Chimeric antigen receptor targeting FLT3

Abstract

The invention relates to a chimeric antigen receptor targeting FLT3. Provided herein are antibodies that specifically bind to Fms-like tyrosine kinase 3 (FLT3); a chimeric antigen receptor (CAR) specifically binding to FLT3; and engineered immune cells (e.g., FLT3 specific CAR-T cells) that express such CARs. The invention also provides the manufacture of the antibodies, CARs and engineered immune cells. The invention also provides the use of the antibodies, CARs and engineered immune cells, for example, for treating conditions associated with malignant cells expressing FLT3 (e.g., cancer).

Description

[0001] This application is a divisional application of the invention patent with application date of May 31, 2018, application number 201880036069.6, and invention name “Chimeric Antigen Receptor Targeting FLT3”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 514,634, filed on June 2, 2017, the contents of which are incorporated herein by reference in their entirety.

[0004] U.S. Provisional Application No. 62 / 514,574, filed on June 2, 2017; and U.S. Provisional Application No. 62 / 660,908, filed on April 20, 2018, both disclosing FLT3-specific antibodies; and PCT application entitled “Antibodies Specific for FLT3 and Uses Thereof” filed on May 31, 2018 are incorporated herein by reference in their entirety. Technical Field

[0005] Provided herein are Fms-like tyrosine kinase 3 (FLT3) specific antibodies; chimeric antigen receptors (CARs) (FLT3 CARs) that specifically bind to FLT3; polynucleotides encoding FLT3 CARs; and engineered immune cells (e.g., FLT3-specific CAR-T cells) comprising FLT3-specific CARs. The present invention further relates to methods for engineering immune cells to express FLT3-specific CARs; and methods for treating conditions associated with FLT3 (e.g., malignant cells expressing FLT3) using FLT3-specific antibodies and FLT3-specific CAR-T cells. Background Art

[0006] Adoptive transfer of immune cells genetically modified to recognize antigens associated with malignant diseases has been shown to hold promise as a novel approach to treating cancer (see, e.g., Brenner et al., Current Opinion in Immunology, 22(2):251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4):299-308 (2008)). T cells can be genetically modified to express a chimeric antigen receptor (CAR), which is a fusion protein consisting of an antigen recognition portion and a T cell activation domain (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993); and Sadelain et al., Curr. Opin. Immunol, 21(2):215-223 (2009)).

[0007] FLT3 is an acute myeloid leukemia (AML) target antigen that is overexpressed on AML patient embryonic cells when compared to healthy cells, and is expressed on most patient cells (Carow et al., February 2, 1996, Blood: 87(3); Birg et al., November 1992, Blood: 80(10)). FLT3 is a gene that is frequently mutated in AML patients, and mutations are associated with poor prognosis (Abu-Duhier et al., Br J Haematol., October 2000; 111(1): 190-5; Yamamoto et al., April 15, 2001; Blood: 97(8)). Small molecule FLT3 inhibitors have shown activity in clinical trials; however, responses are generally short-lived due to acquired resistance. Other kinase inhibitors only treat a certain percentage of patients expressing mutant forms of FLT3, highlighting the urgent need for improved therapies.

[0008] Therefore, there is a need for alternative treatments for cancer, and specifically, malignant diseases involving aberrant expression of FLT3. Provided herein are methods and compositions that address this need. Summary of the invention

[0009] Provided herein is an antibody or an antigen binding fragment thereof that specifically binds to Fms-like tyrosine kinase 3 (FLT3); a chimeric antigen receptor (CAR) that specifically binds to FLT3; and an engineered immune cell (e.g., FLT3-specific CAR-T cell) expressing the CAR. Also provided is the manufacture of the antibody, CAR, and immune cell. Further provided are uses of the antibody, CAR, and engineered immune cell, such as for treating a condition (e.g., cancer) associated with malignant cells expressing FLT3. In one aspect, provided herein is a separation antibody that specifically binds to FLT3, wherein the antibody comprises a heavy chain variable (VH) region having any one of the partial VH sequences listed in Table 2; and / or a light chain variable (VL) region having any one of the partial VL sequences listed in Table 2.

[0010] In another embodiment, provided herein is an isolated antibody that specifically binds to FLT3, wherein the antibody comprises (a) a heavy chain variable (VH) region comprising the complementarity determining region 1 (CDR1), CDR2, and CDR3 sequences listed in Table 3; and / or (b) a light chain variable (VL) region comprising the CDR1, CDR2, and CDR3 sequences listed in Table 3.

[0011] Also provided herein is a chimeric antigen receptor (CAR) that is bound to FLT3. It has been shown that the expression of FLT3-specific CAR in T cells effectively activates T cells after contact with FLT3. The FLT3-specific CAR provided herein binds to human FLT3 and exhibits cytotoxic activity after contact with cells expressing FLT3.

[0012] Therefore, on the other hand, a FLT3-specific chimeric antigen receptor (CAR) is provided herein, comprising an extracellular ligand binding domain, a first transmembrane domain and an intracellular signaling domain, wherein the extracellular domain comprises a single-chain variable fragment (ScFv) bound to the extracellular domain of FLT3. In some embodiments, a FLT3 CAR is provided herein, comprising an extracellular ligand binding domain, a first transmembrane domain and an intracellular signaling domain, wherein the extracellular domain comprises an scFv bound to a specific domain of a FLT3 target, for example, bound to SEQ ID NO: 199 (domain 1), SEQ ID NO: 200 (domain 2), SEQ ID NO: 201 (domain 3), SEQ ID NO: 254 (domain 2-3), SEQ ID NO: 202 (domain 4) or SEQ ID NO: 203 (domain 5), SEQ ID NO: 254 (domain 2-3) or SEQ ID NO: 202 (domain 4).

[0013] In some embodiments, the FLT3-specific CAR comprises an extracellular domain comprising a ScFv, wherein the ScFv binds to domain 4 of the FLT3 target (SEQ ID NO: 202).

[0014] In some embodiments, the FLT3-specific CAR comprises an extracellular domain comprising a ScFv, wherein the ScFv binds to domain 2 of the FLT3 target (SEQ ID NO: 200).

[0015] The extracellular domain of the FLT3-specific CAR comprises an scFv, wherein the scFv comprises a heavy chain variable (VH) region and a light chain variable (VL) region, each of the VH and VL regions comprising three complementarity determining regions (CDRs) specific for FLT3.

[0016] In some embodiments, the VH region comprises (i) a VH complementarity determining region 1 (CDR1) having SEQ ID NO: 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56, 60, 61, 62, 66, 67, 68, 72, 73, 74, 78, 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108, 109, 110, 114, 115, 116 , 120, 121, 122, 126, 127, 128, 132, 133, 134, 138, 139, 140, 294, 295, 296, 300, 301, 305, 306, 307, 311, 312, 316, 317 or 318; (ii) a VH complementarity determining region 2 (CDR2) having SEQ ID NO:40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 297, 298, 302, 303, 308, 309, 313, 314, 319, 320 or 322; and (iii) a VH complementarity determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO:40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, NO:42, 48, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 299, 304, 310, 315 or 321; and / or the VL region comprises (i) a VL complementarity determining region 1 (CDR1) having SEQ ID 172, 175, 178, 181, 184, 187, 190, 193, 196, 324, 329, 332, 334, 341 or 346;and (iii) a VL complementarity determining region 3 (CDR3) having the amino acid sequence set forth in SEQ ID NO: 146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 325, 327, 330, 333, 335, 337, 339, 342, 344, 347 or 349;

[0017] In some embodiments, a FLT3-specific CAR comprising an ScFv is provided, wherein the ScFv comprises a VH region having a sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291 or 293.

[0018] In some embodiments, a FLT3-specific CAR comprising an ScFV is provided, wherein the scFv comprises a VL region having a sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290 or 292.

[0019] In some embodiments, a FLT3-specific CAR comprising an ScFV is provided, wherein the scFv comprises a VH region and a VL region, wherein the VH region has a sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291 or 293; and the VL region has a sequence shown in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, the sequence shown in 290 or 292.

[0020] In some embodiments, the VH region comprises the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291 or 293, or a variant thereof having one or more conservative amino acid substitutions in residues not in a CDR; and / or the VL region comprises SEQ ID NO: The amino acid sequence shown in NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290 or 292, or a variant thereof having one or several amino acid substitutions in amino acids not in a CDR.

[0021] In some embodiments, the VH region comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 90, 91 or 92; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 93 or 94; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 95; and the light chain variable (VL) region comprises the following CDRs: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 171; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 172; and a VLCDR3 comprising the amino acid sequence shown in SEQ ID NO: 173.

[0022] In some embodiments, the VH region comprises the amino acid sequence shown in SEQ ID NO:20, and the VL region comprises the amino acid sequence shown in SEQ ID NO:19.

[0023] In some embodiments, the VH region comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO:43, 44 or 45; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO:46 or 47; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO:48; and the light chain variable (VL) region comprises the following CDRs: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:147; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:148; and a VLCDR3 comprising the amino acid sequence shown in SEQ ID NO:149.

[0024] In some embodiments, the VH region comprises the amino acid sequence shown in SEQ ID NO:4, and the VL region comprises the amino acid sequence shown in SEQ ID NO:3.

[0025] In some embodiments, the VH region comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 49, 44 or 50; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 51 or 52; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 53; and the light chain variable (VL) region comprises the following CDRs: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 150; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 151; and a VLCDR3 comprising the amino acid sequence shown in SEQ ID NO: 152.

[0026] In some embodiments, the VH region comprises the amino acid sequence shown in SEQ ID NO:6, and the VL region comprises the amino acid sequence shown in SEQ ID NO:5.

[0027] In some embodiments, the VH region comprises a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 60, 61 or 62; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 63 or 64; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 65; and the light chain variable (VL) region comprises the following CDRs: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 156; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 157; and a VLCDR3 comprising the amino acid sequence shown in SEQ ID NO: 158.

[0028] In some embodiments, the VH region comprises the amino acid sequence set forth in SEQ ID NO:10, and the VL region comprises the amino acid sequence set forth in SEQ ID NO:9.

[0029] In some embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the AbM definition, or the contact definition of a CDR.

[0030] In some embodiments, the intracellular signaling domain comprises a CD3 zeta signaling domain. In some embodiments, the intracellular signaling domain comprises a 4-1BB domain. In some embodiments, the FLT3-specific CAR comprises a second intracellular signaling domain. In some embodiments, the second intracellular signaling domain comprises a 4-1BB domain.

[0031] In some embodiments, the FLT3-specific CAR disclosed herein may include a stalk domain between the extracellular ligand binding domain and the first transmembrane domain. In some embodiments, the stalk domain is selected from the group consisting of: a CD8α hinge, an IgG1 hinge, and an FcγRIIIα hinge. In some embodiments, the stalk domain is a human CD8α hinge, a human IgG1 hinge, or a human FcγRIIIα hinge.

[0032] In some embodiments, the FLT3-specific CAR disclosed herein may comprise one or more epitopes specific to one or more monoclonal antibodies. In some embodiments, the epitope specific to the monoclonal antibody is a CD20 epitope. In some embodiments, the CD20 epitope comprises the amino acid sequence shown in SEQ ID NO: 229 or SEQ ID NO: 230.

[0033] In some embodiments, the FLT3-specific CAR comprises the amino acid sequence shown in SEQ ID NO: 235, 236, 237 or 242. In some embodiments, the FLT3-specific CAR comprises the amino acid sequence shown in SEQ ID NO: 235. In some embodiments, the FLT3-specific CAR comprises the amino acid sequence shown in SEQ ID NO: 236.

[0034] In some embodiments, the first transmembrane domain comprises a CD8 alpha chain transmembrane domain.

[0035] In some embodiments, the FLT3-specific CAR may comprise another extracellular ligand binding domain that is not specific for FLT3.

[0036] In some embodiments, the one or more extracellular ligand binding domains, the first transmembrane domain, and the one or more intracellular signaling domains are on a single polypeptide.

[0037] In some embodiments, CAR may include a second transmembrane domain, wherein the first transmembrane domain and one or more extracellular ligand binding domains are on a first polypeptide, and wherein the second transmembrane domain and one or more intracellular signaling domains are on a second polypeptide. In an exemplary embodiment, the first transmembrane domain includes a transmembrane domain from an α chain of a high-affinity IgE receptor (FcεRI), and the second transmembrane domain includes a transmembrane domain from a γ or β chain of FcεRI.

[0038] In some embodiments, the CAR may comprise a third polypeptide comprising a third transmembrane domain fused to an intracellular signaling domain from a co-stimulatory molecule, wherein the third transmembrane domain comprises a transmembrane domain from the γ or β chain of FcεRI.

[0039] In another aspect, provided herein is an isolated polynucleotide comprising a nucleic acid sequence encoding a FLT3-specific CAR described herein.

[0040] In another aspect, provided herein is an expression vector comprising a polynucleotide encoding a FLT3-specific CAR described herein.

[0041] On the other hand, an engineered immune cell is provided herein that expresses a FLT3-specific CAR described herein at its cell surface membrane. In some embodiments, the engineered immune cell may include another CAR that is not specific to FLT3. In some embodiments, the engineered immune cell may include a polynucleotide encoding a suicide polypeptide. In some embodiments, the suicide polypeptide is RQR8.

[0042] In some embodiments, the engineered immune cell is derived from an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte.

[0043] In some embodiments, the engineered immune cells may comprise disruption of one or more endogenous genes, wherein the endogenous genes encode TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK), or immune checkpoint proteins such as programmed death-1 (PD-1).

[0044] In some embodiments, the engineered immune cells are obtained from a healthy donor. In some embodiments, the engineered immune cells are obtained from an individual suffering from a disease or disorder.

[0045] On the other hand, an engineered immune cell for use as a medicament is provided herein, which expresses the FLT3 specific CAR described herein at its cell surface membrane. On the other hand, a FLT3 specific antibody for use as a medicament is provided herein. In some embodiments, an immunocyte or FLT3 specific antibody comprising an expression FLT3 specific CAR is provided for use in the treatment of FLT3 related diseases or conditions. In one embodiment, FLT3 related diseases or conditions are hematopoietic cancers, such as lymphomas or leukemias. In some embodiments, the cancer is selected from the group consisting of: multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, macroglobulinemia), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid Granulomatous disease, T cell / histiocyte-rich large B cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B cell lymphoma (leg type), EBV-positive diffuse large B cell lymphoma of the elderly, diffuse large B cell lymphoma associated with inflammation, intravascular large B cell lymphoma, ALK-positive large B cell lymphoma, plasmablastic lymphoma, large B cell lymphoma arising in HHV8-related multicentric Castleman disease, unclassified B cell lymphoma with features between diffuse large B cell lymphoma and Burkitt's lymphoma, unclassified B cell lymphoma with features between diffuse large B cell lymphoma and classical Hodgkin's lymphoma, and other hematopoietic cell-related cancers, for example, ALL or AML.

[0046] In one aspect, provided herein is a cell population comprising an engineered immune cell expressing a FLT-specific CAR described herein, wherein (ii) the cell population comprises a percentage greater than 20%, 30% or 40% of stem cell memory and central memory cells, and / or (iii) the cell population achieves a percentage greater than 10%, 20%, 30% or 40% of lysis of cells expressing FLT3.

[0047] On the other hand, the present invention provides a method for engineering an immune cell that expresses any one of the FLT3-specific CARs described herein, the method comprising: providing an immune cell; and introducing at least one polynucleotide encoding the FLT3-specific CAR into the cell; thereby causing the immune cell to express the FLT3-specific CAR.

[0048] In some embodiments, the method comprises providing an immune cell; introducing into the cell at least one polynucleotide encoding the FLT3-specific CAR; and introducing at least one polynucleotide encoding a CAR that is not specific for FLT3.

[0049] On the other hand, a method for treating a subject with a FLT3-related disease or condition is provided herein, the method comprising: providing an immune cell expressing a FLT3-specific CAR as described herein on the surface; and administering the immune cell to the subject. The present invention also provides a method for treating a subject with a FLT3-related disease or condition, the method comprising providing a FLT3-specific antibody as described herein, and administering the antibody to the subject.

[0050] In some embodiments, a pharmaceutical composition is provided herein, comprising an engineered immune cell expressing a FLT3-specific CAR as described herein. In other embodiments, the present invention provides the following pharmaceutical composition, comprising any one of the FLT3-specific antibodies described herein.

[0051] On the other hand, a method for treating a condition associated with a malignant cell expressing FLT3 in a subject is provided herein, comprising administering an effective amount of a pharmaceutical composition to a subject in need thereof, the pharmaceutical composition comprising an engineered immune cell as described herein or a FLT3-specific antibody as described herein. In some embodiments, the condition is cancer. In some embodiments, cancer is a hematopoietic cancer, such as a lymphoma or leukemia. In some embodiments, cancer is selected from the group consisting of: multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte-based Hodgkin's lymphoma, Kahler's disease and myeloma, plasma cell leukemia, plasma cell tumor, B cell prolymphocytic leukemia, hairy cell leukemia, B cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, primary mediastinal (thymic) large B-cell lymphoma tumor, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, diffuse large B-cell lymphoma associated with inflammation, vascular Large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman's disease, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt's lymphoma, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma, and other hematopoietic cell-related cancers, e.g., ALL or AML.

[0052] In another aspect, provided herein is a method for inhibiting tumor growth or progression in a subject having malignant cells expressing FLT3, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein or a FLT3-specific antibody as described herein to the subject.

[0053] In another aspect, provided herein is a method for inhibiting metastasis of malignant cells expressing FLT3 in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein or a FLT3-specific antibody as described herein to the subject.

[0054] In another aspect, provided herein is a method of inducing tumor regression in a subject having malignant cells expressing FLT3, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an engineered immune cell as described herein or a FLT3-specific antibody as described herein to the subject.

[0055] In some embodiments, any of the above methods further comprises administering one or more other therapeutic agents, such as monoclonal antibodies and / or chemotherapeutic agents. In some embodiments, the monoclonal antibody may be, for example, an antibody that binds to a checkpoint inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, any of the above methods further comprises administering to the subject a receptor tyrosine kinase inhibitor, an mTOR inhibitor, an epigenetic regulator, a proteasome inhibitor, an immunomodulator (such as lenalidomide), a hedgehog inhibitor, or an isocitrate dehydrogenase (IDH) inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Shown are representative FACS plots demonstrating efficient transduction of activated T cells with P3A1 CAR constructs relative to untransduced controls; as well as transduction efficiency of all constructs presented as percentage of T cells expressing CAR and mean fluorescence intensity (MFI).

[0057] Figure 2 Shown is the expression of activation markers CD25 and 4-1BB in T cells transduced with various CAR constructs assessed by flow cytometry.

[0058] Figure 3 The phenotype of CAR-T cells at the end of culture as determined by flow cytometric analysis of the expression of CD62L and / or CD45RO markers is shown.

[0059] Figure 4 The overall experimental design of the long-term killing assay is shown, as well as the response of different CAR-T cells to repeated exposure to target cells measured at day 6 as fold expansion and cytotoxic activity (percent lysis).

