type, age, weight and general condition of the patient, the mode of administration, and the like. Administration may be by injection or other therapeutic means, for example.

The immune effector cells of the invention or compositions containing the cells may also be placed in a suitable kit for use by a clinician. Preferably, the kit may further comprise instructions for use of the compositions of the present invention.

Compared with the prior art, the invention has the following beneficial effects:

the current CAR-T technology for treating chronic lymphocytic leukemia targets CD19 primarily, but CD19CAR-T for clinical treatment of chronic lymphocytic leukemia has a complete remission rate of less than 30%, which may be related to the molecular density of CD19 expression on chronic lymphocytic leukemia tumor cells. The invention provides a novel target CD32b targeting chronic lymphocytic leukemia through CAR-T technology, the protein has wide expression on tumor cells of patients with chronic lymphocytic leukemia, has high expression strength, and has higher antigen density expression compared with CD 19. The CD32b CAR molecule can be used for treating CD32b positive chronic lymphocytic leukemia tumor by modifying T cells and enabling specific CAR-T cells to be highly effective and specific.

The invention overcomes the defects in the prior art, provides a novel CAR molecule through targeting CD32b and proves the effect of the CAR molecule in the treatment of chronic lymphocytic leukemia, and the CAR molecule of the invention endows T cells with the capability of killing tumor cells with CD32b target by targeting CD32b protein on the surface of tumor cells of lymphocytic leukemia and activating signal channels downstream of the T cells through the targeting CD32b protein, thereby providing a brand-new therapeutic means for tumor treatment.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions, such as those described in molecular cloning guidelines, written by J. SammBruk et al, or according to the manufacturer's recommendations.

Example 1 construction of expression vectors for CD32b CAR molecules

In this example, a lentiviral vector expressing a CAR molecule was constructed, using a lentiviral vector pCDH as a backbone plasmid, the structure of which is shown in fig. 2.

Based on the differences in CAR molecules scFv, the inventors constructed two CD32b CAR molecules, scFv1-BBZ and scFv 2-BBZ: wherein the scFv1 and the scFv2 are humanized antibodies of CD32 b. The molecular structure pattern of scFv1-BBZ and scFv2-BBZ is shown in FIG. 1. The CAR molecule as above was constructed into the lentiviral vector pCDH by conventional molecular cloning means.

scFv1-BBZ vector construction scFv1-BBZ nucleic acid fragment was first chemically synthesized into pUC57 vector (Beijing Huada protein Bio Inc.), and digested by XmaI/SalI double digestion to obtain scFv1-BBZ fragment (SEQ ID NO: 11). For example, scFv1 is shown in SEQ ID NO:1(VH), SEQ ID NO:2(V L). For example, BBZ contains CD8hinge (SEQ ID NO:5), CD8TM (SEQ ID NO:6), 4-1BB (SEQ ID NO:7) and CD3 ζ (SEQ ID NO: 8). XmaI/SalI double digestion lentiviral vector pCDH, after electrophoresis the DNA fragment of about 7KB size was recovered from the gel.scFv 1-BBZ vector pCDH was ligated with T4DNA ligase at room temperature for 1-2 hours, followed by transformation of the plasmid named competent cell on the next day, amplified and cultured on L B plasmid on the next day, and the plasmid sequence of BBI/Sal DNA was extracted, and the correct sequence of the digested plasmid was verified by further digestion of the plasmid of the second plasmid pCDH, whether the sequence was obtained.

scFv2-BBZ vector construction, firstly, chemically synthesizing scFv2-BBZ nucleic acid fragment into pUC57 vector (Beijing Hua big protein biology, Ltd.), obtaining scFv2-BBZ fragment (SEQ ID NO:12) by XmaI/SalI double digestion, wherein, the sequence of scFv2 is as SEQ ID NO:3(VH), SEQ ID NO:4(V L). XmaI/SalI double digestion lentiviral vector pCDH, recovering DNA fragment about 7KB size after electrophoresis, connecting scFv2-BBZ and vector pCDH by T4DNA ligase at room temperature for 1-2 hours, then transforming stbl3 competent cells, inoculating single colony to L B amplification culture on the next day, extracting, verifying whether obtained is correct by XmaI/SalI double digestion, and obtaining the digestion result shown in figure 3, third lane, sending the correct plasmid, further verifying whether the sequence of plasmid is correct, sequencing correct sequence of the plasmid pCDH 24-BBZ 2-2.

