Chimeric antigen receptor effector cell with optimized function and application of chimeric antigen receptor effector cell in preparation of composition for treating acute myelogenous leukemia

Abstract

The invention provides a chimeric antigen receptor effector cell with optimized functions and application of the chimeric antigen receptor effector cell in preparation of a composition for treating acute myelogenous leukemia. The invention optimizes and establishes a novel chimeric antigen receptor (CAR) aiming at acute myelogenous leukemia (AML) and an immune effector cell (gene modified 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. More specifically, the present invention relates to a chimeric antigen receptor effector cell with optimized functions and its application in the preparation of a composition for treating acute myeloid leukemia, as well as a corresponding cell therapy. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant blood disease. Clinically, AML is divided into multiple subtypes (M0 - M7) according to the origin and differentiation degree of leukemia cells. The disease has a high mortality rate, a low cure rate, and a poor prognosis. The five-year survival rate of AML patients is low. For relapsed or refractory AML patients (r / r AML), traditional treatment regimens have limited effects, and hematopoietic stem cell transplantation (HSCT) is currently the only viable cure method. However, there are still some patients who relapse after transplantation or have poor treatment effects. The acute myelomonocytic leukemia (M4) / acute monocytic leukemia (M5) subtypes are relatively special types in AML, involving malignant clonal proliferation of monocytic cells and having high invasiveness and aggressiveness. The M4 / M5 subtypes account for about 1 / 3 of AML, and it is generally considered that the curative effect of M4 / M5 is poor in clinical practice.

[0003] In recent years, researchers have developed several different targeted treatment methods, including immune checkpoint inhibitors, T cell receptor-modified T cell therapy, and chimeric antigen receptor-T cell therapy, etc. Chimeric antigen receptor (CAR)-T cell therapy has shown good curative effects in the treatment of B cell acute lymphoblastic leukemia (B-ALL).

[0004] Although preclinical studies have proposed using CD33, CD38, CD123, and other molecules highly expressed on the surface of myeloid tumor cells as target antigens, the feasibility of CAR-T cell therapy in AML still has issues to be determined, and the effect of CAR-T cells targeting these targets in the treatment of r / r AML is poor in clinical trials. CD33 is one of the potential targets for CAR T cell therapy in the treatment of M4 / M5 type AML. However, CD33 is also expressed in normal hematopoietic stem cells, which highlights the urgent need to identify specific targets for M4 / M5 type AML to minimize the risk of potential adverse reactions. Therefore, there is an urgent need in this field to find better ways to change the current situation and obtain effective treatment approaches for this unique cancer disease of AML. Summary of the Invention

[0005] The purpose of the present invention is to provide a chimeric antigen receptor effector cell with optimized functions and its application in the preparation of a composition for treating acute myeloid leukemia.

[0006] In a first aspect of the present invention, there is provided a use of a genetically modified immune effector cell for preparing a medicament 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 expresses a chimeric antigen receptor on its surface; the chimeric antigen receptor comprises, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region and a synergistic factor; wherein, the extracellular binding region comprises a binding protein that specifically recognizes CD64; wherein, the synergistic factor is: interleukin-15 (IL-15), granzyme A (GA), granzyme B (GB), or a combination thereof; preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B, or a combination of interleukin-15 and granzyme A; more preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B.

[0007] In one or more embodiments, the binding protein that specifically recognizes CD64 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 as shown in SEQ ID NO:1.

[0008] In one or more embodiments, the transmembrane region is a sequence comprising the hinge region and transmembrane region of CD8 or CD28; preferably, the transmembrane region is a sequence comprising the hinge region and transmembrane region of CD8 (such as CD8hinge-CD8 TM); more preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO:2, and the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:3.

[0009] In one or more embodiments, the intracellular signaling region comprises 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 comprises 4-1BB and CD3ζ; more preferably, the amino acid sequence of 4-1BB is as shown in SEQ ID NO:4, and the amino acid sequence of CD3ζ is as shown in SEQ ID NO:5.

[0010] In one or more embodiments, the chimeric antigen receptor comprises the following sequentially connected extracellular binding region, transmembrane region and intracellular signaling region: a single-chain antibody against CD64, a CD8 hinge region, a CD8 transmembrane region, 4-1BB, CD3ζ and a synergistic factor.

[0011] In one or more embodiments, the immune effector cells include cells selected from the group consisting of: T lymphocytes, NK cells or NKT cells, or a combination thereof.

[0012] In another aspect of the present invention, there is provided a chimeric antigen receptor (CAR) or a nucleic acid encoding the same, wherein the chimeric antigen receptor comprises, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region and a synergistic factor; wherein the extracellular binding region comprises a binding protein that specifically recognizes CD64; wherein the synergistic factor is interleukin-15, granzyme A, granzyme B, or a combination thereof; preferably, the synergistic factor is a combination of interleukin-15 and granzyme B.

[0013] In another aspect of the present invention, there is provided an expression vector comprising the nucleic acid.

[0014] In one or more embodiments, the expression vector has a pCDH backbone plasmid.

