CAR treatment method for treating acute myelogenous leukemia and application thereof

Abstract

The invention provides a CAR treatment method for treating acute myelogenous leukemia and application of the CAR treatment method. The invention optimizes and establishes a chimeric antigen receptor (CAR) aiming at acute myelogenous leukemia (AML). The chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signal region and an X molecule which are connected in sequence. The invention also provides an immune effector cell modified by the CAR. The invention also discloses a preparation method of the compound and application of the compound in inhibition of acute myelogenous leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of medical biology, and more specifically, the present invention relates to a CAR treatment method for treating acute myeloid leukemia and its application. Background Art

[0002] Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy associated with a wide range of molecular alterations, with high mortality, low cure rate and poor prognosis. Acute myeloid leukemia (AML) is the most common acute leukemia in adults. It is a disease characterized by clonal proliferation from hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs). In the past 40 years, the traditional upfront treatment of AML remains chemotherapy (intensive induction and consolidation chemotherapy) and hematopoietic cell transplantation (HCT). Although most patients respond to chemotherapy, the 5-year overall survival rate for patients under 60 years old can only reach 40%. For patients over 60 years old, it is less than 20%. As the median age at diagnosis is over 70 years old, many elderly AML patients are not suitable for Allo-HCT because they have severe complications, including fatal infections and graft-versus-host disease. All of this highlights the urgency of improving the poor outcomes of acute myeloid leukemia treatment.

[0003] Long-term disease control requires multiple treatments. Conventional treatments are ineffective for patients with relapsed or refractory (r / r) AML, and hematopoietic stem cell transplantation (HSCT) is currently the only curative method. However, many patients still relapse after transplantation or have poor treatment effects.

[0004] A suitable target antigen for CAR-T cell therapy should be highly and uniformly expressed on tumor cells, but absent in normal tissues of most patients. Even after transcriptomic and proteomic analysis of AML cells, it is difficult to find ideal antigens on the surface of AML cells for CAR-T therapy. CAR-T targeting AML cells usually shows severe bone marrow suppression and cytotoxicity to hematopoietic stem cells / whole blood cells. Chimeric antigen receptor (CAR) T cells targeting CD19 have successfully treated patients with relapsed / refractory B-cell acute lymphoblastic leukemia (ALL) and B-cell lymphoma. However, CAR T cell therapy has not been so successful in preclinical and early clinical studies targeting acute myeloid leukemia (AML). This can be partly attributed to the presence of the AML microenvironment, which has an inhibitory effect on the anti-tumor activity of CAR T cells.

[0005] In recent years, several different targeted therapies have been developed, including immune checkpoint inhibitors, T cell receptor-modified T cell therapy, and chimeric antigen receptor-T cell therapy. Among them, chimeric antigen receptor (CAR)-T cell therapy has recently been studied in the treatment of B-cell acute lymphoblastic leukemia (B-ALL).

[0006] There are currently a variety of preclinical studies and clinical trials using CAR-T cell therapy to treat AML, but the efficacy is poor compared to the treatment of B-ALL. In the currently reported clinical trials, CAR-T treatment of (r / r) AML patients for multiple target antigens (including several used in the subsequent present invention: CD33, CD123, CLL1) has a poor cure rate and poor in vivo activity.

[0007] Therefore, the poor efficacy of CAR T cells in the treatment of AML does not seem to be attributed to a specific antigen, but rather represents a general phenomenon. Although several mechanisms have been reported in the literature to explain why myeloid leukemia cells are poorly responsive to CAR T cell therapy, including antigen escape and suppressive microenvironment, the exact molecular mechanisms behind this mechanism remain unknown, and there are currently no established strategies to overcome these obstacles.

[0008] In summary, this field urgently needs to find better ways and methods to change the current situation and obtain effective treatment approaches for AML, a unique cancer disease. Summary of the invention

[0009] The purpose of the present invention is to provide a CAR treatment method for treating acute myeloid leukemia and its application.

[0010] In a first aspect of the present invention, a use of a genetically modified immune effector cell is provided for preparing a drug for inhibiting (including alleviating or treating) acute myeloid leukemia; the genetically modified immune effector cell is transduced with a polynucleotide encoding a chimeric antigen receptor or a chimeric antigen receptor expressed on its surface; the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signaling region and an X molecule connected in sequence; wherein the extracellular binding region comprises a binding protein that specifically recognizes CLL1, CD33 or CD123; wherein the X molecule is c-Jun.

[0011] In one or more embodiments, the X molecule enhances the inhibitory effect of the gene-modified immune effector cells on acute myeloid leukemia and prolongs the survival of the subject (patient).

[0012] In one or more embodiments, the X molecule does not act by counteracting the exhaustion of immune effector cells in acute myeloid leukemia.

[0013] In one or more embodiments, for acute myeloid leukemia, the X molecule does not significantly affect the expression of memory-related genes.

[0014] In one or more embodiments, for acute myeloid leukemia, the X molecule upregulates the expression of cytokines and co-stimulatory molecules by activating the ERK signal of T cells, thereby reversing the low activation state of CAR T cells and promoting the killing and expansion of CAR-T cells.

[0015] In one or more embodiments, the binding protein that specifically recognizes CLL1, CD33 or CD123 is an antibody.

