CAR T cells containing anti-CD33, anti-CLL1, and at least one further CAR anti-CD123 and / or anti-FTL3

Abstract

To provide cells containing anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; and anti-CD123 and / or anti-CAR FLT3 CARs. [Solution] The aforementioned cells can be used in the treatment of diseases such as acute myeloid leukemia (AML). In a first aspect, the present invention provides CAR-expressing cells that target multiple antigens associated with acute myeloid leukemia (AML). These cells target three or four of the following antigens: CD33, CLL-1, CD123, and FLT3. For example, the cells may include anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-CD123 CAR.

Description

Technical Field

[0001] Field of the Invention The present invention relates to cells expressing multiple chimeric antigen receptors (CARs). The aforementioned cells target multiple antigens characteristic of acute myeloid leukemia (AML).

Background Art

[0002] Background of the Invention Acute Myeloid Leukemia Acute myeloid leukemia (AML) is a heterogeneous disease characterized by uncontrolled clonal proliferation of myeloid precursors in the bone marrow and blood, leading to the accumulation of leukemic blasts and severe impairment of normal hematopoiesis. AML is the most common acute leukemia in adults and has the highest mortality rate among all leukemias. It is estimated that 20,830 people in the United States were diagnosed with AML in 2015, and the number of deaths due to AML was estimated to be 10,464. Long-term survivors in AML over the past decade have only slightly increased.

[0003] <00所述の細胞は、急性骨髄球性白血病(AML)に特徴的な複数の抗原を標的にする。

[0004] ​

[0005] Therefore, improvements in the treatment approach for AML are necessary.

[0006] Chimeric antigen receptor Chimeric antigen receptors (CARs) are T cells whose effector function is modified to incorporate the specificity of monoclonal antibodies. CD19-targeted CAR T-cell therapy is highly effective for B-cell malignancies, but CD19-negative escape is a cause of relapse in a significant number of patients.

[0007] CAR T-cell therapy for AML is in the early clinical trial stage. Despite the potential cytotoxicity of various AML antigen-targeting CAR T cells being demonstrated in several preclinical studies, the transition to clinical trials has been delayed. This highlights the inherent challenges in developing CAR-related treatment strategies for AML patients. To date, as listed in Table 1, a small number of relapsed / refractory AML patients have been treated with CAR T-cell immunotherapy through early clinical trials.

[0008] [Table 1] [Brief explanation of the drawing]

[0009] [Figure 1] A schematic diagram illustrating human hematopoiesis.

[0010] [Figure 2] Different binding domain forms of chimeric antigen receptors: (a) Fab CAR form; (b) dAb CAR form; (c) scFv CAR form

[0011] [Figure 3-1] Expression of aCD33 CAR on primary T cells derived from three healthy donors. CARs transduced in T cells were stained with the RQR8 marker gene. The FACS plots were created by pre-gating the viable cell population using eFluor780. [Figure 3-2] Expression of aCD33 CAR on primary T cells derived from three healthy donors. CARs transduced in T cells were stained with the RQR8 marker gene. The FACS plots were created by pre-gating the viable cell population using eFluor780.

[0012] [Figure 4-1] Transduction expression of CAR constructs used in aCD123-VHH CAR screening and confirmation assays. PBMCs were individually stained with soluble human CD123 ectodomains fused to mouse Fc cells fused with anti-CD34-PE antibody (QBend10) or 2×StrepTag2 (Strep-PE secondary stain). All plots were pre-gated to single viable cells using a viability dye (FVD-eFlour780). [Figure 4-2] Transduction expression of CAR constructs used in aCD123-VHH CAR screening and confirmation assays. PBMCs were individually stained with soluble human CD123 ectodomains fused to mouse Fc cells fused with anti-CD34-PE antibody (QBend10) or 2×StrepTag2 (Strep-PE secondary stain). All plots were pre-gated to single viable cells using a viability dye (FVD-eFlour780).

[0013] [Figure 5-1] Transduction expression of CAR constructs used in aCLL1-VHH CAR screening and confirmation assays. PBMCs were individually stained with soluble human CLL1 ectodomains fused to human Fc cells fused with anti-CD34-PE antibody (QBend10) or 2×StrepTag2 (Strep-PE secondary stain). All plots were pre-gated to single viable cells using a viability dye (FVD-eFlour780). [Figure 5-2]Transduced expression of the CAR constructs used in the aCLL1-VHH CAR screening and validation assays. PBMCs were individually stained with soluble human CLL1 ectodomain fused to human Fc fused to anti-CD34-PE antibody (QBend10) or 2×StrepTag2 (Strep-PE secondary stain). All plots were pre-gated on single live cells using a viability dye (FVD-eFlour780).

[0014] [Figure 6] SupT1 transduced with a retroviral vector encoding the relevant antigen. A) Antigen expression on SupT1 cells transduced with CD123, CD33, and CLL1 is shown in blue, while the red peak corresponds to the relevant isotype control. B) Mean antigen density (per cell) within each transduced SupT1 cell using Quantibrite™ beads by flow cytometry.

[0015] [Figure 7-1] Analysis of antigen density using Quantibrite™ beads by flow cytometry. A) Antigen expression on target cells (blue) stained with CD33, CD123, and CLL1 compared to the relevant isotype control (red). The mean antigen density (per cell) was measured for each antigen. [Figure 7-2] Analysis of antigen density using Quantibrite™ beads by flow cytometry. A) Antigen expression on target cells (blue) stained with CD33, CD123, and CLL1 compared to the relevant isotype control (red). The mean antigen density (per cell) was measured for each antigen.

[0016] [Figure 8-1] Example of T cell isolation from target cells using an anti-CD3 antibody on SupT1 NT for aCD33 CAR. [Figure 8-2] Example of T cell isolation from target cells using an anti-CD3 antibody on SupT1 NT for aCD33 CAR. [Figure 8-3] Example of T cell isolation from target cells using anti-CD3 antibody on SupT1 NT for aCD33 CAR. [Figure 8-4] Example of T cell isolation from target cells using anti-CD3 antibody on SupT1 NT for aCD33 CAR.

[0017] [Figure 9-1] Cytotoxicity assays using all aCD33 CARs against different AML cells in 3 donors. Cytotoxicity was measured at different time intervals depending on the cells (HL-60: 24 hours), (SUPT1, MOLM, and THP1: 48 hours). 12783 (aCD19 FMC63 scFv) was used as a negative control and 27983 (aCD33 scFv) was used as a positive control. All data were normalized against non-transduced controls. [Figure 9-2] Cytotoxicity assays using all aCD33 CARs against different AML cells in 3 donors. Cytotoxicity was measured at different time intervals depending on the cells (HL-60: 24 hours), (SUPT1, MOLM, and THP1: 48 hours). 12783 (aCD19 FMC63 scFv) was used as a negative control and 27983 (aCD33 scFv) was used as a positive control. All data were normalized against non-transduced controls.

[0018] [Figure 10-1] 24-hour cytotoxicity assays for all CD123-VHH-CAR T cell constructs. Each condition was tested using a minimum of 3 donors (n = 3) and the median values were shown. The top row - SupT1 NT was used as target cells, the middle row - SupT1 cells were engineered to express high levels of CD123, and the bottom row - AML-derived KG1α was used as the target cell line. Since non-transduced T cells only contribute to antigen-independent cytotoxicity, all data were normalized against non-transduced T cells. CARs were compared by two-tailed paired t-tests. *p < 0.05, **p < 0.01, ***p < 0.001. [Figure 10-2] 24-hour cytotoxicity assays for all CD123-VHH-CAR T cell constructs. Each condition was tested using a minimum of 3 donors (n=3), and median values ​​are shown. Top row - SupT1 NT was used as the target cell line; middle row - SupT1 cells were engineered to express high levels of CD123; bottom row - AML-derived KG1α was used as the target cell line. Since untransduced T cells contribute only to antigen-independent cytotoxicity, all data were normalized to untransduced T cells. CARs were compared by two-way paired t-tests. *p<0.05, **p<0.01, ***p<0.001.

[0019] [Figure 11-1] 48-hour cytotoxicity assays for all CD123-VHH-CAR T cell constructs. Each condition was tested using a minimum of 3 donors (n=3), and the median value is shown. From left to right, the target cell lines SupT1 NT, THP1, and Molm14 were used. Since untransduced T cells contribute only to antigen-independent cytotoxicity, all data were normalized to untransduced T cells. [Figure 11-2] 48-hour cytotoxicity assays for all CD123-VHH-CAR T cell constructs. Each condition was tested using a minimum of 3 donors (n=3), and the median value is shown. From left to right, the target cell lines SupT1 NT, THP1, and Molm14 were used. Since untransduced T cells contribute only to antigen-independent cytotoxicity, all data were normalized to untransduced T cells.

[0020] [Figure 12-1]24-hour cytotoxicity assays for all CLL1-VHH-CAR T cell constructs. Each condition was tested using a minimum of 3 donors (n=3), and median values ​​are shown. Top row - SupT1 NTs were used as target cells, and bottom row - SupT1 cells were manipulated to express high levels of CLL1. Since untransduced T cells contribute only to antigen-independent cytotoxicity, all data were normalized to untransduced T cells. [Figure 12-2] 24-hour cytotoxicity assays for all CLL1-VHH-CAR T cell constructs. Each condition was tested using a minimum of 3 donors (n=3), and median values ​​are shown. Top row - SupT1 NTs were used as target cells, and bottom row - SupT1 cells were manipulated to express high levels of CLL1. Since untransduced T cells contribute only to antigen-independent cytotoxicity, all data were normalized to untransduced T cells.

[0021] [Figure 13-1] 48-hour cytotoxicity assays for all Cll1-VHH-CAR T cell constructs. Each condition was tested using a minimum of 3 donors (n=3), and median values ​​are shown. Top row - SupT1 NT used as target cells, middle row - THP1 patient-derived targets, bottom row - KG1α patient-derived target cell lines. Since untransduced T cells contribute only to antigen-independent cytotoxicity, all data were normalized to untransduced T cells. CARs were compared using a two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns = not significant. [Figure 13-2]48-hour cytotoxicity assays for all Cll1-VHH-CAR T cell constructs. Each condition was tested using a minimum of 3 donors (n=3), and median values ​​are shown. Top row - SupT1 NT used as target cells, middle row - THP1 patient-derived targets, bottom row - KG1α patient-derived target cell lines. Since untransduced T cells contribute only to antigen-independent cytotoxicity, all data were normalized to untransduced T cells. CARs were compared using a two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.

[0022] [Figure 14] IL-2 secretion from co-cultures of aCD33 CAR T cells with target cells in an E:T ratio of 1:1 in two donors (data for donor 6 is not shown). IL-2 production was measured using SupT1 cells and AML-derived cells engineered to express high levels of CD33.

[0023] [Figure 15] IFN-γ production by aCD33 CAR T cells in target cells with a 1:1 E:T ratio in two donors.

[0024] [Figure 16a] Cytokine measurements from cytotoxicity assays using CD123-VHH-CAR T cell constructs at 24 and 48 hours. The assays were performed in a 1:8 ratio for 3 donors (n=3). A) IL-2 measurements, B) IFN-γ measurements. CARs were compared using a two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns = not significant. [Figure 16b] Cytokine measurements from cytotoxicity assays using CD123-VHH-CAR T cell constructs at 24 and 48 hours. The assays were performed in a 1:8 ratio for 3 donors (n=3). A) IL-2 measurements, B) IFN-γ measurements. CARs were compared using a two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.

[0025] [Figure 17a] Cytokine measurements of cytotoxicity assays 24 and 48 hours after using CLL1-VHH-CAR T cell constructs. Performed in a 1:8 ratio for 3 donors (n=3). A) IL-2 measurement, B) IFN-γ measurement. CARs were compared using a two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns = not significant. [Figure 17b] Cytokine measurements of cytotoxicity assays 24 and 48 hours after using CLL1-VHH-CAR T cell constructs. Performed in a 1:8 ratio for 3 donors (n=3). A) IL-2 measurement, B) IFN-γ measurement. CARs were compared using a two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.

[0026] [Figure 18] Growth assays of each aCD33 CAR T cell in different cell lines. Cell 12783 (aCD19 FMC63 scFv) was used as a negative control, and cell 27983 (aCD33 scFv) was used as a positive control. Expression levels were normalized to non-transduced controls. The assays were set to 6 days with an E:T ratio of 1:1. The growth assays were set up using two donors in HL-60 cells.

[0027] [Figure 19] Four-day proliferation assays were performed for all CD123-VHH-CAR T cell constructs. Expression levels were normalized to the CD3+ non-transduced T cell condition. Each condition was tested using a minimum of three donors (n=3).

[0028] [Figure 20]Four-day proliferation assays were performed for all CLL1-VHH-CAR T cell constructs. Expression levels were normalized to the CD3+ non-transduced T cell condition. Each condition was tested using a minimum of three donors (n=3). CARs were compared using a two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns=not significant. [Overview of the project]

[0029] Summary of the embodiments of the invention In a first aspect, the present invention provides CAR-expressing cells that target multiple antigens associated with acute myeloid leukemia (AML). These cells target three or four of the following antigens: CD33, CLL-1, CD123, and FLT3.

[0030] For example, cells may contain anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; and anti-CD123 CARs.

[0031] The cells may contain anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; and anti-FLT3 CARs.

[0032] The cells may contain anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; anti-CD123 CARs; and anti-FLT3 CARs.

