FLT3-car-γδt cell co-expressing cytokine and use thereof

Abstract

The present invention relates to an FLT3-CAR-γδT cell co-expressing a cytokine and a use thereof. Specifically, the present invention provides an engineered CAR-γδT cell targeting FLT3, wherein the CAR-γδT cell co-expresses an IL-2 or IL-7 cytokine, maintains a high stemness level and proliferation capacity, and has a sustained tumor cell killing ability. The FLT3-CAR-γδT cell co-expressing a cytokine of the present invention demonstrates persistent killing ability and anti-tumor activity against acute myeloid leukemia (AML) tumor cells in multiple in vitro and in vivo experiments, has high safety and wide applicability, and has application prospects in the field of AML therapy.

Description

FLT3-CAR-γδT cells co-expressing cytokines and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of immunotherapy, in particular to FLT3-CAR-γδT cells co-expressing cytokines and application thereof. BACKGROUND

[0002] Acute myeloid leukemia (AML) is the most common type of leukemia in adults, characterized by clonal expansion of myeloid blasts in peripheral blood, bone marrow and / or other tissues, accompanied by clinical manifestations such as infection, anemia and bleeding. The incidence of AML increases with age, with a median age of onset of 68 years, and the 5-year overall survival rate of patients over 60 years old is less than 10%.

[0003] Following the success of CD19 CAR-T cells in the clinical treatment of B-cell malignancies, CAR-T cell therapy has been widely developed for the treatment of other malignancies. However, unlike the great achievements of CAR-T cells in lymphoid leukemia, CAR-T cell therapy faces many challenges in acute myeloid leukemia. Tumor immune escape after CAR-T cell therapy is also a major obstacle to CAR-T cell therapy in AML treatment. Studies have reported that the malignant bone marrow microenvironment of AML patients helps the migration and expansion of tumor cells and helps the immune escape of drug-resistant tumor cells. In addition, AML tumor cells can also secrete some soluble cytokines that inhibit T cell proliferation, thereby indirectly inhibiting the efficacy of CAR-T cells.

[0004] γδT cells are another innate subpopulation of T cells different from αβT cells, accounting for only 1-5% of peripheral blood T cells. γδT cells recognize target antigens independent of major histocompatibility complex (MHC) and do not cause graft-versus-host disease (GvHD). Compared with other CAR-T cells, CAR-γδT cells do not need to be genetically edited for allogeneic therapy, and are safer and more applicable, but so far CAR-γδT cells have not shown persistent and effective anti-tumor activity.

[0005] Therefore, there is an urgent need in the art to develop CAR-γδT cells with high and persistent AML tumor inhibitory activity. SUMMARY

[0006] The purpose of the present application is to provide CAR-γδT cells with high and persistent AML tumor inhibitory activity.

[0007] In a first aspect of the present application, a chimeric antigen receptor (CAR) is provided, comprising: an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds to FLT3;

[0008] and the CAR further comprises a cytokine element linked to and co-expressed with the intracellular domain; the cytokine is selected from the group consisting of IL-2, IL-7, or a combination thereof.

[0009] In another preferred embodiment, the CAR has the following structure as shown in Formula I:

[0010] L-EB-H-TM-C-CD3ζ-(A-P)n (I)

[0011] In the formula,

[0012] each “-” is independently a linking peptide or a peptide bond;

[0013] L is nothing or a signal peptide sequence;

[0014] EB is a binding domain that specifically binds to FLT3;

[0015] H is nothing or a hinge region;

[0016] TM is a transmembrane domain;

[0017] C is a costimulatory signaling molecule;

[0018] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0019] A is a self-cleaving protein;

[0020] P is a cytokine;

[0021] n is 0, 1, or 2.

[0022] In another preferred embodiment, n is 1.

[0023] In another preferred embodiment, the L is nothing.

[0024] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of GM-CSF receptor, CD8, CD28, CD4, CD137, or a combination thereof.

[0025] In another preferred embodiment, the L is a signal peptide of a GM-CSF receptor, and the amino acid sequence is shown as SEQ ID NO: 4.

[0026] In another preferred embodiment, the EB is a single-domain antibody (VHH) that specifically targets FLT3, a single-chain antibody (scFv), or a combination thereof.

[0027] In another preferred embodiment, the EB is a single domain antibody (VHH) specifically targeting FLT3, and the amino acid sequence is set forth in SEQ ID NO: 5.

[0028] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of IgG, IgE, CD8, CD28, CD137, or a combination thereof.

[0029] In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of IgG, IgE, CD8, CD28, CD137, or a combination thereof.

[0030] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD8, CD4, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3, or a combination thereof.

[0031] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD8, CD4, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1, LAG-3, or a combination thereof.

[0032] In another preferred embodiment, the C is a costimulatory signaling molecule of a protein selected from the group consisting of CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), OX40, PD1, Dap10, CDS, ICAM-1, or a combination thereof.

[0033] In another preferred embodiment, the C is a costimulatory signaling molecule of a protein selected from the group consisting of CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), OX40, PD1, Dap10, CDS, ICAM-1, or a combination thereof.

[0034] In another preferred embodiment, the self-cleaving protein is selected from the group consisting of T2A, P2A, E2A, F2A, or a combination thereof.

[0035] In another preferred embodiment, the self-cleaving protein is T2A, and the amino acid sequence is set forth in SEQ ID NO: 9.

[0036] In another preferred embodiment, the cytokine is selected from the group consisting of IL-2, IL-7, or a combination thereof.

[0037] In another preferred embodiment, the IL-2 comprises wild type IL-2 and mutant IL-2, or an active fragment thereof.

[0038] In another preferred embodiment, the IL-2 comprises wild type IL-2 and mutant IL-2, or an active fragment thereof.

[0039] In another preferred embodiment, the IL-7 comprises wild type IL-7 and mutant IL-7, or an active fragment thereof.

[0040] In another preferred embodiment, the IL-7 comprises wild type IL-7 and mutant IL-7, or an active fragment thereof.

[0041] In another preferred embodiment, the amino acid sequence of the CAR is set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

[0042] In a second aspect of the present application, there is provided a nucleic acid molecule encoding the chimeric antigen receptor CAR as described in the first aspect of the present application.

[0043] In another preferred embodiment, the nucleic acid molecule is DNA, RNA, or a combination thereof.

[0044] In a third aspect of the present application, there is provided a vector comprising the nucleic acid molecule as described in the second aspect of the present application.

[0045] In another preferred embodiment, the vector is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a transposon, or a combination thereof.

[0046] In another preferred embodiment, the vector is a lentiviral vector.

[0047] In a fourth aspect of the present application, there is provided an engineered immune cell expressing the chimeric antigen receptor CAR as described in the first aspect of the present application.

[0048] In another preferred embodiment, the immune cell comprises a T cell, an NK cell, a macrophage, or a combination thereof.

