Anti-CD123 antibodies, anti-CD123 chimeric antigen receptors and anti-CD123 chimeric antigen receptor T cells

Abstract

The present invention relates to an anti-CD123 monoclonal antibody, or a fragment thereof, that specifically binds to the α chain of the human interleukin 3 (IL3) receptor. Furthermore, the present invention relates to isolated nucleic acid molecules encoding chimeric antigen receptors (CARs), including polypeptides encoded by the nucleic acid molecules, as well as isolated chimeric antigen receptor (CAR) molecules having such antibodies or antibody fragments, antibodies having an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, or a fragment of the antibody. The present invention also relates to vectors having nucleic acid molecules encoding CARs, as well as T cells containing the vectors, and the use of T cells expressing CAR molecules for the treatment of proliferative disorders in mammals, such as cancer.

Description

[Technical field]

[0001] The present invention relates to monoclonal antibodies and uses thereof.

[0002] In particular, the present invention relates to anti-CD123 monoclonal antibodies, or fragments thereof, that specifically bind to the alpha chain of the human interleukin 3 (IL3) receptor.

[0003] The present invention further relates to isolated nucleic acid molecules encoding chimeric antigen receptors (CARs), the CARs including polypeptides encoded by said nucleic acid molecules, as well as isolated chimeric antigen receptor (CAR) molecules having such antibodies or fragments of antibodies, antibodies having an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, or a fragment of said antibody.

[0004] The present invention also relates to a vector carrying a nucleic acid molecule encoding a CAR, as well as a T cell comprising said vector. The present invention also relates to the use of T cells expressing a CAR molecule for the treatment of a proliferative disease in a mammal, such as cancer. Summary of the Invention [Problem to be solved by the invention]

[0005] The fight against cancer is a global challenge. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive cancer. It is a Crohn's disease originating from plasmacytoid dendritic cell (pDC) precursor cells with widespread cutaneous lesions that invariably progress to scattered aggressive leukemia. The median overall survival (OS) for this type of cancer is 8-12 months.

[0006] Both the prognosis and treatment of BPDCN are extremely poor: Almost all patients who receive intensive chemotherapy will relapse within weeks to months, making the chemotherapy ineffective.

[0007] This results in treatment with a fusion protein called SL-401, consisting only of human interleukin 3 (IL3) fused to diphtheria toxin. References: Angelo-Deletara F, Roggie A, Frankel AE, Lamartie B, Seille E, Beachelle S, et al. Sensitivity of blastic plasmacytoid dendritic cell tumors to the interleukin-3 receptor-targeted biologic SL-401 in vivo and in vitro. Hematologica, 2015, 100(2):223-30.

[0008] The present invention improves this situation.

[0009] Applicants discovered that a protein called CD123-, the alpha chain of the interleukin-3 (IL3) receptor, was overexpressed in the cancer cells of all BDPCN patients. References: Garnache-Otto F, Foillard J, Ferran C, Beachelle S, Trimoreau F, Seille E, et al., Expanded diagnostic criteria for plasmacytoid dendritic cell leukemia, British Journal of Hematology, 2009, 145(5):624-36.

[0010] As a result, the present invention focuses on using CD123 as a therapeutic target.

[0011] In this context, and with the aim of developing biotherapeutics directed against CD123, the applicant has succeeded in generating, developing, screening and selecting anti-CD123 monoclonal antibodies (MA) that specifically bind to human CD123. In general, these antibodies can be used therapeutically. More specifically, they can be used therapeutically as a weapon against plasmacytoid dendritic cell neoplasm leukemia and other pathologies expressing CD123.

[0012] A rapidly evolving technology to fight cancer is the use of engineered T cells (or T lymphocytes). These T cells contain so-called chimeric antigen receptors that are specifically directed against target antigens expressed by cancer cells and are commonly referred to as CARs.

[0013] With this in mind, applicants have overcome numerous obstacles to engineer T cells to express a CAR that specifically targets CD123 to specifically and efficiently combat cancer cells using a newly developed anti-CD123 antibody.

[0014] This new therapeutic armamentarium may be applicable to patients with BPDCN or relapsed BPDCN, as well as patients with BPDCN who have been treated with intensive chemotherapy, therapy with SL-401, hematopoietic stem cell allogeneic transplantation, or other therapies. [Means for solving the problem]

[0015] In order to solve the above problem, one embodiment of the present invention is ―9 K under M D Value, 9.0×10 ―4 s -1 Dissociation rate constant K less than off , at least 7 × 10 4 Ms -1 Association rate constant K on An antibody that specifically binds to the antigen CD123, characterized in that it has the antibody is selected from the group consisting of AB1, AB2, AB3, AB4, AB5, and AB6; -AB1 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 25, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 1, and the light chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 26, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 14, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 2; -AB2 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 27, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 15, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 3, and the light chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 28, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 16, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 4; -AB3 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 29, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 5, and the light chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 30, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 6; -AB4 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 31, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 19, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 7, and the light chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 32, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 20, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 8; -AB5 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 9, the light chain comprising a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO: 10, -AB6 is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:35, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:23, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:11, and the light chain comprises a complementarity determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:36, a complementarity determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:24, and a complementarity determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:12.

[0016] According to an embodiment of the invention, AB1 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 85 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 86, the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 25, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 1; the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:2; AB2 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 87 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 88; the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:27, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:15, and a complementarity determining region 3 (CDR3) having at least 90% homology to the amino acid sequence of SEQ ID NO:3; the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:4; AB3 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 89 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 90; the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 29, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) having at least 90% homology to the amino acid sequence of SEQ ID NO: 5; the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 30, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 6; AB4 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 91 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 92; the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 31, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 19, and a complementarity determining region 3 (CDR3) having at least 90% homology to the amino acid sequence of SEQ ID NO: 7; the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 32, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 20, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 8; AB5 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 93 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 94; the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) having at least 90% homology to the amino acid sequence of SEQ ID NO: 9; the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:34, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:22, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:10; AB6 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 95 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 96; the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 11; The light chain comprises a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 36, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 24, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 12.

[0017] According to a preferred embodiment of the invention, AB1 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 85 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 86, The heavy chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 25, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 1, the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 26, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 14, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 2; AB2 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 87 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 88; The heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 27, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 15, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 3; the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 28, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 16, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 4; AB3 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:89 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:90; The heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 29, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 5, the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 30, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 6; AB4 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:91 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:92; The heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 31, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 19, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 7; the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 32, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 20, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 8; AB5 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:93 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:94; The heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 9, the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 10; AB6 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:95 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:96; The heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 11; The light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 36, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 24, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 12.

[0018] According to another embodiment of the invention, the heavy chain of AB1 has a variable region comprising a first framework region (FR1) consisting solely of the amino acid sequence of SEQ ID NO: 37, a second framework region (FR2) consisting solely of the amino acid sequence of SEQ ID NO: 38, a third framework region (FR3) consisting solely of the amino acid sequence of SEQ ID NO: 39, and a fourth framework region (FR4) consisting solely of the amino acid sequence of SEQ ID NO: 40, and the light chain of AB1 has a variable region comprising a first framework region (FR1) consisting solely of the amino acid sequence of SEQ ID NO: 61, a second framework region (FR2) consisting solely of the amino acid sequence of SEQ ID NO: 62, a third framework region (FR3) consisting solely of the amino acid sequence of SEQ ID NO: 63, and a fourth framework region (FR4) consisting solely of the amino acid sequence of SEQ ID NO: 64; The heavy chain of AB2 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 41, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 42, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 43, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 44, and the light chain of AB2 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 65, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 66, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 67, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 68; The heavy chain of AB3 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 45, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 46, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 47, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 48, and the light chain of AB3 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 69, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 70, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 71, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 72; The heavy chain of AB4 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 49, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 50, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 51, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 52, and the light chain of AB4 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 73, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 74, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 75, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 76; The heavy chain of AB5 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 53, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 54, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 55, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 56, and the light chain of AB5 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 77, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 78, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 79, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 80; The heavy chain of AB6 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 57, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 58, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 59, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 60, while the light chain of AB6 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 81, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 82, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 83, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO: 84.

[0019] According to another embodiment of the invention, there is provided an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment comprising an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein the CAR comprises an antibody having an anti-CD123 binding domain, or a fragment of said antibody, The anti-CD123 binding domain comprises the following heavy and light chains: (i) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:25, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:13, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:1, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:2; (ii) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:27, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:15, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:3, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:4; (iii) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:29, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:17, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:5, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:30, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:18, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:6; (iv) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 31, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 19, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 7, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 32, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 20, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 9, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 10; or (vi) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 11, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 36, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 24, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 12.

[0020] According to an embodiment, the anti-CD123 binding domain of the isolated nucleic acid molecule of the invention is selected from the group consisting of an antibody, Fv, scFv, Fab, or other antibody fragment, with scFv being preferred. In another embodiment, the intracellular signaling domain is CD3-zeta (CD3ζ), optionally including a costimulatory domain selected from the group consisting of CD28, 4.1BB, inducible T cell costimulator (ICOS), OX-40, and binding thereof.

[0021] Another embodiment of the present invention is characterized in that an isolated polypeptide molecule is encoded by a nucleic acid molecule as described above.

[0022] A further aspect of the invention is characterized in that the isolated chimeric antigen receptor (CAR) molecule comprises an antibody or an antibody fragment, said antibody or antibody fragment comprising an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, The anti-CD123 binding domain comprises the following heavy and light chains: (i) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:25, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:13, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:1, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:2; (ii) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:27, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:15, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:3, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:4; (iii) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:29, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:17, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:5, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO:30, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO:18, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO:6; (iv) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 31, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 19, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 7, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 32, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 20, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 9, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 10; or (vi) a heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 11, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90% identity to the amino acid sequence of SEQ ID NO: 36, a complementarity determining region 2 (CDR2) having at least 90% identity to the amino acid sequence of SEQ ID NO: 24, and a complementarity determining region 3 (CDR3) having at least 90% identity to the amino acid sequence of SEQ ID NO: 12.

[0023] Another embodiment of the present invention is an isolated chimeric antigen receptor (CAR) molecule, characterized in that said CAR is as described above.

[0024] Another embodiment of the present invention is characterized in that an expression vector comprises a nucleic acid molecule of the present invention, said vector being selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0025] Another embodiment of the invention is characterized in that the modified immune cell comprises a nucleic acid molecule of the invention or a vector of the invention. In one embodiment of the invention, the cell expresses in its membrane a CAR as described above.

[0026] According to one embodiment, the cells are useful as a medicament, preferentially for the treatment of a proliferative disease in a mammal, preferably a human, said proliferative disease being a disease associated with CD123 overexpression, preferably for the treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN).

[0027] A further embodiment of the invention is a pharmaceutical composition comprising the modified immune cells described above and a pharmaceutical excipient.

[0028] Yet another embodiment of the invention is a method for modifying immune cells, comprising transfecting cells with an expression vector as described above.

[0029] A further aspect of the invention features an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the nucleic acid molecule consisting of a sequence having at least 85%, preferentially 90%, more preferentially 95%, and most preferentially 100% identity to the amino acid sequence of SEQ ID NO:97.

[0030] Other characteristics and advantages of the invention will stand out and / or become clear on reading the following description of specific examples, given in an illustrative and non-limiting manner, and on reading the following figures: [Brief description of the drawings]

[0031] [Figure 1] FIG. 2 shows the nucleotide and amino acid sequences of complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) of both the heavy and light chains of the antibody of the present invention; [Diagram 2] FIG. 2 shows the nucleotide and amino acid sequences of framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4) of the heavy chain of the antibody of the present invention; [Diagram 3] FIG. 2 shows the nucleotide and amino acid sequences of framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4) of the light chain of the antibody of the present invention; [Figure 4] FIG. 2 shows the consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the first antibody of the present invention; [Diagram 5] FIG. 2 shows the consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of a second antibody of the present invention; [Figure 6]FIG. 2 shows the consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the third antibody of the present invention; [Figure 7] FIG. 2 shows the consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the fourth antibody of the present invention; [Figure 8] FIG. 5 shows the consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the fifth antibody of the present invention; [Figure 9] FIG. 2 shows the consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the sixth antibody named in the present invention; [Figure 10] Figure 1 shows flow cytometry graphs based on evaluation of CD123 expression on normal hematopoietic cells and BPDCN; [Figure 11] Table and flow cytometry graphs based on evaluation of CD123 expression in BPDCN and other non-pDC acute leukemias; [Figure 12] 1 is a diagram of the design of the chimeric antigen receptor of the present invention, and Western blot and flow cytometry graphs relative to the production of T cells according to the present invention comprising the chimeric antigen receptor of the present invention; [Figure 13] FIG. 1 shows the nucleic acid sequence of a chimeric antigen receptor according to the present invention; [Figure 14] 1 is a flow cytometry graph showing a phenotypic assay and relative expression of CAR T cells according to the invention compared to non-transfected T cells; [Figure 15] Illustrates co-culturing target cells with the chimeric antigen receptor of the present invention and untransfected cells, as well as flow cytometry graphs of the specific cytotoxicity of the CAR of the present invention against BPDCN cell lines and BPDCN patient-derived xenograft cells; [Figure 16] 1 is a flow cytometry graph relative to functional characterization of CD123R CAR T cells of the present invention in co-culture with CD123+ target cells; [Figure 17]Illustrative xenograft model diagrams, bioluminescence imaging and corresponding graphs, and flow cytometry graphs for in vivo anti-tumor activity of T cells of the present invention against BPDCN cell line (CAL-1) and patient-derived xenograft cells (PDX); [Figure 18] 1 is a diagram of a xenograft model of the in vivo antitumor activity of T cells of the present invention against a BPDCN cell line (CAL-1), bioluminescence imaging and a corresponding graph; [Figure 19] Fluorescence graphs of CD123 expression in different cell types, immunohistochemistry images of CD123 expression in tumor and normal tissues, flow cytometry graphs of relative CD123 expression, diagrams of the production of autologous T cells of the invention, and flow cytometry graphs of the relative toxicity of T cells of the invention to CD123 versus cells expressing CD123 at low levels; [Figure 20] Graph showing flow cytometry-based evaluation of T cell toxicity relative to autologous T cells against cell lines (CAL-1 and DAUDI); and [Figure 21] 13A-C are flow cytometry graphs of CD123 T cells of the present invention from a BPDCN patient that induced specific cytotoxicity against autologous BPDCN blasts.

[0032] The figures and descriptions herein contain, for the most part, elements of an explicit nature, and therefore the descriptions and figures are used not only to better understand the invention, but also, where appropriate, to contribute to its definition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] In this description, the term "and / or" should be interpreted as including the relevant cases where one or more may occur. For example, the phrase "proteins or protein sequences can be prepared using standard recombinant and / or synthetic methods" indicates that the protein or protein sequence can be prepared using both standard recombinant and synthetic methods, or that the protein or protein sequence can be prepared using standard recombinant methods, or that the protein or protein sequence can be prepared using synthetic methods. More generally, the term "and / or" used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" and "B". Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0034] The term "comprising" should be interpreted as embracing all features specifically mentioned as well as any additional, unspecified features. More generally, the term "comprising" will be understood to mean the inclusion of a stated step or element, or group of steps or elements, but not the exclusion of other steps or elements, or group of steps or elements. The term "consisting of" is to be interpreted as meaning that no features are present other than those specifically mentioned. Furthermore, the indefinite articles "a" or "an" do not exclude a plurality.

[0035] Reference to "one embodiment," "a particular embodiment," "an embodiment," "an additional embodiment," "another embodiment," or "further embodiment," or to "one embodiment," "a particular embodiment," "an embodiment," "an additional embodiment," "another embodiment," or "further embodiment," or any combination thereof, means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. The articles "a," "an," and "the" are used herein to mean one or to more than one (i.e., at least one) of the grammatical object article.

[0036] The term "gene" refers to a DNA sequence (nucleic acid sequence or nucleotide sequence) that codes for a specific sequence of amino acids. A gene includes all or part of one or more proteins or enzymes. It may also include regulatory DNA sequences (such as promoter sequences) that at least partially determine the conditions under which the gene is expressed. Some genes that are not structural genes are transcribed from DNA to RNA, but none are transcribed into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may contemplate a genomic sequence that codes for a protein, i.e., a sequence including regulators, promoter, intron and exon sequences.

[0037] The term "identical" or "percent" (%), or "identical" in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same or have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are well known in the art. One such non-limiting example of a sequence alignment algorithm is described in Carlin et al., Proceedings of the National Academy of Sciences of the United States of America, 87:2264-2268, 1990. A modification of it was described in Carlin et al., Proceedings of the National Academy of Sciences of the United States of America, 90:5873-5877, 1993. It has also been incorporated into the NBLAST and XBLAST programs (Altschuhl et al., Nucleic Acids Research, 25:3389-3402, 1991). Gapped BLAST can also be used as described in: Altschull et al., Nucleic Acid Research, 25:3389-3402, 1997. BLAST-2, WUBLAST-2 (Altschull et al., Enzymology Laboratory Methods, 266:460-480, 1996), ALIGN, ALIGN-2 (Genentech, South San Francisco, CA) or Megalign (DNASTAR) are other publicly available software programs that can be used to align sequences. Additionally, the Needleman and Wunsch (Journal of Molecular Biology, (48):444-453, 1970) algorithm can be used to determine percent identity between two amino acid sequences (e.g., using either the Blossum 62 matrix or the PAM250 matrix, with gap penalties of 16, 14, 12, 10, 8, 6, or 4 and length penalties of 1, 2, 3, 4, or 5). Alternatively, the percent identity between nucleotide or amino acid sequences can be determined using the algorithm of Mias and Milla (CABIOS, 4:11-17, 1989). For example, percent identity can be determined using the ALIGN program (version 2.0) and using PAM120 with a residue table of length penalty 12 and gap penalty 4. Appropriate parameters for maximum alignment with a particular alignment software can be determined by one of skill in the art. Typically, the default parameters of the alignment software are used.

