CAR T cells comprising anti-CD33, anti-CLL1 and at least one additional anti-CD123 and / or FLT3 CAR

Abstract

The present disclosure provides a cell comprising: an anti-CD33 chimeric antigen receptor (CAR); an anti-CLL1 CAR; and an anti-CD123 and / or anti-CAR FLT3 CAR. The cell can be used in the treatment of diseases such as acute myeloid leukemia (AML).

Description

Field of the Invention

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

[0003] Acute myeloid leukemia

[0004] 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 of all leukemias. In 2015, an estimated 20,830 people were diagnosed with AML in the United States and an estimated 10,460 died from AML. Long-term survival rates for AML have remained small over the past decade.

[0005] Current induction chemotherapy can produce an initial complete remission in almost 70% of young adult patients. However, 43% of patients will eventually relapse, and 18% of patients never achieve complete remission with frontline induction therapy. The 5-year overall survival rate for patients with AML after a first relapse ranges from 7% to 12%. Allogeneic hematopoietic stem cell transplantation (alloHSCT) offers the best chance of curing patients with relapsed or refractory AML. It is the preferred treatment route after a second remission, which can achieve 5-year disease-free survival in 40% to 50% of patients.

[0006] Using current treatment strategies, only approximately half of relapsed patients who previously achieved a complete remission lasting longer than 6 months achieve a second complete remission rate, and only 20% or less of patients with primary refractory disease and those whose initial complete remission lasted less than 6 months achieve a second complete remission rate. Additionally, the considerable morbidity of conventional salvage chemotherapy may worsen the patient's performance status and organ function and reduce the chances of successful alloHSCT.

[0007] Therefore, improved therapeutic approaches are needed to treat AML.

[0008] Chimeric Antigen Receptor

[0009] Chimeric antigen receptors (CARs) transfer the specificity of monoclonal antibodies onto the effector functions of T cells. CAR T-cell therapy targeting CD19 is highly effective in B-cell malignancies, although CD19-negative escape accounts for relapse in a significant proportion of patients.

[0010] CAR T cell therapy for AML is in early clinical testing. Although several preclinical studies have shown the cytotoxic potential of various CAR T cells targeting AML antigens, translation to the clinic has been slow. This highlights the inherent challenges in developing CAR-related therapeutic strategies for AML patients. To date, a small number of patients with relapsed / refractory AML have been treated with CAR T cell immunotherapy through early clinical trials, as shown in Table 1.

[0011] Table 1 : Current Phase I clinical trial of CAR T cell immunotherapy for patients with relapsed / refractory AML

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 -Schematic diagram illustrating hematopoiesis in the human body

[0014] Figure 2 -Different binding domain formats of chimeric antigen receptors

[0015] (a) Fab CAR format; (b) dAb CAR format; (c) scFv CAR format

[0016] Figure 3 - Expression of aCD33 CAR on primary T cells derived from three healthy donors. CAR transduced with T cells were stained with the RQR8 marker gene. FACS dot plots were pre-gated for live cell populations by using eFluor780.

[0017] Figure 4 -Transduction expression of the CAR construct used in aCD123-VHH CAR screening and validation assays.PBMCs were stained (Strep-PE secondary staining) with anti-CD34-PE antibodies (QBend10) or soluble human CD123 extracellular domains fused to 2x StrepTag2 mouse Fc, respectively.All dot plots were pre-gated on live single cells using a viability dye (FVD-eFlour780).

[0018] Figure 5 -Transduction expression of CAR constructs used in aCLL1-VHH CAR screening and validation assays.PBMCs were stained with anti-CD34-PE antibodies (QBend10) or soluble human CLL1 extracellular domains fused to 2x StrepTag2 human Fc (Strep-PE secondary staining).All images were pre-gated on live single cells using a viability dye (FVD-eFlour780).

[0019] Figure 6 - SupT1 transduced with retroviral vectors encoding relevant antigens. Antigen expression on CD123, CD33, and CLL1 transduced SupT1 cells is plotted in blue, while red peaks correspond to relevant isotype controls. TM Average antigen density (per cell) of beads within each transduced SupT1 cell.

[0020] Figure 7 - Flow cytometry using Quantibrite TM Bead analysis of antigen density. Antigen expression on target cells stained with CD33, CD123, and CLL1 (blue) compared to relevant isotype controls (red). The average antigen density (per cell) was measured for each antigen.

[0021] Figure 8 - Example of separation of T cells from target cells by using anti-CD3 antibody on SupT1 NT for aCD33 CAR.

[0022] Fig. 9 - Cytotoxicity assays were performed on different AML cells in three donors using all aCD33 CARs. Cytotoxicity was measured at different time intervals according to 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 standardized relative to non-transduced controls.

[0023] Fig.10 - 24-hour cytotoxicity assay for all CD123-VHH-CAR T cell constructs. Each condition was tested with at least 3 donors (n=3) and the median is indicated. Top row – SupT1 NT was used as target cells, middle row – SupT1 cells were engineered to express high levels of CD123, bottom row – AML-derived KG1α was used as target cell line. All data were normalized to non-transduced T cells, as they contribute only to antigen-independent cytotoxicity. CARs were compared by two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001.

[0024] Fig.11-48-hour cytotoxicity assays for all CD123-VHH-CAR T cell constructs. Each condition was tested with at least 3 donors (n=3), and the median was indicated. From left to right, target cell lines SupT1NT, THP1, and Molm14 were used as target cell lines. All data were normalized relative to non-transduced T cells because they only contribute to antigen-independent cytotoxicity.