[0060] Figure 5Antitumor activity of CAR-T cells expressing P3A1 or P3E10 constructs at two doses using an orthotopic mouse model of human AML is shown. Bioluminescent signals quantified as total flux (photons / second) at different time points are shown.

[0061] Figure 6 Shown is the sensitivity of T cells expressing a FLT3-specific CAR comprising the CD20 epitope at the cell surface to complement-dependent cytotoxicity (CDC) induced by the anti-CD20 antibody rituximab.

[0062] Figure 7 shows the restoration of endogenous myeloid progenitor cells in mice treated with anti-mouse FLT3 CAR T cells expressing the CD20 epitope and subsequently given rituximab. DETAILED DESCRIPTION

[0063] In one aspect, the present invention disclosed herein provides a chimeric antigen receptor (CAR) and an immune cell (e.g., CAR-T cell) comprising a CAR specifically bound to FLT3 (e.g., human FLT3). The present invention also provides polynucleotides encoding these CARs; compositions comprising immune cells expressing these CARs; and methods for manufacturing and using these CARs and immune cells expressing CARs. The present invention also provides methods for treating a subject's pathology-related condition mediated by FLT3 using FLT3-specific CARs as described herein and immune cells expressing these CARs, such as cancer mediated by malignant cells expressing FLT3.

[0064] In one aspect, the invention disclosed herein provides antibodies that specifically bind to FLT3 (e.g., human FLT3). The invention also provides compositions comprising these antibodies and methods of using these antibodies. For example, methods of using these antibodies to treat conditions associated with pathologies mediated by FLT3, such as cancer, in subjects are provided herein.

[0065] General techniques

[0066] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the following literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology (Humana Press); Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JPather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (JP Cellis, ed., 1998) Academic Press; Procedures (Edited by A. Doyle, JB Griffiths and DG Newell, 1993-1998) John Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (Edited by DM Weir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (Edited by JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (Edited by FM Ehrlich, 1996);Ausubel et al., 1987; PCR: The Polymerase Chain Reaction (Mullis et al., 1994); Current Protocols in Immunology (J.E. Coligan et al., 1991); Short Protocols in Molecular Biology (John Wiley & Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 1994); Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

[0067] definition

[0068] As used herein, the term "extracellular ligand binding domain" refers to a polypeptide capable of binding a ligand or capable of interacting with a cell surface molecule. For example, an extracellular ligand binding domain can be selected to recognize a ligand that serves as a cell surface marker on a target cell associated with a particular disease state.

[0069] The term "stem domain" or "hinge domain" is used interchangeably herein and refers to any polypeptide used to connect the transmembrane domain to the extracellular ligand binding domain. Specifically, the stem domain is used to provide more flexibility and accessibility to the extracellular ligand binding domain.

[0070] The term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and directs the cell to perform a specific function.

[0071] As used herein, "costimulatory molecules" refer to cognate binding partners on proliferating immune cells (e.g., T cells) that specifically bind to costimulatory ligands, thereby mediating a costimulatory response by cells, such as, but not limited to, proliferating immune cells (e.g., T cells). Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc.

[0072] "Co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate co-stimulatory signal molecule on a T cell, thereby providing a signal that mediates T cell responses (including, but not limited to, proliferation activation, differentiation, etc.) in addition to the primary signal provided by, for example, binding to the TCR / CD3 complex and the MHC molecule loaded with a peptide. Co-stimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to a toll ligand receptor, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also particularly encompass antibodies that specifically bind to co-stimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0073] "Antibodies" are immunoglobulin molecules that can specifically bind to targets, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only complete polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chains (scFv) and domain antibodies (including, for example, shark and camel antibodies), and fusion proteins comprising antibodies, as well as any other modified configurations of immunoglobulin molecules comprising antigen recognition sites. Antibodies include antibodies of any class, such as IgG, IgA, IgE, IgD or IgM (or their subclasses), and antibodies do not necessarily belong to any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0074] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of a complete antibody that retain the ability to specifically bind to a given antigen (e.g., FLT3). The antigen-binding function of an antibody can be performed by a fragment of a complete antibody. Examples of binding fragments encompassed in the term "antigen-binding fragment" of an antibody include Fab; Fab'; F(ab')2; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; single-domain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1989) and isolated complementary determining regions (CDRs).

[0075] "Specific binding" to an antibody, antigen binding fragment, antibody conjugate or polypeptide of a target (e.g., FLT3 protein) is a well-understood term in the art, and the method for determining the specific binding is also well known in the art. If the reaction or association of a molecule with a specific cell or substance is more frequent, faster, longer lasting and / or with greater affinity than it is with an alternative cell or substance, it is said to exhibit "specific binding". If the binding of an antibody to a target is greater, easier and / or lasts longer than its binding affinity, avidity with other substances, then the antibody "specifically binds" to the target. For example, an antibody that "specifically binds" to a FLT3 epitope is an antibody that has a greater, easier and / or longer lasting binding affinity, avidity with this FLT3 epitope than it has with other FLT3 epitopes or non-FLT3 epitopes. It should also be understood by reading this definition that, for example, an antibody (or part or epitope) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means specific binding.

[0076] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held tightly together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antibody's antigen binding site. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, CDRs may refer to CDRs defined by either approach or by a combination of both approaches.

[0077] The "CDR" of a variable domain is the amino acid residues in the variable region identified according to Kabat, Chothia, a combination of Kabat and Chothia, AbM, the definition of contacts and / or the definition of configuration, or any method of CDR determination known in the art. Antibody CDRs can be identified as hypervariable regions, which were originally defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th edition, National Institutes of Health Public Health Service, Washington, DC. The location of CDRs can also be identified as structural loop structures, which were originally described by Coscia et al. See, for example, Chothia et al., Nature 342:877-883, 1989. Other methods of CDR identification include the "AbM definition", which is a compromise between Kabat and Chothia and uses Oxford Molecular's AbM antibody modeling software (now ) derived; or based on the "contact definition" of CDRs observed for antigen contacts, which is described in MacCallum et al., Journal of Molecular Biology, 262: 732-745, 1996. In another method referred to herein as the "configurational definition" of CDRs, the positions of CDRs can be identified as residues that provide enthalpic contributions to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283: 1156-1166, 2008. In addition, other CDR boundary definitions may not follow one of the above methods exactly, but will still overlap with at least a portion of the Kabat CDRs, but they may be shortened or elongated based on predictions or experimental findings that a particular residue or residue group or even the entire CDR does not significantly affect antigen binding. As used herein, CDRs may refer to CDRs defined by any method known in the art, including a combination of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment containing more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extension, AbM, contact and / or conformational definitions.

[0078] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody group, i.e., the antibodies constituting the group are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. In addition, relative to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. The modifier "monoclonal" represents the characteristics of an antibody such as obtained from a substantially homogeneous antibody group, and is not considered to require the production of antibodies by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by the fusion tumor method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by a recombinant DNA method as described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from a phage library produced using, for example, McCafferty et al., 1990, Nature 348:552-554.

[0079] As used herein, "humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. In one aspect, a humanized antibody is a human immunoglobulin (acceptor antibody) in which residues from the complementary determining regions (CDRs) of the acceptor are replaced with residues from the CDRs of a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) having the desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, a humanized antibody may include residues that are not found in the acceptor antibody or the imported CDR or framework sequences, but are included to further improve and optimize antibody performance. In general, humanized antibodies will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins and all or substantially all of the FR regions are FR regions of human immunoglobulin consensus sequences. Humanized antibodies will also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically at least a portion of a human immunoglobulin. Antibodies with modified Fc regions as described in WO 99 / 58572 are preferred. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that vary relative to the original antibody, also referred to as "derived from" one or more CDRs from one or more CDRs of the original antibody.

[0080] As used herein, "human antibody" means an antibody whose amino acid sequence corresponds to the amino acid sequence of an antibody produced by a human being, and / or an antibody that has been manufactured using any of the techniques for manufacturing human antibodies known to those skilled in the art or disclosed herein. This definition of human antibodies includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a murine light chain and a human heavy chain polypeptide. The antibody can be produced using various techniques known in the art. In one embodiment, human antibodies are selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al., Nature Biotechnology, 14: 309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95: 6157-6162, 1998; Hoogenboom and Winter, Journal of Molecular Biology, 227: 381, 1991; Marks et al., Journal of Molecular Biology, 222: 581, 1991). Human antibodies can also be made by immunization of animals in which human immunoglobulin loci have been transgenically introduced to replace endogenous loci, such as mice in which endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016. Alternatively, human antibodies can be made by immortalizing human B lymphocytes that produce antibodies against a target antigen (the B lymphocytes can be recovered from an individual or from a single cell clone of a cDNA or can be immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1): 86-95, 1991; and U.S. Pat. No. 5,750,373.

[0081] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0082] A "monovalent antibody" contains one antigen binding site per molecule (eg, IgG or Fab). In some cases, a monovalent antibody may have more than one antigen binding site, but the binding sites are from different antigens.

[0083] A "bivalent antibody" contains two antigen binding sites per molecule (e.g., IgG). In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific.

[0084] The antibodies of the present invention can be produced using techniques well known in the art, such as recombinant technology, phage display technology, synthetic technology, or a combination thereof or other techniques readily known in the art (see, e.g., Jayasena, SD, Clin. Chem., 45: 1628-50, 1999 and Fellouse, FA et al., J. Molecular Biology, 373(4): 924-40, 2007).

[0085] As is known in the art, "polynucleotide" or "nucleic acid" as used interchangeably herein refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. The polynucleotide may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. If present, modifications to the nucleotide structure may be imparted before or after chain assembly. The nucleotide sequence may be interspersed with non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by binding to a labeling component. Other types of modifications include, for example, "caps", where one or more naturally occurring nucleotides are replaced by analogs; internucleotide modifications, such as modifications with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), modifications containing side-joining moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with intercalators (e.g., acridine, psoralen, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), modifications containing alkylating agents, modifications with modified bonds (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl group normally present in the sugar may be replaced, for example, with a phosphonate group, a phosphate group, protected with a standard protecting group, or activated to prepare additional linkages with additional nucleotides, or may be bound to a solid support. The 5' and 3' terminal OH may be phosphorylated or substituted with an amine or organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyl groups can also be derived into standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose), pyranose, furanose, sedoheptose, acyclic analogs and abasic nucleoside analogs (such as methyl riboside). One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("methylal"), wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), optionally containing an ether (-O-) linkage, an aryl, an alkenyl, a cycloalkyl, a cycloalkenyl or an arylaldehyde group. All linkages in a polynucleotide need not be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0086] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, alone or in combination.

[0087] As used herein, "substantially pure" refers to a material that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or even at least 99% pure (i.e., free of contaminants).

[0088] "Host cell" includes an individual cell or cell culture that can be or has been a recipient for one or more vectors for incorporation of a polynucleotide insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with one or more polynucleotides of the invention.

[0089] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the Cys226 position or from the amino acid residue at Pro230 to its carboxyl terminus. The numbering of residues in the Fc region is that of the EU index in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institutes of Health, Public Health Service, Bethesda, Maryland, 1991. The Fc region of an immunoglobulin generally comprises two constant regions, CH2 and CH3.

[0090] As used in the art, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), both of which have similar amino acid sequences, differing primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

[0091] As used herein, with respect to antibodies, the term "competition" means that a first antibody or its antigen-binding fragment (or portion) binds to an epitope in a manner sufficiently similar to the binding of a second antibody or its antigen-binding portion, such that in the presence of the second antibody, the result of the binding of the first antibody to its cognate epitope is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. An alternative scheme in which the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may (but not necessarily) be so. That is, the first antibody may inhibit the binding of the second antibody to its epitope, without the second antibody needing to inhibit the binding of the first antibody to its corresponding epitope. However, in the case where each antibody can detectably inhibit the binding of another antibody to its cognate epitope or ligand, whether or not the degree is the same, higher or less, the antibodies are said to "cross-compete" with each other for binding to one or more corresponding epitopes. Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or portion thereof), the skilled artisan will appreciate based on the teachings provided herein that such competing and / or cross-competing antibodies are contemplated and may be suitable for use in the methods disclosed herein.

[0092] As used herein, "autologous" means that the cell, cell line or cell population used to treat a patient originates from the patient.

[0093] As used herein, "allogeneic" means that the cell or population of cells used to treat a patient is not derived from the patient but rather from a donor.

[0094] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing the proliferation of neoplastic or cancerous cells (or destroying them); inhibiting neoplastic cell metastasis; reducing or reducing the size of a tumor expressing FLT3; alleviating a FLT3-related disease (e.g., cancer); reducing the symptoms caused by a FLT3-related disease (e.g., cancer); increasing the quality of life of a patient with a FLT3-related disease (e.g., cancer); reducing the dose of other medications required to treat a FLT3-related disease (e.g., cancer); delaying the progression of a FLT3-related disease (e.g., cancer); curing a FLT3-related disease (e.g., cancer); and / or prolonging the survival of a patient with a FLT3-related disease (e.g., cancer).

[0095] "Relieve" means reducing or improving one or more symptoms compared to not administering FLT3-specific CAR or FLT3-specific CAR-T cells. "Relieve" also includes shortening or reducing the duration of symptoms.

[0096] As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired results. For preventive use, beneficial or desired results include eliminating or reducing risk; reducing severity; or delaying the onset of disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes present during the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing the occurrence of one or more symptoms of a variety of FLT3-related diseases or conditions (such as multiple myeloma) or alleviating them; reducing the dose of other medications required to treat the disease; promoting the effect of another medication; and / or delaying the progression of a patient's FLT3-related disease. The effective dose can be given in one or more administrations. For the purposes of the present invention, the effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment. As understood in clinical situations, the effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result can be achieved or accomplished in combination with one or more other agents.

[0097] "Individual," "patient," or "subject" are used interchangeably herein and are mammals. Mammals include, but are not limited to, humans, monkeys, pigs, other farm animals, sport animals, pets, primates, horses, dogs, cats, rodents, including mice, rats, guinea pigs, and the like. Subjects are mammals and are used interchangeably herein. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a human or a monkey, e.g., a cynomolgus macaque.

[0098] As used herein, "vector" means a construct that is capable of delivering, and in some embodiments, expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0099] As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. An expression control sequence is operably linked to a nucleic acid sequence to be transcribed.

[0100] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any substance that, when combined with an active ingredient, allows the ingredient to remain biologically active and not react with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as oil / water emulsions), and different types of wetting agents. Exemplary diluents for aerosol or parenteral administration include phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 21st edition, Mack Publishing Co., 2005).

[0101] As used herein, the term “k on ” refers to the rate constant for the association of the antibody or ScFv of CAR with the antigen.

[0102] As used herein, the term “k off” refers to the rate constant for the dissociation of the antibody or ScFv of the CAR from the antibody / antigen complex.

[0103] As used herein, the term "K D ” refers to the equilibrium dissociation constant of an antibody-antigen interaction or a ScFv-antigen interaction.

[0104] Reference herein to "about" a certain value or parameter includes (and describes) embodiments for that value or parameter itself. For example, description referring to "about X" includes description of "X." Numerical ranges include the numbers defining the range.

[0105] It should be understood that wherever an embodiment is described herein with the language "comprising," additional similar embodiments described with respect to "consisting of" and / or "consisting essentially of are also provided.

[0106] Where aspects or embodiments of the invention are described in terms of Markush groups or other groupings of alternatives, the invention encompasses not only the entire group recited as a whole, but also each member of the group independently and all possible subgroups of the main group, as well as the main group in which one or more of the group members are absent. The invention also contemplates any group member that specifically excludes one or more of the claimed inventions.

[0107] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In the event of a conflict, the present specification, including definitions, will prevail. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" shall be understood to mean including the stated integer or group of integers but not excluding any other integer or group of integers. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0108] Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0109] FLT3 Antibody

[0110] The present invention provides antibodies that bind to FLT3 [e.g., human FLT3 (e.g., Accession No.: NP_004110 or SEQ ID NO: 198)] and are characterized by any one or more of the following characteristics: (a) treating, preventing, or alleviating one or more symptoms of a condition associated with malignant cells expressing FLT3 (e.g., cancer, such as AML) in a subject; (b) inhibiting tumor growth or progression in a subject (suffering from a malignant tumor expressing FLT3); (c) inhibiting metastasis of cancer (malignant) cells expressing FLT3 in a subject (having one or more malignant cells expressing FLT3); (d) inducing regression (e.g., long-term regression) of tumors expressing FLT3; (e) exerting cytotoxic activity of malignant cells expressing FLT3; (f) blocking the interaction of FLT3 with other factors yet to be identified; and / or (g) inducing a bystander effect that kills or inhibits the growth of nearby malignant cells that do not express FLT3.

[0111] In one aspect, an isolated antibody that specifically binds to FLT3 is provided, wherein the antibody comprises (a) a heavy chain variable (VH) region comprising (i) a VH complementarity determining region 1 (CDR1) comprising the sequence shown in 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56, 60, 61, 62, 66, 67, 68, 72, 73, 74, 78, 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108, 109, 110, 114, 115, 116, 120, 121, 122, 126, 127, 128, 132, 133, 134, 138, 139, 140, 246 or 247; (ii) a VH a CDR2 comprising the sequence shown in SEQ ID NO: 40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 248, 249, 251, 252, 253 or 255; and (iii) a VH CDR3 comprising the sequence shown in SEQ ID NO: NO:42, 48, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 245, 250 or 254; and / or a light chain variable (VL) region comprising (i) a VL CDR1 comprising the sequence shown in SEQ ID NO:144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 257, 261, 263, 265, 268, 270, 273 or 275; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: NO:145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 259, 266 or 271; and (iii) the VL CDR3 comprises the sequence shown in SEQ ID NO:146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 256, 258, 260, 262, 264, 267, 269, 272 or 274.

[0112] In another aspect, an isolated antibody that specifically binds to FLT3 is provided, wherein the antibody comprises: a VH region comprising the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291 or 293; and / or a VL region comprising the sequence shown in SEQ ID NO: NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, the sequence shown in 290 or 292.

[0113] In some embodiments, an isolated antibody that specifically binds to FLT3 is provided, wherein the antibody comprises: a VH region comprising the sequence shown in SEQ ID NO: 275, 289 or 291; and / or a VL region comprising the sequence shown in SEQ ID NO: 274, 288 or 290.

[0114] In some embodiments, an antibody having any one of the partial light chain sequences listed in Table 2 and / or any one of the partial heavy chain sequences listed in Table 2 is provided.

[0115] In some embodiments, the antibodies described herein comprise a constant region. In some embodiments, the antibodies described herein have a human IgG1, IgG2 or IgG2Δa, IgG3 or IgG4 subclass. In some embodiments, the antibodies described herein comprise a glycosylated constant region. In some embodiments, the antibodies described herein comprise a constant region with reduced binding affinity to one or more human Fcγ receptors.

[0116] The antibodies provided herein may encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, single chain (ScFv) and / or humanized antibodies. The antibodies may be murine, rat, human or any other source (including chimeric or humanized antibodies).