[0038] A sequence that is "at least 85% identical to a reference sequence" or "something that is at least 85% identical to a reference sequence" is a sequence that has 85% or more, particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length with the entire length of the reference sequence. In the present context, "percentage of identity" is calculated using pairwise alignment (i.e., two sequences are compared over their entire length). As mentioned above, methods for comparing the identity of two or more sequences are well known in the art. For example, bioinformatics tools or programs such as EMBOSS Needle available on the ebi.ac.uk website can be used to generate pairwise sequence alignments of the present invention. Generally, many bioinformatics tools and programs use the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Molecular Biology. 48:443-453) to find the optimal alignment (including gaps) of two sequences, taking into account their entire length. However, other algorithms may be implemented in various bioinformatics tools or programs.

[0039] More generally, for the purposes of the present invention, "sequence identity" or "sequence homology" is calculated by comparing two aligned sequences in a comparison window. Sequence alignment allows the number of positions (nucleotides or amino acids) common to both of the two sequences in the comparison window to be determined. The number of common positions is then divided by the total number of positions in the comparison window and multiplied by 100 to determine the percentage of homology. The determination of the percentage of sequence identity can be performed manually or by well-known bioinformatics computer programs.

[0040] According to the invention, the percentage of identity between two polypeptides can be calculated using the EMBOSS:Needle(global) program with a "Gap open" parameter equal to 10.0 and a "Gap extend" parameter equal to 0.5.

[0041] A protein having (or consisting of) an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence, or a portion thereof, may contain mutations, such as deletions, insertions and / or substitutions, compared to the reference sequence.

[0042] In the case of substitutions, a protein consisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence (i.e., "homology") from another species other than the reference sequence. Amino acid substitutions may be conservative or non-conservative. A conservative substitution is one in which one amino acid is replaced with another amino acid having a similar side chain, i.e., similar structural and / or similar chemical properties. In this regard, conservative substitutions may occur between: - Amino acids with non-polar side chains: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Leu), proline (Pro), phenylalanine (Phe), methionine (Met), tryptophan (Trp); -Amino acids with uncharged polar side chains: asparagine (Asn), glutamine (Gin), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys); -Amino acids with acidic side chains: aspartic acid (Asp), glutamic acid (Glu); -Amino acids with basic side chains: lysine (Lys), arginine (Arg), histidine (His); -Amino acids with β-branched side chains: threonine (Thr), valine (Val), isoleucine (lie); -Amino acids with aromatic side chains: tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), histidine (His). Non-conservative substitutions among them may occur randomly. Depending on the position of the individual amino acids in the three-dimensional structure of the protein, alternative definitions may be given to conservative and non-conservative substitutions.

[0043] The techniques used in the present invention employ, unless otherwise indicated, methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, genetic and / or biotechnology cell engineering, immunology, and cell biology known to those skilled in the art. Where necessary, such methods are described for illustrative purposes. Most of these and other techniques are described in the literature. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Practice in Molecular Biology (John Wiley & Sons, updated July 2008); Abridged Practice in Molecular Biology: Summary of Methods from the Current Practice in Molecular Biology, John Wiley & Sons, 4th ed., 1999; DNA Cloning: A Practical Approach, Vols. 1 and 2 (IRL Press, Oxford, 1985); Techniques for the Analysis of Complex Genomes, Anand, American Press, 1992; Transcription and Translation, B. Names and S. Higgins, 1984; A Practical Guide to Molecular Cloning, Perval, 1984; Antibodies, Harlow and Lahey, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1998; Current Practice in Immunology, QE Corrigan, AM Kruisbeek, DH Margulies, EM Shevaci and W. Strober, 1991; Annals of Immunology. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred embodiments of the compositions, methods and materials are described herein.

[0044] As understood by those skilled in the art and described herein, an "antibody", also referred to as an "immunoglobulin", comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second and third constant region, and each light chain consists of a variable region and a constant region. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond.

[0045] In mammals, there are two types of light chains, lambda (I) and kappa (k). Furthermore, there are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule, classified as a, d, e, y, and m: IgA, IgD, IgE, IgG, and IgM.

[0046] Each chain contains distinct sequence domains. Light chains contain two domains or regions, a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). More precisely, a complete antibody is in the shape of a "Y". The base or stem of the Y consists of the second and third constant regions (and for IgE and IgM, a fourth constant region) of the two heavy chains linked by disulfide bonds, sometimes called interchain disulfide bonds forming a hinge. Heavy chains y and d have a constant region of three inline Ig domains and a hinge region for added flexibility. Heavy chains m and e have a constant region of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 region" and "CH3 region", respectively. Each arm or branch of the Y contains the variable and first constant regions of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. More generally, the variable regions of the light and heavy chains (VL and VH) determine binding recognition and specificity to the antigen. The constant regions of the light and heavy chains (CL and CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable region. The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant.

[0047] Antibody binding sites are mainly composed of residues from hypervariable or complementarity determining regions (CDRs). Occasionally, residues outside of hypervariable regions or framework region (FR) residues may affect the structure of the entire domain and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of an original immunoglobulin binding site. Each immunoglobulin light and heavy chain has three CDRs, generally named CDR1, CDR2 and CDR3, more specifically, CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Other nomenclatures can be found in the literature. Thus, the CDRs located in the variable region of the antibody heavy chain can be referred to as CDRH1, CDRH2 and CDRH3, while the CDRs located in the variable region of the antibody light chain can be referred to as CDRL1, CDRL2 and CDRL3. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Thus, a conventional antibody antigen-binding site contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions.

[0048] As mentioned above, the light and heavy chain variable regions contain regions called "framework" regions separated by hypervariable CDR regions. The CDRs can be defined or identified as described in Kabat et al., sequences or structures by Choucia et al., sequences by Wu, TT and Kabat, EA, Journal of Experimental Medicine, 132(2):211-50, 1970; Bowden, P. and Rabat, EA, Proceedings of the National Academy of Sciences, 84:2440-2443, 1987; Kabat et al., Sequences of Proteins of Interest in Immunology, United States Department of Health and Human Services, 1991, or structures by Choucia et al., sequences by Choucia, C. and Leschi, AM, Journal of Molecular Biology, 196(4):901-917, 1987; Choucia, C. et al., Nature, 342:877-883, 1989.

[0049] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. More generally, "framework regions" (FRs) are relatively conserved among different immunoglobulins of a single species. The framework region of an antibody is more or less a combination of all the framework regions of the constituent light and heavy chains, i.e., they position and align the CDRs in three-dimensional space when the antibody is folded and active. The CDRs are primarily responsible for binding to the epitope of the antigen. As mentioned above, the CDRs of each chain are called CDR1, CDR2, and CDR3. In fact, the CDRs are numbered sequentially from the N-terminus. The light and heavy chains of an immunoglobulin have four FRs, named FR1, FR2, FR3, and FR4, respectively, more precisely: light chain FR1-L, FR2-L, FR3-L, FR4-L, and light chain FR1-H, FR2-H, FR3-H, FR4-H. Accordingly, the light chain variable domain may be designated as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable region as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Knowing the amino acid sequences of the CDRs, the skilled artisan can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H FR3-H, FR4-H.

[0050] The references in this specification, "V H " or "VH" refers to the variable region of an immunoglobulin heavy chain, including an antibody, Fv, scFv, Fab, or other antibody fragment disclosed herein. L References to " or "VL" refer to the variable region of an immunoglobulin light chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein.

[0051] As used herein, the term "antibody" or "immunoglobulin" refers primarily to conventional antibodies, in particular monoclonal antibodies, and fragments thereof, as well as to single domain antibodies and fragments thereof, in particular the variable heavy chains of single domain antibodies, and also to chimeric, humanized, bispecific or multispecific antibodies. The antibody is preferably a human, murine or humanized antibody.

[0052] A "monoclonal antibody" or "mAb" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the heavy and light chain genes of a single antibody. Monoclonal antibodies are produced by methods well known in the art, for example, by producing hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells, or by production by recombinant techniques, i.e., protein engineering. Monoclonal antibodies include humanized monoclonal antibodies. More generally, monoclonal antibodies are molecules of a single amino acid composition directed against a specific antigen, and are not to be construed as requiring production of the antibody by any particular method other than those known in the art.

[0053] The term "chimeric antibody" in its broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In particular, chimeric antibodies contain the VH and VL domains of an antibody derived from a non-human animal, associated with the CH domain and CL domain of another antibody, in particular a human antibody. The non-human animal can be any animal, such as a mouse, rat, hamster, rabbit, etc. Chimeric antibodies can also represent multispecific antibodies that have specificity for at least two different antigens.

[0054] The term "antibody" or "immunoglobulin" also includes the more recently described and developed "single domain antibodies". Single domain antibodies are antibodies whose complementarity determining regions (CDRs) are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from traditional four-chain antibodies, and engineered single domain antibodies. Single domain antibodies can be from any family, in particular mouse, human or rabbit. Single domain antibodies may be naturally occurring single domain antibodies known as heavy chain antibodies devoid of light chains. In particular, the Camelidae family (e.g. camel, dromedary, llama, alpaca and guanaco) produce heavy chain antibodies naturally devoid of light chains.

[0055] The variable heavy chains of these single domain antibodies that lack light chains are known in the art as "VHH" or "nanobodies". Like conventional VH domains, VHHs contain four framework regions (FRs) and three complementarity determining regions (CDRs). One particular advantage of nanobodies over conventional antibodies is that they are about 10 times smaller than IgG molecules. As a result, properly folded, functional nanobodies can be produced by in vitro expression in high yields. Furthermore, it has been described in the art that nanobodies are very stable and resistant to the action of proteolytic enzymes. The properties and production of nanobodies are reviewed, for example, in Harmsen and DeHard HJ, Applied Microbiology and Biotechnology., 2007, November, 77(l):13-22.

[0056] In a preferred embodiment, the antibody is a humanized antibody (such as a humanized monoclonal antibody) that specifically binds to a surface protein on a tumor cell. A "humanized" antibody is an immunoglobulin that includes a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. Thus, all parts of a humanized immunoglobulin, except for the CDRs, are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. A humanized or other monoclonal antibody may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. A humanized antibody may be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0057] More generally, the term "humanized monoclonal antibody" refers to a monoclonal antibody that is of fully or partially non-human origin and has been modified to replace certain amino acids, particularly in the framework regions of the heavy and light chains, in order to avoid or minimize immune responses in humans. The constant domains of a humanized antibody are most often human CH and CL domains. Various methods for the humanization of antibody sequences are well known in the art; see, for example, the review by Almagro and Franson, 2008, Frontiers in Bioscience. 13:1619-1633. One commonly used method is the so-called CDR grafting or antibody reshaping. This technique involves grafting the CDR sequences of a donor antibody, typically a mouse antibody, onto the framework framework of a human antibody of different specificity. Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of the CDR-grafted non-human antibody, back mutations may be introduced at selected positions of the CDR-grafted antibody in order to retain the binding specificity and affinity of the parent antibody. Identification of possible backmutation positions can be performed using information available in the literature and antibody databases. Candidate amino acid residues for backmutation are typically those located on the surface of the antibody molecule, while hidden or less surface-exposed residues will usually remain unchanged. Resurfacing, another humanization technique to CDR grafting and backmutation, is a method in which surface residues are modified to human residues while retaining the non-surface-exposed residues of non-human origin. Another alternative technique known as directed selection can be used to derive fully human antibodies that preserve the epitopes and binding properties of the parent antibody from antibodies such as murine or rat. Reference: Jesper et al., Biotechnology, 12,899,1994. A further method of humanization is the so-called 4D-humanization. The implementation guidelines for 4D-humanization are described in patent application WO2009032661A1 (or US20110027266) and are exemplified below in the application of 4D humanization to humanize rat antibody variable light and heavy domains (VL and VH).As an example, a rat antibody homology model was typically run in MOE software (v.201 1.10 - Chemical Computing Group, Quebec, Canada) using the PDB structure as a template. Reference: Berman et al., Nucleic Acid Research, 28:235-242, 2000. It was then energy minimized using standard procedures implemented in MOE. Molecular dynamics (MD) simulations were then performed on the minimized 3D homology model of the rat antibody (performed in MOE software) and compared with 49 human models, including those derived from 7 representative light chains (vkl, vk2, vk3, vk4, vlambdal, vlambda2, vlambda3) and 7 representative heavy chains (vhl a, vhl b, vh2, vh3, vh4, vh5, vh6) designed by LGCR / SDI and available in MOE. For example, one model of the linkage (Vkx-Vhx) with the best combination of hydrophobicity, electrostatic components outside the CDRs and sequence identity was selected for 4D-humanization. For pairwise association of the rat antibody variable domain with the selected model, the sequences were typically aligned based on the optimal 3D overlap of the alpha carbons of the corresponding homology models. Undesirable motifs were then considered and altered. Finally, the resulting humanized sequences were blasted for sequence similarity against, for example, the IEDB database (http: / / www.immuneepitope.org; version 2012 / 01 / 30 is accessible locally), ensuring that none of the sequences contained any known B-cell or T-cell epitopes described.

[0058] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences.

[0059] Although amino acid residues that are part of the CDRs will typically not be modified for humanization, in some cases it may be desirable to modify individual CDR amino acid residues, for example to remove glycosylation sites, deamidation sites, or undesired cysteine ​​residues. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of N-glycosylation sites may be achieved by mutating either the Asn or Ser / Thr residue to another residue, particularly by conservative substitution. Deamidation of asparagine and glutamine residues may occur due to factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation when present primarily in Asn-Gly sequences, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such deamidation sites, particularly Asn-Gly, are present in a CDR sequence, it may be desirable to eliminate said site, generally by conservative substitution to remove one of the involved residues. Substitutions in the CDR sequences to remove one of the involved residues are also intended to be encompassed by the present invention.

[0060] The term "antibody" includes antigen-binding fragments thereof. Such "fragments" include portions of intact antibodies, particularly the antigen-binding or variable regions of intact antibodies. Examples of antibody fragments include Fab' fragments, F(ab)'2 fragments, Fv, single chain Fv proteins ("scFv") and portions of full-length antibodies that are responsible for antigen binding, diabodies, diabodies formed from antibody fragments, bispecific and multispecific antibodies. A fragment of a conventional antibody may be a heavy chain antibody or a single domain antibody such as a VHH. The term "Fab" refers to an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, to which about the N-terminal half of the H chain and the entire L chain are bound via disulfide bonds among fragments obtained by treating IgG with the protease papain. The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity, to which a fragment obtained by treating IgG with the protease papain is slightly larger than Fab, to which the fragment is bound via disulfide bonds in the hinge region. "Single-chain Fv" or "scFv" antibody fragments contain the VH and VL domains of an antibody, where these domains are present in either orientation (e.g., VL-VH or VH-VL) in a single polypeptide chain. The single chains can be cloned to form V-region genes of fusion cells specific for a desired target. The production of such fusion cells has become routine. For example, Orlandi et al., Proceedings of the National Academy of Sciences of the United States of America, 1989, 86: 3833-3837, describe techniques that can be used to clone the variable heavy chain (VH) and variable light chain (VL).

[0061] Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. More specifically, single-chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers, which are typically expressed from gene fusions comprising VH and VL encoding genes linked by a peptide-encoding linker. In some embodiments, human scFv fragments comprise CDRs held in the proper structure, particularly by using recombinant gene technology. Bivalent and multivalent antibody fragments may be formed spontaneously by association of monovalent scFvs, or may be formed by coupling monovalent scFvs by a peptide linker, such as a bivalent sc(Fv)2. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" means two dsFvs linked by a peptide linker. The term "bispecific antibody" or "BsAb" generally refers to an antibody that combines the antigen-binding sites of two antibodies in a single molecule. Thus, BsAbs can bind two different antigens simultaneously. As described in EP2050764A1, genetic engineering has been used more and more frequently to design, modify and produce antibodies or antibody derivatives with desired binding properties and effector functions. The term "multispecific antibody" refers to an antibody that combines the antigen-binding sites of two or more antibodies in a single molecule. The term "bispecific antibody" refers to a small antibody fragment with two antigen-binding sites, which fragment comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short for pairing between the two domains on the same chain, the domains are paired with complementary domains of another chain to generate two antigen-binding sites.

[0062] The present invention provides immune effector cells genetically modified with vectors for the expression of chimeric antigen receptors (CARs) capable of redirecting cytotoxicity towards tumor cells. CARs are molecules that combine antibody-based specificity for a target antigen (e.g., tumor antigen) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cell-mediated immune activity. The term "chimeric" as used herein describes being composed of different proteins or portions of DNA from different origins. The main feature of CARs is their ability to redirect immune effector cell specificity, thereby inducing proliferation, cytokine production, phagocytosis or production of molecules capable of mediating cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner, taking advantage of the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands or cell-specific co-receptors.