The sequence structure of the scFv described above is summarized in Table 1.

TABLE 1

Example 2 construction of CD32b CAR-T cells

In this example, the inventors constructed CD32b CAR-T cells.

CAR lentivirus packaging firstly, the lentivirus plasmids pCDH-scFv1-BBZ, pCDH-scFv2-BBZ and pCDH (not containing CD32b CAR control empty vector plasmid) constructed in the example one and the lentivirus system helper packaging plasmids Rev, VSV-G and pMD L are respectively extracted by using MACHEREY-NAGE L endotoxin-free large quality-improving plasmid kit, and 1 × 10 is added into the whole day before transfection⁷One 293T was plated into T75 flasks. The 293T cell medium was changed to 5ml serum-free DMEM medium 20 minutes before transfection. Plasmids were co-transfected into 293T cells using PEI transfection reagent and the cell culture medium was changed to 10ml at 6 hours post transfectionComplete medium DMEM + 10% FBS. Cell supernatants were harvested 48 hours post transfection and 10ml of fresh complete medium was added. Cell supernatants were harvested again for 72 hours and discarded. Centrifuging the collected cell supernatant at 3000rpm for 15min, removing precipitate, filtering the supernatant with 0.45 μm filter membrane, adding 40% PEG-6000 solution to reach concentration of 10%, mixing well, and standing overnight at 4 deg.C. The next day the virus was pelleted by centrifugation at 1500g for 1 hour and resuspended using 0.2ml serum-free T cell culture medium. Standing at-80 deg.C for freezing.

T cell infection by taking blood by a conventional method and isolating T cells using STEMCE LL CD3 sorting kit, adding 0.1g/ml of I L-2 to a serum-free T cell medium and culturing, and stimulating with anti-CD 3/CD28 antibody 48 hours after starting the culture, suspending the activated T cells in the T cell medium, adding the corresponding lentivirus (scFv1-BBZ, scFv2-BBZ or pCDH), then culturing the T cells at 37 ℃ for 8-12 hours, then replacing the cells with a T cell medium containing 0.1g/ml of I L-2, 0.01g/ml of I L-7/I L-15/I L-21, staining the infected T cells with Fc-coupled CD32b protein plus anti-flow Fc antibody 48 hours after the cell replacement, and then detecting the efficiency of molecular infection by flow cytometry.

As shown in FIG. 4, the positivity of scFv1-BBZ and scFv2-BBZ on T cells was observed at approximately 60% -70%, and pCDH was used as a blank control.

Example 3 determination of CD32b CAR-T cell Activity

In this example, assays for CD32b CAR-T cell activity were performed.

1. Identification and construction of CD32b CAR-T target cells

CD32b expression was detected on both human chronic lymphocytic leukemia Mec-1 cell line and Burkitt's lymphoma Raji cell line using CD32b-APC flow antibody.

As shown in FIG. 5, Raji cell line highly expressed CD32b gene, Raji cell line highly expressed GFP-L uciferase gene was constructed by sorting GFP positive cells using lentiviral vector encoding GFP-L uciferase gene, and the expression levels of CD32b and GFP-L uciferase of Raji cell line are shown in FIG. 5.

According to fig. 5, the Raji cell line is positively expressed as CD32 b.