[0117] FLT3-specific CAR and method for producing the same

[0118] Provided herein is a CAR that binds to FLT3 (e.g., human FLT3 (e.g., SEQ ID NO: 198) or accession number: NP_004110). The FLT3-specific CAR provided herein includes a single-chain CAR and a multi-chain CAR. CAR has the ability to redirect T cell specificity and responsiveness toward FLT3 in a non-MHC restricted manner using the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition provides T cells expressing CAR with the ability to recognize antigens that are unrelated to antigen processing, thereby bypassing the main mechanism of tumor escape.

[0119] In some embodiments, the CAR provided herein comprises an extracellular ligand binding domain (e.g., a single-chain variable fragment (scFv)), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular ligand binding domain, the transmembrane domain, and the intracellular signaling domain are in one polypeptide, i.e., in a single chain. Multi-chain CARs and polypeptides are also provided herein. In some embodiments, the multi-chain CAR comprises: a first polypeptide comprising a transmembrane domain and at least one extracellular ligand binding domain; and a second polypeptide comprising a transmembrane domain and at least one intracellular signaling domain, wherein the polypeptides are assembled together to form a multi-chain CAR.

[0120] In some embodiments, FLT3-specific multi-chain CAR is based on high-affinity IgE receptor (FcεRI). FcεRI expressed on mast cells and basophils triggers allergic reactions. FcεRI is a tetrameric complex consisting of a single α subunit, a single β subunit, and two disulfide-linked γ subunits. The α subunit contains an IgE binding domain. The β and γ subunits contain ITAMs that mediate signal transduction. In some embodiments, the extracellular domain of the FcRα chain is missing and replaced by a FLT3-specific extracellular ligand binding domain. In some embodiments, the multi-chain FLT3-specific CAR comprises an ITAM that specifically binds to a scFv, a CD8α hinge, and an FcRβ chain of FLT3. In some embodiments, CAR may or may not include an FcRγ chain.

[0121] In some embodiments, the extracellular ligand binding domain comprises an scFv comprising a light chain variable (VL) region and a heavy chain variable (VH) region of a target antigen-specific monoclonal antibody connected by a flexible linker. Single-chain variable region fragments are obtained by connecting light and / or heavy chain variable regions using a short connecting peptide (Bird et al., Science 242: 423-426, 1988). An example of a connecting peptide is a GS linker having an amino acid sequence (GGGGS) 3 (SEQ ID NO: 232), with a bridge of approximately 3.5 nm between the carboxyl terminus of one variable region and the amino terminus of another variable region. Linkers of other sequences have been designed and used (Bird et al., 1988, supra). In general, the linker can be, for example, a short, flexible polypeptide consisting of about 20 or fewer amino acid residues. The linker can in turn be modified to play other roles, such as attaching a drug or attaching a solid carrier. Single-chain variants can be produced recombinantly or synthetically. For the synthetic production of ScFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell (eukaryotic, such as yeast, plant, insect or mammalian cells; or prokaryotic, such as E. coli). The polynucleotide encoding the relevant ScFv can be obtained by conventional operations, such as ligation of polynucleotides. The resulting ScFv can be isolated using standard protein purification techniques known in the art.

[0122] The FLT3 extracellular domain is composed of five immunoglobulin domains, with domain 5 closest to the cell membrane and domain 1 furthest away (in a linear fashion; Verstraete, 2011 Jul 7; Blood: 118(1)). The sequences of the five FLT3 extracellular domains are listed in Table 1 below.

[0123] Table 1

[0124]

[0125] Domain 2-3 comprises the sequence of domain 2 and domain 3 (SEQ ID NO: 254).

[0126] In some embodiments, the extracellular ligand binding domain of the FLT3-specific CAR described herein binds to domain 1, 2, 3, 4, or 5 of the FLT3 extracellular domain.

[0127] In some embodiments, the extracellular ligand binding domain of the FLT3-specific CAR described herein binds to Domain 1 of the FLT3 extracellular domain.

[0128] In some embodiments, the extracellular ligand binding domain of the FLT3-specific CAR described herein binds to Domain 2 of the FLT3 extracellular domain.

[0129] In some embodiments, the extracellular ligand binding domain of the FLT3-specific CAR described herein binds to domain 3 of the FLT3 extracellular domain.

[0130] In some embodiments, the extracellular ligand binding domain of the FLT3-specific CAR described herein binds to Domain 4 of the FLT3 extracellular domain.

[0131] In some embodiments, the extracellular ligand binding domain of the FLT3-specific CAR described herein binds to domain 5 of the FLT3 extracellular domain.

[0132] In some embodiments, the extracellular ligand binding domain of the FLT3-specific CAR described herein binds to domains 2-3 of the FLT3 extracellular domain.

[0133] In some embodiments, the extracellular ligand binding domain of the FLT3-specific CAR described herein binds to domains 2-3 or 4 of the FLT3 extracellular domain.

[0134] On the other hand, a CAR that specifically binds to FLT3 is provided, wherein the CAR comprises an extracellular ligand binding domain comprising: a VH region having a sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291 or 293; and / or a VL region having a sequence as shown in SEQ ID NO: NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290 or 292. In some embodiments, VH and VL are connected together by a flexible linker. In some embodiments, the flexible linker comprises the amino acid sequence shown in SEQ ID NO:232.

[0135] On the other hand, a FLT3-specific chimeric antigen receptor (CAR) is provided, which comprises an extracellular ligand binding domain, a first transmembrane domain and an intracellular signaling domain, wherein the extracellular domain comprises a single-chain Fv fragment (ScFv) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises (i) a VH complementarity determining region 1 (CDR1) having SEQ ID NO: 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56, 60, 61, 62, 66, 67, 68, 72, 73, 74, 78, 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108, 109, 110, 114, 115, 116 6, 120, 121, 122, 126, 127, 128, 132, 133, 134, 138, 139, 140, 294, 295, 296, 300, 301, 305, 306, 307, 311, 312, 316, 317 or 318; (ii) a VH complementarity determining region 2 (CDR2) having SEQ ID NO:40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 297, 298, 302, 303, 308, 309, 313, 314, 319, 320 or 322; and (iii) a VH complementarity determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO:40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, The amino acid sequence shown in NO:42, 48, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 299, 304, 310, 315 or 321.

[0136] On the other hand, a FLT3-specific chimeric antigen receptor (CAR) is provided, which comprises an extracellular ligand binding domain, a first transmembrane domain and an intracellular signaling domain, wherein the extracellular domain comprises a single-chain Fv fragment (ScFv) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL region comprises (i) a VL complementary determining region 1 (CDR1) having an amino acid sequence as shown in SEQ ID NO: 144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 323, 326, 328, 331, 336, 338, 340, 343, 345, 348 or 350; (ii) a VL complementary determining region 2 (CDR2) having an amino acid sequence as shown in SEQ ID NO: NO:145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 324, 329, 332, 334, 341 or 346; and (iii) a VL complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO:146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 325, 327, 330, 333, 335, 337, 339, 342, 344, 347 or 349.

[0137] On the other hand, a FLT3-specific chimeric antigen receptor (CAR) is provided, which comprises an extracellular ligand binding domain, a first transmembrane domain and an intracellular signaling domain, wherein the extracellular domain comprises a single-chain Fv fragment (ScFv) comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) the VH region comprises (i) a VH complementarity determining region 1 (CDR1) having SEQ ID NO: 37, 38, 39, 43, 44, 45, 49, 50, 54, 55, 56, 60, 61, 62, 66, 67, 68, 72, 73, 74, 78, 79, 80, 84, 85, 86, 90, 91, 92, 96, 97, 98, 102, 103, 104, 108, 109, 110, 114, 115, 116 6, 120, 121, 122, 126, 127, 128, 132, 133, 134, 138, 139, 140, 294, 295, 296, 300, 301, 305, 306, 307, 311, 312, 316, 317 or 318; (ii) a VH complementarity determining region 2 (CDR2) having SEQ ID NO:40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 297, 298, 302, 303, 308, 309, 313, 314, 319, 320 or 322; and (iii) a VH complementarity determining region 3 (CDR3) having an amino acid sequence as shown in SEQ ID NO:40, 41, 46, 47, 51, 52, 57, 58, 63, 64, 69, 70, 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112, 117, 118, 123, 124, 129, NO:42, 48, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113, 119, 125, 131, 137, 143, 299, 304, 310, 315 or 321; and (b) the VL region comprises (i) a VL complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO:144, 147, 150, 153, 156, 159, 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 192, 195, 323, 326, 328, 331, 336, 338, 340, 343, 345, 348 or 350;(ii) a VL complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 145, 148, 151, 154, 157, 160, 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 193, 196, 324, 329, 332, 334, 341 or 346; and (iii) a VL complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in NO:146, 149, 152, 155, 158, 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 191, 194, 197, 325, 327, 330, 333, 335, 337, 339, 342, 344, 347 or 349. ;

[0138] In some embodiments, the CAR of the present invention comprises an extracellular ligand binding domain comprising any one of the partial light chain sequences listed in Table 2 and / or any one of the partial heavy chain sequences listed in Table 2.

[0139] In Table 2, the underlined sequences are CDR sequences according to Kabat, and the sequences in bold are CDR sequences according to Chothia, Apart from Heavy chain CDR2 sequences of P4F6, P4C7, P3A1, P5A3, P9B5, P9F1, P1B4, P1B11, P7H3, P3E10, P1A5, P5F7, P4H11, P15F7, P12B6, P8B6, P14G2, and P7F9 with the Chothia CDR sequences underlined and the Kabat CDR sequences in bold.

[0140] Table 2

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] Also provided herein is a CDR portion of an antigen binding domain of an antibody for FLT3 or a CDR portion of an extracellular ligand binding domain of a CAR for FLT3 (including Chothia, Kabat CDR, and CDR contact region). The determination of CDR regions is well known in the art. It should be understood that in some embodiments, CDR may be a combination of Kabat and Chothia CDRs (also referred to as "combined CR" or "extended CDR"). In some embodiments, CDR is a Kabat CDR. In other embodiments, CDR is a Chothia CDR. In other words, in embodiments with more than one CDR, CDR may be any one of Kabat, Chothia, a combined CDR, or a combination thereof. Table 3 provides examples of CDR sequences provided herein.

[0147] Table 3

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] The present invention encompasses modifications of the CAR and polypeptides of the present invention shown in Table 2, including functionally equivalent CARs with modifications that do not significantly affect their properties and variants with enhanced or reduced activity and / or affinity. For example, the amino acid sequence may be mutated to obtain an antibody with the desired binding affinity to FLT3. The modification of polypeptides is a routine operation in the art and does not need to be described in detail herein. Examples of modified polypeptides include one or more deletions or additions of amino acids with conserved substitutions of amino acid residues, which do not significantly adversely change the functional activity or maturation (enhancement) of the polypeptide to its ligand, or the use of chemical analogs.

[0155] Amino acid sequence insertions include amino and / or carboxyl terminal fusions within the length range of one residue to a polypeptide containing one hundred or more residues, as well as single or multiple amino acid residues inserted within the sequence. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include fusions of enzymes or polypeptides that increase the half-life of antibodies in the blood circulation to the N or C termini of the antibody.

[0156] Substitution variants remove at least one amino acid residue in the antibody molecule and insert different residues in its place. The most relevant sites for substitution mutagenesis include hypervariable regions, but FR changes are also encompassed. Conservative substitutions are shown under the heading "conservative substitutions" in Table 4. If the substitution causes a change in biological activity, more significant changes and screening products named "exemplary substitutions" in Table 4 or as further described below about amino acid classes can be introduced.

[0157] Table 4: Amino Acid Substitutions

[0158]

[0159]

[0160] In some embodiments, provided herein is a CAR comprising an extracellular ligand binding domain that binds to FLT3 and competes with a CAR described herein, including a CAR comprising an extracellular domain that binds to FLT3, wherein the extracellular domain comprises: P4F6, P4C7, P3A1, P5A3, P9B5, P9F1, P1B4, P1B11, P7H3, P3E10, P1A5, P 5F7, P4H11, P15F7, P12B6, P8B6, P14G2, P7F9, P08B06EE, P04A04, P01A05, P08B03, P5F7g, P10A02g, P10A04g, P10A05g, P10A07g, P10B03g, P10B06g, P5F7g2, P5F7g3 and P5F7g4.

[0161] In some embodiments, a CAR specifically binding to FLT3 is provided herein, wherein the CAR comprises a VH region comprising the sequence shown in SEQ ID NO: 4; and / or a VL region comprising the sequence shown in SEQ ID NO: 3. In some embodiments, a CAR specifically binding to FLT3 is provided herein, wherein the CAR comprises a VH region comprising the sequence shown in SEQ ID NO: 6; and / or a VL region comprising the sequence shown in SEQ ID NO: 5. In some embodiments, a CAR specifically binding to FLT3 is provided herein, wherein the CAR comprises a VH region comprising the sequence shown in SEQ ID NO: 10; and / or a VL region comprising the sequence shown in SEQ ID NO: 9. In some embodiments, a CAR specifically binding to FLT3 is provided herein, wherein the CAR comprises a VH region comprising the sequence shown in SEQ ID NO: 20; and / or a VL region comprising the sequence shown in SEQ ID NO: 19.

[0162] In some embodiments, the present invention also provides a CAR comprising a CDR portion of an antibody for a FLT3 antibody based on a CDR contact region. The CDR contact region is a region of an antibody that fully displays the specificity of the antibody to the antigen. In general, the CDR contact region includes CDRs and residue positions in the Vernier zone that are defined to maintain an appropriate loop structure for the antibody to bind to a specific antigen. See, for example, Makabe et al., Journal of Biological Chemistry (J.Biol.Chem.), 283: 1156-1166, 2007. The determination of CDR contact regions is well known in the art.

[0163] The binding affinity (K) of the ligand binding domain of a FLT3-specific CAR as described herein to FLT3, such as human FLT3 (e.g., SEQ ID NO: 198) D ) may be, for example, between about 0.1 and about 1000 nM, such as between about 0.5 nM and about 500 nM, or such as between 1 nM and about 250 nM. In some embodiments, the binding affinity is about any of 1000 nm, 750 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 19 nm, 18 nm, 17 nm, 16 nm, 15 nM, 10 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, or 0.1 nM.

[0164] In some embodiments, the binding affinity (K) of the ligand binding domain of a FLT3-specific CAR as described herein to FLT3 is D ) is about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 20 nM to about 70 nM, about 25 nM to about 75 nM, or about 40 nM to about 110 nM. In one embodiment, the binding affinity of the ScFv described in this paragraph is for human FLT3.

[0165] In some embodiments, the binding affinity (K) of the ScFv of the ligand binding domain of the FLT3-specific CAR described herein to domain 4 of the extracellular domain of human FLT3 is D ) is about 1 nM to about 100 nM, about 10 nM to about 100 nM, about 20 nM to about 100 nM, about 25 nM to about 105 nM, about 40 nM to about 80 nM, about 40 nM to about 100 nM, or about 50 nM to about 100 nM.

[0166] In some embodiments, the binding affinity (K) of the ScFv of the ligand binding domain of the FLT3-specific CAR described herein to domains 2-3 of the extracellular domain of human FLT3 is D ) is about 5 nM to 30 nM, about 5 nM to about 25 nM, about 10 nM to about 30 nM, about 10 nM to about 40 nM, or about 10 nM to about 25 nM.

[0167] In an exemplary embodiment, the K of the ScFv D The measurements were performed as disclosed in Example 1.

[0168] In some embodiments, the binding affinity is less than about any of 1000 nm, 900 nm, 800 nm, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM.

[0169] Monoclonal antibody specific epitope

[0170] In some embodiments, the extracellular domain of any one of the FLT3-specific CARs disclosed herein may include one or more epitopes that are specific to a monoclonal antibody (i.e., specifically recognized by a monoclonal antibody). These epitopes are also referred to herein as mAb-specific epitopes. In these embodiments, the extracellular domain includes VH and VL polypeptides specifically bound to FLT3 and one or more epitopes bound to one or more monoclonal antibodies (mAb). CARs comprising mAb-specific epitopes may be single-chain or multi-chain.

[0171] Including an epitope with specificity for a monoclonal antibody in the extracellular domain of the CAR described herein enables the sorting and exhaustion of engineered immune cells expressing CAR. In certain embodiments, this feature also facilitates the recovery of endogenous cells expressing FLT3 exhausted by administering engineered immune cells expressing CAR.

[0172] Thus, in some embodiments, the invention relates to a method of sorting and / or depleting engineered immune cells endowed with a CAR comprising a mAb-specific epitope; and a method of promoting recovery of endogenous cells expressing FLT3, such as bone marrow progenitor cells.

[0173] Several epitope-monoclonal antibody conjugates are available for generating CARs comprising monoclonal antibody-specific epitopes; specifically, those epitopes that have been approved for medical use, such as the CD20 epitope / rituximab as a non-limiting example.

[0174] In some embodiments, monoclonal antibodies specific for epitopes can be combined with cytotoxic drugs. In addition, CDC cytotoxicity can be promoted by using engineered antibodies with one or more transplant components of the complement system on them. In some embodiments, the activation of CAR-T cells can be regulated by exhausting cells using antibodies that recognize epitopes.

[0175] In addition, the present invention encompasses methods and therapeutic methods of sorting engineered immune cells endowed with FLT3-specific CARs expressing one or more mAb-specific epitopes, wherein activation of engineered immune cells endowed with these CARs is regulated by depleting the cells using antibodies targeting the external ligand-binding domain of the CAR.

[0176] CARs comprising one or more epitopes specifically recognized by monoclonal antibodies are disclosed in WO2016 / 120216, which is incorporated herein by reference in its entirety. The epitope may be selected from any number of epitopes known in the art. In some embodiments, the epitope may be a target of a monoclonal antibody approved for medical use, such as, but not limited to, a CD20 epitope recognized by rituximab. In some embodiments, the epitope comprises the amino acid sequence shown in SEQ ID NO: 229.

[0177] CPYSNPSLC (SEQ ID NO: 229)

[0178] In some embodiments, the epitope may be located between the hinge of the scFv and the CAR. In some embodiments, two instances of the same epitope separated by a linker can be used in the CAR. For example, a polypeptide comprising 2 copies of the epitope shown in SEQ ID NO:229 separated by a linker, such as GSGGGGSCPYSNPSLCSGGGGSCPYSNPSLCSGGGGS (SEQ ID NO:230) can be used in the CAR, located between the light chain variable region and the hinge.