[0016] In one or more embodiments, the antibody is a single chain antibody or a domain antibody.

[0017] In one or more embodiments, the antibody is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0018] In one or more embodiments, the transmembrane region is a sequence comprising the hinge region and the transmembrane region of CD8 or CD28; preferably, the transmembrane region is a sequence comprising the hinge region and the transmembrane region of CD8 (such as CD8hinge-CD8 TM); more preferably, the hinge region amino acid sequence is as shown in SEQ ID NO:5, and the transmembrane region amino acid sequence is as shown in SEQ ID NO:6.

[0019] In one or more embodiments, the intracellular signaling region includes an intracellular signaling region sequence selected from 4-1BB, CD3ζ, FcεRIγ, CD27, CD28, CD134, ICOS, GITR, or a combination thereof; preferably, the intracellular signaling region includes 4-1BB and CD3ζ; more preferably, the 4-1BB amino acid sequence is as shown in SEQ ID NO:7, and the CD3ζ amino acid sequence is as shown in SEQ ID NO:8.

[0020] In one or more embodiments, the chimeric antigen receptor comprises the following sequentially connected extracellular binding region, transmembrane region and intracellular signaling region: single chain antibody of CLL1, CD33 or CD123, CD8 hinge region or transmembrane region, 4-1BB, CD3ζ and X molecule.

[0021] In one or more embodiments, the intracellular signaling region includes a fusion of 4-1BB and CD3ζ, which is a mutant whose amino acid sequence is shown in SEQ ID NO:10.

[0022] In one or more embodiments, the immune effector cells include cells selected from the group consisting of T lymphocytes, NK cells, or NKT cells.

[0023] In another aspect of the present invention, a chimeric antigen receptor (CAR) or a nucleic acid encoding the same is provided, wherein the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signaling region and an X molecule connected in sequence; wherein the extracellular binding region comprises a binding protein that specifically recognizes CLL1, CD33 or CD123; wherein the X molecule is c-Jun.

[0024] In one or more embodiments, the binding protein that specifically recognizes CLL1, CD33 or CD123 is an antibody; preferably, the antibody is a single-chain antibody or a domain antibody; more preferably, the antibody is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0025] In one or more embodiments, the intracellular signaling region includes a fusion of 4-1BB and CD3ζ, which is a mutant whose amino acid sequence is shown in SEQ ID NO:10.

[0026] In one or more embodiments, the present invention also provides an expression vector comprising the nucleic acid.

[0027] In one or more embodiments, the expression vector uses pCDH as a backbone plasmid.

[0028] In one or more embodiments, the intracellular signaling region is connected to the X molecule via a linker, and preferably the linker comprises P2A or a similar molecule.

[0029] In one or more embodiments, the present invention further provides a virus, wherein the virus (such as a lentivirus) comprises the expression vector, or is obtained by packaging the expression vector.

[0030] In another aspect of the present invention, a genetically modified immune effector cell is provided, which inhibits (including alleviating or treating) acute myeloid leukemia, and the cell is transduced with a polynucleotide encoding a chimeric antigen receptor or a chimeric antigen receptor expressed on its surface; the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signaling region and an X molecule connected in sequence; wherein the extracellular binding region comprises a binding protein that specifically recognizes CLL1, CD33 or CD123; wherein the X molecule is c-Jun.

[0031] In one or more embodiments, the binding protein that specifically recognizes CLL1, CD33 or CD123 is an antibody; preferably, the antibody is a single-chain antibody or a domain antibody; more preferably, the antibody is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0032] In one or more embodiments, the intracellular signaling region includes a fusion of 4-1BB and CD3ζ, which is a mutant whose amino acid sequence is shown in SEQ ID NO:10.

[0033] In one or more embodiments, the genetically modified immune effector cells also express cytokines, and the cytokines have characteristics selected from the following group: having immunomodulatory activity or anti-tumor activity, and having the function of enhancing immune effector cells.

[0034] In one or more embodiments, 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.

[0035] In one or more embodiments, the cell also expresses a chemokine receptor (which blocks tumor metastasis); preferably, the chemokine receptor includes: CCR2 or CCR7.

[0036] In one or more embodiments, the cell also expresses an inhibitory molecule (such as siRNA) that can reduce the expression of PD-1 or a protein that blocks PD-L1.

[0037] In one or more embodiments, the cell further expresses a safety switch; preferably, the safety switch comprises: iCaspase-9, Truancated EGFR or RQR8.

[0038] In another aspect of the present invention, a method for preparing a genetically modified immune effector cell is provided, comprising: introducing a polynucleotide encoding a chimeric antigen receptor into an immune effector cell to express the chimeric antigen receptor on the cell surface; the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signaling region and an X molecule connected in sequence; wherein the extracellular binding region comprises a binding protein that specifically recognizes CLL1, CD33 or CD123; wherein the X molecule is c-Jun.

[0039] In another aspect of the present invention, the intracellular signal region includes a fusion of 4-1BB and CD3ζ, which is a mutant and its amino acid sequence is shown in SEQ ID NO:10.

[0040] In another aspect of the present invention, a pharmaceutical composition for inhibiting (including alleviating or treating) acute myeloid leukemia or a medicine kit containing the pharmaceutical composition is provided, wherein the pharmaceutical composition comprises the genetically modified immune effector cells and a pharmaceutically acceptable pharmaceutical carrier or excipient.

