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

Abstract

The present invention relates to monoclonal anti-CD123 antibodies or fragments thereof specifically binding to human interleukin 3 (IL3) receptor alpha chain. Further, the present invention relates to isolated nucleic acid molecules encoding chimeric antigen receptors (CAR), wherein CAR comprises antibodies or antibody fragments including anti-CD123 binding domains, transmembrane domains and intracellular signaling domains comprising at least a stimulating domain, polypeptides encoded by the nucleic acid molecules and isolated chimeric antigen receptors (CAR) molecules comprising such antibodies or antibody fragments. The present invention also relates to vectors comprising nucleic acid molecules encoding CAR, and T cells comprising the vector, and to the use of T cells expressing CAR molecules for treating proliferative diseases (such as cancer) in mammals.

Description

[0001] The present invention relates to monoclonal antibodies and uses thereof. More specifically, the present invention relates to monoclonal anti-CD123 antibodies or fragments thereof that specifically bind to the α chain of human interleukin 3 (IL3) receptor.

[0002] The present invention also relates to 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, a polypeptide encoded by the nucleic acid molecule, and an isolated chimeric antigen receptor (CAR) molecule comprising such an antibody or antibody fragment.

[0003] The present invention also relates to a vector comprising a nucleic acid molecule encoding CAR, and a T cell comprising the vector. The present invention also relates to the use of T cells expressing CAR molecules for treating proliferative diseases such as cancer in mammals.

[0004] Fighting cancer is a global challenge. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive cancer. It is a clonal disease arising from plasmacytoid dendritic cell (pDC) precursors that presents with common skin lesions and always progresses to aggressive leukemic spread. The median overall survival (OS) for this type of cancer is 8 to 12 months.

[0005] The prognosis and treatment of BPDCN are very poor. Intensive chemotherapy is ineffective because almost all patients who receive this treatment will relapse after a few weeks or months.

[0006] A fusion protein called SL-401 provides a lead for therapy, which consists of human interleukin 3 (IL3) fused to diphtheria toxin, see Angelot-Delettre F, Roggy A, Frankel AE, Lamarthee B, Seilles E, Biichle S et al., In vivo and in vitro sensitivity of blasticplasmacytoid dendritic cell neoplasm to SL-401, an interleukin-3receptortargeted biologic agent, Haematologica, 2015, 100(2): 223-30.

[0007] The present invention improves this situation.

[0008] The applicants have found that a protein called CD123, which is the alpha (α) chain of the interleukin-3 (IL3) receptor, is overexpressed in cancer cells of all BDPCN patients, see Garnache-Ottou F, Feuillard J, Ferrand C, Biichle S, Trimoreau F, Seilles E et al., Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia, British journal of haematology, 2009, 145(5): 624-36.

[0009] Therefore, the present invention focuses on the use of CD123 as a therapeutic target.

[0010] In this case, and with the goal of developing a biotherapy for CD123, the applicant has successfully produced, developed, screened and selected anti-CD123 monoclonal antibodies (MA) that specifically bind to human CD123. These antibodies can be used in therapy in general, and more specifically, can be used as weapons against plasmacytoid dendritic cell neoplasm type leukemia and other pathologies expressing CD123.

[0011] A rapidly developing anti-cancer technology uses engineered T cells (or T lymphocytes). These T cells contain so-called chimeric antigen receptors, commonly known as CARs, which are specific for target antigens expressed by cancer cells.

[0012] With this in mind, the applicants overcame many obstacles and used newly developed anti-CD123 antibodies to genetically engineer T cells to express CARs that specifically target CD123, thereby specifically and effectively fighting cancer cells.

[0013] This new therapeutic weapon can be applied to BPDCN patients or patients with relapsed BPDCN, as well as BPDCN patients who have been treated with intensive chemotherapy, SL-401, hematopoietic stem cell allogeneic transplantation or other treatment methods.

[0014] To this end, in a first aspect, the invention features an antibody that specifically binds to the antigen CD123 and has a specificity of no more than 9.5 x 10 -9 M of K D value, not exceeding 9.0x10 -4 s -1 The antibody dissociation rate k off , and at least 7x10 4 Ms -1 The antibody binding rate k on, and wherein the antibody is selected from the group consisting of AB1, AB2, AB3, AB4, AB5 and AB6, wherein

[0015] -AB1 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:25, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:13, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:1, and wherein the light chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:26, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:14, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:2;

[0016] -AB2 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:27, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:15, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:3, and wherein the light chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:28, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:16, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:4;

[0017] -AB3 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:29, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:17, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:5, and wherein the light chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:30, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:18, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:6;

[0018] -AB4 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:31, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:19, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:7, and wherein the light chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:32, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:20, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:8;

[0019] -AB5 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:33, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:21, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:9, and wherein the light chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:34, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:22, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:10; and

[0020] -AB6 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:35, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:23, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:11, and wherein the light chain comprises a complementary determining region 1 (CDR1) having at least 85% identity to the amino acid sequence of SEQ ID NO:36, a complementary determining region 2 (CDR2) having at least 85% identity to the amino acid sequence of SEQ ID NO:24, and a complementary determining region 3 (CDR3) having at least 85% identity to the amino acid sequence of SEQ ID NO:12.

[0021] According to an embodiment of the present 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,

[0022] and wherein the heavy chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:25, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:13, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:1,

[0023] and wherein the light chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:26, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:14, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:2;

[0024] 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,

[0025] and wherein the heavy chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:27, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:15, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:3,

[0026] and wherein the light chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:28, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:16, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:4;

[0027] 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,

[0028] and wherein the heavy chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:29, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:17, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:5,

[0029] and wherein the light chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:30, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:18, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:6;

[0030] 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,

[0031] and wherein the heavy chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:31, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:19, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:7,

[0032] and wherein the light chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:32, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:20, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:8;

[0033] 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,

[0034] and wherein the heavy chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:33, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:21, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:9,

[0035] and wherein the light chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:34, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:22, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:10; and

[0036] 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,

[0037] and wherein the heavy chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:35, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:23, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:11,

[0038] And wherein the light chain comprises a complementary determining region 1 (CDR1) that is at least 90% identical to the amino acid sequence of SEQ ID NO:36, a complementary determining region 2 (CDR2) that is at least 90% identical to the amino acid sequence of SEQ ID NO:24, and a complementary determining region 3 (CDR3) that is at least 90% identical to the amino acid sequence of SEQ ID NO:12.

[0039] According to a preferred embodiment of the present 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,

[0040] and wherein 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,

[0041] and wherein the light chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:2;

[0042] 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,

[0043] and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 27, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 15, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 3,

[0044] and wherein the light chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:4;

[0045] 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,

[0046] and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:29, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO:17, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:5,

[0047] and wherein the light chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:30, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO:18, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:6;

[0048] 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,

[0049] and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:31, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO:19, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:7,

[0050] and wherein the light chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:32, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO:20, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:8;

[0051] 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,

[0052] and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 9,

[0053] and wherein the light chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:34, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO:22, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:10; and

[0054] 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,

[0055] And wherein the heavy chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO:35, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO:23, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:11,

[0056] And wherein the light chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence SEQ ID NO:36, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence SEQ ID NO:24, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence SEQ ID NO:12.

[0057] According to another embodiment of the present invention, the heavy chain of AB1 has a variable region comprising a first framework region (FR1) consisting of an amino acid sequence of SEQ ID NO:37, a second framework region (FR2) consisting of an amino acid sequence of SEQ ID NO:38, a third framework region (FR3) consisting of an amino acid sequence of SEQ ID NO:39, and a fourth framework region (FR4) consisting of an amino acid sequence of SEQ ID NO:40, and wherein the light chain of AB1 has a variable region comprising a first framework region (FR1) consisting of an amino acid sequence of SEQ ID NO:61, a second framework region (FR2) consisting of an amino acid sequence of SEQ ID NO:62, a third framework region (FR3) consisting of an amino acid sequence of SEQ ID NO:63, and a fourth framework region (FR4) consisting of an amino acid sequence of SEQ ID NO:64;

[0058] The heavy chain of AB2 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:41, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:42, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:43, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:44, and wherein the light chain of AB2 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:65, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:66, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:67, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:68;

[0059] The heavy chain of AB3 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:45, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:46, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:47, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:48, and wherein the light chain of AB3 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:69, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:70, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:71, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:72;

[0060] The heavy chain of AB4 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:49, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:50, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:51, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:52, and wherein the light chain of AB4 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:73, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:74, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:75, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:76;

[0061] The heavy chain of AB5 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:53, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:54, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:55, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:56, and wherein the light chain of AB5 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:77, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:78, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:79, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:80;

[0062] The heavy chain of AB6 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:57, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:58, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO:59, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO:60, and wherein the light chain of AB6 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO:81, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO:82, a third framework region (FR3) consisting 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.

[0063] In another aspect, the invention features 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 anti-CD123 binding domain comprises:

[0064] (i) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:25, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:13, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:1, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:26, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:14, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:2;

[0065] (ii) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:27, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:15, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:3, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:28, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:16, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:4;

[0066] (iii) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:29, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:17, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:5, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:30, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:18, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:6;

[0067] (iv) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:31, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:19, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:7, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:32, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:20, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:8;

[0068] (v) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:33, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:21, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:9, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:34, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:22, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:10;

[0069] or

[0070] (vi) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) having at least 90% identity with the amino acid sequence of SEQ ID NO:35, a complementary determining region 2 (CDR2) having at least 90% identity with the amino acid sequence of SEQ ID NO:23, and a complementary determining region 3 (CDR3) having at least 90% identity with the amino acid sequence of SEQ ID NO:11, and the light chain comprising a complementary determining region 1 (CDR1) having at least 90% identity with the amino acid sequence of SEQ ID NO:36, a complementary determining region 2 (CDR2) having at least 90% identity with the amino acid sequence of SEQ ID NO:24, and a complementary determining region 3 (CDR3) having at least 90% identity with the amino acid sequence of SEQ ID NO:12.

[0071] According to an embodiment, the anti-CD123 binding domain of the isolated nucleic acid molecule of the present invention is selected from the group consisting of antibody, Fv, scFv, Fab or other antibody fragments, preferably scFv.

[0072] In another embodiment, the intracellular signaling domain is CD3-zeta (CD3ζ), optionally comprising a co-stimulatory domain selected from the group consisting of CD28, 4.1BB, inducible T-cell co-stimulator (ICOS), OX-40, or a combination thereof.

[0073] Another aspect of the invention features an isolated polypeptide molecule encoded by the above-described nucleic acid molecules.

[0074] A further aspect of the invention is characterized by an isolated chimeric antigen receptor (CAR) molecule comprising 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 anti-CD123 binding domain comprises:

[0075] (i) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:25, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:13, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:1, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:26, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:14, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:2;

[0076] (ii) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:27, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:15, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:3, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:28, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:16, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:4;

[0077] (iii) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:29, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:17, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:5, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:30, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:18, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:6;

[0078] (iv) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:31, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:19, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:7, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:32, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:20, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:8;

[0079] (v) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:33, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:21, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:9, and the light chain comprising a complementary determining region 1 (CDR1) at least 90% identical to the amino acid sequence of SEQ ID NO:34, a complementary determining region 2 (CDR2) at least 90% identical to the amino acid sequence of SEQ ID NO:22, and a complementary determining region 3 (CDR3) at least 90% identical to the amino acid sequence of SEQ ID NO:10;

[0080] or

[0081] (vi) a heavy chain and a light chain, the heavy chain comprising a complementary determining region 1 (CDR1) having at least 90% identity with the amino acid sequence of SEQ ID NO:35, a complementary determining region 2 (CDR2) having at least 90% identity with the amino acid sequence of SEQ ID NO:23, and a complementary determining region 3 (CDR3) having at least 90% identity with the amino acid sequence of SEQ ID NO:11, and the light chain comprising a complementary determining region 1 (CDR1) having at least 90% identity with the amino acid sequence of SEQ ID NO:36, a complementary determining region 2 (CDR2) having at least 90% identity with the amino acid sequence of SEQ ID NO:24, and a complementary determining region 3 (CDR3) having at least 90% identity with the amino acid sequence of SEQ ID NO:12.

[0082] Another aspect of the invention features an isolated chimeric antigen receptor (CAR) molecule, wherein the CAR is as described above.

[0083] Another aspect of the invention features an expression vector comprising a nucleic acid molecule of the invention, wherein the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0084] Another aspect of the present invention is characterized by comprising an engineered immune cell of a nucleic acid molecule of the present invention or a vector of the present invention. In one embodiment of the present invention, the cell expresses CAR as described above on its membrane. According to one embodiment, the cell is used as a medicine, preferably for treating a proliferative disease in a mammal, preferably a human being, wherein the proliferative disease is a disease associated with overexpression of CD123, preferably blastic plasmacytoid dendritic cell tumor (BPDCN).

[0085] Yet another aspect of the invention features a pharmaceutical composition comprising the engineered immune cells described above and a pharmaceutically acceptable excipient.

[0086] Yet another aspect of the invention features a method of engineering an immune cell, the method comprising transducing the cell with an expression vector as described above.

[0087] Yet another aspect of the invention features an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the nucleic acid molecule consists of a sequence that is at least 85%, preferably 90%, more preferably 95%, and most preferably 100% identical to SEQ ID NO:97.

[0088] Other features and advantages of the invention will emerge and / or become clear upon reading the following description, which includes specific examples given in an illustrative and non-limiting manner, and can also be seen from the accompanying drawings, in which:

[0089] Figure 1 The nucleotide sequence and amino acid sequence of the complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) of the heavy chain and light chain of the antibody of the present invention are shown;

[0090] Figure 2 The nucleotide sequence and amino acid sequence of the 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 are shown;

[0091] Figure 3 The nucleotide sequence and amino acid sequence of the 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 are shown;

[0092] Figure 4 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 are shown;

[0093] Figure 5 The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the second antibody of the present invention are shown;

[0094] Figure 6 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 are shown;

[0095] Figure 7 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 are shown;

[0096] Figure 8The 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 are shown;

[0097] Fig. 9 The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the sixth antibody named by the present invention are shown;

[0098] Fig.10 Flow cytometry plots based on the evaluation of CD123 expression on normal hematopoietic cells and BPDCN are shown;

[0099] Fig.11 Tables and flow cytometry plots based on evaluation of CD123 expression in BPDCN and other non-pDC acute leukemias are shown;

[0100] Fig.12 Shown is a schematic diagram of the design of the chimeric antigen receptor of the present invention, as well as a western blot and a flow cytometry diagram related to the production of T cells comprising the chimeric antigen receptor of the present invention according to the present invention;

[0101] Fig.13 The nucleic acid sequence of the chimeric antigen receptor according to the present invention is shown;

[0102] Fig.14 Shown is a flow cytometry graph associated with evaluating the phenotype of CART cells according to the present invention compared to non-transduced T cells;

[0103] Figure 15 shows a schematic diagram of co-culturing target cells with the chimeric antigen receptor of the present invention and non-transduced cells, and flow cytometry graphs associated with the specific cytotoxicity of the CAR of the present invention to BPDCN cell lines and BPDCN patient-derived xenograft cells;

[0104] Fig.16 Shown with and CD123 + Flow cytometry plots related to functional characterization of CD123R CAR T cells of the present invention in target cell co-culture;

[0105] Fig.17 Shown are schematic diagrams of xenograft models, bioluminescent imaging and graphs related thereto, and flow cytometry graphs related to the in vivo anti-tumor activity of T cells of the present invention against BPDCN cell lines (CAL-1) and patient-derived xenograft cells (PDX);

[0106] Fig.18 Shown are schematic diagrams of xenograft models, bioluminescent imaging and graphs thereof related to the in vivo anti-tumor activity of T cells of the present invention against BPDCN cell line (CAL-1);

[0107] Fig.19Shown are fluorescence images associated with CD123 expression in different cell types, immunohistochemical images of CD123 expression in tumors and normal tissues, flow cytometry images associated with CD123 expression, schematic diagrams of the generation of autologous T cells of the present invention, and flow cytometry images of CD123-related toxicity of T cells of the present invention to cells expressing CD123 at low levels;

[0108] Fig. 20 Shown are flow cytometry-based graphs associated with the assessment of autologous T cell toxicity against cell lines (CAL-1 and DAUDI);

[0109] Fig.21 Shown are flow cytometric graphs relating to specific cytotoxicity induced by CD123 T cells of the invention from BPDCN patients against autologous BPDCN blasts.

[0110] The drawings and the description herein generally contain elements of a certain nature. Therefore, the description and the drawings are used not only to better understand the invention but also to help define it when appropriate.

[0111] In this specification, the term "and / or" should be interpreted as including one or more possible situations to which it is connected. For example, the wording "the protein or protein sequence can be prepared using standard recombinant and / or synthetic methods" means 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 specific manifestations: 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).

[0112] The term "comprising" should be interpreted as including all the specifically mentioned features as well as optional, additional, unspecified features. More generally, the terms "comprise", "comprises" and "comprising" will be understood to mean the inclusion of specified steps or elements or groups of steps or elements without excluding any other steps or elements or groups of steps or elements. The term "consisting of" should be interpreted as meaning that in addition to the specifically mentioned features, no other features are present. Furthermore, the indefinite article "a" or "an" does not exclude a plurality.

[0113] When reference is made throughout this specification to "one embodiment," "an embodiment," "a specific embodiment," "an additional embodiment," "another embodiment," or "another embodiment," or "another aspect," "aspect," "aspect," "a specific aspect," "an additional aspect," "another aspect," or "another aspect," and combinations thereof, it means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. The articles "a," "an," and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article.

[0114] The term "gene" refers to a DNA sequence (nucleic acid sequence or nucleotide sequence) encoding a specific amino acid sequence. A gene comprises all or part of one or more proteins or enzymes, and may include a regulatory DNA sequence (e.g., a promoter sequence) that determines, at least in part, under what conditions the gene is expressed. Some genes are not structural genes and can be transcribed into RNA from DNA, but are not translated into amino acid sequences. Other genes can function as regulators of structural genes or regulators of DNA transcription. In particular, the term "gene" may be intended to be used for a genomic sequence encoding a protein, i.e., a sequence comprising regulators, promoters, introns, and exon sequences.

[0115] In the context of two or more nucleic acid sequences or amino acid sequences, the term "identity" or "identity" percentage (%) refers to when comparing or aligning (if necessary, introducing gaps) maximum consistency, two or more sequences or subsequences are identical or have a specific percentage of identical nucleotides or amino acid residues, without considering any conservative amino acid substitutions as part of the sequence identity. Sequence comparison software or algorithms or by visual inspection can be used to measure the identity percentage. Various algorithms and software known in the art can be used to obtain the comparison of amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. 87:2264-2268, 1990, modified in Karlin et al., Proc. Natl. Acad. Sci. 90:5873-5877, 1993, and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1991). Gapped BLAST can also be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480, 1996), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR) are other publicly available software programs that can be used to align sequences. In addition, the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453, 1970) can be used to determine the percent identity between two amino acid sequences (e.g., using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5). Alternatively, the percent identity between nucleotide or amino acid sequences can be determined using the algorithm of Myers and Miller (CABIOS, 4: 11-17, 1989). For example, the ALIGN program (version 2.0) can be used and PAM120 with a residue table, a gap length penalty of 12 and a gap penalty of 4 can be used to determine the percent identity. One skilled in the art can determine the appropriate parameters for maximum alignment by a particular alignment software. Typically, the default parameters of the alignment software are used.

[0116] A sequence "at least 85% identical to a reference sequence" or "something having at least 85% identity to a reference sequence" refers to a sequence having 85% or higher sequence identity, 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 context of this specification, "percent identity" is calculated using pairwise comparisons (i.e., comparisons are made over the entire length of the two sequences). As described above, methods for comparing two or more sequence identities are well known in the art. For example, bioinformatics tools or programs, such as the EMBOSS Needle provided on the ebi.ac.uk website, can be used to generate paired sequence alignments of the present invention. Typically, many bioinformatics tools and programs use the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the best alignment of two sequences (including gaps) when considering their entire length. However, other algorithms may also be implemented in various bioinformatics tools or programs.

[0117] More generally, for purposes of the present invention, "sequence identity" or "sequence homology" is calculated by comparing two aligned sequences in a comparison window. Sequence alignment can determine the number of positions (nucleotides or amino acids) shared by the two sequences in the comparison window. The number of shared positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percent homology. Determining percent sequence identity can be done manually or by using well-known bioinformatics computer programs.

[0118] According to the present invention, the percent identity between two polypeptides can be calculated using the EMBOSS Needle (global alignment) program using a "gap opening" parameter equal to 10.0, a "gap extension" parameter equal to 0.5 and a Blosum62 matrix.

[0119] A protein or portion thereof having an amino acid sequence (or consisting of) that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence may contain mutations, such as deletions, insertions and / or substitutions, compared to the reference sequence.

[0120] In the case of substitutions, a protein consisting of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence derived from another species different from the reference sequence (i.e., "homology"). Amino acid substitutions may be conservative or non-conservative. A conservative substitution is one in which one amino acid is substituted for another amino acid with a similar side chain, i.e., a similar structure and / or similar chemical properties. In this regard, conservative substitutions may occur between:

[0121] - Amino acids with non-polar side chains: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), tryptophan (Trp);

[0122] - Amino acids with uncharged polar side chains: asparagine (Asn), glutamine (Gln), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine ​​(Cys);

[0123] -Amino acids with acidic side chains: aspartic acid (Asp), glutamic acid (Glu);

[0124] -Amino acids with basic side chains: lysine (Lys), arginine (Arg), histidine (His);

[0125] - Amino acids with β-branched side chains: threonine (Thr), valine (Val), isoleucine (Ile);

[0126] -Amino acids with aromatic side chains: tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), histidine (His).