[0049] In another preferred embodiment, the immune cell is autologous.

[0050] In another preferred embodiment, the immune cell is allogeneic.

[0051] In another preferred embodiment, the T cell comprises a γδ T cell, an αβ T cell, or a combination thereof.

[0052] In another preferred embodiment, the T cell is a γδ T cell.

[0053] In another preferred embodiment, the engineered immune cell is a CAR-γδ T cell.

[0054] In a fifth aspect of the present application, there is provided a method of preparing the engineered immune cell as described in the fourth aspect of the present application, comprising the step of transducing the nucleic acid molecule as described in the second aspect of the present application or the vector as described in the third aspect of the present application into an immune cell, thereby obtaining the engineered immune cell.

[0055] In another preferred embodiment, the immune cell is a γδ T cell.

[0056] In another preferred embodiment, the method further comprises a step of detecting the function and effectiveness of the obtained engineered immune cells.

[0057] In a sixth aspect of the present application, a pharmaceutical composition comprising the CAR of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, or the engineered immune cell of the fourth aspect of the present application, and a pharmaceutically acceptable carrier, diluent or excipient is provided.

[0058] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0059] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0060] In another preferred embodiment, the engineered immune cell is a CAR-γδT cell.

[0061] In another preferred embodiment, the concentration of the cells in the pharmaceutical composition is 1 x 10 3 -1 x 10 8 cells / ml, preferably 1 x 10 4 -1 x 10 7 cells / ml.

[0062] In another preferred embodiment, the pharmaceutical composition further comprises other drugs (such as antibody drugs, chemotherapy drugs or other CAR-T drugs) that selectively kill tumor cells.

[0063] In a seventh aspect of the present application, the use of the CAR of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, the engineered immune cell of the fourth aspect of the present application, or the pharmaceutical composition of the sixth aspect of the present application for the preparation of a medicament or preparation for treating a tumor is provided.

[0064] In another preferred embodiment, the tumor is a FLT3-positive tumor.

[0065] In another preferred embodiment, the tumor comprises a hematological tumor, a solid tumor, or a combination thereof.

[0066] In another preferred embodiment, the tumor is acute myeloid leukemia.

[0067] In an eighth aspect of the present application, a method for treating a cancer or a tumor is provided, comprising administering a safe and effective amount of the CAR of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the vector of the third aspect of the present application, the engineered immune cell of the fourth aspect of the present application, or the pharmaceutical composition of the sixth aspect of the present application to a subject in need.

[0068] In another preferred embodiment, the subject comprises a human or a non-human mammal.

[0069] In another preferred embodiment, the non-human mammal comprises a rodent (e.g., mouse, rat), a primate (e.g., monkey).

[0070] In another preferred embodiment, the tumor is a FLT3-positive tumor.

[0071] In another preferred embodiment, the tumor comprises a hematological tumor, a solid tumor, or a combination thereof.

[0072] In another preferred embodiment, the tumor is an acute myeloid leukemia.

[0073] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail below (e.g., in the Examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0074] FIG. 1 shows the purity and subtypes of gd T cells cultured in vitro for 19 days using flow cytometry.

[0075] FIG. 2 shows the preparation and identification of CAR-gd T cells with different structures. A is a schematic diagram of FLT3-CAR, FLT3-IL2-CAR, and FLT3-IL7-CAR structures; B is the positive rate of CAR-gd T cells detected by flow cytometry; C is the expression of CD3 zeta fusion protein detected by Western blotting; D is the content of IL-2 and IL-7 cytokines in the supernatant of gd T cells and CAR-gd T cells cultured in vitro for 5-8 days detected by CBA method; E is the change of the positive rate of CAR-gd T cells at different time points during the in vitro culture process detected by flow cytometry.

[0076] FIG. 3 shows the expression of FLT3 antigen on the surface of different AML tumor cell lines; unstained indicates the control group without staining, and FLT3 represents the experimental group with staining.

[0077] FIG. 4 shows the killing toxicity of CAR-gd T cells with different structures on target cells in vitro; A is the apoptosis of tumor cells detected by flow cytometry after CAR-gd T cells were co-incubated with tumor cells at different effector-to-target ratios for 24 h; B is the release amount of Granzyme B and IFN-γ in the supernatant after CAR-gd T cells were co-incubated with tumor cells for 24 h.

[0078] Figure 5 shows the killing effect of CAR-γδT cells with different structures on primary AML cells, according to the proportion of CD33 + The change in the proportion of tumor cells was used to calculate the killing efficiency.

[0079] Figure 6 shows the results of the in vitro anti-tumor persistence test of FLT3-CAR-γδT cells with different structures; wherein A is a schematic diagram of tumor antigen restimulation (Stimu) experiment; B is the change in killing effect of different CAR-γδT cells during tumor antigen restimulation; C is the proportion of Annexin-V and 7-AAD double negative cells in effector cells detected by flow cytometry after 24h of each tumor antigen stimulation; D is the expression level of Granzyme B, IFN-γ and TNF-α cytokines in the supernatant after each tumor antigen stimulation detected by CBA method.

[0080] Figure 7 shows the expression level of CD45RO and CD62L on the surface of effector cells after 24h of each stimulation, detected by flow cytometry, to characterize the stemness level of effector cells after multiple tumor antigen stimulations.

[0081] Figure 8 shows the in vivo anti-tumor effect of CAR-γδT cells with different structures; wherein A is a schematic diagram of in vivo pharmacodynamic verification scheme; B is the result of mouse live imaging; C is the statistical result of mouse tumor load; D is the change in mouse body weight; E is the survival curve of mice; F is the proportion of total γδT cells in the peripheral blood of mice in each group detected by flow cytometry after 7, 14 and 19 days of CAR-γδT cell transfusion.

[0082] Figure 9 shows the RNA-seq results of CAR-γδT cells after antigen restimulation; wherein A is a differentially expressed gene Wayne diagram of different CAR-γδT cells; B is the KEGG enrichment analysis of up-regulated differential genes of FLT3-IL2-CAR-γδT cells compared with FLT3-CAR-γδT cells (left), and FLT3-IL2-CAR-γδT cells compared with FLT3-IL7-CAR-γδT cells (right); C is a clustering heat map of the expression of differential genes related to cell cycle pathway of different CAR-γδT cells. DETAILED DESCRIPTION

[0083] Through extensive and in-depth research, the inventors provide an engineered FLT3-targeted CAR-gammadelta T cell, which co-expresses IL-2 or IL-7 cytokines, can maintain a high stemness level and proliferation capacity, and has sustained tumor cell killing capacity. In particular, the FLT3-IL2-CAR-gammadelta T cell co-expressing IL-2 of the present application exhibits sustained killing capacity and anti-tumor activity on AML tumor cells in various in vitro and in vivo experiments. The engineered CAR-gammadelta T cell of the present application can be used for allogeneic cell therapy, has high safety and wide applicability. The CAR-gammadelta T cell of the present application has clinical application prospect in treating AML. On this basis, the present application is completed.