[0063] As used herein, the terms "binding domain," "extracellular binding domain," "antigen-specific binding domain," and "extracellular antigen-specific binding domain" are used interchangeably and provide a CAR with the ability to specifically bind to a target antigen of interest. A binding domain may include any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or component thereof). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner of a biological molecule of interest. As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" refer to binding a molecule with a binding affinity greater than background binding to another molecule. A binding domain (or a CAR containing a binding protein or a fusion protein containing a binding domain) may be used to bind to a CAR, e.g., about 10 5 M -1A CAR protein "specifically binds" to a target molecule if it binds to or associates with the target molecule with an affinity or Ka (i.e., the equilibrium association constant of a specific binding interaction with units of 1 / M) or greater. In accordance with the present disclosure, the affinity of a binding domain polypeptide to a CAR protein can be readily determined using conventional techniques such as competitive ELISA (enzyme-linked immunosorbent assay). Furthermore, the qualitative and quantitative determination of binding related to affinity, the so-called K D The value of K D correlates with the affinity and sensitivity of the antibody. D is the k between the antibody and its antigen 0ff / k 0n is the ratio of K D and affinity are inversely proportional. D Since the value is proportional to the antibody concentration (the amount of antibody needed for a particular experiment), K D The lower the value (the lower the concentration), the higher the affinity of the antibody. K D is the antibody association rate of the antibody (k on ) dissociation rate of antibody (k off ) and the dissociation rate of the antibody (k off ) is the rate at which the antibody dissociates from the antigen, and the antibody association rate (k on ) is the speed at which the antibody binds to the antigen. D The k value for a particular antibody / antigen interaction on and k off The ratio of these values ​​is used to calculate K D The affinity of a monoclonal antibody was determined by calculating the equilibrium dissociation constant (K D ), which is used to evaluate and rank the strength of bimolecular interactions. Antibody binding to antigen is a reversible process, and the rate of the binding reaction is proportional to the concentrations of the reactants. At equilibrium, the rate of formation of the [antibody]|[antigen] complex is equal to the rate of dissociation into its components [antibody]+[antigen]. The measurement of the reaction rate constant is the equilibrium or affinity constant (1 / K D ) can be used to define K DThe lower the value, the greater the affinity of the antibody for its target.

[0064] The binding domain of a CAR generally has one or more "hinge regions." The hinge region serves to position the antigen-binding domain away from the effector cell surface to allow proper cell-cell contact, antigen binding and activation.CAR may have one or more hinge regions between the binding domain and the transmembrane domain.As described in the art, the hinge region may be derived from natural, synthetic, semi-synthetic or recombinant sources.

[0065] A "transmembrane domain" is a CAR portion that fuses an extracellular binding portion with an intracellular signaling domain. The transmembrane domain anchors the CAR to the cell membrane of an immune effector cell. As described in the art, the transmembrane region may be natural, synthetic, semi-synthetic, or derived from a recombinant source.

[0066] In a preferred embodiment, the CAR of the present invention comprises an intracellular signaling domain. By "intracellular signaling domain" is meant the portion of the CAR that is involved in introducing the message of effective CAR binding to the target antigen into the immune effector cell and inducing, for example, cytotoxic activity, including activation, cytokine production, proliferation and release of cytotoxic factors to the CAR-bound target cell, or other cellular response triggered by antigen binding to the extracellular CAR domain. By "effector function" is meant the specialized function of a cell. The effector function of a T cell can be an activity including cytolytic activity or secretion of cytokines. Thus, as used herein, "intracellular signaling domain" means a portion of a protein that converts the signal of the "effector function" so that the cell performs a specialized function. As described in the art, the complete intracellular signaling domain or a truncated portion of the intracellular signaling domain may be used. As the skilled artisan will appreciate, an effective truncated portion should convert the effector function signal substantially similar to the complete domain. Herein, the term "intracellular signaling domain" is meant to include any truncated portion of the intracellular signaling domain that can be sufficiently converted into an effector function signal.

[0067] In a preferred embodiment of the present invention, the CAR has a hinge region IgG1-CH2-CH3 (a first portion of IgG1 is located in the membrane and a second portion of IgG1 is located extracellularly) and has CD28 as a transmembrane domain (a first portion of CD28 is located in the membrane and a second portion of CD28 is located intracellularly in the cytoplasm). CD8α can also be used as a transmembrane domain and hinge region. A sequence derived from IgG4 can also be used.

[0068] As known from the art, the signal generated through the T cell receptor (TCR) alone is not sufficient for the complete activation of T cells, and a secondary or costimulatory signal is also required. Thus, it can be said that the activation of T cells is mediated by two different classes of intracellular signaling domains: a primary signaling domain (e.g., TCR / CD3 complex) that initiates antigen-dependent primary activation through the TCR, and a costimulatory signaling domain that acts antigen-independently to provide a secondary signal or any costimulatory signal. In a preferred embodiment, the CAR of the present invention comprises an intracellular signaling domain that comprises one or more "costimulatory signaling domains". Thus, in another preferred embodiment, the isolated nucleic acid molecule of the present invention is encoded with an intracellular signaling domain that comprises at least one costimulatory domain, and thus the resulting intracellular signaling domain therefore comprises at least one costimulatory domain.

[0069] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or an Fc receptor, that provides a second signal necessary for efficient T cell activation / function upon antigen binding.

[0070] The terms "polypeptide", "polypeptide fragment", "peptide" and "protein" are used interchangeably herein and have their conventional meanings, i.e., as sequences of amino acids, unless otherwise specified. A polypeptide is not limited to a particular length and may include a full-length protein sequence or a fragment thereof. A polymer may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acids. Additionally, a polypeptide may have other manipulations or modifications, such as post-translational modifications such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling moiety. More generally, a polypeptide may have any naturally and / or non-naturally occurring modification. Given the fact that the polypeptides of the present invention are based on antibodies, in some embodiments, the polypeptides may occur as single chains or associated chains. The polypeptides may be prepared using any technique known in the art, e.g., recombinant and / or synthetic techniques. More specifically, the polypeptides described herein encompass the CARs of the present disclosure, or sequences having deletions, additions, and / or substitutions from one or more amino acids of the CARs disclosed herein.

[0071] As used herein, "isolated peptide" or "isolated polypeptide" and the like refer to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and its association with other components of a cell. Similarly, an "isolated cell" refers to a cell obtained from an in vivo tissue or organ and that is substantially free of extracellular matrix.

[0072] The term "vector" as used herein refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. More generally, a "vector" refers to a construct capable of delivering and expressing one or more genes or sequences of interest in a host cell. The nucleic acid to be transported or transferred is linked to the vector nucleic acid molecule by any method known in the art, for example, by insertion. The vector may contain a sequence that directs autonomous replication in the cell, or may contain a sequence sufficient to allow integration into the host cell DNA. The present invention also provides a vector comprising a nucleic acid molecule encoding a CAR of the present invention. The vector may be selected from DNA, RNA, a phage vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. The vector of the present invention preferably comprises a promoter, such as the EF-1α promoter. The term "promoter" as used herein refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. As known to those skilled in the art, the RNA polymerase causes transcription of a polynucleotide operably linked to the promoter. In certain embodiments, it may be desirable to express a polynucleotide comprising a CAR from a promoter that provides stable and long-term CAR expression in T cells. Expression of the CAR under the control of a promoter further ensures sufficient expression levels to direct T cells to cells expressing the target antigen.

[0073] Retroviruses are common tools for gene delivery. In some embodiments, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) to cells, preferentially immune cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, which then covalently integrates the genomic DNA into the host genome. Once the virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase, directing the expression of RNA molecules that code for structural proteins and enzymes required to make new viral particles. As a result, T cells transfected with the vectors of the invention can generate stable, long-term and sustained CAR-mediated T cell responses. In certain embodiments, T cells are transfected with a lentiviral vector, i.e., a lentivirus, encoded by the CAR of the invention. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, including long terminal repeats (LTRs), primarily derived from lentiviruses. The term "lentivirus" refers to a group (or genus) of complex retroviruses. Well-known lentiviruses include human immunodeficiency virus (HIV, e.g., types 1 or 2); Visna / Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). The term "self-inactivating" (SIN) vector refers to a replication-deficient vector, e.g., a retroviral or lentiviral vector, in which the 3' LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.

[0074] The terms "purified" and "isolated", when referring to a molecule such as a polypeptide, or an antibody, or a nucleotide sequence of the present invention, mean that the indicated molecule is free of other biological macromolecules of the same type. More specifically, the term "purified" as used herein means that at least 85%, 90%, 95% or 98% by weight of the same type of biological macromolecules are present. More specifically, an "isolated" nucleic acid molecule encoding a particular polypeptide means a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide of interest; however, the molecule may contain some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0075] The terms "antigen," "target," or "target antigen" refer to a molecule or portion of a molecule capable of being bound by an antibody or antibody-like binding protein. Moreover, the term refers to a molecule or a part of a molecule that can be used in animals to produce an antibody, and the antibody can bind to the epitope of that antigen. A target antigen may have one or more epitopes. For each target antigen recognized by an antibody or antibody-like binding protein, the antibody-like binding protein can compete with the intact antibody that recognizes the target antigen.

[0076] The terms "CD123", "IL-3Ra" or "IL-3Rα", "interleukin-3 receptor alpha" or "interleukin-3 receptor α" are used interchangeably herein. 1 IL-3R consists of a ligand-specific α subunit and a signaling common β subunit (also known as CD131) shared by the receptors for interleukin 3 (IL3), colony-stimulating factor 2, colony-stimulating factor (CSF2 / GM-CSF), and interleukin 5 (IL5). Binding of CD123 / IL-3Ra to IL3 is dependent on the β subunit. The β subunit is activated by ligand binding and is required for the biological activity of IL3. All of the above terms for CD123 can refer to any of the protein or nucleic acid sequences set forth herein. The term "CD123 / IL-3Ra" encompasses unprocessed "full-length" CD123 / IL-3Ra, as well as any form of CD123 / IL-3Ra resulting from processing within the cell. The term also encompasses naturally occurring variants of CD123 / IL-3Ra proteins or nucleic acids, such as splice variants, allelic variants and isoforms. The CD123 / IL-3Ra polypeptides and polynucleotides described herein may be isolated from various sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Examples of CD123 / IL-3Ra sequences include, but are not limited to, NCBI reference numbers NP_002174&NM_002183 (protein and nucleic acid sequences for human CD123 variant 1), and NP_001254642&NM_001267713 (protein and nucleic acid sequences for human CD123 variant 2).

[0077] The terms "anti-CD123 antibody", "anti-IL-3Ra antibody" or "anti-IL-3Rα antibody", "antibody that specifically binds to CD123" or "antibody that specifically binds to IL-3Ra / IL-3Rα" refer to an antibody that has sufficient affinity to bind to CD123 so as to be useful as a diagnostic and / or therapeutic agent in targeting CD123. Unless otherwise specified, the extent of binding of an anti-CD123 antibody to unrelated non-CD123 proteins is less than about 10% of the measured binding of the antibody to CD123. More generally, the term "specifically binds" indicates that an antibody binds to an epitope through its antigen-binding domain and that the binding involves a degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" when it binds to its epitope through its antigen-binding domain rather than binding to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody binds to an epitope. For example, antibody "A" is considered to have higher specificity for a given epitope than antibody "B," or antibody "A" is said to bind epitope "C" with higher specificity than related epitope "D." Thus, the antibodies or antigen-binding fragments of the invention specifically bind to the CD123 antigen, and in that respect have higher binding specificity for the CD123 antigen (from any species) than for non-CD123 antigens. More particularly, the antibodies or antigen-binding fragments of the invention specifically bind to the human CD123 antigen, and in that respect have higher binding specificity for the human CD123 antigen than for non-human CD123 antigens (e.g., mouse or rat CD123). Sometimes, the term "preferentially binds" indicates that an antibody specifically binds to an epitope more readily than a related, similar, cognate, or analogous epitope. Thus, an antibody that preferentially binds to a given epitope will more likely bind to the given epitope than to a related epitope, even though such an antibody may cross-react with the related epitope. For example, an antibody or antigen-binding fragment of the invention may preferentially bind to human CD123 antigen over murine CD123.

[0078] According to the above, the terminology for the antibody, CAR, vector or cell of the present invention can be easily derived. In fact, in the general context of the description, it is not necessarily required to express exactly the same as the above label. There may be deviations. For example, when referring to a CAR of a T cell, it is not necessarily specified that the CAR contains a portion that is directed to a specific antigen, i.e., "anti-something". Thus, as a more general point of note, a CAR of the present invention that contains a portion that specifically binds to CD123 can be referred to as a CD123 CAR as well as an anti-CD123 CAR. In a similar manner, a T cell transfected with a CAR that specifically binds to CD123 according to the present invention can be referred to as a CD123 CAR T cell.

[0079] As used herein, "treatment" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include any reduction in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment may include either a reduction in symptoms, or any improvement with respect to the disease or condition. Treatment may also be slowing the progression of the disease or condition. Thus, the term "treatment" does not necessarily mean a complete eradication or cure of the disease or condition, or symptoms associated therewith. With this in mind, the present disclosure provides for the treatment or prevention of BPDCN, comprising administering to a subject in need thereof a therapeutically effective amount of the T cells of the present invention.

[0080] In light of the foregoing, the T cells described herein may be administered alone or as a pharmaceutical composition (also referred to as a pharmaceutical formulation). The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that allows the biological activity of the active ingredient to be effective and that does not contain other components that are unacceptably toxic to the subject when the composition / formulation is administered. The formulation may be sterile. A pharmaceutical composition may comprise the T cells according to the invention alone or in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients. The composition may be administered in a buffer such as neutral buffered saline, phosphate buffered saline; The term "pharmacologically acceptable" or "physiologically acceptable" means compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The compositions of the present invention are preferably formulated for parenteral administration. "Parenteral administration" may mean modes of administration other than enteral and topical administration. Typically, parenteral administration is performed by intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and / or infusion. Preferentially, the CAR-modified T cells or the composition of the invention are administered to the subject by direct injection into the tumor, lymph node, systemic circulation, or site of infection. In general, the invention is useful for treating subjects diagnosed with diseases that overexpress CD123, particularly BPDCN. The treatment comprises removing immune effector cells from the subject, genetically modifying said immune effector cells with a vector comprising a nucleic acid encoding a CAR as contemplated herein, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In a preferred embodiment, the immune effector cells are T cells. The amount and frequency of administration are determined by the patient's physical condition (patient's condition, type and severity of the patient's disease). Appropriate dosages may be determined with the help of animal models, followed by clinical trials. When referring to dosages, the term "effective amount" is an amount sufficient to accomplish a specifically described purpose. The so-called "therapeutically effective amount" of an active ingredient, such as a genetically modified T cell, depends on factors such as the disease and its condition, age, sex, and weight of the patient, and may also depend on the ability of the cells to induce a desired response in the patient.A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transfected therapeutic cells are outweighed by the therapeutically beneficial effects. 4 ~10 9 Cells / kg body weight, preferably 10 5 ~10 6 The therapeutically effective amount may be administered in a dose of cells / kg body weight, including all integer values ​​within these ranges. More generally, the term "therapeutically effective amount" refers to an amount of an antibody, active ingredient, or other drug effective to treat a disease or disorder in a subject. In the case of cancer, a therapeutically effective amount of an agent is effective to reduce the number of cancer cells; reduce tumor size; inhibit cancer cell invasion into peripheral organs (i.e., slow down or stop the cancer to some extent); inhibit tumor metastasis (i.e., slow down or stop the cancer to some extent); inhibit or stop tumor growth to some extent; alleviate to some extent one or more symptoms associated with the cancer; and / or result in a favorable response such as progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or in some cases stable disease state (SD), reduction in progressive disease (PD), reduction in time to progression (TTP), or any combination thereof. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than the therapeutically effective amount, since the prophylactically effective amount is used in subjects prior to or at an earlier stage of disease.

[0081] In certain embodiments, prior to the in vitro manipulation or genetic modification of immune effector cells as described herein, a source of cells is taken from a subject. In certain embodiments, the immune effector cells expressing the CAR of the present invention on their membrane include T cells. T cells can be taken from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus matter, tissue from a site of infection, ascites, pleural effusion, splenic tissue, and tumors. In certain embodiments, T cells can be taken from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation. In another embodiment, cells from an individual's circulating blood are taken by apheresis. Apheresis products generally include lymphocytes. Lymphocytes include T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing. In another embodiment, T cells are isolated from peripheral blood mononuclear cells by lysing red blood cells and removing monocytes. For example, centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells expressing one or several markers, such as CD3, CD4 or CD8, can be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be achieved with a combination of antibodies directed against surface markers that are not expressed on the negatively selected cells.

[0082] In a more general context, the present specification emphasizes cell therapy in which T cells are genetically modified (or genetically modified) ex vivo to express a CAR and the CAR T cells are infused into a patient, preferably a human, in need thereof. The infused cells are capable of killing tumor cells in the patient. As previously mentioned, unlike antibody therapy, T cells containing a CAR are capable of replicating in vivo, resulting in long-term persistence that may lead to sustained tumor control. Furthermore, CARs allow redirection and activation of effector T cells to any bound cell surface molecule by antibody-derived receptors, and are independent of MHC restriction (MHC-restricted antigen recognition; MHC stands for major histocompatibility complex). As previously mentioned, the genetically modified T cells of the present invention are constructed starting from T cells taken from the patient himself (autologous), but can also start from other allogeneic donors to provide allogeneic genetically modified T cells in a bone marrow or peripheral hematopoietic stem cell allogeneic transplantation context (infusion of donor lymphocytes). Said T cells expressing a CAR molecule according to the present invention are useful for treating proliferative diseases in mammals, preferably humans.