[0127] Non-conservative substitutions may occur randomly between the above. Other definitions of conservative or non-conservative substitutions can be given based on the position of the individual amino acids within the three-dimensional shape of the protein.

[0128] Unless otherwise indicated, the techniques used in the present invention are methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, gene and / or biotechnological cell engineering, immunology and cell biology known to those skilled in the art. Where necessary, these methods are described for illustrative purposes. Most of these methods and other techniques are described in the literature. See, e.g., 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 Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley and Sons, 4th ed., 1999; DNA Cloning: A Practical Approach, Vols. I & II (IRL Press, Oxford, 1985); Techniques for the Analysis of Complex Genomes, Anand, Academic Press, 1992; Transcription and Translation, B. Names & S. Higgins, 1984; A Practical Guide to Molecular Cloning, Perbal, 1984; Antibodies, Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1998; Current Protocols in Immunology, QEColigan, AMKruisbeek, DH Margulies, EMSevach and W. Strober, 1991; Annual Review 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 in the field of the present invention. Although any methods or materials similar or equivalent to those described herein can be used to implement or test the present invention, preferred embodiments of the compositions, methods and materials are described herein.

[0129] As understood by those skilled in the art and described herein, an "antibody", also known 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, while each light chain consists of a variable region and a constant region. The two heavy chains are interconnected by disulfide bonds, and each heavy chain is connected to a light chain by a disulfide bond.

[0130] In mammals, light chains are of two types, lambda (I) and kappa (κ). In addition, there are five major heavy chain classes (or isotypes), which are classified as a, d, e, y, and m, which determine the functional activity of the antibody molecule: IgA, IgD, IgE, IgG, and IgM.

[0131] Each chain contains different sequence domains. The light chain includes two domains or regions, the variable domain (VL) and the constant domain (CL). The heavy chain includes four domains, one variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). More precisely, the complete antibody forms a "Y" shape. The base or stem of the Y consists of the second and third constant regions of the two heavy chains (the fourth constant region for IgE and IgM), which are bound together by disulfide bonds, sometimes called interchain disulfide bonds, to form a hinge. Heavy chains y and d have a constant region consisting of three tandem (inline) Ig domains, and a hinge region for increased flexibility; heavy chains m and e have a constant region consisting of four immunoglobulin domains. The second and third constant regions are called "CH2 domains" and "CH3 domains", respectively. Each arm or branch of the Y includes the variable region and the first constant region of a single heavy chain combined with the variable region and constant region 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 (VL) and heavy (VH) chains determine the binding recognition and specificity for the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and consists of the variable portions of one light and one heavy chain. The specificity of an antibody lies in the structural complementarity between the binding site of the antibody and the antigenic determinant. The binding site of an antibody is composed of residues primarily from the hypervariable regions or complementarity determining regions (CDRs). Sometimes, residues from non-hypervariable regions or framework regions (FRs) affect the overall domain structure and thus the binding site. The complementarity determining regions or CDRs refer to the amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light chain and heavy chain of immunoglobulin each have three CDRs, usually named CDR1, CDR2 and CDR3, or more precisely named CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. In the literature, other nomenclatures can be found. Therefore, the CDRs located in the variable domain of the heavy chain of the antibody can be referred to as CDRH1, CDRH2 and CDRH3, while the CDRs located in the variable domain of the light chain of the antibody can be referred to as CDRL1, CDRL2 and CDRL3. Antibodies with different specificities (i.e., having different binding sites for different antigens) have different CDRs. Therefore, a conventional antibody antigen-binding site includes six CDRs, which include a set of CDRs from each of the heavy chain V region and the light chain V region.

[0132] As described above, the light and heavy chain variable regions contain so-called "framework" regions separated by hypervariable CDR regions. CDRs can be defined or identified by methods such as by following sequences, i.e., Kabat et al., Wu, TT and Kabat, EA, J Exp Med, 132(2):211-50, 1970; Borden, P. and Kabat EA, PNAS, 84:2440-2443, 1987; Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991 - or by following structures, i.e., Chothia et al. - Choithia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917, 1987; Choithia, C. et al., Nature, 342:877-883, 1989.

[0133] The sequences of the framework regions of different light or heavy chains are relatively conservative within a species, such as in humans. More generally, "framework regions" (FRs) are relatively conservative in different immunoglobulins of a single species. The framework region of an antibody is, to some extent, a combination of all framework regions of the constituent light and heavy chains, for locating and arranging CDRs in three-dimensional space (i.e., when the antibody is folded and active). CDRs are primarily responsible for binding the epitope of an antigen. As described above, the CDRs of each chain are referred to as CDR1, CDR2, and CDR3. In fact, CDRs are numbered sequentially from the N-terminus. The light and heavy chains of immunoglobulins each have four FRs, respectively named FR1, FR2, FR3, and FR4, or more precisely, FR1-L, FR2-L, FR3-L, FR4-L for the light chain, and FR1-H, FR2-H, FR3-H, FR4-H for the heavy chain. Accordingly, the light chain variable domain can be named (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain can be named (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Knowing the amino acid sequence of the CDRs, those skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0134] The “V H" or "VH" refers to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, Fab, or other antibody fragment disclosed herein. Reference to "V L ” or “VL” refers to the variable region of an immunoglobulin light chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragments disclosed herein.

[0135] As used herein, the term "antibody" or "immunoglobulin" mainly refers to conventional antibodies, particularly monoclonal antibodies and fragments thereof, and single domain antibodies and fragments thereof, particularly variable heavy chains of single domain antibodies, and further refers to chimeric antibodies, humanized antibodies, bispecific antibodies or multispecific antibodies. The antibody is preferably a human antibody, a murine antibody or a humanized antibody.

[0136] "Monoclonal antibody" or "mAb" is an antibody produced by a single clone of B lymphocytes or cells that have been transfected with the heavy and light chain genes of a single antibody. Monoclonal antibodies are produced by methods well known in the art, such as by fusing myeloma cells with immune spleen cells to prepare hybrid antibody-forming cells, or by recombinant technology, i.e., by 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 should not be construed as requiring the production of the antibody by any particular method other than those known in the art.

[0137] The term "chimeric antibody" refers to an engineered antibody that, in its broadest sense, comprises one or more regions from one antibody and one or more regions from one or more other antibodies. In particular, a chimeric antibody comprises a VH domain and a VL domain of an antibody derived from a non-human animal, and a CH domain and a CL domain of another antibody, particularly a human antibody, associated therewith. As non-human animals, any animal can be used, such as mice, rats, hamsters, rabbits, etc. Chimeric antibodies can also refer to multispecific antibodies that are specific for at least two different antigens.

[0138] The term "antibody" or "immunoglobulin" also includes the meaning of "single domain antibodies" which have been recently described and developed. A single domain antibody is an antibody whose complementary determining region (CDR) is part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional four-chain antibodies, and engineered single domain antibodies. Single domain antibodies can be derived from any species, in particular from mice, humans, or rabbits. Single domain antibodies can be naturally generated single domain antibodies, referred to as heavy chain antibodies lacking light chains. In particular, camelid species (such as camels, dromedaries, llamas, alpacas, and guanacos) produce heavy chain antibodies naturally lacking light chains.

[0139] The variable heavy chains of these single-domain antibodies lacking light chains are referred to in the art as "VHH" or "nanobodies". Similar to conventional VH domains, VHHs contain four framework regions (FRs) and three complementary determining regions (CDRs). Compared with conventional antibodies, a special advantage of nanobodies is that they are about ten times smaller than IgG molecules. Therefore, properly folded functional nanobodies can be produced in high yields by in vitro expression. In addition, nanobodies described in the art are very stable and resistant to the effects of proteases. For example, the properties and production of nanobodies have been reviewed in Harmsen and De Haard HJ, Appl.Microbiol.Biotechnol., 2007, November, 77 (1): 13-22.

[0140] In certain preferred embodiments, 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. Therefore, all parts of the humanized immunoglobulin, except for the CDR, are substantially the same as the corresponding parts of the natural human immunoglobulin sequence. Humanization or other monoclonal antibodies may have additional conservative amino acid substitutions, which are substantially unaffected by antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (e.g., referring to U.S. Patent No. 5,585,089).

[0141] More generally, the term "humanized antibody" refers to an antibody of complete or partial non-human origin that has been modified to replace certain amino acids, particularly in the framework regions of heavy and light chains, to avoid or minimize immune responses in humans. The constant domains of humanized antibodies are in most cases human CH and CL domains. There are many methods known in the art for humanizing antibody sequences; for example, see Almagro & Fransson's review, 2008, Front Biosci. 13: 1619-1633. A commonly used method is so-called CDR transplantation or antibody reshaping. The technology includes transplanting the CDR sequence of a donor antibody (usually a mouse antibody) into a framework scaffold of a human antibody with different specificities. Since CDR transplantation can reduce the binding specificity and affinity of non-human antibodies transplanted with CDRs, thereby reducing their biological activity, back mutations can be introduced at selected positions of antibodies transplanted with CDRs to maintain the binding specificity and affinity of the parental antibody. The information provided in the literature and antibody databases can be used to identify the position of possible back mutations. The amino acid residues as candidates for back mutation are usually those residues located on the surface of the antibody molecule, and the residues that are buried or less exposed on the surface are usually not changed. The alternative humanization technology of CDR transplantation and back mutation is surface remodeling, wherein the non-surface exposed residues of non-human origin are retained, and the surface residues are changed to human residues. Another alternative technology is referred to as so-called guided selection, referring to Jespers et al., Biotechnology 12,899,1994, and can be used to derive the epitope and binding properties of the parent antibody from, for example, mouse or rat antibodies. Another humanized method is so-called 4D humanization. 4D humanization scheme is described in patent application WO2009032661A1 (or US20110027266), and 4D humanization is used to humanize rat antibody variable light chain (VL) and heavy chain (VH) domains as examples hereinafter. In one example, a rat antibody homology model is typically completed with MOE software (v. 201 1.10—Chemical Computing Group, Quebec, Canada) using the PDB structure (see Berman et al., Nucleic Acids Research, 28:235-242, 2000) as a template, followed by energy minimization using standard procedures implemented in MOE.Then the minimized 3D homology model of rat antibody (completed with MOE software) was subjected to molecular dynamics (MD) simulation and compared with 49 human models derived from 7 representative light chains (vκ1, vκ2, vκ3, vκ4, vλ1, vλ2, vλ3) and 7 representative heavy chains (vhl a, vhl b, vh2, vh3, vh4, vh5, vh6) designed by LGCR / SDI and provided in MOE. For example, a chain coupling model (Vkx-Vhx) with the best hydrophobicity, electrostatic component and sequence identity outside CDR was selected for 4D humanization design. For the paired association between the rat antibody variable domain and the selected model, the sequence is usually aligned based on the best 3D superposition of the α-carbon of the corresponding homology model. Then the undesirable motif is considered and mutated. Finally, the obtained humanized sequences were compared with, for example, the IEDB database (http: / / www.immuneepitope.org; version 2012 / 01 / 30, which can be accessed locally) for sequence similarity analysis to ensure that all sequences do not contain any known B-cell or T-cell epitopes listed therein.

[0142] For chimeric antibodies, humanization generally involves modification of the framework regions of the variable region sequences.

[0143] The amino acid residues that are part of the CDR are generally not changed with humanization, although in some cases, it may be necessary to change individual CDR amino acid residues, such as removing glycosylation sites, deamidation sites or unwanted cysteine ​​residues. N-linked glycosylation occurs by attaching oligosaccharide chains to asparagine residues in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of N-glycosylation sites can be achieved by mutating Asn or Ser / Thr residues to different residues, particularly by conservative substitutions. Deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such deamidation sites are present in the CDR sequence, especially Asn-Gly, it may be necessary to remove the site, usually by conservative substitution to remove one of the involved residues. Substitutions in the CDR sequence to remove one of the involved residues are also intended to be included in the present invention.

[0144] The term antibody also includes its antigen-binding fragment. Such "fragments" include a portion of a complete antibody, especially the antigen-binding region or variable region of a complete antibody. Examples of antibody fragments include Fab' fragments, F(ab)'2 fragments, Fv, single-chain Fv proteins ("scFv"), and portions of full-length antibodies responsible for antigen binding, diabodies, bispecific and multispecific antibodies formed by antibody fragments. Fragments of conventional antibodies may also be single-domain antibodies, such as heavy chain antibodies or VHH. The term "Fab" means an antibody fragment having a molecular weight of about 50,000 and antigen-binding activity, wherein in a fragment obtained by treating IgG with the protease papain, about half of the N-terminal side of the H chain and the entire L chain are bound together by disulfide bonds. The term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen-binding activity in a fragment obtained by treating IgG with the protease pepsin, which is slightly larger than the Fab bound by the disulfide bonds of the hinge region. "Single-chain Fv" or "scFv" antibody fragments contain the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain and in either orientation (e.g., VL-VH or VH-VL). Single chains can be cloned from the V region genes of a specific hybridoma for the desired target. The production of such hybridomas has become a routine procedure. A technique that can be used to clone the variable region heavy chain (VH) and the variable region light chain (VL) has been described, for example, by Orlandi et al., PNAS, 1989, 86:3833-3837. Typically, the scFv polypeptide also includes a polypeptide linker between the VH and VL domains that enables the scFv to form a structure required for antigen binding. More specifically, a single-chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer that is typically expressed by a gene fusion that includes VH and VL encoding genes connected by a peptide encoding linker. In some embodiments, human scFv fragments include CDRs that maintain appropriate conformations, particularly by using genetic recombination techniques. Divalent and multivalent antibody fragments can be formed spontaneously by the association of monovalent scFv, or can be produced by coupling monovalent scFv by peptide linkers, such as divalent sc(Fv)2."dsFv" is a VH::VL heterodimer stabilized by a disulfide bond. "(dsFv)2" represents two dsFvs coupled 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. Therefore, BsAb can simultaneously bind to two different antigens. Genetic engineering has been increasingly used to design, modify and produce antibodies or antibody derivatives with a series of desired binding properties and effector functions, such as described in EP2050764A1. 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 "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0145] The present invention provides immune effector cells genetically engineered with a vector designed to express a chimeric antigen receptor (CAR), which redirects cytotoxicity to tumor cells. CAR is a molecule that combines antibody-based specificity for target antigens (e.g., tumor antigens) with T cell receptor activating intracellular domains to produce a chimeric protein that exhibits specific anti-tumor cell immune activity. As used herein, the term "chimeric" describes the composition of parts of different proteins or DNA from different sources. The main characteristic of CAR is that they can redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis, or producing molecules that can mediate cell death of target antigen-expressing cells in a manner independent of major histocompatibility (MHC), which utilizes the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0146] 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 CAR with the ability to specifically bind to a target antigen of interest. The 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). The binding domain includes any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner of the biological molecule of interest. As used herein, the term "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specific targeting" describes the binding of one molecule to another molecule with a binding affinity greater than background binding. If the binding domain (or a CAR comprising a binding domain, or a fusion protein comprising a binding domain) is, for example, greater than or equal to about 10 5 M -1A binding domain polypeptide or CAR protein according to the present disclosure can be easily determined using conventional techniques such as competitive ELISA (enzyme-linked immunosorbent assay). In addition, the binding quality associated with affinity, the so-called K, can be determined, quantified and qualified. D Value. K D Related to antibody affinity and sensitivity. K D is the equilibrium dissociation constant between the antibody and its antigen, i.e., k off / k on The ratio of K D Negatively correlated with affinity. K D The value is related to the antibody concentration (the amount of antibody required for a specific experiment), so K D The lower the value (lower the concentration), the higher the affinity of the antibody. D is the antibody dissociation rate (k off ) and the antibody binding rate (k on ), where k off provides information about how quickly an antibody dissociates from its antigen, while k on Provides information about how quickly an antibody binds to its antigen. By measuring the k of a specific antibody / antigen interaction on and k off rate, and then use the ratio of these values ​​to calculate K D value, thereby determining the K D More generally, the affinity of an antibody is the strength with which a single molecule binds to its ligand. It is usually given by the equilibrium dissociation constant (K D ) is measured and reported as a constant used to assess and rank the strength of bimolecular interactions. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentrations of the reactants. At equilibrium, the rate at which the [antibody]|[antigen] complex forms is equal to the rate at which it dissociates into its components [antibody]+[antigen]. Measurement of the reaction rate constant can be used to define the equilibrium or affinity constant (1 / K D ). In short, K D The smaller the value, the greater the affinity of the antibody for its target.

[0147] The binding domain of CAR is usually followed by one or more "hinge regions". The hinge region plays a role in positioning the antigen binding domain away from the effector cell surface to achieve proper cell-to-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, semisynthetic or recombinant sources.

[0148] "Transmembrane domain" is a part of CAR that fuses the extracellular binding part and the intracellular signaling domain. The transmembrane domain anchors CAR to the plasma membrane of immune effector cells. As described in the art, the transmembrane region can be derived from natural, synthetic, semisynthetic or recombinant sources.

[0149] In a preferred embodiment, the CAR of the present invention comprises an intracellular signaling domain. "Intracellular signaling domain" refers to a part of CAR that is involved in transducing information that effectively binds CAR to the target antigen to the inside of the immune effector cell to stimulate effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to target cells bound to CAR, or other cell responses triggered by antigens bound to the extracellular CAR domain. "Effect function" refers to a specialized function of a cell. The effector function of a T cell can be a cytolytic activity or an activity that includes secreting cytokines. Therefore, in this specification, "intracellular signaling domain" refers to a part of a protein that transduces an "effector function" signal that causes the cell to perform a specialized function. As described in the art, a complete intracellular signaling domain or a truncated portion of an intracellular signaling domain can be used. As will be understood by those skilled in the art, an effective truncated portion must transduce an effector function signal in substantially the same manner as the complete domain. Herein, the term "intracellular signaling domain" is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

[0150] In a preferred embodiment of the present invention, CAR has a hinge region IgG1-CH2-CH3 (the first part of IgG1 is located in the membrane, and the second part of IgG1 is located outside the cell) and CD28 as a transmembrane domain (the first part of CD28 is located in the membrane, and the second part of CD28 is located intracellularly in the cytoplasm). CD8α can also be used as a transmembrane domain and hinge region. Sequences derived from IgG4 can also be used.

[0151] As is known in the art, the signal generated by the T cell receptor (TCR) alone is not sufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Therefore, it can be said that the activation of T cells is mediated by two different types of intracellular signaling domains: the main signaling domain that initiates antigen-dependent primary activation through TCR (e.g., TCR / CD3 complex) and the co-stimulatory signaling domain that acts in an antigen-independent manner to provide secondary or any co-stimulatory signal. In a preferred embodiment, the CAR of the present invention comprises an intracellular signaling domain, which comprises one or more "co-stimulatory signaling domains". Thus, in other preferred embodiments, the isolated nucleic acid molecule of the present invention encodes an intracellular signaling domain comprising at least one co-stimulatory domain, so that the resulting intracellular signaling domain therefore comprises at least one co-stimulatory domain.

[0152] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for effective T cell activation / function upon antigen binding.

[0153] In this specification, the terms "polypeptide", "polypeptide fragment", "peptide" and "protein" are used interchangeably and have the conventional meaning, i.e., amino acid sequence, unless otherwise specified to the contrary. The polypeptide is not limited to a specific length and may include a full-length protein sequence or a fragment thereof. The polymer may be linear or branched, it may include modified amino acids, and it may be separated by non-amino acids. In addition, the polypeptide may have post-translational modifications, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a marker component, and more generally, any naturally occurring and / or non-naturally occurring modification. In view of the fact that the polypeptides of the present invention are based on antibodies, in some embodiments, the polypeptides may appear as single chains or associated chains. Any technique known in the art may be used to prepare polypeptides, i.e., recombinant and / or synthetic techniques. More specifically, the polypeptides described herein include CARs of the present disclosure, or sequences having deletions, additions and / or substitutions of one or more amino acids of CARs as disclosed herein.

[0154] As used herein, "isolated peptide" or "isolated polypeptide" etc. refers to the in vitro separation and / or purification of peptide or polypeptide molecules from the cell environment and from the association with other components of the cell. Similarly, "isolated cells" refer to cells obtained from in vivo tissues or organs, essentially free of extracellular matrix.

[0155] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. More generally, "vector" means a construct that can be delivered into a host cell and can express one or more genes or sequences of interest in a host cell. The nucleic acid to be transported or transferred is connected to a carrier nucleic acid molecule in any manner known in the art, such as by insertion. The vector may include a sequence that directs autonomous replication in the cell, or may include a sequence sufficient to allow integration into the host cell DNA. The present invention also provides a vector comprising a nucleic acid molecule encoding the 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 an EF-1α promoter. The term "promoter" as used herein refers to a recognition site of a polynucleotide (DNA or RNA) bound to an RNA polymerase. As known to those skilled in the art, RNA polymerase initiates transcription of a polynucleotide operably connected to a promoter. In a specific embodiment, it may be necessary to express a polynucleotide comprising a CAR from a promoter, which provides stable and long-term CAR expression in T cells. CAR expression under promoter control further ensures sufficient expression levels to direct T cells against cells expressing the target antigen.