[0084] FLT3

[0085] FLT3 (FMS-like tyrosine kinase-3) is a member of the RTK family, which plays an important role in the proliferation, differentiation and survival of hematopoietic stem cells, precursor B cells, etc. It has been found that FLT3, as an important receptor tyrosine kinase in cell signal transduction, can cause abnormal cell proliferation and induce tumor occurrence, especially closely related to the occurrence and development of acute myeloid leukemia (AML). Studies have shown that more than 70% of AML patients and acute lymphoblastic leukemia (ALL) patients have high expression of FLT3.

[0086] The structure of FLT3 protein includes extracellular region, transmembrane region and intracellular region, the five IgG-like structures of the extracellular region are the binding domain of FLT3 receptor and ligand, the transmembrane region is the kinase discontinuous action scope, and the intracellular region is the catalytic region of tyrosine kinase. Under normal circumstances, FLT3 and other kinases have self-inhibitory function and are in an inactive conformation. When a length mutation or a domain activity loop replacement mutation occurs in the membrane proximal region of FLT3, it leads to constitutive FLT3 activation, thereby activating multiple downstream signaling pathways, causing DNA damage and repair defects, and promoting cell proliferation and resistance to apoptosis. FLT3 is one of the most common mutant genes in acute myeloid leukemia (AML), accounting for about 30% of AML patients, of which 20-25% are FLT3-ITD mutant and 5-10% are FLT3-TKD mutant. These mutations lead to poor prognosis and high risk of recurrence in AML patients induced by chemotherapy and allogeneic hematopoietic stem cell transplantation.

[0087] The selection of AML targets is an important part of AML treatment. In addition to FLT3, the treatment targets of AML also include CD123 and CD33, etc. Current studies have shown that compared with CD123 and CD33, the target of FLT3 is safer and has stronger clinical transformation potential. In summary, FLT3 is an important target for treating AML.

[0088] The chimeric antigen receptor CAR of the present application

[0089] Chimeric antigen receptors (CAR) generally consist of an extracellular antigen recognition region, a transmembrane region and an intracellular co-stimulatory signal region. The extracellular segment of CAR can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation, proliferation, cytolytic toxicity and secretion of cytokines, thereby eliminating target cells.

[0090] Specifically, the chimeric antigen receptor (CAR) of the present application comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular region is mainly an antigen binding domain, which is used for recognition and binding with the antigen on the surface of target cells, and is usually a single-chain variable fragment (scFv) of an antibody or a single-domain antibody (also known as nanobody, VHH). The extracellular region can also optionally include a hinge region, which serves as a connection and enhances the flexibility and stability of the structure. The transmembrane region connects the intracellular and extracellular regions of CAR. The intracellular region can include a signaling domain and a costimulatory molecule, which is responsible for signal transmission to further improve the signal transmission capacity of a specific CAR and enable immune cells to have stronger killing ability and survival time.

[0091] The N-terminus of the extracellular domain of the chimeric antigen receptor (CAR) of the present application can optionally comprise a signal peptide sequence. In immature CAR molecules, the function of the signal peptide is mainly to guide the CAR protein to correctly enter the endoplasmic reticulum, so as to perform subsequent folding, modification and transport. After the CAR is successfully positioned on the cell membrane, the signal peptide is usually cut off, so that the mature CAR molecule usually does not contain a signal peptide. In the CAR molecule of the present application, the type of signal peptide is not particularly limited. In an embodiment of the present application, the sequence of the selected signal peptide is as shown in SEQ ID NO: 4.

[0092] In the CAR molecule of the present application, the extracellular binding domain can be a single-domain antibody / nanobody (VHH) or scFv that specifically targets FLT3 antigen. In an embodiment of the present application, the extracellular binding domain is a VHH that specifically targets FLT3 antigen; preferably, the amino acid sequence of the VHH that specifically targets FLT3 antigen is as shown in SEQ ID NO: 5.

[0093] In the present application, the VHH of the present application also includes conservative variants thereof, which means that a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, most preferably at most 3 amino acids in the amino acid sequence of the VHH of the present application with amino acids of similar or similar properties has similar specific targeting FLT3 function as the VHH of the present application.

[0094] For the hinge region and the transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain naturally associated with one of the domains in the CAR is used. In one embodiment of the present application, the hinge region of the CAR molecule is from the IgG4 Fc sequence, having an amino acid sequence as shown in SEQ ID NO: 6; the transmembrane region and the intracellular region of the CAR molecule are from the CD28 protein, having an amino acid sequence as shown in SEQ ID NO: 7.

[0095] The chimeric antigen receptor CAR of the present application also includes a cytokine element linked to the intracellular domain and co-expressed. Cytokine is a small molecular protein that plays a mutual regulatory role between cells, which can regulate the growth and differentiation of cells and effects, thereby regulating immune response. Preferably, the cytokine element is IL-2, IL-7, or a combination thereof; more preferably, the cytokine element is IL-2. It should be understood that the cytokine element of the present application includes wild type, mutant and derivatives thereof (such as truncation or active fragment thereof), as long as the mutant or derivative has similar function to the wild type cytokine.

[0096] For example, the amino acid sequence of IL-2 in the present application is as shown in SEQ ID NO: 10; the IL-2 of the present application also includes a sequence having sequence identity to the sequence shown in SEQ ID NO: 10, preferably a sequence having ≥ 85% (such as ≥ 90%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, ≥ 99%) sequence identity. The amino acid sequence of IL-7 in the present application is as shown in SEQ ID NO: 11; the IL-7 of the present application also includes a sequence having sequence identity to the sequence shown in SEQ ID NO: 11, preferably a sequence having ≥ 85% (such as ≥ 90%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, ≥ 99%) sequence identity.

[0097] In one embodiment of the application, the cytokine element is covalently linked to the intracellular domain via a cleavable peptide, preferably the cleavable peptide is a self-cleaving 2A peptide, including but not limited to T2A, P2A, E2A, F2A. In one embodiment of the application, one or more cytokine elements are covalently linked to the intracellular domain via a cleavable peptide, for example 1, 2, 3 or 4. When multiple cytokine elements are covalently linked to the intracellular domain, the cytokine elements can be the same kind of cytokine or different kinds of cytokine.

