Dual CAR-T cells

Abstract

The present invention relates to novel engineered immune cells that express two CARs directed to two different targets, polynucleotides for preparing the immune cells, pharmaceutical compositions comprising the immune cells, and the use of the immune cells in the treatment of cancer.

Description

[Technical Field]

[0001] Field The present invention relates to the field of cellular immunotherapy, and more particularly to novel engineered immune cells that express two CARs directed against two different targets, which are useful in the treatment of cancer. [Background technology]

[0002] background Approximately one person is diagnosed with blood cancer every three minutes in the United States (US). A total of an estimated 178,520 people will be diagnosed with leukemia, lymphoma, or myeloma in the US in 2020. New cases of leukemia, lymphoma, and myeloma are expected to account for 9.9 percent of the estimated 1,806,590 new cases of cancer diagnosed in the US in 2020 ("Cancer Facts & Figures", 2020, American Cancer Society).

[0003] The development of chimeric antigen receptor (CAR) T-cell therapy for hematological malignancies has been one of the most impressive therapeutic advances in the past decade (Holstein et al., 2020, Clin. Pharmacol. Ther. 107(1):112-122). Indeed, with the rapid development of the field of oncology, adoptive transfer of CAR-T cells has shown remarkable efficacy in the management of hematological malignancies, and several clinical trials have been reported.

[0004] Chimeric antigen receptor ("CAR")-expressing immune cells are cells that have been genetically engineered to express CARs, which are typically designed to recognize specific tumor antigens and kill cancer cells that express those tumor antigens. It is also possible that CAR immune cells may activate the immune system to eliminate tumors. These are generally CAR-expressing T cells ("CAR-T cells"), CAR-expressing natural killer cells ("CAR-NK cells"), or CAR-expressing macrophages.

[0005] CAR is a synthetic receptor consisting of a targeting moiety linked to one or more signaling domains in a fusion molecule. Generally, the binding moiety of CAR is composed of an antigen-binding domain derived from a monoclonal antibody, and the antigen-binding domain is composed of a single-chain variable fragment (scFv), which comprises the light chain variable fragment and heavy chain variable fragment of a monoclonal antibody linked by a flexible linker. The use of binding moieties based on receptor domains or ligand domains has also been successful. The signaling domain of first-generation CARs is derived from the cytoplasmic region of CD3 zeta chain or Fc receptor gamma chain. First-generation CARs have been shown to be successful in redirecting the cytotoxicity of T cells, but this has not been able to bring about long-term proliferation and anti-tumor activity in vivo. To enhance the survival and proliferation of CAR-modified T cells, signaling domains derived from costimulatory molecules, including CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137), have been added, either alone (second generation) or in combination (third generation). CARs have been successful in redirecting T cells to antigens expressed on the surface of tumor cells in a variety of malignancies, including lymphomas and solid tumors (Jena, Dotti et al., 2010, Blood 116(7):1035-1044).

[0006] Adoptive immunotherapy, which involves the transfer of ex vivo generated autologous or allogeneic antigen-specific T cells, is a promising strategy for treating viral infections and cancer, as evidenced by the increasing number of CAR-T cell types approved by the U.S. Food and Drug Administration (FDA) (e.g., Novartis' anti-CD19 CAR-T, tisagenlecleucel (Kymriah™), for the treatment of precursor B-cell acute lymphoblastic leukemia and Kite Pharma's anti-CD19 CAR-T, axicabtageneciloreucel (Yescarta™), for the treatment of several types of large B-cell lymphoma in adult patients).

[0007] Although progress has been made in the research and development of CAR-T cell therapy, there remains a need for improved CAR-T cells that can target a wide range of cancers and that can target recurrent cancers and / or that can target cancers in which the expression of cancer-associated antigens is highly variable and changes over time or during or after treatment. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Holstein et al., 2020, Clin. Pharmacol. Ther. 107(1):112-122 [Non-patent document 2] Jena, Dotti et al., 2010, Blood 116(7):1035-1044 Summary of the Invention

[0009] overview The inventors have developed novel CAR-T cells that target the CD20 and CD22 antigens, which can be activated by tumor cells that express altered levels of CD20 and CD22, and which provide improvements in CAR-T cells over the prior art.

[0010] The first aspect relates to a genetically engineered immune cell that expresses on its cell surface a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20), wherein: a) CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; and b) CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; Includes:

[0011] In one particular aspect, the genetically engineered immune cells are TCR-negative T cells.

[0012] Another aspect relates to a pharmaceutical composition comprising the engineered immune cells, or a cell population comprising the engineered immune cells, and a pharmaceutically acceptable excipient.

[0013] Yet another aspect relates to an isolated polynucleotide comprising: a) a nucleic acid encoding CAR22, wherein CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; a nucleic acid encoding CAR22, comprising: b) a nucleic acid encoding CAR20, wherein CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; A nucleic acid encoding CAR20, comprising:

[0014] Other aspects relate to vectors containing the polynucleotides, and also to host cells containing the polynucleotides or vectors.

[0015] Yet a further aspect relates to a method of preparing engineered immune cells.

[0016] Other aspects relate to engineered immune cells or cell populations for use as pharmaceuticals.

[0017] Another aspect utilizes engineered immune cells for use in treating cancer or inflammatory disorders, particularly cancers or inflammatory disorders associated with expression of CD20 and / or CD22. [The present invention 1001] A genetically engineered immune cell that expresses a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20) on its cell surface, a) CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 11 and comprising the H-CDRs of the amino acid sequences SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a variable light chain (VL) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 12 and comprising the L-CDRs of the amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, and optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; and b) CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 15 and comprising the H-CDRs of the amino acid sequences SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, and a variable light chain (VL) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 16 and comprising the L-CDRs of the amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, and optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; Including, Genetically engineered immune cells. [The present invention 1002] 1001. The genetically engineered immune cell of the present invention, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14 and CAR20 comprises the amino acid sequence of SEQ ID NO: 18. [The present invention 1003] The genetically engineered immune cell of the present invention 1001 or 1002, wherein the extracellular domain of neither CAR20 nor CAR22 comprises a safety switch comprising a rituximab-specific mimotope, such as SEQ ID NO: 22. [The present invention 1004] The CAR is encoded by an exogenous nucleic acid integrated into the genome of the immune cell, and the exogenous nucleic acid is arranged in a 5' to 3' direction as follows: (i) a promoter (such as the EF1α promoter), (ii) a nucleic acid encoding CAR20; (iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19); (iv) a nucleic acid encoding CAR22 Including, This allows the expression of CAR20 and CAR22 to be controlled by the same promoter. A genetically engineered immune cell according to any one of 1001 to 1003 of the present invention. [The present invention 1005] (ii) the nucleic acid comprises a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; and (iv) the nucleic acid comprises a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; The genetically engineered immune cells of the present invention 1004. [The present invention 1006] The genetically engineered immune cell of any one of claims 1004 to 1005, wherein the exogenous nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 32. [The present invention 1007] The genetically engineered immune cell of any of claims 1001 to 1006, which is selected from the group consisting of a T cell, a NK cell, and a macrophage. [The present invention 1008] The genetically engineered immune cell of any of claims 1001 to 1007, which is a T cell, for example a cytotoxic T cell. [The present invention 1009] A genetically engineered immune cell of any of claims 1001 to 1008, which is contained within a cell population, for example, within an immune cell population, in particular, within a T cell population, an NK cell population, and / or a macrophage population. [The present invention 1010] The genetically engineered immune cell of any of 1001 to 1009 of the present invention, which is a TCR-negative T cell. [The present invention 1011] The engineered T cells short hairpin RNA (shRNA) or small interfering RNA (siRNA) directed against polynucleotide sequences encoding components of the TCR 10. The genetically engineered immune cell of the present invention, wherein the genetically engineered immune cell expresses [The present invention 1012] 10. The genetically engineered immune cell of the present invention, wherein the engineered T cell is mutated in its TCR alpha allele and / or TCR beta allele. [The present invention 1013] The engineered T cells At least one allele encoding TCRα, TCRβ, and / or CD3 that is inactivated by a mutation 10 or 10 genetically engineered immune cells of the present invention, [The present invention 1014] The engineered T cells At least one allele selected from β2m, PD1, CTLA4, dCK, CD52, and / or GR that is inactivated Any of 1008 to 1013 genetically engineered immune cells of the present invention, comprising: [The present invention 1015] 15. The genetically engineered immune cell of any of claims 1009 to 1014, wherein the engineered T cell has an inactivated CD52 allele. [The present invention 1016] The genetically engineered immune cell of any of claims 1001 to 1015, wherein the engineered cell, preferably a T cell, is a primary cell. [The present invention 1017] 17. The genetically engineered immune cell of any of claims 1001 to 1016, wherein the engineered cell, preferably a T cell, is a mammalian cell, preferably a human cell. [The present invention 1018] 10. The genetically engineered immune cell of any of claims 1001 to 1017, wherein the engineered cell, preferably a T cell, does not express any additional CARs other than CAR22 and CAR20. [The present invention 1019] An immune cell population comprising the engineered immune cell of any one of 1001 to 1018 of the present invention. [The present invention 1020] A T cell population comprising any one of the engineered T cells of the present invention 1009 to 1018. [The present invention 1021] A pharmaceutical composition comprising any one of the engineered immune cells of the present inventions 1001 to 1018 and a pharmaceutically acceptable excipient. [The present invention 1022] A pharmaceutical composition comprising the immune cell population of the present invention 1019 or 1020 and a pharmaceutically acceptable excipient. [The present invention 1023] A pharmaceutical composition comprising any one of the engineered T cells of the present invention and a pharmaceutically acceptable excipient. [The present invention 1024] A pharmaceutical composition comprising a T cell population of the present invention and a pharmaceutically acceptable excipient. [The present invention 1025] a) a nucleic acid encoding CAR22, wherein CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 11 and comprising the H-CDRs of the amino acid sequences SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a variable light chain (VL) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 12 and comprising the L-CDRs of the amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, and optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; a nucleic acid encoding CAR22, comprising: b) a nucleic acid encoding CAR20, wherein CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 15 and comprising the H-CDRs of the amino acid sequences SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, and a variable light chain (VL) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 16 and comprising the L-CDRs of the amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, and optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; A nucleic acid encoding CAR20, comprising An isolated polynucleotide comprising: [The present invention 1026] A polynucleotide of the present invention 1025, wherein the nucleic acid of a) and the nucleic acid of b) are on a single nucleic acid molecule, and a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, T2A, E2A, or F2A) is located between the nucleic acid of a) and the nucleic acid of b). [The present invention 1027] The polynucleotide of invention 1025 or 1026, which does not include a nucleic acid encoding an additional CAR other than CAR22 and CAR20. [The present invention 1028] Any of the polynucleotides of 1025 to 1027 of the present invention, which does not include a nucleic acid encoding a rituximab-specific mimotope of SEQ ID NO: 22. [The present invention 1029] the nucleic acid of a) comprises a promoter (e.g., an EF1α promoter) that controls the expression of CAR22, wherein CAR22 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; b) the nucleic acid encodes CAR20, wherein CAR20 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; and a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19) is present between the nucleic acid of a) and the nucleic acid of b) to allow for simultaneous expression of CAR20 and CAR22; A polynucleotide of any one of 1025 to 1028 of the present invention. [The present invention 1030] b) the nucleic acid comprises a promoter (such as an EF1α promoter) that controls the expression of CAR20, and CAR20 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; the nucleic acid of a) encodes CAR22, wherein CAR22 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; and a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19) is present between the nucleic acid of b) and the nucleic acid of a) to allow for simultaneous expression of CAR22 and CAR20; A polynucleotide of any one of 1025 to 1028 of the present invention. [The present invention 1031] 1029. An isolated polynucleotide of the present invention comprising the nucleic acid sequence of SEQ ID NO: 31 (CAR22xCAR20 construct). [The present invention 1032] 1030. An isolated polynucleotide of the present invention comprising the nucleic acid sequence of SEQ ID NO: 32 (CAR20xCAR22 construct). [The present invention 1033] A vector comprising any one of the isolated polynucleotides of the present inventions 1025 to 1032. [The present invention 1034] A host cell comprising a vector of the present invention. [This invention 1035] A method for preparing an engineered immune cell of any one of claims 1001 to 1018, comprising the step of introducing a polynucleotide of any one of claims 1025 to 1032 or a vector of claim 1033 into an immune cell. [The present invention 1036] 1018. The engineered immune cell of any of claims 1001 to 1018 for use as a pharmaceutical. [This invention 1037] 19. The engineered immune cell of any of claims 1001 to 1018 for use in treating cancer associated with expression of CD20 and / or CD22. [The present invention 1038] The engineered immune cell of any of claims 1001 to 1018 for use in treating a blood cancer, particularly a CD22- and / or CD20-related blood cancer, more particularly a relapsed or refractory CD22- and / or CD20-related blood cancer, even more particularly an aggressive type of CD22- and / or CD20-related blood cancer. [This invention 1039] The engineered immune cells for use in the present invention 1037 or 1038, wherein the cancer is selected from the group consisting of lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphoid carcinoma, acute myeloid leukemia (AML). [The present invention 1040] The engineered immune cells for use in the present invention 1039, wherein the cancer is non-Hodgkin's lymphoma or acute lymphocytic leukemia. [The present invention 1041] The engineered immune cell for use in any of claims 1037 to 1040, wherein the cancer is associated with low expression of CD20 and / or CD22. [The present invention 1042] The engineered immune cell for use in any of claims 1037 to 1041, wherein the cancer is recurrent non-Hodgkin's lymphoma. [This invention 1043] A method for treating a patient suffering from a cancer associated with expression of CD20 and / or CD22, comprising administering to the patient an effective amount of the engineered immune cell of any of claims 1001 to 1018 or the immune cell population of claim 1019 or 1020. [This invention 1044] The method of treatment of the present invention 1043, wherein the cancer is selected from the group consisting of lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphoid carcinoma, acute myeloid leukemia (AML). [This invention 1045] The method of treatment of any one of claims 1043 to 1044, wherein the cancer is non-Hodgkin's lymphoma or acute lymphocytic leukemia. [The present invention 1046] 1046. The method of any of claims 1043 to 1045, wherein the cancer is associated with low expression of CD20 and / or CD22. [This invention 1047] The treatment method according to any one of claims 1043 to 1046, wherein the cancer is recurrent non-Hodgkin's lymphoma. [Brief explanation of the drawings]