[0156] Retrovirus is a common tool for gene delivery. In some embodiments, retrovirus is used to deliver polynucleotides encoding chimeric antigen receptor (CAR) to cells, preferably 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, and then covalently integrates its 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 and guides the expression of RNA molecules that encode structural proteins and enzymes required to produce new viral particles. Therefore, T cells transduced with the vector of the present invention can produce stable, long-term and continuous CAR-mediated T cell responses. In a specific embodiment, a lentiviral vector encoding a CAR according to the present invention, i.e., a lentivirus, is used to transduce T cells. The term "lentiviral vector" refers to a viral vector or plasmid comprising structural and functional gene elements or parts thereof, including long terminal repeats (LTRs) mainly 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., type 1 or type 2); Vesna-Medie 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-defective vector, such as a retroviral or lentiviral vector, in which the 3'LTR enhancer-promoter region (referred to as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription after the first round of viral replication.

[0157] When referring to a molecule such as a polypeptide or antibody or nucleotide sequence of the present invention, the terms "purified" and "isolated" refer to the presence of the referenced molecule in the absence of other biological macromolecules of the same type. More specifically, the term "purified" as used herein specifically means the presence of at least 85%, 90%, 95% or 98% (by weight) of biological macromolecules of the same type. More specifically, the term "isolated" nucleic acid molecule encoding a specific polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties that do not have a deleterious effect on the basic properties of the composition.

[0158] The term "antigen", "target" or "target antigen" refers to a molecule or a portion of a molecule that can be bound by an antibody or antibody-like binding protein. The term also refers to a molecule or a portion of a molecule that can be used in an animal to produce an antibody that can bind to an epitope of the antigen. A target antigen can 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 an intact antibody that recognizes the target antigen.

[0159] Unless otherwise indicated, the terms "CD123", "IL-3Ra" or "IL-3Rα", "interleukin-3 receptor alpha" or "interleukin-3 receptor α" are used interchangeably herein and refer to any native (human) IL-3Ra or CD123. The CD123 protein is an interleukin 3-specific subunit of a heterodimeric cytokine receptor (i.e., IL-3 receptor or IL-3R). IL-3R consists of a ligand-specific α subunit and a common β subunit for signal transduction (also known as CD131), which is shared by receptors for interleukin 3 (IL3), colony stimulating factor 2 (CSF2 / GM-CSF), and interleukin 5 (IL5). The binding of CD123 / IL-3Ra to IL3 depends on the β subunit. The β subunit is activated by ligand binding and is essential for the biological activity of IL3. All of the aforementioned terms regarding CD123 may refer to the protein or nucleic acid sequence referred to herein. The term "CD123 / IL-3Ra" includes "full-length" unprocessed CD123 / IL-3Ra, as well as any form of CD123 / IL-3Ra produced by intracellular processing. The term also includes 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 can be isolated from a variety of sources, such as from human tissue types or other sources, or prepared by recombinant or synthetic methods. Examples of CD123 / IL-3Ra sequences include, but are not limited to, NCBI accession numbers NP_002174 & NM_002183 (protein and nucleic acid sequences of human CD123 variant 1) and NP_001254642 & NM_001267713 (protein and nucleic acid sequences of human CD123 variant 2).

[0160] 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 binds to CD123 with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent 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 binding of the antibody to CD123 measured. More generally, the term "specifically binds" means that an antibody binds to an epitope through its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope through its antigen binding domain more readily than it binds 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, it can be considered that antibody "A" has a higher specificity for a given epitope than antibody "B", or it can be said that antibody "A" has a higher specificity for binding epitope "C" than its specificity for related epitope "D". Accordingly, the antibody or antigen-binding fragment of the present invention specifically binds to CD123 antigens because its binding specificity for CD123 antigens (from any species) is higher than its binding specificity for non-CD123 antigens. More specifically, the antibody or antigen-binding fragment of the present invention specifically binds to human CD123 antigens because its binding specificity for human CD123 antigens is higher than its binding specificity for non-human CD123 antigens (such as mouse or rat CD123). Sometimes, the term "preferential binding" means that the specific binding of an antibody to an epitope is easier than its binding to a related, similar, homologous or similar epitope. Therefore, an antibody preferentially bound to a given epitope is more likely to bind to the epitope than to a related epitope, even if such an antibody may cross-react with a related epitope. For example, the antibody or antigen-binding fragment of the present invention may preferentially bind to human CD123 antigen relative to mouse CD123.

[0161] According to the above, the terms related to the antibodies, CARs, vectors or cells of the present invention can be easily obtained. In fact, the general context of this specification does not always require adherence to the wording marked above. Some deviations may occur. For example, when referring to the CAR of T cells, it is not always required to explain that the CAR includes a part for a specific antigen, that is, "anti-something". Accordingly, from a broader perspective, the CAR of the present invention comprising a part that specifically binds to CD123 can be referred to not only as anti-CD123 CAR, but also as CD123 CAR. In the same way, T cells transduced with CARs that specifically bind to CD123 according to the present invention can be referred to as CD123 CAR T cells.

[0162] As used herein, "treatment / treatment" includes any beneficial or desired effect on the symptoms or lesions of a disease or pathological condition, and may include any reduction in one or more measurable markers of the disease or condition (e.g., cancer) being treated. Treatment may include symptom relief or any improvement in symptoms of a disease or condition. Treatment may also include delaying the progression of a disease or condition. Therefore, the term "treatment" does not necessarily mean complete eradication or cure of a disease or condition or its associated symptoms. With this in mind, the disclosure of the present invention provides treatment or prevention of BPDCN, comprising administering a therapeutically effective amount of T cells of the present invention to a subject in need thereof.

[0163] In this context, the T cells described herein can be administered alone or as a pharmaceutical composition (also referred to as a pharmaceutical preparation). The term "pharmaceutical composition" or "pharmaceutical preparation" refers to a preparation that exists in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to whom the composition / preparation is administered. This preparation can be sterile. The pharmaceutical composition can contain only T cells according to the present invention, or in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants and stabilizers. The term "pharmaceutically acceptable" or "physiologically acceptable" is used herein to refer to compounds, materials, compositions and / or dosage forms suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, and commensurate with a reasonable benefit / risk ratio, within the scope of reasonable medical judgment. The composition of the present invention is preferably formulated for parenteral administration. "Parenteral administration" may refer to a mode of administration other than enteral and topical administration. Typically, parenteral administration is accomplished by injection, such as intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and / or infusion. Preferably, the CAR-modified T cells or compositions of the present invention are administered to a subject by direct injection into a tumor, lymph node, systemic circulation or infection site. In general, the present invention can be used to treat subjects diagnosed with a disease overexpressing CD123, especially patients with BPDCN. The treatment includes removing immune effector cells from the subject, genetically modifying the immune effector cells with a vector comprising a nucleic acid encoding the CAR covered 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 according to the patient's physical condition (the patient's condition, the type and severity of the patient's disease). An appropriate dose can also be determined with the help of animal models and subsequent clinical trials. When referring to a dose, the term "effective amount" is an amount sufficient to achieve a prescribed purpose. The so-called "therapeutically effective amount" of active ingredients such as genetically modified T cells may depend on factors such as the disease and its state, the patient's age, sex and weight, and the ability of the cells to cause the desired response in the patient. A therapeutically effective amount is also the amount in which any toxic or harmful effects of a virus or transduced therapeutic cell are offset by a therapeutic beneficial effect. In general, a pharmaceutical composition comprising T cells as described herein can be administered in an amount of 104 Up to 10 9 The dosage is 10 cells / kg body weight, preferably 10 5 Up to 10 6 Cells / kg body weight, including all integer values ​​within those ranges. More generally, the term "therapeutically effective amount" refers to the amount of an antibody, active ingredient, or other drug that effectively treats a disease or condition in a subject. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down or stop cancer to some extent) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down or stop cancer to some extent) tumor metastasis; inhibit or stop tumor growth to some extent; alleviate one or more symptoms associated with cancer to some extent; and / or produce a good response, such as increased progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR), or in some cases, stable disease (SD), delayed disease progression (PD), shortened time before progression (TTP), or any combination thereof. "Preventive effective amount" refers to an effective amount that achieves the desired preventive effect within the necessary dosage and time period. Typically, the preventive effective amount is less than the therapeutically effective amount because the preventive dose is used for the subject before the disease or at an earlier stage of the disease.

[0164] In a specific embodiment, before the immune effector cells described herein are manipulated in vitro or genetically modified, a cell source is obtained from a subject. In a specific embodiment, the immune effector cells expressing the CAR of the present invention on their membranes include T cells. T cells can be obtained from a variety of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, any number of techniques known to those skilled in the art, such as Ficoll TM In another embodiment, cells are obtained from the circulating blood of an individual by apheresis. The product of apheresis generally contains lymphocytes, which include T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in a suitable buffer or medium for subsequent processing. In other embodiments, the cells are isolated by lysing red blood cells and depleting mononuclear cells, for example by Percoll. TMGradient centrifugation is used to separate T cells from peripheral blood mononuclear cells. Specific T cell subsets expressing one or more 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 against surface markers that are not expressed on negatively selected cells.

[0165] In a more general case, this specification emphasizes a cell therapy in which T cells are genetically engineered (or genetically modified) to express CAR in vitro, and the CAR T cells are infused into patients in need thereof, preferably humans. The injected cells can kill tumor cells in patients. As described above, and unlike antibody therapy, T cells comprising CAR can replicate in vivo, producing long-term persistence, so that lasting tumor control can be achieved. In addition, CAR allows effector T cells to redirect and activate to any cell surface molecule (after being bound by antibody-derived receptors), and is not restricted by MHC (MHC-restricted antigen recognition; MHC represents major histocompatibility complex). As described above, the genetically engineered T cells of the present invention are constructed from T cells collected from the patient himself (autologous), but they can also be derived from other allogeneic donors to provide allogeneic genetically engineered T cells in a bone marrow or peripheral hematopoietic stem cell allogeneic transplantation environment (donor lymphocyte infusion). These T cells expressing CAR molecules according to the present invention can be used to treat proliferative diseases in mammals, preferably humans.

[0166] In some embodiments of the present invention, polynucleotides or cells containing polynucleotides of the present invention use suicide genes, including the use of inducible suicide genes to reduce the direct toxicity of genetically modified cells (i.e., graft-versus-host disease, GvHD in allogeneic administration) and / or the risk of uncontrolled proliferation. In particular aspects, suicide genes are not immunogenic to hosts containing the polynucleotides or cells. Specific examples of suicide genes that can be used are inducible caspase-9 (iCASP9), herpes simplex virus-thymidine kinase (HSV-tk), CD20, truncated EGFR, caspase-8 or cytosine deaminase. Specific dimerization chemical inducers (CID) can be used to activate caspase-9. Other systems can be activated by metabolic prodrugs (ganciclovir) or by binding antibodies (rituximab, cetuximab).

[0167] The present invention is further described in detail with reference to experimental data and specific examples. Unless otherwise stated, the examples provided are for illustrative purposes only and are not intended to be limiting. Although the following detailed description focuses on BPDCN, the present invention can generally still provide improvements and / or solutions for any disease in which CD123 overexpression occurs.

[0168] The main focus of the present invention is to provide a treatment for blastic plasmacytoid dendritic cell neoplasm (BPDCN).

[0169] BPDCN is a clonal disease originating from plasmacytoid dendritic cell (pDC) precursors, with skin lesions progressing to aggressive leukemia spread, see Blastic Plasmacytoid Dendritic Cell Neoplasm: State of the Art and Prospects, Maria Rosaria Sapienza, Alessandro Pileri, Enrico Derenzini, Federica Melle et al., Cancer, 2019. BPDCN has a poor prognosis, and there is no effective treatment or related reports in the art. Most patients initially responded to intensive chemotherapy, but then relapsed. In the largest series of patients, the median overall survival (OS) was 8 to 12 months. The only exception to this data is for patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT); see Roos-Weil D, Dietrich S, Boumendil A, Polge E, Bron D, Carreras E et al., Stem cell transplantation can provide durable disease control in blastic plasmacytoid dendritic cell neoplasm: a retrospective study from the European Group for Blood and Marrow Transplantation, Blood, 2013, 121(3): 440-6; Pagano L, Valentini CG, Grammatico S, Pulsoni A, Blastic plasmacytoid dendritic cell neoplasm: diagnostic criteria and therapeutical approaches, British journal of haematology, 2016, 174(2): 188-202; Kharfan-Dabaja MA, Lazarus HM, Nishihori T, Mahfouz RA, Hamadani M.Diagnostic and therapeutic advances inblastic plasmacytoid dendritic cell neoplasm:a focus on hematopoietic cell transplantation,Biology of blood and marrow transplantation:journal of theAmerican Society for Blood and Marrow Transplantation,2013;19(7):1006-12; even in patients receiving targeted therapy (Pemmaraju N,Lane AA,Sweet KL,Stein AS,Vasu S,Blum W et al.,Tagraxofusp in Blastic Plasmacytoid Dendritic-Cell Neoplasm,TheNew England journal of medicine,2019;380(17):1628-37). However, most elderly patients do not meet the requirements of this approach. For this type of leukemia, which can be characterized as chemotherapy-refractory leukemia, improved treatment methods are needed. The applicant of the present invention has identified a new therapeutic target. Interleukin (IL) 3 receptor alpha chain (CD123) has been shown to be a valuable target because it is only highly expressed by basophils and pDCs in the body. It is further disclosed in the art that CD123 is also expressed at low levels in normal hematopoietic CD34. + CD38 +It is expressed in progenitor cells, monocytes and endothelial cells, but not in hematopoietic stem cells; see Taussig DC, Pearce DJ, Simpson C, Rohatiner AZ, Lister TA, Kelly G et al. Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia, Blood. 2005; 106(13): 4086-92; see Adhra Al-Mawali1, David Gillis and Ian Lewis, Immunoprofiling of leukemic stem cells CD34+ / CD38- / CD123+delineate FLT3 / ITD-positive clones, Journal of Hematology&Oncology, 2016, 9: 61.

[0170] In order to combat BPDCN, the inventors generally considered that the antigen CD123 is uniformly and highly expressed in BPDCN in both diagnosis and relapse after chemotherapy; see Garnache-Ottou F, Feuillard J, Ferrand C, Biichle S, Trimoreau F, Seilles E et al., Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia, British journal of haematology, 2009; 145 (5): 624-36. Moreover, the inventors also considered that CD123 is a component of the IL-3 receptor, and the IL-3 receptor is a key driver of the pDC lineage required for pDC cell survival. Broughton SE, Dhagat U, Hercus TR, Nero TL, Grimbaldeston MA, Bonder CS, et al., The GM-CSF / IL-3 / IL-5 cytokine receptor family: from ligand recognition to initiation of signaling. Immunological reviews. 2012; 250(1): 277-302; Hara T, Miyajima A., Function and signal transduction mediated by the interleukin 3 receptor system in hematopoiesis, Stem Cells, 1996, 14(6): 605-18; and Testa U, Pelosi E, Frankel A., CD 123 is an embryonic biomarker and a therapeutic target in hematologic malignancies, Biomarker research, 2014, 2(1): 4. The inventors speculate that targeting this IL-3 receptor may induce signaling pathways that are detrimental to BPDCN cells.

[0171] It is reported that CD123 is involved in blast priming in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL); see Ehninger A, Kramer M, Rollig C, Thiede C, Bornhauser M, von Bonin M et al., Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia, Blood cancer journal, 2014, 4: e218. In addition, CD123 is reported to be expressed on CD34+ "leukemia initiating cells (LIC)" components and is associated with adverse clinical effects; see Vergez F, Green AS, Tamburini J, Sarry JE, Gaillard B, Cornillet-Lefebvre P et al., High levels of CD34 + CD38 low / -CD123 + blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study, Haematologica, 2011, 96(12): 1792-8; and 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 not clear whether there are increased numbers of immature LICs in patients. The applicant was also surprised to observe that in the specific BPDCN cases collected by the inventors, low-component blasts expressed higher levels of CD123 than leukemic bulk cells. The applicant then further speculated that if they are LICs, targeting CD123 may eliminate these cells and reduce relapses in BPDCN patients. Then, the applicant actively selected CD123 as a targeted immunotherapy for BPDCN.

[0172] The applicants have further demonstrated the sensitivity of BPDCN cells to SL-401, a fusion protein comprising diphtheria toxin coupled to IL-3, which targets cells expressing CD123 both in vitro and in vivo; see Angelot-Delettre F, Roggy A, Frankel AE, Lamarthee B, Seilles E, Biichle S et al., In vivo and in vitro sensitivity of blastic plasmacytoid dendritic cell neoplasm to SL-401, an interleukin-3 receptor targeted biologic agent, Haematologica, 2015; 100(2): 223-30. In addition, clinical responses have been reported in patients with BPDCN; see Frankel AE, Woo JH, Ahn C, Pemmaraju N, Medeiros BC, Carraway HE et al., Activity of SL-401, a targeted therapy directed to interleukin-3 receptor, in blastic plasmacytoid dendritic cell neoplasm patients, Blood, 2014; 124(3): 385-92.

[0173] As is known in the art, adoptive T cell therapy using chimeric antigen receptors (CARs) is a promising approach for treating hematological malignancies at risk of relapse; see Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR et al., Chimeric antigen receptor-modified T cells for acute lymphoid leukemia, The New England journal of medicine, 2013; 368(16): 1509-18; and Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stetler-Stevenson M et al., Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous Tcells expressing an anti-CD19 chimeric antigen receptor, Journal of clinical oncology: official journal of the American Society of Clinical Oncology, 2015; 33(6): 540-9.

[0174] According to one embodiment of the present invention, CAR has been genetically engineered to be made of a single-chain variable fragment (ScFv) of a monoclonal antibody (MAb) heavy chain and a light chain connected to the intracellular signaling chain of a T cell receptor (TCR). Preferably, the intracellular portion of CAR includes CD3ζ and a costimulatory domain, such as CD28 and 4-1BB, which can better activate and cell signaling. The CAR can be characterized as a third-generation CAR. For more information about CAR: see Andrew D. Fesnak, Carl H. June and Bruce L. Levine's review, Engineered T cells: the promise and challenges of cancer immunotherapy, Nature, Vol. 16, September 2016. ScFV allows antigen recognition independent of major histocompatibility complex (MHC) presentation.

[0175] The efficiency of CAR CD123 in eradicating acute myeloid leukemia in vitro and in several murine patient-derived xenograft models (PDX) has been reported; see Mardiros A, Dos Santos C, McDonald T, Brown CE, Wang X, Budde LE et al., T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia, Blood, 2013, 122(18):3138-48; Gill S, Tasian SK, Ruella 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, Blood, 2014, 123(15):2343-54; Thokala R, Olivares S, Mi T, Maiti S, Deniger D, Huls H et al., Redirecting Specificity of T cells Using the SleepingBeauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VLand VH Domains Targeting CD123+Tumors, PloS one, 2016, 11(8):e0159477; CumminsKD, Gill S.Anti-CD123 chimeric antigen receptor T-cells (CART): an evolving treatment strategy for hematological malignancies, and a potential ace-in-the-hole against antigen-negative relapse, Leukemia&lymphoma, 2018, 59(7):1539-53; Tettamanti S, Marin V, Pizzitola I, Magnani CF, Giordano Attianese GM, Cribioli E et al., Targeting of Acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor, British journal of haematology, 2013, 161(3): 389-401; and Tasian SK, Kenderian SS, Shen F, Ruella M, Shestova O, Kozlowski M, et al., Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute Myeloid leukemia, Blood, 2017; 129(17): 2395-407. Another group of studies showed that CAR CD123 can be used to treat CD123 in established BPDCN cell lines (CAL-1) and mouse models. +Primary BPDCN samples are active. But this CAR is only used in allogeneic cases. It is also the second generation with a suicide system; see Cai T, Galetto R, Gouble A, Smith J, Cavazos A, Konoplev S et al., Pre-Clinical Studies of Anti-CD123 CAR-T Cells for the Treatment of Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), Blood. 2016, 128 (22): 4039. However, more and more clinical trials have shown that patients receiving CAR therapy will experience toxicity. This is a major problem in the development of efficient CAR. The applicant has developed a specific CAR that overcomes the problems of the prior art. More specifically, the applicant has engineered third-generation retroviral and lentiviral CARs at the same time. 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 in vitro for BPDCN cells (BPDCN cell lines, PDX cells, and primary BPDCN cells), as in several mouse BPDCN models in vivo. Evaluation of their effects on CD123 低 The results were unexpectedly promising given the potential cytotoxicity of positive cells.

[0176] According to the present invention, monoclonal antibodies (Mab) against human CD123 are produced. The monoclonal antibodies of the present invention can be used in allogeneic and autologous situations.

[0177] The preparation method of anti-CD123 monoclonal antibody includes immunizing mice with recombinant CD123 protein. More specifically, five mice are immunized with recombinant CD123, and the product number of the recombinant CD123 is 301-R3-025 (available at R&D Systems, Minnesota, USA). Immunization is performed by foot pad and / or intraperitoneal administration. Lymph node and / or spleen B cells are used to produce hybridomas that secrete Mabs. Using CD123 + and CD123 - Cell lines Hybridomas were selected based on the affinity and specificity of the MAb.

[0178] According to the present invention, 6 specific antibodies were selected and named 18B4D5 (also referred to herein as AB1, or sometimes abbreviated 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). Molecular characterization and DNA sequencing were performed according to the SANGER method.

[0179] The VDJ and VJ gene rearrangements were sequenced and the consensus nucleotide sequences were compared with the VQUEST online tool. The sequences were compared with the database and then identified; see Brochet X, Lefranc MP, Giudicelli V. IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and VDJ sequence analysis, Nucleic acids research, 2008; 36(online): W503-8. Figures 1 to 9 The respective sequences (nucleotide sequences and amino acid sequences) of the six antibodies of the present invention are listed in .

[0180] Figure 1 The nucleotide and amino acid sequences of the complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) of the heavy and light chains of the antibodies of the invention (18B4D5, 17F7B2, 11B6E6, 14G12D7, 11A12C6 and 15E11B7, respectively) are shown.