[0098] In one preferred embodiment of the application, the CAR does not contain a cytokine element linked to the intracellular domain, the amino acid sequence of the CAR is shown as SEQ ID NO: 1:

[0099] In one preferred embodiment of the application, the CAR contains an IL-2 element linked to the intracellular domain, the amino acid sequence of the CAR is shown as SEQ ID NO: 2:

[0100] In one preferred embodiment of the application, the CAR contains an IL-7 element linked to the intracellular domain, the amino acid sequence of the CAR is shown as SEQ ID NO: 3:

[0101] γδT cells

[0102] γδT cells (also known as gdT cells) are another innate subpopulation of T cells different from αβT cells, accounting for only 1-5% of peripheral blood T cells. Unlike αβT cells, γδT cells are non-MHC restricted, and γδT cells recognize target antigens independent of MHC, and do not cause graft versus host disease (GvHD).

[0103] According to the δ chain of TCR composition, γδT cells can be further subdivided into four subpopulations of Vδ1T, Vδ2T, Vδ3T and Vδ5T. The Vγ chain paired with the Vδ1 chain is relatively abundant and mainly exists in epithelial tissues such as skin, intestinal tract, spleen and liver; while the Vδ2 chain is usually paired with the Vγ9 chain, so it is called Vγ9Vδ2T cell, which mainly exists in peripheral blood; the more studied γδT cell subpopulations are Vδ1T cells and Vδ2T cells. Vδ1T cells belong to tissue-resident lymphocytes and play a unique role in immune surveillance of human tissues; in addition, the tissue-resident property of Vδ1T cells enables them to have stronger homing and infiltration ability to solid tumors, so compared with other types of immune cells, Vδ1T cells have stronger ability to migrate and infiltrate into tumor sites.

[0104] For other engineered T cells (αβ T cells), if one wants to reduce the risk of graft-versus-host disease, one needs to genetically engineer isolated patient autologous cells or obtain heterologous donor cells, and then inject the CAR-producing immune cells into the patient. But for CAR-γδ T cells, no genetic editing is needed for allogeneic treatment, which is safer and more applicable.

[0105] CAR-γδ T cells have the advantages of rich and diverse killing pathways, MHC independence, and low incidence of graft-versus-host disease, making them safer and more applicable than other CAR-T cells.

[0106] CAR-γδ T cells of the present application

[0107] As used herein, the terms "CAR-γδ T cells of the present application", "CAR-γδ T cells targeting FLT3 of the present application", "CAR-gdT cells of the present application" are used interchangeably, and all refer to the CAR-γδ T cells described in the present application, which can target FLT3 and be used to treat tumors with high expression or positive for FLT3, especially AML.

[0108] In the present application, the nucleic acid molecule encoding the CAR of the present application or the vector containing the nucleic acid molecule encoding the CAR of the present application is introduced into γδ T cells to obtain the CAR-γδ T cells of the present application. Methods for introducing nucleic acid molecules or vectors into immune cells are known in the art, such as electroporation, viral transfection, gene editing, etc., but are not limited thereto.

[0109] The CAR-γδ T cells of the present application are CAR-γδ T cells co-expressing a cytokine element, which have more significant cytotoxicity and anti-tumor activity than cells not expressing a cytokine. In addition, the CAR-γδ T cells of the present application exhibit significant sustained killing capacity during repeated stimulation of tumor antigens, and can maintain a high proportion of stem cell-like memory T cell subpopulations after multiple tumor antigen stimulations. Preferably, the cytokine is IL-2, IL-7, or a combination thereof; more preferably, the cytokine element is IL-2.

[0110] Pharmaceutical composition or preparation

[0111] The present application provides a pharmaceutical composition or preparation, which takes the CAR-γδ T cells of the present application as the active ingredient, and contains a pharmaceutically acceptable carrier, diluent or excipient.

[0112] In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-γδ T cells in the preparation is 1×10 3 -1×108 1 x 105 4 1 x 105 7 1 x 105

[0113] In one embodiment, the formulation can include a buffer such as neutral buffered saline, sulfate buffered saline, and the like; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelator such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulations of the present application are preferably formulated for intravenous administration.

[0114] Therapeutic applications

[0115] In the present application, there is also provided the use of the CAR-γδ T cells of the present application for treating FLT3-positive tumors; preferably, the FLT3-positive tumor is acute myeloid leukemia (AML). The present application provides a method of treating AML, comprising administering to a subject in need thereof the CAR-γδ T cells of the present application or the pharmaceutical composition or formulation of the present application.

[0116] The source of the CAR-γδ T cells of the present application can be autologous or allogeneic. In one embodiment, the source of the CAR-γδ T cells of the present application is allogeneic, and the antigen is recognized by the CAR-γδ T cells in an MHC-unrestricted manner, thus reducing the probability of developing graft versus host disease without the need for genetic engineering.

[0117] In one embodiment, the CAR-γδ T cells of the present application can undergo robust in vivo T cell expansion and can persist for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-γδ T cells induce an immune response specific to the antigen binding domain in the CAR. For example, the CAR-γδ T cells of the present application elicit a specific immune response against cells expressing FLT3.

[0118] The CAR-γδ T cells of the present application can be administered alone or as a pharmaceutical composition in combination with diluents and / or with other components or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present application can include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include a buffer such as neutral buffered saline, sulfate buffered saline, and the like; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelator such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present application are preferably formulated for intravenous administration.

[0119] The pharmaceutical compositions of the present application can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the subject's condition, and the type and severity of the disease - although appropriate dosages can be determined by clinical trials.

[0120] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present application to be administered can be determined by a physician with consideration for a variety of factors which modify the action of drugs, including the age, weight, tumor size, extent of infection or metastasis and individual condition of the patient (subject). It can be generally stated that the pharmaceutical composition comprising the CAR-γδ T cells described herein can be administered at a dosage of 10 3 to 10 8 cells / kg body weight, preferably 10 4 to 10 7 cells / kg body weight (including all integer values within those ranges). The CAR-γδ T cell composition can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques well known in the art of immunotherapy. The optimal dosage and treatment regimen for a particular patient can be readily determined by monitoring the patient's disease signs and adjusting the treatment accordingly by a person skilled in the medical art.

[0121] The administration of the subject composition can be performed in any convenient manner, including by spray, injection, ingestion, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the CAR-γδ T cell composition of the present application is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the CAR-γδ T cell composition of the present application is preferably administered by i.v. injection. The composition of CAR-γδ T cells can be directly infused into a tumor, lymph node or site of infection.

[0122] The main advantages of the present application include:

[0123] (1) The CAR-γδ T cells of the present application are non-MHC restricted, being allogeneic universal CAR-T cells, with a wide applicability.

[0124] (2) The CAR-γδ T cells of the present application co-expressing exogenous cytokines have a significant and persistent in vitro and in vivo killing capacity against AML tumor cells.

[0125] (3) The CAR-γδ T cells of the present application co-expressing exogenous cytokines are able to maintain a high level of stemness and expansion capacity.

[0126] (4) The CAR-γδT cell co-expressing an exogenous cytokine has high safety and clinical application feasibility.