[0015] In one or more embodiments, the intracellular signaling region and the synergistic factor are connected by a linker, preferably the linker comprises P2A or a similar molecule.

[0016] In one or more embodiments, the present invention further provides a virus, such as a lentivirus, comprising the expression vector or obtained by packaging the expression vector.

[0017] In another aspect of the present invention, there is provided a genetically modified immune effector cell that inhibits (including alleviating or treating) acute myeloid leukemia, and 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, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region and a synergistic factor; wherein the extracellular binding region comprises a binding protein that specifically recognizes CD64; wherein the synergistic factor is interleukin-15 (IL-15), granzyme A (Granzyme A; GA), granzyme B (Granzyme B; GB), or a combination thereof; preferably, the synergistic factor is a combination of interleukin-15 and granzyme B, or a combination of interleukin-15 and granzyme A; more preferably, the synergistic factor is a combination of interleukin-15 and granzyme B.

[0018] In one or more embodiments, the genetically modified immune effector cell further expresses a cytokine, and the cytokine has characteristics including being selected from the group consisting of: having immunomodulatory activity or antitumor activity, and enhancing the function of the immune effector cell.

[0019] In one or more embodiments, the cytokine includes (but is not limited to): IL-12, IL-21, IL-2, IL-4, IL-7, IL-9, IL-17, IL-18, IL-23.

[0020] In one or more embodiments, the cell also expresses a chemokine receptor that blocks tumor metastasis; preferably, the chemokine receptor includes: CCR2 or CCR7.

[0021] In one or more embodiments, the cell also expresses an inhibitory molecule (such as siRNA) that reduces PD-1 expression or a protein that blocks PD-L1.

[0022] In one or more embodiments, the cell also expresses a safety switch; preferably, the safety switch includes: iCaspase-9, Truancated EGFR or RQR8.

[0023] In another aspect of the present invention, there is provided a method for preparing a genetically modified immune effector cell, comprising: introducing a polynucleotide encoding a chimeric antigen receptor into an immune effector cell to cause the cell surface to express the chimeric antigen receptor; the chimeric antigen receptor comprises, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region, and a synergistic factor; wherein, the extracellular binding region comprises a binding protein that specifically recognizes CD64; wherein, the synergistic factor is: interleukin-15 (IL-15), granzyme A (Granzyme A; GA), granzyme B (Granzyme B; GB), or a combination thereof; preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B, or a combination of interleukin-15 and granzyme A; more preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B.

[0024] In another aspect of the present invention, there is provided a pharmaceutical composition or a kit containing the pharmaceutical composition for inhibiting (including alleviating or treating) acute myeloid leukemia, the pharmaceutical composition comprising the genetically modified immune effector cell as described above, and a pharmaceutically acceptable pharmaceutical carrier or excipient.

[0025] In one or more embodiments, the kit includes: a container, and the pharmaceutical composition located in the container.

[0026] Other aspects of the present invention will be apparent to those skilled in the art from the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Structural schematic diagrams of the respective elements of the CD64bbz, CD64bbz-IL-15, CD64bbz-GA, and CD64bbz-GB vectors.

[0028] Figure 2 Preparation of different CAR-T cells and analysis of their infection efficiency, analyzed by flow cytometry.

[0029] Figure 3 1. Analysis of the killing efficiency of different CAR-T cells in an in vitro killing model.

[0030] Figure 4 2. Inhibitory effect of different CAR-T cells on the growth of animal tumors (tumors of tumor-bearing mice), in vivo expansion analysis, and animal survival analysis.

[0031] Figure 5 3. Inhibitory effect of different CAR-T cells on the growth of tumors in tumor-bearing mice, in vivo expansion analysis, and animal survival analysis. Detailed implementation manners

[0032] Through in-depth research and screening, the present inventors have optimized and established a novel chimeric antigen receptor (CAR) targeting acute myeloid leukemia (AML), and immune effector cells modified by this CAR (gene-modified immune effector cells). The present invention also discloses its preparation method and its application in inhibiting acute myeloid leukemia.

[0033] Acute myeloid leukemia is a malignant disease characterized by the aggregation of abnormal primitive cells, with a very high mortality rate. After adopting a standardized chemotherapy regimen, only about 40 - 45% of young patients with acute myeloid leukemia and 10 - 20% of elderly patients with acute myeloid leukemia can be cured. The main strategy for treating refractory acute myeloid leukemia is allogeneic stem cell transplantation (SCT). However, the prognosis of patients who have not achieved a complete remission before SCT is extremely poor.

[0034] CAR-T cell therapy has achieved certain efficacy in treating B acute lymphoblastic leukemia (B-ALL), but it has very poor effects in treating acute myeloid leukemia, manifested as the inability of CAR T cells to achieve sufficient amplification efficiency, low / insignificant targeted killing effect, and the subjects showing drug resistance to CAR-T cell therapy, etc. Therefore, there is a great clinical need to obtain a novel CAR T drug that can treat acute myeloid leukemia.