2. Cytokine secretion assay

Either scFv1-BBZ CAR-T cells or control pCDH T cells (effector cells) were mixed with target cells Raji in a 1:1 effective target ratio in 96-well plates. After incubation at 37 ℃ for 24 hours, the expression of each cytokine was examined using flow cytometry.

As shown in FIG. 6, when two CD32 b-targeted CAR-T cells were co-cultured with Raji, the CAR-T cells secreted large amounts of cytokines such as IFN-. gamma.I L-2 and TNF- α.

Example 4 ability of CD32b CAR-T cell bodies to kill CD32b target cells

In this example, the inventors validated the ability of CD32b CAR-T cell bodies to kill CD32b target cells.

Two CD32b CAR-T cells (scFv1-BBZ or scFv2-BBZ) and control T cells (pCDH) were separately combined with CD32b⁺The Raji cells of (1: 5), 1:2, 1:1, 5:1 effective target ratio were seeded in 96-well plates, after 24h of culture, tumor cells were labeled with CD32b flow antibody, apoptotic and necrotic tumor cells were labeled with 7-AAD flow antibody, and cell number was calculated with precision count Beads. The killing efficiency of CAR-T cells against Raji cells was calculated and the results are shown in figure 7. scFv1-BBZCAR-T and scFv2-BBZ CAR-T cells were able to kill Raji cells efficiently, but control pCDH cells were not able to kill both target cells efficiently.

The results show that CAR-T cells constructed by two CAR molecules, namely scFv1-BBZ and scFv2-BBZ, can kill Raji tumor cells positive to CD32b with high efficiency.

Example 5 ability of CAR-T cells to inhibit growth of CD32 b-positive tumors in a mouse model

In this example, the inventors demonstrated the ability of CAR-T cells to inhibit the growth of CD32 b-positive tumors in a mouse model.

Using 5-8 weeks old NSG (NOD-Prkdc)^scidIL2rg^tm1/Bcgen) immunodeficient mice, tail vein injection 3 × 10⁵L uciferase expressing Raji cells, allowed to grow for 5 days, anesthetized mice, and used to test the tumor burden in mice with xenogenisis-200 living body imager, confirming that the tumor burden is smallAfter tumorigenesis in mice, 1 × 10 was injected through tail vein⁶Individual CD32b CAR-T cell scFv1-BBZ or a blank control pCDH were followed by in vivo imaging every 7 days to detect tumor burden in vivo and to record survival.

Results as shown in fig. 8A and 8B, tumors were cleared in mice 7 days after scFv1-BBZ CAR-T cell injection, and survival of all mice was extended to more than 30 days. However, T cells of the blank pCDH were unable to clear tumor cells, and no mice survived at 30 days in this group.

The above results demonstrate that CD32 b-targeted CAR-T cells of the invention are able to efficiently eliminate tumors in a mouse CD32b positive tumor transplant tumor model.

Example 6 detection of the expression of the CD19 and CD32b antigens on tumor cells of patients with Chronic lymphocytic leukemia

The inventor obtains 47 peripheral blood tumor cells of chronic lymphocytic leukemia patients from clinic and detects the antigen expression of CD19 and CD32b on the tumor cells by flow cytometry. As shown in FIG. 9A, the positive rate of CD32b expression in chronic lymphocytic leukemia patients was substantially 100% and slightly higher than the expression of CD19 on tumor cells of chronic lymphocytic leukemia patients.

The inventors also examined the antigen expression density (site intensity) of CD32b and CD19 on the surface of tumor cells derived from these patients with chronic lymphocytic leukemia by flow cytometry. As shown in FIG. 9B, the antigen expression density of CD32B was higher in most patients with chronic lymphocytic leukemia than in CD 19. The high antigen expression density is beneficial to better activating the CAR-T cells, so that the CAR-T cells obtain better killing capability and expansion capability under the stimulation of the antigen.