[0127] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and fractions are weight percentages and weight fractions.

[0128] Materials and methods

[0129] (I) Cell culture:

[0130] AML cell lines (OCI-AML3, THP-1, MOLM-13, MV4-11) and 293T cells were from ATCC. Among them, OCI-AML3, THP-1 and MOLM-13 were cultured in RPMI 1640 medium (Hyclone) containing 10% FBS (allBio); MV4-11 cells were cultured in IMDM medium (Hyclone) containing 10% FBS (allBio); 293T cells were cultured in DMDM medium (Hyclone) containing 10% FBS (allBio).

[0131] After the PBMC cells of healthy donors (Shanghai Miaoshun Biotechnology Co., Ltd.) were resuscitated, they were cultured for one day using Opti Vitro T Cell Serum-Free Medium (ExCell Bio) without cytokines. The PBMCs were sequentially co-incubated with Biotin-Antibody Cocktail and Anti-Biotin MicroBeads (Meitainyi Biotech), and then the non-TCRγ / δ cells and TCRγ / δ cells were separated using a MACS separation column (Meitainyi Biotech). The separated γδT cells were resuspended using Opti Vitro T Cell Serum-Free Medium added with IL-2 (300 IU / mL; Coastal Protein) and IL-21 (30 ng / mL; Coastal Protein), and transferred to a 24-well plate with a cell density of 4×10 6 / well. The γδT cells were activated by human T cell activator CD3 / CD28 magnetic beads (Meitainyi Biotech).

[0132] (II) Construction of CAR-γδT cells:

[0133] CAR sequence targeting FLT3 was designed and synthesized (Guangzhou WISDOM BIOTECH CO., LTD.), then cloned directly into lentivirus vector and lentivirus packaging was performed.

[0134] After two days of γδ T cell activation, lentivirus concentrate was co-incubated with γδ T cells for 24 h, then washed once with PBS buffer, and cell pellets were resuspended in Opti Vitro T cell serum-free medium (containing IL-2 and IL-21). After 7 days of γδ T cell transduction, CAR+ cells were labeled with anti-FLT3-VHH antibody, and then the positive rate was detected using flow cytometry.

[0135] (III) In vitro toxicity detection of CAR-γδ T cells:

[0136] In the AML tumor cell line killing experiment, Violet antibody (Invitrogen) was used to label target cells, which were inoculated into 24-well plates at a specification of 2×10 5 cells / well, and a certain amount of effector cells were added to the corresponding wells at E:T = 1:10, 1:5, and 1:1, respectively. After 24 h of co-incubation in the incubator, the expression levels of 7-AAD and Annexin V proteins on the surface of tumor cells were detected using flow cytometry.

[0137] In the primary AML cell killing experiment, according to the number of primary tumor cells, 1.5-2×10 5 cells / well were added to 24-well plates, and Violet-labeled effector cells were added at an E:T ratio of 1:1, and after 24 h, flow cytometry was used to detect the proportion of CD33 + tumor cells with and without the addition of effector cells.

[0138] (IV) In vitro persistence detection of CAR-γδ T cells:

[0139] In order to study the in vitro persistence of CAR-γδ T cells, an antigen restimulation experiment was designed. Violet antibody was used to label effector cells, which were added to 24-well plates at a specification of 6×10 5 cells / well, and OCI-AML-3 was added to the corresponding plates at an E:T ratio of 1:2, and five groups of parallel experiments were set up at the same time. After 24 h, 6×10 5 cells / well were added to four parallel plates. Flow cytometry was used to detect the expression levels of 7-AAD and Annexin V proteins on the surfaces of effector cells and target cells in the untreated parallel plates. The above operation was repeated every 24 hours until five times of OCI-AML-3 cells were added.

[0140] (V) Cytokine release analysis:

[0141] γδT / CAR-γδT cells were cultured for 5-8 days, and the culture supernatant was collected to detect the contents of IL-2 and IL-7 cytokines according to the operation instruction of CBA kit (BD Biosciences).

[0142] The supernatant of tumor cells co-cultured with effector cells was collected to detect the contents of Granzyme B, IFN-γ and TNF-α cytokines according to the operation instruction of CBA kit (BD Biosciences). The data were analyzed using FACP software (BD Biosciences).

[0143] (VI) Flow cytometry analysis:

[0144] After the γδT / CAR-γδT / primary AML cells were washed with PBS buffer, the cells were co-incubated with corresponding antibodies at 4°C for 30 min, then washed twice with PBS buffer, and finally the expression of corresponding proteins on the cell surface was detected using flow cytometry. These antibodies include anti-human TCR Vδ1 antibody (Invitrogen), anti-human TCR Vδ2 antibody (Biolegend), anti-human TCR-αβ antibody (BD Biosciences), anti-human CD3 antibody (BD Biosciences), anti-human CD45RO antibody (BD Biosciences), and anti-human CD62L antibody (BD Biosciences).

[0145] (VII) Animal experiments:

[0146] To explore the anti-tumor activity of CAR-γδT cells in vivo, 6-8-week-old female NCG (NOD / ShiLtJ Gpt-Prkdcem26Cd52I12rgem26Cd22 / Gpt) mice (Jingchu Yaoke, China) were used to construct an AML model. 1×10 6 OCI-AML3-luciferase cells were injected into the tail vein of the mice on Day 0, and 7×10 6 CAR-γδT cells were reinfused into the tail vein on Day 10. The imaging data were obtained using an IVIS imaging system (PerkinElmer), and the bioluminescence imaging analysis was performed using Living Image software (PerkinElmer). The body weight and survival status of the mice were continuously monitored. The proportion of γδT cells in the peripheral blood of the mice was detected using a flow cytometer on the 7th, 14th and 19th days after the reinfusion of CAR-γδT cells.

[0147] (VIII) RNA-seq:

[0148] According to the antigen repeated stimulation experiment operation process, the number of stimulations is set to 5 times, and the effector target ratio is set to 5:1. The CAR-γδT cells after 5 times of stimulation are sorted by using a full-automatic flow cytometry sorter (SONY), and BGI Genomics is commissioned to perform subsequent transcriptome sequencing.

[0149] (ix) Data analysis:

[0150] In the present application, data are analyzed by using GraphPad Prism 8.4.0 software. Data are expressed as mean ± standard error (Mean ± SEM). The difference between two groups is analyzed by using Student's t-test, and the analysis between multiple groups of data is analyzed by using two-way ANOVA, and survival data are analyzed by using Log-rank (Mantel-Cox). * represents P<0.05; ** represents P<0.01; *** represents P<0.001; **** represents P<0.0001, and ns represents P>0.05. P>0.05 is considered to have no statistical difference.