[0035] Terms

[0036] As used in the present invention, "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 signal transduction domain (also referred to as "intracellular signal region"), wherein the intracellular signal region comprises a functional signal transduction domain derived from a stimulatory molecule and / or a co-stimulatory molecule. For example, the stimulatory molecule can be the ξ chain associated with the T cell receptor complex, and the co-stimulatory molecule can be 4-1BB (CD137) and / or CD28.

[0037] As used in the present invention, the terms "immune cell" and "immune effector cell" are used interchangeably and include: T lymphocytes, NK cells, NKT cells, etc.; preferably, the "immune cell" or "immune effector cell" is a T lymphocyte.

[0038] As used in the present invention, the "intracellular signaling domain" or "intracellular signal region" or "intracellular signal transduction activation region" refers to the intracellular portion of the CAR molecule. The intracellular signaling domain generates signals that can promote the effector functions of CAR immune effector cells (such as CAR T cells). Examples of effector functions include, for example, in CAR T cells, cytolytic activity and helper activity, including cytokine secretion.

[0039] As used in the present invention, the term "operatively 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 nucleic acid sequence of a 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.

[0040] As used herein, the term "construct" refers to a single-stranded or double-stranded DNA molecule that has been artificially intervened to contain DNA fragments combined and arranged in a sequence that does not exist in nature. The "construct" includes an expression vector; alternatively, the "construct" is contained within an expression vector and is part of the expression vector.

[0041] As used in the present invention, the "single-chain antibody (scFv) fragment" refers to an antibody fragment that contains a heavy-chain variable region (VH) and a light-chain variable region (VL) linked by a linker, which associates the two domains to ultimately form an antigen-binding site. The single-chain antibody is preferably an amino acid chain sequence encoded by a single nucleotide chain. The single-chain antibodies used in the present invention can be further modified alone or in combination using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination and / or other modification methods.

[0042] As used in the present invention, "specifically recognize" means that the extracellular binding region (such as a single-chain antibody) of the present invention does not cross-react or substantially does not cross-react with any polypeptide other than the target antigen. The degree of its specificity can be judged by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometry, etc. In the present invention, specific recognition is preferably determined by flow cytometry, and the specific recognition criteria in specific cases can be judged by those of ordinary skill in the art based on the common general knowledge in the art that they have mastered.

[0043] For acute myeloid leukemia, a refractory non-solid tumor with special properties, the inventors 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, making it difficult to apply them 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 or very low tumor cell killing activity. This may be because this target can trigger tumor cells to secrete factors that inhibit immune effector cells. After repeated investigation and screening of a variety of genes, CD64 was determined as the target antigen for preparing CAR, and specific synergistic factors were used to improve its amplification efficiency and / or killing efficiency. The amino acid sequence of CD64 is publicly available under GenBank accession number (GenBank: BC152383.1).

[0044] CD64 is a high-affinity receptor for IgG, which is highly expressed only on activated cells of the myeloid lineage, especially enhanced in acute myeloid leukemia M4 and M5 subtypes, but not expressed on hematopoietic stem cells. As one of the potential target sites for CAR-T therapy of AML, preclinical results show that the in vivo anti-tumor effect is transient and accompanied by a decrease in the number of CAR-T cells in the circulation. Therefore, promoting the clinical application of CD64 CAR-T cell therapy also requires enhancing the killing and duration of CAR-T cells against tumors.

[0045] The research of the present invention shows that when applied to the preparation of CAR effector cells for acute myeloid leukemia involved in the present invention, CD64 can be an ideal therapeutic target for the CAR immune effector cell method. Especially, it can cooperate with synergistic factors and the synergistic effect is very significant. Preparing CAR effector cells with this technical solution can significantly extend the survival period of tumor patients.

[0046] In a preferred embodiment, the binding protein that specifically recognizes and binds to CD64 in the present invention is a single-chain antibody, and preferably its amino acid sequence is listed in Table 1 of the examples. The research of the present inventors shows that the CAR effector cells composed of the two single-chain antibodies described in the present invention retain the highly efficient and selective killing effect against antigen-positive cells.

[0047] The examples of the present invention demonstrate that the CAR-modified effector cells of the present invention can highly selectively eliminate CD64-positive tumor cells. The CAR effector cells against CD64 of the present invention are a new means with high efficiency, low toxicity and strong targeting for the treatment of acute myeloid leukemia.

[0048] Based on the new solution of the present inventors, a CAR expressed on the surface of immune effector cells is provided. The CAR comprises, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region, and a synergistic molecule. The CAR of the present invention combines a specific extracellular binding region and an intracellular signaling region, and further connects them with a synergistic molecule. Expressing the CAR on the surface of immune effector cells can greatly increase the expansion amount of immune effector cells and has a highly specific cytotoxic effect on tumors expressing CD64. The amino acid sequence of the synergistic molecule is listed in Table 1 of the Examples.

[0049] It should be understood that the present invention also includes functional variants of the synergistic molecule element and other functional variants of each element used to prepare the CAR.

[0050] The functional variants of each element include fragments, derivatives, and analogs. As used herein, the terms "fragment", "derivative", and "analog" refer to proteins that substantially retain the same biological function or activity of each element of the present invention. The protein fragments, derivatives, or analogs of the present invention can be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having a substituent group in one or more amino acid residues, or (iii) proteins formed by fusing an additional amino acid sequence to this protein sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this protein or a proprotein sequence, or a fusion protein). According to the definitions herein, these fragments, derivatives, and analogs are within the scope well-known to those skilled in the art.