[0179] In some embodiments, the extracellular binding domain of the CAR comprises VH and VL polypeptides, and one or more mAb-specific epitopes may have different structures, depending on the epitope insertion position and the use of the linker. For example, the extracellular binding domain of a FLT3-specific CAR comprising a mAb-specific epitope may have one of the following structures:

[0180] V1-L1-V2-(L) x -Epitope 1-(L) x -;

[0181] V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -;

[0182] V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -;

[0183] (L) x -Epitope 1-(L) x -V1-L1-V2;

[0184] (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2;

[0185] Epitope 1-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -V1-L1-V2;

[0186] (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x ;

[0187] (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -;

[0188] (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -;

[0189] (L) x -Epitope 1-(L) x -Epitope 2-(L) x -V1-L1-V2-(L) x -Epitope 3-(L) x -;

[0190] (L) x -Epitope 1-(L) x -Epitope 2-(L) x-V1-L1-V2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -;

[0191] V1-(L) x -Epitope 1-(L) x -V2;

[0192] V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x ;

[0193] V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x ;

[0194] V1-(L) x -Epitope 1-(L) x -V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x ;

[0195] (L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2; or,

[0196] (L) x -Epitope 1-(L) x -V1-(L) x -Epitope 2-(L) x -V2-(L) x -Epitope 3-(L) x ;

[0197] in,

[0198] V1 is V L And V2 is V H Or V1 is V H And V2 is V L ;

[0199] L1 is suitable for V H Chain connected to V L The joints of the chain;

[0200] L is a linker comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain may be the same or different from other L occurring in the same extracellular binding domain, such as SGGGG, GGGGS or SGGGGS, and x is 0 or 1 and x is selected independently of the others at each occurrence; and,

[0201] Epitope 1, epitope 2, epitope 3 and epitope 4 are mAb specific epitopes and may be the same or different. In some embodiments, epitope 1, epitope 2 and epitope 4 are mAb specific epitopes having an amino acid sequence of SEQ ID NO: 229, and epitope 3 is a mAb specific epitope having an amino acid sequence of SEQ ID NO: 231.

[0202] In some embodiments, the extracellular binding domain of CAR comprises the sequence

[0203] V1-L1-V2-(L) x -Epitope 1-(L) x -Epitope 2-(L) x -;or,

[0204] (L) x -Epitope 1-(L) x -V1-L1-V2-(L) x -Epitope 2-(L) x -Epitope 3-(L) x -Epitope 4-(L) x -, where V 1、 V 2、 L 1、 L, x and Epitope 1, Epitope 2, Epitope 3 and Epitope 4 are as defined above.

[0205] In some embodiments, any of the FLT3-specific CARs disclosed herein may comprise one or more mAb-specific epitopes selected from the group consisting of a CD52 epitope, a CD20 epitope, a CD3 epitope, a CD41 epitope, a CD25 epitope, a CD30 epitope, an EGFR epitope, a TNFα epitope, a VEGF epitope, a complement protein C5 epitope, a CD11a epitope, a CD33 epitope, an α-4 integrin epitope, an IgE Fc region epitope, an RSV protein F epitope, an IL-6 receptor epitope, a HER2 receptor epitope, an integrin α4β7 epitope, a B cell activating factor (BAFF) epitope, an IL-1β epitope, a RANKL epitope, a CTLA4 epitope, a CD34 epitope, an IL-12 epitope, and / or an IL-23 epitope.

[0206] In some embodiments, the FLT3-specific CAR disclosed herein may comprise one or more mAb-specific epitopes selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegol), daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, or ustekinumab.

[0207] In some embodiments, the FLT3-specific CAR comprises one or more mAb-specific epitopes selected from the epitopes disclosed in Table 5:

[0208] Table 5: Examples of mAb-specific epitopes, such as mimotopes and epitopes with their corresponding mAbs, that can be used in the extracellular binding domain of the FLT3-specific CAR of the present invention

[0209]

[0210] The intracellular signaling domain of the CAR according to the present invention is responsible for the intracellular signaling after the extracellular ligand binding domain binds to the target to cause the activation of immune cells and the immune response. The intracellular signaling domain can activate at least one of the normal effector functions of the immune cells expressing CAR. For example, the effector function of T cells can be cytolytic activity or auxiliary activity (including the secretion of cytokines).

[0211] In some embodiments, the intracellular signaling domain for use in CAR may be, for example, but not limited to, a T cell receptor and a cytoplasmic sequence of a co-receptor that coordinates after antigen receptor binding to initiate signal transduction, and any derivatives or variants of these sequences with the same functional ability and any synthetic sequence. The intracellular signaling domain includes two different categories of cytoplasmic signaling sequences: a sequence that initiates antigen-dependent primary activation and a sequence that acts in an antigen-independent manner to provide a secondary or costimulatory signal. The primary cytoplasmic signaling sequence may include a signaling motif, which is known as an immunoreceptor tyrosine-based activation motif (ITAM). ITAM is a well-defined signaling motif found in the intracytoplasmic tail of various receptors that act as binding sites for syk / zap70-like tyrosine kinases. Examples of ITAM used in the present invention may include ITAM derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d in the form of non-limiting examples. In some embodiments, the intracellular signaling domain of the CAR may comprise a CD3 zeta signaling domain having an amino acid sequence having at least about 70%, such as at least 80%, or at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown in SEQ.ID NO: 210. In some embodiments, the intracellular signaling domain of the CAR of the present invention comprises the domain of a costimulatory molecule.

[0212] In some embodiments, the intracellular signaling domain of the CAR of the present invention comprises a portion of a co-stimulatory molecule selected from the group consisting of fragments of 41BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In some embodiments, the intracellular signaling domain of the CAR of the present invention comprises an amino acid sequence comprising at least about 70%, for example at least 80%, or at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown in SEQ.ID NO: 209 and SEQ.ID NO: 213. In some embodiments, the intracellular signaling domain of the CAR of the present invention comprises an amino acid sequence comprising at least about 70%, for example at least 80%, or at least 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown in SEQ.ID NO: 209 and SEQ.ID NO: 214.

[0213] CAR is expressed on the surface membrane of the cell. Therefore, CAR may include a transmembrane domain. Suitable transmembrane domains of CAR disclosed herein have the following capabilities: (a) expressed on the surface of cells, such as immune cells, such as (but not limited to) lymphocytes or natural killer (NK) cells, and (b) interacting with ligand binding domains and intracellular signaling domains to guide immune cells to predetermined target cells. The transmembrane domain may be derived from a natural or synthetic source. The transmembrane domain may be derived from any membrane-bound or transmembrane protein. As a non-limiting example, a transmembrane polypeptide may be a subunit of a T cell receptor (such as α, β, γ or δ), a subunit chain constituting a CD3 complex, IL-2 receptor p55 (a chain), p75 (β chain) or γ chain, Fc receptor (specifically, Fcγ receptor III) or CD protein polypeptide. Alternatively, the transmembrane domain may be synthetic and may mainly include hydrophobic residues, such as leucine and valine. In some embodiments, the transmembrane domain is derived from human CD8 alpha chain (eg, NP_001139345.1).

[0214] The transmembrane domain is connected to the extracellular ligand binding domain by a stem domain (also referred to as a hinge domain). The stem domain may include up to 300 amino acids, such as 10 to 100 amino acids or 25 to 50 amino acids. The stem region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4 or CD28 or all or part of an antibody constant region. Alternatively, the stem domain may be a synthetic sequence corresponding to a naturally occurring stem sequence or may be a whole synthetic stem sequence. In some embodiments, the stem domain is a part of a human CD8 α chain (e.g., NP_001139345.1). In another specific embodiment, the transmembrane and hinge domains include a part of a human CD8 α chain, for example, it includes at least 70%, or at least 80%, or at least 90%, 95%, 97% or 99% sequence identity with an amino acid sequence selected from a group consisting of SEQ ID NO: 206 and 208. In some embodiments, the stem domain of the FLT3-specific CAR described herein comprises a CD8α hinge, an IgG1 hinge, or an FcγRIIIα hinge. In some embodiments, the stem domain comprises a human CD8α hinge, a human IgG1 hinge, or a human FcγRIIIα hinge. In some embodiments, the CAR disclosed herein may comprise an extracellular ligand binding domain that specifically binds to FLT3, a CD8α human hinge and a transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain.

[0215] Table 6 provides exemplary sequences of domains that can be used in the CARs disclosed herein.

[0216] Table 6: Exemplary sequences of CAR components

[0217]

[0218]

[0219] Downregulation or mutation of the target antigen is usually observed in cancer cells, resulting in antigen loss escape variants. Therefore, in order to counteract tumor escape and make immune cells more specific to the target, FLT3-specific CAR may include one or more additional extracellular ligand binding domains to bind to different elements in the target at the same time, thereby enhancing immune cell activation and function. In some embodiments, the extracellular ligand binding domain can be placed in series on the same transmembrane polypeptide, and optionally, can be separated by a joint. In some embodiments, the different extracellular ligand binding domains may be placed on different transmembrane polypeptides constituting CAR. In some embodiments, the present invention relates to a CAR group, each CAR comprising different extracellular ligand binding domains. Specifically, the present invention relates to a method for engineering immune cells, comprising providing immune cells, and expressing CAR groups at the cell surface, each CAR comprising different extracellular ligand binding domains. In another specific embodiment, the present invention relates to a method for engineering immune cells, comprising providing immune cells, and introducing a polynucleotide encoding a polypeptide into the cell, wherein the polypeptide constitutes each CAR group comprising different extracellular ligand binding domains. A CAR group means at least two, three, four, five, six or more CARs, each comprising a different extracellular ligand binding domain. The different extracellular ligand binding domains according to the present invention can, for example, simultaneously bind to different elements in the target, thereby enhancing immune cell activation and function. The present invention also relates to a separate immune cell comprising a CAR group each comprising a different extracellular ligand binding domain.

[0220] In another aspect, provided herein are polynucleotides encoding any of the CARs and polypeptides described herein. Polynucleotides can be made and expressed by procedures known in the art.

[0221] On the other hand, provided herein is a composition (such as a pharmaceutical composition) comprising any of the cells of the present invention. In some embodiments, the composition comprises a cell comprising a polynucleotide encoding any of the CARs described herein. In yet other embodiments, the composition comprises any one or both of the polynucleotides shown in the following SEQ ID NO: 245 and SEQ ID NO: 246:

[0222] P3E10 heavy chain variable region

[0223] CAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTGAAGAAGCCAGGCAGCTCCGTGAAGGTGTCCTGCAAGGCCTCTGGCGGCACATTCTCTAGCTACGCCATCCAGTGGGTGCGGCAGGCACCAGGACAGGGCCTGGAGTGGATGGGAGGAATCGTGGGAAGCTGGGGCCTGGCAAAC TACGCCCAGAAGTTTCAGGGCAGTGACCATCACCACCGATAAGTCTACAAGCACCGCCTATATGGAGCTGTCCTCTCTGAGGTCCGAGGACACAGCCGTGTACTATTGCGCCACCTCCGCCTTCGGCGAGCTGGCATCTTGGGGACAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:245)

[0224] P3E10 light chain variable region

[0225] GAGATCGTGCTGACACAGAGCCCAGGCACCCTGTCCCTGTCTCCAGGAGAGAGGGCCACACTGTCCTGTAGGGCCAGCCAGTCCGTGCCTTCTAGCCAGCTGGCCTGGTACCAGCAGAAGCCAGGCCAGGCCCCAGACTGCTGATCTATGACGCCTCCTCTAG AGCCACAGGCATCCCAGATAGGTTCTCTGGCAGCGGCTCCGGCACCGACTTTACACTGACCATCTCCAGGCTGGAGCCCGAGGATTTCGCCGTGTACTATTGCCAGCAGTACGGCAGCTCCCCTCTGACATTTGCCAGGGCACCAAGGTGGAGATCAAGG(SEQ ID NO:246)

[0226] In yet other embodiments, the composition comprises either or both of the following polynucleotides shown in SEQ ID NO: 247 and SEQ ID NO: 248:

[0227] P3A1 heavy chain variable region

[0228] CAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTGAAGAAGCCCGGCAGCTCCGTGAAGGTGTCTTGCAAGGCCAGCGGCGGCACATTCTCTAGCTACGATATCTCTTGGGTGAGGCAGGCCCCAGGCCAGGGACTGGAGTGGATGGGAGGAATCATCCCCGTGAGCGGAAGGGCAAACTAC GCACAGAAGTTTCAGGGCCGGGTGACCATCACCACAGACAAGTCCACATCTACCGCCTATATGGAGCTGTCCTCTCTGAGAAGCGAGGATACAGCCGTGTACTATTGCGCCAGAGTGAGGCCTACCTACTGGCCACTGGACTATTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:247)

[0229] P3A1 light chain variable region

[0230] GACATCCAGATGACCCAGTCCCCATCTAGCCTGAGCGCCTCCGTGGGCGATAGAGTGACAATCACCTGTAGGGCCTTCCAGAGCATCTCCTCTTACCTGAATTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGCAGCAAGCTCCCTG CAGTCCGGAGTGCCATCTCGGTTCTCCGGCTCTGGCAGCGGCACAGACTTTACACTGACCATCTCTAGCCTGCAGCCTGAGGATTTCGCCACCTACTATTGCCAGCAGTCCTATTCTACACCACTGACCTTTGGCCAGGGCACAAAGGTGGAGATCAAG(SEQ ID NO:248).

[0231] In yet other embodiments, the composition comprises either or both of the following polynucleotides shown in SEQ ID NO: 249 and SEQ ID NO: 250:

[0232] P9B5 heavy chain variable region

[0233] GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAGCCTGGCGGCTCTCTGAGACTGAGCTGCGCAGCATCCGGCTTCATCTTTGCCTCTTACGCAATGAGCTGGGTGAGGCAGGCCCTGGCAAGGGACTGGAGTGGGTGAGCGAGATCAGCTCCTCTGGCGGCAGCACCACATATGCCGACT CCGTGAAGGGCCGCTTCACAATCAGCCGGGACAACTCTAAGAATACCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCGTGTACTATTGCGCCAGAGATAGAGTGATGGCCGGCCTGGGCTATGACCCATTTGATTACTGGGGCCAGGGCACACTGGTGACCGTGAGTCCC(SEQID NO:249)

[0234] P9B5 light chain variable region

[0235] CAGAGCGTGCTGACCCAGCCACCTTCCGCCTCTGGAACACCCGGCCAGAGGGTGACCATCAGCTGTTCCGGCTCTAGCTCCAACATCGGCTCCAATTACGTGTATTGGTACCAGCAGCTGCCCGGCACAGCCCCTAAGCTGCTGATCTACAGAAACAATCAGAGGCC ATCTGGCGTGCCCGACCGCTTCTCTGGCAGCAAGTCCGGCACCTCTGCCAGCCTGGCAATCAGCGGACTGCGGTCCGAGGACGAGGCCGATTACTATTGCGCAGCATGGGACGATTCCCTGTCTGGAGTGGTGTTTGGCGGCGGCACAAAGCTGACCGTGCTG(SEQ ID NO:250).

[0236] In yet other embodiments, the composition comprises either or both of the following polynucleotides shown in SEQ ID NO: 251 and SEQ ID NO: 252:

[0237] P4C7 heavy chain variable region

[0238] CAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTGAAGAAGCCTGGCAGCTCCGTGAAGGTGTCTTGCAAGGCCAGCGGCGGCACATTCTCTAGCTACACCATTCCTGGGTGCGGCAGGCCCCAGGCCAGGGACTGGAGTGGATGGGAGGAATCATCCCAGCCTTCGGCATCGCCAACTACGC CCAGAAGTTTCAGGGCCGCGTGACAATCACCGCCGACAAGTCCACATCTACCGCCTATATGGAGCTGTCCTCTGCGGAGCGAGGATACCGCCGTGTACTATTGCGCCAAGGGCGGCAGCTACTCCCTGGACTATTTTGATATCTGGGGCCAGGGCACACTGGTGACCGTGAGCTCC(SEQ ID NO:251)

[0239] P4C7 light chain variable region

[0240] GAGATCGTGCTGACACAGTCCCCTGGCACCCTGTCTCTGAGCCCAGGCGAGAGGGCCACACTGTCCTGTAGGGCATCTCAGTACGTGTCCGCCTCTCTGCTGGCCTGGTATCAGCAGAAGCCTGGCCAGGCCCCAAGACTGCTGATCTACGGAGCATCCACA AGAGCCACCGGCATCCCCGACAGGTTCAGCGGCTCCGCCTCTGGAACCGACTTCACCCTGACCATCTCTAGACTGGAGCCTGAGGACTTCGCCGTGTACTATTGCCAGCAGTATGCCAGGTCTAGCACATTTGGCCAGGGCACCAAGGTGGAGATCAAG(SEQ ID NO:252)

[0241] Administration of expression vectors and polynucleotide compositions are further described herein.

[0242] In another aspect, provided herein is a method of making any of the polynucleotides described herein.

[0243] The invention also encompasses polynucleotides complementary to any such sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic DNA) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to DNA molecules in a one-to-one manner; and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may (but not necessarily) be present in the polynucleotides of the invention, and polynucleotides may (but not necessarily) be linked to other molecules and / or supports.

[0244] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof) or may comprise a variant of such a sequence. The polynucleotide variant contains one or more substitutions, additions, deletions, and / or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may be generally assessed as described herein. The variant typically exhibits at least about 70% identity, or at least about 80% identity, or even at least about 90% or 95% identity to a polynucleotide sequence encoding a native antibody or a portion thereof.

[0245] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is identical when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20, typically 30 to about 75 or 40 to about 50 contiguous positions, in which a sequence can be compared to a reference sequence having the same number of contiguous positions after the two sequences have been optimally aligned.

[0246] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This program implements several alignment procedures described in Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO (ed.), Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Vol. 5, Supplement 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pp. 626-645, Methods in Enzymology, Vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor., 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol., 4:406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proceedings of the National Academy of Sciences of the United States of America, 80:726-730.

[0247] Typically, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences within a comparison window of at least 20 positions, wherein for optimal alignment of the two sequences, the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20 percent or less, typically 5 to 15 percent or 10 to 12 percent additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0248] Additionally or alternatively, the variant may be substantially homologous to the native gene, or a portion or complement thereof.Such polynucleotide variants are capable of hybridizing under appropriately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0249] Suitable "appropriately stringent conditions" include pre-washing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization in 5×SSC overnight at 50°C-65°C; followed by washing twice at 65°C for 20 minutes each with 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

[0250] As used herein, "high stringency conditions" or "high stringency conditions" are those that: (1) use low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) use a denaturing agent, such as formamide, e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% polysucrose (Ficoll) / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) at 42°C, and 750 mM sodium chloride, 75 mM sodium citrate; or (3) use 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.5) at 42°C. 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, with washes in 0.2× SSC (sodium chloride / sodium citrate) at 42° C. and in 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1× SSC containing EDTA at 55° C. The skilled artisan will recognize how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length.

[0251] Those of ordinary skill in the art will appreciate that, as described herein, due to the degeneracy of the genetic code, there are a variety of nucleotide sequences encoding polypeptides. Some of these polynucleotides carry minimal homology with the nucleotide sequence of any natural gene. However, the present invention specifically encompasses polynucleotides that vary due to differences in codon usage. In addition, alleles of genes comprising polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that change due to one or more mutations (such as deletions, additions and / or substitutions) of nucleotides. The resulting mRNA and protein may (but not necessarily) have a changed structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and / or database sequence comparisons).

[0252] The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods or PCR. The methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercial DNA synthesizers to produce the desired DNA sequence.