[0018] [Figure 1] Detection of CD20xCD22 CAR or CD22xCD20 CAR. Flow cytometry analysis showing detection of CAR CD20 or CAR CD22 in non-transduced T cells (NT) or in T cells transduced with a CD22 CAR construct, or a CD22xCD20 CAR construct, or a CD20xCD22 CAR construct, using the protocol described in the Examples. [Figure 2] Flow cytometry analysis of various Raji cells obtained after TALEN® treatment and cell sorting for CD20 and CD22. [Figure 3] Lysis percentage of CD20xCD22 CAR T cells, CD22xCD20 CAR T cells, and CD22 CAR T cells from two different donors against Raji cells that express or do not express CD20 and / or CD22 antigens. [Figure 4] Percentage of lysis over time of CD20xCD22 CAR T cells, CD22xCD20 CAR T cells, and CD22 CAR T cells against either (A) Raji cells expressing CD20 and CD22, (B) Raji cells expressing CD22 only, or (C) Raji cells expressing CD20 only. [Figure 5] Dose-dependent control of in vivo tumor burden upon treatment with the indicated doses of CD20xCD22 CAR T cells, CD22xCD20 CAR T cells, and CD22 CAR T cells, as measured by bioluminescence. [Figure 6] Survival curves for animals treated with the indicated doses of CD20xCD22 CAR T cells, CD22xCD20 CAR T cells, and CD22 CAR T cells in a disseminated model of B-cell lymphoma. [Figure 7] Survival curves of animals treated with the indicated doses of CD20xCD22 CAR T cells, CD22xCD20 CAR T cells, and CD22 CAR T cells in a subcutaneous model of B-cell lymphoma. [Figure 8] IFNγ release by CD20xCD22 CAR T cells, CD22xCD20 CAR T cells, CD20 CAR T cells, and CD22 CAR T cells with respect to Daudi cells.

[0019] As used throughout the specification and in the examples, relevant terms are as follows: anti-CD20 CAR = CAR20 = CD20CAR, anti-CD22 CAR = CAR22 = CD22CAR, Anti-CD20 CAR / anti-CD22 CAR = CAR20x22 or CAR22x20 = CD20xCD22 CAR or CD22xCD20 CAR. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art of gene therapy, biochemistry, genetics, and molecular biology.

[0021] In carrying out or testing the present invention, any methods and materials similar or equivalent to those described herein can be used, and suitable methods and materials are described herein.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.Furthermore, the materials, methods, and examples are merely illustrative and, unless otherwise specified, are not intended to be limiting.

[0022] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of those in the art, and such techniques are fully explained in the literature. See, for example, "Current Protocols in Molecular Biology" (Frederick M. AUSUBEL, 2000, Wiley and Sons Inc, Library of Congress, USA); "Molecular Cloning: A Laboratory Manual" 3rd Edition (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); Mullis et al., U.S. Pat. No. 4,683,195; "Nucleic Acid Hybridization" (B.D. Harries and S.J. Higgins, eds., 1984); "Transcription And Translation" (B.D. Hames and S.J. Higgins, eds., 1984); "Culture Of Animal Cells" (R.I. Freshney, Alan R. Liss, Inc., 1987); "Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular "Cloning" (1984); the serial publication "Methods In Enzymology" (first editors J. Abelson and M. Simon, Academic Press, Inc., New York), especially volumes 154 and 155 (Wu et al., eds.) and volume 185 "Gene Expression Technology" (D. Goeddel, ed.); "Gene Transfer Vectors For Mammalian Cells" (J.H. Miller and MPCalos, 1987, Cold Spring Harbor Laboratory); "Immunochemical Methods In Cell And Molecular Biology" (Mayer and Walker, eds., Academic Press, London, 1987); "Handbook Of Experimental Immunology" Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and "Manipulating the Mouse Embryo" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0023] definition As used herein, "pathological condition-associated antigen" refers to an antigen that is present or overexpressed in a given disease.When the antigen is CD20 or CD22, "CD20-associated disease" or "CD22-associated disease" refers to a disease such as cancer or inflammatory disorder, in which CD20 antigen or CD22 antigen is generally present on tumor cells or cells that induce inflammatory response (especially B cells).When the pathological condition is cancer, the pathological condition-associated antigen, i.e., "cancer-associated antigen", can be a tumor antigen as defined herein.

[0024] As used herein, the term "CD20" refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also called membrane-spanning 4-domains, subfamily A, member 1 (MS4A1). Human and mouse amino acid and nucleic acid sequences can be found in public databases, such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found under accession numbers NP_690605.1 and NP_068769.2, and the nucleic acid sequences encoding human CD20 transcript variants 1 and 3 can be found under accession numbers NM_152866.2 and NM_021950.3, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD20 protein. In one aspect, the CD20 protein is expressed in cancer cells. As used herein, "CD20" includes proteins containing mutations, such as point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD20.

[0025] As used herein, the term "CD22" refers to an antigenic determinant known to be detectable on leukemia progenitor cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of human CD22 isoforms 1 to 5 can be found in Accession Nos. NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and nucleic acid sequences encoding human CD22 variants 1 to 5 can be found in Accession Nos. NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one aspect, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD22 protein. In one aspect, the CD22 protein is expressed in cancer cells. As used herein, "CD22" includes proteins that contain mutations, such as point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD22.

[0026] The term "tumor antigen" is intended to encompass "tumor-specific antigens" and "tumor-associated antigens." Tumor-specific antigens (TSAs) are generally present only in tumor cells and not in any other cells, while tumor-associated antigens (TAAs) are present in some types of tumor cells and also in some types of normal cells. As intended herein, tumor antigens also refer to mutant forms of a protein that appear only in tumors, while the non-mutated form is observed in non-tumor tissues.

[0027] The term "extracellular antigen-binding domain," as used herein, refers to an oligopeptide or polypeptide capable of binding to a specific antigen. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular antigen-binding domain may be selected to recognize an antigen that acts as a cell surface marker on target cells associated with a particular pathology. In a particular example, the extracellular antigen-binding domain comprises a single-chain antibody fragment (scFv), which comprises a light chain variable fragment (V) of a monoclonal antibody specific for the target antigen linked by a flexible linker. L ) and heavy chain variable fragment (V H The antigen-binding domain of the CAR expressed on the cell surface of the engineered immune cells described herein can be any domain that binds to a target antigen and is derived from, for example, a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and functional fragments thereof.

