CN111499766B - 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 certain effects 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. 344:149-72]. In recent years, based on the finding that the recognition specificity of Cytotoxic T Lymphocytes (CTL) for target cells depends on T lymphocyte receptors (TCR), scFv of an antibody against a tumor Cell-associated antigen is fused with intracellular signal activation motifs such as CD3 ζ or fceriy of T lymphocyte receptors to form a Chimeric Antigen Receptor (CAR), and is genetically modified on the surface of T lymphocytes by means of, for example, lentiviral infection. Such CAR T lymphocytes are able to selectively target T lymphocytes to tumor cells and specifically kill tumors in a non-limiting manner with a Major Histocompatibility Complex (MHC). CAR T cells are an immunotherapy strategy with good prospects in the field of tumor immunotherapy.

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.

There are many difficulties with current treatments for lymphoid malignancies, particularly recurrence and refractory diseases that are often encountered clinically. Chronic Lymphocytic Leukemia (CLL) is a clinical multi-line therapy that cannot be cured, and neither traditional drug chemotherapy nor emerging small molecule inhibitors can completely eliminate tumors. Therefore, more advanced therapeutic approaches, such as CAR-T cell therapy, need to be explored for CLL high risk patients and patients with multiple relapses. CD19 is one of the cluster differentiation antigens, is an important membrane antigen involved in B cell proliferation, differentiation, activation and antibody production, and is specifically expressed on the surface of B lymphocytes at different differentiation stages, and CD19 antigen is expressed in more than 95% of B cell lymphomas and B lymphocyte leukemias. At present, the CD19CAR-T achieves ideal treatment effect on the acute lymphoblastic leukemia of children, and can induce more than 90% of patients with Acute Lymphoblastic Leukemia (ALL) to completely relieve. However, it is surprising that although CD19 is ubiquitously expressed on CLL tumor cells, multicenter clinical trials have shown that CD19CAR-T can only induce remission in about 26% of CLL patients, which is very different from ALL treatment. This phenomenon, together with the indication that CLL patients require a different treatment regimen than ALL, also suggests that CAR-T treatment efficacy cannot be determined considering only tumor cells indicating high expression of antigen.

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 a suitable gene for CAR-based therapy, and in the extensive research work in this field, many tumor-specific antigens have been 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 by adverse events associated with cellular activity, such as cytokine storms (CRS) and the like. There are currently no reports of CAR T cell therapy for many tumors

In conclusion, the art needs to find a more ideal antigenic molecule for CLL disease 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, fceRI γ, CD27, CD28, CD134, ICOS, GITR, or a combination thereof; preferably, the intracellular signaling region comprises 4-1BB and CD3 zeta; 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 CD32 b-targeted genetically modified immune effector cell.

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 a cytokine having a characteristic selected from the group consisting of: has immunoregulatory activity or antitumor activity, and has the function of enhancing immune effector cells; preferably, the cytokines include (but are not limited to): IL-12, IL-15, IL-21, IL-2, IL-4, IL-7, IL-9, IL-17, IL-18, IL-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 CCR7.

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, truancated EGFR or RQR8.

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 in 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 cell, 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

Figure 1, a schematic representation of the molecular structure of two CD32 b-specific Chimeric Antigen Receptors (CARs) in the examples of the invention.

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

FIG. 3, CD32b CAR molecular cleavage identification electrophoresis results.

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

FIG. 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.

FIGS. 8A-B, results of CD32B CAR-T cell mice killing CD32B positive transplanted tumors in vivo.

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

Figure 10, ability of CD32b CAR-T cell bodies to kill CLL patient-derived tumor cells in animals; wherein BM: bone marrow; RB peripheral blood; SP: spleen; liver: the 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)

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 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 co-stimulatory molecule. For example, the stimulatory molecule may be the ξ chain associated with the T cell receptor complex and the co-stimulatory molecule may be 4-1BB (CD 137) and/or CD18.