[0033] One or more, or all, of the CARs (single or multiple) may contain a domain antibody (dAb) antigen-binding domain.

[0034] A cell may contain one or more tandem chimeric antigen receptors (tanCARs). Each tanCAR may contain a domain antibody (dAb) antigen-binding domain.

[0035] Anti-CD33 CARs containing a domain antibody (dAb) antigen-binding domain may include the following complementarity-determining regions: (i) CDR1-GRTFSMHS (Sequence ID 1); CDR2-VTWSGDTF (Sequence ID 2); CDR3-KDDPYRPAYDY (Sequence ID 3); (ii) CDR1-GRTFSSYV (Sequence ID 4); CDR2-ISWSGGST (Sequence ID 5); CDR3-AAMELRGGSYNYASSRQYDY (Sequence ID 6); (iii) CDR1-EIAFSNFN (sequence number 7); CDR2-ISSHGDTNY (sequence number 8); CDR3-NANDPFLSVSDF (sequence number 9); (iv)CDR1-GSIFSINA(SEQ ID NO: 10);CDR2-ISWSGGST(SEQ ID NO: 5);CDR3-AAISGWGRSIRVGERYEYDY(SEQ ID NO: 11); (v)CDR1-GRTSSSST(sequence number 12);CDR2-ITLSGGST(sequence number 13);CDR3-AARRWSNNRGGYDRAGYDY(sequence number 14);or (vi)CDR1-GRTFSSYA(SEQ ID NO: 15);CDR2-ITWSGGST(SEQ ID NO: 16);CDR3-AMLLRGGLYDYTDYILYNY(SEQ ID NO: 17).

[0036] Anti-CD33 CARs having a domain antibody (dAb) antigen-binding domain may contain one of the sequences shown in Sequence ID No. 18, 19, 20, 21, 22, or 23.

[0037] Anti-CLL-1 CARs containing a domain antibody (dAb) antigen-binding domain may include the following complementarity-determining regions: (i) CDR1-GFTFGNHD (Sequence ID 48); CDR2-IDSGGNVI (Sequence ID 49); CDR3-ATDLDSGAESLESVY (Sequence ID 50); (ii) CDR1-GFAFGSAD (Sequence ID 51); CDR2-IDSGGNTQ (Sequence ID 52); CDR3-TDLDPTTDSLENVY (Sequence ID 53); (iii) CDR1-GRTFSAYF (Sequence ID 54); CDR2-INWNGDSS (Sequence ID 55); CDR3-AADTHGAVGLGSERLYDY (Sequence ID 56); (iv) CDR1-GIGVSSTG (Sequence ID 57); CDR2-IDRDGTT (Sequence ID 58); CDR3-TVVGDYY (Sequence ID 59); (v)CDR1-GFIFGNYD(sequence number 60);CDR2-ISSGGNDI(sequence number 61);CDR3-AADLDPGTDSLDNIH(sequence number 62);or (vi)CDR1-GFTLDYYA(SEQ ID NO: 63);CDR2-ISSSDGST(SEQ ID NO: 64);CDR3-AEAVYYAGVCVAMYDS(SEQ ID NO: 65).

[0038] Anti-CLL-1 CARs having a domain antibody (dAb) antigen-binding domain may contain one of the sequences shown in SEQ ID NOs. 66, 67, 68, 69, 70, or 71.

[0039] Anti-CD123 CARs containing a domain antibody (dAb) antigen-binding domain may include the following complementarity-determining regions: (i)CDR1-GRSINTYA(sequence number 24);CDR2-INYNSRYT(sequence number 25);CDR3-AATSYYPTDYDVASRVATWPS(sequence number 26); (ii) CDR1-GISLNA (sequence number 27); CDR2-IKIGGVS (sequence number 28); CDR3-NTYPPYLNGMDY (sequence number 29); (iii) CDR1-GRSFNTDA (SEQ ID NO: 30); CDR2-ISWDGTRT (SEQ ID NO: 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID NO: 32); (iv)CDR1-GSSISV(sequence code 33);CDR2-ISWSDGNT(sequence code 34);CDR3-AVEPRGWPKGHRY(sequence code 35); (v)CDR1-GSSFSINV(sequence number 36);CDR2-ISWSDGST(sequence number 37);CDR3-AVEPRGWPKGHRY(sequence number 38);or (vi)CDR1-GSIFRINA (SEQ ID NO: 39);CDR2-VNWIGGTT (SEQ ID NO: 40);CDR3-SATDKGGSSRY (SEQ ID NO: 41).

[0040] Anti-CD123 CARs having a domain antibody (dAb) antigen-binding domain may contain one of the sequences shown in SEQ ID NOs: 42, 43, 44, 45, 46, or 47.

[0041] Anti-FLT3 CARs containing a domain antibody (dAb) antigen-binding domain may include the following complementarity-determining regions: (i) CDR1-GIFKTNY (sequence number 72); CDR2-FTNDGST (sequence number 73); CDR3-YGLGH (sequence number 74); (ii) CDR1-GTISSIRY (SEQ ID NO: 75); CDR2-ITSSGNT (SEQ ID NO: 76); CDR3-YTMGY (SEQ ID NO: 77); (iii) CDR1-GIFSTNY (SEQ ID NO: 78); CDR2-FTNDGGT (SEQ ID NO: 79); CDR3-CGLGH (SEQ ID NO: 80); (iv) CDR1-GSISSIRY (sequence number 81); CDR2-ITSSGST (sequence number 82); CDR3-YTMGY (sequence number 83); or (v)CDR1-GIFSTNH (Sequence ID 84); CDR2-FTNDGST (Sequence ID 85); CDR3-YGLGH (Sequence ID 86).

[0042] Anti-FLT3 CARs having a domain antibody (dAb) antigen-binding domain may contain one of the sequences shown in SEQ ID NOs: 87, 88, 89, 90, or 91.

[0043] In a second aspect, the present invention provides nucleic acid constructs encoding multiple CARs. The nucleic acid constructs may encode CARs for three or all four of the following antigens: CD33, CLL-1, CD123, and FLT3. For example, the nucleic acid constructs may encode: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-CD123 CAR.

[0044] Nucleic acid constructs may encode: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-FLT3 CAR.

[0045] Nucleic acid constructs may encode: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-CD123 CAR; and anti-FLT3 CAR.

[0046] In a third aspect, the present invention provides a method for producing cells according to the first aspect of the present invention, comprising the step of transfecting or introducing cells with a nucleic acid construct of the second aspect of the present invention.

[0047] In a fourth aspect, the present invention provides a vector comprising a nucleic acid construct according to a second aspect of the present invention.

[0048] In a fifth embodiment, a vector kit is provided, comprising multiple vectors, each vector encoding a CAR for a target antigen. The kit may include vectors encoding CARs for three or all four of the following target antigens: CD33, CLL-1, CD123, and FLT3. For example, the kit may include: (i) A first vector containing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds to CD33; (ii) A second vector containing a nucleic acid sequence encoding a CAR that binds to CLL1; and (iii) A third vector containing a nucleic acid sequence encoding a CAR that binds to CD123.

[0049] The kit may include: (i) A first vector containing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds to CD33; (ii) A second vector containing a nucleic acid sequence encoding a CAR that binds to CLL1; and (iii) A third vector containing a nucleic acid sequence encoding a CAR that binds to FLT3.

[0050] The kit may include: (i) A first vector containing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds to CD33; (ii) A second vector containing a nucleic acid sequence encoding a CAR that binds to CLL1; (iii) A third vector containing a nucleic acid sequence encoding a CAR that binds to CD123; and (iv) A fourth vector containing a nucleic acid sequence encoding a CAR that binds to FLT3.

[0051] In a sixth aspect, a pharmaceutical composition is provided comprising a plurality of cells according to the first aspect of the present invention together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0052] A seventh aspect provides a method for treating cancer, comprising the step of administering a pharmaceutical composition according to a sixth aspect of the present invention to a subject.

[0053] The cancer could be acute myeloid leukemia (AML).

[0054] Furthermore, this method may include a step of subsequently administering the allogeneic graft to the patient.

[0055] In the eighth aspect, a pharmaceutical composition according to the sixth aspect of the present invention is provided for use in the treatment of cancer.

[0056] A ninth aspect provides the use of cells according to the first aspect of the present invention in the manufacture of a pharmaceutical composition for treating cancer.

[0057] The AML blast phenotype is even more heterogeneous than acute lymphoblastic leukemia (ALL) blasts. Bone marrow hematopoiesis is driven by stem cells that replenish or differentiate their compartments in a stochastic and signal-responsive manner. Similar to normal myeloid stem cells, AML stem cells proliferate or differentiate to result in bone marrow replacement with a range of cells in different differentiated states. AML stem cells can arise along the aforementioned bone marrow hematopoietic ranges and, consequently, have different surface antigen profiles. Furthermore, in a given patient, there may be stem cell nexies or different stem cell hierarchies at different points in ontogeny, all of which contribute to the disease burden (Figure 1).

[0058] To treat the maximum number of patients, the inventors found that AML targeting with chimeric antigen receptors requires simultaneous targeting of multiple antigens along the myeloid cell lineage.

[0059] The OR gate of the present invention offers advantages over current Phase I clinical trials of CAR T cell immunotherapy for relapsed / refractory AML, as shown in Table 1 above. Targeting a single antigen may not eliminate disease-associated stem cell compartments. However, simultaneously targeting multiple myeloid antigens means that AML is treated universally. Immunotherapy using CAR-T cells targeting multiple antigens eradicates disease-associated stem cell compartments regardless of the number and location of the stem cell compartments. Furthermore, targeting multiple antigens reduces the possibility of evasion through antigen downregulation.

[0060] Further aspects Furthermore, the present invention provides a cell composition comprising CAR-expressing cells that express multiple CARs.

[0061] The cell composition may express: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-CD123 CAR.

[0062] The cell composition may express the following: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-FLT3 CAR.

[0063] The cell composition may express the following: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-CD123 CAR; and anti-FLT3 CAR.

[0064] Each cell in the composition may express one CAR type. For example, the composition may comprise one mixture of the following: CD33 CAR-expressing cells, CLL-1 CAR-expressing cells, and CD123 CAR-expressing cells; CD33 CAR-expressing cells, CLL-1 CAR-expressing cells, and FLT3 CAR-expressing cells; or CD33 CAR-expressing cells, CLL-1 CAR-expressing cells, CD123 CAR-expressing cells, and FLT3 CAR-expressing cells.

[0065] Alternatively, at least some of the cells in the composition may express one or more CARs, for example, the composition may include the following combinations: Cells expressing the CD33 CAR / CLL-1 CAR OR gate and CD123 Cells that express CAR; Cells expressing the CD33 CAR / CD123 CAR OR gate and CLL-1 Cells that express CAR; Cells expressing the CD123 CAR / CLL-1 CAR OR gate and CD33 Cells that express CAR; Cells expressing the CD33 CAR / CLL-1 CAR OR gate and cells expressing the FLT3 CAR; Cells expressing the CD33 CAR / FLT3 CAR OR gate and cells expressing the CLL-1 CAR; Cells expressing the FLT3 CAR / CLL-1 CAR OR gate and cells expressing the CD33 CAR; Cells expressing the CD33 CAR / CLL-1 CAR OR gate and CD123 Cells expressing the CAR / FLT3 CAR OR gate; Cells expressing the CD123 CAR / CLL-1 CAR OR gate and CD33 Cells expressing the CAR / FLT3 CAR OR gate; or Cells expressing the CD33 CAR / CD123 CAR OR gate and CLL-1 Cells that express the CAR / FLT3 CAR OR gate.

[0066] At least some of the cells in the composition may express the following tanCARs. For example, the composition may include the following combinations: Cells expressing CD33 / CLL-1 tanCAR and cells expressing CD123 CAR; Cells expressing CD33 / CD123 tanCAR and cells expressing CLL-1 CAR; Cells expressing CD123 / CLL-1 tanCAR and cells expressing CD33 CAR; Cells expressing CD33 / CLL-1 tanCAR and cells expressing FLT3 CAR; Cells expressing CD33 / FLT3 tanCAR and cells expressing CLL-1 CAR; Cells expressing FLT3 / CLL-1 tanCAR and cells expressing CD33 CAR; Cells expressing CD33 / CLL-1 tanCAR and cells expressing CD123 / FLT3 tanCAR; Cells expressing CD123 / CLL-1 tanCAR and cells expressing CD33 / FLT3 tanCAR; or Cells expressing CD33 / CD123 tanCAR and cells expressing CLL-1 / FLT3 tanCAR.

[0067] The following nucleic acid sequences, nucleic acid constructs, vectors, and vector kits, as well as methods, may be used to produce cells of the cell composition according to this embodiment of the present invention.

[0068] The cell composition may be used in the treatment of the disease as described below.