[0041] In one or more embodiments, the medicine kit includes: a container, and the pharmaceutical composition in the container.

[0042] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 , schematic diagram of the structure of each element of CD33 / CLL1 / CD123 bbz-X vector.

[0044] Figure 2 , three The infection efficiency of CAR proteins of various CAR-T cells was analyzed, and the empty plasmid was used as a control.

[0045] Figure 3 , the effect of X-linked CAR-T cells on the survival of mice after administration to tumor-bearing mice, with those without X-linked or empty plasmid as controls.

[0046] Figure 4 CAR-T cells linked to X were administered to tumor-bearing mice and then used for in vivo imaging to detect tumor burden, with CAR-T cells not linked to X or with empty plasmid as controls.

[0047] Figure 5 , in vitro culture and phosphorylation analysis of CD33-bbz-X.

[0048] Figure 6 、CD33bbz MT -X's in vivo anti-tumor ability is demonstrated by in vivo imaging and survival time. DETAILED DESCRIPTION

[0049] The present inventors have optimized and established a chimeric antigen receptor (CAR) for acute myeloid leukemia (AML) through extensive and in-depth research and screening, and immune effector cells modified by the CAR. The present invention also discloses its preparation method and its application in inhibiting acute myeloid leukemia.

[0050] Acute myeloid leukemia is a malignant disease characterized by the accumulation of abnormal primitive cells and has a high mortality rate. Approximately 40-45% of young AML patients and 10-20% of elderly AML patients can be cured after standard chemotherapy regimens. However, the prognosis of patients with refractory or relapsed (r / r) AML is poor, with an overall survival rate of less than 10% after 3 years. The main strategy for treating r / r AML is allogeneic stem cell transplantation (SCT). However, patients who do not achieve complete remission before SCT have a very poor prognosis.

[0051] CAR T therapy has achieved certain efficacy in curing B-ALL, but it is very ineffective in treating AML. CART cannot achieve effective expansion, and AML shows resistance to CAR T therapy. Therefore, there is a great clinical need to obtain a new CAR T drug that can treat refractory recurrent AML.

[0052] the term

[0053] As used in the present invention, "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising an extracellular domain capable of binding to 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 a ξ chain associated with a T cell receptor complex, and the co-stimulatory molecule may be 4-1BB (CD137) and / or CD28.

[0054] As used in the present invention, the "immune cells" and "immune effector cells" are used interchangeably, and include: T lymphocytes, NK cells or NKT cells, etc.

[0055] As used in the present invention, "single-chain antibody (scFv) fragment" refers to an antibody fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker, which associates these two domains to ultimately form an antigen binding site. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. The single-chain antibody used in the present invention can be further modified using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination and / or other modification methods, alone or in combination.

[0056] As used in the present invention, "intracellular signaling domain" or "intracellular signaling region" or "intracellular signaling activation region" refers to the intracellular portion of the 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 cell lytic activity and auxiliary activity, including cytokine secretion.

[0057] As used in the present invention, "specific recognition" means that the extracellular binding region (such as a single-chain antibody) of the present invention does not or substantially does not cross-react with any polypeptide other than the target antigen. The degree of specificity can be determined by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometric analysis, etc. In the present invention, specific recognition is preferably determined by flow cytometry, and the standard of specific recognition in specific cases can be determined by a person skilled in the art based on the common knowledge in the art.

[0058] As used in the present invention, the term "operably linked" or "operably connected" refers to the functional spatial arrangement of two or more nucleic acid / protein regions or nucleic acid / protein sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence, so that transcription of the nucleic acid sequence is guided by the promoter region, and thus the promoter region is "operably linked" to the nucleic acid sequence.

[0059] As used herein, the "construct" refers to a single-stranded or double-stranded DNA molecule that has been artificially intervened to contain DNA fragments combined and arranged according to a sequence that does not exist in nature. The "construct" includes an expression vector; or, the "construct" is contained in an expression vector as a part of the expression vector.

[0060] Target antigen gene

[0061] For acute myeloid leukemia, a refractory non-solid tumor with special properties, the inventors have previously investigated a variety of tumor-related genes and found that a considerable portion of these genes are also expressed in normal cells of some tissues, which are difficult to apply to CAR-modified immune effector cell technology; some tumor-specific genes have good tumor-specific expression characteristics, but the CAR-modified immune effector cells designed based on them have no tumor cell killing activity or very low activity, which may be because the target can induce tumor cells to secrete factors that inhibit immune effector cells. After repeated investigation and screening of a variety of genes, the inventors identified CLL1, CD33 or CD123 as target antigens for designing CAR. The amino acid sequence of CLL1 is disclosed in GenBank Accession No. AAT11783.1; the amino acid sequence of CD33 is disclosed in GenBank Accession No. AAK83654.1; the amino acid sequence of CD123 is disclosed in GenBank Accession No. AAA59148.1.

[0062] The inventors found that CLL1, CD33 or CD123 can be ideal CAR-T therapeutic targets when used in the preparation of CAR effector cells for acute myeloid leukemia in the present invention; among them, the targeted therapeutic effect of CLL1 is particularly excellent, and its synergistic effect after combination with X molecule is the most significant, and some animals can maintain long-term survival; secondly, the synergistic effect of CD33 after combination with X molecule also shows a multiple increase.