[0051] In the present invention, the functional variants also include (but are not limited to): deletions, insertions, and / or substitutions of several (usually 1-20, more preferably 1-10, still more preferably 1-8, 1-5, 1-3, or 1-2) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when substituting amino acids with similar properties or functions, the function of the protein usually does not change. Also, for example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. This term also includes active fragments and active derivatives of the element. The functional variants also include (but are not limited to): derived proteins having a sequence identity of more than 80%, preferably more than 85%, more preferably more than 90%, further more preferably more than 95%, such as more than 98% or more than 99% with the amino acid sequence of the element and retaining its protein activity.

[0052] The present invention also provides polynucleotide sequences encoding each element of the present invention or conservative variant proteins thereof. The polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand.

[0053] The polynucleotides encoding the mature protein of the mutant include: a coding sequence encoding only the mature protein; the coding sequence of the mature protein and various additional coding sequences; the coding sequence of the mature protein (and optional additional coding sequences) and non-coding sequences.

[0054] "Polynucleotides encoding a protein" can be polynucleotides including those encoding this protein, or can also be polynucleotides further including additional coding and / or non-coding sequences.

[0055] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention or the coding sequences of the corresponding elements, and methods for producing the proteins of the present invention by recombinant techniques.

[0056] In the present invention, the polynucleotide sequence can be inserted into a recombinant expression vector. A "recombinant expression vector" can be any plasmid and vector capable of replicating and being stable in a host. An important feature of an expression vector is usually that it contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0057] In the present invention, the synergistic molecule is interleukin-15 (IL-15), granzyme A (Granzyme A; GA), granzyme B (Granzyme B; GB), or a combination thereof; preferably, the synergistic factor is a combination of interleukin-15 and granzyme B. After the synergistic molecule is connected to other elements constituting the CAR, it can effectively exert its activity, and the synergistic effect of the synergistic molecule is very significant. The synergistic effect is particularly manifested in improving the amplification ability and killing efficacy of CAR effector cells, enhancing the therapeutic effect and duration of CAR effector cells on CD64-positive AML cells.

[0058] IL-15 is a pleiotropic cytokine that plays a key role in the homeostasis of innate and adaptive immune cells. In addition, IL-15 can also increase the expression of the anti-apoptotic gene Bcl-2 by regulating metabolic activity, prolong cell survival, increase the number of T cells, enhance its effector function (i.e., break tumor tolerance and activate other tolerant T cells), and promote the precise recognition and localization of effector cells to target tissue cells, thereby significantly improving the therapeutic effect.

[0059] Granzymes are important mediators for immune cells to exert anti-tumor effects. When CAR T cells recognize cancer cells expressing specific antigens, they release factors such as Granzymes to cancer cells, thereby triggering apoptosis. Granzyme A is a trypsin-like enzyme that can induce caspase-independent cell death, mainly relying on cleaving the SET complex to induce cell death. In addition, Granzyme A can also cleave and inactivate high-mobility group box protein 2 (HMGB2) and apurinic / apyrimidinic endonuclease 1 (Ape1), thereby interfering with base excision repair and further disrupting DNA repair; Granzyme B can activate Caspase-3 or directly cleave its substrates BH3 interacting domain death agonist (bid) and caspase-activated DNase inhibitor (ICAD), thereby triggering a cell death cascade and promoting apoptosis of target cells.

[0060] The extracellular binding region contains a protein that specifically recognizes CD64. Expressing this CAR on the surface of immune effector cells can endow the immune effector cells with highly specific cytotoxic effects on tumor cells highly expressing CD64.

[0061] As a preferred embodiment of the present invention, the extracellular binding region is a single-chain antibody (scFv). The scFv is functional. Generally, the single-chain antibody is prepared by preparing the heavy chain variable region (VH) and the light chain variable region (VL) and then operably linking them. Preferably, the VH and VL are linked by a flexible linker.

[0062] 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.

[0063] The transmembrane region of CAR can be selected from the transmembrane regions of proteins such as CD8 or CD28. Human CD8 protein is a heterodimer composed of two chains, αβ or γδ. In a preferred embodiment of the present invention, the transmembrane region is selected from the transmembrane regions of CD8 (CD8α) or CD28. The CD8 hinge region is a flexible region. Therefore, CD8 or CD28 and the transmembrane region plus the hinge region can be used to connect the target recognition domain scFv of CAR and the intracellular signaling region.

[0064] The intracellular signaling region can be selected from the intracellular signaling regions of CD3ζ, FcεRIγ, CD27, CD28, 4-1BB, CD134, ICOS, GITR proteins, and combinations thereof. The CD3 molecule consists of five subunits, among which the CD3ζ subunit (also known as CD3 zeta, abbreviated as Z) contains 3 ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. FcεRIγ is mainly distributed on the surfaces of mast cells and basophils, and it contains one ITAM motif, which is 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 effects generated by their intracellular signal segments cause the continuous proliferation of immune effector cells (mainly T lymphocytes), and can increase the levels of cytokines such as IL-2 and IFN-γ secreted by immune effector cells, and at the same time increase the survival period and anti-tumor effect of CAR immune effector cells in vivo.