[0069] Further aspects of the present invention are summarized in the following numbered paragraphs:

[0070] A1. Domain antibody (dAb) that binds to CD33 and contains the following complementarity-determining region (CDR): (i) CDR1-GRTFSMHS (Sequence ID 1); CDR2-VTWSGDTF (Sequence ID 2); CDR3-KDDPYRPAYDY (Sequence ID 3); (ii) CDR1-GRTFSSYV (Sequence ID 4); CDR2-ISWSGGST (Sequence ID 5); CDR3-AAMELRGGSYNYASSRQYDY (Sequence ID 6); (iii) CDR1-EIAFSNFN (sequence number 7); CDR2-ISSHGDTNY (sequence number 8); CDR3-NANDPFLSVSDF (sequence number 9); (iv)CDR1-GSIFSINA(SEQ ID NO: 10);CDR2-ISWSGGST(SEQ ID NO: 5);CDR3-AAISGWGRSIRVGERYEYDY(SEQ ID NO: 11); (v)CDR1-GRTSSSST(sequence number 12);CDR2-ITLSGGST(sequence number 13);CDR3-AARRWSNNRGGYDRAGYDY(sequence number 14);or (vi)CDR1-GRTFSSYA(SEQ ID NO: 15);CDR2-ITWSGGST(SEQ ID NO: 16);CDR3-AMLLRGGLYDYTDYILYNY(SEQ ID NO: 17).

[0071] A2. The dAb described in paragraph A1, containing one of the sequences shown in sequence numbers 18, 19, 20, 21, 22, or 23.

[0072] A3. Chimeric antigen receptors (CARs) having an antigen-binding domain containing the dAb of paragraph A1 or A2.

[0073] A4. The nucleic acid sequence encoding the dAb described in paragraph A1 or A2, or the CAR described in paragraph A3.

[0074] A5. A vector containing the nucleic acid sequence described in paragraph A4.

[0075] A6. Cells expressing CAR from paragraph A3.

[0076] A7. A method for producing the cells described in paragraph A6, comprising the step of transfecting or introducing cells with the vector described in paragraph A5.

[0077] A8. A pharmaceutical composition containing multiple cells as described in paragraph A6.

[0078] A9. A method for treating cancer, comprising the step of administering the pharmaceutical composition described in paragraph A8 to a subject.

[0079] A10. The method described in paragraph A9, wherein the aforementioned cancer is acute myeloid leukemia (AML).

[0080] A11. The pharmaceutical composition described in paragraph A8 for use in the treatment of cancer.

[0081] A12. Use of cells described in paragraph A6 in the manufacture of pharmaceutical compositions for treating cancer.

[0082] Domain antibodies (dAb) that bind to B1.CD123 and contain the following complementarity-determining regions (CDRs): (i)CDR1-GRSINTYA(sequence number 24);CDR2-INYNSRYT(sequence number 25);CDR3-AATSYYPTDYDVASRVATWPS(sequence number 26); (ii) CDR1-GISLNA (sequence number 27); CDR2-IKIGGVS (sequence number 28); CDR3-NTYPPYLNGMDY (sequence number 29); (iii) CDR1-GRSFNTDA (SEQ ID NO: 30); CDR2-ISWDGTRT (SEQ ID NO: 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID NO: 32); (iv)CDR1-GSSISV(sequence code 33);CDR2-ISWSDGNT(sequence code 34);CDR3-AVEPRGWPKGHRY(sequence code 35); (v)CDR1-GSSFSINV(sequence number 36);CDR2-ISWSDGST(sequence number 37);CDR3-AVEPRGWPKGHRY(sequence number 38);or (vi)CDR1-GSIFRINA (SEQ ID NO: 39);CDR2-VNWIGGTT (SEQ ID NO: 40);CDR3-SATDKGGSSRY (SEQ ID NO: 41).

[0083] B2. The dAb described in paragraph B1, which contains one of the sequences shown in sequence numbers 42, 43, 44, 45, 46, or 47.

[0084] B3. Chimeric antigen receptors (CARs) having an antigen-binding domain containing the dAb of paragraph B1 or B2.

[0085] B4. The nucleic acid sequence encoding the dAb described in paragraph B1 or B2 or the CAR described in paragraph B3.

[0086] B5. A vector containing the nucleic acid sequence described in paragraph B4.

[0087] B6. Cells expressing CAR from paragraph B3.

[0088] B7. A method for producing the cells described in paragraph B6, comprising the step of transfecting or introducing cells with the vector described in paragraph B5.

[0089] B8. A pharmaceutical composition containing multiple cells as described in paragraph B6.

[0090] B9. A method for treating cancer, comprising the step of administering a pharmaceutical composition described in paragraph B8 to a subject.

[0091] B10. The method described in paragraph B9, wherein the aforementioned cancer is acute myeloid leukemia (AML).

[0092] B11. The pharmaceutical composition described in paragraph B8 for use in the treatment of cancer.

[0093] B12. Use of cells described in paragraph B6 in the manufacture of pharmaceutical compositions for treating cancer.

[0094] The present invention provides a domain antibody (dAb) that binds to FLT3 and contains the following complementarity-determining region:

[0095] Domain antibodies (dAb) that bind to C1.FLT3 and contain the following complementarity-determining regions (CDRs): (i) CDR1-GIFKTNY (sequence number 72); CDR2-FTNDGST (sequence number 73); CDR3-YGLGH (sequence number 74); (ii) CDR1-GTISSIRY (SEQ ID NO: 75); CDR2-ITSSGNT (SEQ ID NO: 76); CDR3-YTMGY (SEQ ID NO: 77); (iii) CDR1-GIFSTNY (SEQ ID NO: 78); CDR2-FTNDGGT (SEQ ID NO: 79); CDR3-CGLGH (SEQ ID NO: 80); (iv) CDR1-GSISSIRY (sequence number 81); CDR2-ITSSGST (sequence number 82); CDR3-YTMGY (sequence number 83); or (v)CDR1-GIFSTNH (Sequence ID 84); CDR2-FTNDGST (Sequence ID 85); CDR3-YGLGH (Sequence ID 86).

[0096] C2. A dAb described in paragraph C1, containing one of the sequences shown in sequence numbers 87, 88, 89, 90, or 91.

[0097] C3. Chimeric antigen receptors (CARs) having an antigen-binding domain containing a dAb in paragraph C1 or C2.

[0098] C4. The nucleic acid sequence encoding the dAb described in paragraph C1 or C2 or the CAR described in paragraph C3.

[0099] C5. A vector containing the nucleic acid sequence described in paragraph C4.

[0100] C6. Cells expressing CAR in paragraph C3.

[0101] C7. A method for producing the cells described in paragraph C6, comprising the step of transfecting or introducing cells with the vector described in paragraph C5.

[0102] C8. A pharmaceutical composition containing multiple cells as described in paragraph C6.

[0103] A method for treating cancer, comprising the step of administering a pharmaceutical composition described in paragraph C8 to a subject.

[0104] C10. The method described in paragraph C9, wherein the aforementioned cancer is acute myeloid leukemia (AML).

[0105] C11. Pharmaceutical compositions described in paragraph C8 for use in the treatment of cancer.

[0106] C12. Use of cells described in paragraph C6 in the manufacture of pharmaceutical compositions for treating cancer.

[0107] Domain antibodies (dAb) that bind to D1.CLL1 and contain the following complementarity-determining regions (CDRs): (i) CDR1-GFTFGNHD (Sequence ID 48); CDR2-IDSGGNVI (Sequence ID 49); CDR3-ATDLDSGAESLESVY (Sequence ID 50); (ii) CDR1-GFAFGSAD (Sequence ID 51); CDR2-IDSGGNTQ (Sequence ID 52); CDR3-TDLDPTTDSLENVY (Sequence ID 53); (iii) CDR1-GRTFSAYF (Sequence ID 54); CDR2-INWNGDSS (Sequence ID 55); CDR3-AADTHGAVGLGSERLYDY (Sequence ID 56); (iv) CDR1-GIGVSSTG (Sequence ID 57); CDR2-IDRDGTT (Sequence ID 58); CDR3-TVVGDYY (Sequence ID 59); (v)CDR1-GFIFGNYD(sequence number 60);CDR2-ISSGGNDI(sequence number 61);CDR3-AADLDPGTDSLDNIH(sequence number 62);or (vi)CDR1-GFTLDYYA(SEQ ID NO: 63);CDR2-ISSSDGST(SEQ ID NO: 64);CDR3-AEAVYYAGVCVAMYDS(SEQ ID NO: 65).

[0108] D2. A dAb described in paragraph D1, containing one of the sequences shown in sequence numbers 66, 67, 68, 69, 70, or 71.

[0109] D3. Chimeric antigen receptors (CARs) having an antigen-binding domain containing dAb from paragraph D1 or D2.

[0110] D4. The nucleic acid sequence encoding dAb described in paragraph D1 or D2 or CAR in paragraph D3.

[0111] D5. A vector containing the nucleic acid sequence described in paragraph D4.

[0112] D6. Cells expressing CAR in paragraph D3.

[0113] D7. A method for producing the cells described in paragraph D6, comprising the step of transfecting or introducing cells with the vector described in paragraph D5.

[0114] D8. A pharmaceutical composition containing multiple cells as described in paragraph D6.

[0115] D9. A method for treating cancer, comprising the step of administering a pharmaceutical composition described in paragraph D8 to a subject.

[0116] D10. The method described in paragraph D9, wherein the aforementioned cancer is acute myeloid leukemia (AML).

[0117] D11. The pharmaceutical composition described in paragraph D8 for use in the treatment of cancer.

[0118] D12. Use of cells described in paragraph D6 in the manufacture of pharmaceutical compositions for treating cancer. In certain embodiments, for example, the following are provided: (Item 1) Cells containing anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; and anti-CD123 CARs. (Item 2) Cells containing anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; and anti-FLT3 CARs. (Item 3) Cells as described in item 1 or 2, including anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; anti-CD123 CAR; and anti-FLT3 CAR. (Item 4) A cell according to any of the above items, wherein one or more CARs (single or multiple) include a domain antibody (dAb) antigen-binding domain. (Item 5) Cells described in any of items 1-3, in which each CAR contains a domain antibody (dAb) antigen-binding domain. (Item 6) Cells described in any of items 1-3, containing one or more tandem chimeric antigen receptors (tanCAR(single or multiple)). (Item 7) The cells described in item 6, wherein the tanCAR(s)(single or multiple) comprises a domain antibody (dAb) antigen-binding domain. (Item 8) The aforementioned anti-CD33 CAR has the following complementarity determination region: (i) CDR1-GRTFSMHS (Sequence ID 1); CDR2-VTWSGDTF (Sequence ID 2); CDR3-KDDPYRPAYDY (Sequence ID 3); (ii) CDR1-GRTFSSYV (Sequence ID 4); CDR2-ISWSGGST (Sequence ID 5); CDR3-AAMELRGGSYNYASSRQYDY (Sequence ID 6); (iii) CDR1-EIAFSNFN (Sequence ID 7); CDR2-ISSHGDTN Y (sequence number 8); CDR3-NANDPFLSVSDF (sequence number 9); (iv)CDR1-GSIFSINA(SEQ ID NO: 10);CDR2-ISWSGGST(SEQ ID NO: 5);CDR3-AAISGWGRSIRVGERYEYDY(SEQ ID NO: 11); (v)CDR1-GRTSSSST(sequence number 12);CDR2-ITLSGGST(sequence number 13);CDR3-AARRWSNNRGGYDRAGYDY(sequence number 14);or (vi)CDR1-GRTFSSYA(SEQ ID NO: 15);CDR2-ITWSGGST(SEQ ID NO: 16);CDR3-AMLLRGGLYDYTDYILYNY(SEQ ID NO: 17) A cell having a domain antibody (dAb) antigen-binding domain, as described in any of the above items. (Item 9) The cell described in item 8, wherein the antigen-binding domain contains one of the sequences shown in sequence numbers 18, 19, 20, 21, 22, or 23. (Item 10) The aforementioned anti-CLL1 CAR has the following complementarity determination region: (i) CDR1-GFTFGNHD (Sequence ID 48); CDR2-IDSGGNVI (Sequence ID 49); CDR3-ATDLDSGAESLESVY (Sequence ID 50); (ii) CDR1-GFAFGSAD (Sequence ID 51); CDR2-IDSGGNTQ (Sequence ID 52); CDR3-TDLDPTTDSLENVY (Sequence ID 53); (iii) CDR1-GRTFSAYF (Sequence ID 54); CDR2-INWNGDSS (Sequence ID 55); CDR3-AADTHGAVGLGSERLYDY (Sequence ID 56); (iv) CDR1-GIGVSSTG (Sequence ID 57); CDR2-IDRDGTT (Sequence ID 58); CDR3-TVVGDYY (Sequence ID 59); (v)CDR1-GFIFGNYD(sequence number 60);CDR2-ISSGGNDI(sequence number 61);CDR3-AADLDPGTDSLDNIH(sequence number 62);or (vi)CDR1-GFTLDYYA(SEQ ID NO: 63);CDR2-ISSSDGST(SEQ ID NO: 64);CDR3-AEAVYYAGVCVAMYDS(SEQ ID NO: 65) A cell having a domain antibody (dAb) antigen-binding domain, as described in any of the above items. (Item 11) The cell according to item 10, wherein the antigen-binding domain comprises one of the sequences shown in SEQ ID NOs. 66, 67, 68, 69, 70, or 71. (Item 12) The aforementioned anti-CD123 CAR has the following complementarity determination region: (i)CDR1-GRSINTYA(sequence number 24);CDR2-INYNSRYT(sequence number 25);CDR3-AATSYYPTDYDVASRVATWPS(sequence number 26); (ii) CDR1-GISLNA (sequence number 27); CDR2-IKIGGVS (sequence number 28); CDR3-NTYPPYLNGMDY (sequence number 29); (iii) CDR1-GRSFNTDA (SEQ ID NO: 30); CDR2-ISWDGTRT (SEQ ID NO: 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID NO: 32); (iv)CDR1-GSSISV(sequence code 33);CDR2-ISWSDGNT(sequence code 34);CDR3-AVEPRGWPKGHRY(sequence code 35); (v)CDR1-GSSFSINV(sequence number 36);CDR2-ISWSDGST(sequence number 37);CDR3-AVEPRGWPKGHRY(sequence number 38);or (vi)CDR1-GSIFRINA (Sequence No. 39);CDR2-VNWIGGTT (Sequence ID 40); CDR3-SATDKGGSSRY (Sequence ID 41) Cells as described in item 1, having a domain antibody (dAb) antigen-binding domain. (Item 13) The cell according to item 12, wherein the antigen-binding domain contains one of the sequences shown in SEQ ID NOs: 42, 43, 44, 45, 46, or 47. (Item 14) The aforementioned anti-FTL3 CAR has the following complementarity determination region: (i) CDR1-(sequence number 72); CDR2-(sequence number 73); CDR3-(sequence number 74); (ii) CDR1-(SEQ ID NO: 75); CDR2-(SEQ ID NO: 76); CDR3-(SEQ ID NO: 77); (iii) CDR1-(sequence number 78); CDR2-(sequence number 79); CDR3-(sequence number 80); (iv) CDR1-(SEQ ID NO: 81); CDR2-(SEQ ID NO: 82); CDR3-(SEQ ID NO: 83); (v) CDR1-(sequence number 84); CDR2-(sequence number 85); CDR3-(sequence number 86); or (vi) CDR1-(sequence number 87); CDR2-(sequence number 88); CDR3-(sequence number 89) Cells as described in item 2, having a domain antibody (dAb) antigen-binding domain. (Item 15) The cell according to item 14, wherein the antigen-binding domain contains one of the sequences shown in sequence numbers 90, 91, 92, 93, 94, or 95. (Item 16) Nucleic acid constructs encoding anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; and anti-CD123 CARs. (Item 17) Nucleic acid constructs encoding anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; and anti-FLT3 CARs. (Item 18) Nucleic acid constructs described in item 16 or 17 that encode anti-CD33 chimeric antigen receptors (CARs); anti-CLL1 CARs; anti-CD123 CARs; and anti-FLT3 CARs. (Item 19) A method for producing the cells described in item 1, comprising the step of transfecting or introducing cells with the nucleic acid construct described in item 16. (Item 20) A method for producing the cells described in item 2, comprising the step of transfecting or introducing cells with the nucleic acid construct described in item 17. (Item 21) A vector containing a nucleic acid construct as described in any of items 16-18. (Item 22) It's a Vector kit, (i) A first vector containing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds to CD33; (ii) A second vector containing a nucleic acid sequence encoding a CAR that binds to CLL1; and (iii) A third vector containing a nucleic acid sequence encoding a CAR that binds to CD123. A kit of vectors, including... (Item 23) It's a Vector kit, (i) A first vector containing a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds to CD33; (ii) A second vector containing a nucleic acid sequence encoding a CAR that binds to CLL1; and (iii) A third vector containing a nucleic acid sequence encoding a CAR that binds to FLT3. A kit of vectors, including... (Item 24) A pharmaceutical composition comprising any of the cells described in items 1 to 15, together with a pharmaceutically acceptable carrier, diluent, or excipient. (Item 25) A method for treating cancer, comprising the step of administering a pharmaceutical composition described in item 24 to a subject. (Item 26) The method according to item 25, wherein the cancer is acute myeloid leukemia (AML). (Item 27) The method according to item 26, further comprising the step of administering an allograft to the subject thereafter. (Item 28) A pharmaceutical composition as described in item 24 for use in the treatment of cancer. (Item 29) Use of cells described in any of items 1 to 15 in the manufacture of a pharmaceutical composition for treating cancer. [Modes for carrying out the invention]