[0025] Fig.12 - 24-hour cytotoxicity assay for all CLL1-VHH-CAR T cell constructs. Each condition was tested with at least 3 donors (n=3) and the median is indicated. Top row – SupT1 NT was used as target cells, bottom row – SupT1 cells were engineered to express high levels of CLL1. All data are normalized to non-transduced T cells, as they contribute only to antigen-independent cytotoxicity.

[0026] Fig.13 - 48 h cytotoxicity assay for all Cll1-VHH-CAR T cell constructs. Each condition was tested with at least 3 donors (n=3) and the median is indicated. Top row – SupT1 NT was used as target cells, middle row – THP1 patient-derived target cells, bottom row – KG1α patient-derived target cell line. All data were normalized to non-transduced T cells, as they contribute only to antigen-independent cytotoxicity. CARs were compared by two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns=not significant.

[0027] Fig.14 - IL-2 secretion by aCD33 CAR T cells co-cultured with target cells at a 1:1 E:T ratio in two donors (data not shown for donor 6). IL-2 production was measured using SupT1 cells engineered to express higher levels of CD33 and AML-derived cells.

[0028] Fig.15 - IFN-γ production by aCD33 CAR T cells in target cells at a 1:1 E:T ratio in both donors.

[0029] Figure 16- Cytokine measurements after 24 and 48 hours of cytotoxicity assays using CD123-VHH-CAR T cell constructs. Three donors were performed at a 1:8 ratio (n=3). a) IL-2 measurement b) IFN-γ measurement. CAR was compared by a two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns=no significance.

[0030] Figure 17- Cytokine measurements after 24 and 48 hours of cytotoxicity assays using CLL1-VHH-CAR T cell constructs. Three donors were performed at a 1:8 ratio (n=3). a) IL-2 measurement b) IFN-γ measurement. CARs were compared by two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns=no significance.

[0031] Fig.18 - Proliferation assay for each aCD33 CAR T cell for different cell lines. 12783 (aCD19 FMC63 scFv) was used as a negative control and 27983 (aCD33 scFv) was used as a positive control. Expansion folds were normalized to non-transduced controls. The assay was set up for 6 days at a 1:1 E:T ratio. The proliferation assay was set up in HL-60 cells with 2 donors.

[0032] Fig.19 -4-day proliferation assay for all CD123-VHH-CAR T cell constructs. Expansion folds were normalized to CD3+ non-transduced T cell conditions. Each condition was tested with at least 3 donors (n=3).

[0033] Fig. 20 -4-day proliferation assay for all CLL1-VHH-CAR T cell constructs. Expansion folds were normalized to CD3+ non-transduced T cell conditions. Each condition was tested with at least 3 donors (n=3). CARs were compared by two-way paired t-test. *p<0.05, **p<0.01, ***p<0.001, ns=not significant. SUMMARY OF THE INVENTION

[0035] In the first aspect, the present invention provides cells expressing CAR, which target multiple antigens related to acute myeloid leukemia (AML). The cells target three or four of the following antigens: CD33, CLL-1, CD123 and FLT3.

[0036] For example, the cell can comprise: an anti-CD33 chimeric antigen receptor (CAR); an anti-CLL1 CAR; and an anti-CD123 CAR.

[0037] The cells may comprise: an anti-CD33 chimeric antigen receptor (CAR); an anti-CLL1 CAR; and an anti-FLT3 CAR.

[0038] The cells may comprise: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-CD123 CAR; and anti-FLT3 CAR.

[0039] One or more, or all, of the CARs may comprise a domain antibody (dAb) antigen binding domain.

[0040] The cell may comprise one or more tandem chimeric antigen receptors (tanCARs). The tanCAR or each tanCAR may comprise a domain antibody (dAb) antigen binding domain.

[0041] An anti-CD33 CAR having a domain antibody (dAb) antigen binding domain may comprise the following complementarity determining regions:

[0042] (i) CDR1-GRTFSMHS (SEQ ID No. 1); CDR2-VTWSGDTF (SEQ ID No. 2); CDR3-KDDPYRPAYDY (SEQ ID No. 3);

[0043] (ii) CDR1-GRTFSSYV (SEQ ID No. 4); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAMELRGGSYNYASSRQYDY (SEQ ID No. 6);

[0044] (iii) CDR1-EIAFSNFN (SEQ ID No. 7); CDR2-ISSHGDTNY (SEQ ID No. 8); CDR3-NANDPFLSVSDF (SEQ ID No. 9);

[0045] (iv) CDR1-GSIFSINA (SEQ ID No. 10); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAISGWGRSIRVGERYEYDY (SEQ ID No. 11);

[0046] (v) CDR1-GRTSSSST (SEQ ID No. 12); CDR2-ITLSGGST (SEQ ID No. 13); CDR3-AARRWSSNNRGGYDRAGYDY (SEQ ID No. 14); or

[0047] (vi) CDR1-GRTFSSYA (SEQ ID No. 15); CDR2-ITWSGGST (SEQ ID No. 16); CDR3-AMLLRGGLYDYTDYILYNY (SEQ ID No. 17).

[0048] The anti-CD33 CAR having a domain antibody (dAb) antigen binding domain may comprise one of the sequences shown in SEQ ID No. 18, 19, 20, 21, 22 or 23.