[0063] Since the spatial conformation of proteins affects the whole body, many monoclonal antibodies evolve into single-chain antibodies and often lose their antigen binding activity or specificity. After determining the target antigen, the inventors optimized and determined relatively suitable single-chain antibodies after in-depth research, and their amino acid sequences are listed in Table 1 of the embodiment. The inventors' research shows that the CAR T cells composed of the two single-chain antibodies described in the present invention retain the highly efficient selective killing effect on antigen-positive cells.

[0064] The examples of the present invention demonstrate that the CAR-modified T cells of the present invention can highly selectively eliminate CLL1, CD33 or CD123-positive tumor cells. The CAR T cells of the present invention targeting CLL1, CD33 or CD123 are a new method with high efficiency, low toxicity and strong targeting for the treatment of acute myeloid leukemia.

[0065] Chimeric antigen receptor and nucleic acid encoding same

[0066] The present invention provides a CAR expressed on the surface of an immune effector cell, wherein the CAR comprises an extracellular binding region, a transmembrane region, an intracellular signal region and an X molecule connected in sequence. The CAR of the present invention combines specific extracellular binding regions and intracellular signal regions, and further connects to the X molecule. The CAR is expressed on the surface of immune effector cells, so that the immune effector cells can have a highly specific cytotoxic effect on tumors expressing CLL1, CD33 or CD123. The amino acid sequence of the X molecule is listed in Table 1 of the embodiment. It should be understood that the present invention also includes variants of the X molecule with the same function.

[0067] In the present invention, the X molecule is c-Jun. After the X molecule is connected with other elements constituting CAR, it can effectively exert inhibitory activity, and the synergistic effect of the X molecule is very significant.

[0068] The inventors discovered for the first time that the X molecule has a unique effect on acute myeloid leukemia, which exerts an inhibitory effect by enhancing the genetically modified immune effector cell acute myeloid leukemia and prolongs the survival of the subject (patient). The inventors' research results also confirmed that for acute myeloid leukemia, the X molecule does not work by resisting the exhaustion of immune effector cells; the X molecule also does not significantly affect the expression of memory-related genes. Different from these effects, the X molecule upregulates the expression of cytokines and co-stimulatory molecules by activating the ERK signal of T cells, thereby reversing the low activation state of CAR T cells and promoting the killing and amplification of CAR-T cells. This reflects that for acute myeloid leukemia, a refractory disease, the X molecule promotes the inhibitory effect of immune effector cells on the disease through a unique effect.

[0069] The inventors found through sequencing that the dysfunction of CAR T cells exposed to myeloid tumors was not caused by CART exhaustion. The overexpression of X did not play the role of resisting CART cell exhaustion as reported, and did not significantly affect the expression of memory-related genes. Through in-depth exploration of the mechanism of action of molecule X, the inventors found that X did not resist CART cell exhaustion in the AML tumor microenvironment, but activated the ERK signal of T cells to upregulate the expression of cytokines and co-stimulatory molecules, thereby reversing the low activation state of CAR T cells and restoring the killing and amplification of CAR-T cells. This new mechanism is the first disclosure of the inventors and provides theoretical support for its greater clinical application.

[0070] The extracellular binding region comprises a protein that specifically recognizes CLL1, CD33 or CD123. The CAR is expressed on the surface of immune effector cells, so that the immune effector cells have a highly specific cytotoxic effect on tumor cells that highly express CLL1, CD33 or CD123.

[0071] As a preferred embodiment of the present invention, the extracellular binding region is a single-chain antibody (scFv). The scFv is functional. Usually, the single-chain antibody is prepared by preparing a heavy chain variable region (VH) and a light chain variable region (VL), and then operatively connecting them. Preferably, the VH and VL are connected by a flexible linker.

[0072] In the chimeric antigen receptor of the present invention, the single-chain antibody can be operably linked to the hinge region sequence and the transmembrane region sequence, and then operably linked to the intracellular signaling region. In a preferred embodiment of the present invention, the hinge region is the CD8 hinge region.

[0073] The transmembrane region of CAR can be selected from the transmembrane region of proteins such as CD8 or CD28. Human CD8 protein is a heterodimer composed of two chains of αβ or γδ. In a preferred embodiment of the present invention, the transmembrane region is selected from the transmembrane region of CD8 (CD8a) or CD28. The CD8 hinge region is a flexible region, so CD8 or CD28 and the transmembrane region plus the hinge region can be used to connect the target recognition domain scFv of CAR to the intracellular signaling region.

[0074] The intracellular signal region can be selected from the intracellular signal region of CD3ζ, FcεRIγ, CD27, CD28, 4-1BB, CD134, ICOS, GITR protein, and a combination thereof. The CD3 molecule consists of five subunits, of which the CD3ζ subunit (also known as CD3 zeta, referred to as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. FcεRIγ is mainly distributed on the surface of mast cells and basophils, which contains an ITAM motif, similar to CD3ζ in structure, distribution and function. In addition, CD28, 4-1BB, and CD134 are co-stimulatory signal molecules. After binding to their respective ligands, the co-stimulatory effect generated by their intracellular signal segments causes the continuous proliferation of immune effector cells (mainly T lymphocytes), and can increase the level of cytokines such as IL-2 and IFN-γ secreted by immune effector cells, while improving the survival cycle and anti-tumor effect of CAR immune effector cells in vivo.