[0151] Example 1: Construction and detection of CAR-γδT

[0152] (I) Purity detection of γδT cells cultured in vitro

[0153] In order to verify the purity of the γδT cells cultured in vitro, the purity and subtype of the γδT cells at the end of the in vitro culture were detected by using flow cytometry. The results are shown in Figure 1, wherein, CD3 + TCRαβ - is a γδT cell, accounting for 99.35%; CD3 + TCR Vδ1 + is a Vδ1 type γδT cell, accounting for 73.46%; CD3 + TCR Vδ2 + is a Vδ2 type γδT cell, accounting for 15.31%; CD3 + TCR Vδ1 - TCR Vδ2 - is other type γδT cell, accounting for 10.62%. The above results show that high-purity γδT cells mainly in Vδ1 type are obtained by the culture method of the present application.

[0154] (ii) Construction and verification of CAR-γδT targeting FLT3

[0155] The FLT3 VHH nano sequence is used to construct a CAR vector targeting FLT3, and is named FLT3-CAR. The CAR structure uses CD28 as the co-stimulatory domain and CD3ζ as the intracellular signal domain. The cytokine IL-2 or IL-7 protein sequence is added to the FLT3-CAR structure, and is named FLT3-IL2-CAR and FLT3-IL7-CAR, respectively. The specific structure is shown in FIG. 2A.

[0156] The amino acid sequence of FLT3-CAR is shown in SEQ ID NO: 1; the amino acid sequence of FLT3-IL2-CAR is shown in SEQ ID NO: 2; and the amino acid sequence of FLT3-IL7-CAR is shown in SEQ ID NO: 3.

[0157] Lentivirus concentrates of the three CAR structures are prepared using 293T cells, and γδT cells are transduced to obtain FLT3-CAR-γδT, FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT cells, respectively. The expression rates of the three CAR-γδT cells are detected by flow cytometry and Western blotting when the γδT cells are cultured to Day 9 after lentivirus transduction. The results show that the three CARs can be stably expressed on the surface of γδT cells (FIGS. 2B-2C). In addition, the three different CARs can be continuously and stably expressed on γδT cells during the in vitro culture of CAR-γδT cells (FIG. 2E).

[0158] To further verify whether FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT cells can normally secrete the respective co-expressed cytokines, the contents of IL-2 and IL-7 cytokines in the supernatants of γδT cells and CAR-γδT cells cultured in vitro for Day 5-Day 8 are detected by CBA. The results show that FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT cells can secrete IL-2 and IL-7 cytokines, respectively (FIG. 2D).

[0159] The above results show that the FLT3-CAR-γδT, FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT engineered γδT cells are successfully constructed, and FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT cells can successfully secrete the co-expressed cytokines.

[0160] Example 2: Cytotoxic effect of CAR-γδT cells on target cells

[0161] (I) Detection of AML tumor cell surface antigen expression

[0162] To understand the expression of FLT3 antigen on the surface of AML tumor cell lines, the expression of surface antigens on common AML tumor cell lines was detected. The results are shown in Figure 3. The four AML tumor cell lines all highly expressed FLT3 antigen.

[0163] (ii) Killing effect of CAR-γδT cells on AML cell lines in vitro

[0164] To explore the anti-tumor effect of different CAR-γδT cells on AML cell lines in vitro, three kinds of CAR-γδT cells were co-incubated with OCI-AML-3, THP-1, MOLM-13 and MV4-11 tumor cells, respectively. The results showed that FLT3-IL2-CAR-γδT cells exhibited superior tumor killing effect at effector-to-target ratios of 1:10, 1:5 and 1:1, especially at an effector-to-target ratio of 1:1, FLT3-IL2-CAR-γδT cells could almost completely eliminate OCI-AML3, THP-1 and MV4-11 tumor cells (Figure 4A). The killing effect of FLT3-IL2-CAR-γδT was better than that of FLT3-IL7-CAR-γδT and FLT3-CAR-γδT.

[0165] The expression levels of Granzyme B and IFN-γ in the supernatant after co-incubation of effector cells and tumor cells were also detected. Compared with the FLT3-CAR-γδT group, the FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT groups had higher cytokine release, and the FLT3-IL2-CAR-γδT group had the strongest cytokine release, which was consistent with the killing results (Figure 4B).

[0166] The above results show that FLT3-IL2-CAR-γδT cells exhibit the most superior cytotoxicity to various AML tumor cell lines in vitro.

[0167] Example 3: Killing effect of CAR-γδT cells on primary AML cells

[0168] To explore the killing effect of different CAR-γδT cells on primary AML cells in vitro, peripheral blood samples from 7 patients with acute myeloid leukemia were collected, and the expression of antigens on the surface of each sample was detected using flow cytometry. The results are shown in Table 1.

[0169] Table 1. Information of patients with acute myeloid leukemia

[0170] CAR-γδT cells were co-incubated with primary tumor cells at an effector to target ratio of 1:1, and the killing rate was calculated and counted according to the change in the proportion of CD33+ tumor cells before and after killing.

[0171] The experimental results are shown in Figure 5, and the killing statistics show that the killing effect of FLT3-IL2-CAR-γδT cells on primary AML cells is significantly higher than that of each of the other groups. The above data show that the FLT3-IL2-CAR-γδT cells constructed by the present application can exhibit a significant cytotoxic effect on patient-derived primary AML cells, and have the potential to treat AML in clinical treatment.

[0172] Example 4: Persistent killing ability of CAR-γδT cells

[0173] To explore the anti-tumor persistence of CAR-γδT cells with different structures, an antigen repetitive stimulation experiment was designed to simulate the long-term tumor stimulation environment in vivo, and the persistent killing ability of different CAR-γδT cells on AML tumor cells was compared. The experimental design is shown in Figure 6A, and tumor antigen was added every 24 hours to stimulate CAR-γδT cells, and the killing efficiency of CAR-γδT cells stimulated by antigen was detected for five days.

[0174] The results show that with the increasing number of tumor antigen stimulations, the killing effect of γδT cells and FLT3-CAR-γδT cells on OCI-AML3 cells decreased sharply, the killing efficiency of FLT3-IL7-CAR-γδT cells showed a slow downward trend, and FLT3-IL2-CAR-γδT cells still had a high killing efficiency after experiencing multiple antigen stimulations (Figure 6B).

[0175] At the same time, the viability of γδT cells and CAR-γδT cells after each antigen stimulation was analyzed. The results show that CAR-γδT cells carrying cytokines always maintain a high viability during the process of repeated tumor antigen stimulation, while the viability of γδT cells and FLT3-CAR-γδT cells rapidly decreases after experiencing three tumor antigen stimulations (Figure 6C). This may be one of the reasons for the poor killing persistence of the γδT group and the FLT3-CAR-γδT group.