[0049] An anti-CLL-1 CAR having a domain antibody (dAb) antigen binding domain may comprise the following complementarity determining regions:

[0050] (i) CDR1-GFTFGNHD (SEQ ID No. 48); CDR2-IDSGGNVI (SEQ ID No. 49); CDR3-ATDLDSGAESLESVY (SEQ ID No. 50);

[0051] (ii) CDR1-GFAFGSAD (SEQ ID No. 51); CDR2-IDSGGNTQ (SEQ ID No. 52); CDR3-TDLDPTTDSLENVY (SEQ ID No. 53);

[0052] (iii) CDR1-GRTFSAYF (SEQ ID No. 54); CDR2-INWNGDSS (SEQ ID No. 55); CDR3-AADTHGAVGLGSERLYDY (SEQ ID No. 56);

[0053] (iv) CDR1-GIGVSSTG (SEQ ID No. 57); CDR2-IDRDGTT (SEQ ID No. 58); CDR3-TVVGDYY (SEQ ID No. 59);

[0054] (v) CDR1-GFIFGNYD (SEQ ID No. 60); CDR2-ISSGGNDI (SEQ ID No. 61); CDR3-AADLDPGTDSLDNIH (SEQ ID No. 62); or

[0055] (vi) CDR1-GFTLDYYA (SEQ ID No. 63); CDR2-ISSSDGST (SEQ ID No. 64); CDR3-AEAVYYAGVCVAMYDS (SEQ ID No. 65).

[0056] The anti-CLL-1CAR having a domain antibody (dAb) antigen binding domain can comprise one of the sequences shown in SEQ ID No.66, 67, 68, 69, 70 or 71.

[0057] An anti-CD123 CAR having a domain antibody (dAb) antigen binding domain may comprise the following complementarity determining regions:

[0058] (i) CDR1-GRSINTYA (SEQ ID No. 24); CDR2-INYNSRYT (SEQ ID No. 25); CDR3-AATSYYPTDYDVASRVATWPS (SEQ ID No. 26);

[0059] (ii) CDR1-GISLNA (SEQ ID No. 27); CDR2-IKIGGVS (SEQ ID No. 28); CDR3-NTYPPYLNGMDY (SEQ ID No. 29);

[0060] (iii) CDR1-GRSFNTDA (SEQ ID No. 30); CDR2-ISWDGTRT (SEQ ID No. 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32);

[0061] (iv) CDR1-GSSISV (SEQ ID No. 33); CDR2-ISWSDGNT (SEQ ID No. 34); CDR3-AVEPRGWPKGHRY (SEQ ID No. 35);

[0062] (v) CDR1-GSSFSINV (SEQ ID No. 36); CDR2-ISWSDGST (SEQ ID No. 37); CDR3-AVEPRGWPKGHRY (SEQ ID No. 38); or

[0063] (vi) CDR1-GSIFRINA (SEQ ID No. 39); CDR2-VNWIGGTT (SEQ ID No. 40); CDR3-SATDKGGSSRY (SEQ ID No. 41).

[0064] The anti-CD123 CAR having a domain antibody (dAb) antigen binding domain may comprise one of the sequences shown in SEQ ID No. 42, 43, 44, 45, 46 or 47.

[0065] An anti-FLT3 CAR having a domain antibody (dAb) antigen binding domain may comprise the following complementarity determining regions:

[0066] (i) CDR1-GIFKTNY (SEQ ID No. 72); CDR2-FTNDGST (SEQ ID No. 73); CDR3-YGLGH (SEQ ID No. 74);

[0067] (ii) CDR1-GTISSIRY (SEQ ID No. 75); CDR2-ITSSGNT (SEQ ID No. 76); CDR3-YTMGY (SEQ ID No. 77);

[0068] (iii) CDR1-GIFSTNY (SEQ ID No. 78); CDR2-FTNDGGT (SEQ ID No. 79); CDR3-CGLGH (SEQ ID No. 80);

[0069] (iv) CDR1-GSISSIRY (SEQ ID No. 81); CDR2-ITSSGST (SEQ ID No. 82); CDR3-YTMGY (SEQ ID No. 83); or

[0070] (v) CDR1-GIFSTNH (SEQ ID No. 84); CDR2-FTNDGST (SEQ ID No. 85); CDR3-YGLGH (SEQ ID No. 86).

[0071] The anti-FLT3 CAR having a domain antibody (dAb) antigen binding domain may comprise one of the sequences shown in SEQ ID No. 87, 88, 89, 90 or 91.

[0072] In a second aspect, the present invention provides nucleic acid constructs encoding multiple CARs. Nucleic acid constructs can encode three or all four CARs for the following antigens: CD33, CLL-1, CD123 and FLT3. For example, nucleic acid constructs can encode: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1CAR; and anti-CD123 CAR.

[0073] The nucleic acid construct may encode: an anti-CD33 chimeric antigen receptor (CAR); an anti-CLL1 CAR; and an anti-FLT3 CAR.

[0074] The nucleic acid construct may encode: an anti-CD33 chimeric antigen receptor (CAR); an anti-CLL1 CAR; and an anti-CD123 CAR; and an anti-FLT3 CAR.

[0075] In a third aspect, the present invention provides a method for preparing a cell according to the first aspect of the present invention, comprising the step of transducing or transfecting the cell with a nucleic acid construct according to the second aspect of the present invention.

[0076] In a fourth aspect, the present invention provides a vector comprising the nucleic acid construct according to the second aspect of the present invention.