[0181] Figure 2 The nucleotide sequences and amino acid sequences of the framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4) of the heavy chain of the antibodies of the invention (18B4D5, 17F7B2, 11B6E6, 14G12D7, 11A12C6 and 15E11B7, respectively) are shown.

[0182] Figure 3 The nucleotide sequences and amino acid sequences of the framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4) of the light chain of the antibodies of the invention (18B4D5, 17F7B2, 11B6E6, 14G12D7, 11A12C6 and 15E11B7, respectively) are shown.

[0183] Figure 4The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the first antibody AB1 of the present invention (named 18B4D5 or B4D5) are shown. The amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted from left to right in reading order.

[0184] Figure 5 The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the second antibody AB2 of the present invention, designated as 17F7B2, are shown. The amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted from left to right in reading order.

[0185] Figure 6 The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the third antibody AB3 of the present invention, designated as 11B6E6, are shown. The amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted from left to right in reading order.

[0186] Figure 7 The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the fourth antibody AB4 of the present invention, designated as 14G12D7, are shown. The amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted from left to right in reading order.

[0187] Figure 8 The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the fifth antibody AB5 of the present invention, designated as 11A12C6, are shown. The amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted from left to right in reading order.

[0188] Fig. 9 The consensus amino acid sequence of the light chain and the consensus amino acid sequence of the heavy chain of the sixth antibody AB6 of the present invention, designated as 15E11B7, are shown. The amino acids corresponding to CDR1, CDR2 and CDR3 are highlighted from left to right in reading order.

[0189] The sequence homology of each of the heavy chain VH and JH and the light chain VK and JK is shown in Table 1

[0190]

[0191]

[0192] Table 1: Sequence homology of variable chains.

[0193] The affinity of the antibodies of the present invention to CD123 was tested on a streptavidin biosensor. DSeven different concentrations of CD123 were used on the immobilized antibodies of the invention. The results were analyzed according to the Octet system available from ForteBio.

[0194] The antibody affinity results are shown in Table 2.

[0195] Load Sample ID Concentration (nM) answer KD(M) <![CDATA[k on (1 / Ms)]]> <![CDATA[K dis (1 / s)]]> Full R^2 AB1(18B4D5) 50 0,6972 <1.0E-12 1,22E+05 <1.0E-07 0,9988 AB1(18B4D5) 25 0,4202 <1.0E-12 1,22E+05 <1.0E-07 0,9988 AB1(18B4D5) 12,5 0,2301 <1.0E-12 1,22E+05 <1.0E-07 0,9988 AB1(18B4D5) 6,25 0,12 <1.0E-12 1,22E+05 <1.0E-07 0,9988 AB5(11A12C6) 12,5 0,4158 <1.0E-12 3,26E+05 <1.0E-07 0,9885 AB5(11A12C6) 6,25 0,2815 <1.0E-12 3,26E+05 <1.0E-07 0,9885 AB5(11A12C6) 3,13 0,1349 <1.0E-12 3,26E+05 <1.0E-07 0,9885 AB3(11B6E6) 50 0,3164 9,368E-09 7,01E+04 6,57E-04 0,9973 AB3(11B6E6) 25 0,1794 9,368E-09 7,01E+04 6,57E-04 0,9973 AB3(11B6E6) 12,5 0,0895 9,368E-09 7,01E+04 6,57E-04 0,9973 AB3(11B6E6) 6,25 0,0349 9,368E-09 7,01E+04 6,57E-04 0,9973 AB4(14G12D7) 100 0,3965 8,843E-09 3,67E+04 3,25E-04 0,9986 AB4(14G12D7) 50 0,2206 8,843E-09 3,67E+04 3,25E-04 0,9986 AB4(14G12D7) 25 0,1342 8,843E-09 3,67E+04 3,25E-04 0,9986 AB2(17F7B2) 50 0,5066 2,552E-09 3,46E+05 8,82E-04 0,9747 AB2(17F7B2) 25 0,34 2,552E-09 3,46E+05 8,82E-04 0,9747 AB2(17F7B2) 12,5 0,2645 2,552E-09 3,46E+05 8,82E-04 0,9747 AB2(17F7B2) 6,25 0,1486 2,552E-09 3,46E+05 8,82E-04 0,9747 AB6(15E11B7) 25 0,3052 2,485E-09 3,32E+05 8,26E-04 0,9829 AB6(15E11B7) 12,5 0,1552 2,485E-09 3,32E+05 8,26E-04 0,9829 AB6(15E11B7) 6,25 0,0878 2,485E-09 3,32E+05 8,26E-04 0,9829

[0196] Table 2: Antibody affinities.

[0197] Cells for implementing the present invention, particularly cells for the purposes of embodiments and results herein, are obtained from humans. More precisely, primary cells from BPDCN patients (DC-2008-713 and DC-2016-2791 collections) are used for CD123 expression assessment, cytotoxicity assays, and primary cell expansion in NSG mice to produce PDX (primary derived xenografts), see Philippe L. et al., Bortezomib as a new therapeutic approach for blasticplasmacytoid dendritic cell neoplasm.Haematologica, 2017. Features of these cases have been described according to current recommendations for the diagnosis of BPDCN established by the applicants, see Garnache-Ottou F, Feuillard J, Ferrand C, Biichle S, Trimoreau F, Seilles E et al., Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia, British journal of haematology, 2009, 145(5): 624-36 and Swerdlow, SH, Campo, E., Harris, NL, Jaffe, ES, Pileri, SA, Stein, H., Thiele, J., Arber, DA, Hasserjian, RP, Le Beau, MM, Orazi, A. & Siebert, R. (2017) WHO classification of tumors of haematopoeitic and lymphoid tissues. In: WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues. (Editors: Swerdlow, SH, Campo, E., Harris, NL, Jaffe, ES, Pileri, SA, Stein, H., Thiele, J., Arber, DA, Hasserjian, RP, Le Beau, MM, Orazi, A. & Siebert, R.), pp. 98-106. IARC, Lyon, France. PDX cells P127B, P25B, CM60915, F13-CAL1 and F13-LAN were cultured in vitro in appropriate culture media.Two types of CD123. + BPDCN cell lines (GEN 2.2 and CAL-1, Dr. Maeda, Nagasaki University, Japan) and CD123 - Cell lines (Daudi, Jurkat, HL-60, REH). K562 CD123 cell lines were produced by lentiviral transduction to express CD123. In order to study the expression of CD123, cells derived from normal blood (n=19) and bone marrow (n=4), acute leukemia (myeloid AML, n=3) and lymph (B-ALL, n=3; T-ALL, n=3) were obtained from healthy donors and / or patients. In the present invention, peripheral blood mononuclear cells (PBMC) were used to produce genetically modified T cells. By Ficoll TM Gradient density centrifugation (using commercially available Ficoll-Paque TM PBMCs were isolated from healthy donors or cord blood samples obtained at the French Blood Center (EFS B / FC, France) collection.

[0198] Unless otherwise stated, the statistical analysis herein is expressed as mean. More specifically, the results of MFI and MFIR are expressed as mean [minimum-maximum], and all other results are expressed as mean ± standard deviation. All statistical analyses in the embodiments are performed using GraphPad Prism 7 (GraphPad software, San Diego, CA, USA). Comparisons between the two groups are evaluated by Student's t test. Survival studies are evaluated by Kaplan-Meier curves and log-rank (Mantel-Cox) tests.

[0199] Using flow cytometry, the expression profile of CD123 on normal hematopoietic cells and BPDCN cells was tested. The expression profile of CD123 in normal cells and BPDCN patient cells was determined using the antibody B4D5 (AB1) of the present invention and a commercial anti-CD123 antibody (6H6). More specifically, normal blood and bone marrow cells, i.e., normal pDC (targeted with CD304), monocytes (CD14 + ), lymphocytes (CD45 +高 / SSC 低 ), granulocytes (CD45 + 、SSC 高 and FSC 高 ) and CSH / progenitor cells (CD34 + ) and BPDCN patient cells (CD45 +低 、CD304 +) were compared. The mean fluorescence intensity (MFI) of CD123 was divided by the mean fluorescence intensity of the isotype control mAb to calculate the mean fluorescence intensity ratio (MFIR). When the MFIR was higher than 2, the cell was considered to be positive for CD123 expression; see Garand R, Robillard N., Immunophenotypic characterization of acute leukemias and chronic lymphoproliferative disorders: practical recommendations and classifications. Hematology and cell therapy, 1996; 38 (6): 471-86. CD123 was highest on normal pDC and BPDCN (130.6 [118.4-146.8], n = 5 and 136 [102.1-158.1], n = 5, respectively). Low expression was detected on 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 multinuclear cells (1.1 [1.1-1.2], n=3) did not express CD123.

[0200] Fig.10 Flow cytometry plots based on the evaluation of CD123 expression on normal hematopoietic cells and BPDCN are shown. In particular, flow cytometry plots based on the evaluation of CD123 expression on normal hematopoietic cells and BPDCN using the B4D5 antibody compared to the commercial antibody 6H6 are shown. The isotype control is presented in white, while the anti-CD123 B4D5 is presented in dark. Normal cells were evaluated in peripheral blood and bone marrow as described above, see Fig.10 A, For BPDCN cell lines (CAL-1 and GEN 2.2), PDX (P127B-M5), and primary cells from BPDCN patients, see Fig.10 B. In addition, CD123 expression was assessed by MFIR (ratio of mean fluorescence intensity of CD123 to that of isotype control mAb) 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). In addition to the CD34 population, data from blood and bone marrow samples (n=4 and 7) were combined because we observed no difference in the expression of CD123 levels, see Fig.10 C.

[0201] In addition, several non-pDC acute leukemias were tested with the B4D5 monoclonal antibody. Applicants demonstrated that BPDCN expressed higher levels of CD123 than the AML, B or T lymphocyte acute leukemias tested.

[0202] Fig.11 Shown are tables and flow cytometry graphs based on the evaluation of CD123 expression in BPDCN and other non-pDC acute leukemias. Fig.11 The expression of CD123 was evaluated by MFIR (the ratio of the mean fluorescence intensity of CD123 divided by the mean fluorescence intensity of the isotype control mAb) on lymphocytes (n=12), monocytes (n=12), granulocytes (n=5), and normal pDCs (n=7) using the 6H6 commercial antibody, see Fig.11 A. As we observed no differences in CD123 levels, data from blood and bone marrow samples were combined. Expression on the CD34 population was assessed on the bone marrow. Fig.11 Also shown is flow cytometric assessment of CD123 expression on B-LAL, T-LAL and AML cell lines and patient primary cells using the CD123 B4D5 antibody in dark and an isotype control in white, see Fig.11 B. CD123 expression levels in non-pDC acute leukemias are lower than those in BPDCN patients, see Fig.11 B.

[0203] Fig.12 Shown is a schematic diagram of the design of the chimeric antigen receptor of the present invention, as well as western blots and flow cytometry images related to the production of T cells comprising the chimeric antigen receptor of the present invention according to the present invention.

[0204] Fig.12 The design and production of CD123 CAR T cells according to the present invention are shown. Anti-CD123 CAR T cells were constructed using the antibody AB1 of the present invention. The CAR of the present embodiment of the present invention is based on the scFv of B4D5 (AB1), a specific domain (CD3ζ) activated by T cells, and two co-activation domains (CD28 and 4.1BB). The sequence encoding CAR, the 2A sequence (encoding 2A self-cleavage peptide), and the screening marker ΔCD19 (truncated CD19) are cloned into the pSFG retroviral vector (SFG.CARCD123.IRES.ΔCD19 vector). Retroviral supernatant is produced by a PG13 packaging cell line transfected with SFG.CAR CD123.IRES.ΔCD19 vector.

[0205] According to another embodiment of the present invention, a lentiviral construct was also generated: the same CAR sequence was controlled by the EF1α promoter, and downstream was a T2A ribosomal skipping sequence and a truncated human CD19 transduction marker.

[0206] Fig.12 A shows an embodiment of a retrovirus and a lentivirus. More specifically, Fig.12 Item A shows a schematic diagram of a third-generation retroviral anti-CD123 CAR vector (CD123R CAR) and a third-generation lentiviral anti-CD123 CAR vector (CD123LCAR). The scFv CD123 derived from the B4D5 antibody of the present invention is fused with T cell signaling domains CD28, 4.1BB, and CD3ζ. Screening markers (ΔCD19) are also indicated.

[0207] The genetically modified primary T cells in the present invention were generated by viral transduction. Prior to transduction, PBMCs were activated for two days with CD3 / CD28 Dynabeads (Life Technologies, California, USA). Recombinant human fibronectin (retronectin) (overnight in PBS at 4°C, 1.2 μg / cm 2 Retroviral transduction was performed in 6-well plates coated with 1% paraformaldehyde (Takara, Japan). Then, cells were transduced with retroviral particles by centrifugation (1.5 h, 2000 g, 10 °C). For lentiviral transduction, cells were transduced by centrifugation at 800 g, 32 °C for 1 h with an MOI of 10. Flow cytometry was performed to identify CD3 + The transduction efficiency was determined by measuring the expression of the ΔCD19 cell surface marker on the cells. The reverse transcription-transduced positive cells were magnetically labeled with microbeads (CD19 microbeads, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol and loaded onto On the column. For T cells obtained by retroviral transduction, the transduced T cells will be named CD123R CAR, and the T cells obtained by lentiviral transduction will be named CD123L CAR. From the same donor, untransduced T cells (called C0) were collected and cultured similarly to the above, but without viral transduction: these cells were used as controls in all experiments. CD123 CAR T cells were expanded for 21 days and cryopreserved.

[0208] For the described embodiments of the present invention, unless otherwise stated, cytotoxicity can be evaluated based on transduced T cells derived from BPDCN patients. More specifically, T cells from BPDCN patients (DC 2016-2791) were activated from PBMCs using human CD3 / CD28 Dynabeads and magnetic selection. The negative fraction contains BPDCN blasts (blood cell differential count is 60%), which can be used to evaluate the cytotoxicity of CAR and B lymphocytes. In addition, as previously described, T cells were transduced with lentiviral constructs. After 10 days, transduction efficiency and CAR expression were determined by flow cytometry as described. The cytotoxic activity of BPCDN blasts was evaluated for 24 hours at an E:T ratio of 5:1.

[0209] For the described embodiments of the present invention, unless otherwise indicated, immunophenotyping can be performed as follows. Antibody binding is detected on an LSR Fortessa flow cytometer or BD FACS CANTO II and analyzed using Diva software (BD Biosciences, New Jersey, USA). Appropriately matched isotype controls are included in the analysis. Briefly, T cells are stained for CD3, CD4, CD8, CD19. CD3 + / CD19 + T cells are CD123 CAR T cells. BPDCN cells, cell lines, and PDX were characterized by CD123 staining using B4D5 (AB1 used to construct the CAR) and commercial antibodies (i.e., 6H6) for additional results. T cells were CD45 高 / SSC 低 , and granulocytes are CD45 + / SSC 高 / FSC 高 ), CD304 (normal pDC), CD14 (monocytes) and CD34 (CSH / progenitor cells) to analyze normal blood and bone marrow samples. BPDCN samples from patients were stained by CD56, CD4, CD303, CD304, CD45. Some embodiments of the present invention relate to a BPDCN mouse model. Accordingly, in the BPDCN mouse model, BD TruCount tubes were used to monitor the mother cell count (anti-human hCD45, hCD123) and T cells (C0 and CD123 CAR) (hCD3, hCD19) in blood cell samples every week. In humanized mice, peripheral blood was analyzed by flow cytometry (Attune NxT flow cytometer, Life Technologies) to quantify human cells: total leukocytes (hCD45 + ), monocytes (CD45 / SSC +中 、hCD33+ ), B lymphocytes (hCD19 + ), T lymphocytes (hCD3 + )、pDC(hCD123 + 、hCD304 + ) and CSH / progenitor cells in the bone marrow after mouse sacrifice (CD34 + ). The monoclonal antibodies used to characterize the cell surface phenotype are shown in Table 3.

[0210]

[0211]

[0212]

[0213] Table 3: Monoclonal antibodies used to characterize cell surface phenotypes.

[0214] Some embodiments described herein also involve in vitro cytotoxicity assays and functional analysis. To this end, C0 or CD123 CAR T cells (1.10 6 The labeled cells were then cultured with target cells (CAL-1, GEN 2.2, DAUDI, HL-60, K562 CD123, and PDX) at the appropriate effector to target (E:T) ratio at 37°C for 24 hours or 15 hours (PDX). After co-culture, cells were labeled with 7-AAD (Beckman Coulter), anti-CD3, anti-CD19 (to evaluate C0 or CAR T cells), and anti-CD123 to evaluate target cells (7-AAD + CD123 + Cell death was detected using BD Cytofix / Cytoperm according to the manufacturer's protocol. TM Plus Fixation / Permeabilization Solution Kit and BD GolgiPlug TM CD107a (BD Biosciences) PE Degranulation assay (clone H4A3, BD Biosciences) and IFNγ PEThe functional properties of CD123 CAR T cells were tested in vitro by intracellular production (clone B27, BD Biosciences). The co-culture supernatant was analyzed by CBA human Th1 / Th2 / Th17 cytokine kit (BD Biosciences) according to the manufacturer's protocol. The cytotoxicity of CAR to HMEC adherent cell lines or primary monocytes was evaluated using the MTT cell proliferation test (Sigma-Aldrich, Missouri, USA) according to the manufacturer's protocol.

[0215] Some embodiments described herein also relate to tissue microarrays and immunohistochemistry. The specificity of B4D5 (AB1) antibody is analyzed by tissue microarray, which includes 90 tissue cores (30 organs) of three individual donors of each organ (US Biomax, Rockville, United States). Immunostaining is detected using UltraView Universal DAB Detection Kit (Ventana, USA), and images are analyzed using NDP.view 2.0 software. Staining intensity is graded as follows: negative (0), low staining (1+), moderate staining (2+) or positive staining (3+), high staining (4+). High CD123 (CAL-1) or negative (Daudi) expression cell lines are used as positive or negative controls, respectively. For each tissue (colon, stomach, muscle, esophagus, skin and thyroid) showing CD123 staining, CD123 expression on paraffin-embedded normal tissues from three patients is controlled by immunohistochemistry (IHC), and compared with the skin from two BPDCN patients.

[0216] In addition, some embodiments described herein relate to in vivo models to evaluate the efficiency of CAR CD123. To this end, NOD / SCID / IL2Rγc-deficient (NSG) mice (6-8 weeks old, The Jackson laboratory, Sacramento, CA, USA) were irradiated (2.5 Gy) and intravenously inoculated with an appropriate number of CAL-1 expressing luciferase (1 10 6 / mice) or PDX cells (2·10 6 / mice). Engraftment and quantification of BPDCN cells have been described; see Philippe L, Ceroi A, Bole-Richard E, Jenvrin A, Biichle S, Perrin S et al., Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neoplasm, Haematologica, 2017, 102(11): 1861-8. For the CAL-1 model, T cells (untransduced and transduced) were injected into the tail vein on day 3. Engraftment was monitored weekly by BLI measurements: mice received 3 mg of fluorescein (VivoGlo fluorescein, Cat. No. P1043, Promega, Wisconsin, USA) intraperitoneally within 10 minutes before imaging (IVIS Lumina Series III, Perkin Elmer, Massachusetts, USA). In the PDX model, mice received T cells when BPDCN blasts were detected in blood samples. Monitor blast counts every 10 days by flow cytometry using anti-hCD45, anti-CD123, and BD Trucount tubes.

[0217] Some other embodiments described herein involve evaluating CAR for CD123 低 Toxicity to cells. For this purpose, a humanized mouse model was used. More specifically, a humanized mouse model (female hCD34-implanted NOD / Shi-scid / IL-2Rγnull immunodeficient mouse strain: hu-NOG) was used to study the in vivo toxicity of the CAR of the present invention to normal human hematopoietic cells (n=3 donors). On day -15, two humanized mice were sacrificed; T cells were harvested from the spleen and transduced to obtain CD123 CAR T cells or C0. On day 0, two humanized mice (generated from the same donor) were injected retro-orbitally with 20·10 6C0 or CD123 CAR. On days -5 and 15, the total number of leukocytes, monocytes, B and T lymphocytes, pDCs, and basophils in the blood were quantified, and HSC / progenitor cells in the bone marrow obtained after sacrifice were quantified (only on day 15). In order to evaluate the in vitro cytotoxicity of CAR T cells against normal autologous CD34 cells, magnetically sorted CD34+ cells (CD34 microbeads, Miltenyi Biotec) and CD123 CAR obtained from the bone marrow of sacrificed humanized mice can be used. Alternatively, C0 produced by T cells obtained from the same humanized mice (n=3) as above can be used. Co-culture was performed under the same conditions as the BPDCN cells described, with an E:T ratio of 5:1. The live hCD34 after co-culture with CD123 CAR or C0 was compared. + The same experiment was performed using CD34 cells magnetically sorted from cord blood and co-cultured with CAR CD123 or C0 generated from the same cord blood T lymphocytes (n=3).

[0218] Fig.12 Transduction efficiencies are shown in Fig.12 B. Transduction efficiency was assessed by quantifying T cells co-expressing CD3 and CD19. The variable transduction levels observed using retroviral constructs averaged 17.8% ([3-57%], n=43). This may indicate that transduction capacity is variable and can be controlled by using magnetic selection in order to efficiently utilize CAR T cells (the purity of transduced T cells was 90% ± 9.5%, [60-98.5%], n=43). More precisely, Fig.12 Item B shows the representative phenotype CD3 / CD19 of non-transduced T cells (C0) compared with transduced T cells of the present invention (CD123 CAR T cells or anti-CD123 CAR T cells) before and after magnetic cell separation of T cells of the present invention. Lentiviral transduction showed good transduction efficiency (91.2% ± 5.22, n = 6), see Fig.12 B.