[0119] Detailed explanation Chimeric antigen receptor This invention relates to cells that express multiple chimeric antigen receptors on their cell surface.

[0120] Classical chimeric antigen receptors (CARs) are chimeric type I transmembrane proteins in which an extracellular antigen-recognition domain (binder) is connected to an intracellular signaling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but can be based on other forms including an antibody-like antigen-binding site. A spacer domain is usually required to separate the binder from the membrane and allow for proper orientation. A common spacer domain used is the Fc of IgG1. Smaller spacers, such as a stalk derived from CD8α depending on the antigen, or even just the IgG1 hinge, may be sufficient in some cases. The transmembrane domain anchors the protein within the cell membrane and connects the spacer to the endodomain.

[0121] Early CAR designs had endodomains derived from either the γ chain of FcεR1 or the intracellular portion of CD3ζ. Consequently, these first-generation receptors were sufficient to transmit immunological signal 1 and induce T cell killing of congeneral target cells, but they could not sufficiently activate T cells to proliferate and survive. To overcome this limitation, composite endodomains were constructed as follows: fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ yields a second-generation receptor capable of simultaneously transmitting activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is the CD28 costimulatory domain, which delivers the most potent costimulatory signal (i.e., immunological signal 2 that induces T cell proliferation). Several other receptors have also been described, including those with TNF receptor family endodomains (such as the closely related OX40 and 41BB that transmit survival signals). Here we describe even more potent third-generation CARs with endodomains capable of transmitting activation, proliferation, and survival signals.

[0122] When a CAR binds to a target antigen, this binding transmits an activation signal to the T cells expressing the CAR. Therefore, the CAR directs the specificity and cytotoxicity of T cells towards tumor cells expressing the targeted antigen.

[0123] A CAR typically includes: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain containing or associated with a signaling domain.

[0124] A CAR may have the following general structure: Antigen-binding domain - spacer domain - transmembrane domain - intracellular signal transduction domain (endodomain).

[0125] antigen-binding domain The antigen-binding domain is part of the chimeric receptor that recognizes the antigen. In classical CARs, the antigen-binding domain contains a single-chain variable fragment (scFv) derived from a monoclonal antibody (see Figure 2c). CARs have also been produced that have a domain antibody (dAb) antigen-binding domain or a VHH antigen-binding domain (see Figure 2b) or in the Fab CAR form (Figure 2a). FabCARs consist of two chains: a chain with an antibody-like light chain variable region (VL) and a constant region (CL); and a chain with a heavy chain variable region (VH) and a constant region (CH). One of the chains also contains a transmembrane domain and an intracellular signaling domain. The association between CL and CH assembles the receptor.

[0126] The antigen-binding domain(s) of a CAR may be a single-domain binder, also known as a "dAb," "VHH," "domain antibody," or "nanobody."

[0127] Conventional IgG molecules consist of two heavy and two light chains. The heavy chain contains three constant domains and one variable domain (VH); the light chain contains one constant domain and one variable domain (VL). The naturally functioning antigen-binding unit is formed by the non-covalent association of the VH and VL domains. This association is mediated by a hydrophobic framework region.

[0128] Single-domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first type of single-domain antibody was engineered from heavy-chain antibodies found in camelids, lacking the light chain and CH1 domain of classical antibodies. These heavy-chain antibodies contain a single antigen-binding domain, the VHH domain. Additionally, cartilaginous fish possess heavy-chain antibodies (IgNAR, "novel immunoglobulin antigen receptor"), from which single-domain antibodies called VNAR fragments can be obtained. Another approach involves splitting a dimeric variable domain derived from common immunoglobulin G (IgG) from humans or mice into monomers. Although most studies of single-domain antibodies are currently based on heavy-chain variable domains, light-chain-derived nanobodies have also been shown to specifically bind to target epitopes.

[0129] Single-domain antibodies can be obtained by immunizing dromedary camels, camels, llamas, alpacas, or sharks with the desired antigen and subsequently isolating mRNA encoding heavy-chain antibodies. Reverse transcription and polymerase chain reaction can produce gene libraries of single-domain antibodies. Screening techniques such as phage display and ribosome display can help identify clones that bind to the antigen. Alternatively, single-domain antibodies can be produced from common mouse or human IgG with four strands.

[0130] This invention relates to the targeting of multiple antigens. This can be done by several approaches (including OR gates and tanCARs (described in more detail below)). Antigen-binding domains of domain antibodies are particularly well suited to such approaches because they are separate and do not tend to ligate. These domains are not very complex, which means that expression and folding are less likely to be impaired, and the sequences encoding such CARs / tanCARs do not require much space on the viral vector genome.

[0131] TanCAR The cells of the present invention may contain TanCAR.

[0132] Bispecific CARs, known as tandem CARs or TanCARs, are developed to simultaneously target two or more cancer-specific markers. In TanCARs, the extracellular domain contains two antigen-binding specificity sites linked by a linker in tandem. Thus, both of the two binding specificity sites (scFvs) are linked to a single transmembrane portion: one scFv is close to the membrane, and the other is distal. When a TanCAR binds to one or both of the target antigens, this transmits an activation signal to the cell expressing the TanCAR.

[0133] Grada et al (2013, Mol Ther Nucleic Acids 2:e105) describes a TanCAR containing a CD19-specific scFv, followed by a Gly-Ser linker, and then a HER2-specific scFv. The HER2-scFv was located near the membrane, while the CD19-scFv was located distally. The TanCAR was shown to induce distinct T-cell reactivity to each of the two tumor-limiting antigens. This arrangement was chosen because the respective lengths of HER2 (632aa / 125Å) and CD19 (280aa, 65Å) were helpful for spatial arrangement. It was also known that the HER2 scFv binds to the four most distal loops of HER2.

[0134] The cells of the present invention may contain a TanCAR comprising two antigen-binding specificity sites in tandem. The tanCAR may bind to one of the following antigen pairs: CD33 and CD123; CD33 and CLL-1, CD33 and FLT-3; CD123 and CLL-1; CD123 and FLT-3; CLL1 and FLT-3.

[0135] In each of these antigen pairs, the antigen-binding domains can be located in any order within the molecule. For example, for the target antigen pair CD33 and CD123, the CD33-binding antigen-binding domain may be located close to the membrane and the CD123-binding antigen-binding domain may be located distal to the membrane; or the CD123-binding antigen-binding domain may be located close to the membrane and the CD33-binding antigen-binding domain may be located distal to the membrane.

[0136] The cells of the present invention may include a combination of tanCAR and a CAR containing a single antigen-specific site such as scFv-CAR or dAb CAR. In this respect, the cells have the following three antigen specificities: two antigen specificities for TanCAR and one antigen specificity for scFv or dAb CAR. The cells may include, for example, one of the combinations shown in Table 2.

[0137] [Table 2]

[0138] The cells of the present invention may contain two tanCARs. For example, the cells may contain the double tanCARs shown in Table 3.

[0139] [Table 3]

[0140] ORGATE "Logic gate" CAR combinations are described in WO2015 / 075469, WO2015 / 075470, and WO2015 / 075470. A CAR logic gate is a CAR combination that, when expressed by cells such as T cells, can detect a specific expression pattern of at least two target antigens. If at least two target antigens are arbitrarily identified as antigen A and antigen B, the three possible options are as follows:

[0141] "OR gate" - T cells are triggered when either antigen A or antigen B is present on the target cell. "AND gate" - T cells are triggered when both antigens A and B are present on the target cell. "AND NOT GATE" - T cells are triggered when antigen A is present alone on the target cell, but not when both antigens A and B are present on the target cell.

[0142] Engineered T cells expressing these CAR combinations can be tuned to be highly specific to cancer cells based on the specific expression (or lack thereof) of two or more markers in cancer cells.

[0143] An OR gate contains two or more CARs, each directed towards a distinct target antigen expressed by the target cell. The advantage of an OR gate is that the effectiveness is increased because the efficacy is antigen A + antigen B, thus increasing the number of effectively targetable antigens on the target cell. This is particularly important for antigens expressed at fluctuating or low densities on the target cell, as the level of a single antigen may be below the threshold required for CAR-T cells to effectively target it. Also, OR gates avoid antigen avoidance. For example, some lymphomas and leukemias become CD19-negative after CD19 is targeted: if this phenomenon occurs, using an OR gate that targets CD19 in combination with another antigen provides a "backup" antigen.

[0144] The cells of the present invention may express a triple OR gate containing three CARs. For example, the cells may express the following: CARs that bind to CD33, CARs that bind to CLL-1; and CARs that bind to CD123 or CARs that bind to CD33, CARs that bind to CLL-1, and CARs that bind to FLT3.

[0145] The cells of the present invention may express a quadruple OR gate containing four CARs. For example, the cells may express: a CAR that binds to CD33; a CAR that binds to CLL-1; a CAR that binds to CD123; and a CAR that binds to FLT3.

[0146] In the triple and quadruple OR gates of the present invention, one or more CARs (singular or plural) can be dAb CARs. In particular, all CARs in a cell can be dAb CARs.

[0147] target antigen The antigen-binding domain(s) of the CAR, or one of them, may specifically bind to one of the following target antigens: CD33, CD123, CLL-1, and FLT-3.

[0148] CD33 CD33 is a myeloid differentiation antigen that is presented on some normal B cells, activated T cells, and natural killer cells, but is not expressed on pluripotent hematopoietic stem cells or outside the hematopoietic system. CD33 is found on at least a subset of blast cells in almost all acute myeloid leukemia (AML). On average, 10⁴ molecules / leukemia cell of CD33 is not very abundant, but levels vary considerably from patient to patient.