[0253] In order to prepare polynucleotides using recombinant methods, polynucleotides comprising the desired sequence can be inserted into a suitable vector, and the vector can in turn be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing exogenous polynucleotides using direct uptake, endocytosis, transfection, F-pairing or electroporation. After introduction, the exogenous polynucleotides can be maintained in the cell in the form of a non-integrating vector (such as a plasmid) or integrated into the host cell genome. Therefore, the amplified polynucleotides can be separated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

[0254] Alternatively, PCR allows DNA sequence reproduction. PCR technology is well known in the art and is described in U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and in PCR: The Polymerase Chain Reaction, Mullis et al., eds., Birkauswer Press, Boston, 1994.

[0255] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and transcribes the DNA into RNA, the RNA can then be isolated using methods well known to those skilled in the art, such as described in Sabrook et al., 1989 (supra).

[0256] Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector can be changed according to the host cell intended to be used, suitable cloning vectors generally have the ability to self-replicate, can have a single target of a specific restriction endonuclease and / or can carry genes for markers that can be used to select clones containing vectors. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors (such as pSA3 and pAT28). These and various other cloning vectors can be purchased from commercial suppliers (such as BioRad, Strategene, and Invitrogen).

[0257] An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the invention. This implies that the expression vector must be replicable in a host cell as an episome or as a component of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, retroviruses), cosmids, and one or more expression vectors disclosed in PCT Publication No. WO 87 / 04462. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as ribosome binding sites, translation start sites, and stop codons, are also generally required.

[0258] Vectors containing the polynucleotides of interest can be introduced into host cells by any of a variety of suitable means, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; liposome transfection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of the vector or polynucleotide to be introduced will generally depend on the characteristics of the host cell.

[0259] The polynucleotide encoding the FLT3-specific CAR disclosed herein may be present in an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell; or a viral vector for transfecting an insect host cell, such as a baculovirus vector; or a plasmid or viral vector for transfecting a mammalian host cell, such as a lentivirus). In some embodiments, the polynucleotide or vector may include a nucleic acid sequence encoding a ribosome skipping sequence, such as, but not limited to, a sequence encoding a 2A peptide. The 2A peptide identified in the foot-and-mouth disease virus subgroup of the picornavirus allows the ribosome to "jump" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codon (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). "Codon" means three nucleotides that are translated into an amino acid residue by the ribosome on mRNA (or on the sense strand of a DNA molecule). Thus, two polypeptides can be synthesized from a single, continuous open reading frame in an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. The ribosome skipping mechanism is well known in the art and is known to be used by several vectors for expressing several proteins encoded by a single messenger RNA.

[0260] To direct the transmembrane polypeptide into the secretory pathway of the host cell, in some embodiments, a secretory signal sequence (also referred to as a leader sequence, prepro sequence or presequence) is provided in the polynucleotide sequence or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, that is, the two sequences are combined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. The secretory signal sequence is usually located 5' of the nucleic acid sequence encoding the relevant polypeptide, but some secretory signal sequences may be located at other positions in the relevant nucleic acid sequence (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). In some embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 204 or 215. Those skilled in the art will recognize that, given the degeneracy of the genetic code, there may be considerable sequence variation in these polynucleotide molecules. In some embodiments, the nucleic acid sequences of the present invention are codon-optimized for expression in mammalian cells, such as in primate (e.g., human or monkey) cells. Codon optimization refers to the exchange of codons in related sequences that are rare in highly expressed genes of a given species by codons that are common in highly expressed genes of the species, which, when exchanged, encode amino acids.

[0261] Methods for engineering immune cells

[0262] Provided herein is a method for preparing immune cells for immunotherapy. In some embodiments, the method comprises obtaining immune cells, introducing CAR according to the present invention into immune cells, and amplifying the cells. In some embodiments, the present invention relates to a method for engineering immune cells, comprising: providing cells, and expressing at least one CAR as described above at the cell surface. The method for engineering immune cells is described in, for example, PCT patent application publication Nos. WO / 2014 / 039523, WO / 2014 / 184741, WO / 2014 / 191128, WO / 2014 / 184744, and WO / 2014 / 184143, each of which is incorporated herein by reference in its entirety. In some embodiments, the method comprises: transfecting cells with at least one polynucleotide encoding CAR as described above; and expressing polynucleotides in cells.

[0263] Before engineering immune cells, cell sources can be obtained from subjects via a variety of non-limiting methods. Cells can be obtained from a variety of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection sites, ascites, pleural effusion, spleen tissue and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art can be used. In some embodiments, cells can be derived from healthy donors or donors with diseases or conditions, for example, individuals diagnosed with cancer or individuals diagnosed with infection. In some embodiments, cells can be part of a mixed cell population exhibiting different phenotypic characteristics.

[0264] In some embodiments, for stable expression in a cell, the polynucleotide is present in a lentiviral vector.

[0265] In some embodiments, the method further comprises a step of genetically modifying cells by interfering or inactivating the expression of, for example, but not limited to, components of TCR, at least one gene of a target of an immunosuppressant, an HLA gene, and / or an immune checkpoint protein such as PDCD1 or CTLA-4. Interfering or inactivating a gene means that the relevant gene is not expressed in the form of a functional protein. In some embodiments, the interfered or inactivated gene is selected from a group consisting of, for example, but not limited to, TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (DCK), PD-1, and CTLA-4. In some embodiments, the method comprises inactivating one or more genes by introducing a rare cutting endonuclease that can selectively inactivate a gene by selective DNA cleavage into a cell. In some embodiments, the rare cutting endonuclease may be, for example, a transcription activator-like effector nuclease (TALE-nuclease) or a Cas9 endonuclease.

[0266] In some embodiments, an additional catalytic domain is used with a rare cutting endonuclease to enhance its ability to inactivate the target gene. For example, an additional catalytic domain may be a DNA end processing enzyme. Non-limiting examples of DNA end processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' alkaline exonucleases, 5' flap endonucleases, helicases, phosphatases, hydrolases, and non-template-dependent DNA polymerases. Non-limiting examples of the catalytic domain include a protein domain selected from the group consisting of or a catalytically active derivative of the protein domain: hExoI (EXO1_HUMAN), yeast ExoI (EXO1_YEAST), Escherichia coli ExoI, human TREX2, mouse TREX1, human TREX1, cattle TREX1, rat TREX1, terminal deoxynucleotidyl transferase (TdT) human DNA2, yeast DNA2 (DNA2_YEAST). In some embodiments, the additional catalytic domain may have 3'-5'-nuclease activity, and in some embodiments, the additional catalytic domain is TREX, for example, TREX2 catalytic domain (WO2012 / 058458). In some embodiments, the catalytic domain is encoded by a single-chain TREX polypeptide. The additional catalytic domain may be fused to a nuclease fusion protein or a chimeric protein. In some embodiments, the additional catalytic domain is fused using, for example, a peptide linker.

[0267] In some embodiments, the method further comprises the step of introducing an exogenous nucleic acid into the cell, the nucleic acid comprising at least one sequence homologous to a portion of the target nucleic acid sequence, so that homologous recombination occurs between the target nucleic acid sequence and the exogenous nucleic acid. In some embodiments, the exogenous nucleic acid comprises a first portion and a second portion homologous to the 5' and 3' regions of the target nucleic acid sequence, respectively. The exogenous nucleic acid may also comprise a third portion between the first portion and the second portion, which is not homologous to the 5' and 3' regions of the target nucleic acid sequence. After the target nucleic acid sequence is cleaved, a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid. In some embodiments, homologous sequences of at least about 50 bp, more than about 100 bp, or more than about 200 bp can be used in the donor matrix. The exogenous nucleic acid may be, for example, (but not limited to) about 200 bp to about 6000 bp, for example, about 1000 bp to about 2000 bp. The common nucleic acid homology is located in the upstream and downstream regions of the flanking break site, and the nucleic acid sequence to be introduced is located between the two arms.

[0268] In some embodiments, the nucleic acid comprises, in sequence, a first homologous region for a sequence upstream of the cleavage; a sequence for inactivating a target gene selected from the group consisting of: TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (DCK) and immune checkpoint proteins, such as programmed death-1 (PD-1); and a second homologous region for a sequence downstream of the cleavage. The polynucleotide introduction step may be performed simultaneously with, before or after the introduction or expression of a rare cutting endonuclease. Depending on the position of the target nucleic acid sequence in which the break event has occurred, the exogenous nucleic acid may be used to knock out a gene, for example, when the exogenous nucleic acid is located in the open reading frame of a gene, or for introducing a new related sequence or gene. Sequence insertion using the exogenous nucleic acid may be used to modify an existing target gene by correcting or replacing a gene (allele exchange as a non-limiting example); or for upregulating or downregulating the expression of a target gene (promoter exchange as a non-limiting example), target gene correction or replacement. In some embodiments, inactivation of a gene selected from the group consisting of TCRα, TCRβ, CD52, GR, DCK, and immune checkpoint proteins can be performed at a precise genomic location targeted by a specific TALE-nuclease, wherein the specific TALE-nuclease catalyzes cleavage, and wherein the exogenous nucleic acid sequentially comprises at least one homology region and a sequence that inactivates a target gene selected from the group consisting of TCRα, TCRβ, CD52, GR, DCK, and immune checkpoint proteins, and the sequence is integrated by homologous recombination. In some embodiments, several genes can be inactivated sequentially or simultaneously by using several TALE-nucleases, respectively, and specifically, targeting a determined gene and several specific polynucleotides for specific gene inactivation.

[0269] In some embodiments, the method comprises inactivating one or more additional genes selected from the group consisting of: TCRα, TCRβ, CD52, GR, DCK, and immune checkpoint proteins. In some embodiments, gene inactivation can be accomplished by: introducing at least one rare-cutting nuclease into the cell so that the rare-cutting nuclease specifically catalyzes the cleavage of a target sequence of the cell genome; and optionally, introducing an exogenous nucleic acid into the cell, the exogenous nucleic acid sequentially comprising a first homologous region for a sequence upstream of the cleavage, a sequence inserted into the genome of the cell, and a second homologous region for a sequence downstream of the cleavage; wherein the introduced exogenous nucleic acid inactivates the gene and integrates at least one exogenous polynucleotide sequence encoding at least one related recombinant protein. In some embodiments, the exogenous polynucleotide sequence is integrated into a gene encoding a protein selected from the group consisting of: TCRα, TCRβ, CD52, GR, DCK, and immune checkpoint proteins.

[0270] On the other hand, the step of genetically modifying cells may include: modifying T cells by inactivating at least one gene expressing a target of an immunosuppressant; and optionally expanding the cells in the presence of an immunosuppressant. An immunosuppressant is an agent that suppresses immune function through one of several mechanisms of action. An immunosuppressant can weaken the degree and / or severity of an immune response. Non-limiting examples of immunosuppressants include calcineurin inhibitors, targets of rapamycin, interleukin-2α chain blockers, inhibitors of monophosphate inosine dehydrogenase, inhibitors of dihydrofolate reductase, corticosteroids, and immunosuppressive antimetabolites. Some cytotoxic immunosuppressants work by inhibiting DNA synthesis. Other immunosuppressants can work via the activation of T cells or by inhibiting the activation of helper cells. The method according to the present invention allows for conferring T cell immunosuppression resistance for immunotherapy by inactivating the target of an immunosuppressant in T cells. As a non-limiting example, the target of an immunosuppressant can be a receptor of an immunosuppressant, such as (but not limited to) CD52, glucocorticoid receptor (GR), FKBP family gene members, and cyclophilin family gene members.

[0271] In some embodiments, the genetic modification of the method involves expressing a rare-cutting endonuclease in a provided cell to be engineered, such that the rare-cutting endonuclease specifically catalyzes cleavage in a target gene, thereby inactivating the target gene. In some embodiments, the method of engineering cells comprises at least one of the following steps: providing T cells such as by cell culture or by a blood sample; selecting a gene expressing a target of an immunosuppressant in the T cell; introducing a rare-cutting endonuclease into the T cell, the endonuclease being capable of selectively inactivating by DNA cleavage, for example, by causing a double-strand break in a gene encoding a target of an immunosuppressant; and, optionally, amplifying the cells in the presence of an immunosuppressant.

[0272] In some embodiments, the method comprises: providing T cells, such as from cell culture or from a blood sample; selecting a gene in the T cells, wherein the gene expresses a target of an immunosuppressant; transfecting the T cells with a nucleic acid encoding a rare-cutting endonuclease that is capable of selectively inactivating the gene encoding the target of the immunosuppressant by DNA cleavage, such as by double-strand breaks; and expressing the rare-cutting endonuclease in the T cells; and, optionally, expanding the cells in the presence of an immunosuppressant.

[0273] In some embodiments, the rare cutting endonuclease specifically targets CD52 or GR. In some embodiments, the gene selected for inactivation encodes CD52, and the immunosuppressive therapy comprises a humanized antibody targeting the CD52 antigen. In some embodiments, the gene selected for inactivation encodes GR, and the immunosuppressive therapy comprises a corticosteroid such as dexamethasone. In some embodiments, the gene selected for inactivation is a FKBP family gene member or a variant thereof, and the immunosuppressive therapy comprises FK506, which is also called Tacrolimus or fujimycin. In some embodiments, the FKBP family gene member is FKBP12 or a variant thereof. In some embodiments, the gene selected for inactivation is a cyclophilin family gene member or a variant thereof, and the immunosuppressive therapy comprises cyclosporine.

[0274] In some embodiments, the rare-cutting endonuclease can be, for example, a meganuclease, a zinc finger nuclease, or a TALE-nuclease (TALEN). In some embodiments, the rare-cutting endonuclease is a TALE-nuclease.

[0275] Also provided herein is a method for engineering T cells suitable for immunotherapy, wherein the method comprises: genetically modifying T cells by inactivating at least an immune checkpoint protein. In some embodiments, the immune checkpoint protein is, for example, PD-1 and / or CTLA-4. In some embodiments, the method of genetically modifying cells comprises: modifying T cells by inactivating at least one immune checkpoint protein; and expanding the cells. Immune checkpoint proteins include (but are not limited to) programmed death 1 (PD-1, also known as PDCD1 or CD279, accession number: NM_005018), cytotoxic T lymphocyte antigen 4 (CTLA-4, also known as CD152, gene bank accession number AF414120.1), LAG3 (also known as CD223, accession number: NM-002286.5), Tim3 (also known as HAVCR2, gene bank accession number: JX049979.1), BTLA (also known as CD272, accession number: NM-181780.3), BY55 (also known as CD160, GenBank Accession No.: CR541888.1), TIGIT (also known as VSTM3, Accession No.: NM-173799), B7H5 (also known as C10orf54, a homolog of the mouse vista gene, Accession No.: NM-022153.1), LAIR1 (also known as CD305, GenBank Accession No.: CR542051.1), SIGLEC10 (GenBank Accession No.: AY358337.1), 2B4 (also known as CD244, Accession No.: NM-001166664.1), which directly inhibit immune cells. For example, CTLA-4 is a cell surface protein expressed on certain CD4 and CD8 T cells; when engaged to antigen presenting cells through its ligands (B7-1 and B7-2), T cell activation and effector function are inhibited.

[0276] In some embodiments, the method for engineering cells comprises at least one of the following steps: providing T cells, such as by cell culture or by a blood sample; introducing a rare-cutting endonuclease into the T cells, the endonuclease being capable of selectively inactivating a gene encoding an immune checkpoint protein by DNA cleavage, such as by double-strand breaks; and expanding the cells. In some embodiments, the method comprises: providing T cells, such as by cell culture or by a blood sample; transfecting the T cells with a nucleic acid encoding a rare-cutting endonuclease, the endonuclease being capable of selectively inactivating a gene encoding an immune checkpoint protein by DNA cleavage, such as by double-strand breaks; expressing the rare-cutting endonuclease in the T cells; and expanding the cells. In some embodiments, the rare-cutting endonuclease specifically targets a gene selected from the group consisting of: PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, TCRα, and TCRβ. In some embodiments, the rare-cutting endonuclease may be a meganuclease, a zinc finger nuclease, or a TALE-nuclease. In some embodiments, the rare-cutting endonuclease is a TALE-nuclease.

[0277] In some embodiments, the present invention may be particularly applicable to allogeneic immunotherapy. In such embodiments, cells may be modified by a method comprising: inactivating at least one gene encoding a component of a T cell receptor (TCR) in a T cell; and expanding the T cell. In some embodiments, the genetic modification of the method relies on expressing a rare-cutting nuclease in a provided cell to be engineered, so that the rare-cutting nuclease specifically catalyzes the cleavage in a target gene, thereby inactivating the target gene. In some embodiments, the method for engineering cells comprises at least one of the following steps: providing T cells such as by cell culture or by a blood sample; introducing a rare-cutting nuclease into a T cell, the nuclease being able to selectively inactivate at least one gene encoding a component of a T cell receptor (TCR) by DNA cleavage, such as by double-strand breaks; and expanding the cell.

[0278] In some embodiments, the method comprises: providing T cells, such as from cell culture or from a blood sample; transfecting the T cells with a nucleic acid encoding a rare-cutting endonuclease that is capable of selectively inactivating at least one gene encoding a component of a T cell receptor (TCR) by DNA cleavage, such as by double-strand breaks; expressing the rare-cutting endonuclease in the T cells; sorting the transformed T cells that do not express the TCR on the cell surface; and expanding the cells.

[0279] In some embodiments, the rare cutting endonuclease may be a meganuclease, a zinc finger nuclease, or a TALE-nuclease. In some embodiments, the rare cutting endonuclease is a TALE-nuclease. In some embodiments, the TALE-nuclease recognizes and cleaves a sequence encoding TCR alpha or TCR beta. In some embodiments, the TALE-nuclease comprises a polypeptide sequence selected from the amino acid sequence shown in SEQ ID NO: 219, 220, 221, 222, 223, 224, 225, or 226.

[0280] TALE-nuclease polypeptide sequence:

[0281] Repeat TRAC_T01-L

[0282] (SEQ ID NO:219).

[0283] Repeat TRAC_T01-R

[0284] (SEQ ID NO:220).

[0285] Repeat TRBC_T01-L

[0286] (SEQ ID NO:221).

[0287] Repeat TRBC_T01-R

[0288] (SEQ ID NO:222).

[0289] Repeat TRBC_T02-L

[0290] (SEQ ID NO:223).

[0291] Repeat TRBC_T02-R

[0292] (SEQ ID NO:224).

[0293] Repeat CD52_T02-L

[0294] (SEQ ID NO:225).

[0295] Repeat CD52_T02-R

[0296] (SEQ ID NO:226).

[0297] On the other hand, another step of genetically modifying cells can be a method for expanding T cells lacking TCRα, which comprises introducing pTα (also known as pre-TCRα) or a functional variant thereof into T cells, and allowing the cells to expand, optionally via stimulation of the CD3 complex. In some embodiments, the method comprises: a) transfecting cells with a nucleic acid encoding at least one fragment of pTα to support CD3 surface expression; b) expressing the pTα in cells; and c) allowing the cells to expand, optionally via stimulation of the CD3 complex.

[0298] Also provided is a method of preparing T cells for immunotherapy, comprising steps for a method of expanding T cells. In some embodiments, the pTα polynucleotide sequence may be introduced randomly or by homologous recombination. In some embodiments, the insertion may be associated with the inactivation of the TCRα gene.