[0077] In a fifth aspect, a kit of vectors is provided, comprising a plurality of vectors, each vector encoding a CAR for a target antigen. The kit may include vectors encoding three or all four CARs for the following target antigens: CD33, CLL-1, CD123, and FLT3. For example, the kit may include:

[0078] (i) a first vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds CD33;

[0079] (ii) a second vector comprising a nucleic acid sequence encoding a CAR that binds to CLL1; and

[0080] (iii) a third vector comprising a nucleic acid sequence encoding a CAR that binds CD123.

[0081] The kit may contain:

[0082] (i) a first vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds CD33;

[0083] (ii) a second vector comprising a nucleic acid sequence encoding a CAR that binds to CLL1; and

[0084] (iii) a third vector comprising a nucleic acid sequence encoding a CAR that binds to FLT3.

[0085] The kit may contain:

[0086] (i) a first vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) that binds CD33;

[0087] (ii) a second vector comprising a nucleic acid sequence encoding a CAR that binds to CLL1;

[0088] (iii) a third vector comprising a nucleic acid sequence encoding a CAR that binds CD123; and

[0089] (iii) a fourth vector comprising a nucleic acid sequence encoding a CAR that binds to FLT3.

[0090] In a sixth aspect, a pharmaceutical composition is provided, comprising a plurality of cells according to the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0091] In a seventh aspect, there is provided a method for treating cancer, comprising the step of administering to a subject a pharmaceutical composition according to the sixth aspect of the present invention.

[0092] The cancer may be acute myeloid leukemia (AML).

[0093] The method may further involve the step of subsequently administering the allograft to the subject.

[0094] In an eighth aspect, there is provided a pharmaceutical composition according to the sixth aspect of the present invention for use in treating cancer.

[0095] In a ninth aspect, there is provided use of the cell according to the first aspect of the invention in the preparation of a pharmaceutical composition for treating cancer.

[0096] The phenotype of AML blasts is more heterogeneous than that of acute lymphoblastic leukemia (ALL) blasts. Myelopoiesis is driven by stem cells, which are stochastic and in response to cues to replenish their compartments or differentiate. Similar to normal myeloid stem cells, AML stem cells multiply or differentiate, resulting in bone marrow replacement with a range of cells in different differentiation states. AML stem cells can occur along the spectrum of myelopoiesis and therefore have different surface antigen profiles. In addition, in a given patient, there may be a hierarchy of stem cell nexi or different stem cells at different points in individual development, all of which contribute to the disease burden ( Figure 1 ).

[0097] To treat the largest number of patients, the inventors discovered that targeting of AML via chimeric antigen receptors requires simultaneous targeting of multiple antigens based on myeloid lineage.

[0098] The OR gate of the present invention provides advantages over the current Phase I clinical trials of CAR T cell immunotherapy for patients with relapsed / refractory AML shown in Table 1 above. Targeting a single antigen may not eliminate the stem cell compartment associated with the disease. However, simultaneously targeting multiple myeloid antigens means that the treatment is universal in AML. Immunotherapy using CAR-T cells targeting multiple antigens eradicates the disease stem cell compartment, regardless of the number and location of the stem cell compartment. Targeting multiple antigens also reduces the possibility of escape caused by antigen downregulation.

[0099] Other aspects

[0100] The present invention also provides a cell composition comprising a CAR-expressing cell, which expresses multiple CARs.

[0101] The composition of cells can express: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-CD123 CAR.

[0102] The composition of cells can express: an anti-CD33 chimeric antigen receptor (CAR); an anti-CLL1 CAR; and an anti-FLT3 CAR.

[0103] The composition of cells can express: anti-CD33 chimeric antigen receptor (CAR); anti-CLL1 CAR; and anti-CD123 CAR; and anti-FLT3 CAR.

[0104] The cells of the composition can each express one CAR type. For example, the composition can include a mixture of one of the following:

[0105] cells expressing CD33 CAR, cells expressing CLL-1CAR, and cells expressing CD123 CAR;

[0106] CD33 CAR-expressing cells, CLL-1 CAR-expressing cells, and FLT3 CAR-expressing cells; or

[0107] Cells expressing CD33 CAR, cells expressing CLL-1 CAR, cells expressing CD123 CAR, and cells expressing FLT3 CAR.

[0108] Alternatively, at least some of the cells of the composition may express more than one CAR, for example, the composition may comprise a combination of:

[0109] cells expressing CD33 CAR / CLL-1CAR or gate and cells expressing CD123 CAR;

[0110] cells expressing CD33 CAR / CD123 CAR or gate and cells expressing CLL-1CAR;

[0111] cells expressing CD123 CAR / CLL-1CAR or gate and cells expressing CD33 CAR;

[0112] cells expressing CD33 CAR / CLL-1CAR or gate and cells expressing FLT3 CAR;

[0113] cells expressing CD33 CAR / FLT3 CAR or gate and cells expressing CLL-1CAR;

[0114] cells expressing FLT3 CAR / CLL-1CAR or gate and cells expressing CD33 CAR;

[0115] cells expressing CD33 CAR / CLL-1CAR or gate and cells expressing CD123 CAR / FLT3 CAR or gate;

[0116] cells expressing CD123 CAR / CLL-1CAR or gate and cells expressing CD33 CAR / FLT3 CAR or gate; or

[0117] Cells expressing CD33 CAR / CD123 CAR or gate and cells expressing CLL-1CAR / FLT3 CAR or gate.