[0075] In the embodiments of the present invention, the preferred CARs of the present inventors are provided (scFv1-CD8-4-1BB-CD3ζ, scFv2-CD8-4-1BB-CD3ζ). In addition, it should be understood that, based on the optimized CAR of the present invention, CARs with some changes or modifications may also be included in the present invention.

[0076] As one aspect of the present invention, CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during cellular processing and localization of CAR to the cell membrane.

[0077] The present invention also includes nucleic acids encoding the CAR. The nucleic acid sequence of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The nucleic acid codons encoding the amino acid sequence of the CAR protein of the present invention may be degenerate, that is, a variety of degenerate nucleic acid sequences encoding the same amino acid sequence are included in the scope of the present invention. Degenerate nucleic acid codons encoding corresponding amino acids are well known in the art.

[0078] The present invention also includes variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. The variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it.

[0079] Expression constructs and immune effector cells

[0080] The present invention also provides a genetically modified immune effector cell, which is transduced with the nucleic acid of the present invention or the recombinant plasmid containing the nucleic acid or the virus containing the plasmid. The cell is a cell or a cell containing the cell, preferably a T cell or a cell population containing a T cell.

[0081] Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used in the present invention. Non-viral transduction methods include electroporation and transposon methods.

[0082] The present invention also provides an expression construct (vector) comprising the above-mentioned nucleic acid encoding the chimeric antigen receptor protein expressed on the surface of immune effector cells, including a viral vector or a non-viral vector.

[0083] The non-viral vector system, such as the Sleeping Beauty system or the PiggyBac transposon system, has a significantly higher transduction efficiency than conventional electroporation. The combined application of the nucleofector transfection instrument and the Sleeping Beauty transposon system has been reported [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10):OF1-10.] This method has both high transduction efficiency and can achieve site-specific integration of the target gene. In addition, mRNA transfection technology can also be used.

[0084] A variety of vectors for viral packaging can be applied to the present invention, including, for example, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc., and also include viral vectors formed after further transformation on the basis of these viral vectors. In a specific embodiment of the present invention, the backbone vector used in the present invention is a lentiviral plasmid vector pCDH. In the pCDH vector, the expression of the CAR molecule is driven by transcriptional expression by the EF1 promoter, and the CAR lentiviral vector plasmid is co-transfected with HEK293T cells in the presence of auxiliary packaging plasmids Rev, VSV-G and pMDL, and can be packaged as a lentivirus with a CAR molecule. In addition, it should be understood that although the pCDH vector is preferred in the present invention, other types of vectors are also available, as long as active CAR and immune effector cells modified therefrom can be ultimately obtained.

[0085] The present invention also includes viruses packaged from viral vectors. The viruses may be lentiviruses, adenoviruses, adeno-associated viruses, etc., and also include viruses formed after further modification on the basis of these viruses. The viruses of the present invention include viruses with infectious power after packaging, and also include viruses to be packaged that contain the necessary components for packaging as infectious viruses. It should be understood that although lentiviruses are preferably used in the present invention, other viruses known in the art that can be used to transduce exogenous genes into immune effector cells and their corresponding plasmid vectors can also be used in the present invention.

[0086] In one embodiment of the present invention, the transduction method of the immune effector cell modified by the chimeric antigen receptor gene is based on the transduction method of virus such as slow virus. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, and can shorten the time for in vitro cultured immune effector cells to reach clinical grade quantity. On the surface of the transgenic immune effector cell, the transduced nucleic acid is expressed on its surface by transcription and translation. By carrying out in vitro cytotoxic experiments on various cultured tumor cells, it is proved that the immune effector cell modified by the chimeric antigen receptor gene of the present invention has a highly specific tumor cell killing effect (also known as cytotoxicity). Therefore, the nucleic acid encoding the chimeric antigen receptor protein of the present invention, the plasmid comprising the nucleic acid, the virus comprising the plasmid and the transduced genetically modified immune effector cell with the above-mentioned nucleic acid, plasmid or virus can be effectively used for the immunotherapy of acute myeloid leukemia.

[0087] The immune cells of the present invention may also carry coding sequences of exogenous cytokines; the cytokines include but are not limited to: IL-12, IL-15 or IL-21. These cytokines have immunomodulatory or anti-tumor activities, can enhance the functions of effector T cells and activated NK cells, or directly exert anti-tumor effects. Therefore, those skilled in the art can understand that the use of these cytokines helps the immune cells to function better.

[0088] The immune cells of the present invention may also express another chimeric antigen receptor in addition to the above-mentioned chimeric antigen receptor, which may not contain CD3ζ but contains the intracellular signaling domain of CD28, the intracellular signaling domain of CD137, or a combination of the two.

[0089] The immune cells of the present invention may also express chemokine receptors, including but not limited to CCR2. Those skilled in the art will appreciate that the CCR2 chemokine receptor can allow CCR2 in the body to competitively bind to it, which is beneficial for blocking tumor metastasis.