[0299] Different functional variants of pTα can be used. A "functional variant" of a peptide refers to a molecule or fragment thereof that is substantially similar to a complete peptide. A "fragment" of pTα or a functional variant thereof refers to any subset of a molecule, i.e., a peptide that is shorter than the full-length pTα. In some embodiments, pTα or a functional variant may be, for example, a full-length pTα or a C-terminally truncated pTα form. The C-terminally truncated pTα lacks one or more residues at the C-terminus. As a non-limiting example, the C-terminally truncated pTα form lacks 18, 48, 62, 78, 92, 110 or 114 residues from the C-terminus of the protein. The amino acid sequence variant of the peptide can be prepared by mutation of the DNA encoding the peptide. The functional variant includes, for example, a deletion or insertion or substitution of a residue in the amino acid sequence. Any combination of deletion, insertion and substitution may also be performed to obtain the final construct, provided that the final construct has the desired activity, specifically, repairing a functional CD3 complex. In an exemplary embodiment, at least one mutation is introduced into different pTα forms as described above to affect dimerization. As a non-limiting example, the mutated residue may be W46R, D22A, K24A, R102A or R117A of at least human pTα protein, or the position may be aligned on the pTα family or homolog member using the CLUSTALW method. For example, pTα or its variants as described above include mutated residues W46R or mutated residues D22A, K24A, R102A and R117A. In some embodiments, the pTα or variants are also fused with signal transduction domains, such as CD28, OX40, ICOS, CD27, CD137 (4-1BB) and CD8 as non-limiting examples. The extracellular domain of pTα or variants as described above may be fused with a fragment of TCRα protein, specifically, the transmembrane and intracellular domains of TCRα. The pTα variants may also be fused with the intracellular domain of TCRα.

[0300] In some embodiments, pTalpha forms can be fused to an extracellular ligand binding domain. In some embodiments, pTalpha or a functional variant thereof is fused to a single-chain antibody fragment (scFv) comprising light and heavy variable fragments of a target antigen-specific monoclonal antibody connected by a flexible linker.

[0301] The term "T cell lacking TCR alpha" refers to an isolated T cell lacking the expression of a functional TCR alpha chain. This can be accomplished by different means, as a non-limiting example, by engineering the T cell so that it does not express any functional TCR alpha on the cell surface; or by engineering the T cell so that it produces very few functional TCR alpha chains on the surface; or by engineering the T cell to express a mutation or truncated form of the TCR alpha chain. Cells lacking TCR alpha can no longer be amplified via the CD3 complex. Therefore, in order to address this problem and allow cells lacking TCR alpha to proliferate, pT alpha or its functional variants are introduced into the cell, thereby repairing the functional CD3 complex. In some embodiments, the method further comprises introducing a rare cutting endonuclease into the T cell, the endonuclease being able to selectively inactivate a gene encoding a component of a T cell receptor (TCR) by DNA cleavage. In some embodiments, the rare cutting endonuclease is a TALE-nuclease.

[0302] In some embodiments, the polynucleotide encoding the polypeptide according to the present invention can be mRNA, which is directly introduced into the cell, for example, by electroporation. In some embodiments, the cytoPulse technology can be used to transiently infiltrate living cells to deliver substances into the cell. Parameters can be adjusted to determine conditions for high transfection efficiency and very low mortality.

[0303] Also provided herein is a method for transfecting T cells. In some embodiments, the method comprises: contacting the T cell with the RNA and applying an agile pulse sequence to the T cell, which consists of: (a) an electric pulse with a voltage ranging from about 2250 to 3000 V per centimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval between the electric pulses of steps (a) and (b) of about 0.2 to 10 ms; (d) an electric pulse with a voltage ranging from about 2250 to 3000 V, a pulse width of about 100 ms and a pulse interval between the electric pulse of step (b) and the first electric pulse of step (c) of about 100 ms; and (e) four electric pulses with a voltage of about 325 V, a pulse width of about 0.2 ms and a pulse interval of 2 ms between each of the four electric pulses. In some embodiments, the method of transfecting a T cell comprises contacting the T cell with an RNA and applying an agile pulse sequence to the T cell, comprising: (a) an electric pulse having a voltage of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900, or 3000 V per centimeter; (b) a pulse width of 0.1 ms; (c) and the pulse interval between the electric pulses of steps (a) and (b) is about 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1 (d) an electric pulse having a voltage range of about 2250, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000 V, with a pulse width of 100 ms and a pulse interval of 100 ms between the electric pulse of step (b) and the first electric pulse of step (c); and (e) 4 electric pulses having a voltage of about 325 V, with a pulse width of about 0.2 ms and a pulse interval of about 2 ms between each of the 4 electric pulses. Any value included in the numerical range described above is disclosed in the present application. The electroporation medium may be any suitable medium known in the art. In some embodiments, the conductivity of the electroporation medium is in the range of about 0.01 to about 1.0 millisiemens.

[0304] In some embodiments, as non-limiting examples, the RNA encodes a rare-cutting endonuclease, a monomer of a rare-cutting endonuclease (such as a half-TALE-nuclease), a CAR, at least one component of a multi-chain chimeric antigen receptor, pTα or a functional variant thereof, an exogenous nucleic acid and / or an additional catalytic domain.

[0305] Engineered immune cells

[0306] The present invention also provides engineered immune cells comprising any of the CAR polynucleotides described herein. In some embodiments, CAR can be introduced into immune cells as a transgene via a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selection marker, which provides identification and / or selection of cells receiving the vector.

[0307] After the polynucleotide encoding the CAR polypeptide is introduced into the cell, the CAR polypeptide can be synthesized in situ in the cell. Alternatively, the CAR polypeptide can be produced extracellularly and then introduced into the cell. The method of introducing a polynucleotide construct into a cell is known in the art. In some embodiments, a stable transformation method can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, a transient transformation method can be used to transiently express a polynucleotide construct and a polynucleotide construct that is not integrated into the genome of the cell. In other embodiments, a viral-mediated method can be used. Polynucleotides can be introduced into cells by any suitable means, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc. Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or particle bombardment. Polynucleotides may be included in a vector, such as a plasmid vector or a viral vector.

[0308] Also provided herein are isolated cells and cell lines obtained by the above-mentioned methods for engineering cells provided herein. In some embodiments, the isolated cells comprise at least one CAR as described above. In some embodiments, the isolated cells comprise a CAR population, each CAR comprising a different extracellular ligand binding domain.

[0309] Also provided herein is an isolated immune cell obtained according to any of the methods described above. Any immune cell capable of expressing heterogeneous DNA can be used for the purpose of expressing the relevant CAR. In some embodiments, the immune cell for expressing any of the CARs described herein is a T cell. In some embodiments, the immune cell for expressing CAR can be derived from, for example, (but not limited to) stem cells. The stem cell may be an adult stem cell, a non-human embryonic stem cell, more specifically, a non-human stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell. Representative human stem cells are CD34+ cells.

[0310] In some embodiments, the engineered immune cells expressing the FLT3-specific CAR of the present invention on its cell surface membrane include a percentage greater than 10%, 20%, 30%, 40%, 50% or 60% of stem cell memory and central memory cells. In some embodiments, the engineered immune cells expressing the FLT3-specific CAR of the present invention on its cell surface membrane include a percentage of about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 15% to about 50%, about 15% to about 40%, about 20% to about 60% or about 20% to about 70% of stem cell memory and central memory cells.

[0311] In some embodiments, the percentages of stem cell memory and central memory cells are measured as disclosed in Example 2d.

[0312] The immune cell for expressing any of the CARs described herein may also be a dendritic cell, a killer dendritic cell, a mast cell, a NK cell, a B cell, or a T cell selected from the group consisting of: an inflammatory T lymphocyte, a cytotoxic T lymphocyte, a regulatory T lymphocyte, or a helper T lymphocyte. In some embodiments, the cell may be derived from a group consisting of CD4+T lymphocytes and CD8+T lymphocytes.

[0313] In one embodiment, the immune cell is an inflammatory T lymphocyte expressing any one of the CARs described herein. In one embodiment, the immune cell is a cytotoxic T lymphocyte expressing any one of the CARs described herein. In one embodiment, the immune cell is a regulatory T lymphocyte expressing any one of the CARs described herein. In one embodiment, the immune cell is a helper T lymphocyte expressing any one of the CARs described herein.

[0314] Also provided herein is a cell line obtained by a transformed T cell according to any of the above methods. Also provided herein is a modified cell resistant to immunosuppressive therapy. In some embodiments, the isolated cell according to the present invention comprises a polynucleotide encoding a CAR.

[0315] The immune cells of the present invention can be activated and expanded before or after genetic modification of the T cells using methods generally described in, for example, but not limited to, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo. Typically, the T cells of the present invention can be expanded, for example, by contacting with agents that stimulate the CD3 TCR complex and co-stimulatory molecules on the surface of the T cells to form an activation signal for the T cells. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA) or mitogenic lectin-like phytohemagglutinin (PHA) can be used to form an activation signal for the T cells.

[0316] In some embodiments, the T cell population can be stimulated in vitro by contacting, for example, an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody fixed to a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ion carrier. For the co-stimulation of auxiliary molecules on the surface of T cells, a ligand that binds to auxiliary molecules is used. For example, under conditions suitable for stimulating T cell proliferation, the T cell population can contact anti-CD3 antibodies and anti-CD28 antibodies. Conditions suitable for T cell culture include suitable culture media (e.g., minimal essential medium or RPMI culture medium 1640 or X-vivo 5 (Lonza)) that may contain factors required for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGFp and TNF or any other additives known to skilled technicians for cell growth. Other additives for cell growth include, but are not limited to, surfactants, plasma proteins, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20, optimizers, and added amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or one or more cytokines in an amount sufficient for T cell growth and expansion. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and not in cell cultures to be infused into a subject. Target cells are maintained under conditions required to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to different stimulation times may exhibit different characteristics.

[0317] In some embodiments, the cells of the invention can be expanded by co-culturing with tissues or cells. After the cells are administered to a subject, the cells can also be expanded in vivo, for example in the blood of the subject.

[0318] In some embodiments, the isolated cells according to the present invention comprise an inactivated gene selected from the group consisting of: CD52, GR, PD1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRα and TCRβ; and / or express CAR, multi-chain CAR and / or pTα transgene. In some embodiments, the isolated cells include polynucleotides encoding polypeptides constituting multi-chain CARs. In some embodiments, the isolated cells according to the present invention comprise two inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TC Rβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ; and / or expressing CAR, multi-chain CAR and pTα transgene.

[0319] In some embodiments, by inactivating the TCRα gene and / or the TCRβ gene, the TCR is rendered non-functional in the cell according to the present invention. In some embodiments, a method for obtaining modified cells derived from an individual is provided, wherein the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independently of the MHC signaling pathway and are easily obtained by this method are included in the scope of the present invention. The modified cells disclosed herein can be used to treat individuals who need to fight host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD); therefore, within the scope of the present invention, there is a method for treating individuals who need to fight host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), which comprises treating the individual by administering an effective amount of modified cells to the individual, wherein the modified cells comprise inactivated TCRα and / or TCRβ genes.

[0320] In some embodiments, immune cells are engineered to be resistant to one or more chemotherapeutic drugs. The chemotherapeutic drug may be, for example, a purine nucleotide analog (PNA), so that immune cells are suitable for cancer treatment combined with adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, and cytarabine, which are in separate or combined forms. PNA is metabolized into single, di, and triphosphate PNAs by deoxycytidine kinase (dCK). Its triphosphate form competes with ATP for DNA synthesis, acts as a pro-apoptotic agent, and is an effective inhibitor of ribonucleotide reductase (RNR) involved in the manufacture of trinucleotides. Provided herein are FLT3-specific CAR-T cells comprising an inactivated dCK gene. In some embodiments, dCK knockout cells are obtained by transfecting T cells using polynucleotides encoding specific TAL-nucleases for dCK genes, such as by electroporation of mRNA. dCK knockout FLT3-specific CAR-T cells are resistant to PNAs, including, for example, clofarabine and / or fludarabine, and maintain T cell cytotoxic activity against cells expressing FLT3.

[0321] In some embodiments, the isolated cells or cell lines of the invention may comprise pTalpha or a functional variant thereof. In some embodiments, the isolated cells or cell lines may be further genetically modified by inactivating the TCRalpha gene.

[0322] In some embodiments, the CAR-T cell comprises a polynucleotide encoding a suicide polypeptide, such as RQR8. See, for example, WO2013153391A, which is incorporated herein by reference in its entirety. In the CAR-T cell comprising the polynucleotide, the suicide polypeptide is expressed at the surface of the CAR-T cell. In some embodiments, the suicide polypeptide comprises the amino acid sequence shown in SEQ ID NO: 227.

[0323] CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 227).

[0324] The suicide polypeptide may further comprise a signal peptide at the amino terminus. In some embodiments, the suicide polypeptide comprises the amino acid sequence shown in SEQ ID NO: 228.

[0325] MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV (SEQ ID NO: 228).

[0326] When the suicide polypeptide is expressed on the surface of the CAR-T cell, the binding of rituximab to the rituximab epitope of the polypeptide causes cell lysis. Each polypeptide expressed on the cell surface can bind more than one rituximab molecule. Each rituximab epitope of the polypeptide can bind to a separate rituximab molecule. The deletion of FLT3-specific CAR-T cells can be performed in vivo, for example, by administering rituximab to the subject. The decision to delete the metastatic cell may be caused by the undesirable effects attributable to the metastatic cell detected in the subject, such as when an unacceptable level of toxicity is detected.

[0327] In some embodiments, after administration to a patient, an engineered immune cell expressing any of the FLT3 specific CARs described herein at the cell surface can reduce, kill or lyse the patient's endogenous cells expressing FLT3. In one embodiment, the reduction or lysis percentage of the cell of the endogenous cell expressing FLT3 or the cell line expressing FLT3 realized by the engineered immune cell expressing any of the FLT3 specific CARs described herein is at least about or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In one embodiment, the reduction or lysis percentage of the endogenous cells expressing FLT3 or the cell line expressing FLT3 achieved by the engineered immune cells expressing any of the FLT3-specific CARs described herein is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75% or about 25% to about 60%. In one embodiment, the endogenous cells expressing FLT3 are endogenous bone marrow cells expressing FLT3.

[0328] In one embodiment, the percentage reduction or lysis of target cells, such as cell lines expressing FLT3, achieved by engineered immune cells expressing a FLT3-specific CAR of the present invention at the cell surface membrane can be measured using the assay disclosed in Example 2e.

[0329] Method for sorting CAR-positive immune cells

[0330] In one aspect, a method for in vitro sorting of an immune cell population is provided, wherein a subset of the immune cell population comprises an engineered immune cell expressing any one of a FLT3-specific CAR, wherein the FLT3-specific CAR comprises an epitope specific for a monoclonal antibody described herein. The method comprises contacting the immune cell population with a monoclonal antibody specific for the epitope, and selecting immune cells bound to the monoclonal antibody to obtain a cell population rich in engineered immune cells expressing a FLT3-specific CAR.

[0331] In some embodiments, the monoclonal antibody specific for the epitope is optionally bound to a fluorophore. In this embodiment, the step of selecting cells bound to the monoclonal antibody can be performed by fluorescence activated cell sorting (FACS). In some embodiments, the monoclonal antibody specific for the epitope is optionally bound to a magnetic particle. In this embodiment, the step of selecting cells bound to the monoclonal antibody can be performed by magnetic activated cell sorting (MACS).

[0332] In some embodiments, the extracellular binding domain of the CAR comprises a mAb-specific epitope of SEQ ID NO: 262. In some embodiments, the extracellular binding domain of the CAR comprises a mAb-specific epitope of SEQ ID NO: 262, and the antibody used to contact the immune cell population is QBEND-10.

[0333] In some embodiments, the extracellular binding domain of the CAR comprises a mAb-specific epitope of SEQ ID NO: 229. In some embodiments, the extracellular binding domain of the CAR comprises a mAb-specific epitope of SEQ ID NO: 229, and the antibody used to contact the immune cell population is rituximab.

[0334] In some embodiments, the population of immune cells expressing FLT3 CAR obtained when using the method described above for in vitro sorting of immune cells comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of immune cells expressing FLT3 CAR. In some embodiments, the population of immune cells expressing FLT3 CAR obtained when using the method described above for in vitro sorting of immune cells expressing CAR comprises at least 85% of immune cells expressing FLT3 CAR.

[0335] In some embodiments, as is routinely appreciated by those skilled in the art, the mAb used in the in vitro sorting method is previously bound to a load (such as a column) or beads. In some embodiments, the immune cell expressing CAR is a T cell.

[0336] According to the present invention, cells administered to a recipient can be enriched in vitro from a source population. Methods for amplifying a source population are well known in the art and may include selecting cells expressing an antigen (such as the CD34 antigen) using a combination of density centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence activated cell sorting, all of which are known to those skilled in the art.

[0337] Flow cytometry is a method commonly used in the art and well known to those of ordinary skill in the art to sort and quantify specific cell types in a cell population. In general, flow cytometry is a method for quantifying components or structural features of cells, primarily by optical means. Since different cell types can be distinguished by quantifying structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0338] Flow cytometric analysis involves two basic steps: 1) labeling selected cell types with one or more labeled markers, and 2) determining the number of labeled cells relative to the total number of cells in the population.

[0339] The main method of labeling cell types is by having labeled antibodies that bind to markers expressed by specific cell types. Antibodies are labeled directly with fluorescent compounds or indirectly using, for example, a fluorescently labeled secondary antibody that recognizes the primary antibody.

[0340] In some embodiments, the method used to sort CAR-expressing immune cells is magnetic activated cell sorting (MACS).

[0341] Magnetic activated cell sorting (MACS) is a method for separating various cell populations by using superparamagnetic nanoparticles and columns, depending on cell population surface antigens (CD molecules). It takes a few simple steps to obtain pure cell populations. Cells in a single cell suspension are magnetically labeled with microbeads. The sample is applied to a column consisting of ferromagnetic spheres, which is covered with a cell-friendly coating that allows rapid and gentle separation of cells. Unlabeled cells pass through while magnetically labeled cells remain in the column. The flow-through can be collected as an unlabeled cell fraction. After a short washing step, the column is removed from the separator and the magnetically labeled cells are eluted from the column.

[0342] In some embodiments, the mAb used in the method for sorting CAR-expressing immune cells is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, bentuximab, cetuximab, infliximab, rituximab, bevacizumab, becelizumab, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranbizumab, tosilimab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 or ustekinumab. In some embodiments, the mAb is rituximab. In another embodiment, the mAb is QBEND-10.