[0083] In some embodiments of the present invention, the polynucleotides or cells carrying the polynucleotides of the present invention use suicide genes, including inducible suicide genes, to reduce the risk of direct toxicity (i.e., graft-versus-host disease in allogeneic administration, GvHD) and / or uncontrolled proliferation of the genetically modified cells. In certain embodiments, the suicide genes are not immunogenic to the host carrying the polynucleotide or cells. Specific examples of suicide genes that can be used are inducible caspase-9 (iCASP9), herpes simplex thymidine kinase (HSV-tk), CD20, truncated EGFR, caspase-8 or cytosine deaminase. Caspase-9 can be activated using specific chemically induced dimerization (CID). Other systems can be activated by metabolizing prodrugs (ganciclovir), or by binding antibodies (rituximab, cetuximab).

[0084] The present invention will be described in more detail with reference to experimental data and examples. Although the following detailed description focuses on BPDCN, the present invention can generally provide improvements and / or solutions for any disease in which overexpression of CD123 occurs. The main focus of the present invention is to provide a treatment for blastic plasmacytoid dendritic cell neoplasm (BPDCN).

[0085] BPDCN is a Crohn's disease originating from plasmacytoid dendritic cell (pDC) precursors with cutaneous lesions that progress to disseminated aggressive leukemia. Reference: Blastic plasmacytoid dendritic cell neoplasms: current status and perspectives, Maria Rosaria Sapienza, Alessandro Pirelli, Enrico De Renzini, Federica Melle et al., Cancer, 2019. The prognosis of BPDCN is poor, and there are no effective treatments or reports on it in the art. Most patients initially respond to intensive chemotherapy but then relapse. The median overall survival (OS) in the largest series of subjects is 8-12 months. And even in patients treated with targeted therapy, the only exception to the data concerns patients who have undergone allogeneic hematopoietic stem cell transplantation (allo-HSCT). References: Ruch-Weil D, Dietrich S, Boumendil A, Polgi E, Bron D, Carreras E et al., Stem cell transplantation can provide sustained disease control in blastic plasmacytoid dendritic cell neoplasms: European Group of Blood and Marrow Transplantation, Blado, 2013, 121(3): 440-6; Pagano L, Valentini CG, Grammatico S, Pulsoni A, Blastic plasmacytoid dendritic cell neoplasms: diagnostic criteria and therapeutic approaches, British Journal of Haematology, 2016, 174(2): 188-202; Karfan-Dabaja MA, Lazarus HM, Nishihori T, Mahfouz RA, Hamadani M., Advances in diagnosis and treatment in blastic plasmacytoid dendritic cell neoplasms: biology of blood and bone marrow transplantation with a focus on hematopoietic cell transplantation: Journal of the American Society of Blood and Marrow Transplantation, 2013, 19(7), 1006-12; Penmarj N, Raa A. AA, Sweet KL, Stein AS, Vas S, Bloom W et al., Tagraxofusp for blastic plasmacytoid dendritic cell neoplasms, New England Journal of Medicine, 2019, 380(17), 1628-37. However, most elderly patients are not eligible for this approach. Improved therapies are needed for leukemias that can be considered chemotherapy-resistant leukemias. The applicants of the present invention have identified a new therapeutic target. The interleukin (IL) 3 receptor α chain (CD123) has proven to be a valuable target, as it is highly expressed in vivo only on basophils and pDCs.The art further discloses that CD123 is also expressed at low levels on normal hematopoietic CD34+, CD34+ progenitor cells, monocytes and endothelial cells, but not on hematopoietic stem cells. References: Taussig DC, Pierce DJ, Simpson C, Rohatiner AZ, Lister TA, Kelly G, et al., Hematopoietic stem cells expressing multiple myeloid markers: Implications for hematologic origin and targeted therapy of acute myeloid leukemia, Brad, 2005, 106(13):4086-92; Adra Al-Mawaleel, David Gillis and Ian Lewis, Immunoprofile of leukemic stem cells delineating FLT3 / ITD positive clones CD34+ / CD38- / CD123+, Journal of Hematology & Oncology, 2016, 9:61.

[0086] To combat BPDCN, the inventors have generally considered that the antigen CD123 is uniformly and highly expressed in BPDCN both at diagnosis and in relapsed cases after chemotherapy. References: Garnache-Otto F, Foillard J, Ferran C, Beachelle S, Trimoreau F, Seille E, et al., Expanded diagnostic criteria for plasmacytoid dendritic cell leukemia, British Journal of Hematology, 2009, 145(5):624-36. Furthermore, the inventors considered that CD123 is a component of the IL-3 receptor, and that the IL-3 receptor is a key driver of pDC lineage, which is necessary for pDC survival. References: Broughton SE, Durgat U, Hercules TR, Nero TL, Grimbaldiston MA, Bondar CS, et al., The GM-CSF / IL-3 / IL-5 cytokine receptor family: from gund recognition to initiation of signaling, Immunological Reviews, 2012, 250 (1):277-302; Hara T, Miyajima A., Function and signaling through the interleukin-3 receptor system in hematopoiesis, Stem Cells, 1996, 14(6):605-18; and Testa U, Pelosi E, Frankel A., Membrane biomarkers and therapeutic targets in hematological malignancies, Biomarker Research, 2014, 2(1):4. We hypothesized that targeting this IL-3 receptor may induce deleterious signaling pathways for BPDCN cells.

[0087] CD123 has been reported to be involved in initiating blast crisis in acute myeloid leukemia (AM and acute lymphoblastic leukemia (ALL)). Reference: Ehinger A, Kramer M, Lorig C, Teed C, Bornhauser M, von Bonan M, et al., Cell surface detection levels and distribution of CD33 and CD123 in acute myeloid leukemia, Blood Cancer Journal, 2014, 4:e218. Furthermore, it has been reported that CD123 is expressed on CD34+ "leukemia-initiating cell (LIC)" components, with adverse clinical consequences. References: Bergize F, Green AS, Tamburini J, Sally JE, Gaillard B, Cornillet-Lefèbvre P, et al., High CD34+CD381ow / -CD123+ blasts predict adverse outcome in acute myeloid leukemia: the West-East Group on Acute Leukemia and Hematological Disorders (GOELAMS) study, Hematologica, 2011, 96(12):1792-8; Reya T, Morrison SJ, Clarke MF, Weissman IL, Stem cells, cancer, and cancer stem cells, Nature, 2001, 414(6859):105-11. However, in BPDCN, it is unclear whether there is an increased number of immature LICs present in patients. Applicant was surprised to find that in certain BPDCN cases collected by the inventors, higher levels of CD123 expression were observed in the low component of blast cells than in the leukemic bulk cells. Applicant then hypothesized that targeting CD123, if present, may eliminate these cells and reduce recurrence in BPDCN patients. Applicant then proactively selected CD123 as a targeted immunotherapy for BPDCN.

[0088] Furthermore, applicants have demonstrated the sensitivity of BPDCN cells to SL-401, a fusion protein containing diphtheria toxin linked to IL-3 that targets cells expressing CD123 in vivo and in vitro. References: Angelo-Deletara F, Roggie A, Frankel AE, Lamartie B, Seille E, Beachelle S, et al., Sensitivity of blastic plasmacytoid dendritic cell neoplasms to the interleukin-3 receptor-targeted biologic SL-401 in vivo and in vitro, Hematologica, 2015, 100(2):223-30. Clinical responses have also been reported in patients with BPDCN. Reference: Frankel AE, Wu JH, Ahn C, Penmarj N, Medeiros BC, Carrollway HE et al., Activity of SL-401, a targeted therapy against the interleukin-3 receptor in patients with blastic plasmacytoid dendritic cell neoplasms, Blood, 2014, 124(3):385-92.

[0089] As known in the art, adoptive T cell therapy using chimeric antigen receptors (CARs) is a promising method to treat hematological malignancies with risk of relapse. References: Grupp SA, Karos M, Barrett D, Aplen R, Porter DL, Reingard SR, et al., Chimeric antigen receptor modified T cells for the treatment of acute lymphoblastic leukemia, New England Journal of Medicine, 368(16)1509-18; Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stettler-Stevenson M, et al., Autologous T cells expressing anti-CD19 chimeric antigen receptors can be used to effectively treat chemotherapy-refractory diffuse large B cell lymphoma and indolent B cell malignancies. Journal of Clinical Oncology: Proceedings of the American Society of Clinical Oncology, 2015,33(6),540-9.

[0090] According to an embodiment of the present invention, CARs are genetically engineered to be made of single chain variable fragments (ScFv) of heavy and light chains of monoclonal antibodies (MAbs) linked to the intracellular signaling chain of the T cell receptor (TCR). Preferably, the intracellular portion of the CAR contains costimulatory domains such as CD3ζ, CD28 and 4-1BB, which allow for better activation and cell signaling. CARs are considered as third generation CARs. See the review by Andrew D. Fesnak, Carl H. June and Bruce L. Levine, T cell engineering: Promises and challenges of cancer immunotherapy, Nature, Vol. 16, September 2016, for more details on CARs. ScFvs can recognize antigens independent of major histocompatibility complex (MHC) presentation.

[0091] There have been reports on the efficacy of CAR CD123 in eradicating acute myeloid leukemia in vitro and in mouse patient xenograft models (PDX). References: Mardiros A, dos Santos C, McDonald T, Brown CE, Ou X, Budde LE, et al. T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector function and antitumor efficacy against human acute myeloid leukemia, Vlad, 2013, 122(18):3138-48; Gill S, Tacian SK, Luella M, Shestova O, Li Y, Porter DL, et al. Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells, Vlad, 2014, 123(15):2343-54; Tocara R, Olivares S, Mi T, Mighty S, Deniger D, Hulse H, et al. Using the Sleeping Beauty system to express chimeric antigen receptors with mix-and-match VL and VH domains to target CD123+ tumors. Restoring T cell specificity using the CD123-specific chimeric antigen receptor T cell (CART) system, PLOS One, 2016, 11(8):e0159477;Cummins KD, Gill S.,Anti-CD123 chimeric antigen receptor T cells (CART): an evolving approach to the treatment of hematological malignancies and a secret weapon against antigen-negative relapse,Leukemia and Lymphoma, 2018, 59(7):1539-53;Tetamanti S, Marin V, Pichitola I, Magnani CF, Giordano Attianese GM, Crivioli E et al.,Targeting acute myeloid leukemia with cytokine-induced killer cells restored by a novel CD123-specific chimeric antigen receptor, British Journal of Hematology, 161(3):389-401;Tassian SK, Kendarian SS, Shen F, Luella M, Shestova O, Kozlovsky M et al., Optimization of chimeric antigen receptor T cell elimination in a mouse xenograft model of human acute myeloid leukemia, Brad, 2017;129(17):2395-407. Another group showed that CAR CD123 was active against an established BPDCN cell line (CAL-1) and CD123+ primary BPDCN samples in a mouse model.However, this CAR is only used in allogeneic situations. There is a second generation with a suicide system. References: Sai T, Garrett R, Double A, Smith J, Kavasos A, Konoplev S, et al., Preclinical study of anti-CD123 CAR-T cells against blastoid plasmacytoma (BPDCN), Brad, 2016, 128(22):4039. However, growing clinical trials show that patients receiving CAR therapy develop toxicity. This is a major problem in developing effective CARs. The applicant has developed a special CAR to overcome the problems of the prior art. More specifically, the applicant has modified both third generation retroviral and lentiviral CARs. The CAR of the present invention overcomes the problems of the prior art, especially those related to cytotoxicity. The CAR of the present invention has improved functional activity against BPDCN cells (BPDCN cell lines, PDX cells and primary BPDCN cells) in vivo and in vitro in several mouse models of BPDCN. CD123. ロー Evaluation of their potential cytotoxicity against positive cells is highly promising.

[0092] According to the present invention, monoclonal antibodies (Mabs) against human CD123 have been produced. The monoclonal antibodies of the present invention can be used in both allogeneic and autologous cases.

[0093] The method for producing anti-CD123 monoclonal antibodies involved immunization of mice with recombinant CD123 protein. Five mice, available reference number #301-R3-025, were immunized with recombinant CD123 (available from R&D Systems, Minnesota, USA). Immunization was performed via footpad and / or intraperitoneal injection. Lymph node and / or splenic B cells were used to produce Mab-secreting fusion cells. CD123 + and CD123 ― The cell lines were used to select fusion cells based on MAb affinity and specificity.

[0094] According to the present invention, six specific antibodies were selected and designated 18B4D5 (also referred to herein as AB1, or sometimes as B4D5), 17F7B2 (also referred to herein as AB2), 11B6E6 (also referred to herein as AB3), 14G12D7 (also referred to herein as AB4), 11A12C6 (also referred to herein as AB5) and 15E11B7 (also referred to herein as AB6), respectively, and their molecular characteristics and DNA sequences were determined according to the SANGER method.

[0095] VDJ and VJ gene rearrangements were sequenced and identified after alignment of consensus nucleotide sequences against the IMGT® database using the Vision Quest (VQUEST) online tool. Reference: Brochette X, Lefranc MP, Giudicelli V., IMGT / V-QUEST: a highly customized and integrated system of IGs and TRs for standardized VJ and VDJ sequence analysis, Nucleic Acids Research, 2008;36(WebSequence):W503-8. The sequences (nucleotide and amino acid sequences) of each of the six antibodies of the invention are listed in Figures 1-9.

[0096] FIG. 1 shows the nucleotide and amino acid sequences of complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) of both the heavy and light chains of the antibodies of the invention 18B4D5, 17F7B2, 11B6E6, 14G12D7, 11A12C6 and 15E11B7.

[0097] FIG. 2 shows the nucleotide and amino acid sequences of framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4) of the heavy chains of antibodies of the present invention 18B4D5, 17F7B2, 11B6E6, 14G12D7, 11A12C6 and 15E11B7, respectively.

[0098] FIG. 3 shows the nucleotide and amino acid sequences of framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4) of the light chain of antibodies of the present invention 18B4D5, 17F7B2, 11B6E6, 14G12D7, 11A12C6 and 15E11B7, respectively.

[0099] 4 shows the consensus amino acid sequence of the heavy chain and the consensus amino acid sequence of the light chain of the first antibody AB1 of the present invention, designated 8B4D5 or B4D5. Amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted in reading order from left to right, respectively.

[0100] 5 shows the consensus amino acid sequence of the heavy chain and the consensus amino acid sequence of the light chain of the second antibody of the invention, AB2, designated 17F7B2. Amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted in reading order from left to right, respectively.

[0101] 6 shows the consensus amino acid sequence of the heavy chain and the consensus amino acid sequence of the light chain of the third antibody of the invention, AB3, designated 11B6E6, with the amino acids corresponding to CDR1, CDR2 and CDR3 highlighted in reading order from left to right, respectively.

[0102] 7 shows the consensus amino acid sequence of the heavy chain and the consensus amino acid sequence of the light chain of the fourth antibody of the invention, AB4, designated 14G12D7. Amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted in reading order from left to right, respectively.

[0103] 8 shows the consensus amino acid sequence of the heavy chain and the consensus amino acid sequence of the light chain of the fifth antibody of the invention, AB5, designated 11A12C6, in which the amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted in reading order from left to right, respectively.

[0104] 9 shows the consensus amino acid sequence of the heavy chain and the consensus amino acid sequence of the light chain of the hexabody AB6 of the invention, designated 15E11B7. Amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted in reading order from left to right, respectively.

[0105] The sequence homologies for the heavy chain VH and JH, and the light chain VK and JK are shown in Table 1.

[0106] [Table 1]

[0107] The affinity of the antibodies of the present invention for CD123 is examined using a streptavidin biosensor. D To determine this value, seven different concentrations of CD123 were used on the immobilized antibodies of the invention. The results were analyzed by the Octet system provided by ForteBio. The antibody affinity results are shown in Table 2.

[0108] [Table 2]

[0109] The cells used in the study of the present invention, particularly in the application of the examples and results herein, were obtained from humans. In more detail, primary cells from BPDCN patients (DC-2008-713 and DC-2016-2791 collections) were used for CD123 expression assessment, cytotoxicity analysis, and expansion of primary cells in NSG mice to generate PDXs (primary derived xenografts). Reference: Philip L. et al., Bortezomib as a novel therapeutic approach for blastic plasmocytoid dendritic cell tumors, Hematologica, 2017. These cases were characterized according to the current recommendations for the diagnosis of BPDCN established by the applicant. References: Garnache-Otto F, Foillard J, Ferran C, Beachre S, Trimoreau F, Seilles E, et al., Expanded diagnostic criteria for plasmacytoid dendritic cell leukemia, British Journal of Hematology, 2009, 145(5):624-36. Swerdlow, SH, Campo, E., Harris, NL, Yahoo, ES, Pirelli, SA, Stein, H., Thiele, J., Arbour, DA, Hassaj, RP, Le Beau, MM, Orazi, A. & Siebert, R. (2017), WHO classification of neoplasms of the blood and lymphoid system. In the WHO classification of neoplasms of the blood and lymphoid system (edited by Swerdlow, SH, Campo, E., Harris, NL, Yahoo, ES, Pirelli, SA, Stein, H., Thiele, J., Arbour, DA, Hassage, RP, Le Beau, MM, Orazi, A. & Siebert, R.), pages 98-106, Lyon, France. PDX cells P127B, P25B, CM60915, F13-CAL1 and F13-LA were cultured ex vivo in the appropriate culture medium. CD123 + BPDCN cell lines (GEN2.2 and CAL-1, Dr. Maeda, Nagasaki University, Japan) and CD123 ―There are two cell lines: Daudi, Jurkat, HL-60, and REH. The K562 CD123 cell line was generated by lentiviral transfection to express CD123. To study CD123 expression, cells from normal blood (n=19) and bone marrow (n=4), acute leukemia (myeloid AML, n=3) and lymphoma (B-ALL, n=3; T-ALL, n=3) were obtained from healthy donors and / or patients. In the present invention, peripheral blood mononuclear cells (PBMCs) were used to generate genetically modified T cells. PBMCs were obtained from healthy donor or umbilical cord blood samples collected at the French Blood Center (EFS B / FC, Besançon, France) by Ficoll (FICOLL trademark) gradient density centrifugation (commercially available FICOLL-PAQUE). TM It was isolated by

[0110] Unless otherwise stated, statistical analyses herein are expressed as mean values. All statistical analyses in the described embodiments were performed using GraphPad Prism 7 (GraphPad software Inc., San Diego, CA, USA). Comparisons between two populations were evaluated by Student's t test. Survival studies were evaluated by Kaplan-Meier curves and log-rank (Mantel-Cox) tests.