[0090] The immune cells of the present invention may also express siRNA that can reduce the expression of PD-1 or a protein that blocks PD-L1. It will be appreciated by those skilled in the art that competitively blocking the interaction between PD-L1 and its receptor PD-1 is beneficial for restoring anti-tumor T cell responses, thereby inhibiting tumor growth.

[0091] The immune cells of the present invention may also express a safety switch; preferably, the safety switch includes: iCaspase-9, Truancated EGFR or RQR8. As mentioned above, CAR therapy is currently challenged by the complexity of its production and adverse events associated with cell activity, such as cytokine storm (CRS). Therefore, drugs or therapies that can effectively regulate CAR-T, such as setting a safety switch, are more preferred.

[0092] In a specific embodiment of the present invention, the inventor screened out molecule X through in vivo experiments, which has a significantly enhanced anti-tumor effect compared with the control group and significantly prolonged the survival of mice. This shows that overexpression of X has a very strong and sustained effect in restoring the anti-AML activity of CAR T cells.

[0093] Pharmaceutical composition

[0094] The genetically modified immune effector cells of the present invention can be used to prepare compositions, particularly pharmaceutical compositions. In addition to an effective amount of the immune effector cells, the composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that when the molecular entities and compositions are appropriately administered to animals or humans, they do not produce adverse, allergic or other adverse reactions.

[0095] Specific examples of some substances that can 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, ethylcellulose and methylcellulose; tragacanth powder; 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; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.

[0096] The composition of the present invention can be prepared into various dosage forms as required, and the physician can determine the dosage that is beneficial to the patient according to factors such as the patient type, age, weight, general disease condition, and administration method. The administration method can be, for example, injection or other treatment methods.

[0097] The immune effector cells of the present invention or the composition containing the cells can also be placed in an appropriate medicine box for the convenience of use by clinicians. Preferably, the medicine box can also contain instructions for use of the composition of the present invention.

[0098] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0099] Example 1. Construction of CD33 / CLL1 / CD123 bbz-X vector

[0100] For acute myeloid leukemia (AML), the inventors conducted in-depth analysis and screening, using CD33, CLL1, and CD123 as targets for CAR.

[0101] The nucleotide sequences of the target fragments CD33 scFv, CLL1 scFv, and CD123 scFv were synthesized, loaded into the pUC57 vector and sequence verified. The corresponding amino acid sequence structures are summarized in Table 1.

[0102] Table 1

[0103]

[0104]

[0105] The synthesized target fragment was cut from the pUC57 vector by restriction endonuclease (Xmal / Sal1), and the lentiviral vector pCDH was cut with the same restriction endonuclease. The target fragment and the vector fragment were separated by agarose gel electrophoresis, and then the cut gel fragment was recovered and the DNA fragment concentration was measured by NanoDrop spectrophotometer. After calculating the amount of vector and fragment, the target fragment was connected to the lentiviral vector pCDH by T4 ligase, and the ligation product was transformed into competent cells. The next day, a single colony was inoculated into LB for amplification culture, and the plasmid was extracted.

[0106] The vector CD33 / CLL1 / CD123 bbz-X was successfully constructed through first-generation sequencing verification. The vector structure diagram is shown in Figure 1 shown.

[0107] Example 2. Lentiviral packaging and preparation of CD33 / CLL1 / CD123 bbz-X CAR-T cells

[0108] 1. CAR Lentivirus Packaging

[0109] First, the MACHEREY-NAGEL endotoxin-free large extraction plasmid kit was used to extract the constructed lentiviral plasmids pCDH-CD33bbz-X, pCDH-CD123bbz-X, pCDH-CLL1bbz-X and pCDH (empty vector plasmid) as well as the lentiviral system auxiliary packaging plasmids Rev, VSVG and pMDL.

[0110] One day before transfection, 1×10 7 293T cells were plated in T75 culture flasks. 20 minutes before transfection, the 293T cell culture medium was replaced with 5 ml serum-free DMEM culture medium. The plasmid was co-transfected into 293T cells using PEI transfection reagent, and the cell culture medium was replaced with 10 ml complete culture medium DMEM + 10% FBS + 1% PS 6 hours after transfection. The cell supernatant was collected 48 hours after transfection and 10 ml of fresh complete culture medium was added. The cell supernatant was collected again after 72 hours and the cells were discarded. After centrifugation of the collected cell supernatant at 3000rpm for 15min, the precipitate was removed, the supernatant was filtered with a 0.45μm filter membrane, and then 40% PEG-6000 solution was added to a concentration of 10%, mixed evenly and incubated overnight at 4 degrees. The next day, centrifugation was performed at 1500g for 1 hour to precipitate the virus, and the virus was resuspended in 0.2 ml serum-free T cell culture medium. Store frozen at -80℃ for later use.

[0111] 2. CAR-T cell preparation and infection analysis

[0112] T cells were isolated using the STEMCELL T cell sorting kit, 0.1g / ml IL-2 was added to the T cell culture medium, and stimulated with anti-CD3 / CD28 antibodies. 48 hours after the start of culture, the activated T cells were suspended in the T cell culture medium, and the corresponding lentivirus (pCDH-CD33bbz-X, pCDH-CD123bbz-X, pCDH-CLL1bbz-X or pCDH) and polybrene were added and gently mixed. T cells were cultured at 37°C for 8-12 hours. The cells were then replaced with T cell culture medium containing 0.1g / ml IL-2. After 48 hours, the infection efficiency of the CAR protein was detected by flow cytometry.