[0028] "Chimeric antigen receptor" or "CAR" generally refers to a synthetic receptor containing a targeting moiety linked to one or more signaling domains in a single fusion molecule. As defined herein, the term "chimeric antigen receptor" encompasses both single-chain CARs and multi-chain CARs. Typically, the binding moiety of a CAR consists of a single-chain antibody antigen-binding domain (scFv), which comprises the light chain and variable fragment of a monoclonal antibody linked by a flexible linker. The use of binding moieties based on receptor or ligand domains has also been successful. The signaling domain of first-generation CARs is derived from the cytoplasmic region of the CD3 zeta chain or the Fc receptor gamma chain. First-generation CARs have been shown to successfully redirect T cell cytotoxicity. However, this failed to result in long-term proliferation and antitumor activity in vivo. To enhance the survival rate and proliferation of CAR-modified T cells, signaling domains derived from costimulatory molecules, including CD28, OX-40 (CD134), and 4-1BB (CD137), have been added alone (second generation) or in combination (third generation). CARs are not necessarily single-chain polypeptides; multi-chain CARs are also possible. Regarding the structure of multi-chain CARs, the signaling domain and the costimulatory domain are arranged on separate polypeptide chains, as described, for example, in WO2014039523. Such multi-chain CARs can be derived from FcεRI by replacing the high-affinity IgE-binding domain of the α chain of FcεRI with an extracellular ligand-binding domain, such as an scFv, while fusing the N- and / or C-terminal tails of the β and / or γ chains of FcεRI with the signaling domain and the costimulatory domain, respectively. The extracellular ligand-binding domain serves to redirect the specificity of T cells toward cellular targets, while the signaling domain activates immune cell responses.

[0029] By "immune cell" is meant a cell of hematopoietic origin that is functionally involved in initiating and / or executing an innate and / or adaptive immune response, such as, for example, a typically CD45-positive cell, a CD3-positive cell, or a CD4-positive cell. The immune cell described herein may be a dendritic cell, a killer dendritic cell, a mast cell, a macrophage, a natural killer cell (NK cell), a cytokine-induced killer cell (CIK cell), or 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, a γδ T cell, or a natural killer T cell ("NKT cell").

[0030] "Allogeneic" means that the cells are of donor origin or are generated and / or differentiated from stem cells, allowing for infusion into a patient with a different haplotype.

[0031] Such immune cells are generally engineered to reduce alloreactivity and / or be more durable in the patient host. More specifically, the method of engineering allogenic immune cells can include reducing or inactivating the TCR expression of T cells or stem cells that will give rise to T cells. This can be achieved by various sequence-specific reagents, such as by gene silencing or gene editing techniques (nucleases, base editing, shRNA, RNAi, etc.).

[0032] By "donor-derived" it is meant that the T cells do not necessarily have to be derived directly from the donor as fresh cells, but may be derived from stem cells or cell lines obtained from an original donor that is not the patient being treated (i.e., has a different haplotype).

[0033] By "primary cell" or "primary cells" is meant cells obtained directly from living tissue (e.g., biopsy material) and established for a limited time in vitro, meaning that they have undergone only a limited number of population doublings. Primary cells are contrasted with persistent tumorigenic cell lines or artificially immortalized cell lines. Non-limiting examples of such cell lines include CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; and Molt 4 cells.

[0034] Primary immune cells can be obtained from numerous sources, including, but not limited to, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and can also be obtained from tumors, such as tumor-infiltrating lymphocytes. In some embodiments, the immune cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infection. In another embodiment, the cells are part of a mixed population of immune cells that exhibit distinct phenotypic characteristics, such as a mixed population of immune cells comprising CD4-positive cells, CD8-positive cells, and CD56-positive cells. Primary immune cells are provided from a donor or patient by various techniques known in the art, such as leukapheresis techniques, as reviewed by Schwartz J. et al. ("Guidelines on the use of therapeutic apheresis in clinical practice—evidence-based approach from the Writing Committee of the American Society for Apheresis: the sixth special issue" (2013) J Clin Apher. 28(3):145-284).

[0035] Stem cell-derived immune cells are also considered primary immune cells in the present invention, specifically those derived from induced pluripotent stem cells (iPS) [Yamanaka, K. et al. (2008). "Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors". Science. 322 (5903): 949-53]. Lentiviral expression of reprogramming factors has been used to induce multipotent cells from human peripheral blood cells [Staerk, J. et al. (2010). "Reprogramming of human peripheral blood cells to induced pluripotent stem cells". Cell Stem Cell. 7 (1): 20-4], [Loh, YH. et al. (2010). "Reprogramming of T cells from human peripheral blood". Cell Stem Cell. 7 (1): 15-9].

[0036] Immune cells can be derived from human embryonic stem cells by techniques known in the art that do not involve the destruction of human embryos [Chung et al. (2008). "Human Embryonic Stem Cell lines generated without embryo destruction". Cell Stem Cell 2(2):113-117].

[0037] "Genetic manipulation" refers to any method aimed at introducing genetic material into, modifying, and / or removing genetic material from a cell. "Gene editing" refers to genetic manipulation that allows genetic material to be added, removed, or changed at a specific location (locus) in the genome, including point mutations. Gene editing generally requires sequence-specific reagents.

[0038] "Identity" refers to the sequence identity between two nucleic acid molecules or two polypeptides.Identity can be determined by comparing a certain position in each sequence that can be aligned for comparison purposes.If a certain position in the compared sequences is occupied by the same base, the molecules are identical at that position.The degree of similarity or identity between nucleic acid sequences or amino acid sequences is a function of the number of identical or matching nucleotides at the position shared by the nucleic acid sequences.To calculate the identity between two sequences, various alignment algorithms and / or alignment programs can be used, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wisconsin), and they can be used, for example, with default settings. For example, polypeptides and polynucleotides encoding such polypeptides that have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to the specific polypeptides described herein and preferably exhibit substantially the same function are contemplated. Unless otherwise specified, the present invention encompasses polypeptides and polynucleotides that have the same function as the polypeptides and polynucleotides described herein and that are at least 80% identical, generally at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 97% identical to the polypeptides and polynucleotides described herein.

[0039] As used herein, the terms "patient" or "subject" and "donor" include all members of the animal kingdom, including non-human primates and humans.

[0040] The present invention is based on the surprising observation that tumor cells that express low levels of the CD20 antigen and low levels of the CD22 antigen, and that are unable to effectively activate CAR-T cells that target either CD20 or CD22, were surprisingly able to activate dual CAR-T cells that express both a CD20-targeting CAR and a CD22-targeting CAR.

[0041] Without intending to be bound by this theory, in these conditions, the total number of CD20 and CD22 antigen molecules per tumor site represents a threshold above which dual CAR-T cells but not single CAR-T cells are activated.

[0042] This can be expressed as follows: X = density of CD20 antigen molecules on the surface of the tumor, recognized by anti-CD20 CAR (CAR20); Y = density of CD22 antigen molecules on the surface of the tumor, recognized by anti-CD22 CAR (CAR22); X + Y = Z = density of CD20 and CD22 antigen molecules on the surface of the tumor; T = density of antigen molecules bound to T cells that triggers T cell activation = threshold required for T cell activation If Z > T, T cell activation occurs.

[0043] Table 1. Schematic diagram showing the relationship between CAR T cell activation and the expression levels of the target antigens CD20 and CD22. TIFF0007766674000001.tif54161

[0044] Thus, one advantage of the T cells of the invention that express both CAR20 and CAR22 (dual "CAR20x22-T cells" or dual "CAR22x20-T cells") is that they are useful for immunotherapy to target a broad population of tumor cells with different expression levels of CD20 and / or CD22 antigens. Thus, not only tumor cells that express low levels of CD20 antigen and high levels of CD22 antigen, but also tumor cells that express high levels of CD20 antigen and low levels of CD22 antigen can be targeted (and therefore killed) by the dual CAR22x20-T cells of the invention, but surprisingly, cells that express low levels of CD20 antigen and CD22 antigen can also be targeted and therefore killed.

[0045] Another advantage of the dual CAR-T cells of the present invention utilizes their utility in immunotherapy to treat tumors that change over time or treatment by expressing more or less of the CD20 and CD22 antigens.

[0046] Another advantage of the dual CAR-T cells of the present invention utilizes their utility in immunotherapy to treat cancers characterized by low expression of the CD20 and CD22 antigens.

[0047] Yet another advantage of the dual CAR-T cells of the present invention relates to the synergistic effect of the two CARs, which may be due to supporting and strengthening the immune synapse between the T cell and its target tumor cell, which may allow for the production of higher levels of effector cytokines.

[0048] In addition to the above-mentioned advantages, the dual CAR-T cells of the present invention are also useful for avoiding relapse and / or antigen escape of CD20-associated and CD22-associated cancers.

[0049] As detailed above, what is observed and described herein may be broadly applicable; thus, dual CAR-T cells that target two different tumor-associated antigens may be useful for immunotherapy targeting a broad range of tumor cell populations with different expression levels of tumor-associated antigens.

[0050] A further surprising effect of one aspect of the present invention relates to an observation made by the inventors of an immune cell that expresses CAR20 and CAR22 on its cell surface and has exogenous nucleic acid integrated into the genome of the cell, wherein the exogenous nucleic acid is arranged 5' to 3' as follows: (i) the promoter; (ii) a nucleic acid encoding CAR20; (iii) a nucleic acid encoding a self-cleaving peptide; (iv) a nucleic acid encoding CAR22 Including, immune cells, whereby the same promoter controls the expression of CAR20 and CAR22; The above immune cells show more potent tumor reduction in vivo compared to the following immune cells: 1. An immune cell that expresses CAR20 and CAR22 on its cell surface and has exogenous nucleic acid integrated into the genome of the cell, wherein the exogenous nucleic acid is arranged 5' to 3' as follows: (i) the promoter; (ii) a nucleic acid encoding CAR22; (iii) a nucleic acid encoding a self-cleaving peptide; (iv) a nucleic acid encoding CAR20 including immune cells.

[0051] Immune cells expressing anti-CD20 and anti-CD22 CARs The immune cells described herein are equipped with two synthetic chimeric antigen receptors (CARs) that target the CD20 and CD22 antigens, respectively.

[0052] - Anti-CD22 CAR (CAR22) and anti-CD20 CAR (CAR20) The immune cells described herein are loaded with two types of synthetic CARs that confer greater specificity to the immune cells for certain cells, such as tumors containing cells that express the CD20 and / or CD22 antigens, or for inflammatory cells that express the CD20 and / or CD22 antigens.

[0053] Generally, the recombinant chimeric antigen receptor is encoded by an exogenous polynucleotide, which is introduced into cells using a viral vector, such as in one of the transduction steps mentioned elsewhere in this application. The recombinant receptor encoded by the exogenous polynucleotide can also be introduced into cells in the form of a plasmid or PCR product.

[0054] Generally, a CAR polypeptide comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, where the intracellular domain comprises a costimulatory domain and / or a primary signaling domain, and the antigen-binding domain binds to an antigen associated with a pathology.