[0111] Using flow cytometry, the profile of CD123 expression on normal hematopoietic cells and BPDCN cells was examined. Using the inventive B4D5 (AB1) antibody and a commercially available anti-CD123 antibody (6H6), the profile of CD123 expression on normal cells and BPDCN patient cells was determined. More specifically, the profile of CD123 expression on normal blood and bone marrow cells, i.e., normal pDCs (targeted with CD304), monocytes (targeted with CD14 + ), lymphocytes (CD45 +ハイ / SSC ロー ), granulocytes (CD45 + , S.S.C. ハイ and FSC ハイ) and CSH / progenitor cells (CD34+) were transfected with BPDCN patient cells (CD45 +ロー , CD304 + ) compared to the mean fluorescence intensity ratio (MFIR) of CD123. The mean fluorescence intensity ratio (MFIR) was calculated by dividing the mean fluorescence intensity (MFI) of CD123 by the one of the isotype control mAb. Cells were considered positive for CD123 expression when they had an MFIR higher than 2. Reference: Garand R, Robillard N., Immunophenotypic features of acute leukemia and chronic lymphoproliferative disorders: practical recommendations and classification Hematology and Cellular Therapy, Hematology and Cellular Therapy, 1996;38(6):471-86. CD123 was highest in normal pDCs and BPDCNs (130.6 [118.4-146.8], n=5 and 136 [102.1-158.1], n=5, respectively). Low expression was detected in monocytes (20.7 [4.5–51.1], n = 7) and CD34+ cells (23.7 [19.2–32.2], n = 4), whereas lymphocytes (1.4 [1–2.4], n = 7) and polymorphonuclear cells (1.1 [1.1–1.2], n = 3) did not express CD123.

[0112] Figure 10 shows flow cytometry graphs based on evaluation of CD123 expression on normal hematopoietic cells and BPDCN. In particular, flow cytometry graphs based on evaluation of CD123 expression on normal hematopoietic cells and BPDCN using BB4D5 antibody in comparison with the commercial antibody 6H6. Isotype control appears in white and anti-CD123 B4D5 appears in black. As previously described, normal cells were evaluated in peripheral blood and bone marrow (see Figure 10A), BPDCN cell lines (CAL-1 and GEN 2.2), PDX (P127B-M5) and primary cells of BPDCN patients (see Figure 10B). Additionally, CD123 expression was assessed by MFIR (ratio of mean fluorescence intensity of CD123 with one of the isotype control mAbs) using anti-CD123 B4D5 and 6H6 commercial antibodies on lymphocytes (n=7 and 12), monocytes (n=7 and 12), granulocytes (n=3 and 5), and normal pDCs (n=5 and 7). For the CD34 population, data from blood and bone marrow samples (n=4 and 7) were combined, since no differences in CD123 level expression were observed in Figure 10C.

[0113] Additionally, several non-pDC acute leukemias were tested with the B4D5 monoclonal antibody, and Applicants determined that BPDCN expressed higher levels of CD123 than AML, B or T lymphocytic acute leukemias.

[0114] Figure 11 shows a table and flow cytometry graphs based on the evaluation of CD123 expression in BPDCN and other non-pDC acute leukemias. Figure 11 shows CD123 expression assessed by MFIR (ratio of mean fluorescence intensity of CD123 divided by 1 of isotype control mAb) using 6H6 commercial antibody on lymphocytes (n=12), monocytes (n=12), granulocytes (n=5), and normal pDC (n=7) (see Figure 11 A). Data from blood and bone marrow samples (n=4 and 7) were combined since no difference in CD123 level expression was observed. Expression on CD34 population is evaluated on bone marrow. Figure 11 also shows flow cytometry evaluation of CD123 expression on B-LAL, T-LAL, and AML cell lines, where CD123 expression on patient primary cells using CD123 B4D5 antibody appears in black and isotype control appears in white (see Figure 11 B). The expression level of CD123 in non-pDC acute leukemia is lower than that in the one in BPDCN patients (see Figure 11 B).

[0115] FIG. 12 shows a diagram of the design of a Western blot and flow cytometry graphs for the chimeric antigen receptor of the present invention, the generation of T cells according to the present invention comprising the chimeric antigen receptor of the present invention.

[0116] FIG. 12 shows the design and production of CD123 CAR T cells according to the invention. Anti-CD123 CAR T cells were constructed using the antibody AB1 of the invention. The CAR of this embodiment of the invention is based on the scFv of B4D5 (AB1), a specific domain of T cell activation (CD3ζ) and two coactivation domains (CD28 and 4.IBB). The sequences encoding the CAR, the 2A sequence (encoding the 2A self-cleaving peptide) and the selection marker ACD19 (truncated CD19) were cloned into the pSFG retroviral vector (SFG.CAR CD123.IRES.ACD19 vector). Retroviral supernatant was produced from the PG13 packaging cell line transfected with the SFG.CAR CD123.IRES.ΔCD19 vector.

[0117] According to another embodiment of the invention, a lentiviral construct was also produced: the same CAR sequence is under the control of the EFlα promoter, downstream is a T2A ribosomal skip sequence and a truncated human CD19 transfection marker.

[0118] Both retroviral and lentiviral embodiments are shown in Figure 12A. In more detail, a schematic diagram of a third generation retroviral anti-CD123 CAR vector (CD123R CAR) and a third generation Reltivir-based anti-CD123 CAR vector (CD123L CAR) is shown in Figure 12. The scFv CD123 derived from the B4D5 antibody of the invention is fused to the T cell signaling domains CD28, 4IBB and CD3ζ. The selection marker (ΔCD19) is also shown. Genetically modified primary T cells of the invention were generated by viral transfection. PBMCs were activated with CD3 / CD28 Dynabeads (Life Technologies, CA, USA) for 2 days prior to transfection. 6-well-Retronectin (1.2 μg / cm in PBS) was used to transfect the PBMCs. 2 Retroviral transfection was performed in 6-well retronectin-coated plates (Takara, Japan) and incubated overnight at 4°C. The cells were then spun down with retroviral particles (1.5 h, 2000g, 10°C). For lentiviral transfection, the cells were spun down at 800g with MOI=10, lh, 32°C. Transfection efficiency was evaluated using CD3 +The expression of ΔCD19 cell surface marker on the cells was measured by performing flow cytometry analysis. The positively reverse transduced cells were magnetically labeled with microbeads (CD19 Microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany) and loaded onto a MACS® column according to the manufacturer's protocol. For T cells obtained by retroviral transfection, the transduced T cells are designated CD123R CAR, and for T cells obtained by lentiviral transfection, the transduced T cells are designated CD123L CAR. From the same donor, non-transduced T cells (called C0) were harvested and cultured as above, but without viral transfection: these cells serve as controls in all experiments. CD123 CAR T cells were expanded for 21 days and cryopreserved.

[0119] For the purposes of the described embodiments of the present invention, unless otherwise specified, cytotoxicity can be evaluated based on BPDCN patient-derived transfected T cells. More specifically, T cells (DC 2016-2791) from BPDCN patients are activated from PBMCs using human CD3 / CD28 Dynabeads and magnetic selection. The negative fraction includes BPDCN blasts (60% by differential count) that are used to evaluate CAR and B lymphocyte cytotoxicity. In addition, T cells are transfected with lentiviral constructs as described above. After 10 days, transfection efficiency and CAR expression are measured by flow cytometry as described above. Cytotoxic activity was evaluated against BPDCN blasts at an E:T ratio of 5:1 at 24 hours.

[0120] For the purposes of the described embodiments of the present invention, unless otherwise stated, immunophenotypic assays can be performed as follows: Antibody binding was detected on an LSR Fortessa flow cytometer or BD FACS CANTO II and analyzed with Diva software (BD Biosciences, NJ, USA). Appropriately matched isotype controls were included for analysis. Briefly, T cells were stained for CD3, CD4, CD8, CD19. CD3 + / CD19 + The T cells are CD123 CAR T cells. BPDCN cells, cell lines and PDXs are characterized with CD123 staining using B4D5 (AB1 used in CAR construction) and commercial antibodies (i.e. 6H6) to obtain more results. Anti-CD45 (T cells are CD45 ハイ / SSC ロー and granulocytes are CD45 + / SSC ハイ / FSC ハイ Normal blood and bone marrow specimens are analyzed with CD56, CD4, CD303, CD304, CD45, CD305 (normal pDC), CD14 (monocytes), and CD34 (CSH / progenitor cells). Patient-derived BPDCN samples are stained with CD56, CD4, CD303, CD304, CD45. Some embodiments of the present invention refer to the BPDCN mouse model. Thus, in the BPDCN mouse model, blast counts (anti-human hCD45, hCD123) and T cells (C0 and CD123 CAR) (hCD3, hCD19) are monitored weekly in blood cell samples using BD TruCount tubes. In humanized mice, peripheral blood is analyzed by flow cytometry (Attune NxT flow cytometer, Life Technologies) and total white blood cells (hCD45, hCD123) in bone marrow after mouse sacrifice are analyzed. + ), monocytes (CD45 / SSC +ミディアム , hCD33 + ), B lymphocytes (hCD19 + ), T lymphocytes (hCD3 + ), pDC (hCD123 + , hCD304 + ) and CSH / progenitor cells (CD34 +The monoclonal antibodies used for characterization of the cell surface phenotype are shown in Table 3.

[0121] [Table 3] JPEG0007645824000004.jpg115170

[0122] Some embodiments described herein further refer to in vitro cytotoxicity assessment and functional analysis. For this, C0 or CD123 CAR T cells (1·10 6 Cells (1000 x 1000 cells / mL) are labeled with the cell proliferation dye eFluor 450 (Thermo Fisher Scientific, Ecubren, Switzerland). The labeled cells are then co-cultured at appropriate effector / target (E:T) ratios with target cells (CAL-1, GEN 2.2, DAUDI, HL-60, K562 CD123 and PDX) for 24 h or 15 h (PDX) at 37 °C. After co-culture, target cells (7-AAD + CD123 + To assess cell death in 100% mouse C0 or CAR T cells, cells are labeled with 7-AAD (Beckman Coulter), anti-CD3, anti-CD19 (to assess C0 or CAR T cells) and anti-CD123. PE For degranulation assessment, the functionality of CD123 CAR T cells was tested and transfected with GolgiPlug (BD GolgiPlug™) according to the manufacturer's protocol. TM BD Biosciences with BD Cytofix / Cytoperm TM ) plus fixation and permeabilization solution kit, PEIntracellular production was performed. Co-culture supernatants were analyzed by CAR Human Thl / Th2 / Thl7 Cytokine Kit (BD Biosciences) according to the manufacturer's protocol. CAR cytotoxicity against HMEC adherent cell lines or primary monocytes was assessed using the MTT cell proliferation assay (Sigma-Aldrich, MO, USA) according to the manufacturer's protocol.

[0123] Some embodiments described herein further refer to tissue microarrays and immunohistochemistry. The specificity of the B4D5 (AB1) antibody is analyzed by tissue microarrays (Tissue MicroArrays) containing 90 tissue cores (30 organs) from three individual donors per organ (US Biomax, Rockville, USA). Immunostaining was detected using the UltraView Universal DAB Detection Kit (Ventana, USA) and image analysis was performed with NDP.view2 software. Staining intensity is graded in the order of negative (0), weak staining (1+), moderate staining (2+), or positive staining (3+), strong staining (4+). CD123 (CAL-1) high or negative (Daudi)-expressing cell lines were used as positive or negative controls, respectively. For each tissue that showed CD123 staining (colon, stomach, muscle, esophagus, skin, and thyroid), CD123 expression was controlled by immunohistochemistry (IHC) on paraffin-embedded normal tissues from three patients and compared with skin from two BPDCN patients.

[0124] Furthermore, some embodiments described herein refer to an in vivo model for evaluating CAR CD123 efficiency, in which NOD / SCID / IL2Rγc-deficient (NSG) mice (6-8 weeks old, Jackson Laboratory, Sacramento, CA, USA) were irradiated (2.5 Gy) and injected with CAL-1 (1·10 6 / mouse) or PDX cells (2 10 6T cells were inoculated intravenously with an appropriate number of luciferase expressing T cells (1000 / mouse). Engraftment and quantification of BPDCN cells was described. Philippe L, Céléry A, Beauret-Richard E, Jennevrin A, Beachelle S, Perrin S, et al., Bortezomib as a novel treatment for blastic plasmacytic dendritic cell neoplasms, Hematologica, 2017, 102(11): 861-8. In the CAL-1 model, T cells (untransfected or transfected) were injected into the tail vein on day 3. Engraftment was monitored weekly by BLI measurements: mice were given 3 mg luciferin (VivoGlo Luciferin, #P1043, Promega, WI, USA) intraperitoneally within 10 min before imaging (IVIS Lumina Series III, PerkinElmer, MA, USA). In the PDX model, mice were given T cells if BPDCN blasts were detected in blood samples. Blast counts will be monitored every 10 days by flow cytometry using anti-hCD45, anti-CD123 and BD TruCount tubes.

[0125] Some other embodiments described herein include CD123 ロー This refers to the evaluation of CAR toxicity on normal human hematopoietic cells. For this, a humanized mouse model is used. More specifically, a humanized mouse model (female hCD34 engrafted mouse NOD / Shi-scid / IL-2Rγnull immunodeficient mouse strain: hu-NOG) is used to study the in vivo toxicity of the CAR of the invention in normal human hematopoietic cells (n=3 donors). On day 15, two humanized mice were sacrificed; T cells were taken from the spleen and transfected to obtain CD123 CAR T cells or C0. On day 0, two humanized mice (generated from the same donor) were injected with 20·10 6C0 or CD123 CAR are injected retro-orbitally. On days 5 and 15, total leukocytes, monocytes, B and T lymphocytes, pDCs and basophils are quantified in blood and HSC / progenitor cells in bone marrow after sacrifice (only on day 15). For the assessment of ex vivo cytotoxicity of CAR T-cells against normal autologous CD34 cells, magnetically sorted CD34+ cells (CD34 microbeads, Miltenyi Biotec) obtained from bone marrow of sacrificed humanized mice and CD123 CAR can be used. Alternatively, C0 produced from T cells obtained from the same humanized mice (n=3) as above can be used. Co-culture is performed in the same conditions as described for BPDCN cells with an E:T ratio of 5:1. The number of viable hCD34+ after co-culture with CD123 CAR or C0 is compared. The same experiments are performed using magnetically sorted CD34 cells from cord blood co-cultured with CAR CD123 or CO generated from the same cord blood T lymphocytes (n=3).

[0126] Figure 12 shows the transfection efficiency (see Figure 12B). Transfection efficiency was assessed by quantification of T cells co-expressing CD3 and CD19. Variable transfection levels were observed with retroviral constructs, with an average of 17.8% ([3-57%], n=43). This can indicate a variable transfection capacity that can be controlled using magnetic selection to effectively use CAR T cells (purity of transfected T cells was 90% + 9.5% ([60-98.5%], n = 43). More specifically, item B of Figure 12 shows the representative phenotype CD3 / CD19 of non-transfected T cells (C0) compared to transfected T cells of the invention (CD123 CAR T cells or anti-CD123 CAR T cells) before and after magnetic cell separation for T cells of the invention. Lentiviral transfection shows good transfection efficiency (91.2% + 5.22, n = 6) (see Figure 12B).

[0127] For the purposes of the present invention, the evaluation of the expression of the CAR construct in transfected cells can be performed by Western blotting. More specifically, non-transfected T cells (C0) and CD123 CAR T cells according to the invention are lysed by sonication in RIPA buffer supplemented with a protease inhibitor cocktail (Complete Mini EDTA-free; Roche, France). Equal amounts of protein were fractionated by SDS-PAGE and electrotransferred onto a PVDF membrane (Bio-Rad, CA, USA). The membrane was probed overnight with a primary antibody (diluted 1:1000) bound to the human CD3 ζ chain (51-6527GR, BD Biosciences). As an internal loading control, an antibody recognizing β-actin (clone AC15, #A5441, Sigma-Aldrich, MO, USA) was used (diluted 1:10). 6 ) is added. To perform immunodetection, a secondary antibody is added (diluted 1:6000): sheep anti-mouse IgG (#515-035-062, Jackson, PA, USA). Chemiluminescence is detected by adding the appropriate detection reagent (Clarity™, Western ECL Blotting Substrates, Bio-Rad, Cressier, Switzerland) and using a camera and Bio-ID software (Villebar-Roumat, Collégien-France, France).