[0149] The extracellular portion of CD33 contains two immunoglobulin domains, while the intracellular portion contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The amino acid sequence of human CD33 is available from Uniprot acceptance number P20138.

[0150] Several commercially available antibodies against CD33 are known (WM-53, P67.6, HIM3-4 (Thermofisher), etc.).

[0151] The present invention provides a domain antibody (dAb) that binds to CD33 and includes the following complementarity-determining region: (i) CDR1-GRTFSMHS (Sequence ID 1); CDR2-VTWSGDTF (Sequence ID 2); CDR3-KDDPYRPAYDY (Sequence ID 3); (ii) CDR1-GRTFSSYV (Sequence ID 4); CDR2-ISWSGGST (Sequence ID 5); CDR3-AAMELRGGSYNYASSRQYDY (Sequence ID 6); (iii) CDR1-EIAFSNFN (sequence number 7); CDR2-ISSHGDTNY (sequence number 8); CDR3-NANDPFLSVSDF (sequence number 9); (iv)CDR1-GSIFSINA(SEQ ID NO: 10);CDR2-ISWSGGST(SEQ ID NO: 5);CDR3-AAISGWGRSIRVGERYEYDY(SEQ ID NO: 11); (v)CDR1-GRTSSSST(sequence number 12);CDR2-ITLSGGST(sequence number 13);CDR3-AARRWSNNRGGYDRAGYDY(sequence number 14);or (vi)CDR1-GRTFSSYA(SEQ ID NO: 15);CDR2-ITWSGGST(SEQ ID NO: 16);CDR3-AMLLRGGLYDYTDYILYNY(SEQ ID NO: 17).

[0152] Anti-CD33 dAb may contain one of the sequences shown in sequence numbers 18, 19, 20, 21, 22, or 23.

[0153] Sequence ID 18 (CD33 dAb P1.E4 - 44738) QVQLESGGGLVQAGGSLRLSCAASGRTFSMHSMGWFRQAPGKEREFVAAVTWSGDTFAYADFVKGRFTISRGIAKNTLYLQMNSLKPEDTAVYYCAAKDDPYRPAYDYWGQGTQVTVSS Sequence ID 19 (CD33 dAb P1.H3- 44739) QVQLQESGGGLVQAGGSLRLLSCAASGRTFSSYVMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAMELRGGSYNYASSRQYDYWGQGTQVTVSS Sequence ID 20 (CD33 dAb P1.G8 - 44742) QVQLQESGGGLVQTGGSLTLSCAASEIAFSNFNMGWYRQGSGKQRTLVAQISSHGDTNYLDSMKGRFTISRDNNKKTVYLQMNALKPEDTAVYYCNANDPFLSVSDFWGQGTQVTVSS Sequence ID 21 (CD33 dAb P2.A7 - 46173) QVQLQQSGGGLVQAGGSLRLSCAASGSIFSINAMGWFRQAPGKEREFVAAISWSGGSTYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAISGWGRSIRVGERYEYDYWGQGTQVTVSS Sequence ID 22 (CD33 dAb P2.B12 - 46174) QVQLQESGGGLVQAGGSLLSCAASGRTSSSSTMAWFRQAPGKEREFVAAITLSGGSTHYADSAKGRFTISRESAKNTVYLQMNSLKPEDTADYYCAARRWSNNNRGGYDRAGYDYWGQGTQVTVSS Sequence ID 23 (CD33 dAb P2.F2 - 46176) QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAMLLRGGLYDYTDYILYNYWGQGTQVTVSS

[0154] Furthermore, the present invention provides the following: A CAR containing a CD33 dAb that acts as an antigen-binding domain; A nucleic acid sequence that codes for such a dAb or CAR.

[0155] CD123 CD123 is the transmembrane α subunit (IL-3Ra) of the interleukin-3 receptor and, together with CD131, forms high-affinity IL-3R. Upon binding to IL-3, IL-3R promotes cell proliferation and survival. CD123 is normally expressed at high levels on plasmacytoid dendritic cells and basophils. CD123 is expressed at low levels on monocytes, eosinophils, and myeloid dendritic cells. The amino acid sequence of human CD123 is available from the following NCBI reference sequence: NP_002174.1.

[0156] Several commercially available antibodies against CD123 are known (e.g., 6H6 and 5B11 (ThermoFisher)).

[0157] The present invention provides a domain antibody (dAb) that binds to CD123 and includes the following complementarity-determining region: (i)CDR1-GRSINTYA(sequence number 24);CDR2-INYNSRYT(sequence number 25);CDR3-AATSYYPTDYDVASRVATWPS(sequence number 26); (ii) CDR1-GISLNA (sequence number 27); CDR2-IKIGGVS (sequence number 28); CDR3-NTYPPYLNGMDY (sequence number 29); (iii) CDR1-GRSFNTDA (SEQ ID NO: 30); CDR2-ISWDGTRT (SEQ ID NO: 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID NO: 32); (iv)CDR1-GSSISV(sequence code 33);CDR2-ISWSDGNT(sequence code 34);CDR3-AVEPRGWPKGHRY(sequence code 35); (v)CDR1-GSSFSINV(sequence number 36);CDR2-ISWSDGST(sequence number 37);CDR3-AVEPRGWPKGHRY(sequence number 38);or (vi)CDR1-GSIFRINA (SEQ ID NO: 39);CDR2-VNWIGGTT (SEQ ID NO: 40);CDR3-SATDKGGSSRY (SEQ ID NO: 41).

[0158] Anti-CD123 dAb may contain one of the sequences shown in sequence numbers 42, 43, 44, 45, 46, or 47.

[0159] Sequence ID 42 (CD123 dAb H11 45897) QVQLQESGGGLVQAGGSLRLSCTASGRSINTYAMAWFRQAPGKEREFVASINYNSRYTHYVDSVKGRFTISRDNTKNTLFLQMDSLNREDTAVYYCAATSYYPTDYDVASRVATWPSWGQGTQVTVSS Sequence ID 43 (CD123 dAb F8 45888) QVQLQESGGGLVQAGESLRLTCAVSGISLNAMGWYRQAPGKQLREWVAVIKIGGVSNYAVSVKGRFTISRDNAKNTIYLQMNSLKPEDTGVYYCNTYPPYLNGMDYWGKGTLVTVSS Sequence ID 44 (CD123 dAb A7 45865) QVQLQQSGGGLVQAGGSLRLSCAFSGRSFNTDAVAWFRQAPGKEREFVAAISWDGTRTYYADSAKGRFTISRDNAKNTVYLQMNSLNSEDTAVYYCAAEPQKAWPIGTSAAGFRSWGQGTQVTVSS Sequence ID 45 (CD123 dAb B4 45868) QVQLQESGGGSVQSGGSLRLSCAASGSSISVMGWFRQAPGKEREFVAAIISWSDGNTNYADSVNGRFSVSRDNTKNTVYLQMNSLKPEDTAIYYCAVEPRGWPKGHRYWGQGTQVTVSS Sequence ID 46 (CD123 dAb A10 45866) QVQLQESGGSSVQAGGSLRLLSCAASGSSFSINVMGWFRQAPGKEREFVAAISWSDGSTNYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAIYYCAVEPRGWPKGHRYWGQGTQVTVSS Sequence ID 47 (CD123 dAb C11 45874) QVQLQESGGGLVQAGGSLLSCAASGSIFRINAMGWFRQAPGKEREFVTAVNWIGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYFCSATDKGGSSRYWGQGTQVTVSS

[0160] Furthermore, the present invention provides the following: A CAR containing a CD123 dAb as an antigen-binding domain; A nucleic acid sequence that codes for such a dAb or CAR.

[0161] FLT3 FMS-like tyrosine kinase 3 (FLT3) (receptor tyrosine kinase (RTK)) is a membrane-bound receptor with an endogenous tyrosine kinase domain. FLT3 consists of an immunoglobulin-like extracellular ligand-binding domain, a transmembrane domain, a near-membrane dimerizing domain, and a highly conserved intracellular kinase domain separated by a kinase insert. FLT3 belongs to the class III subfamily of RTKs, which includes structurally similar members such as the c-FMS receptor, c-KIT receptor, and PDGF receptor. FLT3 is primarily expressed on myeloid and lymphoid progenitor cells, with altered expression in more mature monocyte lineages.

[0162] FLT3 expression has been reported in lymphohematopoietic organs such as the liver, spleen, thymus, and placenta. Under non-stimulated conditions, the FLT3 receptor exists as a non-phosphorylated monomer with an inactive kinase moiety. When this receptor interacts with the FLT ligand (FL), its conformation changes, causing it to unfold and expose the dimerization domain, leading to receptor-to-receptor dimerization. This receptor dimerization is a preparatory step for the activation of tyrosine kinase enzymes, which lead to phosphorylation at various sites within the intracellular domain. The amino acid sequence of human FLT3 is available from the following NCBI reference sequence: NP_004110.2.

[0163] FLT3 is biologically important in some cases of AML, and mutations in FLT3 are the most common mutations in this disease. These mutations typically lead to constitutive activation. Some cases of AML respond to small molecule inhibition of FLT3.

[0164] Several commercially available antibodies against FLT3 are known (e.g., A2F10 and BV10A4H2 (ThermoFisher)).

[0165] The present invention provides a domain antibody (dAb) that binds to FLT3 and contains the following complementarity-determining region: (i) CDR1-GIFKTNY (sequence number 72); CDR2-FTNDGST (sequence number 73); CDR3-YGLGH (sequence number 74); (ii) CDR1-GTISSIRY (SEQ ID NO: 75); CDR2-ITSSGNT (SEQ ID NO: 76); CDR3-YTMGY (SEQ ID NO: 77); (iii) CDR1-GIFSTNY (SEQ ID NO: 78); CDR2-FTNDGGT (SEQ ID NO: 79); CDR3-CGLGH (SEQ ID NO: 80); (iv) CDR1-GSISSIRY (sequence number 81); CDR2-ITSSGST (sequence number 82); CDR3-YTMGY (sequence number 83); or (v)CDR1-GIFSTNH (Sequence ID 84); CDR2-FTNDGST (Sequence ID 85); CDR3-YGLGH (Sequence ID 86).

[0166] Anti-FLT3 dAb may contain one of the sequences shown in SEQ ID NOs: 87, 88, 89, 90, or 91.

[0167] Sequence ID 87 (FLT3 dAb B5) QVQLQQSGGGLVQAGGSLLSCAASGIFKTNYMAWYRQAPGKQRELVAAFTNDGSTLYGDSVKGRFTISRDDAKYTVSLQMNSLKPEDTAVYYCYGLGHWGQGTQVIVSSEPKTPKPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA Sequence ID 88 (FLT3 dAb G3) QVQLQESGGGLVQAGGSLRLLSCAASGTISSIRYMNWYRQAPGKQREVVAYITSSGNTNYADSVKGRFTISRDNAKNTVYLQMDNLKPEDTAAYYCYTMGYWGQGTQVTVSSEPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA Sequence ID 89 (FLT3 dAb H5) QVQLQESGGGLVQAGGSLLSCAASGIFSTNYMVWCRQAPGKQRELVAAFTNDGGTLYADSLKGRFSISQDNAKNTVLLLMNSLKPEDTAVYYCCGLGHWGRGTKVTVSSEPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA Sequence ID 90 (FLT3 dAb D12) QVQLQESGGGLVQAGGSLRLLSCAASGSISSIRYMNWYRQAPGKQRESVAWITSSGSTNYADSVQGRFTISRDNAKNTVYLQMDNLKPEDTAVYYCYTMGYWGQGTQVTVSSEPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA Sequence ID 91 (FLT3 dAb F10) QAQVQLQESGGGLVQAGGSLRLSCAASGIFSTNHMAWYRQAPGKQRELVAAFTNDGSTLYGDSVKGRFVISRDNAKYTVFLQMNSLKPEDTAVYYGLGHWGQGTQVTVSSEPKTPKPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0168] Furthermore, the present invention provides the following: A CAR containing an FLT3 dAb as an antigen-binding domain; A nucleic acid sequence that codes for such a dAb or CAR.

[0169] CLL1 Human C-type lectin-like molecule-1 (CLL-1, MICL, or CLEC12A) is a type II transmembrane glycoprotein and a member of a large family of C-type lectin-like receptors involved in immune regulation. The intracellular domain of CLL-1 contains an ITIM motif and a PI-3 kinase binding site. The expression pattern of CLL-1 in hematopoietic cells is restricted and is particularly observed in myeloid cells derived from peripheral blood and bone marrow. The amino acid sequence of human CLL1 is available from Uniprot acceptance number Q5QGZ9.

[0170] Several antibodies against CLL-1 are described, for example, in WO2009051974, WO2013169625, WO2016205200, and WO2016040868.

[0171] The present invention provides a domain antibody (dAb) that binds to CLL1 and contains the following complementarity-determining region: (i) CDR1-GFTFGNHD (Sequence ID 48); CDR2-IDSGGNVI (Sequence ID 49); CDR3-ATDLDSGAESLESVY (Sequence ID 50); (ii) CDR1-GFAFGSAD (Sequence ID 51); CDR2-IDSGGNTQ (Sequence ID 52); CDR3-TDLDPTTDSLENVY (Sequence ID 53); (iii) CDR1-GRTFSAYF (Sequence ID 54); CDR2-INWNGDSS (Sequence ID 55); CDR3-AADTHGAVGLGSERLYDY (Sequence ID 56); (iv) CDR1-GIGVSSTG (Sequence ID 57); CDR2-IDRDGTT (Sequence ID 58); CDR3-TVVGDYY (Sequence ID 59); (v)CDR1-GFIFGNYD(sequence number 60);CDR2-ISSGGNDI(sequence number 61);CDR3-AADLDPGTDSLDNIH(sequence number 62);or (vi)CDR1-GFTLDYYA(SEQ ID NO: 63);CDR2-ISSSDGST(SEQ ID NO: 64);CDR3-AEAVYYAGVCVAMYDS(SEQ ID NO: 65). Anti-CLL-1 dAb may contain one of the sequences shown in SEQ ID NOs. 66, 67, 68, 69, 70, or 71.