[0176] To explore the ability of γδT / CAR-γδT cells to release functional cytokines during repeated stimulation of tumor antigens, the supernatant of the effector cells co-incubated with tumor cells during repeated stimulation of tumor antigens was collected. The results showed that the Granzyme B, IFN-γ and TNF-α cytokines of the γδT group and the FLT3-CAR-γδT group were always at a low release level, while the FLT3-IL2-CAR-γδT group could still release higher levels of cytokines after experiencing multiple tumor antigen stimulations compared to the FLT3-IL7-CAR-γδT group (Figure 6D). Stable release of high levels of cytokines is one of the reasons for maintaining the high and efficient killing ability of cells, and this result is consistent with the FLT3-IL2-CAR-γδT having the most persistent killing ability.

[0177] Compared with other memory T lymphocytes, stem cell-like memory T cells exhibit more significant self-renewal and proliferation capacity, and can timely supplement more memory and effector T cell subpopulations, which also makes stem cell-like memory T cells more suitable for immunotherapy. Therefore, the expression levels of memory differentiation markers of γδT, FLT3-CAR-γδT, FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT cells after each tumor antigen stimulation were detected by flow cytometry. Among them, CD45RO - CD62L + is stem cell-like memory T cells (T SCM ); CD45RO + CD62L + is central memory T cells (T CM ); CD45RO + CD62L - is effector memory T cells (T EM ); CD45RO - CD62L - is terminally differentiated T cells (T EMRA ).

[0178] As shown in Figure 7 and Table 2, after experiencing multiple tumor antigen stimulations, the memory phenotypes of γδT, FLT3-CAR-γδT and FLT3-IL7-CAR-γδT cells changed similarly, mainly showing a decrease in the proportion of stem cell-like memory T cell subpopulations, while the proportion of stem cell-like memory T cell subpopulations of FLT3-IL2-CAR-γδT cells remained at a high level and did not decrease with an increase in the number of tumor antigen stimulations.

[0179] Table 2. Proportion of stem cells of CAR-γδT in each group after the 4th and 5th stimulations (%) Table 2. Proportion of stem cells of CAR-γδT in each group after the 4th and 5th stimulations (%)

[0180] Example 5: Anti-tumor effect of CAR-γδT cells in vivo

[0181] To compare the anti-tumor abilities of CAR-γδT cells with different structures in vivo, a CDX mouse model of AML was constructed using NCG immunodeficient mice, and the experimental scheme is shown in FIG. 8A. First, the mice were injected with OCI-AML3-luciferase cells in the tail vein on Day 0, and then the CAR-γδT cells were transfused on Day 10. Subsequently, the mice were subjected to live fluorescence imaging at regular intervals, and the survival of the mice was continuously monitored.

[0182] The results of live imaging showed that the FLT3-IL2-CAR-γδT group significantly inhibited the growth of tumor cells in mice compared with other CAR-γδT groups (FIGS. 8B-8C), and the body weight of the mice did not decrease significantly (FIG. 8D). In addition, the survival curve statistical results showed that the CAR-γδT group effectively prolonged the survival of tumor-bearing mice compared with the PBS group and the γδT group, and the FLT3-IL2-CAR-γδT group had the best effect, which could significantly prolong the survival of mice to more than 68 days (FIG. 8E).

[0183] At the same time, the content of total γδT cells in the peripheral blood of mice was also detected on Day 7, Day 14, and Day 19 after CAR-γδT cell transfusion. The results of flow cytometry detection showed that only the FLT3-IL2-CAR-γδT group could detect the presence of γδT cells in the peripheral blood 19 days after CAR-γδT cell transfusion (FIG. 8F), which indicated that the co-expression of IL-2 cytokine could prolong the survival of FLT3-IL2-CAR-γδT cells in mice and maintain their expansion ability.

[0184] The above results showed that FLT-IL2-CAR-γδT cells can significantly inhibit the growth of tumors in mice, prolong the survival of mice, and have good safety.

[0185] Example 6: Establishment and preliminary analysis of gene expression profile data

[0186] To study the reasons for the differences in anti-tumor activity of the three CAR-γδT cells, RNA-seq was performed on FLT3-CAR-γδT, FLT3-IL2-CAR-γδT, and FLT3-IL7-CAR-γδT cells, and a simple comparison of their gene expression was made. The specific method is as follows:

[0187] First, FLT3-CAR-γδT, FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT cells were prepared. The CAR-γδT cells were repeatedly stimulated with OCI-AML3 cells, and tumor cells were added every 24 hours. The effector-to-target ratio was set to 5:1, and the stimulation number was set to 4. After repeated tumor antigen stimulation, CAR + cells were sorted by flow cytometry, and then were sent for RNA-seq.

[0188] The sequencing results showed that FLT3-IL2-CAR-γδT cells had the most different genes compared with FLT3-CAR-γδT (Figure 9A). KEGG enrichment analysis results showed that the up-regulated genes in FLT3-IL2-CAR-γδT cells were significantly concentrated in the cell cycle pathway (Figure 9B) compared with FLT3-CAR-γδT and FLT3-IL7-CAR-γδT cells, such as PCNA, MCM5, MCM6, MCM7 and other genes promoting cell proliferation (Figure 9C), which suggested that FLT3-IL2-CAR-γδT cells still had good proliferation ability after being subjected to multiple tumor antigen stimulation, which was consistent with the in vivo expansion results of CAR-γδT cells.

[0189] In summary, co-expression of IL-2 increased the expression of proliferation-related genes in FLT3-IL2-CAR-γδT cells, which might be one of the reasons why FLT3-IL2-CAR-γδT cells had more significant anti-tumor activity.

[0190] Discussion

[0191] Various CAR-T products have been successful in the treatment of B lymphoid leukemia and lymphoma. However, unlike B lymphoid leukemia, CAR-T therapy still faces major challenges in the treatment of AML patients, such as immunosuppressive tumor microenvironment, antigen escape and CAR-T quality problems, which leads to the slow development of CAR-T therapy in the treatment of AML.

[0192] In recent years, CAR-γδT cells have attracted much attention as one of the potential alternatives to CAR-T cells. Unlike traditional CAR-T cells, CAR-γδT cells can not only recognize tumor cells through CAR, but also recognize tumor cells through intrinsic receptors and activate rich tumor killing pathways; activated γδT cells can also secrete various pro-inflammatory factors and chemokines to create an inflammatory environment. These advantages of CAR-γδT cells can effectively reduce tumor immune escape and improve the tumor microenvironment. More importantly, γδT cells are MHC-unrestricted and have a low risk of GvHD, which greatly improves the safety and applicability of CAR-γδT cells compared to CAR-T cells, and reduces the quality problems of the product.

[0193] Studies have shown that FLT3 is a specific antigen of AML, which is highly expressed on most AML tumor cells, but is lowly expressed or not expressed in normal tissues. Based on this, the inventors of the present application selected FLT3 as a target to construct CAR-γδT cells and carried out preclinical efficacy studies on AML.