[0113] The results are as follows Figure 2 As shown, the CAR proteins of the three CAR-T cells all have good infection efficiency, among which CLL1bbz-X has the best effect, and CD33-bbz-X is also very ideal.

[0114] 3. CAR-T cells inhibit tumor growth and prolong survival in mouse models

[0115] 5-8 week-old NOD-Prkdcscid Il2rgtm1 / Bcgen (NSG) mice (Beijing Biocytogen) were used for tail vein injection of 5×10 5 The mice were then inoculated with luciferase-expressing acute myeloid leukemia U937 cells and the tumors were allowed to grow for 5 days. The tumor burden in the mice was then measured using a Xenogen IVIS-200 in vivo imager after the mice were anesthetized.

[0116] After confirming that the tumor had formed in the mouse, 1×10 6 CAR-T cells (CD33-bbz-X, CD123-bbz-X, CLL1-bbz-X) or blank control pCDH. Afterwards, in vivo imaging was performed every 7-10 days to detect the tumor burden in vivo and the survival period was recorded.

[0117] The results are as follows Figure 3 and Figure 4 As shown, compared with the plasmid control and the group without X molecules, the three types of X-linked CAR-T cells can significantly improve the survival of mice after being administered to mice, among which the survival of CD33-bbz-X is increased by about 4 times compared with the group without X molecules; the survival of CD123-bbz-X is increased by about 25% compared with the group without X molecules; surprisingly, about 40% of the mice in the CLL1-bbz-X group can obtain a longer survival time.

[0118] Example 3: In vitro culture and phosphorylation analysis of CD33-bbz-X

[0119] 1. In vitro culture

[0120] In a 96-well concave plate, U937 tumor cells and two CAR-T cells, CD33-bbz and CD33-bbz-X, were co-cultured for 16 hours, and then the cells were taken for flow cytometry to observe the expression of CD86, 4-1BB, CD28, and IL-21 of CD33-bbz and CD33-bbz-X in the co-culture system.

[0121] The results are as follows Figure 5 A, shows that CD33-bbz-X has higher expression of CD86, 4-1BB, CD28, and IL-21 than CD33-bbz, and there is a significant statistical difference. Especially for CD86, its expression is extremely improved; and the expression of 4-1BB and CD28 is also very significantly improved.

[0122] The increased expression of CD86, 4-1BB, CD28, and IL-21 indicates that the CD33-bbz-X of the present invention has high expression efficiency and high activity.

[0123] The expression of cytokines and co-stimulatory molecules of CD33-bbz-X of the present invention is significantly improved, thereby reversing the low activation state of CART cells and promoting the killing and expansion of CAR-T cells.

[0124] 2. Phosphorylation experiment

[0125] Under 1:1 E:T conditions, CAR-T and tumor cells were co-cultured in vitro for 12 hours. Subsequently, CAR+T cells were sorted. The sorted CAR-T cells were then stimulated with U937 cells at 37°C for 15-30 minutes, and the cells were incubated with FVD (eBioscience) as a live / dead marker. Subsequently, the inventors fixed the CAR-T cells with 4% paraformaldehyde at 25°C for 20 minutes. The cells were washed in staining buffer (PBS) containing 1% BSA, and frozen methanol was added to a final concentration of 90% (v / v) and placed on ice for 30 minutes. After washing with 1% BSA in PBS, the cells were stained at 25°C for 60 minutes using the following antibodies: PE Cy7 phos-ERK1 / 2 (Thr202 / Tyr204, Biolgend).

[0126] Flow cytometry analysis revealed that the expression of phosphorylated ERK1 / 2 in CD33-bbz-X was higher than that in CD33-bbz, and the difference was statistically significant.

[0127] The results are as follows Figure 5 B, shows that CD33-bbz-X of the present invention has very high activity.

[0128] Example 4, CD33bbz MT Establishment of -X and analysis of anti-tumor effect of CAR T cells in vivo

[0129] 1. CD33bbz MT -X establishment

[0130] The intracellular region of 33bbz (including 4-1BB and CD3ζ) was mutated, and all lysine K in the amino acid sequence of the intracellular region was mutated to arginine R.

[0131] 4-1BB and CD3ζ original amino acid sequence (SEQ ID NO:9)

[0132] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0133] MT mutant amino acid sequence (SEQ ID NO: 10)

[0134] R RGR RR LLYIF R QPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV R FSRSADAPAY R QGQNQLYNELNLGRREEYDVLD R RRGRDPEMG R PRR R NPQEGLYNELQ R DRMAEAYSEIGM R GERRRG R GHDGLYQGLSTAT R DTYDALHMQALPPR

[0135] 2. CD33bbz MT -X's anti-tumor ability in vivo

[0136] Female NSG mice aged 6-8 weeks were injected with 5*10 5 U937-luc tumor cells, 5 days later, were injected with PCDH, 33bbz and 33bbz MT -X, 3 mice per group, each mouse was injected with 1*10 6CAR-T cells were then used for in vivo imaging. After that, images were taken weekly and survival was observed.

[0137] like Figure 6 As shown, the data of in vivo imaging and survival showed that compared with the control group PCDH and CD33bbz, CD33bbz MT -X's anti-tumor ability in vivo was significantly enhanced and the survival period was significantly prolonged.