[0065] In the embodiments of the present invention, the CAR ( Figure 1 ) preferred by the inventors is provided. In addition, it should be understood that based on the optimized CAR of the present invention, CARs with some changes or modifications can also be included in the present invention.

[0066] As a mode of the present invention, the CAR further includes 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 (such as scFv) during the cell processing of the CAR and its targeting to the cell membrane.

[0067] The present invention also includes nucleic acids encoding the CAR. The nucleic acid sequences of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can 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 can be degenerate, that is, multiple degenerate nucleic acid sequences encoding the same amino acid sequence are included within the scope of the present invention. Degenerate nucleic acid codons corresponding to the amino acids are well known in the art.

[0068] The present invention also includes variants of the above polynucleotides, which encode polypeptides or polypeptide fragments, analogs, and derivatives having the same amino acid sequence as the present invention. Such variants of the polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes.

[0069] The present invention also provides genetically modified immune effector cells, which are transduced with the nucleic acid of the present invention or with the above-mentioned recombinant plasmid containing the nucleic acid of the present invention or a virus containing the plasmid. The cells are cells or cell populations containing the cells, preferably T cells or cell populations containing T cells.

[0070] Conventional nucleic acid transduction methods 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.

[0071] The present invention also provides an expression construct (vector) containing the above-mentioned nucleic acid encoding a chimeric antigen receptor protein expressed on the surface of immune effector cells, including viral vectors or non-viral vectors.

[0072] The non-viral vector systems described above, such as the Sleeping Beauty system or PiggyBac transposon system and other transposon systems, have a much higher transduction efficiency than ordinary 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 not only has a high transduction efficiency but also can achieve site-specific integration of the target gene. In addition, mRNA transfection technology can also be applied.

[0073] A variety of vectors for virus packaging can be applied in the present invention, such as lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc., and also include virus vectors formed by further modification on the basis of these virus vectors. In a specific embodiment of the present invention, the backbone vector used is the lentiviral plasmid vector pCDH. In the pCDH vector, the expression of the CAR molecule is driven by the EF1α promoter for transcriptional expression. The CAR lentiviral vector plasmid can be co-transfected into HEK293T cells in the presence of the helper packaging plasmids Rev, VSV-G, and pMDL, and then packaged into lentivirus carrying the 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 by it can be finally obtained.

[0074] The present invention also includes viruses packaged by viral vectors. The viruses can be lentiviruses, adenoviruses, adeno-associated viruses, etc., and also include viruses formed by further modification based on these viruses. The viruses of the present invention include the packaged infectious viruses, and also include the viruses to be packaged containing the necessary components for packaging into 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 foreign genes into immune effector cells and their corresponding plasmid vectors can also be used in the present invention.

[0075] In one embodiment of the present invention, the method for transducing immune effector cells modified with a chimeric antigen receptor gene is based on the transduction method of viruses such as lentiviruses. On the surface of the transgenic immune effector cells, the transduced nucleic acid is expressed on the surface through transcription and translation. Through in vitro cytotoxicity experiments on various different cultured tumor cells, it is demonstrated that the immune effector cells modified with the chimeric antigen receptor gene of the present invention have a highly specific tumor cell killing effect (also known as cytotoxicity). Therefore, 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 cells transduced with the above nucleic acid, plasmid or virus can be effectively used for the immunotherapy of acute myeloid leukemia.

[0076] The immune cells of the present invention can also express another chimeric antigen receptor in addition to the above-mentioned chimeric antigen receptor. This receptor may not contain CD3ζ, but contains the intracellular signaling domain of CD28, the intracellular signaling domain of CD137, or a combination of the two.

[0077] The immune cells of the present invention can also express chemokine receptors; the chemokine receptors include, but are not limited to, CCR2. Those skilled in the art can understand that the CCR2 chemokine receptor can competitively bind to CCR2 in the body, which is beneficial for blocking tumor metastasis.

[0078] The immune cells of the present invention can also express siRNA that can reduce the expression of PD-1 or a protein that blocks PD-L1. Those skilled in the art can understand that competitively blocking the interaction between PD-L1 and its receptor PD-1 is beneficial for restoring the anti-tumor T cell response, thereby inhibiting tumor growth.

[0079] The immune cells of the present invention can also express a safety switch; preferably, the safety switch includes: iCaspase-9, Truancated EGFR or RQR8. As mentioned above, the current CAR therapy is still challenged by the complexity of its production and adverse events related to cell activity, such as cytokine release syndrome (CRS), etc. Therefore, drugs or therapies that can effectively regulate CAR-T, such as setting a safety switch, are more preferred.