[0055] Although the anti-CD20 CAR and anti-CD22 CAR described herein are not limited to a specific CAR structure, nucleic acids that can be used to genetically engineer immune cells generally encode CARs that include: an extracellular antigen-binding domain that binds to an antigen associated with a pathology, a hinge, a transmembrane domain, and an intracellular domain that includes a stimulatory domain and / or a primary signaling domain. Typically, the extracellular antigen-binding domain is an scFv that includes the variable heavy chain (VH) and variable light chain (VL) of an antibody that binds to a specific antigen (e.g., a tumor antigen) linked via a linker. The transmembrane domain can be, for example, the transmembrane domain of CD8α or the transmembrane domain of 4-1BB. The stimulatory domain can be, for example, the stimulatory domain of 4-1BB. The primary signaling domain can be, for example, the signaling domain of CD3ζ.

[0056] In one embodiment, to avoid any recombination events within a construct comprising polynucleotides encoding two CARs comprising identical domains, the nucleotide sequences used to encode the same amino acid sequence (e.g., the same transmembrane domain, the same stimulatory domain) present twice in the construct are optimized using codon usage and code degeneracy such that the nucleotide sequences are different.

[0057] Table 2: Sequences of various domains typically present in CARs TIFF0007766674000002.tif142162

[0058] In one aspect, an antigen-binding domain specific for CD20 comprises a variable heavy chain (VH) and a variable light chain (VL) connected by a linker, wherein the VH chain comprises the H-CDRs of SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, and the VL chain comprises the L-CDRs of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52.

[0059] For example, an antigen-binding domain specific for CD20 comprises a variable heavy chain (VH) and a variable light chain (VL) linked by a linker (forming the scFv of SEQ ID NO: 17), wherein the VH and VL chains comprise the H-CDRs of SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, and the L-CDRs of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52.

[0060] The anti-CD20 CAR described herein can include: i) an antigen-binding domain specific for CD20, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 15 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 16 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain;

[0061] In one particular aspect, the CAR that targets the CD20 antigen present on tumor cells and that is expressed by the engineered immune cells described herein is as described in Tables 3 and 4.

[0062] Table 3: VH and VL sequences contained in the scFvs of the anti-CD20 CARs described herein and shown in the Examples section TIFF0007766674000003.tif37161

[0063] Table 4: Structures of anti-CD20 CARs described herein and shown in the Examples section TIFF0007766674000004.tif35162

[0064] In one aspect, an antigen-binding domain specific for CD22 comprises a variable heavy chain (VH) and a variable light chain (VL) connected by a linker, wherein the VH chain comprises the H-CDRs of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and the VL chain comprises the L-CDRs of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.

[0065] For example, an antigen-binding domain specific for CD22 comprises a variable heavy chain (VH) and a variable light chain (VL) linked by a linker (forming the scFv of SEQ ID NO: 13), wherein the VH and VL chains comprise the H-CDRs of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and the L-CDRs of SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.

[0066] The anti-CD22 CAR described herein can include: i) an antigen-binding domain specific for CD22, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 11 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 12 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain;

[0067] In one particular aspect, CARs that target the CD22 antigen present on tumor cells and that are expressed by the engineered immune cells described herein are described in Tables 5 and 6 below and in the Examples section.

[0068] Table 5. VH and VL sequences contained in the scFvs of the anti-CD22 CARs described herein and shown in the Examples section. TIFF0007766674000005.tif37161

[0069] Table 6. Anti-CD22 CAR structures described herein and shown in the Examples section TIFF0007766674000006.tif35162

[0070] - Immune cells expressing CAR20 and CAR22 The engineered immune cells described herein are equipped with two synthetic chimeric antigen receptors (CARs), which target the CD20 and CD22 antigens, respectively, as described herein.

[0071] In one particular example, the immune cells do not express any other CARs that target other antigens other than CD20 and CD22. More specifically, the immune cells do not express any other CARs other than CAR22 and CAR20 described herein.

[0072] The immune cells may be, for example, dendritic cells, killer dendritic cells, mast cells, macrophages, NK cells, cytokine-induced killer (CIK) cells, or may be B cells or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory or helper T lymphocytes, gamma delta T cells, NKT cells, and tumor-infiltrating lymphocytes (TILL).

[0073] In one particular example, the immune cells engineered to express two CARs are selected from the group consisting of T cells, NK cells, and macrophages.

[0074] In a more specific example, the immune cells expressing the two CARs are T cells, for example cytotoxic T cells.

[0075] In one comprehensive example, the immune cell is comprised within a cell population, for example, within an immune cell population, in particular, within a T cell population, a NK cell population, and / or a macrophage population.

[0076] In one particular example, the immune cells are T cells for use in off-the-shelf immunotherapy.

[0077] In one particular example, the genetically engineered immune cells are T cells that are TCR negative (do not express TCRα on their cell surface).

[0078] In one particular example, the engineered T cells: short hairpin RNA (shRNA) or small interfering RNA (siRNA) directed against polynucleotide sequences encoding components of the TCR is expressed.

[0079] In another particular example, the engineered T cell is mutated in its TCR alpha and / or beta alleles.

[0080] In particular, engineered T cells At least one allele encoding TCRα, TCRβ, and / or CD3 that is inactivated by a mutation may have:

[0081] In yet a further example, the engineered T cells comprise: At least one allele selected from β2m, PD1, CTLA4, dCK, CD52, and / or GR that is inactivated It has.

[0082] In yet a further example, the engineered immune cells do not express the rituximab-specific mimotope of SEQ ID NO:22.

[0083] One particular aspect relates to an engineered T cell that expresses CAR22 and CAR20 on its cell surface, where CAR22 comprises a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 12, and CAR20 comprises a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 16.

[0084] Even more specifically, genetically engineered T cells are provided that express CAR22 and CAR20 on their cell surface, where CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and CAR20 comprises the amino acid sequence of SEQ ID NO: 18.

[0085] One particular aspect relates to engineered T cells that express CAR22 and CAR20 on their cell surface, where CAR22 comprises a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 12, and CAR20 comprises a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 16, and the engineered T cells have at least one allele encoding TCRα, TCRβ, and / or CD3 that is mutationally inactivated.

[0086] One particular aspect relates to a genetically engineered T cell that expresses CAR22 and CAR20 on its cell surface, wherein CAR22 comprises a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 12, and CAR20 comprises a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 16, and wherein the engineered T cell has at least one allele encoding TCR alpha, TCR beta, and / or CD3 that is mutationally inactivated, and also has at least one allele selected from CD52 and beta2m that is inactivated, wherein in particular cases, the engineered T cell has TCR alpha and / or TCR beta, and CD52 that are mutationally inactivated.

[0087] Even more specifically, genetically engineered T cells are provided that express CAR22 and CAR20 on their cell surface, where CAR22 comprises the amino acid sequence of SEQ ID NO: 14, CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and the engineered T cells have at least one allele encoding TCRα, TCRβ, and / or CD3 that is mutationally inactivated.

[0088] Even more specifically, genetically engineered T cells are provided that express CAR22 and CAR20 on their cell surface, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14, CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and the engineered T cells have at least one allele encoding TCR alpha, TCR beta, and / or CD3 that is mutationally inactivated, and also have at least one allele selected from CD52 and beta2m that is inactivated, wherein in certain cases, the engineered T cells have TCR alpha and / or TCR beta, and CD52 that are mutationally inactivated.

[0089] Additionally, as described above, also provided herein are genetically engineered immune cells that express CAR22 and CAR20 on their cell surface, wherein the CAR is encoded by an exogenous nucleic acid integrated into the genome of the immune cell, and the exogenous nucleic acid is arranged 5' to 3' as follows: (i) a promoter (such as the EF1α promoter), (ii) a nucleic acid encoding CAR20; (iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19); (iv) a nucleic acid encoding CAR22 Including, This allows the expression of CAR20 and CAR22 to be controlled by the same promoter.

[0090] More specifically, as described above, genetically engineered immune cells are also provided that express CAR22 and CAR20 on their cell surface, wherein the exogenous nucleic acid is, 5' to 3': (i) a promoter controlling the expression of CAR20 (e.g., the EF1α promoter); (ii) a nucleic acid encoding CAR20, wherein the CAR20 comprises a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; (iii) a nucleic acid encoding a self-cleaving peptide (e.g., P2A of SEQ ID NO: 19); (iv) a nucleic acid encoding CAR22, wherein CAR22 comprises a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9. Including, This allows the expression of CAR20 and CAR22 to be controlled by the same promoter.

[0091] Alternatively, as described above, also provided herein are genetically engineered immune cells that express CAR22 and CAR20 on their cell surface, wherein the CAR is encoded by an exogenous nucleic acid integrated into the genome of the immune cell, and the exogenous nucleic acid is arranged 5' to 3' as follows: (i) a promoter (such as the EF1α promoter), (ii) a nucleic acid encoding CAR22; (iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19); (iv) a nucleic acid encoding CAR20 Including, This allows the expression of CAR20 and CAR22 to be controlled by the same promoter.

[0092] More specifically, also provided are genetically engineered immune cells that express CAR22 and CAR20 on their cell surface, as described above, wherein the exogenous nucleic acid comprises, from 5' to 3': (i) a promoter controlling the expression of CAR22 (e.g., the EF1α promoter); (ii) a nucleic acid sequence encoding CAR22, wherein CAR22 comprises a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; (iii) a nucleic acid encoding a self-cleaving peptide (e.g., P2A of SEQ ID NO: 19); (iv) A nucleic acid encoding CAR20, wherein the CAR20 comprises a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9.

[0093] Method for preparing genetically engineered immune cells As described herein, immune cells to be genetically engineered to express CAR20 and CAR22 can be prepared by introducing one or more exogenous polynucleotides encoding the CAR.Polynucleotides can be introduced into cells by transduction using viral vectors.Polynucleotides can also be introduced into cells in the form of plasmids or PCR products.

[0094] Stable expression of CARs, particularly CAR20 and CAR22 as described herein, in the above-mentioned immune cells can be achieved, for example, using viral vectors (e.g., lentiviral vectors, retroviral vectors, adeno-associated viral (AAV) vectors), or using transposon / transposase systems, or by integration of plasmids or PCR products. Other approaches include direct electroporation of mRNA.

[0095] To deliver both CARs simultaneously to a cell, one or more polynucleotides encoding the anti-CD22 CAR (CAR22) and anti-CD20 CAR (CAR20) described herein can have various structures, for example, as follows: (a) a polycistronic arrangement, in which two transcription units are both controlled by a single promoter, have the same direction of transcription, and are separated by a "self-cleaving" peptide, such as a 2A peptide (e.g., P2A, T2A, E2A, F2A), (b) a bidirectional arrangement in which each of the two transcription units is controlled by an independent promoter and is transcribed in opposite directions in a head-to-head configuration; and (c) Monocistronic arrangement, in which both scFV transcripts are separated by a genomic spacer.