[0219] For the purposes of the present invention, the expression of CAR constructs in transduced cells can be evaluated by Western blotting. More specifically, in RIPA buffer supplemented with a protease inhibitor cocktail (complete mini-mixture without EDTA; Roche, France), untransduced T cells (C0) and CD123 CAR T cells according to the present invention were lysed by ultrasonic treatment. Equal amounts of protein were separated by SDS-PAGE and electrotransferred to PVDF membranes (Bio Rad, California, USA). The membrane was probed overnight (51-6527GR, BD Biosciences) with the first antibody binding to human CD3ζ chain (diluted at 1:1000). Antibodies recognizing β-actin were added (at 1:10 6 For immunodetection, the secondary antibody was added (1:6000 dilution): sheep anti-mouse IgG (Cat. No. 515-035-062, Jackson, Pennsylvania, USA). The appropriate detection reagents (Clarity TM Western ECL blotting substrate, Bio-Rad, Cressier, Switzerland), and chemiluminescence was detected by using a camera and Bio-ID software (Wilber-Lourmat, College, France).

[0220] Fig.12 Also shown is a western blot, see Fig.12 C. The expression of the CAR of the present invention was verified by immunoblotting in three experiments ( Figure 2 C). A lower band (16 kDa) corresponding to the endogenous CD3ζ chain was detected in transduced and non-transduced T cell lysates, while an additional band appeared at 55 kDa in CD123 CAR T cells, corresponding to the theoretical size of the CD123 CAR construct. This band was absent in non-transduced T cells. More precisely, Fig.12 Item C shows western blot analysis of CD123 CAR protein expression (55 kDa, compared to endogenous CD3ζ (16 kDa), CD3ζ fused to the CAR construct) in whole cell lysates derived from genetically modified T cells (CAR) compared to no expression in C0.

[0221] Fig.12 Flow cytometric analysis is also shown, see Fig.12 D and Fig.12E. Flow cytometry analysis was performed using biotin-conjugated CD123 protein to confirm the surface expression of CD123 CAR on T cells. The fixation of CD123 protein occurred only on transduced T cells (16% ± 11.5 for CD123R CAR, n = 8, and 31.5% ± 12.2 for CD123L CAR, n = 9), and 0% on non-transduced T cells. Fig.12 Item D shows the detection of CAR expression on the cell surface by flow cytometry: using biotin-conjugated CD123 protein and streptavidin PE CD123 CAR and C0 were stained. More precisely, Fig.12 Item D shows an example of expression, and in Fig.12 The average of a total of 17 different experiments (n=8 for CD123R CAR and n=9 for CD123L CAR) reported is shown in E. No expression was detected in non-transduced cells.

[0222] More generally, for the purposes of the present invention, the evaluation of CAR surface expression in transduced cells was completed by flow cytometry. More precisely, the expression of anti-CD123 scFV on the cell surface of transduced T cells was examined by flow cytometry. T cells (100 000 cells) were stained with 0.25 μg of biotinylated CD123 protein for 1 hour at room temperature and then stained with PE-conjugated streptavidin (BD Biosciences). CD123 was then evaluated in CD3 compared to C0. + CD19 + Fixation of CAR cells.

[0223] Fig.13 The nucleic acid sequence of the CAR according to the present invention is shown.

[0224] CAR is constructed with the antibody AB1 of the present invention. Nucleic acids corresponding to signal peptide, tag HA, heavy chain, hinge 1, light chain, hinge 2, CD28, 4.1BB and CD3z are highlighted from left to right in reading order with different shades.

[0225] Accordingly, an object of the present invention is an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the nucleic acid molecule consists of a sequence that is at least 85% identical, preferably at least 90% identical, more preferably at least 95% identical to SEQ ID NO:97.

[0226] Another object of the present invention is an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the nucleic acid consists of SEQ ID NO:97.

[0227] Fig.14Shown are flow cytometry graphs associated with evaluating the phenotype of CAR T cells according to the present invention compared to untransduced T cells.

[0228] 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 difference in CD4 / CD8 ratio was observed between untransduced and transduced T cells. Fig.14 Item A shows the percentage of the CD8 population (as opposed to the CD4 population) in untransduced T cell C0 and genetically modified cell (GMC) CD123R CAR T cells in 10 experiments based on 10 different donors. Overall, there was no significant difference in the re-division of the two populations (CD4 and CD8) in transduced and untransduced cells (ns, p = 0.57), see Fig.14 A. Fig.14 Panel B shows a representative example of flow cytometric analysis of memory T cell phenotype based on the expression of CD45RA and CD45RO as well as CD95 and CCR7, see Fig.14 B. Fig.14 Items C and D of show the re-division of CD4 and CD8 T subsets in CD123R CAR T cells (GMC) compared with C0 for three donors, respectively. TEFF: effector T cells; TEM: effector memory T cells; TCM: central memory T cells; TSCM: memory T cell stem cells.

[0229] Figure 15 shows a schematic diagram of co-culturing target cells with the chimeric antigen receptor of the present invention and untransduced cells, as well as flow cytometry graphs associated with the specific cytotoxicity of the CAR of the present invention against BPDCN cell lines and BPDCN patient-derived xenograft cells (also against CD123-negative cells, showing targeting specificity).

[0230] More generally, Figure 15 shows the specific cytotoxicity of CAR of the present invention to BPDCN cell lines and BPDCN patient-derived xenograft cells. For each graph, the mean and SD are indicated.

[0231] Item A of Figure 15 shows a general schematic diagram of co-culturing C0 and CD123 CAR T cells with BPDCN cells on the left, followed by flow cytometry results on the right. To evaluate the cytotoxicity of CD123 CAR T cells (e.g., the ability to induce apoptosis of target cells), target cells were co-cultured with effector T cells (C0 or CD123 CAR) at an E:T ratio of 10:1 (CD123R CAR) or 5:1 (CD123L CAR) or an E:T ratio of 1:0 (control) alone for 16 hours (PDX) or 24 hours (CAL-1, GEN2.2, DAUDI, REH), see Figure 15A. BPDCN cell death can be detected using eDye cell proliferation targeting T cells and 7-AAD staining. Live target cells are 7-AAD - CD123 + (gray). By comparing the viable target cells (7AAD - / CD123 + ) to calculate the percentage of consumption. Since the T lymphocytes used for CAR production are allogeneic to the target cells, cytotoxicity (related to the allogeneity of T cells) is obtained under C0 culture conditions. This is compared with the cytotoxicity obtained in CAR CD123 culture. The specific cytotoxicity under C0 or CD123 CAR conditions represents the % of target cell death compared to the percentage of death under the condition of a single target.

[0232] Item B of Figure 15 shows the expression of CD123 in BPDCN cell lines (CAL-1 and GEN 2.2) detected by flow cytometry, Item C of Figure 15 shows the specific cytotoxicity of CD123R CAR T cells to CAL-1 and GEN 2.2 after co-culture at an E:T ratio of 10:1 (CAL-1, n=22 and GEN 2.2, n=10), Item D of Figure 15 shows the expression of CD123 in BPDCN PDX cells detected by flow cytometry, and Item E of Figure 15 shows the cytotoxicity of CD123 CAR T cells to the indicated CD123+PDX cells (n=9 for P127B-M5 and n=6 for P25B-M1). CD123R CAR T cells exhibited potent antigen-specific cytotoxicity when cultured with the following cell lines: CAL-1 cell line (69.1% ± 27.4 depletion when cultured with CD123R CAR, and 22.2% ± 11.8 depletion when cultured with C0, n = 22, p < 0.0001); GEN2.2 cell line (65.7% ± 26.4 when cultured with CD123R CAR, and 25.3% ± 12.2 when cultured with C0, n = 10, p = 0.0004), and PDX cells (68% ± 22.2 when cultured with CD123R CAR, and 13% ± 29.5 when cultured with C0, n = 15), see Figures 15B, 15C, 15D, and 15E.

[0233] 15 F shows the absence of CD123 expression in DAUDI and REH cell lines by flow cytometry on the left, and the percentage of viability of these cell lines (DAUDI, n=6 and REH, n=4) after co-culture with C0 and CD123 CAR on the right. - The cell lines DAUDI and REH were not cytotoxic, see Figure 15F.

[0234] Item G of FIG. 15 shows a graph of the percentage of cell death when CAL-1 was incubated with B4D5 antibody for 1 h before co-culturing with C0 or CD123R CAR T cells.

[0235] The specific cytotoxicity of CD123R CAR T cells was confirmed by pre-incubating CAL-1 cells with B4D5 CD123 antibodies (AB1). The cytotoxicity of CD123R CAR T cells induced by pre-incubation was inhibited, n = 5. Inhibition of CARCD123 cytotoxicity resulted in a death level similar to that observed with C0 using CD123R CAR (27.2% ± 13.7 for CD123CAR and 17.2% ± 11 for C0, (ns, p = 0.2368, n = 5), see Figure 15G). In fact, CD123R CAR T cells showed in vitro inhibition of CD123 + Specific cytotoxicity to leukemic cells.

[0236] Item H of Figure 15 shows the specific cytotoxicity of CD123L CAR to CAL-1 (E:T=10:1, n=3 or E:T=5:1, n=5), GEN2.2 (E:T=5:1, n=3) and 2 PDXs (E:T=5:1, P127B-M3, n=3 and F13-CAL-1-M2, n=3).

[0237] The cytotoxicity assay was validated using CD123L CAR at E:T = 10:1 and 5:1 ratios, see Figure 15H. For CAL-1, when the E:T ratio was equal to 5:1, a depletion of 94.6% ± 6.1 with CD123L CAR was observed, while the depletion with C0 was 16.6% ± 18.7 (p < 0.0001, n = 5); for GEN 2.2: the depletion of CD123L CAR was 96.7% ± 4.9, while the depletion of C0 was 7% ± 11.9 (p = 0.0032, n = 3); for PDX: the depletion of CD123L CAR was 87.5% ± 6.8, while the depletion of C0 was 21.2% ± 22.7 (p < 0.0001, n = 6), see Figure 15H.

[0238] Item I of FIG. 15 shows that after co-culture with C0 and CD123L CAR T cells, CD123 - The viability percentage of the cell lines (DAUDI, n=3 and HL-60, n=4) was also not observed for CD123 - Toxicity of cell lines HL-60 and DAUDI (viability of HL-60 was 73.8±7.3 with CD123L CAR, while viability was 72.5±15.5 with C0, n=4; viability of DAUDI was 93±9.9 with CD123L CAR, while viability was 94.1±8.4 with C0, n=3), see Figure 3 I.

[0239] Fig.16 The results show that the CD123R CAR T cells of the present invention have a positive correlation with CD123 + Flow cytometry plots associated with functional characterization in target cell co-cultures.

[0240] Fig.16 Panel A shows a representative degranulation assay detected by CD107a expression of CD123R CAR after co-culture with CAL-1 cell line at an E:T ratio of 10:1 for 6 hours compared to untransduced cells (C0). Fig.16 Item B shows a histogram representing the mean + / - 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 cell degranulation is due to its expression of a certain amount of CD107a (CD123RCAR is 10.7% ± 7.6, while C0 is 2.4 ± 1.3, p = 0.0253, n = 6), see Fig.16 A and Fig.16 B.

[0241] Fig.16 Panel C shows staining of the intracellular cytokine IFN-γ after 24 hours of co-culture with CAL-1. Fig.16 The D item shows a histogram representing the mean + / - SD of 3 experiments, *p = 0.0314. CD123R CAR T cells secrete IFN-γ in the intracellular compartment (11% ± 4.7 for CD123R CAR and 2% ± 0.7 for C0, p = 0.0314, n = 3) ( Figure 4 C&D), indicating CD123R CAR T cell activation, see Fig.16 C and Fig.16 D.

[0242] Fig.16 Item E shows a representative cytokine microbead array human Th1 / Th2 / Th17 analysis in the co-culture supernatant (from top to bottom: IL-2; IL-6; IL-4; IL-10; TNF; IFN-γ; IL-17a-data are displayed in color, where red = IL-2; orange = IL-6; yellow = IL-4; green = IL-10; blue = TNF; dark blue = IFN-γ; purple = IL-17a). Higher levels of IFN-γ and IL10 (CBA analysis) were found in the supernatant of co-culture with CD123-CAR (IL-10: CD123RCAR is 91.4pg / mL ± 19.4, relative to C0 is 31.6 ± 6.8, n = 5), see Fig.16 E.

[0243] Fig.16The F column shows a histogram representing the mean of IFN-γ secretion by C0 and CD123R CAR after co-culture with different target cells, n = 3. A significant increase in IFN-γ secretion was observed (288pg / mL ± 70 for CD123 CAR, 147pg / mL ± 67 for C0, n = 5). This secretion is specific because CD123R CAR T cells have a high secretion rate when co-cultured with CD123 - The DAUDI cell line does not secrete IFN-γ when co-cultured, see Fig.16 F.

[0244] Fig.16 Item G shows a graph of IFN-γ secretion under conditions of inhibition of IFN-γ secretion measured by CBA analysis of CD123R CAR after co-culture with CAL-1 pre-incubated with B4D5 antibody for 1 hour, n = 5. When CAL-1 cells were pre-incubated with the CD123 B4D5 antibody of the present invention, IFN-γ production by CD123R CAR T cells was reduced to the level of cytotoxicity observed using C0, see Fig.16 G. IL10 also increased, but at a lower level (91pg / mL±19 for CD123 CAR and 31±6 for C0, n=5), see Fig.16 E.

[0245] Fig.17 Shown are a schematic diagram of the xenograft model, bioluminescent imaging and graphs related thereto, and flow cytometry graphs related to the in vivo anti-tumor activity of the T cells of the invention against the BPDCN cell line (CAL-1) and primary derived xenograft cells (PDX).

[0246] More generally, Fig.17 The in vivo anti-tumor activity of the CD123L CAR T cells of the present invention against CAL-1 and PDX (primary derived xenografts or patient-derived xenografts) is shown.

[0247] Fig.17 A shows a general schematic diagram of the xenograft model. The model is based on 6-8 week old NSG mice that are transfected with the CAL-1 luciferase cell line (1·10 6 ) or PDX (2·10 6 ) were irradiated (2.5 Gy) one day before intravenous injection. In the CAL-1 model, hCD45 was detected in blood samples on day 3 or when flow cytometry was performed. + / CD123 + When BPDCN cells were used, C0 T cells or T cells containing the CD123L CAR of the present invention were injected into the tail vein (5 or 10×10 6 / mice), see Fig.17 A.

[0248] Fig.17 Panel B shows images of bioluminescence imaging analysis before and during 33 days of CAR anti-CD123 or C0 treatment. Cross marks indicate dead mice. Fig.17 Item C shows a quantitative (cts / s) graph of bioluminescence detected in mice treated with C0 (dashed line) and CD123L CAR (solid line). Compared with CD123L CAR, the luminescence reflecting leukemic infiltration in mice treated with C0 was higher. This demonstrates the ability of the CD123L CAR of the present invention to control and / or treat BPDCN. The total survival of mice treated with one dose of CD123L CAR showed high survival compared to mice treated with C0 (41 compared to 26, 5 mice / group, p=0.0017, log-rank test), see Fig.17 B and Fig.17 C.

[0249] Fig.17 Item D of shows a mouse overall survival analysis graph. Obviously, the survival of mice treated with the CAR of the present invention was significantly improved compared with mice treated with C0 (n=5 mice / group). The CD123L CAR of the present invention controls BPDCN in a dose-dependent manner. This is supported by the fact that the dose of 10×10 6 The CD123L CAR ratio dose was 5×10 6 The CD123L CAR induced better mouse survival. In addition, it was supported by lower tumor burden (for 5×10 6 T cells is 41, while for 10×10 6 T cells were 55, 5 mice / group, p = 0.0173, log-rank test), see Fig.17 D.

[0250] Using CD123R CAR, the retroviral form of the present invention, further verified Fig.17 The same previous experiments were relevant.

[0251] Accordingly, Fig.18 Xenograft models, bioluminescent imaging and graphs thereof are shown relating to the in vivo anti-tumor activity of the T cells of the present invention against the BPDCN cell line (CAL-1).

[0252] More generally, Fig.18 The in vivo antitumor activity of CD123R CAR T cells against CAL-1 is shown. Fig.18 Panel A shows a general schematic diagram of the xenograft model: CAL-1 luciferase (1×106 NSG mice were irradiated (2.5 Gy) one day before the first day of the experiment. On the third day, C0 and GMC (10×10 6 ), see Fig.18 A. Fig.18 Item B shows bioluminescent imaging images before and after CD123R CAR T cells or C0 treatment after CAL-1 luciferase transplantation, see Fig.18 B. Fig.18 Item C of Figure 3 shows a mouse overall survival analysis graph. Survival analysis showed that mice treated with CD123R CAR had significant survival (n=5 mice) compared to mice treated with C0.

[0253] Therefore, the CAR of the present invention effectively treats BPDCN.

[0254] Further examination revealed Fig.17 The CAR lentiviral format of the present invention is shown.

[0255] More specifically, the results were confirmed in PDX models, see Fig.17 A.

[0256] Fig.17 Figure E shows a representative example of blood immunostaining performed during C0 or CD123L CAR therapy. Human T cells and primary BPDCN cells can be distinguished based on hCD45 expression. C0 (hCD45 高 / CD123 - / CD3 + CD19 - ) or CD123L CAR (hCD45 高 / CD123 - / CD3 + CD19 + ) in gray (data also shown in color: blue); BPDCN cells (hCD45 低 / CD123 + ) in black (data are also shown in color: pink). Fig.17 Panel F shows a graph of mean BPDCN cell counts in the blood of mice injected with LAN PDX during treatment with C0 (dark line, or blue line when data are displayed in color) or CD123 CAR (gray line or red line when data are displayed in color). Fig.17Item G shows a graph showing the overall survival of PDX xenograft mice. Compared with mice treated with C0, mice treated with CD123L CAR had higher survival (p=0.01). More specifically, in the PDX F13-LAN model, BPDCN cells were detected in all mouse blood samples 78 days after injection: one group received 5×10 6 CD123L CAR (mean number of BPDCN cells: 24 mother cells / μL [1-56], n = 8), while the other group received 5×10 6 The C0 of 26 blasts / μL [1-66], n=8. The mice were monitored during 25 days by blood analysis using absolute count (trucount) analysis. On day 7, BPDCN cells and T cells in mouse blood samples were quantified, see Fig.17 E and Fig.17 F. During follow-up, treatment with CD123L CAR was shown to control the number of circulating BPDCN cells, see Fig.17 C, and prolonged the overall survival of mice (31 days for CD123L CAR treatment and 20 days for C0 treatment, P = 0.0103), see Fig.17 G. Overall, the results obtained in the mouse model using BPDCN cell lines and PDX cells showed that CAR CD123L according to the present invention can control BPDCN in vivo.

[0257] The CAR retroviral format of the invention has been demonstrated in experiments similar to the lentiviral format shown.

[0258] Fig.19 Shown are fluorescence images associated with CD123 expression in different cell types, immunohistochemical images of CD123 expression in tumors and normal tissues, flow cytometry images associated with CD123 expression, schematic diagrams of the generation of autologous T cells of the present invention, and flow cytometry images of CD123-associated toxicity of T cells of the present invention to cells expressing CD123 at low levels.

[0259] More generally, Fig.19 Shown is an evaluation of the toxicity of the CD123L CAR T cells of the present invention to cells expressing CD123 at low levels.

[0260] It was shown above that anti-CD123 antibodies (B4D5 of the present invention and commercial antibody 6H6) showed tight staining of CD123, see Fig.10 C. Accordingly, the 6H6 antibody was used to compare CD123 expression on normal hematopoietic and endothelial cell lines (HMEC) and compared with BPDCN.

[0261] Fig.19 Item A of FIG shows a graph of CD123 expression in different cell lines. The expression of CD123 was studied using the 6H6 commercial antibody to compare BPDCN (n=60) with lymphocytes (n=31), monocytes (n=22), normal pDC (n=30), CD34 cells in normal bone marrow (n=16), and endothelial HMEC cell lines (n=4) in blood and bone marrow samples. The results are expressed as MFI, see Fig.19 A. Compared with BPDCN cells (MFI = 11677 [2962-33439], n = 60) and normal pDC (MFI = 34352 [17822-76610], n = 30), low expression was detected on 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). The CD123 CAR of the present invention is effective in vivo for CD123 低 The cytotoxicity of cells is lower than that of CD123 高 Cytotoxicity in BPDCN cells.

[0262] In order to evaluate the expression of CD123 on tissue, AB1 (or 18B4D5 antibody) of the present invention is used to carry out tissue microarray.For this reason, the frozen tissue array of the U.S. Food and Drug Administration standard is used, including 90 tissue cores (30 kinds of organs) of 3 individual donors of each organ (US Biomax, Rockville, MD, USA).Use UltraView universal DAB detection kit (Ventana, USA) to detect immunostaining.Images are collected and analyzed with NDP.view 2.0 software.Staining intensity is graded as follows: negative (0), weak staining (1+), moderate (2+), positive (3+), strong (4+).High CD123 (Cal-1) or negative (Daudi) expression cell lines are used as positive or negative controls, respectively.

[0263] Table 4 shows the results of tissue microarray.

[0264]

[0265]

[0266] Table 4: Tissue microarray results.

[0267] The results showed that CD123 was not significantly expressed on specific tissue cells, and only some endothelial cells in lymph nodes, stomach, colon, muscle, esophagus, skin and thyroid expressed CD123.