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 a heavy chain variable region (VH) and a light chain variable region (VL) linked by a linker (linker) that associates the two domains to ultimately form an antigen binding site. Single chain antibodies are preferably a sequence of amino acids encoded by a single nucleotide chain. The single chain antibodies used in the present invention may be further modified using conventional techniques known in the art, such as amino acid deletions, insertions, substitutions, additions, and/or recombinations and/or other modifications, either alone or in combination.

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 immune effector function of a CAR immune effector cell (e.g., a CAR T cell). 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 location 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, and thus, 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 that do not occur in nature. The "construct" includes an expression vector; alternatively, the "construct" is included in an expression vector as part of an 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, possibly because the target can trigger tumor cells to secrete factors which have an inhibitory effect on the immune effector cells. Through repeated investigation and screening of a wide variety of genes, the present inventors identified the CD32b gene as the target gene for designing CARs. The amino acid sequence of CD32b, disclosed in GenBank accession No. AF543826.1.

The inventor finds that the antigen density of CD32b on CLL is obviously higher than that of molecules such as CD19, the expression is more extensive, and the CD32b is a very ideal CAR-T therapeutic target. The inventors also found that relatively high antigen density (site diversity) of CD32b on CLL enhances CAR-T tumor killing, releases more anti-tumor cytokines, and increases CAR-T activation. On the basis, the benzene inventors further carry out optimized design and provide an anti-CD 32b CAR-T targeting a new target CD32b, which can kill CLL tumor cells more effectively.

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 studies by the present inventors, it was not known whether CD32b could be successfully used against chronic lymphocytic leukemia. Because the spatial structure of the protein appears to be moving in tandem and moving the whole body, many monoclonal antibodies evolve into single-chain antibodies, and the antigen binding activity or specificity is lost. 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 highly specific cytotoxic effects of immune effector cells on CD32b expressing tumors.

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 highly specific cytotoxic effects of immune effector cells on CD32 b-highly expressing tumor cells.

In a preferred embodiment of the present invention, the extracellular binding region is a single chain antibody (scFv). The scFv is functional. As a more preferred mode of the present invention, the antibody against human CD32b is a single chain antibody scFv1 (comprising SEQ ID NO:1 (VH), SEQ ID NO:2 (VL)) or scFv2 (comprising SEQ ID NO:3 (VH), SEQ ID NO:4 (VL)). Preferably, the VH and VL are linked by a flexible linker, for example a linker having the amino acid sequence ggsggggsggggs, to form 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 embodiment of the present invention, the hinge region is a CD8hinge region.

The transmembrane region of the CAR may be selected from the transmembrane regions of proteins such as CD8 or CD 28. The human CD8 protein is a heterodimer, consisting of two chains, either α β or γ δ. In a preferred embodiment of the invention, the transmembrane region is selected from the transmembrane region of CD8 (CD 8 a) or CD 28. 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 scFv of the CAR to the intracellular signaling region.

The intracellular signaling region can be selected from the group consisting of intracellular signaling regions of CD3 ζ, fc ε RI γ, CD27, CD28,4-1BB, CD134, ICOS, GITR proteins, and combinations thereof. The CD3 molecule consists of five subunits, where the CD3 zeta subunit (also known as CD3 zeta, Z for short) contains 3 ITAM motifs, which are important signaling regions in the TCR-CD3 complex. Fcsri γ is distributed primarily on the surface of mast cells and basophils, contains an ITAM motif and is similar in structure, distribution and function to CD3 ζ. In addition, CD28,4-1BB, CD134 is a costimulatory signaling molecule, whose costimulation by the intracellular signaling segment upon binding to the respective ligand causes the sustained proliferation of immune effector cells (mainly T lymphocytes) and is capable of increasing the level of cytokines such as IL-2 and IFN- γ secreted by immune effector cells, as well as increasing the survival cycle and antitumor effect of CAR immune effector cells in vivo.

In embodiments of the invention, we provide a preferred CAR of the present inventors (scFv 1-CD8-4-1BB-CD3 ζ, scFv2-CD8-4-1BB-CD3 ζ). It will also be understood that CARs with some alterations or modifications based on the optimized CARs of the invention may also be included in the invention, e.g., the CARs of the invention may be selected from, but are 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 in SEQ ID NO. 5, the sequence of the transmembrane region is shown in SEQ ID NO. 6, the sequence of the intracellular signal region 4-1BB is shown in SEQ ID NO. 7, and the sequence of CD3 zeta is shown in 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 form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially changing the function of the encoded polypeptide.