[0096] As used herein, "polycistronic" mRNA refers to a single messenger RNA that contains two or more coding sequences (i.e., cistrons) and encodes multiple proteins. Polycistronic mRNAs may contain any element known in the art that allows for the translation of two or more genes from the same mRNA molecule, including, but not limited to, self-cleaving peptides such as P2A, T2A, E2A, and F2A elements, or IRES elements.

[0097] The self-cleaving peptide contained in the polynucleotide described herein may be selected from a 2A peptide, a 2A-like peptide, a P2A peptide, an E2A peptide, an F2A peptide, or a T2A peptide, and may specifically be a 2A peptide, more specifically, a P2A peptide of SEQ ID NO: 19, a T2A peptide of SEQ ID NO: 38, an E2A peptide of SEQ ID NO: 39, or an F2A peptide of SEQ ID NO: 40, and even more specifically, a P2A peptide of SEQ ID NO: 19.

[0098] IRES refers to an "internal ribosome entry site" and refers to any IRES that allows transcription and subsequent translation of a coding sequence inserted into a gene that may be used herein. For example, the IRES contained in the polynucleotides described herein may have SEQ ID NO: 37.

[0099] As described herein, CAR20 and CAR22 can be encoded by two nucleic acids, wherein: 1) one nucleic acid encodes CAR20, and CAR20 is i) an antigen-binding domain specific for CD20, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 15 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 16 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; and 2) another nucleic acid encodes CAR22, and CAR22 is i) an antigen-binding domain specific for CD22, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 11 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 12 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; Includes:

[0100] In one example, CAR22 and CAR20 are encoded by two independent nucleic acids, a) and b), wherein: (1) The nucleic acid a) encodes CAR22, and CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; and (2) Nucleic acid b) encodes CAR20, and CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; Includes:

[0101] In one particular example, an isolated polynucleotide is provided comprising: a) a nucleic acid encoding CAR22, wherein CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; a nucleic acid encoding CAR22, comprising: b) a nucleic acid encoding CAR20, wherein CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; A nucleic acid encoding CAR20, comprising:

[0102] In a further example, the nucleic acid of a) and the nucleic acid of b) are on a single nucleic acid molecule, and the isolated polynucleotide comprises a nucleic acid sequence encoding a self-cleaving peptide (e.g., P2A, T2A, E2A, or F2A) located between the nucleic acid of a) and the nucleic acid of b).

[0103] Thus, in one general aspect, a polynucleotide is disclosed herein, the polynucleotide comprising, from 5' to 3': (i) a promoter (such as the EF1α promoter), (ii) a nucleic acid encoding CAR20 as described herein; (iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19); (iv) a nucleic acid encoding CAR22 as described herein. Including, This allows the expression of CAR20 and CAR22 to be controlled by the same promoter.

[0104] Additionally, a polynucleotide is also disclosed herein, the polynucleotide comprising, from 5' to 3': (i) a promoter (such as the EF1α promoter), (ii) a nucleic acid encoding CAR22 as described herein; (iii) a nucleic acid encoding a self-cleaving peptide (such as P2A of SEQ ID NO: 19); (iv) a nucleic acid encoding CAR20 described herein Including, This allows the expression of CAR20 and CAR22 to be controlled by the same promoter.

[0105] In a particular example, the isolated polynucleotides described herein do not include nucleic acids encoding additional CARs other than CAR22 and CAR20.

[0106] In one particular example, the isolated polynucleotides described herein do not include nucleic acids encoding the rituximab-specific mimotope of SEQ ID NO:22.

[0107] In one example, the isolated polynucleotides encoding CAR20 and CAR22 described herein are: a) a promoter (such as an EF1α promoter) that controls expression of CAR20, followed by a nucleic acid encoding CAR20, wherein CAR20 is: i) an antigen-binding domain specific for CD20, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 15 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 16 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; a promoter controlling the expression of CAR20, comprising: b) a nucleic acid encoding CAR22, wherein CAR22 is i) an antigen-binding domain specific for CD22, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 11 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 12 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; A nucleic acid encoding CAR22, comprising Including, Here, the nucleic acid a) and the nucleic acid b) are on one nucleic acid molecule, and a nucleic acid sequence encoding a self-cleaving peptide (e.g., P2A, T2A, E2A, or F2A) is located between the nucleic acid a) and the nucleic acid b).

[0108] In another example, the isolated polynucleotides encoding CAR20 and CAR22 described herein are: a) a promoter (such as an EF1α promoter) that controls expression of CAR22, followed by a nucleic acid encoding CAR22, wherein CAR22 is: i) an antigen-binding domain specific for CD22, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 11 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 12 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; a promoter controlling the expression of CAR22, comprising: b) a nucleic acid encoding CAR20, wherein CAR20 is i) an antigen-binding domain specific for CD20, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 15 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 16 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; A nucleic acid encoding CAR20, comprising Including, Here, the nucleic acid a) and the nucleic acid b) are on one nucleic acid molecule, and a nucleic acid sequence encoding a self-cleaving peptide (e.g., P2A, T2A, E2A, or F2A) is located between the nucleic acid a) and the nucleic acid b).

[0109] In yet a further example, the isolated polynucleotides encoding CAR20 and CAR22 described herein include: a) a nucleic acid encoding CAR22, comprising a promoter (e.g., an EF1α promoter) that controls expression of CAR22, wherein CAR22 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; b) a nucleic acid encoding CAR20, wherein CAR20 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; and c) A nucleic acid encoding a self-cleaving peptide (e.g., P2A of SEQ ID NO: 19) positioned between the nucleic acids of a) and b) to enable simultaneous expression of CAR20 and CAR22.

[0110] In yet a further example, an isolated polynucleotide encoding CAR20 and CAR22 described herein is provided, comprising: a) a nucleic acid encoding CAR20, comprising a promoter (e.g., an EF1α promoter) that controls expression of CAR20, wherein CAR20 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; b) a nucleic acid encoding CAR22, wherein CAR22 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; and c) A nucleic acid encoding a self-cleaving peptide (e.g., P2A of SEQ ID NO: 19) positioned between the nucleic acids of a) and b) to enable simultaneous expression of CAR22 and CAR20.

[0111] In yet a further example, an isolated polynucleotide encoding CAR20 and CAR22 is provided, which comprises the nucleic acid sequence of SEQ ID NO: 31 (CAR22xCAR20 construct).

[0112] In another specific example, an isolated polynucleotide encoding CAR20 and CAR22 is provided, which comprises the nucleic acid sequence of SEQ ID NO: 32 (CAR20xCAR22 construct).

[0113] Additionally, vectors comprising any of the isolated polynucleotides described herein are also disclosed herein.

[0114] Additionally, host cells containing the vectors described herein are also disclosed herein.

[0115] The immune cell population to be engineered is generally extracted from the patient's or healthy donor's blood by apheresis and further engineered to express a chimeric antigen receptor on their surface. Alternatively, the immune cell population to be engineered may be derived from umbilical cord blood cells or stem cells, which are further engineered to express a chimeric antigen receptor on their surface.

[0116] CAR-expressing immune cells may be derived from the patient's immune cells or from the immune cells of a compatible donor, which cells have been engineered to express a specific CAR on their surface.

[0117] CAR-expressing immune cells may also be derived from stem cells, such as iPS cells, of such patient origin or of a compatible donor origin, or may be derived from tumor-infiltrating lymphocytes (TILLs).

[0118] In other aspects, the CAR-expressing immune cells are so-called "off-the-shelf" immune cell compositions, whereby immune cells that do not specifically belong to the patient to be treated have been engineered to express a CAR and to be suitable for use in allogeneic therapeutic treatment.

[0119] "Allogeneic" means that the cells are of donor origin or are generated and / or differentiated from stem cells, allowing for infusion into a patient with a different haplotype.

[0120] Such immune cells are generally engineered to reduce alloreactivity and / or be more durable in the patient host. More specifically, the method of engineering allogenic immune cells can include reducing or inactivating the TCR expression of T cells or stem cells that will give rise to T cells. This can be achieved by various sequence-specific reagents, such as by gene silencing or gene editing techniques (nucleases, base editing, RNAi, etc.).

[0121] Previously, the Applicant has made available robust protocols and gene editing strategies for generating allogeneic therapeutic-grade T cells from PBMCs, in particular by providing highly safe and specific endonuclease reagents in the form of TALE nucleases (TALEN®). ネガティブThe generation of T cells, so-called "universal T cells," has been achieved and has been successfully infused into patients to reduce graft-versus-host disease (GVhD) (Poirot et al. 2015, Cancer. Res.75 (18): 3853-3864; Qasim et al., 2017, Science Translational 9(374)). Also, for example, inactivation of constituent TCRs or β2m in primary T cells can be combined with inactivation of additional genes encoding checkpoint inhibitor proteins, as described, for example, in WO2014184744.

[0122] In a further example, the engineered immune cells can be further modified to render them resistant to at least one immunosuppressant, for example by inactivating CD52, the target of anti-CD52 antibodies (e.g., alemtuzumab), as described, for example, in WO2013176915.

[0123] In a further example, the engineered immune cells can be further modified to confer resistance to and / or chemotherapeutic agents, particularly purine analogue agents, for example by inactivating DCK, as described in WO201575195.

[0124] In a further example, the engineered immune cells can be further modified to improve their persistence or longevity in the patient, particularly by inactivating genes encoding one or more components of MHC-I, such as HLA or β2m, as described, for example, in WO2015136001 or by Liu et al. (2017, Cell Res 27:154-157).

[0125] In still further examples, the engineered immune cells are mutated to improve their CAR-dependent immune activation, in particular to reduce or suppress expression of immune checkpoint proteins and / or their receptors, e.g., PD1 or CTLA4, as described in WO2014184744.

[0126] Pharmaceutical Compositions One aspect relates to a pharmaceutical composition comprising a genetically engineered immune cell that expresses a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20) on its cell surface, as described herein, and a pharmaceutically acceptable excipient.

[0127] Further, also disclosed are pharmaceutical compositions comprising a population of immune cells, including genetically engineered immune cells that express a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20) on their cell surface, as described herein, and a pharmaceutically acceptable excipient.

[0128] One particular aspect relates to a pharmaceutical composition comprising the genetically engineered T cells described herein and a pharmaceutically acceptable excipient.

[0129] A further particular aspect relates to a pharmaceutical composition comprising a population of T cells, including the genetically engineered T cells described herein, and a pharmaceutically acceptable excipient.

[0130] Another specific aspect relates to a pharmaceutical composition comprising engineered T cells that express CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein CAR22 comprises a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 12, and CAR20 comprises a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 16.