[0118] At least some cells of the composition may express tanCAR, as described below. For example, the composition may comprise a combination of:

[0119] cells expressing CD33 / CLL-1tanCAR and cells expressing CD123 CAR;

[0120] cells expressing CD33 / CD123 tanCAR and cells expressing CLL-1CAR;

[0121] cells expressing CD123 / CLL-1tanCAR and cells expressing CD33 CAR;

[0122] cells expressing CD33 / CLL-1tanCAR and cells expressing FLT3 CAR;

[0123] cells expressing CD33 / FLT3 tanCAR and cells expressing CLL-1CAR;

[0124] cells expressing FLT3 / CLL-1tanCAR and cells expressing CD33 CAR;

[0125] cells expressing CD33 / CLL-1tanCAR and cells expressing CD123 / FLT3tanCAR;

[0126] Cells expressing CD123 / CLL-1tanCAR and cells expressing CD33 / FLT3tanCAR; or

[0127] Cells expressing CD33 / CD123 tanCAR and cells expressing CLL-1 / FLT3 tanCAR.

[0128] The nucleic acid sequences, nucleic acid constructs, vectors and kits of vectors and methods described below can be used to prepare cells of the cell composition of this aspect of the invention.

[0129] As described below, the cell compositions can be used in methods of treating disease.

[0130] Further aspects of the invention are summarized in the following numbered paragraphs:

[0131] A1. Domain antibodies (dAbs) that bind CD33 and comprise the following complementarity determining regions (CDRs):

[0132] (i) CDR1-GRTFSMHS (SEQ ID No. 1); CDR2-VTWSGDTF (SEQ ID No. 2); CDR3-KDDPYRPAYDY (SEQ ID No. 3);

[0133] (ii) CDR1-GRTFSSYV (SEQ ID No. 4); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAMELRGGSYNYASSRQYDY (SEQ ID No. 6);

[0134] (iii) CDR1-EIAFSNFN (SEQ ID No. 7); CDR2-ISSHGDTNY (SEQ ID No. 8); CDR3-NANDPFLSVSDF (SEQ ID No. 9);

[0135] (iv) CDR1-GSIFSINA (SEQ ID No. 10); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAISGWGRSIRVGERYEYDY (SEQ ID No. 11);

[0136] (v) CDR1-GRTSSSST (SEQ ID No. 12); CDR2-ITLSGGST (SEQ ID No. 13); CDR3-AARRWSSNNRGGYDRAGYDY (SEQ ID No. 14); or

[0137] (vi) CDR1-GRTFSSYA (SEQ ID No. 15); CDR2-ITWSGGST (SEQ ID No. 16); CDR3-AMLLRGGLYDYTDYILYNY (SEQ ID No. 17).

[0138] A2. The dAb according to paragraph A1, which comprises one of the sequences shown as SEQ ID No. 18, 19, 20, 21, 22 or 23.

[0139] A3. A chimeric antigen receptor (CAR) having an antigen binding domain comprising a dAb according to paragraph A1 or A2.

[0140] A4. Nucleic acid sequence encoding a dAb according to paragraph A1 or A2 or a CAR according to paragraph A3.

[0141] A5. A vector comprising the nucleic acid sequence according to paragraph A4.

[0142] A6. A cell expressing the CAR according to paragraph A3.

[0143] A7. A method for preparing a cell according to paragraph A6, comprising the step of transducing or transfecting the cell with a vector according to paragraph A5.

[0144] A8. A pharmaceutical composition comprising a plurality of cells according to paragraph A6.

[0145] A9. A method for treating cancer, comprising the step of administering to a subject a pharmaceutical composition according to paragraph A8.

[0146] A10. The method according to paragraph A9, wherein the cancer is acute myeloid leukemia (AML).

[0147] A11. A pharmaceutical composition according to paragraph A8, for use in treating cancer.

[0148] A12. Use of the cell according to paragraph A6 in the preparation of a pharmaceutical composition for treating cancer.

[0149] B1. Domain antibodies (dAbs) that bind to CD123 and comprise the following complementarity determining regions (CDRs):

[0150] (i) CDR1-GRSINTYA (SEQ ID No. 24); CDR2-INYNSRYT (SEQ ID No. 25); CDR3-AATSYYPTDYDVASRVATWPS (SEQ ID No. 26);

[0151] (ii) CDR1-GISLNA (SEQ ID No. 27); CDR2-IKIGGVS (SEQ ID No. 28); CDR3-NTYPPYLNGMDY (SEQ ID No. 29);

[0152] (iii) CDR1-GRSFNTDA (SEQ ID No. 30); CDR2-ISWDGTRT (SEQ ID No. 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32);

[0153] (iv) CDR1-GSSISV (SEQ ID No. 33); CDR2-ISWSDGNT (SEQ ID No. 34); CDR3-AVEPRGWPKGHRY (SEQ ID No. 35);

[0154] (v) CDR1-GSSFSINV (SEQ ID No. 36); CDR2-ISWSDGST (SEQ ID No. 37); CDR3-AVEPRGWPKGHRY (SEQ ID No. 38); or

[0155] (vi) CDR1-GSIFRINA (SEQ ID No. 39); CDR2-VNWIGGTT (SEQ ID No. 40); CDR3-SATDKGGSSRY (SEQ ID No. 41).

[0156] B2. The dAb according to paragraph B1, which comprises one of the sequences shown as SEQ ID No: 42, 43, 44, 45, 46 or 47.

[0157] B3. A chimeric antigen receptor (CAR) having an antigen binding domain comprising a dAb according to paragraph B1 or B2.

[0158] B4. A nucleic acid sequence encoding a dAb according to paragraph B1 or B2 or a CAR according to paragraph B3.

[0159] B5. A vector comprising the nucleic acid sequence according to paragraph B4.