[0172] Sequence ID 66 (CLL-1 dAb 44548) QVQLQQSGGGLVQPGGSLRLSCVGSGFTFGNHDMSWVRQAPGKEVEFVAGIDSGGNVIVYEEVVKGRFTISRDNAKNTLYLQMDGLKPEDAGMYFCATDLDSGAESLESVYHGQGTQVTVSS Sequence ID 67 (CLL-1 dAb 44544) QVQLQESGGGLVESGGSLRISCTGFGFAFGSADMSWVRQAPGKEVEFVAGIDSGGNTQTYEDTVKGRFTISRDNAKNTLYLQMNSLQSEDAGVYFCATDLDPTTDSLENVYHGQGTQVIVSS Sequence ID 68 (CLL-1 dAb 44538) QVQLQESGGGLVQTGDSLRLSCVASGRTFSAYFMGWFRQAPGKEREFVSAINWNGDSSWYRDSVKGRFTVSRDNAKNTVYLQMNSLEPEDTAVYYCAADTHGAVGLGSERLYDYWGQGTQVTVSS SEQ ID NO: 69 (CLL-1 dAb 44546) QVQLQESGGGVVQAGGSLRLSCAVSGIGVSSTGMGWSRQTPGKQVELVALIDRDGTTNYADTVKGRFTISKDNSKNMVYLQMNSLKPEDTALYHCTVVGDYYWGQGTQVTVSS SEQ ID NO: 70 (CLL-1 dAb 44545) QVQLQQSGGGLVQPGGSLRLSCVGSGFIFGNYDMSWVRQAPGKEVEFVAGISSGGNDIVYEDAVKGRFSISRDNARNTVYLDMASVKPEDAGVYYCAADLDPGTDSLDNIHHGQGTQVFVSS SEQ ID NO: 7l (CLL-1 dAb 44536) QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTAYADSVKGRFTISRDNAKNSVYLQMNSLKPEDTAVYYCAEAVYYAGVCVAMYDSWGQGTQVTVSS

[0173] Furthermore, the present invention provides the following: A CAR comprising such a CD123 dAb as an antigen-binding domain; A nucleic acid sequence encoding such a dAb or CAR.

[0174] Spacer Classical CARs contain a spacer sequence that connects the antigen-binding domain to the transmembrane domain and spatially separates the antigen-binding domain from the endodomain. The flexible spacer allows the antigen-binding domain to be oriented in different directions to facilitate binding.

[0175] A spacer can be used to dimerize two CAR-forming polypeptide chains. The two polypeptide chains may contain, for example, one or more cysteine ​​residues suitable for forming disulfide crosslinks (one or more). Commonly used spacers include the IgG1 Fc region, the IgG1 hinge, or the human CD8 stalk. The hinge spacer may contain the sequence shown in Sequence ID No. 92.

[0176] Sequence ID 92 (Hinge Spacer) EPKSCDKTHTCPPCP

[0177] Spacers can be selected to suit the target antigen (i.e., the position and orientation of the epitope on the target antigen, as well as the distance of the target epitope from the target cell membrane). In OR gates, different spacers can be used to suit different relative positions of the target epitopes and to avoid cross-pairing between the two CARs.

[0178] transmembrane domain A transmembrane domain (TM) is a part of a CAR that crosses the membrane. A TM can be any protein structure that is thermodynamically stable within the membrane. Typically, it is an α-helix composed of several hydrophobic residues. The TM domain of any TM protein can be used to supply the TM portion of a chimeric receptor. The sequence and full length of a protein's TM domain can be determined by those skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Alternatively, an artificially designed TM domain may be used.

[0179] End domain The endodomain is the signaling portion of the CAR. The endodomain may be part of the intracellular domain of the CAR, or it may associate with the intracellular domain. After antigen recognition, the receptors cluster, the native CD45 and CD148 are excluded from the synapse, and the signal is transmitted to the cell. The most commonly used endodomain component is the endodomain component of CD3-zeta, which contains three ITAMs. This transmits an activation signal to T cells after binding to the antigen. CD3-zeta may not provide a sufficiently qualified activation signal, and further co-stimulatory signaling may be required. Co-stimulatory signals promote T cell proliferation and survival. There are two main types of co-stimulatory signals: those belonging to the Ig family (CD28, ICOS) and those belonging to the TNF family (OX40, 41BB, CD27, GITR, etc.). For example, chimeric CD28 and OX40 can be used with CD3-zeta to transmit proliferation / survival signals, or all three can be used together.

[0180] The end domain may include the following: (i) ITAM-containing end domains (such as CD3 zeta-derived end domains); and / or (ii) Co-stimulatory domains (such as CD28 or ICOS-derived end domains); and / or (iii) Domains that transmit survival signals (e.g., TNF receptor family endodomains (OX-40, 4-1BB, CD27, or GITR, etc.)).

[0181] Several systems have been described in which the antigen recognition portion resides on a separate molecule derived from the signal transduction portion (e.g., those described in WO015 / 150771; WO2016 / 124930; and WO2016 / 030691). Therefore, the cells of the present invention can express a CAR system comprising an antigen-binding component containing one or more antigen-binding domains and a transmembrane domain (which can interact with individual intracellular signal transduction components containing signal transduction domains).

[0182] Since CARs can contain signal peptides, when the signal peptide is expressed inside the cell, the newly synthesized protein moves to the endoplasmic reticulum and then to the cell surface where it is expressed. The signal peptide can be located at the amino terminus of the molecule.

[0183] Suicide gene Furthermore, the cells of the present invention may express suicide genes.

[0184] Suicide genes are genetically encoded mechanisms that allow for the selective destruction of adoptive cells, such as T cells, when faced with unacceptable toxicity (e.g., on-target off-tumor toxicity, cytokine release syndrome (CRS), or neurotoxicity).

[0185] When treating acute myeloid leukemia (AML) using the cells of the present invention, the treatment may cause the patient to develop persistent or permanent myelopyesfunction. It is possible to rescue the patient from this condition using allogeneic transplantation, such as allogeneic hematopoietic stem cell transplantation (alloHSCT). Furthermore, the incorporation of suicide genes into CAR-expressing cells allows for the removal of CAR-expressing cells to prevent CAR-mediated graft rejection when using grafts.

[0186] The cells of the present invention may contain one of the suicide genes previously tested in clinical studies (such as herpes simplex virus thymidine kinase (HSV-TK) or inducible caspase 9 (iCasp9)).

[0187] WO2013 / 153391 describes a compact sort-suicide gene containing a CD20 epitope that can selectively kill cells expressing a polypeptide using rituximab. The cells of the present invention may express a suicide gene having the sequence shown in SEQ ID NO: 93.

[0188] Sequence ID 93 CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

[0189] WO2016 / 135470 describes a suicide gene that dimerizes in the presence of a dimerization-inducing chemical (CID), such as rapamycin or a rapamycin analog, that causes caspase-induced apoptosis in cells.

[0190] The suicide gene may have the following structure: Ht1-HT2-Casp Here, Ht1 and Ht2 are heterodimerization domains, one of which contains the FK506-binding protein (FKBP) and the other contains the FRB domain of mTOR; and Casp is the caspase 9 domain.

[0191] The suicide gene may have the sequence shown in SEQ ID NO: 94 or a variant thereof having 90, 95, or 99% sequence identity.

[0192] SEQ ID NO: 94 (FRB-FKBP12-L3-dCasp9)

Chemical formula

[0193] Nucleic acid construct The nucleic acid sequence encoding the CAR may have the following structure: AgB-spacer-TM-endo Here, AgB is a nucleic acid sequence encoding the antigen-binding domain of the CAR; spacer is a nucleic acid sequence encoding the spacer of the CAR; TM is a nucleic acid sequence encoding the transmembrane domain of the CAR; endo is a nucleic acid sequence that encodes the end domain of CAR.

[0194] The nucleic acid sequence encoding tanCAR may have the following structure: AgB1-linker-AgB2-spacer-TM-endo Here, AgB1 is a nucleic acid sequence that encodes the first antigen-binding domain of tanCAR; The linker is the nucleic acid sequence that codes for the linker of tanCAR; AgB2 is a nucleic acid sequence that encodes the second antigen-binding domain of tanCAR; The spacer is the nucleic acid sequence that codes for the spacer in tanCAR; TM is a nucleic acid sequence that encodes the transmembrane domain of tanCAR; endo is a nucleic acid sequence that encodes the endo-domain of tanCAR.

[0195] The nucleotide sequence encodes a polypeptide that, when expressed in cells, expresses a first and second antigen-binding domain in tandem on the cell surface.

[0196] The linker may be, or may include, a Gly-Ser movable linker.

[0197] The antigen-binding domain(s) of a CAR or tanCAR may be, for example, scFv(s) or dAb(s).

[0198] The present invention provides a nucleic acid construct that encodes a triple OR gate containing three CARs.

[0199] A nucleic acid construct encoding a triple OR gate may have the following structure: AgBD1-spacer1-TM1-endo1-coexpr1-AgBD2-spacer2-TM2-endo2-coexpr2-AgBD3-spacer3-TM3-endo3 Here, AgBD1 is a nucleic acid sequence that encodes the antigen-binding domain of the first CAR; spacer1 is a nucleic acid sequence that encodes the spacer of the first CAR; TM1 is a nucleic acid sequence that encodes the transmembrane domain of the first CAR; endo1 is a nucleic acid sequence that encodes the end domain of the first CAR; coexpr1 and coexpr2 may be the same or different, and are nucleic acid sequences that enable the simultaneous expression of the first, second, and third CARs; AgBD2 is a nucleic acid sequence that encodes the antigen-binding domain of the second CAR; spacer2 is a nucleic acid sequence that encodes the spacer for the second CAR; TM2 is a nucleic acid sequence that encodes the transmembrane domain of the second CAR; endo2 is a nucleic acid sequence that encodes the end domain of the second CAR; AgBD3 is a nucleic acid sequence that encodes the antigen-binding domain of the third CAR; spacer3 is a nucleic acid sequence that encodes the spacer for the third CAR; TM3 is a nucleic acid sequence that encodes the transmembrane domain of the third CAR; endo3 is a nucleic acid sequence that encodes the end domain of the third CAR.

[0200] The antigen-binding domains of the first, second, and third CARs may be, for example, scFv or dAb. In particular, all three CARs may have a dAb antigen-binding domain.

[0201] The present invention provides a nucleic acid construct that encodes a quadruple OR gate containing four CARs.

[0202] A nucleic acid construct encoding a quadruple OR gate may have the following structure: AgBD1-spacer1-TM1-endo1-coexpr1-AgBD2-spacer2-TM2-endo2-coexpr2-AgBD3-spacer3-TM3-endo3-coexpr3-AgBD4-spacer4-TM4-endo4 Here, AgBD1 is a nucleic acid sequence that encodes the antigen-binding domain of the first CAR; spacer1 is a nucleic acid sequence that encodes the spacer of the first CAR; TM1 is a nucleic acid sequence that encodes the transmembrane domain of the first CAR; endo1 is a nucleic acid sequence that encodes the end domain of the first CAR; coexpr1, coexpr2, and coexpr3 may be the same or different, and are nucleic acid sequences that enable the simultaneous expression of the first, second, third, and fourth CARs; AgBD2 is a nucleic acid sequence that encodes the antigen-binding domain of the second CAR; spacer2 is a nucleic acid sequence that encodes the spacer for the second CAR; TM2 is a nucleic acid sequence that encodes the transmembrane domain of the second CAR; endo2 is a nucleic acid sequence that encodes the end domain of the second CAR; AgBD3 is a nucleic acid sequence that encodes the antigen-binding domain of the third CAR; spacer3 is a nucleic acid sequence that encodes the spacer for the third CAR; TM3 is a nucleic acid sequence that encodes the transmembrane domain of the third CAR; endo3 is a nucleic acid sequence that encodes the end domain of the third CAR; AgBD4 is a nucleic acid sequence that encodes the antigen-binding domain of the fourth CAR; spacer4 is a nucleic acid sequence that codes for the spacer of the fourth CAR; TM4 is a nucleic acid sequence that encodes the transmembrane domain of the fourth CAR; and endo4 is a nucleic acid sequence that encodes the end domain of the fourth CAR.

[0203] The antigen-binding domains of the first, second, third, and fourth CARs may be, for example, scFv or dAb. In particular, all four CARs may have a dAb antigen-binding domain.