[0131] Even more specifically, a pharmaceutical composition is provided comprising engineered T cells that express CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and CAR20 comprises the amino acid sequence of SEQ ID NO: 18.

[0132] A further particular aspect relates to a pharmaceutical composition comprising a T cell population comprising engineered T cells that express CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein CAR22 comprises a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 12, and CAR20 comprises a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 16.

[0133] A further particular aspect relates to a pharmaceutical composition comprising a T cell population comprising engineered T cells that express CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and CAR20 comprises the amino acid sequence of SEQ ID NO: 18.

[0134] Another specific aspect relates to a pharmaceutical composition comprising engineered T cells that express CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein CAR22 comprises a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 12, and CAR20 comprises a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 16, and wherein the T cells have at least one allele encoding TCRα, TCRβ, and / or CD3 that is mutationally inactivated, and / or the engineered T cells have at least one allele selected from β2m and CD52 that is inactivated.

[0135] Even more specifically, a pharmaceutical composition is provided comprising engineered T cells that express CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14, CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and the T cells have at least one allele encoding TCRα, TCRβ, and / or CD3 that is mutationally inactivated.

[0136] Even more specifically, pharmaceutical compositions are provided comprising engineered T cells that express CAR22 and CAR20 on their cell surface and a pharmaceutical excipient, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14, CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and the T cells have at least one allele encoding TCRα, TCRβ, and / or CD3 that is mutationally inactivated, and / or the engineered T cells have at least one allele selected from β2m and CD52 that is inactivated.

[0137] A further specific aspect relates to a pharmaceutical composition comprising a T cell population comprising engineered T cells that express CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein CAR22 comprises a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 12, and CAR20 comprises a VH of SEQ ID NO: 15 and a VL of SEQ ID NO: 16, and the T cells have at least one allele encoding TCRα, TCRβ, and / or CD3 that is mutationally inactivated, and / or the engineered T cells have at least one allele selected from β2m and CD52 that is inactivated.

[0138] A further particular aspect relates to a pharmaceutical composition comprising a T cell population comprising genetically engineered T cells that express CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14, CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and the T cells have at least one allele encoding TCRα, TCRβ, and / or CD3 that is mutationally inactivated.

[0139] A further specific aspect relates to a pharmaceutical composition comprising a T cell population comprising genetically engineered T cells that express CAR22 and CAR20 on their cell surface, and a pharmaceutical excipient, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14, CAR20 comprises the amino acid sequence of SEQ ID NO: 18, and the T cells have at least one allele encoding TCRα, TCRβ, and / or CD3 that is mutationally inactivated, and / or the engineered T cells have at least one allele selected from β2m and CD52 that is inactivated.

[0140] Additionally, also provided herein are genetically engineered immune cells expressing CAR20 and CAR22 as described herein for use as pharmaceuticals.

[0141] Further, also provided herein are immune cell populations comprising engineered immune cells that express CAR20 and CAR22, as described herein, for use as pharmaceuticals.

[0142] Treatment method Another aspect relates to a method of treating cancer and / or an inflammatory disorder, the method comprising administering to a patient in need thereof engineered immune cells expressing CAR20 and CAR22, as described herein.

[0143] One similar aspect relates to engineered immune cells expressing CAR20 and CAR22, as described herein, for use in methods of treating cancer and / or inflammatory disorders.

[0144] One similar aspect relates to immune cell populations, including engineered immune cells expressing CAR20 and CAR22, described herein, for use in methods of treating cancer and / or inflammatory disorders.

[0145] A similar aspect relates to the use of engineered immune cells that express CAR20 and CAR22, as described herein, or to the use of a population of immune cells that includes engineered immune cells that express CAR20 and CAR22, as described herein, to prepare a medicament.

[0146] A similar aspect relates to the use of engineered immune cells expressing CAR20 and CAR22, as described herein, or to the use of a population of immune cells comprising engineered immune cells expressing CAR20 and CAR22, as described herein, to prepare a medicament for treating cancer and / or an inflammatory disorder.

[0147] The treatment can be for treating cancer, including hematological cancers, such as hematological cancers selected from lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL) (also known as acute lymphoblastic leukemia), acute lymphoid carcinoma, acute myeloid leukemia (AML), particularly CD22- and / or CD20-associated hematological cancers, more particularly relapsed / refractory CD22- and / or CD20-associated hematological cancers, and even more particularly aggressive forms of CD22- and / or CD20-associated hematological cancers.

[0148] Treatment may involve the prevention or attenuation of inflammatory disorders associated with CD20 and / or CD22.

[0149] In one particular aspect, the treatment is for treating patients with relapsed / refractory NHL.

[0150] In a further particular aspect, the treatment is for treating a patient suffering from a cancer with low expression of the CD20 and CD22 antigens.

[0151] In one particular aspect, the treatment is for treating a patient who has previously undergone treatment with rituximab, an anti-CD20 antibody that is the standard of care in the treatment of NHL.

[0152] "Non-Hodgkin's lymphoma (NHL)" is a term used to describe a diverse group of blood cancers that all share one characteristic: they arise from lymphocytes. More than 60 distinct NHL subtypes have been identified and assigned names, called "diagnostic names," by the World Health Organization (WHO).

[0153] In particular, the term "non-Hodgkin's lymphoma (NHL)" includes the following diagnostic names for non-Hodgkin's lymphoma (NHL): 1. Mature B-cell lymphoma (approximately 85%-90% of NHL cases): Aggressive: Diffuse large B-cell lymphoma (DLBCL) (31%), mantle cell lymphoma (MCL) (which can present as aggressive or indolent) (6%), lymphoblastic lymphoma (2%), Burkitt lymphoma (BL) (2%), primary mediastinal (thymic) large B-cell lymphoma (PMBCL) (2%), transformed follicular lymphoma and transformed mucosa-associated lymphoid tissue (MALT) lymphoma, double-hit or triple-hit high-grade B-cell lymphoma (HBL), primary cutaneous DLBCL (leg type), primary central nervous system DLBCL, primary central nervous system (CNS) lymphoma, acquired immunodeficiency syndrome (AIDS)-related lymphoma Indolent: Follicular lymphoma (FL) (22%), marginal zone lymphoma (MZL) (8%), chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL) (6%), gastric mucosa-associated lymphoid tissue (MALT) lymphoma (5%), lymphoplasmacytic lymphoma (1%), Waldenström's macroglobulinemia (WM), nodal marginal zone lymphoma (NMZL) (1%), splenic marginal zone lymphoma (SMZL) 2. Mature T-cell and natural killer (NK) cell lymphoma (approximately 10%–15% of NHL cases) Aggressive: peripheral T-cell lymphoma (PTCL), not otherwise specified (6%), systemic anaplastic large cell lymphoma (ALCL) (2%), lymphoblastic lymphoma (2%), hepatosplenic gamma delta T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma (SPTCL), enteropathic intestinal T-cell lymphoma, primary cutaneous anaplastic large cell lymphoma · Indolent: Cutaneous T-cell lymphoma (CTCL) (4%), mycosis fungoides (MF), Sézary syndrome (SS), angioimmunoblastic T-cell lymphoma (AITL), adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma (ENK / TCL) · Nasal type

[0154] The foregoing description of the invention provides manners and processes of making and using the invention to enable any person skilled in the art to make and use the same, this enabling capability being provided with particularity with respect to the subject matter of the appended claims, which form a part of the present description.

[0155] When numerical limits or ranges are recited herein, the endpoints are included. Moreover, all values ​​and all subranges within a numerical limit or range are also expressly included, as if they were explicitly recited.

[0156] Having broadly described the invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the claimed invention.