[0160] B6. A cell expressing a CAR according to paragraph B3.

[0161] B7. A method for preparing a cell according to paragraph B6, comprising the step of transducing or transfecting the cell with a vector according to paragraph B5.

[0162] B8. A pharmaceutical composition comprising a plurality of cells according to paragraph B6.

[0163] B9. A method for treating cancer, comprising the step of administering to a subject a pharmaceutical composition according to paragraph B8.

[0164] B10. The method according to paragraph B9, wherein the cancer is acute myeloid leukemia (AML).

[0165] B11. A pharmaceutical composition according to paragraph B8, for use in treating cancer.

[0166] B12. Use of the cell according to paragraph B6 in the preparation of a pharmaceutical composition for treating cancer.

[0167] The present invention provides domain antibodies (dAbs) that bind to FLT3 and comprise the following complementarity determining regions:

[0168] C1. A domain antibody (dAb) that binds FLT3 and comprises the following complementarity determining regions (CDRs):

[0169] (i) CDR1-GIFKTNY (SEQ ID No. 72); CDR2-FTNDGST (SEQ ID No. 73); CDR3-YGLGH (SEQ ID No. 74);

[0170] (ii) CDR1-GTISSIRY (SEQ ID No. 75); CDR2-ITSSGNT (SEQ ID No. 76); CDR3-YTMGY (SEQ ID No. 77);

[0171] (iii) CDR1-GIFSTNY (SEQ ID No. 78); CDR2-FTNDGGT (SEQ ID No. 79); CDR3-CGLGH (SEQ ID No. 80);

[0172] (iv) CDR1-GSISSIRY (SEQ ID No. 81); CDR2-ITSSGST (SEQ ID No. 82); CDR3-YTMGY (SEQ ID No. 83); or

[0173] (v) CDR1-GIFSTNH (SEQ ID No. 84); CDR2-FTNDGST (SEQ ID No. 85); CDR3-YGLGH (SEQ ID No. 86).

[0174] C2. The dAb according to paragraph C1, which comprises one of the sequences shown as SEQ ID No. 87, 88, 89, 90 or 91.

[0175] C3. A chimeric antigen receptor (CAR) having an antigen binding domain comprising a dAb according to paragraph C1 or C2.

[0176] C4. A nucleic acid sequence encoding a dAb according to paragraph C1 or C2 or a CAR according to paragraph C3.

[0177] C5. A vector comprising the nucleic acid sequence according to paragraph C4.

[0178] C6. Cells expressing the CAR according to paragraph C3.

[0179] C7. A method for preparing a cell according to paragraph C6, comprising the step of transducing or transfecting the cell with a vector according to paragraph C5.

[0180] C8. A pharmaceutical composition comprising a plurality of cells according to paragraph C6.

[0181] C9. A method for treating cancer, comprising the step of administering to a subject a pharmaceutical composition according to paragraph C8.

[0182] C10. The method according to paragraph C9, wherein the cancer is acute myeloid leukemia (AML).

[0183] C11. A pharmaceutical composition according to paragraph C8, for use in treating cancer.

[0184] C12. Use of the cell according to paragraph C6 in the preparation of a pharmaceutical composition for treating cancer.

[0185] D1. A domain antibody (dAb) that binds CLL1 and comprises the following complementarity determining regions (CDRs):

[0186] (i) CDR1-GFTFGNHD (SEQ ID No. 48); CDR2-IDSGGNVI (SEQ ID No. 49); CDR3-ATDLDSGAESLESVY (SEQ ID No. 50);

[0187] (ii) CDR1-GFAFGSAD (SEQ ID No. 51); CDR2-IDSGGNTQ (SEQ ID No. 52); CDR3-TDLDPTTDSLENVY (SEQ ID No. 53);

[0188] (iii) CDR1-GRTFSAYF (SEQ ID No. 54); CDR2-INWNGDSS (SEQ ID No. 55); CDR3-AADTHGAVGLGSERLYDY (SEQ ID No. 56);

[0189] (iv) CDR1-GIGVSSTG (SEQ ID No. 57); CDR2-IDRDGTT (SEQ ID No. 58); CDR3-TVVGDYY (SEQ ID No. 59);

[0190] (v) CDR1-GFIFGNYD (SEQ ID No. 60); CDR2-ISSGGNDI (SEQ ID No. 61); CDR3-AADLDPGTDSLDNIH (SEQ ID No. 62); or

[0191] (vi) CDR1-GFTLDYYA (SEQ ID No. 63); CDR2-ISSSDGST (SEQ ID No. 64); CDR3-AEAVYYAGVCVAMYDS (SEQ ID No. 65).

[0192] D2. The dAb according to paragraph D1, comprising one of the sequences shown as SEQ ID No. 66, 67, 68, 69, 70 or 71.

[0193] D3. A chimeric antigen receptor (CAR) having an antigen binding domain comprising a dAb according to paragraph D1 or D2.

[0194] D4. A nucleic acid sequence encoding a dAb according to paragraph D1 or D2 or a CAR according to paragraph D3.

[0195] D5. A vector comprising the nucleic acid sequence according to paragraph D4.

[0196] D6. Cells expressing the CAR according to paragraph D3.

[0197] D7. A method for preparing a cell according to paragraph D6, comprising the step of transducing or transfecting the cell with a vector according to paragraph D5.

[0198] D8. A pharmaceutical composition comprising a plurality of cells according to paragraph D6.

[0199] D9. A method for treating cancer, comprising the step of administering to a subject a pharmaceutical composition according to paragraph D8.