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 combined application OF a nuclear inductor transfectator and the Sleeping Beauty transposon system has been reported [ Davies JK., et al. Combining CD19 redirection and amplification to generation stator-specific human T cells for inductive cell therapy OF B-cell amplification, cancer Res,2010, 70 (10): OF1-10 ], so that the method not only has higher transduction efficiency, but also can realize the site-directed integration OF target genes. In addition, mRNA transfection techniques may be used.

Various vectors for viral packaging can be used in the present invention, including, for example, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and the like, as well as viral vectors formed by further engineering on the basis of these viral vectors. In a particular embodiment of the invention, the backbone vector used in the invention is the lentiviral plasmid vector pCDH (more specifically pCDH-CMV-MCS-EF 1-Puro). In pCDH vectors, expression of the CAR molecule is driven by the EF1 promoter for transcriptional expression, and the CAR lentiviral vector plasmids are co-transfected with HEK293T cells in the presence of helper packaging plasmids Rev, VSV-G and pMDL, i.e., can be packaged as lentiviruses with the CAR molecule. It is further understood that although pCDH vectors are preferred in the present invention, other types of vectors are also useful, as long as an active CAR and immune effector cells modified therefrom are ultimately obtained.

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 known in the art and their corresponding plasmid vectors useful for 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 cytotoxic experiments on various cultured tumor cells prove that the immune effector cells modified by the anti-CD 32b chimeric antigen receptor genes have a highly 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 can also carry the coding sequence of exogenous cell factors; such cytokines include, but are not limited to: IL-12, IL-15, IL-21, and the like. These cytokines have immunomodulatory or anti-tumor activity, and can enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects. Thus, it will be appreciated by those skilled in the art that the use of these cytokines may contribute to the better functioning of the immune cells.

The immune cell 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 an intracellular signaling domain of CD28, an 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, CCR2. It will be appreciated by those skilled in the art that the CCR2 chemokine receptor allows CCR2 in vivo to competitively bind with it, which 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. It will be appreciated by those skilled in the art that competitive blockade of the interaction of PD-L1 with its receptor PD-1 facilitates restoration of an anti-tumor T cell response, 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 RQR8. 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 effectively modulate 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 its surface, the amino acid sequence of the CAR is shown in 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 particular 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 CD32b positive tumor growth in animals.

In one example, the inventors co-incubate CD32b CAR-T cells with a CD32b expressing tumor cell line Raji, and the results show that CD32b CAR-T cells are capable of producing IFN- γ, IL-2 and TNF- α in large amounts upon stimulation of target cells. This example demonstrates the good specific targeting of CD32b CAR-T cells, capable of activating T cell killing function under CD32b stimulation.

In another example, the inventors demonstrated that CD32b CAR-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 CD32 b-positive tumor cell line Raji ^scid IL2rg ^tm1 /Bcgen) immunodeficient animals subcutaneous transplantation tumor model. The results of treating the tumor-bearing animals with the CD32b CAR-T cell scFv1-BBZ of the invention show that the CD32b CAR-T cells can effectively eliminate CD32b positive tumor cells, and the animals in the treatment group all survive for 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 carboxymethylcellulose, ethyl cellulose and methyl cellulose; powdered 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; a flavoring 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. The administration may be, for example, by injection or other therapeutic means.

The immune effector cells of the invention or compositions containing the cells may also be placed in a suitable kit to facilitate 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 target of CAR-T technology for the treatment of chronic lymphocytic leukemia is mainly CD19, but the complete remission rate of CD19CAR-T for clinical treatment of chronic lymphocytic leukemia is 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 for targeting chronic lymphocytic leukemia through CAR-T technology, and the protein has wide expression on tumor cells of patients with chronic lymphocytic leukemia, high expression intensity and higher antigen density expression compared with CD19. The CD32b CAR molecule can be used for treating CD32b positive chronic lymphocytic leukemia tumors by modifying T cells and enabling specific CAR-T cells to be efficient and specific.