[0080] In a specific embodiment of the present invention, the inventors modified CD64 bbz (CD64 scFV-4-1BB-CD3ζ) cells by co-overexpressing CD64 bbz and IL-15, and the results significantly promoted the activation, proliferation, and survival of CAR-T cells. The inventors verified through in vivo experiments that the co-expression of CD64 bbz and IL-15 had significantly enhanced anti-tumor effects and CAR-T expansion ability compared to the control groups (single expression of CD64bbz and blank control pCDH), and significantly prolonged the survival period of animals. This indicates that the overexpression of the synergistic molecule has a very powerful and sustained effect in the anti-AML aspect of CD64 CAR-T cells. In addition, in order to further improve the anti-tumor effect and sustained effect of CART cells, the inventors co-expressed granzyme A or granzyme B (preferably granzyme B) on the basis of CD64bbz-IL-15. In vivo and in vitro experiments demonstrated that the co-expression of CD64bbz-IL-15 and granzyme A or granzyme B was significantly superior to CD64bbz in terms of anti-tumor effect and duration, especially the co-expression effect with granzyme B was extremely excellent.

[0081] The co-expression of CD64 bbz and IL-15 and the expression of granzyme A or granzyme B (preferably granzyme B) on this basis can enhance the anti-tumor effect, which was first discovered and disclosed by the inventors, and provides theoretical support for its greater clinical application.

[0082] The gene-modified immune effector cells of the present invention can be applied to the preparation of a composition, especially a pharmaceutical composition. The said composition may include, in addition to an effective amount of the said immune effector cells, a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that when the molecular entity and the composition are appropriately administered to an animal or a human, they do not produce adverse, allergic, or other untoward reactions.

[0083] Specific examples of some substances that can be used as pharmaceutically acceptable carriers or their components 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; 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 Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting aids; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffer solutions, etc.

[0084] The composition of the present invention can be made into various dosage forms as needed, and the physician can determine the dosage beneficial to the patient according to factors such as the type, age, weight and general disease condition of the patient, and the administration method, etc. The administration method can be, for example, injection or other treatment methods.

[0085] The immune effector cells of the present invention or the composition containing the cells can also be placed in a suitable kit for the use of clinicians. Preferably, the kit can also contain an instruction manual for explaining the usage method of the composition of the present invention.

[0086] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out according to the conditions described in, for example, "Molecular Cloning: A Laboratory Manual", Third Edition, edited by J. Sambrook et al., published by Science Press, or according to the conditions recommended by the manufacturer.

[0087] Example 1. Vector construction

[0088] In this example, CD64bbz, CD64bbz-IL-15, CD64bbz-GA, and CD64bbz-GB vectors were established.

[0089] The vector structure is as Figure 1 shown, and the sequences of each element are shown in Table 1 and inserted into the restriction enzyme cleavage site of the pCDH vector. The antigen-binding domain of the vector is derived from the single-chain fragment variable (scFv) region of the CD64 antibody.

[0090] Table 1

[0091]

[0092]

[0093] The CD64 scFv synthesized on the pUC57 vector and the target fragments of molecules IL-15 / GA / GB (Suzhou Genewiz Biotechnology Co., Ltd.) were cut at specific positions at both ends of the target gene by restriction enzymes (NEB); the lentiviral vector pCDH was specifically digested with the same restriction enzymes, and the target fragments and vector fragments were separated by agarose gel electrophoresis.

[0094] The excised gel fragments were recovered (TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0), and the concentration of DNA fragments was measured by NanoDrop spectrophotometer. After adding the vector and fragments in proportion, the target fragments were ligated to the lentiviral vector pCDH by T4 ligase, and the ligation products were transformed into Stable competent cells (Bomed Biotech). They were placed in a shaker at 37°C and cultured with shaking at 200 rpm for 60 minutes. Then, 100 μl of the bacterial solution was taken and spread on an LB agar plate (ampicillin-resistant). After inverted culture in an incubator at 37°C for 12 - 18 hours, single colonies were picked into liquid LB medium and cultured with shaking at 200 rpm and 37°C for 16 hours, and then plasmids were extracted.

[0095] Verified by Sanger sequencing, the vectors CD64bbz, CD64bbz-IL-15, CD64bbz-GA, and CD64bbz-GB were successfully constructed.

[0096] Example 2: Lentivirus packaging and CAR-T cell preparation

[0097] In this example, lentivirus was used for packaging and CD64bbz, CD64bbz-IL-15, CD64bbz-GA, CD64bbz-GB, and pCDH T cells were prepared.

[0098] 1. CAR lentivirus packaging

[0099] First, endotoxin-free large-scale plasmid extraction kits (MACHEREY-NAGEL) were used to extract the lentiviral plasmids CD64bbz, CD64bbz-IL-15, CD64bbz-GA, CD64bbz-GB, and pCDH (empty vector plasmid), as well as the lentiviral packaging helper plasmids Rev, VSV-G, and pMDL with correct sequencing sequences.