[0204] Furthermore, the present invention provides nucleic acid constructs encoding scFv / dAb CARs and tanCARs. In this embodiment, the nucleic acid construct has the following structure: AgB1-linker-AgB2-spacer1-TM1-endo1-coexpr-AgB3-spacer2-TM2-endo2 Here, AgB1 is a nucleic acid sequence that encodes the first antigen-binding domain of tanCAR; The linker is the nucleic acid sequence that codes for the linker of tanCAR; AgB2 is a nucleic acid sequence that encodes the second antigen-binding domain of tanCAR; spacer1 is the nucleic acid sequence that encodes the spacer of tanCAR; TM1 is a nucleic acid sequence that encodes the transmembrane domain of tanCAR; endo1 is a nucleic acid sequence that encodes the endodomain of tanCAR; coexpr is a nucleic acid sequence that allows for the simultaneous expression of CAR and tanCAR; AgB3 is a nucleic acid sequence that encodes the antigen-binding domain of CAR. spacer2 is a nucleic acid sequence that codes for the CAR spacer; TM2 is a nucleic acid sequence that encodes the transmembrane domain of CAR; endo2 is a nucleic acid sequence that encodes the endodomain of CAR; Also, the following structure: AgB1-spacer1-TM1-endo1-coexpr-AgB2-linker-AgB3-spacer2-TM2-endo2 Here, AgB1 is a nucleic acid sequence that encodes the antigen-binding domain of CAR. spacer1 is a nucleic acid sequence that codes for the CAR spacer; TM1 is a nucleic acid sequence that encodes the transmembrane domain of CAR; endo1 is a nucleic acid sequence that encodes the end domain of CAR; coexpr is a nucleic acid sequence that allows for the simultaneous expression of CAR and tanCAR; AgB2 is a nucleic acid sequence that encodes the first antigen-binding domain of tanCAR; The linker is the nucleic acid sequence that codes for the linker of tanCAR; AgB3 is a nucleic acid sequence that encodes the second antigen-binding domain of tanCAR; spacer2 is the nucleic acid sequence that encodes the spacer of tanCAR; TM2 is a nucleic acid sequence that encodes the transmembrane domain of tanCAR; endo2 is a nucleic acid sequence that encodes the endodomain of tanCAR. It may have.

[0205] Furthermore, the present invention provides nucleic acid constructs encoding two tanCARs. In this embodiment, the nucleic acid constructs may have the following structure: AgB1-linker1-AgB2-spacer1-TM1-endo1-coexpr-AgB3-linker2-AgB4-spacer2-TM2-endo2 Here, AgB1 is a nucleic acid sequence that encodes the first antigen-binding domain of the first tanCAR; linker1 is the nucleic acid sequence that encodes the linker of the first tanCAR; AgB2 is a nucleic acid sequence that encodes the second antigen-binding domain of the first tanCAR; spacer1 is the nucleic acid sequence that encodes the spacer of the first tanCAR; TM1 is a nucleic acid sequence that encodes the transmembrane domain of the first tanCAR; endo1 is a nucleic acid sequence that encodes the end domain of the first tanCAR; coexpr is a nucleic acid sequence that allows for the simultaneous expression of the first and second tanCARs; AgB3 is a nucleic acid sequence that encodes the first antigen-binding domain of the second tanCAR; linker2 is the nucleic acid sequence that codes for the second tanCAR linker; AgB4 is a nucleic acid sequence that encodes the second antigen-binding domain of the second tanCAR; spacer2 is the nucleic acid sequence that encodes the second tanCAR spacer; TM2 is a nucleic acid sequence that encodes the second transmembrane domain of tanCAR; endo2 is a nucleic acid sequence that encodes the end domain of the second tanCAR.

[0206] Furthermore, the nucleic acid construct of the present invention may include a nucleic acid sequence that codes for a suicide gene.

[0207] As used herein, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” are intended to be synonymous with each other.

[0208] Those skilled in the art will understand that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, those skilled in the art should understand that, using routine techniques, it is possible to substitute nucleotides that do not affect the polypeptide sequence encoded by the polynucleotides described herein in order to reflect the codon usage frequency of any particular host organism in which the polypeptide is expressed.

[0209] The nucleic acids of the present invention may comprise DNA or RNA. The nucleic acids of the present invention may be single-stranded or double-stranded. The nucleic acids of the present invention may also be polynucleotides comprising nucleotides synthesized or modified within the nucleic acids of the present invention. Several different types of modifications to oligonucleotides are known in the art. These modifications include methylphosphonate and phosphorothioate skeletons, and the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. It should be understood that for the uses described herein, polynucleotides may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity and extend the lifespan of the polynucleotide of interest.

[0210] The terms “variant,” “homogram,” or “derivative” relating to a nucleotide sequence include any substitution, modification, alteration, replacement, deletion, or addition of one (or more) nucleic acids to or from the sequence.

[0211] In the above structure, "coexpr" is a nucleic acid sequence capable of co-expressing two polypeptides as separate entities. coexpr may be a sequence encoding a cleavage site so that the nucleic acid construct produces both polypeptides linked by the cleavage site(s). The cleavage site(s) may be self-cleaving so that when the polypeptides are produced, they are immediately cleaved into individual peptides without requiring any external cleavage activity.

[0212] The cleavage site can be any sequence that allows the two polypeptides to separate.

[0213] The term "cleavage" is used herein for convenience, but depending on the cleavage site, peptides can be separated into individual entities by mechanisms other than classical cleavage. For example, for the foot-and-mouth disease virus (FMDV) 2A autocleavage peptide (see below), various models have been proposed to explain the following "cleavage" activity: proteolytic activity by host cell proteinases, autoproteolytic activity, or translational effect (Donnelly et al. (2001) J. Gen. Virol. 82:1027-1041). The exact mechanism of such "cleavage" is not important to the purposes of the present invention, as long as the protein is expressed as an individual entity when the cleavage site is located between protein-coding nucleic acid sequences.

[0214] The cleavage sites may be, for example, furin cleavage sites, tobacco ecchi disease virus (TEV) cleavage sites, or they may encode self-cleaving peptides.

[0215] A "self-cleaving peptide" refers to a peptide that, when a polypeptide containing a protein or self-cleaving peptide is produced, functions to be immediately "cleaved" or separated into individual and isolated first and second polypeptides without requiring any external cleavage activity.

[0216] Self-cleaving peptides can be 2A self-cleaving peptides derived from aphthoviruses or cardioviruses. Primary 2A / 2B cleavage in aphthoviruses and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In aphthoviruses (such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus), the 2A region is a short segment of about 18 amino acids that, together with the N-terminal residue (conserved proline residue) of protein 2B, exhibits an autonomous element capable of mediating its own C-terminus "cleavage" (Donelly et al. (2001) above).

[0217] "2A-like" sequences have been found in picornaviruses other than aftviruses or cardioviruses, "picornavirus-like" insect viruses, type C rotaviruses, and repetitive sequences in Trypanosoma spp and bacterial sequences (Donnelly et al. (2001) above).

[0218] The cleavage site may contain a 2A-like sequence as shown in sequence number 95. Sequence ID 95: RAEGRGSLLTCGDVEENPGP

[0219] vector Furthermore, the present invention provides a vector or a kit of vectors comprising one or more nucleic acid sequences(s) encoding one or more chimeric antigen receptors(s) of the cells of the present invention. Using such vectors, nucleic acid sequences(s) can be introduced into host cells so that the host cells express CARs or each(s)(s).

[0220] The vector may be, for example, a plasmid or viral vector (such as a retroviral vector or lentiviral vector), or a transposon-based vector or synthetic mRNA.

[0221] The vector may be transfected or transduced into cells such as T cells or NK cells.

[0222] cell The present invention provides cells containing multiple chimeric antigen receptors.

[0223] The cells could be cytolytic immune cells (such as T cells or NK cells).

[0224] T cells, or T lymphocytes, are a type of lymphocyte that plays a central role in cellular immunity. They can be distinguished from other lymphocytes (such as B cells and natural killer cells (NK cells)) by the presence of T cell receptors (TCRs) on their cell surface. Various types of T cells exist, as summarized below.

[0225] Helper T cells (TH cells) assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes (TH1, TH2, TH3, TH17, Th9, or TFH) that secrete different cytokines to facilitate different types of immune responses.

[0226] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected cells and tumor cells and are also involved in graft rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of all nucleated cells. CD8+ cells can be inactivated into an anergic state via IL-10, adenosine, and other molecules secreted by regulatory T cells, thereby preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0227] Memory T cells are a subset of antigen-specific T cells that are maintained over a long period after recovery from infection. Memory T cells rapidly expand into numerous effector T cells upon re-exposure to their congener antigen, thus "remembering" past infections in the immune system. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0228] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their main roles are to suspend T-cell immunity toward the termination of the immune response and to suppress autoreactive T cells that have evaded negative selection processes within the thymus.

[0229] Two main classes of CD4+ Treg cells (endogenous Treg cells and adaptive Treg cells) are described.

[0230] Endogenous Treg cells (also known as CD4+CD25+FoxP3+Treg cells) originate in the thymus and are involved in the interaction of developing T cells with both myeloid (CD11c+) dendritic cells and plasmacytoid (CD123+) dendritic cells activated by TSLP. Endogenous Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can inhibit the development of regulatory T cells and can lead to the fatal autoimmune disease IPEX.

[0231] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0232] The cells may be natural killer cells (or NK cells). NK cells can form part of the innate immune system. NK cells respond rapidly to endogenous signals from virus-infected cells in an MHC-dependent manner.

[0233] NK cells (belonging to the congenital lymphoid cell group) are defined as large granular lymphocytes (LGLs) and constitute a third cell type differentiated from common lymphoid progenitor cells that produce B lymphocytes and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus before entering the circulation.

[0234] The cells of the present invention may be any of the cell types described above.

[0235] T cells or NK cells according to the first aspect of the present invention can be generated ex vivo in hematopoietic stem cell transplantation from the patient's own peripheral blood (first party), or from donor peripheral blood (second party) or peripheral blood from an unrelated donor (third party).

[0236] Alternatively, the T cells or NK cells of the first aspect of the present invention may be derived from the ex vivo differentiation of induced progenitor cells or embryonic progenitor cells into T cells or NK cells. Alternatively, immortalized cells that retain lytic function and can act as therapeutic agents. T cell lines may be used.

[0237] In all of these embodiments, chimeric polypeptide-expressing cells are generated by introducing DNA or RNA encoding the chimeric polypeptide by one of several means (including transduction using a viral vector, transfection using DNA or RNA).

[0238] The cells of the present invention may be subject-derived ex vivo T cells or NK cells. The T cells or NK cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The T cells or NK cells may be activated and / or proliferated by treatment with, for example, an anti-CD3 monoclonal antibody before being transduced with a nucleic acid encoding a molecule that yields the chimeric polypeptide of the first aspect of the present invention.

[0239] The T or NK cells of the present invention can be produced by the following: (i) Isolation of samples containing T or NK cells from the subjects listed above or other sources; and (ii) Transduction or transfection of T cells or NK cells using nucleic acid constructs, vectors, or vector kits of the present invention.

[0240] Subsequently, T or NK cells can be purified (for example, they can be selected based on the expression of the antigen-binding domain of the antigen-binding polypeptide).

[0241] Pharmaceutical composition The present invention also relates to a pharmaceutical composition comprising a plurality of cells according to the present invention.

[0242] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such formulations may be in a form suitable for, for example, intravenous infusion.

[0243] Treatment method The present invention provides a method for treating a disease, comprising the step of administering the cells of the present invention (for example, in the pharmaceutical composition described above) to a subject.

[0244] The method for treating diseases relates to the therapeutic use of cells of the present invention. In this specification, cells may be administered to a subject already having a disease or condition in order to alleviate, reduce, or improve at least one symptom associated with the disease, and / or to delay, reduce, or block the progression of the disease.

[0245] This method may include the following steps: (i) A step of isolating a sample containing T or NK cells; (ii) Transfecting or transfecting such cells with a nucleic acid sequence or vector provided by the present invention; (iii)(ii) A step of administering cells derived from (ii) to the subject.

[0246] Samples containing T cells or NK cells can be isolated, for example, from the subject or other sources mentioned above. T cells or NK cells can be isolated from the patient's own peripheral blood (first source), or from donor peripheral blood (second source) or peripheral blood from an unrelated donor (third source) in hematopoietic stem cell transplantation.

[0247] The present invention may include the following steps: (i) A step of administering cells according to the first aspect of the present invention to a subject; (ii) The step of administering allogeneic hematopoietic stem cell grafts (alloHSCT) to the subject as described above.

[0248] For example, the present invention may include the following steps: (i) A step of administering cells according to the first aspect of the present invention to a subject; (ii) The process of monitoring the bone marrow dysplasia of the subject as described above. (ii) If bone marrow malformation is detected, administer an allogeneic hematopoietic stem cell graft (alloHSCT) to the subject as described above.

[0249] Myeloid metaplasia is a clinical and pathological syndrome characterized by extramedullary hematopoiesis, which constantly occurs in the spleen and almost always in the liver, splenomegaly (usually hepatomegaly), and anemia accompanied by immature red and white blood cells in the peripheral blood.

[0250] The present invention provides cells for use in the treatment and / or prevention of disease.

[0251] The present invention also relates to the use of the cells of the present invention in the manufacture of pharmaceuticals for the treatment and / or prevention of disease.

[0252] The disease to be treated by the acute myelocytic leukemia (AML) method of the present invention may be a cancerous disease. In particular, the aforementioned disease may be acute myelocytic leukemia (AML).