[0157] Specific Aspects 1. A genetically engineered T cell that expresses a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20) on its cell surface, a) CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; and b) CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; and c) TCR negative; Genetically engineered T cells. 2. The genetically engineered T cell of embodiment 1, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14, and CAR20 comprises the amino acid sequence of SEQ ID NO: 18. 3. The genetically engineered T cell of any one of aspects 1-2, wherein the extracellular domain of neither CAR20 nor CAR22 comprises the rituximab-specific mimotope of SEQ ID NO: 22. 4. The genetically engineered T cell of any one of aspects 1 to 3, which expresses a short hairpin RNA (shRNA) or a short interfering RNA (siRNA) directed against a polynucleotide sequence encoding a component of a TCR. 5. The genetically engineered T cell of any one of aspects 1 to 3, which is mutated in its TCR alpha allele and / or TCR beta allele. 6. The genetically engineered T cell of any one of aspects 1 to 3 and 5, wherein the T cell has at least one allele encoding TCR alpha, TCR beta, and / or CD3 that is mutationally inactivated. 7. The genetically engineered T cell of any one of aspects 1 to 6, which has an inactivated CD52 allele. 8. The genetically engineered T cell of any one of aspects 1 to 7, which has an inactivated β2m allele. 9. The genetically engineered T cell of any one of aspects 1 to 8, wherein the T cell has an inactivated PD1 allele. 10. The genetically engineered T cell of any one of aspects 1 to 9, having an inactivated CTLA4 allele. 11. The genetically engineered T cell of any one of aspects 1 to 10, which has an inactivated dCK allele. 12. The genetically engineered T cell of any one of aspects 1 to 11, which has an inactivated GR allele. 13. The genetically engineered T cell of any one of aspects 1 to 12, wherein the engineered immune cell is a cytotoxic T cell. 14. The genetically engineered T cell of any one of aspects 1 to 13, which is comprised in a T cell population. 15. The genetically engineered T cell of any one of aspects 1 to 14, which is a primary cell. 16. The genetically engineered T cell of any one of aspects 1 to 15, which is a mammalian cell, preferably a human cell. 17. The genetically engineered T cell of any one of aspects 1-16, which does not express an additional CAR other than CAR22 and CAR20. 18. A population of T cells comprising the engineered T cells of any one of aspects 1 to 17. 19. A pharmaceutical composition comprising the engineered T cells of any one of aspects 1 to 17 and a pharmaceutically acceptable excipient. 20. A pharmaceutical composition comprising the population of T cells according to embodiment 18 and a pharmaceutically acceptable excipient. 21. a) A nucleic acid encoding CAR22, wherein CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) of SEQ ID NO: 11 and a variable light chain (VL) of SEQ ID NO: 12, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; a nucleic acid encoding CAR22, comprising: b) a nucleic acid encoding CAR20, wherein CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) of SEQ ID NO: 15 and a variable light chain (VL) of SEQ ID NO: 16, optionally including a leader sequence; ·CD8α-derived hinge domain at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; A nucleic acid encoding CAR20, comprising An isolated polynucleotide comprising: 22. The polynucleotide of embodiment 21, wherein the nucleic acid of a) and the nucleic acid of b) are on a single nucleic acid molecule, and a nucleic acid sequence encoding a self-cleaving peptide (e.g., P2A, T2A, E2A, or F2A) is located between the nucleic acid of a) and the nucleic acid of b). 23. The polynucleotide of embodiment 21 or 22, which does not comprise a nucleic acid encoding an additional CAR other than CAR22 and CAR20. 24. The polynucleotide of any one of aspects 21 to 23, which does not comprise a nucleic acid encoding a rituximab-specific mimotope of SEQ ID NO: 22. 25. The nucleic acid of a) comprises a promoter (e.g., an EF1α promoter) that controls the expression of CAR22, and CAR22 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; b) the nucleic acid encodes CAR20, wherein CAR20 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; and a nucleic acid encoding a self-cleaving peptide (e.g., P2A of SEQ ID NO: 19) is present between the nucleic acid of a) and the nucleic acid of b) to allow for simultaneous expression of CAR20 and CAR22; A polynucleotide according to any one of embodiments 21 to 24. 26. The nucleic acid of b) comprises a promoter (e.g., an EF1α promoter) that controls the expression of CAR20, wherein CAR20 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 17, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; a) the nucleic acid encodes CAR22, wherein CAR22 is composed of a signal peptide of SEQ ID NO: 1, an scFv of SEQ ID NO: 13, a CD8α hinge of SEQ ID NO: 4, a CD8α transmembrane domain of SEQ ID NO: 6, a 4-1BB costimulatory domain of SEQ ID NO: 8, and a CD3ζ signaling domain of SEQ ID NO: 9; and a nucleic acid encoding a self-cleaving peptide (e.g., P2A of SEQ ID NO: 19) is present between the nucleic acid of a) and the nucleic acid of b) to allow for simultaneous expression of CAR22 and CAR20; A polynucleotide according to any one of embodiments 21 to 24. 27. The isolated polynucleotide of aspect 25, comprising the nucleic acid sequence of SEQ ID NO: 31 (CAR22xCAR20 construct). 28. The isolated polynucleotide of embodiment 26, comprising the nucleic acid sequence of SEQ ID NO: 32 (CAR20xCAR22 construct). 29. A vector comprising the isolated polynucleotide of any one of aspects 21 to 28. 30. A host cell comprising the vector of embodiment 29. 31. A method for preparing an engineered T cell according to any one of aspects 1 to 17, the method comprising the step of introducing a polynucleotide according to any one of aspects 21 to 28 or a vector according to aspect 29 into an immune cell. 32. The engineered T cell of any one of aspects 1 to 17 for use as a medicament. 33. The engineered T cell of any one of aspects 1 to 17 for use in treating a cancer associated with expression of CD20 and / or CD22. 34. The engineered T cell of any one of aspects 1 to 17 for use in treating a hematological cancer, particularly a CD22- and / or CD20-associated hematological cancer, more particularly a relapsed or refractory CD22- and / or CD20-associated hematological cancer, even more particularly an aggressive form of a CD22- and / or CD20-associated hematological cancer. 35. The engineered T cell for use according to aspect 33 or 34, wherein the cancer is selected from the group consisting of lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphoid carcinoma, acute myeloid leukemia (AML). 36. The engineered T cell for use according to aspect 35, wherein the cancer is non-Hodgkin's lymphoma or acute lymphocytic leukemia. 37. The engineered T cell for use according to any one of aspects 33 to 36, wherein the cancer is associated with low expression of CD20 and / or CD22. 38. The engineered T cell for use according to any one of aspects 33 to 37, wherein the cancer is recurrent non-Hodgkin's lymphoma. 39. A method of treating a patient suffering from a cancer associated with expression of CD20 and / or CD22, comprising administering to the patient an effective amount of the engineered T cell of any one of aspects 1 to 17 or the population of T cells of aspect 18. 40. The method of treatment of aspect 39, wherein the cancer is selected from the group consisting of lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphoid carcinoma, and acute myeloid leukemia (AML). 41. A method of treatment according to aspect 39 or 40, wherein the cancer is non-Hodgkin's lymphoma or acute lymphocytic leukemia. 42. A method of treatment according to any one of aspects 39 to 41, wherein the cancer is associated with underexpression of CD20 and / or CD22. 43. A method of treatment according to any one of aspects 39 to 42, wherein the cancer is recurrent non-Hodgkin's lymphoma. [Example]

[0158] The examples provided herein describe how to generate dual CAR-T cells that target the CD20 and CD22 antigens ("CD20xCD22" CAR-T cells or "CD20xCD22" CAR-T cells) and demonstrate the ability of the dual CAR-T cells to lyse tumor cells that under-express or do not express CD22 and / or CD20.

[0159] Example 1 Generation of CD20 and CD22 dual CAR constructs and their regulation To compare expression, activity, and potency, two configurations of dual CARs were designed and assembled into recombinant lentiviral vectors. The first construct (CD22xCD20) contained an EF1α promoter driving the expression of the first CD22 CAR, which is composed of a signal peptide (SEQ ID NO: 1), an scFv of SEQ ID NO: 13, a CD8α hinge domain (SEQ ID NO: 4) and TM domain (SEQ ID NO: 6), a 4-1BB costimulatory domain (SEQ ID NO: 8), and a CD3ζ signaling domain (SEQ ID NO: 9). This first CAR is followed by the self-cleaving peptide P2A (SEQ ID NO: 19), which enables the expression of the second CD20 CAR, which is composed of a signal peptide (SEQ ID NO: 1), an anti-CD20 scFv of SEQ ID NO: 17, the hinge domain (SEQ ID NO: 4) and TM domain (SEQ ID NO: 6) of CD8α, a 4-1BB costimulatory domain (SEQ ID NO: 8), and a CD3ζ signaling domain (SEQ ID NO: 9). To avoid any recombination events within the rLV construct, the nucleotide sequences used to encode the same amino acid sequence twice in the construct were optimized using codon usage and code degeneracy such that the nucleotide sequences were different. The second dual construct (CD20xCD22) contains the exact same sequence (nucleotide and amino acids), but the CD20 CAR is located immediately after the EF1α promoter.

[0160] For comparison, CD22 or CD20 CARs were constructed in recombinant lentiviral vectors containing an EF1α promoter driving the expression of a single-car targeting CD22 (SEQ ID NO: 14) or a single-car targeting CD20 (SEQ ID NO: 18).

[0161] Example 2 Generation of CD20xCD22 dual CAR-T cells or CD22xCD20 dual CAR-T cellsCryopreserved PBMCs from at least three different donors were used. PBMCs were thawed at 37°C, washed with OpTmizer medium supplemented with 5% AB human serum and recombinant human interleukin-2 (rhIL-2, 350 IU / ml), and resuspended in the medium for overnight incubation in a 5% CO2 incubator at 37°C. Cells were then activated with anti-CD3 / CD28-coated beads in OpTmizer medium supplemented with 5% AB human serum (also supplemented with 5% CTS™ Immune Cell SR during the expansion phase) and recombinant human interleukin-2 (rhIL-2, 350 IU / ml) for 3 days in a CO2 incubator. The expanded T cells were then transduced with lentiviral particles expressing either the CD20xCD22 CAR or the CD22xCD20 CAR (SEQ ID NO: 31 and SEQ ID NO: 32, respectively) in the presence of Lentiboost (SB-P-LV-101-12, Mayflower bioscience) at an MOI of 15 (MOI stands for multiplicity of infection). The expanded T cells were also transduced with lentiviral particles expressing either the CD20 CAR alone or the CD22 CAR alone (SEQ ID NO: 14 and SEQ ID NO: 18) at an MOI of 5. Two days after transduction, cells were electroporated with four mRNAs using the AgilePulse Max system, two of which encoded the TRAC_T01 TALEN arms (SEQ ID NO: 23 and SEQ ID NO: 24) and two of which encoded the CD52_T01 TALEN arms (SEQ ID NO: 25 and SEQ ID NO: 26). Cells were resuspended in culture medium, incubated at 30°C for 16–18 hours, and then expanded at 37°C with occasional adjustments to cell concentration after adding fresh culture medium. On the final day of culture (18 days after thawing), T cells were used for various assays or frozen in freezing medium (90% FBS, 10% DMSO). Cells were kept frozen at -150°C until use.

[0162] Example 3 Detection of CD20 and CD22 dual CAR-T cells To detect CD20 CAR, CD20 recombinant protein fused with His tag (Acro #CD0-H52H3, SEQ ID NO: 20) was used in combination with APC-labeled anti-His antibody (BioLegend #362605).To detect CD22 CAR, CD22-Fc protein (SEQ ID NO: 21) was used in combination with anti-Fcγ subclass 1 tag Cy3 (Jackson ImmunoResearch #115-165-205).

[0163] The various CAR T cells (untransduced, CD20xCD22 CAR, CD22xCD20 CAR, or CD22 CAR) generated in Example 1 were incubated with 100 ng of CD22-Fc protein and 200 ng of CD20-His protein, washed, and further incubated with anti-Fcγ (50 ng) or anti-His (50 ng), and then fixed in PFA 2%. The cells were then analyzed by flow cytometry.

[0164] The results in Figure 1 demonstrate that transduction of CD22 CAR alone resulted in 40% CD22 CAR-positive T cells, while transduction of CD22xCD20 CAR or CD20xCD22 CAR resulted in 39% and 25% double CAR-positive T cells, respectively. These results were reproducible using three different donors. Interestingly, CD22xCD20 resulted in a higher MFI for CD22 CAR staining, while CD20xCD22 resulted in a higher MFI for CD20 CAR staining.

[0165] Example 4 Cell lines used to test CD20 and CD22 dual CAR-T cells The Raji cell line expresses high levels of CD20 and CD22. This cell line was modified to express the luciferase gene and used as a positive control, i.e., as target cells expressing both CD20 and CD22. In addition, the Raji cell line was treated with CD22 TALEN and / or CD20 TALEN. Briefly, Raji cells were electroporated with mRNA encoding CD22 TALEN arms (SEQ ID NO: 27 and SEQ ID NO: 28) and mRNA encoding CD20 TALEN (SEQ ID NO: 29 and SEQ ID NO: 30) using the AgilePulse Max system. Cells were incubated in RPMI 1640, 10% fetal bovine serum (FBS), and 1% penicillin / streptavidin (culture medium) at 30°C for 16–18 hours and then expanded in fresh culture medium in a 37°C, 50% CO2 incubator until sorting. Different cell populations were purified using anti-biotin microbeads and either anti-CD20-biotin, anti-CD22-biotin, or both (Biolegend) to obtain different cell populations: i) expressing CD20 but not CD22; ii) expressing CD22 but not CD20; or iii) expressing neither CD20 nor CD22. Figure 2 shows the phenotypes of these selected populations.