[0343] Therapeutic applications

[0344] The isolated cells obtained by the method described above or the cell lines derived from the isolated cells expressing FLT3-specific CAR can be used as medicaments. Similarly, the FLT 3-specific antibodies disclosed herein can be used as medicaments. In some embodiments, such agents can be used to treat FLT3-related diseases or conditions. In some embodiments, FLT3-related diseases or conditions comprise malignant cells expressing FLT3, such as cancer. In some embodiments, cancer is a hematopoietic cancer, such as lymphoma or leukemia. In some embodiments, cancer is multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte-dominant Hodgkin's lymphoma, Kahler's disease and myeloma, plasma cell leukemia, plasma cell tumor, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL) , chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, primary mediastinal (thymic) large B-cell lymphoma lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell associated with inflammation Lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma with features between diffuse large B-cell lymphoma and Burkitt's lymphoma, unclassified B-cell lymphoma with features between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma, or other hematopoietic cell-related cancers. In an exemplary embodiment, the cancer is AML. In an exemplary embodiment, the cancer is ALL.

[0345] In some embodiments, the isolated cells or cell lines or antibodies derived from the isolated cells according to the present invention can be used in the manufacture of a medicament for treating cancer in a subject in need thereof.

[0346] Also provided herein are methods for treating a subject. In some embodiments, the method comprises providing an immune cell of the present invention to a subject in need. In some embodiments, the method comprises administering a transformed immune cell of the present invention to a subject in need. In some embodiments, the method comprises administering an antibody of the present invention to a subject in need.

[0347] In some embodiments, T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.

[0348] The treatment methods of the present invention can be used to alleviate, cure or prevent. The method of the present invention can be part of an autologous immunotherapy or part of an allogeneic immunotherapy treatment. The present invention is particularly suitable for allogeneic immunotherapy. T cells from donors can be converted into non-alloreactive cells using standard protocols and reproduced on demand, thereby producing CAR-T cells that can be administered to one or several subjects. The CAR-T cell therapy can be obtained in the form of a "ready-made" therapeutic product.

[0349] Cells that can be used with the disclosed methods are described in the previous section. Treatment can be used to treat subjects diagnosed with, for example, cancer. Treatable cancers include, for example, but not limited to, cancers involving B lymphocytes, including any of the cancers listed above. The types of cancers to be treated with the CAR and CAR-T cells of the present invention include, but are not limited to, certain leukemias or lymphoid malignancies. Also included are adult tumors / cancers and pediatric tumors / cancers. In some embodiments, treatment can be combined with one or more anti-cancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiotherapy.

[0350] In certain embodiments, treatment may be given to a subject who has undergone immunosuppressive therapy. In fact, the present invention generally relies on a cell or cell group, which has been resistant to at least one immunosuppressant due to the gene inactivation of the receptor encoding the immunosuppressant. In this regard, immunosuppressive therapy should assist in selecting and amplifying T cells according to the present invention in a subject. The administration of cells or cell groups according to the present invention can be carried out in any suitable manner, including by aerosol inhalation, injection, injection, infusion, implantation or transplantation. Compositions described herein may be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection or intraperitoneally. In certain embodiments, the cell composition of the present invention is administered by intravenous injection.

[0351] In some embodiments, administering a cell or cell population may comprise administering, for example, about 10 4 to about 10 9In some embodiments, administering cells or cell populations may comprise administering, for example, about 10 cells per kilogram of body weight. 5 Up to 10 6 Cells, including all integer values ​​of the number of cells within those ranges. Cells or cell groups can be administered in one or more doses. In some embodiments, the effective amount of cells can be administered in a single dose. In some embodiments, the effective amount of cells can be administered in more than one dose over a period of time. The timing of administration is at the discretion of the managing physician and depends on the clinical condition of the subject. Cells or cell groups can be obtained from any source, such as a blood bank or a donor. Although individual needs may vary, the determination of the optimal range of effective amounts of a given cell type for a specific disease or condition is within the technical scope of the art. An effective amount means an amount that provides a therapeutic or preventive benefit. The dose administered will depend on the age, health status and weight of the recipient, the type of concurrent treatment (if any), the frequency of treatment, and the nature of the desired effect. In some embodiments, an effective amount of cells or a composition comprising the cells is administered parenterally. In some embodiments, administration may be intravenous administration. In some embodiments, administration may be performed directly by injection in a tumor.

[0352] In some embodiments of the invention, cells are administered to an individual in combination with (e.g., before, simultaneously with, or after) any number of related therapeutic modalities, including but not limited to treatment with agents such as monoclonal antibody therapy, CCR2 antagonists (e.g., INC-8761), antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment (for MS patients) or efalizumab treatment (for psoriasis patients), or other treatments for PML patients. In some embodiments, FLT3-specific CAR-T cells are administered to a subject in conjunction with one or more of the following: anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, or PF-06801591), anti-PD-L1 antibodies (e.g., averumab, atezolizumab, or durvalumab), anti-OX40 antibodies (e.g., PF-04518600), anti-4-1BB antibodies (e.g., PF-05082566), anti-MCSF antibodies (e.g., PD-0360324), anti-GITR antibodies, and / or anti-TIGIT antibodies. A FLT3-specific CAR comprising the amino acid sequence shown in SEQ ID NO: 236 is administered to a subject in conjunction with the anti-PD-L1 antibody averumab. In other embodiments, the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil) and FK506, antibodies or other immunoablative agents (such as CAMPATH), anti-CD3 antibodies or other antibody therapies, cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and / or radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is essential for growth factor-induced signal transduction (rapamycin) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In further embodiments, the T cells of the invention can be used in combination with receptor tyrosine kinase inhibitors (such as Midostaurin and Sunitinib), mTOR inhibitors (such as rapamycin and everolimus), epigenetic regulators (such as Vormostat), proteasome inhibitors (such as Bortezomib), immunomodulators (such as Lenalidomide), Hedgehog inhibitors (such as Erismodegib) and PF-04449913 or isocitrate dehydrogenase (IDH) inhibitors (such as AG-120 and AG-221).In another embodiment, the cell composition of the invention is administered to a subject in conjunction with (e.g., before, simultaneously with, or after) a bone marrow transplant, T cell ablation therapy using, for example, a chemotherapeutic agent (e.g., fludarabine, external beam radiation therapy (XRT), cyclophosphamide) or an antibody (e.g., OKT3 or CAMPATH), and in some embodiments, after B cell ablation therapy (e.g., an agent reactive with CD20, e.g., Rituxan). For example, in some embodiments, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after the transplant, the subject receives an infusion of the expanded immune cells of the invention. In some embodiments, the expanded cells are administered before or after surgery.

[0353] In some embodiments, a method is provided for depleting engineered immune cells expressing FLT3-specific CAR from an individual to whom the cells are administered. Depletion can be performed by inhibition or elimination.

[0354] In one aspect, a method of depleting an engineered immune cell expressing a FLT3-specific CAR comprising an epitope specific for a monoclonal antibody comprises contacting the engineered immune cell with a monoclonal antibody specific for the epitope.

[0355] In some embodiments, the method of exhaustion from a subject of an engineered immune cell expressing FLT3-specific CAR containing an epitope specific to a monoclonal antibody includes administering a monoclonal antibody specific to the epitope to the subject. In these embodiments, a monoclonal antibody specific to the epitope present in the extracellular domain of CAR is administered to the subject to eliminate or suppress the activity of the engineered immune cells expressing CAR from the subject. In one aspect, the exhaustion of engineered immune cells expressing CAR allows the endogenous population of cells expressing FLT3 to be restored.

[0356] In one aspect, the present invention relates to a method for promoting the recovery of endogenous cells expressing FLT3 in a subject given an engineered immune cell expressing FLT3-specific CAR on the cell surface, wherein the FLT3-specific CAR comprises an epitope specific to a monoclonal antibody, and the method comprises administering a monoclonal antibody specific to the epitope to the subject. In some embodiments, the endogenous cells expressing FLT3 are endogenous bone marrow cells expressing FLT3. In one aspect, the term "recovery" refers to increasing the number of endogenous cells expressing FLT3. The number of endogenous cells expressing FLT3 may be increased due to an increase in the proliferation of endogenous cells expressing FLT3 and / or due to the reduction of endogenous cells expressing FLT3 by the engineered immune cells expressing FLT3 CAR. In some embodiments, administering a monoclonal antibody to a subject depletes the engineered immune cells expressing FLT3 CAR in the subject and increases the number of endogenous cells expressing FLT3, such as endogenous bone marrow progenitor cells expressing FLT3. In one embodiment, administration of the monoclonal antibody to a subject increases the number of endogenous cells expressing FLT3 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% compared to the number of endogenous cells expressing FLT3 prior to administration of the monoclonal antibody.

[0357] In one aspect, a method of treating a FLT3-mediated disorder in a subject is provided, the method comprising: (a) administering to the subject engineered immune cells that express a FLT3-specific CAR at the cell surface that comprises one or more epitopes specific for one or more monoclonal antibodies; and (b) thereafter depleting the engineered immune cells from the subject by administering to the subject one or more monoclonal antibodies specific for the epitopes.

[0358] In some embodiments, the mAb used in the method for depleting engineered immune cells expressing a CAR is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab, cetuximab, infliximab, rituximab, bevacizumab, becelimumab, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tosilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10, ustekinumab, and a combination thereof.

[0359] In some embodiments, the epitope for which the monoclonal antibody is specific (mAb-specific epitope) is a CD20 epitope or mimotope, e.g., SEQ ID NO. 229, and the mAb specific for the epitope is rituximab.

[0360] In some embodiments, the step of administering a monoclonal antibody to a subject comprises administering a monoclonal antibody to the subject. In some embodiments, the amount of the epitope-specific mAb administered to the subject is sufficient to eliminate at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the immune cells expressing CAR in the subject.

[0361] In some embodiments, the step of administering a monoclonal antibody to a subject comprises infusing 375 mg / m 2 Rituximab.

[0362] In some embodiments, when immune cells expressing a CAR comprising a mAb-specific epitope (CAR-expressing immune cells) are depleted in a CDC assay using an epitope-specific mAb, the amount of live CAR-expressing immune cells decreases, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0363] In some embodiments, the cytotoxic drug is coupled to an epitope-specific mAb for depletion of CAR-expressing immune cells. By combining the targeting ability of monoclonal antibodies with the cell-killing ability of cytotoxic drugs, antibody-drug conjugates (ADCs) provide sensitive discrimination between healthy and diseased tissues when compared to the use of individual drugs. Several ADCs have been approved for marketing; their manufacturing techniques, especially utilizing linkers, are extensively presented in the prior art (Payne, G. (2003) Cancer Cell 3:207-212; Trail et al. (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278).

[0364] In some embodiments, epitope-specific mAbs are pre-conjugated with molecules that can promote complement-dependent cytotoxicity (CDC). Thus, the complement system aids or supplements the ability of antibodies to clear pathogens from an organism. When stimulated by one of several stimuli, an activation cascade is triggered due to the large-scale amplification of the response and the activation of the membrane attack complex of killer cells. Different molecules can be used to conjugate mAbs, such as glycans [Courtois, A, Gac-Breton, S., Berthou, C, Guezennec, J., Bordron, A. and Boisset, C. (2012), Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling, Electronic Journal of Biotechnology ISSN: 0717-3458; http: / / www.ejbiotechnology.info DOI: 10.2225 / voll5-issue5].

[0365] Reagent test kit

[0366] The present invention also provides kits for use in the methods of the present invention. The kits of the present invention include one or more containers that contain a polynucleotide encoding an FLT3-specific CAR as described herein, an engineered immune cell comprising a polynucleotide encoding an FLT3-specific CAR, or an FLT-specific antibody; and instructions for use according to any of the methods of the present invention described herein. Typically, these instructions include a description of administering the engineered immune cell for the above-described therapeutic treatment.

[0367] Instructions related to the use of the engineered immune cells or antibodies as described herein generally include information about the dosage, dosing schedule, and route of administration of the intended treatment. The containers can be unit dose, bulk (e.g., multi-dose packaging), or sub-unit dose. While the instructions supplied in the kits of the present invention are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), machine-readable instructions (e.g., instructions carried on a disk or CD-ROM storage disk) are also acceptable.

[0368] The kit of the present invention is in a suitable packaging form. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (e.g., sealed polyester film or plastic bags), etc. It also encompasses packaging for combination with a specific device (e.g., an inhaler, a nasal administration device (e.g., a nebulizer), or an infusion device (e.g., a small pump). The kit may have a sterile access hole (e.g., a container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access hole (e.g., a container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a FLT3-specific CAR or a FLT3-specific antibody. The container may further include a second pharmaceutically active agent.

[0369] The kit may optionally provide additional components (such as a buffer) and instructional information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0370] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In fact, various modifications of the present invention other than those shown and described herein will be apparent to those skilled in the art from the foregoing description, and the modifications are within the scope of the appended claims.

[0371] Representative materials of the present invention were deposited on June 1, 2017 at the American Type Culture Collection at 10801 University Boulevard, Manassas, Virginia, 20110-2209. Vector P3E10-VL with ATCC Accession No. PTA-124228 is a polynucleotide encoding the light chain variable region of P3E10, and vector P3E10-VH with ATCC Accession No. PTA-124227 is a polynucleotide encoding the heavy chain variable region of P3E10. The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder. This ensures that the deposited living culture will be maintained for 30 years from the date of deposit. The deposit will be made by ATCC under the terms of the Budapest Treaty and is subject to a contract between Pfizer, Inc. and ATCC which ensures that progeny of the deposited cultures will be available to the public in perpetuity and without restriction after the issuance of the associated U.S. patent or the public disclosure of any U.S. or foreign patent application, whichever occurs first, and that progeny will be available to such persons as the Commissioner of the United States Patent and Trademark may determine, including those entitled thereto, pursuant to 35 U.S.C. section 122 and regulations of the Commissioner thereunder (including 37 CFR section 1.14, specifically referring to 886 OG 638).

[0372] The assignee of this application has agreed that if a culture of the material in the deposit dies or is lost or destroyed when grown under suitable conditions, the material will be promptly replaced upon notice with another of the same. The availability of the deposited material should not be construed as a license to practice the invention in violation of the rights granted by any governmental authority under its patent laws.

[0373] Examples

[0374] Example 1: Determination of the kinetics and affinity of human FLT3 / FLT3 antibody interactions at 37°C

[0375] This example measures the kinetics and affinity of various anti-FLT3 antibodies at 37° C. All experiments were performed on a Biacore T200 surface plasmon resonance biosensor (GE Lifesciences, Piscataway NJ).

[0376] Sensor chip preparation was performed at 25°C with a running buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.4. Anti-human Fc sensor chips were obtained by activating all flow cells of a Biacore CM4 sensor chip with a 1:1 (v / v) mixture of 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 7 minutes. Anti-human Fc reagent (goat anti-human IgG Fc, Southern Biotech catalog #2081-01) was diluted to 30 μg / mL in 10 mM sodium acetate (pH 4.5) and injected into all flow cells at 20 μL / min for 7 minutes. All flow cells were blocked at 10 μL / min with 100 mM ethylenediamine in 150 mM borate buffer (pH 8.5) for 7 minutes.

[0377] Experiments were performed at 37°C with a running buffer of 10mM HEPES, 150mM NaCl, 0.05% (v / v) Tween-20, pH 7.4, 1mg / mL BSA. FLT3 antibodies were captured from undiluted supernatants at a flow rate of 10 μL / min downstream of flow cells (flow cells 2, 3, and 4) for 1 minute. Different antibodies were captured on each flow cell. Flow cell 1 was used as a reference surface. After capturing the FLT3 antibody, analytes (buffer, hFLT3) were injected on all flow cells at 30 μL / min for two minutes. After analyte injection, dissociation was monitored for 10 minutes followed by re-generation of all flow cells with three 1-minute injections of 75mM phosphoric acid. For each captured FLT3 antibody, the following analyte injections were performed: buffer, 11nM hFLT3, 33nM hFLT3, 100nM hFLT3, and 300nM hFLT3. Buffer cycles were collected for each captured FLT3 antibody for double referencing purposes (double referencing as described in Myszka, DG, Improving biosensor analysis., J. Mol. Recognit. 12, 279-284 (1999)). The double reference sensorgrams were overall fit to a simple 1:1 Langmuir with mass transport binding model.

[0378] The kinetic and affinity parameters for testing anti-FLT3 antibodies are shown in Table 7. The antibodies shown in Table 7 share the same VH and VL regions as the CARs shown in Table 8 with the same names.

[0379] Table 7

[0380]

[0381]

[0382] Example 2: Generation of FLT3-specific CAR-T cells

[0383] a) Plasmid

[0384] The following codon-optimized FLT3 CAR sequences listed in Table 8 below were synthesized and subcloned into a lentiviral vector such as pLVX EF1a-IRES-Puro (Clontech) using EcoRI (5') and MluI (3') restriction sites (thus removing the IRES-Puro cassette).

[0385] Table 8: Exemplary FLT3-specific CARs

[0386]

[0387]

[0388]

[0389]

[0390] In some experiments, the sequence encoding the CAR was preceded by a sequence encoding a fluorescent protein such as blue fluorescent protein (BFP) and separated from the sequence by a sequence encoding a self-cleaving 2A peptide. Lentivirus was generated using psPAX2, an HIV-1 gag-pol packaging plasmid, and pMD2.G, a VSV-G expression plasmid.

[0391] b) T cell activation and lentiviral transduction

[0392] Unaffected T cells were isolated from human peripheral blood mononuclear cells using a pan T cell isolation kit (Miltenyi Biotec) and activated for three days with anti-human CD2, CD3 and CD28 antibodies (T cell activation / expansion kit - Miltenyi Biotec). Lentiviral vectors (LV) were produced by transient transfection of subconfluent HEK-293T / 17 (American Type Culture Collection (ATCC)) cells in 6-well plates. Briefly, pLVX, psPAX2 and pMD2.G plasmids were transfected at a 4:3:1 ratio using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The next day, the culture medium was replaced with T cell culture medium (5% human AB serum in X-vivo-15 medium (Lonza)), and 48 hours after transfection, the LV supernatant was collected and filtered through a 0.45 μm syringe filter (Millipore). 0.25×10 6 Activated T cells were inoculated in T cell culture medium containing 40ng / ml IL-2 and transduced by adding the same volume of fresh LV supernatant. LVs containing the following CAR constructs were used in these experiments: P1A5, P1B4, P3A1, P3E10, P4C7, P4H11, P7H3, P9B5, P12B6, and P14G2. Cells were cultured for three days at 37°C and 5% CO2 and used for flow cytometry analysis or expanded in fresh T cell culture medium containing 20ng / ml IL-2.

[0393] c) Measurement of transduction efficiency by flow cytometry

[0394] To determine the transduction efficiency of different FLT3 CAR lentiviral constructs, flow cytometry analysis was performed on T cells 72 hours after transduction. Briefly, single cells were gated using forward scatter and side scatter parameters. In other cases, cells expressing CAR were detected by staining with anti-CD20 antibody rituximab followed by FITC-conjugated anti-human IgG, which binds to the CD20 epitope in the CAR molecule. Figure 1 It was shown that activated T cells were efficiently transduced with different FLT3 CAR constructs at varying efficiencies (30% to 70%).