[0268] Fig.19 Item B shows immunohistochemical images using the B4D5 antibody on tumor tissue (BPDCN) and normal tissue. In order to compare the levels of CD123 expression on endothelial cells in these tissues with the levels of CD123 expression on BPDCN, IHC was performed on 3 normal tissues using the AB1 (18B4D5) antibody of the present invention. The expression was compared with the expression levels obtained on the skin lesions of BPDCN patients (n=2). High levels of CD123 expression were obtained on the skin lesions of 2 patients, as well as low expression on the endothelial cells of the tested tissues, see Fig.19 B. More specifically, BPDCN patients 1 and 2 showed high expression of CD123 in the dermal blastic infiltration of BPDCN cells. CD123 immunostaining of different healthy tissues showed low levels of CD123 expression on endothelial cells (arrows). Specific tissue cells were CD123 negative. Low nonspecific labeling was found in gastric and thyroid secretions.

[0269] Both methods showed that monocytes, CD34 + The low level of CD123 on cells and endothelial cells indicates that the "on target off tumor" potential of the CAR CD123 of the present invention is low.

[0270] Fig.19 Item C shows representative expression of CD123 K532. More specifically, representative expression of CD123 on live K562 CD123 cell lines cultured alone or with C0 or CD123 CAR at an E:T ratio of 5:1 for 24 hours is shown. CD123 - The region is derived from the natural K562 line (which is CD123 - ) definition, CD123 低 Regions correspond to maximal expression on monocytes and HMEC cells. CD123 阳性 represents the expression level of CAL-1 and CD123 高 Represents a level higher than CAL-1. This modeling shows the relationship between the expression level of CD123 on target cells and the killing efficiency of CD123L CAR, using K562 cell lines expressing different levels of CD123 (K562 CD123). As described, the cell lines were co-cultured with C0 or CD123 CAR or cultured alone. According to the CD123 expression level of K562 cells, i.e., CD123 - 、CD123 低 (corresponding to the highest expression of CD123 on monocytes and endothelial HMEC cells), CD123 + / 高(corresponding to the expression level of CD123 on CAL-1 and higher), compare the persistence percentage of K562 cells in the whole population, see Fig.19 C.

[0271] Fig.19 D shows the percentage of cells in each subgroup of K562. When cells were cultured with CAR CD123, K562 CD123 低 The percentage of cells did not decrease, while K562 CD123 阳性 / 高 The cells disappeared; K562 CD123 - More specifically, the distribution of K562 cells associated with each expression level was the same when cultured alone or co-cultured with C0, whereas the proportion of positive / highly positive CD123 cells was significantly reduced when K562 CD123 was co-cultured with CD123L CAR (n=3, p=0.0001) (thus, the percentage of CD123- cells increased), see Fig.19 D. Moreover, for CD123 低 The percentage of cells persisting was similar between cells cultured with C0 or CD123L CAR, suggesting that CD123L CAR has a significant effect on CD123 低 The cells have no significant toxicity. This demonstrates the ability of the CD123L CAR of the present invention to kill CD123 high-expressor cells, while having no effect or little effect on weak-expressor cells.

[0272] Fig.19 Item E of shows a graph related to inhibition of target cell proliferation. The cytotoxicity of CD123L CAR T cells to endothelial cell line HMEC and primary monocytes was evaluated. Proliferation inhibition after 18 hours of co-culture was evaluated using a proliferation test (MTT cell proliferation test). The percentage of cell growth inhibition is provided. CAL-1 was used as a CD123 + Positive control, and DAUDI was used as CD123 - Negative control, see Fig.19 E.

[0273] Fig.19 Item F shows the + Schematic representation of the experimental generation of autologous CD123L CAR T cells in transplanted humanized mice. Fig.19Item G shows the toxicity graph of CD123L CAR to CD34 cells. Two sources of CD34 cells were used. CD123L CAR T cells were generated from humanized mice. In addition, umbilical cord blood was used to generate CD123L CAR T cells, which were cultured with CD34+ cells sorted from the same umbilical cord blood, **p=0.0019. Fig.19 Panel H shows the expression of CD123 in CD34 from mouse bone marrow after co-culture with autologous CAR T cells or C0. + Representative expression on cells. CD123LCAR induces CD123 低 The low lysis level of HMECs expressing CD123L demonstrates the potential low toxicity of CD123L CAR to endothelial cells. + Cells, so the in vitro cytotoxicity of CD123L CAR T cells was studied using the above two strategies. First, umbilical cord blood sorted T cells were transduced to produce CD123L CAR or C0 T cells and co-cultured with autologous umbilical cord blood sorted CD34+ cells; no CD34+ cell depletion due to CD123L CAR was observed (CD123LCAR depletion was 5.7% ± 5.1, and C0 depletion was 11.7% ± 10.2, n = 3, p = 0.41), see Fig.19 G. Second, human T cells sorted from humanized mice are used to generate CD123L CAR or C0, which are co-cultured with autologous CD34 cells, see Fig.19 F. Fig.19 G and Fig.19 H. Co-culture with CD123L CAR T cells or C0 cells resulted in low depletion of CD34+ cells (12.7% ± 16.3 for CD123L CAR and 2.7% ± 3.1 for C0, (ns, p = 0.3542, n = 3)), see Fig.19 G. Focus on CD34 + / CD123 + Subpopulations (representing 38% and 16% of CD34+ cells, n=2) were observed with only CD34 + / CD123 + Cells were killed by CD123L CAR (82.1% ± 0.6 depletion), while CD34 + / CD123 - The cells remained viable (consumption was 3.5% ± 4.9), see Fig.19 G and Fig.19 H.

[0274] In addition, the reactivity of CD123L CAR to autologous hematopoietic cells in vivo was studied in the above humanized mouse model. Human CD123 CAR T cells or C0 were generated from human T cells. The transduction efficiency was 55.4% ± 9.2 (n = 3), and the efficiency of this CD123L CAR T cell was verified in vitro before the mouse was reinfused.

[0275] Fig. 20 Figures related to the evaluation of the toxicity of CD123L CAR T cells from humanized mice to CAL-1 and HL-60 based on flow cytometry are shown. More specifically, the toxicity of CD123L CAR T cells obtained from humanized mice to autologous CAL-1 and CD123 negative HL-60 cells is shown. The depletion of CAL-1 cells when using CAR CD123 was 98.6% (± 0.5%, n = 3), while it was only 0.7% (± 0.6, n = 3) when using C0, and it was observed that C0 and CD123L CAR had no significant effect on CD123. - There were no differences between the depletions of the HL-60 cell lines.

[0276] Fig.19 Item I shows a representative example of the in vivo toxicity of CD123L CAR in humanized mice. For each mouse, the fold change (FC) of the human hematopoietic population (blood: B and T lymphocytes, monocytes, pDC) between D-5 and D15 was measured. There was no significant difference in the human immune cell population (T and B lymphocytes, monocytes, pDC, basophils) between the two groups of mice (C0 and CD123L CAR). Taking the first day -5 as a reference, the different immune active cell subsets (total hCD45 + Fold changes of 447 cells (n=24), 45 cells (n=24), 29 cells (n=3), 51 cells (n=10), 28 cells (n=29). Cell counts were performed from peripheral blood samples collected retro-orbitally, and a representative example is shown, n=3.

[0277] Fig.19 Item J shows the percentage of CD34+ cells in the bone marrow of humanized mice 15 days after injection of C0 or CD123L CAR T cells, n = 3 donors. Briefly, the FC of mice injected with CD123L CAR and mice injected with C0 were compared, see Fig.19 I and Fig.19 J.

[0278] In three experiments, no differences in the number of T and B lymphocytes, monocytes, and pDCs were observed between the two groups of mice (C0 and CD123L CAR). Bone marrow CD34 was analyzed only at D15. +cells and compared between mice injected with C0 and CD123 CARL (n=3): no expression of human CD45 + A significant decrease in the percentage of CD34 cells is seen in Fig.19 J.

[0279] These data confirm that the CD123L CAR of the present invention is effective for CD123 低 Cytotoxicity to hematopoietic cells was absent or at least very low. Furthermore, the data also confirmed the effects of CD123 + In vivo, no effects on circulating monocytes, lymphocytes, and pDCs, as well as on bone marrow CD34 + Significant impact on cells.

[0280] Fig.21 Flow cytometry plots related to specific cytotoxicity induced by CD123 CAR T cells from BPDCN patients on autologous BPDCN blasts are shown. The results showed that the patient's autologous CD123L CAR T cells successfully eliminated primary BPDCN blasts in vitro without cytotoxicity to normal blood cells (B lymphocytes and T lymphocytes).

[0281] Fig.21 Item A of shows a graph from a flow cytometry phenotyping assay, where the phenotype CD3 / CD19 of untransduced T cells (C0) is compared to the transduced CD123L CAR T cells of the present invention. For one BPDCN patient, effective CD123 CAR T cells and C0 were generated using autologous T cells. After 9 days, a transduction efficiency of greater than 79.2% was achieved, see Fig.21 A. This efficiency is similar to that achieved in healthy donor T cells, see Fig.12 B.

[0282] Fig.21 Panel B shows the use of biotin-conjugated CD123 protein and streptavidin PE Flow cytometry plot of CD123 CAR cell surface expression. This is a control for CD123 CAR cell surface expression using biotin-conjugated CD123 protein, see Fig.21 B.

[0283] Fig.21 Panel C shows a plot of the patient negative fraction after CD3 / CD28 magnetic selection / activation. Grey data (pink in colored data) represent blasts (CD45 低 、CD3 - 、CD19 - 、CD303 + 、CD123高 ), dark gray (blue in the color data) represents T lymphocytes (CD45 高 、CD3 + 、CD19 - 、CD303 - 、CD123 - ), light grey (orange in the color data) represents B lymphocytes (CD45 高 、CD3 - 、CD19 + 、CD303 - 、CD123 - ). More specifically, the functional activity of autologous CAR T cells against patient BPDCN blasts was tested. The blast fraction was assembled from the negative fraction of T cell selection. It consisted of BPDCN blasts (grey / pink population: CD45 低 / CD3 - / CD19 - / CD303 + / CD123 高 ), T cells (blue / dark gray group: CD45 高 / CD3 + / CD19 - / CD303 - / CD123 - ) and B cells (orange / light grey population: CD45 高 / CD3 - / CD19 + / CD303 - / CD123 - ) composition, see Fig.21 C. CD123L CAR and C0 were co-cultured with this autologous blast fraction at an E:T ratio of 5:1 for 24 hours.

[0284] Fig.21 Item D shows a flow cytometry graph and a cell viability graph. It shows the specific cytotoxicity of CD123L CAR T cells to autologous BPDCN blasts after 24h of co-culture at an E:T ratio of 5:1 (orange = B cells; pink = BPDCN blasts; blue = T cells, gray = C0 or CD123L CAR). More specifically, 95% of BPDCN blasts (CD123 + ) is cleared, but has no cytotoxicity to CD123 cells (T cells and B cells). Since patients do not have monocytes in the blood, it is impossible to evaluate the toxicity of autologous monocytes. However, taken together, these results show that the autologous CD123L CAR T cells of the present invention selectively and effectively clear BPDCN mother cells in vitro.

[0285] As mentioned above, BPDCN is a clonal disease that progresses to an aggressive leukemia in most cases, and the prognosis is very poor. Improved treatment methods are needed. Although BPDCN cells are sensitive to chemotherapy, patients still relapse and become resistant to second-line treatment. The present invention provides targeted therapies for CD123, which is a "key antigen" highly expressed in BPDCN patients. More specifically, the present invention provides a BPDCN treatment method using a therapy based on a specifically designed chimeric antigen receptor (CAR). The present invention can selectively target and kill BPDCN cells in patients. Moreover, the present invention provides long-term remission of BPDCN.

[0286] Accordingly, the present invention provides genetically engineered T cells of third-generation CARs with specific recognition and / or specific binding to CD123. The CAR of the present invention is constructed using a retroviral or lentiviral strategy. Both retroviral and lentiviral strategies can mediate anti-tumor activity. T cells expressing the CD123CAR of the present invention can specifically lyse BPDCN cells. More specifically, T cells expressing the CD123CAR of the present invention can specifically lyse different BPDCN targets, i.e., BPDCN cell 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 targets, cells activate effector functions in vitro, such as IFN-γ release and CD107a degranulation. The functionality of the CAR of the present invention is specific to the CD123 expression of target cells. In fact, when the expression of CD123 is blocked by pre-incubation with the anti-CD123 antibody of the present invention (such as AB1), both cytotoxicity and cytokine secretion are reversed. In addition, a surprising effect of the present invention is that when the CAR of the present invention is cultured with CD123 - When cell lines (Daudi, REH, HL-60) are co-cultured, cytotoxicity (or substantially no) and / or cytokine secretion (or substantially no) does not occur. In vivo, T cells expressing the CAR of the present invention show anti-leukemia activity against the BPDCN line. This positive effect of the present invention provides long-term survival for subjects (e.g., mice) affected by BPDCN.

[0287] The prior art discloses agents targeting CD123, i.e., antibodies [see Li F, Sutherland MK, Yu C, Walter RB, Westendorf L, Valliere-Douglass J et al., Characterization of SGN-CD123A, A Potent CD123-Directed Antibody-Drug Conjugate for Acute Myeloid Leukemia, Molecular cancer therapeutics, 2018, 17(2): 554-64; Xie LH, Biondo M, Busfield SJ, Arruda A, Yang X, Vairo G et al., CD123 target validation and preclinical evaluation of ADCC activity of anti-CD123 antibody CSL362 incombination with NKs from AML patients in remission, Blood cancer journal, 2017, 7(6): e567; and Kovtun Y, Jones GE, Adams S, Harvey L, Audette CA, Wilhelm A et al., A CD123-targeting antibody-drug conjugate, IMGN632, designed to eradicate AML while sparing normal bone marrow cells, Blood advances, 2018, 2(8):848-58], fusion protein [Frankel AE, Woo JH, Ahn C, Pemmaraju N, Medeiros BC, Carraway HE et al., Activity of SL-401, a targeted therapy directed to interleukin-3receptor, in blasticplasmacytoid dendritic cell neoplasm patients, Blood, 2014; 124(3):385-92], BiTes [Pizzitola I, Anjos-Afonso F, Rouault-Pierre K, Lassailly F, Tettamanti S, Spinelli O, et al;Chimeric antigen receptors against CD33 / CD123 antigens efficiently target primary acute myeloid leukemia cells in vivo, Leukemia, 2014, 28(8):1596 - 605; Chu SY, Pong E, Chen H, Phung S, Chan EW, Endo NA et al., Immunotherapy with Long-Lived Anti-CD123×Anti-CD3 Bispecific Antibodies Stimulates Potent T Cell-Mediated Killing of Human AML Cell Lines and of CD123+ Cells in Monkeys: A Potential Therapy for Acute Myelogenous Leukemia, Blood, 2014, 124(21):2316;and Ruella M, Barrett DM, Kenderian SS, Shestova O, Hofmann TJ, Perazzelli J, et al., Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies, The Journal of clinical investigation, 2016, 126(10):3814-26], DART [Al-Hussaini M, Rettig MP, Ritchey JK, Karpova D, Uy GL, Eissenberg LG, et al., Targeting CD123 in acute myeloid leukemia using a T-cell-directed dual-affinity retargeting platform, Blood, 2016, 127(1):122-31], triple antibodies (triplebodies) or even CAR T cells [Gill S, Tasian SK, Ruella 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, Blood, 2014, 123(15):2343-54;

[0013] and Tettamanti S, Marin V, Pizzitola I, Magnani CF, Giordano Attianese GM, Cribioli E et al., Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor, British journal of haematology, 2013, 161(3): 389-401]. Although some of these studies have shown that anti-CD123 therapeutics are effective to a certain extent for AML, various negative effects on normal hematopoiesis are still reported, see Pemmaraju N, Lane AA, Sweet KL, Stein AS, Vasu S, Blum W et al., Tagraxofusp in Blastic Plasmacytoid Dendritic-Cell Neoplasm, The New England journal of medicine, 2019, 380(17): 1628-37. A recent study of 32 BPDCN patients using SL-401 (IL3 conjugated to diphtheria toxin) as first-line treatment showed that 72% of patients had a complete clinical response and a 52% survival rate at 24 months (including 45% of patients who received an allogeneic transplant). In the 15 patients who had been previously treated, the response rate was 67% and the median overall survival was 8.5 months. However, in this study, 78% of the patients experienced negative side effects (grade 3 or higher), resulting in the death of 3 patients. Therefore, toxicity is a major problem. By addressing this problem, the present invention greatly improves this situation. ;

[0288] The active CAR of the present invention was tested in clinical trials of BPDCN patients (as well as AML patients). No major toxic events and / or adverse effects were reported.

[0289] Tissue microarray (TMA) and immunohistochemistry (IHC) confirmed that when the antibodies of the present invention (e.g., 18B4D5) were used to generate CAR, the expression of CAR in CD123 低 There is no cytotoxicity or cytotoxicity is low in the cell. Moreover, CARCD123L of the present invention does not produce or produces very weak cytotoxicity to HMEC endothelial cell line.

[0290] In vivo data collected from humanized mice injected with the autologous CAR CD123 of the present invention showed that mice treated with the CD123 CAR of the present invention had significant effects on blood subsets (total CD45 + Cells, T and B cells and monocytes) have no significant effect. The prior art reports problems that may be caused by CAR T cells. The so-called cytokine release syndrome (CRS) is common, and studies have shown that monocytes are the main source of IL-1 and IL-6 during CRS, see Giavridis T, van der Stegen SJC, Eyquem J, Hamieh M, Piersigilli A, Sadelain M., CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1blockade, Nat Med, 2018, 24 (6): 731-8; and Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M et al., Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells, Nat Med, 2018, 24 (6): 739-48. The data provided by the present invention lead the inventors to believe that the potential low-level monocyte depletion caused by the CD123 CAR T cells of the present invention in vivo may be involved in limiting CRS (because monocytes express low levels of CD123).

[0291] In order to control the CAR of the present invention and for the purpose of human treatment, one embodiment of the T cell of the present invention includes a suicide gene, namely the system iCASP9 / rimiducid, see Warda W, Larosa F, Neto Da Rocha M, TradR, Deconinck E, Fajloun Z et al. CML Hematopoietic Stem Cells Expressing IL1RAP CanBe Targeted by Chimeric Antigen Receptor-Engineered T Cells, Cancer research, 2019, 79(3): 663-75.

[0292] The present invention also provides anti-CD123 CAR T cells, which are capable of specifically targeting and killing CD123 of BPDCN in vitro and in vivo. + progenitor cells and have normal CD123 低 Safety profile evaluated on cells. The lentiviral strategy allowed good and reproducible transgene expression and good activity in vitro and in vivo. In clinical application and / or clinical development, the lentiviral form of the CAR of the present invention is preferred, see Zufferey R, Donello JE, Trono D, Hope TJ., Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. Journal of virology, 1999, 73(4): 2886-92; Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM., Development of a self-inactivating lentivirus vector, Journal of virology, 1998, 72(10): 8150-7; Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L et al., Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. Journal of virology, 1998, 72(12): 9873-80.

[0293] Accordingly, the objects of the present invention are defined in the appended claims.Another object of the present invention is defined in the appended claims with reference to sequence homology rather than sequence identity.

[0294] Accordingly, an embodiment of the present invention relates to an antibody selected from the group consisting of AB1, AB2, AB3, AB4, AB5 and AB6, wherein

[0295] AB1 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:25, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:13, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:1, and wherein the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:26, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:14, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:2;

[0296] AB2 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:27, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:15, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:3, and wherein the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:28, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:16, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:4;

[0297] AB3 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:29, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:17, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:5, and wherein the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:30, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:18, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:6;

[0298] AB4 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:31, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:19, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:7, and wherein the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:32, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:20, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:8;

[0299] AB5 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:33, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:21, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:9, and wherein the light chain comprises a complementarity determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:34, a complementarity determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:22, and a complementarity determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:10; and

[0300] AB6 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementary determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:35, a complementary determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:23, and a complementary determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:11, and wherein the light chain comprises a complementary determining region 1 (CDR1) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:36, a complementary determining region 2 (CDR2) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:24, and a complementary determining region 3 (CDR3) having at least 90% homology, preferably 100% homology to the amino acid sequence of SEQ ID NO:12.

[0301] 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, 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 anti-CD123 binding domain comprises:

[0302] (i) a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO: 1, and the light chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO: 2;

[0303] (ii) a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO:3, and the light chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO:4;

[0304] (iii) a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO:5, and the light chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO:6;

[0305] (iv) a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO:7, and the light chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO:8;

[0306] (v) a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO: 9, and the light chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO: 10;

[0307] or

[0308] (vi) a heavy chain and a light chain, the heavy chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO:11, and the light chain comprising a complementarity determining region 3 (CDR3) having at least 90% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO:12.

[0309] Another embodiment of the present invention is the above CAR, but with reference to sequence homology rather than sequence identity.

[0310] Another object of the present invention is to include the expression vector of the nucleic acid molecule as defined above, and the engineered immune cell (such as T cell) comprising the nucleic acid molecule as defined above. According to the present invention, the cell can be used as a medicament and / or for treating a proliferative disease in a mammal, preferably in humans, wherein the proliferative disease is a disease associated with CD123 overexpression, preferably blastic plasmacytoid dendritic cell tumor (BPDCN). In addition, the present invention further relates to a pharmaceutical composition comprising an engineered immune cell as defined above. Furthermore, the present invention relates to a method for engineering an immune cell, the method including transfecting cells with an expression vector as defined above.