[0100] One day before transfection, human embryonic kidney cells (HEK293 T) in a 10-cm culture dish at passage 1 / 3 were passaged into a new 10-cm culture dish so that they reached 80 - 90% confluence on the day of transfection. 30 minutes before transfection, the three helper plasmids (Rev, VSV-G, and pMDL) and the target plasmids (CD64bbz-IL-15, CD64bbz-GA, CD64bbz-GB, CD64bbz, pCDH) were added to opti-MEM medium in a certain proportion, mixed well, and then a quantitative PEI transfection reagent was added to form a plasmid mixture. Before transfection, the complete medium (DMEM + 10% FBS + 1% PS) in HEK293T cells was replaced with 4 ml of serum-free DMEM medium, and then the plasmid mixture was added to the 293T culture dish at 80 - 90% confluence.

[0101] Replace the cell culture medium with 10 ml of complete medium 6 hours after transfection. Collect the cell supernatant 48 hours after transfection. After centrifuging the collected cell supernatant at 3000 rpm for 15 min, remove the precipitate, and filter the supernatant through a 0.45 μm filter membrane. Store the lentivirus at -80 °C for later use.

[0102] 2. Preparation of CD64bbz, CD64bbz-IL-15, CD64bbz-IL-15 + CD64bbz-GA, CD64bbz-IL-15 + CD64bbz-GB CAR-T cells and pCDH cells

[0103] Use the STEMCELL T cell sorting kit (EasySep TM Human CD3 Positive Selection KitII) to isolate T cells from peripheral blood, culture them in complete T cell medium (T cell medium + 10% FBS + 1% PS) supplemented with 100 U / ml of IL-2, and stimulate them with anti-CD3 / CD28 antibodies.

[0104] After 48 hours of cell stimulation, resuspend the activated T cells in T cell medium, add the corresponding lentivirus and 1‰ polybrene, and gently mix. Centrifuge the cell suspension at 4 °C and 2000 rpm for 1 hour and 20 minutes, then culture the T cells at 37 °C for 8 - 12 hours. Then replace the cell medium with complete T cell medium containing 100 U / ml of IL-2. For cells that require secondary infection, perform transfection twice within 72 hours after stimulation using the same method. Detect the infection efficiency of CAR by flow cytometry 48 hours after transfection.

[0105] The results are as Figure 2 shown. According to Figure 2 , the infection efficiency of each type of CAR T cell is between 35% - 45%. Among them, the infection efficiency of the group using the combination of IL-15 and GA or GB is significantly higher than that of the group using IL-15 alone.

[0106] Example 3. Analysis of the killing activity of different CAR-T cells

[0107] Use the U937 AML cell line with high expression of CD64 antigen as the target cell.

[0108] Resuspend the target cells (U937-GFP) overexpressing green fluorescent protein (GFP) (at a density of 10,000 - 100,000 cells / ml), and take 50 μl and add it to a 96-well plate. Add 50 μl of effector cells according to an appropriate E:T ratio (1:3; 1:5; 1:10), with three parallels in each group, and there are three parallels with only target cells.

[0109] After culturing the plate in an incubator at 37 °C and 5% CO2 for 16 hours, resuspend by pipetting. After aspirating 50 μl of cell suspension from each well, stain with Counting beads and perform flow cytometry detection. Calculate the percentage of cell lysis and plot the killing curve.

[0110] The results are as Figure 3 shown. The CD64bbz-IL-15 + CD64bbz-GB group showed the most ideal killing effect. When the effector-to-target ratio E:T was 1:5, compared with the CD64bbz group, the improvement in the killing effect of the CD64bbz-IL-15 + CD64bbz-GB group was very significant (**P < 0.01). When the effector-to-target ratio E:T was 1:10, compared with other groups, the improvement in the killing effect of the CD64bbz-IL-15 + CD64bbz-GB group was extremely significant (****P < 0.0001).

[0111] Therefore, CD64bbz-IL-15 + CD64bbz-GB CAR-T cells showed better killing efficiency in the in vitro killing model, especially in the case of high tumor burden.

[0112] Example 4. Inhibitory effect of different CAR-T cells on animal tumor growth

[0113] 1. Analysis of in vivo expansion and survival rate

[0114] Conduct animal experiments to analyze the inhibitory effect of CD64bbz and CD64bbz-IL-15 CAR-T cells on tumors in tumor-bearing mice, using cells transfected with an empty vector plasmid group (pCDH) as a control.

[0115] Use 5 - 8-week-old NOD-Prkdc scid Il2rg tm1 / Bcgen (NSG) mice (Beijing Biocytogen) immunodeficient mice. Inject 3 × 10 5 U937 cells overexpressing luciferase (U937-luc) via the tail vein, and allow the tumor cells to grow in the mice for 5 days. Then, after anesthetizing the mice, use a live imaging instrument to detect the tumor burden in the mice and analyze the tumor formation situation.

[0116] After confirming tumor formation in mice, 1.5×10 6 effector cells or blank control pCDH were injected via the tail vein. Thereafter, in vivo imaging was performed every 7 - 10 days to detect the tumor burden in the body and the continuous effect of CART expansion, and the survival period was recorded.

[0117] The results are as Figure 4 shown. According to Figure 4 , the tumor burden and the number of CAR T cells were recorded from day 0 to 21. The results showed that the tumor burden in the CD64bbz - IL - 15 group was significantly lower than that in the CD64bbz group (**P<0.01); the number of CAR T cells in the CD64bbz - IL - 15 group was significantly higher than that in the CD64bbz group. Compared with the blank control group and the CD64bbz group, the survival period of the CD64bbz - IL - 15 group was significantly better.