[0395] d) FLT3FLT3 CAR-T cells differentiate during culture but maintain a high percentage of memory T cell subsets

[0396] The expression of activation markers CD25 and 4-1BB (CD137) on CAR-T cells during culture is associated with an increase in T cell exhaustion and differentiation to an effector memory phenotype (Long et al., 2015). In this experiment, T cells transduced with P1A5, P1B4, P3A1, P3E10, P4C7, P4H11, P7H3, P9B5, P12B6, and P14G2CAR were stained with CD25 and CD137 antibodies and analyzed by flow cytometry ten days after stimulation. The degree of T cell activation was determined by the percentage of CAR-T cells positive for CD25 and 4-1BB. T cell populations that were not transduced (NT) were included for comparison ( Figure 2 Expression of some FLT3 CARs (P1A5, P12B6, and P14G2) is associated with an “activated” state ( Figure 2 ), while expression of other FLT3 CARs including P9B5, P3E10, P4C7, and P3A1 produced amounts of activation markers comparable to those of untransduced cells ( Figure 2 ). Higher expression of activation markers translates into increased differentiation of CAR-T cells, which can be assessed by measuring the expression of CD62L and CD45RO on their cell surface by day 15 after stimulation. In this experiment, CAR-T cells were stained with the markers using specific antibodies, and the cells were classified into four different categories or subpopulations with increasing degrees of differentiation: 1) CD62L 高 D45RO 低 Stem cell memory (Tscm) cells; 2) CD62L 高 CD45RO 高 Central memory (Tcm) cells; CD62L 低 D45RO 高 Effector memory (Tem) cells; and CD62L 低 CD45RO 低 Effector (Teff) cells. Figure 3 Representative FACS plots showing P9B5 CAR-T cells (poorly differentiated) and P14G2 CAR-T cells (highly differentiated) and T cell subset compositions of all different FLT3 CAR-T cells at day 15 post-stimulation. By the end of culture, CAR-T cells exhibiting an "activated" phenotype also displayed a more differentiated phenotype (i.e., a higher percentage of Tem cells and a lower percentage of Tscm cells) ( Figure 3Specifically, expression of the P9B5, P3A1, P7H3, and P3E10FLT3 CARs was associated with a high percentage of stem cell memory and central memory cells, which correlated with increased persistence and anti-tumor efficacy in ALL and CLL subjects (Xu et al., Blood 2014; 123(24): 3750-3759).

[0397] e) In vitro proliferation and long-term killing activity assay

[0398] The ability of CAR-T cells to proliferate and lyse target cells expressing homologous antigens is an effective way to assess their in vitro activity. In order to monitor target cell viability by fluorescence, Molm13 cells expressing FLT3 are stably transduced to express the luciferase gene. These Luc+ target cells were then cultured with FLT3 CAR-T cells, and the killing activity and proliferation of CAR-T cells were measured by fluorescence and viable cell counting, respectively. For more stringent assays designed to emphasize effector cells (CAR-T cells), new target cells are regularly added to the initial culture, and the long-term killing activity of CAR-T cells is determined by fluorescence at the end of the culture ( Figure 4 ). Consistent with the higher proliferation capacity of stem cell memory and central memory T cells, P9B5, P3A1, P7H3, and P3E10 CAR-T cell populations showed higher target-dependent expansion and long-term in vitro cytotoxic activity, while CAR-T cells with a higher degree of differentiation, such as P12B6 and P14G2, expanded poorly in the presence of target cells and had reduced killing activity.

[0399] f) Activity of FLT3-specific CAR-T cells in orthotopic mouse assay

[0400] This example illustrates the use of Eol1 orthotopic model to treat AML with FLT3 CAR-T cells. In vivo efficacy studies of FLT3 CAR-T cells were performed in orthotopic models using Eol1 cells expressing luciferase and GFP. Three hundred thousand Eol1 Luc2AGFP cells were injected intravenously into 6-8 week-old female CB17 / SCID animals via the tail vein. D-luciferin (Morton Grove, IL, Regis Technologies) was injected intraperitoneally (15 mg / mL, 200 μL per animal), followed by anesthesia with isoflurane, and subsequent whole-body bioluminescence imaging (BLI) was used to monitor tumor burden. The bioluminescent signal emitted by the interaction between luciferase expressed by tumor cells and luciferin was captured by imaging using IVIS Spectrum CT (Perkin Elmer, MA) and quantified as total flux (photons / second) using LivingImage 4.4 (Caliper Life Sciences, Alameda, CA).

[0401] When the total flux reached an average of 30E6 for all animals (10th day after tumor implantation), the animals were randomly divided into three groups. One of the following cells was given to each group: 1) untransduced T cells were used as controls, 2) FLT3 CAR-T cells expressing P3E10 ("P3E10 V1") or 3) FLT3 CAR-T cells expressing P3A1 ("P3A1v1"). All 1-3 cells have TCRα. FLT3 CAR-T cells were prepared as described in the above examples. FLT3 CAR constructs P3E10 and P3A1 are shown in Table 8 above. A single dose of 2.5 or 5 million control (NT) or FLT3 CAR-T (P3E10 or P3A1) cells was given by rapid injection via the tail vein. When its total body weight loss exceeded 15% (an indicator of the Eol1 in situ model), the animal was killed.

[0402] The results of the study are summarized in Figure 5 Compared with negative controls, a single dose of 5 million P3E10 or P3A1 FLT3 CAR-T cells or 2.5 million P3E10 FLT3 CAR-T cells produced lower total flux from days 13-43 after tumor implantation. Therefore, treatment with FLT3 CAR-T cells inhibited tumor progression compared with negative controls.

[0403] These results demonstrate that FLT3 CAR-T cells effectively inhibit tumor progression.

[0404] Example 3: Depletion of FLT3-specific CAR T cells expressing the CD20 epitope

[0405] a) FLT3-specific CAR T cells expressing the CD20 epitope are sensitive to complement-dependent cytotoxicity (CDC) in the presence of rituximab

[0406] The sensitivity of FLT3-specific CAR T cells to complement in the presence of anti-CD20 antibodies was assessed in vitro using CDC assays. For this experiment, T cells transduced with lentiviral constructs to express FLT3 CARs containing CD20 epitopes in the extracellular domain were mixed with 25% complement (AbD serotec) and incubated at 37°C and 5% CO2 for 4 hours in the presence or absence of rituximab (100 μg / mL). The exhaustion of FLT3 CAR-T cells was determined by flow cytometry analysis using biotinylated FLT3 protein. Figure 6 We show that cells expressing FLT3CAR and CD20 epitopes were efficiently depleted in the presence of antibodies and complement, whereas control FLT3-specific CAR T cells that did not express the CD20 epitope were minimally affected.

[0407] These results demonstrate that FLT3-specific CAR-T cells expressing the CD20 epitope can be depleted in vitro in the presence of rituximab and complement.

[0408] b) Depletion of FLT3 CAR T cells after rituximab administration restores FLT3-expressing bone marrow progenitor cells in NSG mice

[0409] The ability of the anti-CD20 antibody rituximab to mediate depletion of FLT3-specific CAR T cells expressing the CD20 epitope and promote bone marrow recovery was tested in mice. In this experiment, mice were treated with T cells expressing a FLT3 CAR that binds to the mouse FLT3 protein on the surface of bone marrow progenitor cells and contains the CD20 epitope recognized by rituximab (α-mouse FLT3 CAR T) or T cells expressing a control CAR in which binding to the mouse FLT3 protein was negligible. Flow cytometric analysis of bone marrow cells was used to demonstrate CAR T cell cytotoxic activity against hematopoietic progenitor cells expressing FLT3, and a reduction in progenitor cells was seen compared to mice receiving control CAR T cells or untreated mice. ( Figure 7 , Panel B). After confirming the killing activity of CAR T cells, mice were given rituximab for four consecutive days, and circulating residual CAR T cells were counted by flow cytometry on day 13. Figure 7 Panel C shows that these mice had depletion of FLT3 CAR T cells in the blood compared to the control group that did not receive rituximab. Finally, flow cytometric analysis of bone marrow cells demonstrated that mice that received only rituximab (day 20) showed bone marrow recovery ( Figure 7 , Figure area D).

[0410] This experiment demonstrated that rituximab-dependent depletion of FLT3 CAR T cells expressing the CD20 epitope mitigated damage to FLT3-expressing tissues such as the bone marrow, allowing for rapid recovery of the bone marrow from residual progenitor cells.

[0411] Although the disclosed teachings about various applications, methods, kits and compositions have been described, it should be understood that various changes and modifications can be made without departing from the teachings herein and the inventions required below. The above examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. Although the present teachings have been described in terms of these exemplary embodiments, it will be readily understood by those skilled in the art that a large number of changes and modifications to these exemplary embodiments are possible without excessive experimentation. All such changes and modifications are within the scope of the existing teachings.

[0412] All references cited herein (including patents, patent applications, papers, texts, etc., and references cited therein (to the extent they are not incorporated)) are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials (including but not limited to defined terms, term usage, described techniques, etc.) differs from or conflicts with this application, this application shall prevail.

[0413] The foregoing description and examples detail certain specific embodiments of the present invention and describe the best mode contemplated by the inventor. However, it should be understood that no matter how detailed the foregoing is in text, the present invention can be practiced in many ways, and the present invention should be interpreted according to the appended claims and any equivalents thereof.

Claims

1. A Fms-like tyrosine kinase 3 (FLT3)-specific chimeric antigen receptor (CAR), comprising an extracellular ligand binding domain, a first transmembrane domain, and an intracellular signaling domain, wherein the extracellular ligand binding domain comprises a single-chain variable fragment (scFv), which comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein (a) the VH region comprises: a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 49, 44 or 50; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 51 or 52 and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 53; and The VL region comprises: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 150; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 151; and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 152; or (b) the VH region comprises (i) a VH complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 43, 44 or 45; (ii) a VH complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 46 or 47, and (iii) a VH complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 48; and The VL region comprises (i) a VL complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 147; (ii) a VL complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 148 and (iii) a VL complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 149; or (c) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 84, 85 or 86; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 87 or 88 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 89; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 168; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 169 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 170; or (d) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 60, 61 or 62; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 63 or 64 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 65; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 156; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 157 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 158; or (e) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 72, 73 or 74; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 75 or 76 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 77; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 162; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 163 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 164; or (f) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 78, 79 or 80; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 81 or 82 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 83; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 165; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 166 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 167; or (g) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 96, 97 or 98; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 99 or 100 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 101; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 174; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 175 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 176; or (h) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 102, 103 or 104; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 105 or 106 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 107; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 177; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 178 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 179; or (i) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 114, 115 or 116; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 117 or 118 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 119; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 183; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 184 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 185; or (j) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 120, 121 or 122; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 123 or 124 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 125; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 186; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 187 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 188; or (k) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 126, 127 or 128; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 129 or 130 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 131; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 189; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 190 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 191; or (l) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 132, 133 or 134; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 135 or 136 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 137; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 192; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 193 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 194; or (m) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 138, 139 or 140; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 141 or 142 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 143; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 195; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 196 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 197; or (n) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 294, 295 or 296; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 297 or 298 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 299; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 323; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 324 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 325; or (o) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 300, 295 or 301; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 302 or 303 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 304; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 326; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 324 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 327; or (p) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 305, 306 or 307; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 308 or 309 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 310; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 328; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 329 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 330; or (q) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 311, 295 or 312; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 313 or 314 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 315; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 331; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 332 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 333; or (r) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 319 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 331; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 334 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 335; or (s) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 319 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 336; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 332 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 337; or (t) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 319 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 338; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 332 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 339; or (u) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 319 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 340; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 341 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 342; or (v) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 319 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 343; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 341 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 344; or (w) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 319 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 345; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 346 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 347; or (x) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 319 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 348; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 341 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 349; or (y) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 322 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 331; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 334 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 335; or (z) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 316, 317 or 318; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 322 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 350; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 332 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 339; or (aa) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 138, 317 or 140; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 322 or 320 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 321; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 350; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 332 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 339; or (bb) the VH region comprises: a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 108, 109 or 110; a VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 111 or 112; and a VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 113; and The VL region comprises: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 180; a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 181; and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 182; or (cc) the VH region comprises (i) a VH complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 37, 38 or 39; (ii) a VH complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 40 or 41 and (iii) a VH complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 42; and The VL region comprises (i) a VL complementarity determining region 1 (CDR1) having the amino acid sequence shown in SEQ ID NO: 144; (ii) a VL complementarity determining region 2 (CDR2) having the amino acid sequence shown in SEQ ID NO: 145 and (iii) a VL complementarity determining region 3 (CDR3) having the amino acid sequence shown in SEQ ID NO: 146; or (dd) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 54 or 55 or 56; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 57 or 58 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 59; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 153; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 154 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 155; or (ee) the VH region comprises (i) a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 66, 67 or 68; (ii) a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 69 or 70 and (iii) a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 71; and The VL region comprises (i) a VL CDR1 having the amino acid sequence shown in SEQ ID NO: 159; (ii) a VL CDR2 having the amino acid sequence shown in SEQ ID NO: 160 and (iii) a VL CDR3 having the amino acid sequence shown in SEQ ID NO:

161.

2. The FLT3-specific CAR according to claim 1, wherein the single-chain variable fragment (ScFv) comprises: a heavy chain variable (VH) region having the sequence shown in SEQ ID NO:20 and a light chain variable (VL) region having the sequence shown in SEQ ID NO:19; (a) the VH region comprises the sequence shown in SEQ ID NO:6 and the VL region comprises the sequence shown in SEQ ID NO:5; or (b) the VH region comprises the sequence shown in SEQ ID NO:4 and the VL region comprises the sequence shown in SEQ ID NO:3; or (c) the VH region comprises the sequence shown in SEQ ID NO: 26 and the VL region comprises the sequence shown in SEQ ID NO: 25; or (d) the VH region comprises the sequence shown in SEQ ID NO: 18 and the VL region comprises the sequence shown in SEQ ID NO: 17; or (e) the VH region comprises the sequence shown in SEQ ID NO: 10 and the VL region comprises the sequence shown in SEQ ID NO: 9; or (f) the VH region comprises the sequence shown in SEQ ID NO: 16 and the VL region comprises the sequence shown in SEQ ID NO: 15; or (g) the VH region comprises the sequence shown in SEQ ID NO: 22 and the VL region comprises the sequence shown in SEQ ID NO: 21; or (h) the VH region comprises the sequence shown in SEQ ID NO: 24 and the VL region comprises the sequence shown in SEQ ID NO: 23; or (i) the VH region comprises the sequence shown in SEQ ID NO: 26 and the VL region comprises the sequence shown in SEQ ID NO: 25; or (j) the VH region comprises the sequence shown in SEQ ID NO: 28 and the VL region comprises the sequence shown in SEQ ID NO: 27; or (k) the VH region comprises the sequence shown in SEQ ID NO:30 and the VL region comprises the sequence shown in SEQ ID NO:29; or (1) the VH region comprises the sequence shown in SEQ ID NO: 32 and the VL region comprises the sequence shown in SEQ ID NO: 31; or (m) the VH region comprises the sequence shown in SEQ ID NO: 34 and the VL region comprises the sequence shown in SEQ ID NO: 33; or (n) the VH region comprises the sequence shown in SEQ ID NO:36 and the VL region comprises the sequence shown in SEQ ID NO:35; or (o) the VH region comprises the sequence shown in SEQ ID NO: 265 and the VL region comprises the sequence shown in SEQ ID NO: 264; or (p) the VH region comprises the sequence shown in SEQ ID NO: 267 and the VL region comprises the sequence shown in SEQ ID NO: 266; or (q) the VH region comprises the sequence shown in SEQ ID NO: 269 and the VL region comprises the sequence shown in SEQ ID NO: 268; or (r) the VH region comprises the sequence shown in SEQ ID NO: 271 and the VL region comprises the sequence shown in SEQ ID NO: 270; or (s) the VH region comprises the sequence shown in SEQ ID NO: 273 and the VL region comprises the sequence shown in SEQ ID NO: 272; or (t) the VH region comprises the sequence shown in SEQ ID NO: 275 and the VL region comprises the sequence shown in SEQ ID NO: 274; or (u) the VH region comprises the sequence shown in SEQ ID NO: 277 and the VL region comprises the sequence shown in SEQ ID NO: 276; or (v) the VH region comprises the sequence shown in SEQ ID NO: 279 and the VL region comprises the sequence shown in SEQ ID NO: 278; or (w) the VH region comprises the sequence shown in SEQ ID NO: 281 and the VL region comprises the sequence shown in SEQ ID NO: 280; or (x) the VH region comprises the sequence shown in SEQ ID NO: 283 and the VL region comprises the sequence shown in SEQ ID NO: 282; or (y) the VH region comprises the sequence shown in SEQ ID NO: 285 and the VL region comprises the sequence shown in SEQ ID NO: 284; or (z) the VH region comprises the sequence shown in SEQ ID NO: 287 and the VL region comprises the sequence shown in SEQ ID NO: 286; or (aa) the VH region comprises the sequence shown in SEQ ID NO: 289 and the VL region comprises the sequence shown in SEQ ID NO: 288; or (bb) the VH region comprises the sequence shown in SEQ ID NO: 291 and the VL region comprises the sequence shown in SEQ ID NO: 290; or (cc) the VH region comprises the sequence shown in SEQ ID NO: 293 and the VL region comprises the sequence shown in SEQ ID NO: 292; or (dd) the VH region comprises the sequence shown in SEQ ID NO:2 and the VL region comprises the sequence shown in SEQ ID NO:1; or (ee) the VH region comprises the sequence shown in SEQ ID NO:8 and the VL region comprises the sequence shown in SEQ ID NO:7; or (ff) the VH region comprises the sequence shown in SEQ ID NO: 12 and the VL region comprises the sequence shown in SEQ ID NO: 11; or (gg) the VH region comprises the sequence shown in SEQ ID NO:14 and the VL region comprises SEQ ID NO:

13.

3. A FLT3-specific CAR according to any one of claims 1 to 2, wherein the extracellular ligand binding domain comprises one or more epitopes specific for a monoclonal antibody.

4. A polynucleotide comprising a nucleic acid sequence encoding the FLT3-specific CAR according to any one of claims 1 to 2. An expression vector comprising the polynucleotide according to claim 4 .

6. An engineered immune cell expressing the FLT3-specific CAR according to any one of claims 1 to 2.

7. A method for engineering immune cells expressing FLT3-specific CAR, comprising: a. Provide immune cells; and b. introducing into the cell at least one polynucleotide encoding a FLT3-specific CAR according to any one of claims 1 to 2; Thereby, the immune cells express the FLT3-specific CAR.

8. A pharmaceutical composition comprising the engineered immune cell according to claim 5.

9. Use of an effective amount of the pharmaceutical composition according to claim 8 or the engineered immune cell according to claim 6 in the preparation of a medicament for treating acute myeloid leukemia (AML) in a subject in need thereof.

10. A method for in vitro sorting of an immune cell population, wherein the subset of the immune cell population Comprising an engineered immune cell expressing a FLT3-specific chimeric antigen receptor (CAR) according to claim 3, the method comprising: a. contacting the immune cell population with a monoclonal antibody specific for the epitope; and b. Selecting the immune cells that bind to the monoclonal antibody to obtain a cell population enriched in engineered immune cells.

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