[0253] Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells, characterized by the rapid growth of abnormal cells that develop in the bone marrow and blood, interfering with normal blood cells. Symptoms may include fatigue, shortness of breath, easy bruising and bleeding, and an increased risk of infection. Diagnosis is usually based on bone marrow aspiration and blood tests. As an acute leukemia, AML progresses rapidly, and if left untreated, death typically occurs within weeks or months.

[0254] The cells of the present invention may be capable of killing target cells such as cancer cells. Target cells can be characterized by the expression of one or more target antigens, such as one, two, three, or all four of the following: CD33, CD123, CLL-1, and FLT3.

[0255] The cells and pharmaceutical compositions of the present invention may be used for the treatment and / or prevention of the above-mentioned diseases.

[0256] The present invention will be further illustrated here by examples, which are intended to assist those skilled in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. [Examples]

[0257] Example 1 - Design and generation of cells expressing single CAR and triple OR gates Generate a panel of nucleic acid constructs that encode CARs as follows: Single CAR construct expressing a CD123 CAR with an RQR8-2A-V5CD123CAR-V5 tag Single CAR construct expressing an FLT3 CAR with the RQR8-2A-V5FLT3CAR-V5 tag Single CAR construct expressing a CD33 CAR with the RQR8-2A-HACD33CAR-HA tag Single CAR construct expressing a CLL1 CAR with the RQR8-2A-FLAGCLL1CAR-FLAG tag. Triple CAR construct expressing a CD123 CAR with a RQR8-2A-V5 tag; a CD33 CAR with an HA tag; and a CLL1 CAR with a FLAG tag. Triple CAR construct expressing an FLT3 CAR with a RQR8-2A-V5 tag; a CD33 CAR with an HA tag; and a CLL1 CAR with a FLAG tag.

[0258] All constructs co-express the sort-suicide gene RQR8, as described in WO2013 / 153391.

[0259] All CARs are dAb CARs that have a second-generation endodomain containing CD3ζ and 4-1BB co-stimulation domains.

[0260] Peripheral blood-derived CD4+ and CD8+ T cells were transduced with constructs. Single CAR expression was detected by co-staining of T cells with CAR-specific tag antibodies (against V5, HA, or FLAG) and QBEND10 (to detect RQR8 expression). Figure 3 shows the expression of aCD33 CAR. Figure 4 shows the expression of aCD123 CAR. Figure 5 shows the expression of aCLL1 CAR.

[0261] Triple CAR expression is detected by simultaneous staining of T cells with all three CAR-specific tag antibodies (against V5, HA, and FLAG) and QBEND10 (to detect RQR8 expression).

[0262] Example 2 - FACS-based kill assay (FBK) The ability of cells expressing single CARs to kill target cells expressing individual antigens CD123, CLL1, and CD33 was investigated using a FACS-based cell death assay. This assay was performed using SupT1 cell lines engineered to express the desired antigens and literature-derived human patient cell lines (Molm-14, KG1α, HL-60, K562, and THP-1) whose pre-use antigen expression homogeneity had been investigated (Figures 6 and 7).

[0263] T cells were co-cultured with target cells in a 1:1 ratio. 5 × 10⁶ cells per well. 4 Transduced T cells and target cells were used in ratios of 1:1, 1:2, 1:4, or 1:8 in a 96-well plate with a total volume of 0.2 ml for the assay. Co-cultures were prepared after normalization of transduction efficiency. FBK was performed 24 or 48 hours after incubation.

[0264] The FBK results are shown in Figures 8 and 9 for aCD33, Figures 10 and 11 for aCD123, and Figures 12 and 13 for aCLL1.

[0265] All single aCD33 dab CARs showed potent cytotoxic activity against each CD33-expressing AML cell compared to the negative control. Dose-response death was as expected, with higher effector-target ratios (1:1) resulting in more target death. The aCD19 FMC63 CAR showed some levels of low-background cytotoxic activity in some cells. No significant differences in cytotoxicity levels were observed among the CD33 single-domain CARs. In all cells, a death response of approximately 50–60% was observed, even using the lowest E:T ratio.

[0266] CD123-VHH-CAR-2 showed a significant difference over CD123-VHH-CAR_1 at 1:2 and 1:4 ratios compared to SupT1 CD123 (p=0.0205 N=4 and p=0.0012 n=4, respectively). On the other hand, only CD123-VHH-CAR_2 showed a significant difference over CD123-VHH-CAR_3 compared to SupT1 CD123 at 1:4 (p=0.0194, n=4). For co-cultures maintained for 24 hours, potent CD123-specific cytotoxic activity was observed across all CD123 CARs, and there were no significant differences between constructs at any ratio (Figure 10). However, significant antigen-independent basal cytotoxicity was observed in CD123- / SupT1 NT cells only at the 1:1 ratio. As expected, it showed a dose-response behavior in which target survival increased as the E:T ratio increased. All constructs exhibited 70% targeted lysis in SupT1 CD123 and KG1a cell lines in the absence of basal antigen-independent cytotoxicity (E:T, 1:2). Significant nonspecific cytotoxicity against SupT1 NT was observed for all constructs in 48-hour co-cultures at 1:1 and 1:2 ratios. At 1:8, no significant basal cytotoxicity against CD123- / SupT1 NT was observed for any CAR construct, and all CAR constructs achieved 70% targeted lysis against Molm14 and THP1 (Figure 11).

[0267] No significant differences were observed for SupT1 CLL1 with any VHH binder exhibiting potent CLL1-specific cytotoxicity (Figure 12). This trend was observed across all E:T ratios, and single VHH cells did not show increased activity. Basal activity was significant for all CARs incubated with CLL1- / SupT1 cells at a 1:1 ratio for 48 hours, with only CLL1-VHH-CAR-3 showing significant basal activity at an E:T ratio of 1:2 (p=0.0425, n=7). All CARs exhibited CLL-specific cytotoxicity when incubated with THP1 and KG1a cells, and VHH-CARs 2 and 5 increased VHH CAR death (Figure 13). The only CAR showing significant CLL1-specific cytotoxicity to THP1 was CAR-5, more so than CAR-3 at an E:T ratio of 1:2 (p=0.0133, n=7). There was no significant difference in CLL1-specific cytotoxicity between CAR2 and CAR5 across all ratios using all cell lines.

[0268] Example 3 - Cytokine Release IL-2 secretion is an indicator of T cell activation and was evaluated using the supernatant of a cytotoxic co-culture assay. Cytokine secretion for single CARs was analyzed along with related controls to investigate the production of IL-2 and IFNγ as markers of T cell activation. IL-2 and IFNγ production was detected by ELISA.

[0269] The results of the cytokine release assays are shown in Figures 14 and 15 for aCD33, in Figure 16 for aCD123, and in Figure 17 for aCLL1.

[0270] When aCD33 CAR T cells were exposed to CD33-positive cells, higher IL-2 secretion was observed. All aCD33 sdAb CAR T cells produced relatively more IL-2 than non-transduced controls and negative controls. IL-2 secretion levels were relatively low when using the aCD33 scFv positive control. When using the FMC63 negative control, IL-2 production levels were similar to those of the aCD33 scFv positive control. IFNγ production for aCD33 CAR T cells was significantly higher than that of the negative control. All aCD33 sdAb CARs produced similar levels of IFN-γ in both engineered SupT1 cells and AML-derived cells (Figure 15). sdAb CD33.6 maintained higher levels of IFN-γ production in all cells. In contrast, IFN-γ production of sdAb CD33.2 was lower compared to all other sdAb CD33 CARs.

[0271] IFN-γ levels produced by aCD33 scFv were lower when challenged in THP1 cells and MOLM14 cells. The difference in IFN-γ production between engineered SupT1-derived cells and AML-derived cells was not very large. Furthermore, FMC63 scFv negative controls produced the same level of IFN-γ (approximately 10,000 pg / ml) as aCD33 scFv positive controls on MOLM14 cells.

[0272] All aCD123 CAR T cells produced IL-2 when exposed to CD123+ cells, but there was no significant difference in IL-2 production compared to the VHH-CAR-T cell construct. VHH-CAR-2 and 6 produced the highest levels of IL-2 in Molm14 (mean approximately 1.77 × 10⁴ and 1.63 × 10⁴ pg / ml), while VHH-CAR-4 produced the lowest levels of IL-2 in THP1 (mean approximately 3.5 × 10³ pg / ml). Despite no significant differences observed in cytotoxicity assays over both 24 and 48 time courses, the VHH-CAR-construct showed improved IL-2 production compared to the scFv CAR positive control.

[0273] All aCD123 CARs produced similar levels of IFNγ compared to SupT1 CD123 and Molm14 (mean approximately 3.4 × 10³ and 4.0 × 10⁴ pg / ml, respectively). There were no significant differences in IFNγ production among the CD123 CAR constructs across all constructs. Similar to IL-2 production, VHH-CAR-6 consistently produced higher cytokine levels across all constructs, but the differences were not significant. The scFv control CAR produced similar levels of IFNγ as the VHH CAR, which was consistent with the trend observed for targeted lysis in cytotoxic assays. IFNγ production was highest with VHH-CAR-6 using Molm14 (mean approximately 4.0 × 10⁴ pg / ml), while it was lowest with VHH-CAR-4 using THP1 (mean approximately 1.45 × 10³ pg / ml).

[0274] Antigen-specific IL-2 production was observed in all CLL1-VHH-CAR constructs using SupT1 CLL1 and THP1, but there were no significant differences between the constructs (Figure 17, a). However, despite the cell death data indicating antigen-specific cytotoxicity, antigen-specific IL-2 production was not observed in CARs co-cultured with KG1a. This finding can be associated with the very low expression level of CLL1 on the cell surface of KG1a cells (638 / cell, Figure 7). A similar trend was observed in IFNγ production, and SupT1 All CAR constructs showed antigen-specific IFNγ production for CLL1 and THP1, but there were no significant differences between constructs (Figure 17, b). In addition, KG1a produced relatively low levels of IFNγ, while CARs that functioned well in cytotoxic assays produced higher levels of cytokines (VHH-CAR 2, 4, and 5).

[0275] Example 4 - Growth assay (PA) To measure proliferation, the same panel of CAR-expressing T cells described in Example 1 was labeled with the dye Cell Trace Violet (CTV) (a fluorescent dye that is hydrolyzed and retained within cells). CTV is excited by a 405 nm (violet) laser, and its fluorescence can be detected in a Pacific blue channel. The CTV dye was reconstituted to 5 mM with DMSO. T cells were placed in PBS at a rate of 2 × 10⁶ 6 The cells were resuspended in 1 ml / ml of CTV, and 1 ul / ml of CTV was added. The T cells were incubated with CTV at 37°C for 20 minutes. Subsequently, the cells were quenched with 5V complete medium. After 5 minutes of incubation, the T cells were washed and resuspended in 2 ml of complete medium. Further incubation at room temperature for 10 minutes resulted in acetic acid hydrolysis, and the dye was retained.

[0276] Labeled T cells were co-cultured with target cells for 4 days. 5 × 10⁶ cells per well. 4 Assays were performed in 96-well plates with a total volume of 0.2 ml using transduced T cells and an equal number of target cells (ratio 1:1). At four time points on the same day, T cells were analyzed by flow cytometry to measure the dilution of CTV that occurred as T cells divided. The number of T cells present at the end of co-culture was calculated and expressed as a multiple compared to the number of T cells introduced.

[0277] The results of the proliferation assays are shown in Figure 18 for aCD33, Figure 19 for aCD123, and Figure 20 for aCLL1.

[0278] A significant difference in the expression level of aCD33 CAR T cells was observed at 6 days. A higher expression level was observed in aCD33 sdAb CAR T cells compared to non-transduced controls and negative controls (FMC63 scFv). It should be noted that the proliferation levels of positive controls (aCD33 scFv) were lower than those of aCD33 sdAb CAR T cells in all AML cells. CAR T cell expression levels were significantly higher in SupT1 cells artificially transduced with CD33 compared to other cells.

[0279] There were no significant differences among all CD123-VHH-CAR constructs across all conditions.

[0280] Antigen-specific proliferation was observed in all CLL1-VHH-CAR constructs using CLL1+ target cell lines; however, there was no significant difference in proliferation capacity between SupT1 CLL1 and THP1. For KG1a co-cultures, CLL1-VHH-CAR 2 and 5 were significantly more favorable to proliferation than CLL1-VHH-CAR 1, 2, and 3 across all ratios (p-values ​​ranged from <0.05 to <0.01, n=7). CLL1-VHH-CAR-5 was significantly more favorable to proliferation than CLL1-VHH-CAR-2 when using KG1a (p=0.0445, n=7).

[0281] All six aCD33 single-dab CARs showed similar levels of response to target cells in all proliferation assays, cytotoxicity assays, and cytokine production assays compared to the negative CAR control (FMC63 scFv). The sdAb CD123 CAR demonstrated dose-response efficacy against engineered SupT1 CD123 cells and other AML-derived cells (MOLM14, THP1, KG1a). The sdAb CLL1 CAR was also effective against CLL1+ cells. Results from the Phase I study demonstrated that each single-domain binder (CD123, CD33, and CLL1) effectively acted as a second-generation CAR against AML.

[0282] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention is described in conjunction with certain preferred embodiments, it should be understood that the invention described in the claims should not be unduly limited to such specific embodiments. In fact, various modifications of the style of description for carrying out the invention that will be apparent to those skilled in the art in molecular biology or related fields are intended to fall within the scope of the following claims.

Claims

[Claim 1] The object, method, or system described herein and in the drawings.

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