[0138] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A use of a genetically modified immune effector cell for preparing a drug for inhibiting acute myeloid leukemia; the genetically modified immune effector cell is transduced with a polynucleotide encoding a chimeric antigen receptor or a chimeric antigen receptor expressed on its surface; the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signal region and an X molecule connected in sequence; in, The extracellular binding region comprises a binding protein that specifically recognizes CLL1, CD33 or CD123; Wherein, the X molecule is c-Jun.

2. The use according to claim 1, characterized in that The binding protein that specifically recognizes CLL1, CD33 or CD123 is an antibody; preferably, the antibody is a single-chain antibody or a domain antibody; more preferably, the antibody is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

3. The use according to claim 1, characterized in that The transmembrane region is a sequence comprising the hinge region and the transmembrane region of CD8 or CD28; preferably, the transmembrane region is a sequence comprising the hinge region and the transmembrane region of CD8; more preferably, the hinge region amino acid sequence is as shown in SEQ ID NO:5, and the transmembrane region amino acid sequence is as shown in SEQ ID NO:6; and / or The intracellular signal region includes an intracellular signal region sequence selected from 4-1BB, CD3ζ, FcεRIγ, CD27, CD28, CD134, ICOS, GITR, or a combination thereof; preferably, the intracellular signal region includes 4-1BB and CD3ζ; more preferably, the 4-1BB amino acid sequence is as shown in SEQ ID NO:7, and the CD3ζ amino acid sequence is as shown in SEQ ID NO:8; Preferably, the chimeric antigen receptor comprises the following sequentially connected extracellular binding region, transmembrane region and intracellular signaling region: single-chain antibody of CLL1, CD33 or CD123, hinge region or transmembrane region of CD8, 4-1BB, CD3ζ and X molecule; Preferably, the intracellular signaling region comprises a fusion of 4-1BB and CD3ζ, which is a mutant, and its amino acid sequence is shown in SEQ ID NO:

10.

4. The use according to claim 1, characterized in that The immune effector cells include cells selected from the following groups or a combination thereof: T lymphocytes, NK cells or NKT cells.

5. A chimeric antigen receptor or a nucleic acid encoding the same, wherein the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signaling region and an X molecule connected in sequence; wherein: The extracellular binding region comprises a binding protein that specifically recognizes CLL1, CD33 or CD123; wherein the X molecule is c-Jun; Preferably, the binding protein that specifically recognizes CLL1, CD33 or CD123 is an antibody; preferably, the antibody is a single-chain antibody or a domain antibody; more preferably, the antibody is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; Preferably, the intracellular signaling region comprises a fusion of 4-1BB and CD3ζ, which is a mutant, and its amino acid sequence is shown in SEQ ID NO:

10.

6. A genetically modified immune effector cell that inhibits acute myeloid leukemia, wherein the cell is transduced with a polynucleotide encoding a chimeric antigen receptor or expresses a chimeric antigen receptor on its surface; the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signaling region and an X molecule connected in sequence; wherein, The extracellular binding region comprises a binding protein that specifically recognizes CLL1, CD33 or CD123; wherein the X molecule is c-Jun; Preferably, the binding protein that specifically recognizes CLL1, CD33 or CD123 is an antibody; preferably, the antibody is a single-chain antibody or a domain antibody; more preferably, the antibody is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; Preferably, the intracellular signaling region comprises a fusion of 4-1BB and CD3ζ, which is a mutant, and its amino acid sequence is shown in SEQ ID NO:

10.

7. The genetically modified immune effector cell according to claim 6, characterized in that The cells also express cytokines, and the cytokines have characteristics selected from the following groups: having immunomodulatory activity or anti-tumor activity, and having the function of enhancing immune effector cells; preferably, the cytokines include: IL-12, IL-15, IL-21, IL-2, IL-4, IL-7, IL-9, IL-17, IL-18, IL-23.

8. The genetically modified immune effector cell according to claim 6, characterized in that The cell also expresses chemokine receptors; preferably, the chemokine receptors include: CCR2 or CCR7; The cells also express inhibitory molecules that reduce PD-1 expression or proteins that block PD-L1; or The cell also expresses a safety switch; preferably, the safety switch comprises: iCaspase-9, Truancated EGFR or RQR8.

9. A method for preparing genetically modified immune effector cells, comprising: A polynucleotide encoding a chimeric antigen receptor is introduced into an immune effector cell so that the chimeric antigen receptor is expressed on the cell surface; the chimeric antigen receptor comprises an extracellular binding region, a transmembrane region, an intracellular signal region and an X molecule connected in sequence; wherein the extracellular binding region comprises a binding protein that specifically recognizes CLL1, CD33 or CD123; wherein the X molecule is c-Jun; Preferably, the intracellular signaling region comprises a fusion of 4-1BB and CD3ζ, which is a mutant, and its amino acid sequence is shown in SEQ ID NO:

10.

10. A pharmaceutical composition for inhibiting acute myeloid leukemia or a medicine kit containing the pharmaceutical composition, wherein the pharmaceutical composition comprises the genetically modified immune effector cells according to any one of claims 6 to 8, and a pharmaceutically acceptable pharmaceutical carrier or excipient.

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