[0200] D10. The method according to paragraph D9, wherein the cancer is acute myeloid leukemia (AML).

[0201] D11. A pharmaceutical composition according to paragraph D8, for use in treating cancer.

[0202] D12. Use of the cell according to paragraph D6 in the preparation of a pharmaceutical composition for treating cancer. DETAILED DESCRIPTION OF THE INVENTION

[0204] Chimeric Antigen Receptor

[0205] The present invention relates to cells expressing multiple chimeric antigen receptors on the cell surface.

[0206] Classical chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that connects an extracellular antigen recognition domain (conjugate) to an intracellular signaling domain (intracellular domain). The conjugate is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other forms containing antibody-like antigen binding sites. A spacer domain is usually required to separate the conjugate from the membrane and orient it appropriately. The common spacer domain used is the Fc of IgG1. Depending on the antigen, a more compact spacer can be sufficient, such as a stem from CD8α, or even a separate IgG1 hinge. The transmembrane domain anchors the protein in the cell membrane and connects the spacer to the intracellular domain.

[0207] Early CAR designs have intracellular domains derived from the intracellular portion of the γ chain of FcεR1 or CD3ζ. As a result, these first-generation receptors transmit immune signal 1, which is enough to trigger T cells to kill associated target cells, but cannot fully activate T cells to proliferate or survive. In order to overcome this limitation, a composite intracellular domain is constructed: the intracellular portion of the T cell co-stimulatory molecule is fused to the intracellular portion of CD3ζ, resulting in a second-generation receptor, which can transmit activation and co-stimulatory signals simultaneously after antigen recognition. The most commonly used co-stimulatory domain is the co-stimulatory domain of CD28. This provides the most powerful co-stimulatory signal-i.e., immune signal 2, which triggers T cell proliferation. Some receptors have also been described, including TNF receptor family intracellular domains, such as closely related OX40 and 41BB, which transmit survival signals. Even more powerful third-generation CARs have now been described, which have intracellular domains capable of transmitting activation, proliferation and survival signals.

[0208] When the CAR binds to the target antigen, this results in the transmission of an activation signal to the T cell on which it is expressed. The CAR thus directs the specificity and cytotoxicity of the T cell to the tumor cell expressing the targeted antigen.

[0209] A CAR generally comprises: (i) an antigen binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain that comprises or is associated with a signaling domain.

[0210] A CAR may have the following general structure:

[0211] Antigen binding domain – spacer domain – transmembrane domain – intracellular signaling domain (intracellular domain).

[0212] Antigen binding domain

[0213] The antigen binding domain is the part of the chimeric receptor that recognizes the antigen. In a classic CAR, the antigen binding domain comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody (see Figure 2c). CARs have also been generated using domain antibodies (dAbs) or VHH antigen binding domains (see Figure 2 b); or in Fab CAR format ( Figure 2 a). FabCAR contains two chains: one with an antibody-like light chain variable region (VL) and a constant region (CL); and one with a heavy chain variable region (VH) and a constant region (CH). One chain also contains a transmembrane domain and an intracellular signaling domain. The association between CL and CH causes the assembly of the receptor.

[0214] The antigen binding domain of a CAR may be a single domain binder, also referred to as a "dAb," "VHH," "domain antibody," or "nanobody."

[0215] Conventional IgG molecules consist of two heavy chains 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 natural functional antigen-binding unit is formed by the non-covalent association of the VH and VL domains. This association is mediated by the hydrophobic framework region.

[0216] Single-domain antibodies are antibody fragments composed of a single monomeric variable antibody domain. The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids that lack the light chain and CH1 domains of classical antibodies. These heavy-chain antibodies contain a single antigen-binding domain, the VHH domain. Cartilaginous fish also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to divide the dimeric variable domains of common immunoglobulin G (IgG) from humans or mice into monomers. Although most of the current research on single-domain antibodies is based on heavy-chain variable domains, it has also been shown that nanobodies derived from light chains can specifically bind to target epitopes.

[0217] Single domain antibodies can be obtained by immunizing dromedaries, camels, llamas, alpacas or sharks with the desired antigen and then isolating the mRNA encoding the heavy chain antibody. By reverse transcription and polymerase chain reaction, a gene library of single domain antibodies can be generated. Screening techniques such as phage display and ribosome display help identify clones that bind to the antigen. Alternatively, single domain antibodies can be made from a common mouse or human IgG with four chains.

[0218] The present invention relates to targeting of multiple antigens. This can be achieved by a variety of methods, including or gates and tanCAR (as described in more detail below). Domain antibody antigen binding domains are particularly suitable for such methods because they are discrete and have no tendency to be connected. They are also less complex, which means that expression and folding are less likely to be impaired, and the sequences encoding such CAR / tanCAR require less space on the viral vector genome.

[0219] TANCAR

[0220] The cells of the present invention may comprise TanCAR.

[0221] Bispecific CARs called tandem CARs or TanCARs have been developed to simultaneously target two or more cancer-specific markers. In TanCAR, the extracellular domain contains two antigen-binding specificities in series, joined by a linker. Therefore, both binding specificities (scFv) are connected to a single transmembrane portion: one scFv is juxtaposed to the membrane, and the other scFv is at a distal position. When TanCAR binds to either or both target antigens, this results in the transmission of an activation signal to cells on which it is expressed.