[0166] Example 5 Cytotoxicity of CD20 and CD22 Dual CAR T Cells CD20xCD22 CAR T cells, CD22xCD20 CAR T cells, and CD22 CAR T cells generated in Example 1 and derived from three different donors were tested for their cytotoxic capacity against various Raji cells generated in Example 4.

[0167] T cells were thawed, and viable cells were counted using a NucleoCounter® instrument (Chemometec NC-250). Different CAR T cells were co-cultured with different luciferase-expressing Raji cells (CD20+CD22+, CD20+CD22-, CD22-CD20+, and CD20-CD22-) at various effector / target ratios in 96-well plates for 4 to 16 hours (37°C in a 5% CO2 incubator). At the end of the incubation period, released luciferase was measured using the One-Glo® kit (Promega #E6110) according to the supplier's protocol. Figure 3 shows that for all donors used, CD22 CAR T cells were able to effectively lyse CD20+CD22+ Raji and CD20-CD22+ Raji clones, while CD22xCD20 or CD20xCD22 CAR T cells were also able to produce the same level of lysis as the CD22 CAR; however, CD22xCD20 or CD20xCD22 CAR T cells were also able to produce the same level of lysis as the CD20+CD22- Raji clone.

[0168] The killing capabilities of the CD20xCD22 CAR T cells, CD22xCD20 CAR T cells, and CD22 CAR T cells generated in Example 1 were also tested in reloading or serial killing assays. Various CAR T cells were thawed, counted, and incubated with various Raji cells (CD20+CD22+, CD20-CD22+, CD20+CD22-) at a 1:1 ratio of CAR-positive T cells to Raji cells. After 3 days of incubation, half of the wells were used to measure released luciferase using the One-Glo® kit (Promega #E6110), while the other half were transferred to a new plate containing Raji cells for further incubation. Luciferase measurements were repeated on days 7, 10, 14, and 17.

[0169] Figure 4 shows that CD22xCD20 CAR T cells or CD20xCD22 CAR T cells were able to effectively kill all tested Raji cells, while CD22 CAR T cells were able to effectively kill only CD20+CD22+ Raji cells and CD20-CD22+ Raji cells. Surprisingly, CD22 CAR T cells had low activity against CD20+CD22- Raji cells, suggesting that CD22 expression was not completely disrupted in this clone. This also suggests that the dual CAR was able to effectively kill target tumor cells with low expression of both target antigens.

[0170] Table 7. List of TALENs used TIFF0007766674000007.tif107129

[0171] Example 6 Tumor burden control in an in vivo dissemination model of B cell lymphoma Daudi cells expressing CD20 and CD22 were modified to express luciferase and GFP. Daudi cells were intravenously injected into NSG immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, Jackson Laboratories). Seven days after tumor implantation, CD20xCD22 CAR T cells (1 million and 3 million), CD22xCD20 CAR T cells (1 million and 3 million), and CD22 CAR T cells (10 million) were intravenously injected into individual mice. Bioluminescence signals (BLI) upon D-luciferin injection were monitored twice weekly until day 60 after CAR T cell injection, and the values ​​are shown in Figure 5. For all CAR T cell treatment conditions tested, BLI signals were reduced compared to vehicle controls or untransduced T cells. Surprisingly, at a low dose of 1 million CAR+ T cells, CD20xCD22 CAR T cells had potent killing activity that was higher than that of CD22xCD20 CAR T cells.

[0172] Example 7 Therapeutic efficacy in an in vivo disseminated B-cell lymphoma model In Example 6, animals infused with Daudi cells, followed by intravenous infusion of CD20xCD22 CAR T cells (1 million), CD22xCD20 CAR T cells (1 million), and CD22 CAR T cells (10 million), were monitored for survival over a 60-day period. As shown in Figure 6, animals treated with any of the tested CAR T cells survived longer than animals treated with either vehicle or non-transduced T cells (NTD). Additionally, and surprisingly, animals treated with CD20xCD22 CAR T cells survived longer than animals treated with CD22xCD20 CAR T cells.

[0173] Example 8 Therapeutic efficacy in an in vivo disseminated B-cell lymphoma model The Raji cell line described in Example 2 was subcutaneously injected into NSG immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ, Jackson Laboratories). All three cell lines were injected simultaneously into each animal; specifically, Raji WT cells were injected into one flank, Raji CD22- cells were injected into the other flank, and CD20- cells were injected into a third flank. One week after tumor injection, CD20xCD22 CAR T cells (3 million and 8 million), CD22xCD20 CAR T cells (3 million and 8 million), and CD22 CAR T cells (8 million) were intravenously injected, and the animals were monitored for survival. As shown in Figure 7, animals treated with CD22 CAR T cells died of disease within a short period of time because they harbored CD22- tumor cells that could not be targeted by the CD22 CAR T cells. Treatment with 3 million and 8 million dual CAR T cells shows that while both treatments were effective, animals treated with CD20xCD22 surprisingly survived for a longer period of time than animals treated with CD22xCD20.

[0174] Example 9 IFNγ release of CD20 and CD22 dual CAR T cells Daudi cells expressing CD20 and CD22 were modified to express luciferase and GFP. These Daudi cells were incubated overnight with CD20xCD22+ CAR T cells, CD22xCD20+ CAR T cells, CD22+ CAR T cells, or CD20+ CAR T cells at a 1:1 (effector:target) ratio. The following day, the plates were centrifuged, and the supernatants were collected. IFN-γ levels released upon tumor incubation with CAR T cells were quantified using a human IFN-γ Quantikine ELISA kit (R&D Systems, DIF50C) according to the manufacturer's instructions. As a positive control, CAR-T cells were incubated with phorbol myristate acetate (PMA, Sigma-Aldrich P8139) and ionomycin (Sigma-Aldrich I0634). Final IFN-γ release values ​​were normalized to those of the positive control. Figure 8 shows that dual CD20xCD22 CAR-T cells and dual CD22xCD20 CAR T cells were activated upon antigen recognition on tumor cells and released higher levels of IFNγ than single CAR (CD20 or CD22) T cells, demonstrating the synergistic benefit of using CD20 and CD22 dual CAR T cells.

Claims

1. A genetically engineered immune cell that expresses a chimeric antigen receptor (CAR) specific for CD22 (CAR22) and a chimeric antigen receptor specific for CD20 (CAR20) on its cell surface, a) CAR22 is i) an antigen-binding domain specific for CD22, comprising a variable heavy chain (VH) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 11 and comprising the H-CDRs of the amino acid sequences SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43, and a variable light chain (VL) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 12 and comprising the L-CDRs of the amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, and optionally including a leader sequence; - Hinge domain derived from CD8α at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; and b) CAR20 is i) an antigen-binding domain specific for CD20, comprising a variable heavy chain (VH) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 15 and comprising the H-CDRs of the amino acid sequences SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, and a variable light chain (VL) comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 16 and comprising the L-CDRs of the amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, and optionally including a leader sequence; - Hinge domain derived from CD8α at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; Including, CAR22 and CAR20 are encoded by exogenous nucleic acids integrated into the genome of the immune cell, and the exogenous nucleic acids are arranged in a 5' to 3' direction as follows: (i) a promoter (e.g., the EF1α promoter); (ii) a nucleic acid encoding CAR20; (iii) a nucleic acid encoding a self-cleaving peptide (e.g., P2A of SEQ ID NO: 19); (iv) a nucleic acid encoding CAR22 whereby expression of CAR20 and CAR22 is controlled by the same promoter; Genetically engineered immune cells.

2. 2. The genetically engineered immune cell of claim 1, wherein CAR22 comprises the amino acid sequence of SEQ ID NO: 14 and CAR20 comprises the amino acid sequence of SEQ ID NO:

18.

3. The genetically engineered immune cell of claim 1 or 2, wherein the exogenous nucleic acid comprises the nucleic acid sequence of SEQ ID NO:

32.

4. The genetically engineered immune cell of any one of claims 1 to 3, wherein the immune cell is selected from the group consisting of a T cell, a NK cell, and a macrophage.

5. The genetically engineered immune cell of any one of claims 1 to 4, which is a TCR-negative T cell.

6. The engineered T cells At least one allele encoding TCRα, TCRβ, and / or CD3 that is inactivated by a mutation 6. The genetically engineered immune cell of claim 5, comprising:

7. The engineered T cells At least one allele selected from β2m, PD1, CTLA4, dCK, CD52, and / or GR that is inactivated The genetically engineered immune cell of any one of claims 5 to 6, comprising:

8. A pharmaceutical composition comprising the engineered immune cells of any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. 1. An isolated polynucleotide encoding CAR20 and CAR22, comprising, from 5' to 3': a) a promoter (such as the EF1α promoter) that controls expression of CAR20, followed by a nucleic acid encoding CAR20, wherein CAR20 is: i) an antigen-binding domain specific for CD20, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 15 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 16 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, optionally including a leader sequence; - Hinge domain derived from CD8α at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; A promoter that controls the expression of CAR20, comprising: b) a nucleic acid encoding CAR22, wherein CAR22 is i) an antigen-binding domain specific for CD22, comprising: a variable heavy chain (VH) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 11 and comprising the H-CDRs of amino acid sequences SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43; and a variable light chain (VL) comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 12 and comprising the L-CDRs of amino acid sequences SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46, optionally including a leader sequence; - Hinge domain derived from CD8α at least one extracellular domain comprising ii) the transmembrane domain from CD8α, and iii) an intracellular domain containing the 4-1BB stimulatory domain and the CD3ζ signaling domain; a nucleic acid encoding CAR22, Including, wherein the nucleic acid a) and the nucleic acid b) are on one nucleic acid molecule, and a nucleic acid sequence encoding a self-cleaving peptide (such as P2A, T2A, E2A, or F2A) is located between the nucleic acid a) and the nucleic acid b). Isolated polynucleotides.

10. 10. The isolated polynucleotide of claim 9, comprising the nucleic acid sequence of SEQ ID NO: 32 (CAR20xCAR22 construct).

11. A vector comprising the isolated polynucleotide of any one of claims 9 to 10.

12. 12. An ex vivo method for preparing an engineered immune cell according to any one of claims 1 to 7, comprising introducing a polynucleotide according to any one of claims 9 to 10 or a vector according to claim 11 into an immune cell.

13. The engineered immune cell of any one of claims 1 to 7 for use as a medicament.

14. 8. The engineered immune cell of any one of claims 1 to 7 for use in the treatment of cancer associated with expression of CD20 and / or CD22.

15. 15. The engineered immune cell for use of claim 14, wherein the cancer is selected from the group consisting of lymphoma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), leukemia, multiple myeloma (MM), B-chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), acute lymphoid carcinoma, acute myeloid leukemia (AML).

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