[0118] 2. In vivo expansion and survival rate analysis

[0119] The animal operation process was as described in "1" above. The inhibitory effects of CD64bbz, CD64bbz - IL - 15 + CD64bbz - GA, and CD64bbz - IL - 15 + CD64bbz - GB CAR - T cells on tumors in tumor - bearing mice were analyzed. The cells transfected with the empty vector plasmid group (pCDH) were used as the control. In vivo imaging was performed on days 0 - 28 to detect the tumor burden in the body and the continuous effect of CAR T expansion, and the survival period was observed ( Figure 5 ).

[0120] According to Figure 5 , the tumor burden and the number of CAR T cells were recorded from day 0 to 21. After adoptive transfer, the tumor in the CD64bbz - IL - 15 + CD64bbz - GB group was always in a low - burden state compared with other groups, and the number of CART cells in the CD64bbz - IL - 15 + CD64bbz - GB group was significantly higher than that in other groups.

[0121] Compared with the blank control group and the CD64bbz group, the survival periods of the CD64bbz - IL - 15 + CD64bbz - GA and CD64bbz - IL - 15 + CD64bbz - GB groups were both significantly prolonged.

[0122] Therefore, both CD64bbz - IL - 15 + CD64bbz - GA and CD64bbz - IL - 15 + CD64bbz - GB cells can better inhibit tumor growth in the mouse model, significantly prolong the survival period of mice, and more preferably the CD64bbz - IL - 15 + CD64bbz - GB group.

[0123] The above results indicate that the combined application of IL-15 with GA or GB can greatly enhance the inhibitory effect of CAR T cells on tumor growth.

[0124] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited herein as references, just as if each document was cited separately as a reference.

Claims

1. Use of a genetically modified immune effector cell for preparing a medicament for inhibiting acute myeloid leukemia; the genetically modified immune effector 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, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region and a synergistic factor; Wherein, The extracellular binding region contains a binding protein that specifically recognizes CD64; Among them, the synergistic factor is: interleukin-15, granzyme A, granzyme B, or a combination thereof; preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B, or a combination of interleukin-15 and granzyme A; more preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B.

2. The use according to claim 1, characterized in that, The binding protein that specifically recognizes CD64 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 as shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that, The transmembrane region is a sequence containing the hinge region and transmembrane region of CD8 or CD28; preferably, the transmembrane region is a sequence containing the hinge region and transmembrane region of CD8; more preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO:2, and the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:3; and / or The intracellular signaling region includes intracellular signaling region sequences 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 amino acid sequence of 4-1BB is as shown in SEQ ID NO:4, and the amino acid sequence of CD3ζ is as shown in SEQ ID NO:

5.

4. The use according to claim 3, characterized in that, The chimeric antigen receptor includes an extracellular binding region, a transmembrane region, and an intracellular signaling region connected in the following order: a single-chain antibody against CD64, the hinge region of CD8, the transmembrane region of CD8, 4-1BB, CD3ζ, and a synergistic factor.

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

6. A chimeric antigen receptor or a nucleic acid encoding the same, the chimeric antigen receptor comprises, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region and a synergistic factor; wherein, The extracellular binding region contains a binding protein that specifically recognizes CD64; among them, the synergistic factor is: interleukin-15, granzyme A, granzyme B, or a combination thereof; preferably, the synergistic factor is a combination of interleukin-15 and granzyme B; Preferably, the binding protein that specifically recognizes CD64 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 as shown in SEQ ID NO:

1.

7. A genetically modified immune effector cell that inhibits acute myeloid leukemia, 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, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region and a synergistic factor; Wherein, The extracellular binding region contains a binding protein that specifically recognizes CD64; Among them, the synergistic factor is: interleukin-15, granzyme A, granzyme B, or a combination thereof; preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B, or a combination of interleukin-15 and granzyme A; more preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B.

8. The genetically modified immune effector cell according to claim 7, characterized in that, The binding protein that specifically recognizes CD64 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 as shown in SEQ ID NO:

1.

9. A method for preparing a genetically modified immune effector cell, comprising: Introduce a polynucleotide encoding a chimeric antigen receptor into immune effector cells, such that the chimeric antigen receptor is expressed on the cell surface; the chimeric antigen receptor comprises, in sequential connection: an extracellular binding region, a transmembrane region, an intracellular signaling region, and a synergistic factor; wherein the extracellular binding region comprises a binding protein that specifically recognizes CD64; wherein the synergistic factor is: interleukin-15, granzyme A, granzyme B, or a combination thereof; preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B, or a combination of interleukin-15 and granzyme A; more preferably, the synergistic factor is: a combination of interleukin-15 and granzyme B.

10. A pharmaceutical composition for inhibiting acute myeloid leukemia or a kit containing the pharmaceutical composition, the pharmaceutical composition comprises the genetically modified immune effector cell according to claim 7 or 8, and a pharmaceutically acceptable pharmaceutical carrier or excipient.

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