[0128] Figure 12 also shows a Western blot (see Figure 12C). The expression of the CAR of the invention was verified by immunoblot in three experiments (Figure 12C). A low band (16 kDa) corresponding to the endogenous CD3 ζ chain was detected in transfected and non-transfected T cell lysates, while an additional band appears in CD123 CAR T cells at 55 kDa, corresponding to the theoretical size of the gdCD123 CAR construct. This band is not present in non-transfected T cells. More specifically, item C of Figure 12 shows a Western blot analysis of CD123 CAR protein expression (55 kDa, CD3 ζ fused to the CAR construct compared to endogenous CD3 ζ (16 kDa)) in cell lysates derived from genetically modified T cells (CAR), compared to no expression in C0.

[0129] Figure 12 also shows cytometric flow analysis (see Figure 12D and Figure 12E). Surface expression of CD123 CAR on T cells was confirmed by flow cytometric analysis using biotin-conjugated CD123 protein. Fixation of protein CD123 occurred only on transfected T cells (for CD123R CA, R16%+11.5, n=8, for CD123L CA, 31.5%+12.2, n=9) versus 0% of non-transfected. Item D of Figure 12 shows CAR cell surface expression by flow cytometry: CD123 CAR and C0 were combined with biotin-conjugated CD123 protein plus streptavidin. PE (Streptavidin PE ) More specifically, examples of expression are shown in FIG. 12 section D, and average values ​​from a total of 17 different experiments (n=8 for CD123R CAR, n=9 for CD123L CAR) are shown in FIG. 12 section E. No expression was detected in non-transfected cells.

[0130] More generally, for the purposes of the present invention, the evaluation of the surface expression of CAR in transfected cells is performed by flow cytometry. More specifically, the expression of anti-CD123 scFV on the cell surface of transfected T cells is examined by flow cytometry. T-cells (100,000 cells) are stained with 0.25 μg biotinylated CD123 protein at room temperature for 1 hour, followed by PE-conjugated ptavidin (BD Biosciences). Then, CD3 + CD19 + Immobilization of CD123 on CAR cells will be assessed.

[0131] Figure 13 shows the nucleic acid sequence of a CAR according to the present invention. The CAR is constructed with the monoclonal antibody AB1 of the present invention. The nucleic acids corresponding to the peptide signal, Tag HA, heavy chain, hinge 1, light chain, hinge 2, CD28, 4.IBB, and CD3ζ are each highlighted in a different color from left to right in reading order.

[0132] Thus, an object of the present invention is an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), which consists of a sequence having at least 85%, preferentially 90%, more preferentially 95% identity to SEQ ID NO: 97.

[0133] Another object of the present invention is an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the nucleic acid consists of the amino acid sequence of SEQ ID NO:97.

[0134] Figure 14 shows flow cytometry graphs for phenotypic assay of CAR T cells according to the invention compared to non-transfected T cells.

[0135] The CAR CD123 cell product of this embodiment comprises a mixture of CD4 T cells (58% [22.6-84.8%], n=23) and CD8 T cells (39.6% [16.1-74.9%], n=23). No differences in the CD4 / CD8 ratio are observed between non-transduced and transduced T cells. Item A of Figure 14 shows the percentage of the CD8 population (vs. the CD4 population) in T cells C0 and genetically modified cell (GMC) CD123R CAR T cells in 10 experiments based on 10 different donors. Overall, there is no significant difference in the redistribution of both populations (CD4 and CD8) between transfected and non-transfected cells (ns, p=0,57) (see Figure 14A). Item B of Figure 14 shows a representative example of flow cytometry analysis of memory T cell phenotype based on expression of CD45RA and CD45RO, as well as CD95 and CCR7 (see Figure 14B). Items C and D of Figure 4 show the redistribution of T subpopulations of CD4 and CD8 CD123R CAR T cells (GMC) compared to C0 of three donors, respectively. TEFF: effector T cells, TEM: effector memory T cells, TCM: central memory T cells, TSCM: memory T cells.

[0136] FIG. 15 shows a diagram of targeted cells with the chimeric antigen receptor of the invention and untransfected cells, as well as the specific cytotoxicity of the CAR of the invention against BPDCN cell lines and BPDCN patient-derived xenograft cells, as well as flow cytometry graphs against CD123 negative cells demonstrating the specificity of targeting.

[0137] More generally, Figure 15 shows the specific cytotoxicity of the CARs of the invention against BPDCN cell lines and BPDCN patient-derived xenograft cells. For each graph, the mean and standard deviation are shown.

[0138] The diagram shown on the left in item A of Figure 15 is the general principle of co-culturing C0 and CD123 CAR T cells against BPDCN cells, followed by flow cytometry results on the right. To evaluate CD123 CAR T cytotoxicity (e.g., ability to induce apoptosis in target cells), target cells were co-cultured with effector T cells (C0 or CD123 CAR) for 16h (PDX) or 24h (CAL-1, GEN 2.2, DAUDI, REH) at an E:T ratio of 10:1 (CD123R CAR) or 5:1 (CD123L CAR) or alone 1:0 (control) (see Figure 15A). T cells are targeted using eDye cell proliferation and 7-AAD staining to allow detection of BPDCN cell death. Live target cells were stained with 7-AAD. - CD123 + (gray). Live target cells (7AAD - / CD123 + The percentage of attrition was calculated by comparing the number of cells that died from CAR production with the culture medium alone. As the T lymphocytes used for CAR production are allogeneic to the target cells, a cytotoxicity is obtained in C0 culture conditions (linked to the allogeneicity of the T cells) which is compared to the cytotoxicity obtained with CAR CD123 cultures. The specific cytotoxicity of the C0 or CD123 CAR conditions represented the % of target cell death compared to the % of death in the target alone condition.

[0139] Item B of Figure 15 shows CD123 expression in BPDN cell lines (CAL-1 and GEN2.2) by flow cytometry, item C of Figure 15 shows CDR CAR T cell specific cytotoxicity against CAL-1 and GEN2.2 co-cultured at an E:T ratio of 10:1 (CAL-1, n=22 and GEN2.2, n=10), item D of Figure 15 shows CD123 expression in BPDCN PDXs cells by flow cytometry, and item E of Figure 15 shows cytotoxicity of CD123 CAR T cells against CD123+ PDXs cells (against P127B-M5, n=9, and against P25B-M1, n=6). CD123R CAR T cells demonstrated strong antigen-specific cytotoxicity when co-cultured with CAL-1 cell line (69.1% ± 27.4 by CD123R CAR vs. 22.2% ± 11.8 by C0 attrition, n=22, p<0.0001), GEN 2.2 cell line (65.7% ± 26.4 by CD123R CAR vs. 25.3% ± 12.2 by C0, n=10, p=0.0004), and PDX cells (68% ± 22.2 by CD123R CAR vs. 13% ± 29.5 by C0, n=15) (see Figure 15B, C, D, and E).

[0140] The diagram on the left in item F of Figure 15 shows the lack of CD123 expression in AUDI and REH cell lines by flow cytometry, and the diagram on the right shows the viability of these cell lines (DAUDI, n=6 and REH, n=4) after co-culture with C0 and CD123. - No cytotoxicity was observed in the cell lines DAUDI and REH (see FIG. 15F).

[0141] Item G of Figure 15 shows a graph of cell death in conditions where CAL-1 is cultured with B4D5 antibody for lh before co-culture with C0 or CD123R CAR T cells. Confirmation of the specific cytovalue of CD123R CAR T cells is performed by pre-incubating CAL-1 cells with B4D5 CD123 antibody (AB1). Pre-incubation induces inhibition of cytotoxicity of CD123R CAR T cells, n=5. Inhibition of CAR CD123 cytotoxicity leads to a level of death by CD123R CAR similar to that observed with C0 (27.2% ± 13.7 for CD123 CAR, 17.2% ± 11 for C0, (ns, p=0.2368, n=5)) (see Figure 15G). Indeed, in vitro CD123 + It exhibits specific cytotoxicity against leukocytes.

[0142] Section H of Figure 15 shows the specific cytotoxicity of CD123L CAR against CAL-1 (E:T=10:1, n=3 or E:T=5:1, n=5), GEN2.2 (E:T=5:1, n=3), and 2PDX (E:T=5:1, P127B-M3, n=3 and F13-CAL-1-M2, n=3). Cytotoxicity tests were validated using CD123L CAR at E:T=10:1 and 5:1 ratios (see Figure 15H). For CAL-1, when the E:T ratio is equal to 5:1, an attrition of 94.6% ± 6.1 is observed for CD123L CAR versus 16.6% ± 18.7 for C0 (p<0.0001, n=5), GEN2.2: an attrition of 96.7% ± 4.9 for CD123L CAR versus 7% ± 11.9 for C0 (p=0.0032, n=3); PDX: an attrition of 87.5% ± 6.8 for CD123L CAR versus 21.2% ± 22.7 for C0 (p<0.0001, n=6) (see Figure 15H).

[0143] Item I of Figure 15 shows the viability of CD123 cell lines (DAUDI, n=3 and HL-60, n=4) after co-culture with C0 and CD123L CAR T cells. Again, no toxicity is observed for the CD123 cell lines, HL-60 and DAUDI (for CD123CAR, HL-60 viability 73.8±7.3 vs. for C0, 72.5±15.5, n=4; for CD123CAR, DAUDI viability 93±9.9 vs. for C0, 94.1±8.4, n=3) (see Figure 3I).

[0144] Figure 16 shows CD123 + 1 shows flow cytometry graphs comparing functional characterization of CD123R CAR T cells of the invention in co-culture with target cells.

[0145] Item A of FIG. 16 shows a representative degranulation assay with CD107a expression of CD123R compared to non-transfected cells (C0) after 6h of co-culture with CAL-1 cell line with E:T of 10:1. Item B of FIG. 16 shows a histogram representing the mean value + / - SD of 6 experiments, *p=0.0253. To determine the effector function of CD123R CAR T cells, different parameters were measured after co-culture. CD123R CAR T cells degranulate as they expressed a constant amount of CD107a (10.7% ± 7.6 for CD123R CAR, 2.4 ± 1.3 for C0, p=0.0253, n=6, cf) (see FIG. 16A and FIG. 16B).

[0146] Item C of Figure 16 shows the stained intracellular cytokine IFN-y after 24 hours of co-culture with CAL-1. Item D of Figure 16 shows a histogram representing the mean value + / - SD of three experiments, *p=0.0314. CD123R CAR T cells secrete IFN-g in the intracellular compartment (11% ± 4.7 for CD123R CAR, 2% ± 0.7 for C0, p=0.0314, n=3) (Figure 4C&D), indicating activation of CD123R CAR T cells (see Figure 16C and Figure 16D).

[0147] Item E of Figure 16 shows 16 representative cytokine bead arrays of human Thl / Th2 / Thl7 analysis in co-culture supernatants (from top to bottom: IL-2; IL-6; IL-4; IL-10; TNF; IFN-γ; IL-17a - for colors, red=IL-2; orange=IL-6; yellow=IL-4; green=IL-10; blue=TNF; dark blue=IFN-Y; purple=IL-17a). Higher levels of IFN-γ and IL-10 (CBA analysis) were found in the supernatants of co-cultures with CD123 CAR (IL-10: 91.4 pg / mL ± 19.4 with CD123R CAR vs. 31.6 ± 6.8 with C0, n = 5) (see Figure 16E).

[0148] Section 16F shows histograms representing the mean secretion of IFN-g for CD123R and C0 after co-culture with different target cells, n=3. A significant increase in IFN-gamma secretion (288pg / ml±70 for CD123 CAR and 147pg / ml±67 for C0, n=5) was observed. Secretion is typical of CD123R CAR T-cells, since they do not secrete IFN-gamma in co-culture with the CD123 DAUDI cell line (see Figure 16F).

[0149] Item G of Figure 16 shows a graph of IFN-γ secretion in conditions measured by CBA analysis of CD123 CAR after co-culture with CAL-1 pre-incubated with B4D5 antibody, n=5. IFN-γ produced by CD123R CAR T cells was reduced when CAL-1 cells pre-incubated with CD123 B4D5 antibody of the invention leading to the level of cytotoxicity observed with C0 (see Figure 16G). IL10 was also increased, but at lower levels (91 pg / mL ± 19 for CD123 CAR, 31 ± 6 for C0, n=5) (see Figure 16E).

[0150] FIG. 17 shows a xenograft model, bioluminescence imaging and associated graphs, and flow cytometry graphs for the in vivo anti-tumor activity of T cells of the present invention against BPDCN cell line (CAL-1) and primary derived xenograft cells (PDX).

[0151] More generally, Figure 17 shows in vivo anti-cancer drug activity against CAL-1 and PDX (primary-derived xenografts or patient-derived xenografts).

[0152] Item A of Figure 17 shows a general diagram of the xenograft model. This model uses the CAL-1 luciferase cell line (1·10 6 ) or PDX(2 10 6 ) Based on 6-8 week old NSG mice that are irradiated (2.5 Gy) 1 day before intravenous injection. C0 T cells or T cells containing the CD123L CAR of the invention were either expressed in the CAL-1 model or in blood samples by flow cytometry using CD45 + / CD123 + When BPDCN cells were detected, they were cultured in the tail vein on day 3 (5 or 10 10 6 / mouse) is injected.

[0153] Item B of FIG. 17 shows pictures of bioluminescence imaging analysis both before and during 33 days of CAR anti-CD123 or C0 therapy. Crosses indicate dead mice. Item C of FIG. 17 shows a diagram of quantification of bioluminescence (acts / s) detected in mice treated with C0 (dotted line) and CD123L CAR (solid line). The brightness reflecting leukemia infiltration is higher in those treated with C0 compared to CD123L CAR. This demonstrates the ability of the CD123L CAR of the present invention to suppress and / or treat BPCN. The overall survival of mice treated with a dose of CD123L CAR shows a higher survival rate compared to C0 treated mice (41 vs. 26, 5 mice / group, p=0.0017 log-rank test) (see FIG. 17B and C).

[0154] Item D of FIG. 17 shows a diagram of the overall survival analysis of mice. It appears clear that a significant increase in survival occurs in mice treated with the CAR of the present invention compared to mice treated with C0 (n=5 mice per group). The CD123L CAR of the present invention suppresses BPDCN in a dose-dependent manner. This is consistent with a 10·10 increase in survival of CD123L CAR. 6 The input amount is CD123L CAR5·10 6 This was supported by the superior survival of 100% of patients with 100% CI, which was further supported by the low tumor burden (5·10 6 41 to 10 10 in T cells 6 55 of T cells, 5 mice / population, p=0.0173, log-rank test) (see Figure 17D).

[0155] The same experiments described above in relation to Figure 17 are further validated with the CD123R CAR, i.e., the retroviral version of the invention.

[0156] Thus, FIG. 18 shows a xenograft model, bioluminescence imaging and graphs of the in vivo anti-tumor activity of T cells of the present invention against a BPDCN cell line (CAL-1).

[0157] More generally, Figure 18 shows the in vivo antitumor activity of CD123R CAR T cells against CAL-1. Item A of Figure 18 shows a schematic of the xenograft model: NSG mice were treated with CAL-1 luciferase injection (1·10 6 On the third day, the mice were irradiated with C0 and GMC (10 10 6 ) (see FIG. 18A). Item B of FIG. 18 shows bioluminescence imaging after CD123R CAR T-cell or C0 treatment following CAL-1 luciferase implantation. Item C of FIG. 18 shows a diagram of the overall survival analysis of the mice. In the survival analysis, mice treated with CD123R CAR show significant survival compared to mice treated with C0 (n=5 mice).

[0158] As a result, the CAR of the present invention is an effective treatment for BPDCN.

[0159] As shown in FIG. 17, the Le CAR lentiviral version of the present invention was further tested.

[0160] More specifically, the results are confirmed in PDX models (see Figure 17A).

[0161] Item E of Figure 17 shows representative examples of blood immunoassays performed upon C0 or CD123L CAR treatment. Human T cells and primary BPDCN cells are distinguishable based on hCD45 expression. C0 (hCD45 ハイ / CD123 - / CD3+ CD19- or CD123L CAR (hCD45 ハイ / CD123- / CD3+ CD19+) are grey (data are in colour: blue), and BPDCN cells (hCD45 ロー / CD123 + ) are in black (pink if in color). Item F of Figure 17 shows a graph of the average number of BPDCN cells in the blood of mice injected with LAN PDX during treatment with C0 (black line, or blue line if data is in color) or CD123 CAR (gray line, or red line if data is in color). Item G of Figure 17 shows a graph showing the overall survival of PDX xenografted mice. Item G of Figure 17 shows the overall survival of PDX xenografted mice. Compared to mice treated with C0, mice treated with CD123L CAR have a higher survival rate (p=0,01). More specifically, in the PDFX 13-LAN model, 78 days after injection, BPDCN cells are detectable in the blood of all mice: 5·10 6 one group received 5 10 CD123L CAR (mean BPDCN cells: 24 blasts / μL [1-56], n=8) and the other 5 10 6C0 (26 blasts / μL [1-66], n=8). Mice are monitored by blood analysis using a traquant analysis for 25 days. On day 7, BPDCN cells and T-cells are quantified in mouse blood samples (see Figure 17E and F). During the follow-up, it was possible to suppress the number of circulating BPDCN cells when treated with CD123L CAR (see Figure 17C) and a higher overall survival of mice (31 days of CD123L CAR treatment vs. 20 days of C0 treatment, p=0.0103) was shown (see Figure 17G). Overall, the results obtained in the model with BPDCN lines and PDX cells showed that the inventive CAR CD123L allows the suppression of BPDCN in vivo.