Example 7 ability of CD32b CAR-T cell bodies to kill tumor cells derived from C LL patients in animals

After 1.5cGy irradiation of 5-8 weeks old NSG mice for 12-24h, 2-4 × 10 is infused⁷PBMC (peripheral blood mononuclear cells) from C LL patients, 48h examined tumor burden in peripheral blood with greater than 1% CD32b⁺The cells are considered to be tumorous and subsequent experiments can be performed.

The tumorigenic animals were grouped, 3 mice per group, infused with 5 × 10⁵After 15-18 days, each organ tissue (including bone marrow, peripheral blood, spleen and liver) of the mouse is taken, and CAR-T and tumor conditions are detected in a flow mode.

As a result, as shown in FIG. 10, the amount of T cells in the scFv1-CAR-T group was significantly higher than that in the control group, and the amount of tumor cells was reduced to 0. This result demonstrates that CD32 b-targeted CAR-T cells of the invention are able to eliminate tumor cells in mice with great efficiency.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala

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Arg Asn Gly Asp Ser Asp Tyr Tyr Ser Gly Met Asp Tyr Trp Gly Gln

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Gly Thr Thr Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro

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Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

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Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

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Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

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Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

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Ser Ser Met Asp Ser Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val

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Gly Gly Ile Ile Pro Ala Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

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Ala Arg Glu Gln Asp Pro Glu Ser Gly Tyr Gly Gly Tyr Pro Tyr Glu

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Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr

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Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

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Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

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Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

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Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

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Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

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Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

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Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

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Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

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Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

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Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

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Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

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Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

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gtgaaggtct cctgcaaggc ttctggttac acctttacca actactggat acactgggtg 540

cgacaggccc ctggacaagg gcttgagtgg atgggagtga ttgatccttc tgatacttat 600

ccaaattaca ataaaaagtt caagggcaga gtcaccatga ccacagacac atccacgagc 660

acagcctaca tggagctgag gagcctgaga tctgacgaca cggccgtgta ttactgtgcg 720

agaaacggtg attccgatta ttactctggt atggactact gggggcaagg gaccacggtc 780

accgtctcct caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960

ggggtccttc tcctgtcact ggttatcacc ctttactgca aacggggcag aaagaaactc 1020

ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1080

tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1140

aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1200

ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1260

gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440

atgcaggccc tgccccctcg ctaa 1464

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atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgagctacg aactgaccca gccgctgagc gtgagcgtgg ccctgggcca gaccgcgagg 120

attacctgta gcggcgataa catcccgcag cattctgttc attggtacca gcagaaaccg 180

ggccaggcgc cggtgctggt gatctacgac gacactgaac gtccgagcgg catcccggaa 240

cgttttagcg gttcgaacag cggcaacacc gcgaccctga ccattagcag ggcccaggcg 300

ggcgacgaag cggattatta ctgctcttct tgggactctt ctatggactc tgttgtgttt 360

ggcggcggca cgaagttaac cgtcctaggt ggcggtggct cgggcggtgg tgggtcgggt 420

ggcggcggat ctcaggtgca attggtgcag agcggtgccg aagtgaaaaa accgggcagc 480

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gtgcgccagg ccccaggcca gggcctcgag tggatgggcg gtatcatccc ggctttcggc 600

actgcgaact acgcccagaa atttcagggc cgggtgacca ttaccgccga tgaaagcacc 660

agcaccgcct atatggaact gagcagcctg cgcagcgaag atacggccgt gtattattgc 720

gcgcgtgaac aggacccgga aagcggttac ggtggttacc cgtatgaagc tatggatgtt 780

tggggccaag gcaccctggt gactgttagc tcaaccacga cgccagcgcc gcgaccacca 840

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 960

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1020

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1080

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gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 1200

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1260

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1320

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1380

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1440

gacacctacg acgcccttca catgcaggcc ctgccccctc gctaa 1485

Claims (13)

1. A chimeric antigen receptor comprising, connected in sequence: an extracellular binding region, a transmembrane region, and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes CD32 b.