[0194] In the present application, FLT3-CAR-γδT, FLT3-IL2-CAR-γδT and FLT3-IL7-CAR-γδT cells were successfully constructed, and the stability of CAR expression was verified. Currently, FDA-approved FLT3 inhibitors only have therapeutic effects on patients with FLT3 mutations, and patients are prone to relapse after treatment, which is far from meeting the clinical needs. The FLT3-targeted CAR-γδT cells constructed by the inventors of the present application have high killing efficiency on FLT3 mutant and wild-type AML tumor cell lines, accompanied by high levels of Granzyme B and IFN-γ release. This suggests that the FLT3-targeted CAR-γδT cells of the present application have a more extensive application prospect than FLT3 inhibitors. Importantly, the inventors found in the killing experiment of primary AML cells that FLT3-IL2-CAR-γδT cells not only showed superior killing effect on primary AML cells with high expression of FLT3 antigen, but also exhibited certain cytotoxicity on primary AML cells with low expression of FLT3 antigen.

[0195] In the tumor antigen repeated stimulation experiment, the inventors found that FLT3-IL2-CAR-γδT cells had unexpectedly excellent effect on killing persistence. FLT3-IL2-CAR-γδT cells can maintain a high cell viability during the process of tumor antigen repeated stimulation, and can also stably and continuously release high levels of functional cytokines, which enhances the killing persistence. Studies have shown that stem cell-like memory T cells can self-renew, have strong proliferation ability, and can timely supplement memory T cell subpopulation and effector T cell subpopulation. After experiencing multiple tumor antigen stimulations, the proportion of stem cell-like memory T cell subpopulation in the rest of the groups decreased, while the proportion of stem cell-like memory T cell subpopulation in FLT3-IL2-CAR-γδT cells remained relatively stable, which may be one of the reasons for maintaining the killing persistence of FLT3-IL2-CAR-γδT cells.

[0196] In mouse experiments, the inventors found that FLT3-IL2-CAR-γδT cells can significantly inhibit the growth of tumor cells in AML tumor-bearing mice in vivo, and significantly prolong the survival of mice, and the presence of γδT cells can still be detected in peripheral blood 19 days after the reinfusion of CAR-γδT cells. The preliminary RNA-seq analysis results also suggest that co-expression of IL-2 up-regulates the genes related to cell cycle in FLT3-IL2-CAR-γδT cells, which is consistent with the in vivo results.

[0197] In summary, the inventors successfully constructed FLT3-targeting CAR-γδT cells with different structures, and screened out CAR-γδT cells with persistent anti-tumor effect (FLT3-IL2-CAR-γδT cells carrying IL-2) through a series of in vitro and in vivo experiments, which provides a new strategy and method for the clinical treatment of AML patients.

[0198] Sequence

[0199] FLT3-CAR amino acid sequence (SEQ ID NO: 1):

[0200] FLT3-IL2-CAR amino acid sequence (SEQ ID NO: 2):

[0201] FLT3-IL7-CAR amino acid sequence (SEQ ID NO: 3):

[0202] Signal peptide amino acid sequence (SEQ ID NO: 4):

[0203] FLT3 Nanobody sequence (SEQ ID NO: 5):

[0204] Fc amino acid sequence (SEQ ID NO: 6):

[0205] CD28 transmembrane + intracellular region (SEQ ID NO: 7):

[0206] CD3 zeta sequence (SEQ ID NO: 8):

[0207] T2A sequence (SEQ ID NO: 9):

[0208] IL-2 amino acid sequence (SEQ ID NO: 10):

[0209] IL-7 amino acid sequence (SEQ ID NO: 11):

[0210] All documents referred to in this disclosure are incorporated herein by reference as if each were individually incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that these equivalents ypes do not depart from the scope of the application. Accordingly, the application is not to be restricted except in the spirit of the claims that follow.

Claims

A chimeric antigen receptor, CAR, characterized in that, The CAR comprises: an antigen binding domain, a transmembrane domain, and an intracellular domain, wherein the antigen binding domain specifically binds to FLT3; and the CAR further comprises a cytokine element linked to and co-expressed with the intracellular domain; the cytokine is selected from the group consisting of IL-2, IL-7, or a combination thereof. The chimeric antigen receptor CAR as claimed in claim 1, characterized in that, The structure of the CAR is shown in Formula I: L-EB-H-TM-C-CD3ζ-(A-P)n (I) In the formula, each “-” is independently a linking peptide or a peptide bond; L is nothing or a signal peptide sequence; EB is a binding domain that specifically binds to FLT3; H is nothing or a hinge region; TM is a transmembrane domain; C is a costimulatory signaling molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ; A is a self-cleaving protein; P is a cytokine; n is 0, 1, or 2. The chimeric antigen receptor CAR as claimed in claim 2, characterized in that, The EB is a single-domain antibody (VHH) that specifically targets FLT3, a single-chain antibody (scFv), or a combination thereof. The chimeric antigen receptor CAR as claimed in claim 2, characterized in that, The EB is a single-domain antibody (VHH) that specifically targets FLT3, and the amino acid sequence thereof is shown in SEQ ID NO:

5. The chimeric antigen receptor CAR as claimed in claim 1, characterized in that, The amino acid sequence of the CAR is shown in SEQ ID NO: 2 or SEQ ID NO:

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric antigen receptor CAR of claim 1. A vector, characterized in that, The vector contains the nucleic acid molecule of claim 6. An engineered immune cell, characterized in that, The engineered immune cell expresses the chimeric antigen receptor CAR of claim 1. The engineered immune cell of claim 8, wherein The engineered immune cell is a CAR-γδT cell. A method of making an engineered immune cell as claimed in claim 8, characterized in that, The method comprises the step of: transducing the nucleic acid molecule of claim 6 or the vector of claim 7 into an immune cell, thereby obtaining the engineered immune cell. The method of claim 10, wherein The immune cell is a γδT cell. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the CAR of claim 1, the nucleic acid molecule of claim 6, the vector of claim 7, or the engineered immune cell of claim 8, and a pharmaceutically acceptable carrier, diluent, or excipient. Use of a CAR as claimed in claim 1, a nucleic acid molecule as claimed in claim 6, a vector as claimed in claim 7, an engineered immune cell as claimed in claim 8, or a pharmaceutical composition as claimed in claim 12, characterized in that, The pharmaceutical composition is used for preparing a medicament or a preparation for treating a tumor. Use according to claim 13, characterized in that The tumor is acute myeloid leukemia. A method of treating cancer or a tumor, characterized by, The method comprises: administering a safe and effective amount of the CAR of claim 1, the nucleic acid molecule of claim 6, the vector of claim 7, the engineered immune cell of claim 8, or the pharmaceutical composition of claim 12 to a subject in need thereof.

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