[0162] The CAR retroviral version of the present invention has been validated in experiments similar to those shown for the lentiviral version.

[0163] FIG. 19 shows a fluorescence graph of CD123 expression in different cell types, immunohistochemistry images of CD123 expression in tumor and normal tissues, a flow cytometry graph of CD123 expression, a diagram of the production of autologous T cells of the invention, and a flow cytometry graph of the relative CD123 toxicity of T cells of the invention against cells expressing CD123 at low levels.

[0164] More generally, Figure 19 shows an assessment of the toxicity of the CD123L CAR T cells of the invention against cells expressing low levels of CD123.

[0165] As previously described, anti-CD123 antibodies (B4D5 of the present invention and the commercially available antibody 6H6) show similar staining with CD123 (see FIG. 10C). Therefore, 6H6 antibody was used to compare CD123 expression on normal hematopoietic and endothelial cell lines (HMEC) compared to BPDCN.

[0166] FIG. 19, item A, shows a graph of CD123 expression in different cell lines. The 6H6 commercial antibody was used to compare CD123 expression in BPDCN (n=60) to CD34 cells (n=16) in lymphocytes (n=31), monocytes (n=22), normal pDC (n=30), normal bone marrow and endothelial HMEC cell lines (n=4) in blood and bone marrow samples. The results are shown in MFI (see Figure 19A). Lower expression is detected in monocytes (MFI=1704[778-4578], n=22), endothelial cell lines (MFI=1252[602-2283], n=4), and CD34+ cells (MFI=874[326-1424], n=16) compared to BPDCN cells (MFI=11677[2962-33439], n=60) and normal pDCs (MFI=34352[17822-76610], n=30). The CD123 CAR of the present invention enhances CD123 expression in vivo. ハイ CD123 from BPDCN cells ロー It has less cytotoxicity to cells.

[0167] To evaluate CD123 expression on tissues, tissue microarrays were performed using the AB1 (or 18B4D5 antibody) of the present invention. For this purpose, US Food and Drug Administration standard frozen tissue arrays (US Biomax, Rockville, MD, USA) containing 90 tissue cores (30 organs) of three individual donors per organ were used. Immunostaining was detected using the UltraView Universal DAB Detection Kit (Ventana, USA). Images were acquired and analyzed with NDP.view 2.0 software. Staining intensity was graded in the following order: negative (0), weak staining (1+), moderate (2+), positive (3+), and strong (4+). High CD123 (Cal-1) or negative (Daudi) expressing cell lines were used as positive or negative controls, respectively. Table 4 shows the results of the tissue microarray.

[0168] [Table 4]

[0169] The results show that there was no significant expression of CD123 on specific tissue cells, and only some endothelial cells in lymph nodes, stomach, colon, muscle, esophagus, skin and thyroid expressed CD123.

[0170] Item B of FIG. 19 shows pictures of immunohistochemistry with B4D5 antibody in tumor tissue (BPDCN) and normal tissue. To compare the level of CD123 expression in endothelial cells in said tissues with that in BPDCN, IHC was performed in three normal tissues with the AB1 (18B4D5) antibody of the invention. Expression is compared with the expression level obtained in skin lesions of BPDCN patients (n=2). Two patients have high levels of CD123 expression in skin lesions and low levels in endothelial cells of the tested tissues (see FIG. 19B). In more detail, BPDCN patient 1 and patient 2 show high CD123 expression in intradermal blastic infiltrates of BPDCN cells. CD123 immunostaining of different healthy tissues shows low levels of CD123 expression in endothelial cells (arrows). Specific tissue cells are CD123 negative. Low non-specific labeling is seen in gastric and thyroid secretions.

[0171] Both approaches showed low levels of CD123 on monocytes, CD34+ cells and endothelial cells, indicating low "on-tumor targeting" potential of the inventive CAR CD123.

[0172] Item C of Figure 19 shows a representative expression of CD123 K532. More specifically, a representative expression of CD123 in live K562 CD123 line cultured alone or with C0 or CD123 CAR for 24 hours at an E:T ratio of 5:1 is shown. The CD123 region is defined by the unique K562 line (which is CD123) and corresponds to the maximum expression in monocytes and HMEC cells. CD123 POS represents the level of CAL-1 expression, and CD123 ハイrepresents a higher level than CAL-1. This modeling shows the relationship between CD123 expression levels on target cells and the killing efficiency of CD123L CAR using K562 cell lines expressing different levels of CD123 (K562 CD123). As mentioned above, cell lines are co-cultured with C0 or CD123 CAR or cultured alone. Comparisons were made of the percentage of the total population of persistent K562 cells according to their level of CD123 expression, i.e. CD123 - , CD123 ロー (corresponding to maximum CD123 expression on monocytes and endothelial HMEC cells), CD123 + / ハイ (Comparable to the levels of CD123 expression in CAL-1 and others) (see FIG. 19C).

[0173] Item D of Figure 19 shows a graph of the percentage of cells in each subgroup of K562. For n=3, when cells were cultured with CAR CD123, K562 CD123 ロー The percentage of K562 CD123 cells was not decreased. pos / ハイ Cells disappeared; K562 CD123 - More specifically, whereas the distribution of K562 cells with respect to each expression level was similar when co-cultured alone or with C0, the percentage of positive / high positive CD123 cells was significantly decreased (n=3, p=0.0001) when K562 CD123 was co-cultured with CD123L CAR (resulting in an increased percentage of CD123 cells) (see FIG. 19D). Furthermore, CD123 ロー For cells, the percentage of persister cells was similar during culture with C0 or CD123L CAR, with the CD123L CAR showing a higher persister percentage than the C123L CAR. ロー No significant cytotoxicity was observed against the cells, demonstrating the ability of the CD123L CAR of the present invention to kill highly CD123-expressing cells with no or very little effect on weakly expressing cells.

[0174] Item E of Figure 19 shows a graph for inhibition of target cell proliferation. The cytotoxicity of CD123L CAR T was evaluated on endothelial cell line HMEC and primary monocytes. Inhibition of proliferation after 18 hours of co-culture is evaluated using a proliferation test (MTT cell proliferation test). The percentage of inhibition of cell proliferation is shown. CAL-1 is used as a CD123 positive control and DAUDI as a CD123 negative control (see Figure 19E).

[0175] Item F of Figure 19 shows human CD34 + Figure 19 shows a diagram of the experimental generation of autologous CD123L CAR T-cells from transplanted humanized mice. Item G of Figure 19 shows a graph of the cytotoxicity of CD123L CAR against CD34 cells. Two sources of CD34 cells are used. CD123L CAR T-cells were produced from humanized mice. In addition, umbilical cord blood was used to produce CD123L CAR T-cells cultured with CD34+ cells sorted from the same umbilical cord blood, **p is 0.0019.

[0176] Item H of Figure 19 shows representative expression of CD123 on CD34+ cells from mouse bone marrow after co-culture with autologous CAR T cells or C0. The CD123L CAR expresses CD123 on HMECs. ローinduced low lysis levels of CD123L CAR, indicating the potential low toxicity of CD123L CAR to endothelial cells. Because CD123 is expressed at low levels on CD34+ cells, the in vitro cytotoxicity of CD123L CAR T cells was examined using the two strategies. First, T cells sorted from umbilical cord blood were transfected to produce CD123L CAR or C0 T cells and co-cultured with CD34+ cells sorted from autologous umbilical cord blood; no depletion of CD34+ cells by CD123L CAR was observed (5.7% ± 5.1 for depletion of CD123L CAR, 11.7% ± 10.2 for depletion of C0, n = 3, p = 0.41) (see Figure 19G). Second, human T cells sorted from humanized mice were used to produce CD123L CAR or C0 co-cultured with autologous CD34+ cells (see Figure 19F, G and H). Co-culture with CD123L CAR T cells or C0 cells leads to a low attrition of CD34+ cells (12.7% ± 16.3 for CD123L CAR and 2.7% ± 3.1 for C0, ns, p = 0.3542, n = 3) (see Figure 19G). Focusing on the CD34+ / CD123+ subpopulation (representing 38% and 16% of CD34+ cells, n = 2), it is observed that only CD34 / CD123+ cells are killed by CD123L CAR (82.1% ± 0.6 attrition), whereas CD34+ / CD123 cells remain viable (3.5% ± 4.9 attrition) (see Figures 19G and H).

[0177] Furthermore, we studied CD123L CAR reactivity against autologous hematopoietic cells in vivo in the humanized mouse model described above. Human CD123 CAR T cells or C0 were generated from human T cells. The transfection efficiency was 55.4% ± 9.2 (n = 3), and the efficiency of this CD123L CAR T-cells was verified ex vivo before reinfusion.

[0178] Figure 20 shows a graph based on flow cytometry for the evaluation of the toxicity of CD123L CAR T cells from humanized mice against CAL-1 and HL-60. More specifically, the toxicity of CD123L CAR T cells obtained from humanized mice against autologous CAL-1 and CD123 negative HL-60 cells is shown. It is observed that CAR CD123 attenuates CAL-1 cells by 98.6% (±0.5%, n=3) while C0 attenuates only 0.7% (±0.6, n=3), with no difference between C0 and CD123L CAR attenuation against CD123 HL-60 cell line.

[0179] Item 1 of Figure 19 shows a graph of a representative example of in vivo toxicity of CD123L CAR in humanized mice. For each mouse, the fold change (FC) of human hematopoietic populations between D-5 and D15 (blood: B and T lymphocytes, monocytes, pDCs) is measured. No significant differences are found between the two mouse groups (C0 and CD123L CAR) in human immune cell populations (T, B cells, monocytes, pDCs, basophils). Day 5 is used as a reference, and the FC of each immune cell subpopulation (total hCD45 + The fold change in the number of IL-17 positive cells (IL-1, B cells, T cells, monocytes) is calculated. n=3 cell counts are performed from peripheral blood collected by retro-orbital samples. A representative example is shown.

[0180] Item J of Figure 19 shows a graph of the percentage of CD34+ cells in humanized mice 15 days after C0 or CD123L CAR T-cell injection from n=3 donors. Briefly, the FC of mice injected with CD123L CAR was compared to the FC of mice injected with C0 (see Figures 19I and J).

[0181] In three experiments, no differences in the numbers of T, B lymphocytes, monocytes, or pDCs were observed between the two mouse groups (C0 and CD123L CAR). Only at D15 did bone marrow CD34 +When cells were analyzed and compared between C0-injected and CD123 Curl-injected mice (n=3), no significant decrease in the percentage of CD34+ in human CD45+ cells was observed (see FIG. 19J).

[0182] These data suggest that CD123 ロー The cytotoxicity of the CD123L CAR of the present invention is confirmed to be absent or at least extremely low on hematopoietic cells.Furthermore, the data confirms the selective cytotoxicity on CD123+ subpopulation.In vivo, no significant effect is observed on circulating monocytes, lymphocytes and pDCs as well as bone marrow CD34+ cells.

[0183] Figure 21 shows flow cytometry and graphs of BPDCN patient-derived CD123 CAR T cells induced specific cytotoxicity against autologous BPDCN blasts. The results show that the patient's autologous CD123L CAR T cells successfully eliminated primary BPDCN blasts ex vivo without cytotoxicity against normal blood cells (B and T lymphocytes).

[0184] Section A of Figure 21 shows a graph of a flow cytometry phenotype assay comparing untransfected T cells (C0) and transfected CD123L CAR T cells of the invention for CD3 / CD19 phenotype. In one BPDCN patient, efficient CD123 CAR T cells and C0 were generated using autologous T cells. A transfection efficiency of over 79.2% was obtained after 9 days (see Figure 21A), which is similar to that achieved with T cells from healthy donors (see Figure 21B).

[0185] Item B of FIG. 21 shows biotin-conjugated CD123 protein plus streptavidin. PE A flow cytometry graph of CD123 CAR cell surface expression is shown with a control for CD123 CAR cell surface expression with biotin-conjugated CD123 protein (see FIG. 21B).

[0186] Figure 21, item C, is a graph of the patient negative fraction after CD3 / CD28 magnetic selection / activation. The grey data (color: pink) represent blast cells (CD45 ロー , CD3 - , CD19 - , CD303 + , CD123 ハイ ), and dark grey data (blue in color) are T lymphocytes (CD45 ハイ , CD3 + , CD19 - , CD303 - , CD123 - ), and light gray data (orange in color) represent B lymphocytes (CD45 ハイ , CD3 - , CD19 + , CD303 - , CD123 - ) represents the functional activity of autologous CAR T cells against the patient's BPDCN blasts. The blast fraction is composed of the negative fraction of T cell selection. This represents the BPDCN blasts (grey / pink population: CD45 ロー / CD3 - / CD19 - / CD303 + / CD123 ハイ ), T cells (blue / dark grey population: CD45 ハイ / CD3 + / CD19 - / CD303 - / CD123 - ) and B cells (orange / light grey population: CD45 ハ / CD3 - / CD19 + / CD303 - / CD123 - ) (see Figure 21C). CD123L CAR and C0 were co-cultured with autologous blast fraction for 24 h at an E:T ratio of 5:1.

[0187] Item D of Figure 21 shows a flow cytometry graph and cell viability diagram, which shows the specific cytotoxicity of CD123L CAR T cells against autologous BPCN blasts after 24 h of co-culture with (orange = B cells; pink = BPCN blasts; blue = T cells, grey = C0 or CD123L CAR) at an E:T ratio of 5:1. More specifically, CD123 - BPDCN blasts (CD123 + ) is observed. Patients lack monocytes in their blood, precluding toxicity assessment of autologous monocytes. Taken together, these results demonstrate that the autologous CD123L CAR T cells of the present invention are selectively effective in depleting BPDCN blasts ex vivo.

[0188] As mentioned above, BPDCN is a clonal disease that progresses in most cases to aggressive leukemia with extremely poor prognosis. Improved treatment is needed. BPDCN cells are sensitive to chemotherapy, but patients nevertheless relapse and are resistant to second-line treatment. The present invention provides targeted therapy against CD123, a "key antigen" highly expressed in BPDCN patients. More specifically, the present invention is used for BPDCN treatment using a specifically designed chimeric antigen receptor (CAR)-based therapy. The present invention allows for selective targeting and eradication of BPDCN cells in patients. Furthermore, the present invention provides long-lasting remission in BPDCN.

[0189] Thus, the present invention provides T cells genetically modified with a third generation CAR that specifically recognizes and / or specifically binds to CD123. The CAR of the present invention is produced using a retroviral or lentiviral method. The retroviral or lentiviral method mediates antitumor activity. T cells expressing the CD123 CAR of the present invention can specifically lyse BPDCN cells. More specifically, T cells expressing the CD123 CAR of the present invention can specifically lyse different BPDCN targets, namely BPDCN lines CAL-1 and GEN2.2 and / or BPDCN PDX and primary BPDCN patient cells. When the CAR of the present invention is cultured with these target cells, effector functions in vitro are activated, such as IFN-γ release, CD107a degranulation, etc. The functionality of the CAR of the present invention is specific to CD123 expression by the target cells. Indeed, when CD123 expression is blocked by pre-culture with the anti-CD123 antibody of the present invention (e.g., AB1), cytotoxicity and cytokine secretion are reversed. Furthermore, there is a surprising effect of the present invention in that the CAR of the present invention is - When co-cultured with BPDCN strains (Daudi, REH, HL-60), there is no (or virtually no) cytotoxicity and / or secretion of cytokines. In vivo, T cells expressing the CAR of the present invention exhibit anti-leukemic activity against BPDCN strains. The positive effect of the present invention is that it allows long-term survival of subjects (e.g., mice) affected by BPDCN.