[0311] Another embodiment of the invention features an engineered immune cell comprising a nucleic acid molecule as described in the claims, wherein the cell is a T cell or a natural killer cell.

[0312] Other definitions of the present invention may include an antibody that specifically binds to the antigen CD123, wherein the antibody is selected from the group consisting of AB1, AB2, AB3, AB4, AB5 and AB6, and wherein

[0313] 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;

[0314] 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;

[0315] 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;

[0316] 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;

[0317] 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; and

[0318] 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;

[0319] and wherein the antibody has a -9 M of K D value, not exceeding 9.0x10 -4 s -1 The antibody dissociation rate k off , and at least 7x10 4 Ms -1 The antibody binding rate k on .

[0320] In one embodiment, the CD123 binding domain is a scFv comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising an amino acid sequence having at least 1, 2 or 3 modifications but not more than 30, 20 or 10 modifications to the amino acid sequence 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 the heavy chain variable region comprises an amino acid sequence having at least 1, 2 or 3 modifications but not more than 30, 20 or 10 modifications to the amino acid sequence of the light 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:14, SEQ ID NO:15 The amino acid sequence of the heavy chain variable region of 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 has at least 1, 2 or 3 modifications but not more than 30, 20 or 10 modifications.

[0321] In another embodiment, the CD123 binding domain is a scFv comprising (i) a light chain variable region comprising a complementarity determining region 1 (CDR1) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical 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, a complementarity determining region 2 (CDR2) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24, and a complementarity determining region 2 (CDR2) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical 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 (ii) a heavy chain variable region comprising a complementary determining region 1 (CDR1) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical 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:22. NO:23 has a complementary determining region 2 (CDR2) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical, and a complementary determining region 3 (CDR3) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical, or 100% identical 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.

[0322] In one embodiment, the anti-CD123 binding domain is connected to the transmembrane domain via a hinge region. Preferably, the encoded CAR comprises the transmembrane domain of CD28.

[0323] In one embodiment, the hinge region comprises the hinge sequence of IgG1, or a sequence with 95-99% identity thereto. In a further embodiment, the hinge region comprises the hinge sequence of IgG4, or a sequence with 95-99% identity thereto. In a further embodiment, the hinge region may also comprise the CH2-CH3 region of IgG1 or IgG4 or CD8α, or a sequence with 95-99% identity thereto.

[0324] In a preferred embodiment, at least one co-stimulatory domain of the functional intracellular signaling domain is obtained from one or more proteins selected from 4.1BB and / or a co-stimulatory domain obtained from CD3ζ. Sequence Listing <110> UNIVERSITÉ BOURGOGNE FRANCHE-COMTÉ <120> New monoclonal anti-CD123 antibody <130> UNIVERSITÉ BOURGOGNE FRANCHE-COMTÉ | CAS 1 FR [122119] <160> 175 <170> BiSSAP 1.3.6 <210> 1 <211> 6 <212> PRT <213> Mouse <220> <223> HC-CDR3 "18B4D5 <400> 1 Ala Arg Gly Leu Asp Tyr 1 5 <210> 2 <211> 9 <212> PRT <213> Mouse <220> <223> LC-CDR3 "18B4D5 <400> 2 His Gln Leu His Arg Ser Pro Trp Thr 1 5 <210> 3 <211> 11 <212> PRT <213> Mouse <Mus, genus> <220> <223> HC-CDR3 "17F7B2 <400> 3 Thr Lys Gly Tyr Arg Tyr Asp Trp Phe Ala Tyr 1 5 10 <210> 4 <211> 9 <212> PRT <213> Mouse <Mus, genus> <220> <223> LC-CDR3 "17F7B2 <400> 4 Ser Gln Asn Thr His Val Pro Phe Thr 1 5 <210> 5 <211> 12 <212> PRT <213> Mouse <Mus, genus> <220> <223> HC-CDR3 "11B6E6 <400> 5 Val Arg Ala His Gly Tyr Tyr Phe Ala Met Asp Tyr 1 5 10 <210> 6 <211> 8 <212> PRT <213> Mouse <Mus, genus> <220> <223> LC-CDR3 "11B6E6 <400> 6 Lys Gln Ser Tyr Asn Leu Phe Thr 1 5 <210> 7 <211> 6 <212> PRT <213> Mouse <220> <223> HC-CDR3 "14G12D7 <400> 7 Thr Arg Gly Val Asp Tyr 1 5 <210> 8 <211> 9 <212> PRT <213> Mouse <220> <223> LC-CDR3 "14G12D7 <400> 8 Leu Gln Phe Ser Gly Tyr Pro Phe Thr 1 5 <210> 9 <211> 12 <212> PRT <213> Mouse <220> <223> HC-CDR3 "11A12C6 <400> 9 Val Arg Gly Asp Gly Tyr Tyr Trp Tyr Phe Asp Val 1 5 10 <210> 10 <211> 8 <212> PRT <213> Mouse <220> <223> LC-CDR3 "11A12C6 <400> 10 Lys Gln Ser Tyr Ser Leu Phe Thr 1 5 <210> 11 <211> 11 <212> PRT <213> Mouse <220> <223> HC-CDR3 "15E11B7 <400> 11 Gly Gly Gly Val Arg Gly Ala Trp Phe Ala Phe 1 5 10 <210> 12 <211> 9 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR3 "15E11B7 <400> 12 Gln His Ser Ala Asp Leu Pro Tyr Thr 1 5 <210> 13 <211> 7 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "18B4D5 <400> 13 Ile Ser Phe Ser Gly Ser Thr 1 5 <210> 14 <211> 3 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR2 "18B4D5 <400> 14 Ser Thr Ser 1 <210> 15 <211> 8 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "17F7B2 <400> 15 Phe Tyr Pro Gly Lys Ser Asp Thr 1 5 <210> 16 <211> 3 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR2 "17F7B2 <400> 16 Lys Val Ser 1 <210> 17 <211> 7 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "11B6E6 <400> 17 Ile Trp Thr Gly Gly Gly Thr 1 5 <210> 18 <211> 3 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR2 "11B6E6 <400> 18 Trp Ala Ser 1 <210> 19 <211> 7 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "14G12D7 <400> 19 Ile Ile Tyr Ser Gly Thr Thr 1 5 <210> 20 <211> 3 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR2 "14G12D7 <400> 20 Ser Thr Ser 1 <210> twenty one <211> 7 <212> PRT <213> Mouse <220> <223> HC-CDR2 "11A12C6 <400> twenty one Ile Trp Thr Gly Gly Gly Thr 1 5 <210> twenty two <211> 3 <212> PRT <213> Mouse <220> <223> LC-CDR2 "11A12C6 <400> twenty two Trp Ala Ser 1 <210> twenty three <211> 8 <212> PRT <213> Mouse <220> <223> HC-CDR2 "15E11B7 <400> twenty three Ile Asn Pro Tyr Asn Asp Tyr Thr 1 5 <210> twenty four <211> 3 <212> PRT <213> Mouse <220> <223> LC-CDR2 "15E11B7 <400> twenty four Leu Ala Ser 1 <210> 25 <211> 9 <212> PRT <213> Mouse <220> <223> HC-CDR1 "18B4D5 <400> 25 Gly Tyr Ser Ile Thr Ser Asp Tyr Thr 1 5 <210> 26 <211> 7 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "18B4D5 <400> 26 Ser Ser Ile Ser Ser Ser Tyr 1 5 <210> 27 <211> 8 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-CDR1 "17F7B2 <400> 27 Gly Tyr Ser Phe Thr Ser Tyr Trp 1 5 <210> 28 <211> 11 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "17F7B2 <400> 28 Gln Ser Leu Val His Ser Asn Gly Ile Thr Tyr 1 5 10 <210> 29 <211> 8 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-CDR1 "11B6E6 <400> 29 Gly Phe Ser Leu Thr Ser Tyr Asp 1 5 <210> 30 <211> 12 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "11B6E6 <400> 30 Gln Ser Leu Leu Asn Ser Ser Thr Arg Lys Asn Tyr 1 5 10 <210> 31 <211> 9 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-CDR1 "14G12D7 <400> 31 Gly Tyr Ser Val Thr Ser Asp Tyr Ala 1 5 <210> 32 <211> 7 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "14G12D7 <400> 32 Ser Ser Val Arg Ser Ser Tyr 1 5 <210> 33 <211> 8 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-CDR1 "11A12C6 <400> 33 Gly Phe Ser Leu Thr Ser Tyr Asp 1 5 <210> 34 <211> 12 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "11A12C6 <400> 34 Gln Ser Leu Phe Ser Ser Ser Ala Arg Lys Asn Tyr 1 5 10 <210> 35 <211> 8 <212> PRT <213> Mouse <Mus, genus> <220> <223> HC-CDR1 "15E11B7 <400> 35 Gly Tyr Ser Phe Thr Gly Tyr Phe 1 5 <210> 36 <211> 10 <212> PRT <213> Mouse <Mus, genus> <220> <223> LC-CDR1 "15E11B7 <400> 36 Lys Ser Val Ser Thr Ser Gly Tyr Asn Tyr 1 5 10 <210> 37 <211> 25 <212> PRT <213> Mouse <Mus, genus> <220> <223> HC-FR1 "18B4D5 <400> 37 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr 20 25 <210> 38 <211> 17 <212> PRT <213> Mouse <Mus, genus> <220> <223> HC-FR2 "18B4D5 <400> 38 Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly 1 5 10 15 Tyr <210> 39 <211> 38 <212> PRT <213> Mouse <Mouse, genus> <220> <223> HC-FR3 "18B4D5 <400> 39 Ser Tyr Asn Pro Ser Leu Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr 1 5 10 15 Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> 40 <211> 42 <212> PRT <213> Mouse <Mouse, genus> <220> <223> HC-FR4 "18B4D5 <400> 40 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala 1 5 10 15 Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser 20 25 30 Ser Val Thr Leu Gly Cys Leu Val Lys Ser 35 40 <210> 41 <211> 25 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR1 "17F7B2 <400> 41 Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser 20 25 <210> 42 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR2 "17F7B2 <400> 42 Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Ala <210> 43 <211> 38 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR3 "17F7B2 <400> 43 Thr Tyr Asn Gln Lys Phe Lys Gly Lys Ala Lys Leu Thr Ala Val Thr 1 5 10 15 Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn Glu Asp 20 25 30 Ser Ala Val Tyr Tyr Cys 35 <210> 44 <211> 21 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR4 "17F7B2 <400> 44 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro 1 5 10 15 Pro Ser Val Tyr Ser 20 <210> 45 <211> 25 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR1 "11B6E6 <400> 45 Gln Val Lys Leu Glu Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser 20 25 <210> 46 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR2 "11B6E6 <400> 46 Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly 1 5 10 15 Val <210> 47 <211> 38 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR3 "11B6E6 <400> 47 Asn Tyr Asn Ser Ala Phe Met Ser Arg Leu Ser Ile Ser Lys Asp Asn 1 5 10 15 Ser Lys Ser Gln Val Phe Leu Lys Met Phe Ser Leu Gln Thr Asp Asp 20 25 30 Thr Ala Ile Tyr Tyr Cys 35 <210> 48 <211> 42 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR4 "11B6E6 <400> 48 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala 1 5 10 15 Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser 20 25 30 Ser Val Thr Leu Gly Cys Leu Val Lys Ser 35 40 <210> 49 <211> 25 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR1 "14G12D7 <400> 49 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr 20 25 <210> 50 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR2 "14G12D7 <400> 50 Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly 1 5 10 15 Tyr <210> 51 <211> 38 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR3 "14G12D7 <400> 51 Ser Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr 1 5 10 15 Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> 52 <211> 21 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR4 "14G12D7 <400> 52 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro 1 5 10 15 Pro Ser Val Tyr Ser 20 <210> 53 <211> 25 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR1 "11A12C6 <400> 53 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser 20 25 <210> 54 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR2 "11A12C6 <400> 54 Ile Asn Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Leu Gly 1 5 10 15 Val <210> 55 <211> 38 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR3 "11A12C6 <400> 55 Asn Tyr Asn Ser Ala Phe Met Ser Arg Leu Ser Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp 20 25 30 Thr Ala Ile Tyr Tyr Cys 35 <210> 56 <211> 21 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR4 "11A12C6 <400> 56 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Pro 1 5 10 15 Pro Ser Val Tyr Ser 20 <210> 57 <211> 25 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR1 "15E11B7 <400> 57 Glu Val Gln Leu Glu Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Asp 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser 20 25 <210> 58 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR2 "15E11B7 <400> 58 Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly 1 5 10 15 Arg <210> 59 <211> 38 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR3 "15E11B7 <400> 59 Phe Asn Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys 1 5 10 15 Ser Ser Ser Thr Ala His Met Glu Leu Leu Ser Leu Thr Ser Glu Asp 20 25 30 Ser Ala Val Tyr Tyr Cys 35 <210> 60 <211> 42 <212> PRT <213> Mouse <Mus musculus> <220> <223> HC-FR4 "15E11B7 <400> 60 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Glu Ala Lys Thr Thr Ala 1 5 10 15 Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser 20 25 30 Ser Val Thr Leu Gly Cys Leu Val Lys Ser 35 40 <210> 61 <211> 26 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR1 "18B4D5 <400> 61 Asp Ile Val Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser 20 25 <210> 62 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR2 "18B4D5 <400> 62 Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile 1 5 10 15 Tyr <210> 63 <211> 36 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR3 "18B4D5 <400> 63 Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 64 <211> 20 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR4 "18B4D5 <400> 64 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 1 5 10 15 Thr Val Ser Thr 20 <210> 65 <211> 26 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR1 "17F7B2 <400> 65 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 20 25 <210> 66 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR2 "17F7B2 <400> 66 Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 67 <211> 36 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR3 "17F7B2 <400> 67 Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly 20 25 30 Val Tyr Phe Cys 35 <210> 68 <211> 20 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR4 "17F7B2 <400> 68 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 1 5 10 15 Thr Val Ser Thr 20 <210> 69 <211> 26 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR1 "11B6E6 <400> 69 Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Arg Cys Lys Ser Ser 20 25 <210> 70 <211> 17 <212> PRT <213> Rat <Mouse, genus> <220> <223> LC-FR2 "11B6E6 <400> 70 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 71 <211> 36 <212> PRT <213> Rat <Mouse, genus> <220> <223> LC-FR3 "11B6E6 <400> 71 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 72 <211> 20 <212> PRT <213> Rat <Mouse, genus> <220> <223> LC-FR4 "11B6E6 <400> 72 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 1 5 10 15 Thr Val Ser Thr 20 <210> 73 <211> 26 <212> PRT <213> Rat <Mouse, genus> <220> <223> LC-FR1 "14G12D7 <400> 73 Glu Ile Val Ile Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser 20 25 <210> 74 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR2 "14G12D7 <400> 74 Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp Ile 1 5 10 15 Tyr <210> 75 <211> 36 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR3 "14G12D7 <400> 75 Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Ala Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 76 <211> 20 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR4 "14G12D7 <400> 76 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 1 5 10 15 Thr Val Ser Thr 20 <210> 77 <211> 26 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR1 "11A12C6 <400> 77 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser 20 25 <210> 78 <211> 17 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR2 "11A12C6 <400> 78 Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 79 <211> 36 <212> PRT <213> Mouse <Mus musculus> <220> <223> LC-FR3 "11A12C6 <400> 79 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Gly Ser Val Gln Ala Glu Asp Leu Ala 20 25 30 Val Tyr Tyr Cys 35 <210> 80 <211> 20 <212> PRT <213> Mouse <Mus, genus> <220> <223> LC-FR4 "11A12C6 <400> 80 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 1 5 10 15 Thr Val Ser Thr 20 <210> 81 <211> 26 <212> PRT <213> Mouse <Mus, genus> <220> <223> LC-FR1 "15E11B7 <400> 81 Asp Ile Val Leu Thr Gln Ser Thr Thr Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser 20 25 <210> 82 <211> 17 <212> PRT <213> Mouse <Mus, genus> <220> <223> LC-FR2 "15E11B7 <400> 82 Methionine, Histidine, Tryptophan, Tyrosine, Glutamine, Glutamine, Lysine, Proline, Glycine, Glutamine, Proline, Proline, Lysine, Leucine, Leucine, Isoleucine 1 5 10 15 Tyrosine <210> 83 <211> 36 <212> PRT <213> Mouse <Mus, genus> <220> <223> LC-FR3 "15E11B7 <400> 83 Asparagine, Leucine, Glutamic acid, Serine, Glycine, Valine, Proline, Alanine, Arginine, Phenylalanine, Serine, Glycine, Serine, Glycine, Serine, Glycine 1 5 10 15 Threonine, Aspartic acid, Phenylalanine, Threonine, Leucine, Asparagine, Isoleucine, Histidine, Proline, Valine, Glutamic acid, Glutamic acid, Glutamic acid, Aspartic acid, Alanine, Alanine 20 25 30 Threonine, Tyrosine, Tyrosine, Cysteine 35 <210> 84 <211> 20 <212> PRT <213> Mouse <Mus, genus> <220> <223> LC-FR4 "15E11B7 <400> 84 Phenylalanine, Glycine, Glycine, Glycine, Threonine, Lysine, Leucine, Glutamic acid, Isoleucine, Lysine, Arginine, Alanine, Aspartic acid, Alanine, Alanine, Proline 1 5 10 15 Threonine, Valine, Serine, Threonine 20 <210> 85 <211> 144 <212> PRT <213> Mouse <Mus, genus> <220> <223> Total HC "18B4D5 <400> 85 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Thr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Phe Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Leu Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 100 105 110 Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys 115 120 125 Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Ser 130 135 140 <210> 86 <211> 118 <212> PRT <213> Rat <Mouse, genus> <220> <223> Shared LC "18B4D5 <400> 86 Asp Ile Val Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Ile Ser Ser Ser 20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Leu His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala 100 105 110 Ala Pro Thr Val Ser Thr 115 <210> 87 <211> 128 <212> PRT <213> Rat <Mouse, genus> <220> <223> Shared HC "17F7B2 <400> 87 Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Phe Tyr Pro Gly Lys Ser Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Gly Tyr Arg Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Ser 115 120 125 <210> 88 <211> 122 <212> PRT <213> Mouse <Mouse, genus> <220> <223> Shared LC "17F7B2 <400> 88 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn 85 90 95 Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Thr 115 120 <210> 89 <211> 149 <212> PRT <213> Mouse <Mus musculus> <220> <223> Shared HC "11B6E6 <400> 89 Gln Val Lys Leu Glu Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Phe Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Arg Ala His Gly Tyr Tyr Phe Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Ser 145 <210> 90 <211> 122 <212> PRT <213> Mouse <Mouse, genus> <220> <223> Shared LC "11B6E6 <400> 90 Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Arg Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Ser Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala Asp Ala Ala Pro Thr Val Ser Thr 115 120 <210> 91 <211> 123 <212> PRT <213> Mouse <Mouse, genus> <220> <223> Shared HC "14G12D7 <400> 91 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Val Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ile Tyr Ser Gly Thr Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Thr Arg Gly Val Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 100 105 110 Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Ser 115 120 <210> 92 <211> 118 <212> PRT <213> Mouse <Mouse, genus> <220> <223> Shared LC "14G12D7 <400> 92 Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Arg Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Leu Gln Phe Ser Gly Tyr Pro 85 90 95 Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala 100 105 110 Ala Pro Thr Val Ser Thr 115 <210> 93 <211> 128 <212> PRT <213> Mouse <Mus musculus> <220> <223> Shared HC "11A12C6 <400> 93 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Asn Trp Ile Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 Ser Arg Leu Ser Ile Ser Arg Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Arg Gly Asp Gly Tyr Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Ser 115 120 125 <210> 94 <211> 121 <212> PRT <213> Mouse <Mus musculus> <220> <223> Shared LC "11A12C6 <400> 94 Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly Glu 1 5 10 15 Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Phe Ser Ser Ser 20 25 30 Ala Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75 80 Gly Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln Ser 85 90 95 Tyr Ser Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Thr 115 120 <210> 95 <211> 149 <212> PRT <213> Mouse <Mus musculus> <220> <223> Shared HC "15E11B7 <400> 95 Glu Val Gln Leu Glu Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Asp 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Phe Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asn Pro Tyr Asn Asp Tyr Thr Phe Asn Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala His 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Gly Gly Gly Val Arg Gly Ala Trp Phe Ala Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Glu Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Ser 145 <210> 96 <211> 121 <212> PRT <213> Mouse <Mus musculus> <220> <223> Shared LC "15E11B7 <400> 96 Asp Ile Val Leu Thr Gln Ser Thr Thr Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Asn Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Ala 85 90 95 Asp Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Thr 115 120 <210> 97 <211> 1533 <212> DNA <213> Human <220> <223> CAR-18B4D5 nucleic acid sequence <400> 97 atgaaactgc ccgtgagact gctggtgctg atgttttgga ttcccgcttc ctcgagcgac 60 ccatacgatg ttccagatta cgccatcctg tctgatgtgc agcttcagga gtcgggacct 120 ggcctggtga aaccttctca gtctctgtcc ctcacctgca ctgtcactgg ctactcaatc 180 accagtgatt atacctgga ctggattcgg cagtttccag gaaacaaact ggagtggatg ggctacataa gcttcagtgg tagcactagc tacaacccat ctctcaaaag tcgactctct atcacccgag acacatccaa gaaccagttc ttcctgcagt tgaattctgt gactactgag 420. cacacagcca catattactg tgcaagaggg ctcgactact ggggccaagg caccactctc acagtctcct caggaggcgg ttcaggcgga ggtggctctg gcggtggcgg atcgataatg 480 540. tccagaggac aaattgttct cacccagtct ccagcaatca tgtctgcatc tcttgggga cgggtcacca tgacctgcac tgccagctca stablett ccagttactt gtactggtac 600 cagcagaagc caggctcctc ccccaaactc tggatttata gcacatccaa cctggcttct ggagtcccag ctcgcttcag tggcagtggg tctgggacct cttactctct cacaatcagc 720 agcatggagg ctgaagatgc tgccacttat tactgccacc agttgcatcg ttccccgtgg 780 acgttcggtg gaggcacca gctggaatc aaagagccca aatcttgtga caaaactcac acatgcccac cgtgccctga tcccaaattt tgggtgctgg tggtggttgg tggagtcctg 900 gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 960 agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1020 aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc 1080 agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1140 gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1200 gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1260 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag gcgtggccgg 1320 gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1380 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1440 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1500 gacgcccttc acatgcaggc cctgccccct cgc 1533 <210> 98 <211> 75 <212> DNA <213> Mouse <Mouse, genus> <220> <223> HC-FR1 "18B4D5" DNA <400> 98 gaggtcaagc tgcaggagtc aggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcact 75 <210> 99 <211> 75 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR1 "17F7B2" DNA <400> 99 gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc cgtgaagatg 60 tcctgcaagg cttct 75 <210> 100 <211> 75 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR1 "11B6E6" DNA <400> 100 caggtcaagc tggaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatt 60 acctgcactg tctct 75 <210> 101 <211> 75 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR1 "14G12D7" DNA <400> 101 gaggttcagc tgcaggagtc tggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcact 75 <210> 102 <211> 75 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR1 "11A12C6" DNA <400> 102 caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatt 60 acctgcactg tctct 75 <210> 103 <211> 75 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR1 "15E11B7" DNA <400> 103 gaggttcagc tggagcagtc tggacctgag ctggtgaagc ctggggattc agtgaagata 60 tcctgcaagg cttct 75 <210> 104 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR2 "18B4D5" DNA <400> 104 tggaactgga tccggcagtt tccaggaaac aaactggagt ggatgggcta c 51 <210> 105 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR2 "17F7B2" DNA <400> 105 atgcactggg taaaacagag gcctgggcag ggtctagaat ggattggtgc t 51 <210> 106 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR2 "11B6E6" DNA <400> 106 ataagctgga ttcgccagcc accaggaaag ggtctggagt ggcttggagt a 51 <210> 107 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR1 "14G12D7" DNA <400> 107 tggaactgga tccggcagtt tcccggaaac aaactggagt ggatgggcta c 51 <210> 108 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR2 "11A12C6" DNA <400> 108 ataaactgga ttcgccagac accaggaaag ggtctggagt ggcttggagt a 51 <210> 109 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR2 "15E11B7" DNA <400> 109 atgaactggg tgaaacagag ccatggaaag agccttgagt ggattggacg t 51 <210> 110 <211> 114 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR3 "18B4D5" DNA <400> 110 agctacaacc catctctcaa aagtcgactc tctatcactc gagacacatc caagaaccag 60 ttcttcctgc agttgaattc tgtgactact gaggacacag ccacatatta ctgt 114 <210> 111 <211> 114 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR3 "17F7B2" DNA <400> 111 acctacaacc agaagttcaa gggcaaggcc aaactgactg ccgtcacatc cgccagtact 60 gcctacatgg agctcagcag cctgacaaat gaggactctg cggtctatta ctgt 114 <210> 112 <211> 114 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR3 "11B6E6" DNA <400> 112 aattataatt cagctttcat gtccagactg agcatcagca aggacaactc caagagccaa 60 gttttcttaa aaatgttcag tctgcaaact gatgacacag ccatatatta ctgt 114 <210> 113 <211> 114 <212> DNA <213> Mouse <Mouse, genus> <220> <223> HC-FR3 "14G12D7" DNA <400> 113 agctacaacc catctctcaa aagtcgaatc tctatcactc gagacacatc caagaaccag 60 ttcttcctgc agttgaattc tgtgactact gaggacacag ccacatatta ctgt 114 <210> 114 <211> 114 <212> DNA <213> Mouse <Mouse, genus> <220> <223> HC-FR3 "11A12C6" DNA <400> 114 aattataatt cagctttcat gtccagactg agcatcagca gggacaactc caagagccag 60 gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatatatta ctgt 114 <210> 115 <211> 114 <212> DNA <213> Mouse <Mouse, genus> <220> <223> HC-FR3 "15E11B7" DNA <400> 115 ttcaacaacc agaagttcaa ggacaaggcc acattgactg tagacaaatc ctcgagcaca 60 gcccacatgg agctcctgag cctgacatct gaggactctg cagtctatta ttgt 114 <210> 116 <211> 120 <212> DNA <213> Mouse <Mouse, genus> <220> <223> HC-FR4 "18B4D5" DNA <400> 116 tggggccaag gcaccactct cacagtctcc tcagccaaaa caacagcccc atcggtctat 60 ccactggccc ctgtgtgtgg agatacaact ggctcctcgg tgactctagg atgcctggtc 120 <210> 117 <211> 64 <212> DNA <213> Mouse <Mouse, genus> <220> <223> HC-FR4 "17F7B2" DNA <400> 117 tggggccaag ggactctggt cactgtctct gcagccaaaa cgacaccccc atctgtctat 60 tcca 64 <210> 118 <211> 126 <212> DNA <213> Mouse <Mouse, genus> <220> <223> HC-FR4 "11B6E6" DNA <400> 118 tggggtcaag gaacctcagt caccgtctcc tcagccaaaa caacagcccc atcggtctat 60 ccactggccc ctgtgtgtgg agatacaact ggctcctcgg tgactctagg atgcctggtc 120 aagtcc 126 <210> 119 <211> 64 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR4 "14G12D7" DNA <400> 119 tggggccaag gcaccactct cacagtctcc tcagccaaaa cgacaccccc atctgtctat 60 tcca 64 <210> 120 <211> 64 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR4 "11A12C6" DNA <400> 120 tggggcgcag ggaccacggt caccgtctcc tcagccaaaa cgacaccccc atctgtctat 60 tcca 64 <210> 121 <211> 127 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR4 "15E11B7" DNA <400> 121 tggggccaag ggactctggt cactgtctct gaagccaaaa caacagcccc atcggtctat 60 ccactggccc ctgtgtgtgg agatacaact ggctcctcgg tgactctagg atgcctggtc 120 aagtcca 127 <210> 122 <211> 78 <212> DNA <213> Mouse <Mouse, genus> <220> <223> LC-FR1 "18B4D5" DNA <400> 122 gatattgtga tgacacagtc tccagcaatc atgtctgcat ctcttgggga acgggtcacc 60 atgacctgca ctgccagc 78 <210> 123 <211> 78 <212> DNA <213> Mouse <Mouse, genus> <220> <223> LC-FR1 "17F7B2" DNA <400> 123 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagt 78 <210> 124 <211> 78 <212> DNA <213> Mouse <Mouse, genus> <220> <223> LC-FR1 "11B6E6" DNA <400> 124 gatattgtgc tgacacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60 atgaggtgca aatccagt 78 <210> 125 <211> 78 <212> DNA <213> Mouse <Mouse, genus> <220> <223> LC-FR1 "14G12D7" DNA <400> 125 gaaattgtga tcacccagtc tccagcaatc atgtctgcat ctccagggga aaaggtcacc 60 atgacctgca gggccagc 78 <210> 126 <211> 78 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR1 "11A12C6" DNA <400> 126 gatattgtga tgacacagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60 atgagctgca aatccagt 78 <210> 127 <211> 78 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR1 "15E11B7" DNA <400> 127 gatattgtgc tgacacagtc tacaacttcc ttagctgtgt ctctgggcca gagggccacc 60 atctcatgca gggccagc 78 <210> 128 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-FR2 "18B4D5" DNA <400> 128 ttgtactggt accagcagaa gccaggatcc tcccccaaac tctggattta t 51 <210> 129 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR2 "17F7B2" DNA <400> 129 ttacattggt acctgcagaa gccaggccag tctccaaagc tcctgatcta c 51 <210> 130 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR2 "11B6E6" DNA <400> 130 ttggcttggt accagcagaa accagggcag tctcctaaac tgctgatcta c 51 <210> 131 <211> 51 <212> DNA <213> Mouse <Mus musculus> <400> 131 ttgcactggt accagcagaa gtcaggtgcc tcccccaaac tctggattta t 51 <210> 132 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR2 "11A12C6" DNA <400> 132 ttggcttggt accagcagag accagggcag tctcctaaac tgctgatcta c 51 <210> 133 <211> 51 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR2 "15E11B7" DNA <400> 133 atgcactggt accaacagaa accaggacag ccacccaaac tcctcatcta t 51 <210> 134 <211> 108 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR3 "18B4D5" DNA <400> 134 aacctggctt ctggagtccc agctcgcttc agtggcagtg ggtctgggac ctcttactct 60 ctcacaatca gcagcatgga ggctgaagat gctgccactt attactgc 108 <210> 135 <211> 108 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR3 "17F7B2" DNA <400> 135 aaccgatttt ctggggtccc agacaggttc agtggcagtg gatctgggac agatttcaca 60 ctcaagatca gcagagtgga ggctgaggat ctgggagttt atttctgc 108 <210> 136 <211> 108 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR3 "11B6E6" DNA <400> 136 actagggaat ctggggtccc tgatcgcttc acaggcagtg gatctgggac agatttcact 60 ctcaccatca gcagtgtgca ggctgaagac ctggcagttt attactgc 108 <210> 137 <211> 108 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR3 "14G12D7" DNA <400> 137 aacttggctt ctggagtccc tgctcgcttc agtggcagtg ggtctgggac ctcttactct 60 ctcacaatca gcagtgtgga ggctgaagat gctgccactt attactgc 108 <210> 138 <211> 108 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR3 "11A12C6" DNA <400> 138 accagggaat ctggggtccc tgatcgcttc acaggcagtg gatctgggac agatttcact 60 ctcaccatcg gcagtgtgca ggctgaagac ctggcagttt attactgc 108 <210> 139 <211> 108 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR3 "15E11B7" DNA <400> 139 aacctcgaat ctggggtccc tgccaggttc agtggcagtg ggtctgggac agacttcacc 60 ctcaacatcc atcctgtgga ggaggaggat gctgcaacct attactgt 108 <210> 140 <211> 60 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR4 "18B4D5" DNA <400> 140 ttcggtggag gcaccaagct ggaaatcaaa cgggctgatg ctgcaccaac tgtatccacc 60 <210> 141 <211> 60 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR4 "17F7B2" DNA <400> 141 ttcggctcgg ggacaaagtt ggaaataaaa cgggctgatg ctgcaccaac tgtatccacc 60 <210> 142 <211> 61 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR4 "11B6E6" DNA <400> 142 ttcggctcgg ggacaaagtt ggaaataaaa cgggctgatg ctgcaccaac tgtatccacc 60 a 61 <210> 143 <211> 60 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR4 "14G12D7" DNA <400> 143 ttcggagggg ggaccaagct ggaaataaaa cgggctgatg ctgcaccaac tgtatccacc 60 <210> 144 <211> 60 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR4 "11A12C6" DNA <400> 144 ttcggctcgg ggacaaagtt ggaaataaaa cgggctgatg ctgcaccaac tgtatccacc 60 <210> 145 <211> 60 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-FR4 "15E11B7" DNA <400> 145 ttcggagggg ggaccaagct ggaaataaaa cgggctgatg ctgcaccaac tgtatccacc 60 <210> 146 <211> 18 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR3 "18B4D5" DNA <400> 146 gcaagagggc tcgactac 18 <210> 147 <211> 33 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR3 "17F7B2" DNA <400> 147 acaaagggct ataggtacga ctggtttgct tat 33 <210> 148 <211> 36 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR3 "11B6E6" DNA <400> 148 gtgagagctc acggctatta ctttgctatg gactac 36 <210> 149 <211> 18 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR3 "14G12D7" DNA <400> 149 acaagggggg ttgactac 18 <210> 150 <211> 36 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR3 "11A12C6" DNA <400> 150 gtaagaggtg atggttacta ctggtacttc gatgtc 36 <210> 151 <211> 33 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR3 "15E11B7" DNA <400> 151 ggaggggggg tacgaggggc ctggtttgct ttc 33 <210> 152 <211> 21 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "18B4D5" DNA <400> 152 ataagcttca gtggtagcac t 21 <210> 153 <211> 24 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "17F7B2" DNA <400> 153 ttttatcctg gaaaaagtga tact 24 <210> 154 <211> 21 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "11B6E6" DNA <400> 154 atatggactg gtggaggcac a 21 <210> 155 <211> 21 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "14G12D7" DNA <400> 155 ataatctaca gtggtaccac t 21 <210> 156 <211> 21 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "11A12C6" DNA <400> 156 atatggactg gtggaggcac a 21 <210> 157 <211> 24 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR2 "15E11B7" DNA <400> 157 attaatcctt acaatgatta tact 24 <210> 158 <211> 27 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR1 "18B4D5" DNA <400> 158 ggctactcaa tcaccagtga ttatacc 27 <210> 159 <211> 24 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR1 "17F7B2" DNA <400> 159 ggctacagct ttaccagcta ctgg 24 <210> 160 <211> 24 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR1 "11B6E6" DNA <400> 160 gggttctcat taaccagcta tgat 24 <210> 161 <211> 27 <212> DNA <213> Mouse <Mus musculus> <220> <223> HC-CDR1 "14G12D7" DNA <400> 161 ggctactcag tcaccagtga ttatgcc 27 <210> 162 <211> 24 <212> DNA <213> Mouse <Mus, genus> <220> <223> HC-CDR1 "11A12C6" DNA <400> 162 gggttctcat taaccagcta tgat 24 <210> 163 <211> 24 <212> DNA <213> Mouse <Mus, genus> <220> <223> HC-CDR1 "15E11B7" DNA <400> 163 ggttactcat ttactggcta cttt 24 <210> 164 <211> 27 <212> DNA <213> Mouse <Mus, genus> <220> <223> LC-CDR3 "18B4D5" DNA <400> 164 caccagttgc atcgttcccc gtggacg 27 <210> 165 <211> 27 <212> DNA <213> Mouse <Mus, genus> <220> <223> LC-CDR3 "17F7B2" DNA <400> 165 tctcaaaata cacatgttcc attcacg 27 <210> 166 <211> 24 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR3 "11B6E6" DNA <400> 166 aagcaatctt ataatctatt cacg 24 <210> 167 <211> 27 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR3 "14G12D7" DNA <400> 167 ctgcagttca gtggttaccc gttcacg 27 <210> 168 <211> 24 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR3 "11A12C6" DNA <400> 168 aagcaatctt atagtctatt cacg 24 <210> 169 <211> 27 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR3 "15E11B7" DNA <400> 169 cagcacagtg cggatcttcc ttacacg 27 <210> 170 <211> 21 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "18B4D5" DNA <400> 170 tcaagtataa gttccagtta c 21 <210> 171 <211> 33 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "17F7B2" DNA <400> 171 cagagccttg tacacagtaa tggaatcacc tat 33 <210> 172 <211> 37 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "11B6E6" DNA <400> 172 cagagtctgc tcaacagtag cacccgaaag aactact 37 <210> 173 <211> 21 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "14G12D7" DNA <400> 173 tcaagtgtac gttccagtta c 21 <210> 174 <211> 36 <212> DNA <213> Mouse <Mus musculus> <220> <223> LC-CDR1 "11A12C6" DNA <400> 174 cagagtctgt tcagcagtag tgcccgaaag aactac 36 <210> 175 <211> 30 <212> DNA <213> Mouse <Mouse, genus> <220> <223> LC-CDR1 "15E11B7" DNA <400> 175 aaaagtgtca gtacatctgg ctataattat 30