2. The chimeric antigen receptor according to claim 1, wherein said protein that specifically recognizes CD32b is an antibody; preferably, the antibody is a single chain antibody or a domain antibody; more preferably, the antibody is a single chain antibody, the heavy chain of which has the amino acid sequence shown in SEQ ID NO. 1 and the light chain of which has the amino acid sequence shown in SEQ ID NO. 2, or the heavy chain of which has the amino acid sequence shown in SEQ ID NO. 3 and the light chain of which has the amino acid sequence shown in SEQ ID NO. 4.

3. The chimeric antigen receptor according to claim 1, wherein the transmembrane region is a sequence comprising a hinge region and a transmembrane region of CD8 or CD 28; preferably, the transmembrane region is a sequence comprising the hinge region and the transmembrane region of CD 8; more preferably, the hinge region has an amino acid sequence shown in SEQ ID NO. 5, and the transmembrane region has an amino acid sequence shown in SEQ ID NO. 6; or

The intracellular signaling region comprises an intracellular signaling region sequence selected from 4-1BB, CD3 ζ, FcRIγ, CD27, CD28, CD134, ICOS, GITR, or a combination thereof; preferably, the intracellular signaling region comprises 4-1BB and CD3 ζ; more preferably, the 4-1BB has the amino acid sequence shown in SEQ ID NO. 7, and the CD3 zeta has the amino acid sequence shown in SEQ ID NO. 8.

4. The chimeric antigen receptor according to claim 1, wherein said chimeric antigen receptor comprises an extracellular binding domain, a transmembrane domain and an intracellular signaling domain connected in this order as follows: a single chain antibody that specifically recognizes CD32b, a CD8hinge region, a CD8 transmembrane region, 4-1BB, and CD3 ζ; preferably, the chimeric antigen receptor has an amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10; preferably, the immune effector cells comprise cells selected from the group consisting of: t lymphocytes, NK cells or NKT cells.

5. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1 to 4; preferably, it has the nucleotide sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12.

6. An expression vector comprising the nucleic acid of claim 5.

7. A virus comprising the expression vector of claim 6.

8. Use of the chimeric antigen receptor of any one of claims 1 to 4, the nucleic acid of claim 5, the expression vector of claim 6, or the virus of claim 7 for the preparation of genetically modified immune effector cells targeted to CD32 b.

9. A genetically modified immune effector cell transduced with the nucleic acid of claim 5, the expression vector of claim 6 or the virus of claim 7; or, which expresses on its surface the chimeric antigen receptor of any one of claims 1 to 4.

10. The immune effector cell of claim 9, comprising a cell selected from the group consisting of: t lymphocytes, NK cells or NKT cells; or

The cell also expresses a cytokine having a property selected from the group consisting of immunomodulatory activity or antitumor activity and an immune effector cell-enhancing function, preferably, the cytokine comprises I L-12, I L-15, I L-21, I L-2, I L-4, I L-7, I L-9, I L-17, I L-18, I L-23, or

The cell also expresses a chemokine receptor; preferably, the chemokine receptors comprise: CCR2 or CCR 7; or

The cell also expresses a molecule that reduces the expression of PD-1 or a protein that blocks PD-L1, or

The cell also expresses a safety switch; preferably, the safety switch comprises: iCaspase-9, Truanated EGFR or RQR 8.

11. Use of the genetically modified immune effector cell of claim 9 in the preparation of a medicament for inhibiting chronic lymphocytic leukemia, Burkitt's lymphoma.

12. A pharmaceutical composition for inhibiting chronic lymphocytic leukemia or Burkitt's lymphoma comprising the genetically modified immune effector cell of claim 9 and a pharmaceutically acceptable carrier or excipient.

13. A kit for inhibiting chronic lymphocytic leukemia, comprising: a container, and the pharmaceutical composition of claim 11 in the container; or

Which comprises the following steps: a container, and the genetically modified immune effector cell of any one of claims 9 to 10 located in the container.

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