The art has not yet provided CAR T targeting CD32b, and the present inventors succeeded for the first time in finding a target gene CD32b suitable for CAR T cells from among a number of tumor-associated genes, and successfully generated CAR T cells targeting CD32 b. The present invention overcomes the deficiencies of the prior art by providing a novel CAR molecule that targets CD32b and demonstrates its role in the treatment of chronic lymphocytic leukemia. The CAR molecule of the invention endows the T cell with the capacity of killing tumor cells with CD32b targets by targeting CD32b protein on the surface of the lymphocyte leukemia tumor cells and activating a downstream signal channel of the T cell, thereby providing a brand-new treatment means for tumor treatment. In the present examples, the CAR T cells established in the present invention exhibited very excellent killing effect on CLL tumor cells.

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 scFv2-BBZ: wherein scFv1 and scFv2 are humanized antibody of CD32 b. The molecular structural 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.

Construction of scFv1-BBZ vector: first, the scFv1-BBZ nucleic acid fragment was chemically synthesized into a pUC57 vector (Beijing Huada protein biol., ltd.) and subjected to double digestion with XmaI/SalI to obtain the scFv1-BBZ fragment (SEQ ID NO: 11). Wherein, the scFv1 has the sequence shown as SEQ ID NO 1 (VH) and SEQ ID NO 2 (VL); 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 enzyme digestion of lentiviral vector pCDH, electrophoresis, gel recovery of about 7KB size DNA fragment. The scFv1-BBZ and the vector pCDH were ligated using T4DNA ligase at room temperature for 1-2 hours, followed by transformation of stbl3 competent cells. The next day, a single colony is inoculated to LB amplification culture, and plasmids are extracted. The XmaI/SalI double digestion was used to verify the correctness of the resulting plasmid, see FIG. 3, lane two. The correct plasmid is sent to sequence, and whether the sequence is correct or not is further verified. The plasmid with the correct sequence was named pCDH-scFv1-BBZ.

Construction of scFv2-BBZ vector: first, chemically synthesizing scFv2-BBZ nucleic acid fragment into pUC57 vector (Beijing Huada protein biology, ltd.), and obtaining scFv2-BBZ fragment (SEQ ID NO: 12) by XmaI/SalI double digestion; wherein, the scFv2 has the sequence shown in SEQ ID NO. 3 (VH) and SEQ ID NO. 4 (VL). XmaI/SalI double digestion of lentiviral vector pCDH, gel recovery of about 7KB DNA fragment after electrophoresis. scFv2-BBZ was ligated with vector pCDH using T4DNA ligase at room temperature for 1-2 hours, followed by transformation of stbl3 competent cells. The next day, a single colony is inoculated to LB amplification culture, and plasmids are extracted. The XmaI/SalI double digestion was used to verify the correctness of the resulting plasmid, see FIG. 3, third lane. The correct plasmid is sent to sequence, and whether the sequence is correct or not is further verified. The plasmid with the correct sequence was named pCDH-scFv2-BBZ.

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.

Packaging of CAR lentivirus: first, the lentiviral plasmids pCDH-scFv1-BBZ, pCDH-scFv2-BBZ and pCDH (CD 32b CAR-free control empty vector plasmid) and lentiviral system helper packaging plasmids Rev, VSV-G and pMDL constructed in example one were extracted respectively using MACHEREY-NAGEL endotoxin-free large quality-improving plasmid kit. 1X 10 day before transfection ⁷ One 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 complete medium DMEM +10% FBS 6 hours after transfection. 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. And (5) placing at-80 ℃ for freezing storage for later use.