Claims

1. An antibody that specifically binds to the antigen CD123, wherein the antibody is selected from the group consisting of AB1 and AB5, and wherein AB1 comprises a heavy chain and a light chain, and wherein 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, and wherein the light chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 26, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 14, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 2; and AB5 comprises a heavy chain and a light chain, and wherein the heavy chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 9, and wherein the light chain comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 34, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:

10.

2. The antibody of claim 1, wherein AB1 comprises a heavy chain variable region having at least 85% identity to the amino acid sequence of SEQ ID NO: 85 and a light chain variable region having at least 85% identity to the amino acid sequence of SEQ ID NO: 86, and wherein the heavy chain variable region 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, And wherein the light chain variable region comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence SEQ ID NO: 26, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence SEQ ID NO: 14, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence SEQ ID NO:

2.

3. The antibody of claim 1, wherein AB5 comprises a heavy chain variable region that is at least 85% identical to the amino acid sequence of SEQ ID NO: 93 and a light chain variable region that is at least 85% identical to the amino acid sequence of SEQ ID NO: 94, and wherein the heavy chain variable region comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 9, And wherein the light chain variable region comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence SEQ ID NO: 34, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence SEQ ID NO:

10.

4. The antibody of claim 1, wherein AB1 comprises a heavy chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 85 and a light chain variable region having at least 90% identity to the amino acid sequence of SEQ ID NO: 86, and wherein the heavy chain variable region 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, And wherein the light chain variable region comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence SEQ ID NO: 26, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence SEQ ID NO: 14, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence SEQ ID NO:

2.

5. The antibody of claim 1, wherein AB5 comprises a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 93 and a light chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 94, and wherein the heavy chain variable region comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 33, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 9, And wherein the light chain variable region comprises a complementarity determining region 1 (CDR1) consisting of the amino acid sequence SEQ ID NO: 34, a complementarity determining region 2 (CDR2) consisting of the amino acid sequence SEQ ID NO: 22, and a complementarity determining region 3 (CDR3) consisting of the amino acid sequence SEQ ID NO:

10.

6. The antibody according to any one of the preceding claims, wherein the heavy chain of AB1 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO: 37, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO: 38, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO: 39, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO: 40, and wherein the light chain of AB1 has a variable region comprising a first framework region (FR1) consisting of the amino acid sequence of SEQ ID NO: 61, a second framework region (FR2) consisting of the amino acid sequence of SEQ ID NO: 62, a third framework region (FR3) consisting of the amino acid sequence of SEQ ID NO: 63, and a fourth framework region (FR4) consisting of the amino acid sequence of SEQ ID NO: 64; and wherein the heavy chain of AB5 has a variable region comprising a first framework region (FR1) consisting of an amino acid sequence of SEQ ID NO: 53, a second framework region (FR2) consisting of an amino acid sequence of SEQ ID NO: 54, a third framework region (FR3) consisting of an amino acid sequence of SEQ ID NO: 55, and a fourth framework region (FR4) consisting of an amino acid sequence of SEQ ID NO: 56, and wherein the light chain of AB5 has a variable region comprising a first framework region (FR1) consisting of an amino acid sequence of SEQ ID NO: 77, a second framework region (FR2) consisting of an amino acid sequence of SEQ ID NO: 78, a third framework region (FR3) consisting of an amino acid sequence of SEQ ID NO: 79, and a fourth framework region (FR4) consisting of an amino acid sequence of SEQ ID NO:

80.

7. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein the anti-CD123 binding domain is a scFv and comprises: (i) a heavy chain complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 25, a heavy chain complementary determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 13, a heavy chain complementary determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 1, a light chain complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 26, a light chain complementary determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 14, and a light chain complementary determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 2; or (ii) a heavy chain complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 33, a heavy chain complementary determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 21, a heavy chain complementary determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 9, a light chain complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 34, a light chain complementary determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 22, and a light chain complementary determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:

10.

8. The isolated nucleic acid molecule of claim 7, wherein the intracellular signaling domain is CD3-zeta (CD3ζ).

9. The isolated nucleic acid molecule of claim 8, wherein the intracellular signaling domain comprises a co-stimulatory domain selected from the group consisting of CD28, 4.1BB, inducible T-cell co-stimulator (ICOS), OX-40, or a combination thereof.

10. An isolated polypeptide molecule encoded by the nucleic acid molecule of any one of claims 7 to 9.

11. An isolated chimeric antigen receptor (CAR) molecule comprising an anti-CD123 binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein the anti-CD123 binding domain is a scFv and comprises: (i) a heavy chain complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 25, a heavy chain complementary determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 13, a heavy chain complementary determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 1, a light chain complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 26, a light chain complementary determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 14, and a light chain complementary determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 2; or (ii) a heavy chain complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 33, a heavy chain complementary determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 21, a heavy chain complementary determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO: 9, a light chain complementary determining region 1 (CDR1) consisting of the amino acid sequence of SEQ ID NO: 34, a light chain complementary determining region 2 (CDR2) consisting of the amino acid sequence of SEQ ID NO: 22, and a light chain complementary determining region 3 (CDR3) consisting of the amino acid sequence of SEQ ID NO:

10.

12. An isolated chimeric antigen receptor (CAR) molecule, wherein the CAR is as defined in any one of claims 8 to 9.

13. An expression vector comprising a nucleic acid molecule as defined in any one of claims 7 to 9, wherein the vector is a DNA or RNA vector.

14. An expression vector comprising a nucleic acid molecule as defined in any one of claims 7 to 9, wherein the vector is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector.

15. An engineered immune cell comprising the nucleic acid molecule of any one of claims 7 to 9 or the vector of claim 13 or 14.

16. Use of the immune cell according to claim 15 in the preparation of a medicament for treating blastic plasmacytoid dendritic cell neoplasm (BPDCN) or acute myeloid leukemia (AML) in a mammal.

17. The use according to claim 16, wherein the mammal is a human.

18. A pharmaceutical composition comprising the engineered immune cells according to claim 15 and a pharmaceutically acceptable excipient.

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