[0190] The art discloses agents, i.e. antibodies, that target CD123. References: Li RB, Wu C, Walter MK, Valliere-Douglas J, et al., Characterization of the CD123-directed antibody-drug conjugate SGN-CD123A for acute myeloid leukemia, Molecular Cancer Circulation, 2018, 17(2):554-64; Sha LH, Biondo M, Busfield SJ, Arruda A, You X, Vairo G, et al., CD123 target validation and preclinical evaluation of ADCC activity by the combination of anti-CD123 antibody CSL362 with NK from AML patients in remission, Blood Cancer Journal, 2017, 7(6); Kovtun Y, Jones GE, Adams S, Harvey L, Audette CA, Wilhelm A et al.,Antibody-drug conjugate targeting CD123, IMGN632, designed to eradicate AML while sparing normal myeloid cells,Blood Advances,2018,2(8):848-58;fusion proteins [Frankel AE, Wu JH, Ahn C, Penmarj N, Medeiros BC, Carrollway HE et al.,Activity of SL-401, a targeted therapeutic agent against interleukin-3 receptor in patients with blastic plasmacytic dendritic cell neoplasms,Blood,124(3):385-92],BiTes [Pichitola I, Anjos-Afonso F, Rassairi F, Tettamanti S, Spinelli O et al.,A chimeric antigen receptor for CD33 / CD123 antigen efficiently targets primary acute myeloid leukemia cells in vivo,Lukemyia,28(8):1596-605;Chu SY, Peng E, Chin H, Hung S, Chan EW, Endo NA et al., Immunotherapy with a long-lived anti-CD123 anti-CD3 bispecific antibody effectively promotes T cell-mediated killing of human AML cell lines and CD123+ cells in monkeys: both possible targets for acute myeloid leukemia, Blood, 2014, 124(21):2316;and Luella M, Barrett DM, Kendarian SS, Shestova O, Hoffman TJ, Perazzelli J, et al., Dual targeting of CD19 and CD123 prevents relapse of antigenic loss after CD19-directed immunotherapy, Journal of Clinical Investigation, 2016, 126(10):3814-26], DART [Al-Hussaini M, Rettig MP, Rettig-Ritchie JK, Karpova D, Yui GL, Eisenberg LG, et al., Targeting CD123 in acute myeloid leukemia with a T cell-directed dual affinity retargeting platform, Blood, 2016, 127(1):122-31], triple body or CAR T cells [Gill S, Tacian SK, Luella M, Shestova O, Li Y, Porter RD, et al., Dual targeting of CD19 and CD123 prevents relapse of antigenic loss after CD19-directed immunotherapy, Journal of Clinical Investigation, 2016, 126(10):3814-26], DL et al., Targeting and myeloablation in preclinical trials of human acute myeloid leukemia using chimeric antigen receptor-modified T cells, Blood, 123(15):2343-54;and Tettamanti S, Marin V, Pizzittola I, Magnani CF, Giordano Attianese GM, Crivioli E, et al., Targeting acute myeloid leukemia with cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor, British Journal of Hematology: 2013, 161(3):389-401. Some of these studies show the efficacy of anti-CD123 therapy for AML, but the various negative effects on normal hematopoiesis remain to be reported. References: Penmarj N, Laa AA, Sweet KL, Stein AS, Vas S, Bloom W, et al., Tagraxofusp in blastic plasmacytic dendritic cell neoplasms, New England Journal of Medicine, 2019, 380(17):1628-37. A recent study using SL-401 (IL3 related to diphtheria toxin) in 32 patients with BPDCN in the first-line setting achieved complete clinical response in 72% of patients and 52% of patients alive at 24 months (including 45% of patients who underwent allogeneic transplant). In 15 previously treated patients, the response rate was 67% and the median overall survival was 8.5 months. However, negative side effects (grade 3 or higher) were observed in 78% of patients, leading to the death of three patients in the study. As a result, toxicity is a major concern. This problem has been significantly improved by the present invention;

[0191] The active CAR of the present invention is undergoing clinical trials in BPDCN patients (and also in AML patients). No serious toxic events and / or negative effects have been reported.

[0192] Tissue microarray (TMA) and immunohistochemistry (IHC) showed that when CARs were produced using the antibodies of the present invention (e.g., 18B4D5), CD123 ロー It is confirmed that no or low cytotoxicity occurs in cells. Furthermore, no or low cytotoxicity occurs in endogenous cell lines of HMECs by the CAR CD123L of the present invention.

[0193] In vivo data collected from humanized mice injected with the autologous CAR CD123 of the present invention shows no significant effect on total CD45+ cells, T and B cells, and blood subpopulations of monocytes for mice treated with the CD123 CAR of the present invention.The art has reported problems potentially induced by CAR T cells.The so-called cytokine release syndrome (CRS) is frequent, and there are studies showing that monocytes are the main source of IL-1 and IL-6 during CRS development. References: Giavridis T, van der Stegen SJC, Eychem J, Hamier M, Piezilli A, Sadelain M., CAR T cell-induced cytokine release syndrome is mediated by macrophages and attenuated by IL-1 blockade, Nature Medicine, 2018, 24(6):731-8; and Norelli M, Camisa B, Barbiera G, Falcone L, Pueavedrji A, Genova M, et al., Monocyte-derived IL-1 and IL-6 are differentially required for cytokine release syndrome and CAR T cell-induced neurotoxicity, Nature Medicine, 2018, 24(6):739-48. The present inventors provide data that lead them to believe that a potential low level of monocyte depletion in vivo by the present CD123 CAR T-cells may be involved in limiting CRS (as monocytes express low levels of CD123).

[0194] To tailor the CAR of the invention for therapeutic purposes in humans, an embodiment of the T cells of the invention includes a suicide gene, i.e., the system iCASP9 / rimiduside. References: Warda W, La Rosa F, Neto da Rocha M, Trad R, Decooninck E, Fajloun Z, et al., IL1RAP-expressing CML hematopoietic stem cells can be targeted by chimeric antigen receptor-modified T cells, Cancer Research, 2019, 79(3):663-75.

[0195] The present invention further provides a method for the detection and treatment of BPDCN CD123+ blasts in vitro and in vivo. ローThe present invention provides anti-CD123 CAR T cells with a safety profile evaluated for cells. The lentivirus method allows good reproducible transgene expression, which has good activity in vitro and in vivo. In clinical application and clinical development, the CAR of the present invention is preferably in lentivirus version. References:Zufrey R, Donello JE, Trono D, Hope TJ, Woodchuck hepatitis virus posttranscriptional regulatory elements promote expression of genes delivered by retroviral vectors, Journal of Virology, 1999;73(4):2886-92;Miyoshi H, Blommer U, Takahashi M, Gage FH, Verma IM, Development of self-inactivating lentiviral vectors, Journal of Virology, 1998;72(10):8150-7;Zufrey R, Dal T, Mandel RJ, Bukovski A, Kiiros D, Nardini L, et al., Safe and efficient in vivo gene expression with self-inactivating lentiviral vectors, Journal of Virology, 1998;72(12):9873-80.

[0196] The objects of the present invention are therefore defined in the appended claims. Further objects of the present invention are defined in the appended claims with reference to sequence homology instead of sequence identity.

[0197] Thus, an embodiment of the present invention is directed to an antibody selected from the group consisting of AB1, AB2, AB3, AB4, AB5 and AB6, wherein AB1 has a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 25, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 1, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 26, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 14, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 2; AB2 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 27, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 15, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 3, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 28, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 16, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 4; AB3 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 29, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 5, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 30, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 6; AB4 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 31, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 19, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 7, and a light chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 32, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 20, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100% identity to the amino acid sequence of SEQ ID NO: 8; AB5 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 9; and the light chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 10; AB6 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 11, and the light chain comprising a complementarity determining region 1 (CDR1) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 36, a complementarity determining region 2 (CDR2) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 24, and a complementarity determining region 3 (CDR3) having at least 90%, preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 12.

[0198] Another object of the present invention is an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) or an isolated chimeric antigen receptor (CAR) molecule, characterized in that said CAR comprises an antibody or an antibody fragment comprising an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and comprises an antibody having an anti-CD123 binding domain, or a fragment of said antibody, The anti-CD123 binding domain consists of the following binding domains: (i) a heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO:1, and a light chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO:2; (ii) a heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 3, and a light chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 4; (iii) a heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO:5, and a light chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO:6; (iv) a heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 7, and a light chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90%, preferably 100%, identity to the amino acid sequence of SEQ ID NO: 9, and a light chain comprising a complementarity determining region 3 (CDR3) having at least 90%, preferably 100%, identity to the amino acid sequence of SEQ ID NO: 10; or (vi) a heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 11, and a light chain comprising a complementarity determining region 3 (CDR3) having at least 90%, and preferentially 100%, identity to the amino acid sequence of SEQ ID NO: 12; Another embodiment of the present invention is a CAR as described above, but with reference to sequence homology instead of sequence identity.

[0199] Another object of the present invention is an expression vector consisting of a nucleic acid molecule as defined above, as well as modified immune cells (such as T cells) consisting of said nucleic acid molecule as defined above. According to the present invention, the cells can be used as a medicament for the treatment of a proliferative disease in a mammal, preferably a human, wherein the proliferative disease is a disease associated with CD123 overexpression, preferably blastic plasmacytoid dendritic cell neoplasm (BPDCN). Furthermore, the present invention is directed to a pharmaceutical composition consisting of the modified immune cells as defined above. Furthermore, the present invention is directed to a method of modifying immune cells comprising transfection of the cells with an expression vector as described above.

[0200] Another embodiment of the invention features an engineered immune cell comprising a claimed nucleic acid molecule, where the cell is a T cell or a natural killer cell.

[0201] Other definitions of the present invention may include antibodies that specifically bind to the antigen CD123, selected from the group consisting of AB1, AB2, AB3, AB4, AB5 and AB6, AB1 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 85 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 86; AB2 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO:87 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO:88; AB3 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO:89 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO:90; AB4 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO:91 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO:92; AB5 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO:93 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO:94; AB6 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO:95 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO:96; In addition, the antibody is 9.5×10 ―9 K under M D Value, 9.0×10 ―4 s -1 Dissociation rate less than K off , at least 7 × 10 4 Ms -1 Association rate constant K on has.

[0202] In one embodiment, the CD123 binding domain is an scFv, the scFv comprising an amino acid sequence having 1, 2 or 3 or more, and up to 30, 20 or 10 modifications of the light chain variable region of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36. and a heavy chain variable region having an amino acid sequence having 1, 2 or 3 or more and 30, 20 or 10 or less recombinations of the heavy chain variable region of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35.

[0203] In one embodiment, the CD123 binding domain is an scFv, and the scFv has (i) a complementarity determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, or SEQ ID NO:36, or SEQ ID NO:24; and a complementarity determining region 3 (CDR4) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. 3) a light chain variable region consisting of: and (ii) a complementarity determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, or SEQ ID NO:35, and a complementarity determining region 2 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. and a complementarity determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

[0204] In one embodiment, the anti-CD123 binding domain is linked to the transmembrane domain by a hinge region.Preferably, the encoded CAR comprises the transmembrane domain of CD28.

[0205] In one embodiment, the hinge region comprises an IgG1 hinge sequence or a sequence having 95 to 99% identity thereto. In a further embodiment, the hinge region comprises an IgG4 hinge sequence or a sequence having 95 to 99% identity thereto. In a further embodiment, the hinge region may be composed of the CH2-CH3 region of IgG1 or IgG4 or CD8α, or a sequence having 95 to 99% identity thereto.

[0206] In a preferred embodiment, at least one costimulatory domain of the functional intracellular signaling domain is obtained from one or more proteins selected from the group consisting of costimulatory domains obtained from 4.IBB and / or CD3ζ.

Claims

1. An antibody that specifically binds to the antigen CD123, the antibody is selected from the group consisting of only AB1, AB2, AB3, AB5, and AB6; The antibody was 9.5×10 -９ K less than M Ｄ value, 9.0 x 10 -４ s -1 Dissociation rate K less than off And at least 7 x 10 ４ Ms. -1 Association rate constant K on having wherein AB1 comprises a heavy chain and a light chain, the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:25, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:13, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:1, and the light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:2; AB2 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:27, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:15, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:3, and the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:4; AB3 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:29, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:17, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:5, and the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:30, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:18, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:6; AB5 comprises a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 9, and the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 10; AB6 is an antibody comprising a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 11, and the light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 36, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 24, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:

12.

2. 2. The antibody of claim 1, wherein AB1 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 85 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 86; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 25, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 1; The light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:2: AB2 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 87 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO: 88; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 27, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 15, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 3; the light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:4; AB3 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO:89 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO:90; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 29, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 5; the light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 30, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 6; AB5 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO:93 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO:94; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 9; the light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 10; AB6 comprises a heavy chain having at least 85% identity to the amino acid sequence of SEQ ID NO:95 and a light chain having at least 85% identity to the amino acid sequence of SEQ ID NO:96; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 11; The light chain of this antibody comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 36, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 24, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:

12.

3. 3. The antibody of claim 1, wherein AB1 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 85 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 86; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 25, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) consisting solely of SEQ ID NO: 1; The light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:2: AB2 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 87 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 88; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:27, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:15, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:3; the light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:4; AB3 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:89 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:90; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 29, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 5; the light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 30, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 6; AB5 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:93 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:94; The heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 9; the light chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:34, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:22, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:10; AB6 comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO:95 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO:96; the heavy chain comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 11; The light chain of this antibody comprises a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 36, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 24, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:

12.

4. The antibody according to any one of claims 1 to 3, The heavy chain of AB1 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 37, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 38, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 39, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 40; The light chain of AB1 has a variable region comprising a first framework region (FR1) consisting solely of the amino acid sequence of SEQ ID NO:61, a second framework region (FR2) consisting solely of the amino acid sequence of SEQ ID NO:62, a third framework region (FR3) consisting solely of the amino acid sequence of SEQ ID NO:63, and a fourth framework region (FR4) consisting solely of the amino acid sequence of SEQ ID NO:64; The heavy chain of AB2 has a variable region including a first framework region (FR1) consisting of only the amino acid sequence of SEQ ID NO: 41, a second framework region (FR2) consisting of only the amino acid sequence of SEQ ID NO: 42, a third framework region (FR3) consisting of only the amino acid sequence of SEQ ID NO: 43, and a fourth framework region (FR4) consisting of only the amino acid sequence of SEQ ID NO: 44; The light chain of AB2 has a variable region comprising a first framework region (FR1) consisting solely of the amino acid sequence of SEQ ID NO:65, a second framework region (FR2) consisting solely of the amino acid sequence of SEQ ID NO:66, a third framework region (FR3) consisting solely of the amino acid sequence of SEQ ID NO:67, and a fourth framework region (FR4) consisting solely of the amino acid sequence of SEQ ID NO:68; The heavy chain of AB3 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO: 45, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO: 46, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO: 47, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO: 48; The light chain of AB3 has a variable region comprising a first framework region (FR1) consisting solely of the amino acid sequence of SEQ ID NO:69, a second framework region (FR2) consisting solely of the amino acid sequence of SEQ ID NO:70, a third framework region (FR3) consisting solely of the amino acid sequence of SEQ ID NO:71, and a fourth framework region (FR4) consisting solely of the amino acid sequence of SEQ ID NO:72; The heavy chain of AB5 has a variable region including a first framework region (FR1) consisting of only the amino acid sequence of SEQ ID NO:53, a second framework region (FR2) consisting of only the amino acid sequence of SEQ ID NO:54, a third framework region (FR3) consisting of only the amino acid sequence of SEQ ID NO:55, and a fourth framework region (FR4) consisting of only the amino acid sequence of SEQ ID NO:56; The light chain of AB5 has a variable region comprising a first framework region (FR1) consisting solely of the amino acid sequence of SEQ ID NO: 77, a second framework region (FR2) consisting solely of the amino acid sequence of SEQ ID NO: 78, a third framework region (FR3) consisting solely of the amino acid sequence of SEQ ID NO: 79, and a fourth framework region (FR4) consisting solely of the amino acid sequence of SEQ ID NO: 80; The heavy chain of AB6 has a variable region including a first framework region (FR1) consisting only of the amino acid sequence of SEQ ID NO:57, a second framework region (FR2) consisting only of the amino acid sequence of SEQ ID NO:58, a third framework region (FR3) consisting only of the amino acid sequence of SEQ ID NO:59, and a fourth framework region (FR4) consisting only of the amino acid sequence of SEQ ID NO:60; The light chain of AB6 is an antibody having a variable region including a first framework region (FR1) consisting solely of the amino acid sequence of SEQ ID NO: 81, a second framework region (FR2) consisting solely of the amino acid sequence of SEQ ID NO: 82, a third framework region (FR3) consisting solely of the amino acid sequence of SEQ ID NO: 83, and a fourth framework region (FR4) consisting solely of the amino acid sequence of SEQ ID NO:

84.

5. 1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR consisting of an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain; The anti-CD123 binding domain comprises: (i) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 25, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 1; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:2; (ii) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 27, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 15, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 3; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:4; (iii) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 29, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 5; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:30, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:18, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:6; (iv) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 31, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 19, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 7; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 32, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 20, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 9; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 10; or (vi) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 11; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:36, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:24, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:

12.

6. 6. The isolated nucleic acid molecule of claim 5, wherein the anti-CD123 binding domain is selected from the group consisting of an antibody, Fv, scFv, Fab, or other antibody fragment, preferably an scFv.

7. 7. The isolated nucleic acid molecule of claim 5, wherein the intracellular signaling domain is CD3-zeta (CD3ζ), and optionally comprises a costimulatory domain selected from the group consisting of CD28, 4.1BB, inducible T cell costimulatory factor (ICOS), OX-40 and binding thereof.

8. An isolated polypeptide molecule encoded by the isolated nucleic acid molecule of any one of claims 5 to 7.

9. An isolated chimeric antigen receptor (CAR) molecule, said CAR consisting of an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain; The anti-CD123 binding domain comprises: (i) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 25, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 1; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:2; (ii) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 27, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 15, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 3; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:4; (iii) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 29, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 17, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 5; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:30, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:18, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:6; (iv) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 31, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 19, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 7; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 32, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 20, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 8; (v) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 9; and a light chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 10; or (vi) a heavy chain comprising a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO: 35, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO: 23, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO: 11; and a complementarity determining region 1 (CDR1) consisting solely of the amino acid sequence of SEQ ID NO:36, a complementarity determining region 2 (CDR2) consisting solely of the amino acid sequence of SEQ ID NO:24, and a complementarity determining region 3 (CDR3) consisting solely of the amino acid sequence of SEQ ID NO:

12.

10. 8. An isolated chimeric antigen receptor (CAR) molecule, which is a CAR according to any one of claims 6 or 7.

11. An expression vector comprising the nucleic acid molecule according to any one of claims 5 to 7, The vector is an expression vector selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

12. A modified immune cell comprising a nucleic acid molecule or an inventive vector according to any one of claims 5 to 7.

13. A cell according to claim 12, which is useful as a pharmaceutical agent.

14. A cell as described in any one of claims 12 or 13, which is useful as a medicine and is preferentially useful for the treatment of a proliferative disease in a mammal, preferably a human, said proliferative disease being a disease associated with CD123 overexpression, preferably useful for the treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN).

15. A pharmaceutical composition comprising the modified immune cells of any one of claims 12 to 14 and a pharmaceutical excipient.

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