T cell infection: blood was collected by a conventional method and T cells were separated by STEMCELL CD3 sorting kit, cultured by adding 0.1g/ml of IL-2 to a serum-free T cell culture medium, and stimulated with anti-CD 3/CD28 antibody. 48 hours after the start of the culture, the activated T cells were suspended in T cell medium and the corresponding lentivirus (scFv 1-BBZ, scFv2-BBZ or pCDH) was added. The T cells were then cultured at 37 ℃ for 8-12 hours. The cells were then plated out with T cell media containing 0.1g/ml IL-2,0.01g/ml IL-7/IL-15/IL-21. After 48 hours, infected T cells were stained with Fc-conjugated CD32b protein plus anti-Fc flow antibody, and then the CAR molecule infection efficiency was examined using flow cytometry.

As a result, as shown in FIG. 4, the positive rates of scFv1-BBZ and scFv2-BBZ on T cells were observed at approximately 60% to 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-APC flow antibodies were used to detect CD32b expression on both human chronic lymphocytic leukemia Mec-1 cell line and Burkitt's lymphoma Raji cell line.

As a result, as shown in FIG. 5, the Raji cell line highly expressed the CD32b gene. And (3) using a lentiviral vector for encoding the GFP-Luciferase gene, sorting GFP positive cells, and constructing a Raji cell line of the high-table GFP-Luciferase gene. The expression levels of CD32b and GFP-Luciferase in the Raji cell line are shown in FIG. 5.

According to FIG. 5, the Raji cell line is positively expressed for CD32 b.

2. Cytokine secretion assay

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

The results are 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., IL-2 and TNF-. Alpha..

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

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

Two CD32b CAR-T cells (scFv 1-BBZ or scFv 2-BBZ) and control T cells (pCDH) were contacted with CD32b, respectively ⁺ The Raji cells of (a) are cultured in a medium at an effective target ratio of1, 5,1,1, 1,5. The killing efficiency of CAR-T cells against Raji cells was calculated and the results are shown in figure 7.scFv1-BBZ CAR-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 for CD32b efficiently.

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

In this example, the inventors demonstrated the ability of CAR-T cells to inhibit CD32b positive tumor growth in a mouse model.

NSG (NOD-Prkdc) with an age of 5 to 8 weeks was used ^scid IL2rg ^tm1 /Bcgen) immunodeficient mice, 3X 10 by tail vein injection ⁵ The tumor growth of each Raji cell expressing Luciferase was allowed to grow for 5 days, and after anesthetizing the mice, the tumor burden in the mice was examined using a xenogenic IVIS-200 in vivo imager, confirming that tumors had formed in the mice, and then 1X 10 cells were injected via the tail vein ⁶ Individual CD32b CAR-T cells scFv1-BBZ or blank 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 30 days or more. However, T cells from the blank control pCDH were unable to clear tumor cells, and no mice survived in this group at 30 days.

The above results demonstrate that CD32 b-targeting CAR-T cells of the invention are able to efficiently eliminate tumors in a mouse CD32 b-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 the peripheral blood tumor cells of 47 chronic lymphocytic leukemia patients from clinic and detects the antigen expression of CD19 and CD32b on the tumor cells by flow cytometry. As a result, 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 than that of CD19 in most patients with chronic lymphocytic leukemia. 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 CLL patient-derived tumor cells in animals

After 1.5cGy irradiation of 5-8 week-old NSG mice for 12-24h, 2-4X 10 infusion ⁷ PBMC (peripheral blood mononuclear cells) from one CLL patient, 48h to detect 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 × 10 ⁵ 15-18 days later, taking each organ tissue (including bone marrow, peripheral blood, spleen and liver) of the mouse, and carrying out flow detection on the CAR-T and the tumor condition.

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 very high efficiency.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or 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 appended claims of the present application.

Sequence listing

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Val Leu Val Ile Tyr Asp Asp Thr Glu Arg Pro Ser Gly Ile Pro Glu

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Arg Ala Gln Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Trp Asp

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

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Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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

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cgacaggccc ctggacaagg gcttgagtgg atgggagtga ttgatccttc tgatacttat 600

ccaaattaca ataaaaagtt caagggcaga gtcaccatga ccacagacac atccacgagc 660

acagcctaca tggagctgag gagcctgaga tctgacgaca cggccgtgta ttactgtgcg 720

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gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320

aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440

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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

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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

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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 (24)

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 CD32b, and the protein that specifically recognizes CD32b is an antibody; the antibody is a single-chain antibody, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain is shown as SEQ ID NO. 2, or the amino acid sequence of the heavy chain is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain is shown as SEQ ID NO. 4.

2. 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.

3. The chimeric antigen receptor according to claim 2, wherein the transmembrane region is a sequence comprising a hinge region and a transmembrane region of CD 8.

4. The chimeric antigen receptor according to claim 3, wherein the amino acid sequence of the hinge region is shown in SEQ ID NO. 5, and the amino acid sequence of the transmembrane region is shown in SEQ ID NO. 6.

5. The chimeric antigen receptor of claim 1, wherein the intracellular signaling region is selected from the group consisting of an intracellular signaling region sequence of 4-1bb, cd3 ζ, fcepsilonray, CD27, CD28, CD134, ICOS, GITR, or combinations thereof.

6. The chimeric antigen receptor according to claim 5, wherein the intracellular signaling region is 4-1BB and CD3 ζ.

7. The chimeric antigen receptor according to claim 6, wherein the amino acid sequence of 4-1BB is shown in SEQ ID No. 7 and the amino acid sequence of CD3 ζ is shown in SEQ ID No. 8.

8. 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 linked in the following order: a single chain antibody that specifically recognizes CD32b, a CD8hinge region, a CD8 transmembrane region, 4-1BB, and CD3 zeta.

9. The chimeric antigen receptor according to claim 8, wherein the amino acid sequence of said chimeric antigen receptor is represented by SEQ ID No. 9 or SEQ ID No. 10.

10. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1 to 9.

11. The nucleic acid of claim 10, having a nucleotide sequence as set forth in SEQ ID No. 11 or SEQ ID No. 12.

12. An expression vector comprising the nucleic acid of claim 10 or 11.

13. A virus comprising the expression vector of claim 12.

14. Use of a chimeric antigen receptor or a nucleic acid encoding the same for the preparation of a genetically modified immune effector cell targeted to CD32 b; wherein said chimeric antigen receptor comprises, 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 CD32b, and the protein that specifically recognizes CD32b is an antibody; the antibody is a single-chain antibody, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain is shown as SEQ ID NO. 2, or the amino acid sequence of the heavy chain is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain is shown as SEQ ID NO. 4.

15. A genetically modified immune effector cell transduced with the nucleic acid of claim 10 or 11, the expression vector of claim 12 or the virus of claim 13; or, the surface of which expresses the chimeric antigen receptor of any one of claims 1 to 9.

16. The immune effector cell of claim 15, selected from the group consisting of: t lymphocytes, NK cells or NKT cells.

17. The immune effector cell of claim 15, wherein the cell further expresses a cytokine having immunomodulatory or anti-tumor activity that enhances the function of the immune effector cell; the cytokine is selected from: IL-12, IL-15, IL-21, IL-2, IL-4, IL-7, IL-9, IL-17, IL-18 or IL-23.

18. The immune effector cell of claim 15, wherein the cell further expresses a chemokine receptor; the chemokine receptor is selected from the group consisting of: CCR2 or CCR7.

19. The immune effector cell of claim 15, wherein the cell further expresses a molecule that reduces PD-1 expression or a protein that blocks PD-L1.

20. The immune effector cell of claim 15, wherein the cell further expresses a safety switch; the safety switch is selected from: iCaspase-9, truanated EGFR or RQR8.

21. Use of a genetically modified immune effector cell of any one of claims 15 to 20 in the manufacture of a medicament for inhibiting chronic lymphocytic leukemia or Burkitt's lymphoma.

22. A pharmaceutical composition for inhibiting chronic lymphocytic leukemia or Burkitt's lymphoma comprising the genetically modified immune effector cell of any one of claims 15 to 20 and a pharmaceutically acceptable carrier or excipient.

23. A kit for inhibiting chronic lymphocytic leukemia comprising: a container, and the pharmaceutical composition of claim 22 in the container.

24. A kit for inhibiting chronic lymphocytic leukemia comprising: a container, and a genetically modified immune effector cell of any one of claims 15 to 20 positioned in the container.

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