[0222] Grada et al. (2013, Mol Ther Nucleic Acids 2:e105) described a TanCAR that includes a CD19-specific scFv, followed by a Gly-Ser linker, and then a HER2-specific scFv. The HER2-scFv is in a proximal position, while the CD19-scFv is in a distal position. The TanCAR was shown to induce different T cell reactivity against each of the two tumor-restricted antigens. This arrangement was chosen because HER2 and CD19 The respective lengths of are suitable for this spatial arrangement. It is also known that HER2 scFv binds to the four most distal loops of HER2.

[0223] The cells of the invention may comprise a TanCAR comprising two antigen binding specificities in series. 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.

[0224] In each of these antigen pairs, the antigen binding domains can be in any order in the molecule. For example, for the target antigen pair CD33 and CD123, the antigen binding domain that binds CD33 can be juxtaposed to the membrane, while the antigen binding domain that binds CD123 can be distal to the membrane; or the antigen binding domain that binds CD123 can be juxtaposed to the membrane, while the antigen binding domain that binds CD33 can be distal to the membrane.

[0225] The cell of the present invention can include a combination of tanCAR and a CAR with a single antigen specificity such as scFv-CAR or dAbCAR. In this regard, the cell has three antigen specificities: TanCAR has two, and scFv or dAb CAR has one. The cell can, for example, include one of the combinations shown in Table 2.

[0226] Table 2

[0227] Tan CAR specificity scFv / dAb CAR specificity CD33 / CLL-1 CD123 CD33 / CD123 CLL-1 CLL-1 / CD123 CD33 CD33 / CLL-1 FLT3 CD33 / FLT3 CLL1 CLL-1 / FLT3 CD33

[0228] The cell of the present invention may comprise two tanCARs. For example, the cell may comprise a dual tanCAR as shown in Table 3.

[0229] Table 3

[0230] TanCAR1 specificity TanCAR2 specificity CD33 / CLL-1 CD123 / FLT3 CD33 / CD123 CLL-1 / FLT3 CD33 / FLT-1 CLL-1 / CD123

[0231] OR Gate

[0232] "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 a cell (such as a T cell), is capable of detecting a specific expression pattern of at least two target antigens. If at least two target antigens are arbitrarily designated as antigen A and antigen B, three possible options are as follows:

[0233] “OR GATE” – T cells are triggered when either antigen A or antigen B is present on the target cell

[0234] “AND GATE” – T cells trigger only when both antigens A and B are present on the target cell

[0235] “AND NOT GATE” – When antigen A alone is present on the target cell, the T cell triggers, but when both antigen A and B are present on the target cell, the T cell does not trigger

[0236] Engineered T cells expressing these CAR combinations can be tailored to have strong specificity for cancer cells based on the specific expression (or lack of expression) of two or more markers.

[0237] The "OR gate" contains two or more CARs, each of which is directed against a different target antigen expressed by the target cell. The advantage of the OR gate is that the effective targetable antigen on the target cell is increased because it is actually antigen A + antigen B. This is particularly important for antigens expressed at variable or low density on the target cell, because the level of a single antigen may be below the threshold required for effective targeting by CAR-T cells. Moreover, it avoids the phenomenon of antigen escape. For example, some lymphomas and leukemias become CD19 negative after CD19 targeting: if this happens, the use of an OR gate that targets CD19 in combination with another antigen provides a "backup" antigen.

[0238] The cells of the present invention can express a triple OR gate comprising three CARs. For example, the cells can express:

[0239] a CAR that binds to CD33, a CAR that binds to CLL-1; and a CAR that binds to CD123, or

[0240] CAR that binds CD33, CAR that binds CLL-1; and CAR that binds FLT3.

[0241] The cells of the present invention can express a quadruple or gate comprising four CARs. For example, the cells can express: a CAR incorporating CD33, a CAR incorporating CLL-1; a CAR incorporating CD123; and a CAR incorporating FLT3.

[0242] In the triple and quadruple OR gates of the present invention, one or more CARs can be dAb CARs. Specifically, all CARs of the cell can be dAb CARs.

[0243] Target Antigen

[0244] The antigen binding domain of the CAR or one of them can specifically bind to one of the following target antigens: CD33, CD123, CLL-1 and FLT-3.

[0245] CD33

[0246] CD33 is a myeloid differentiation antigen that is displayed on some normal B cells as well as activated T cells and natural killer cells, but is not expressed on pluripotent hematopoietic stem cells or outside the hematopoietic system. It is found on at least a subset of blasts in nearly all acute myeloid leukemias (AML). With an average of 104 molecules / leukemic cell, CD33 is not abundant, but levels vary widely among individual patients.

[0247] 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 Accession No. P20138.

[0248] Several commercially available antibodies against CD33 are known, such as WM-53, P67.6, HIM3-4 (Thermofisher).

[0249] The present invention provides domain antibodies (dAbs) that bind CD33 and comprise the following complementarity determining regions:

[0250] (i) CDR1-GRTFSMHS (SEQ ID No. 1); CDR2-VTWSGDTF (SEQ ID No. 2); CDR3-KDDPYRPAYDY (SEQ ID No. 3);

[0251] (ii) CDR1-GRTFSSYV (SEQ ID No. 4); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAMELRGGSYNYASSRQYDY (SEQ ID No. 6);

[0252] (iii) CDR1-EIAFSNFN (SEQ ID No. 7); CDR2-ISSHGDTNY (SEQ ID No. 8); CDR3-NANDPFLSVSDF (SEQ ID No. 9);

[0253] (iv) CDR1-GSIFSINA (SEQ ID No. 10); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAISGWGRSIRVGERYEYDY (SEQ ID No. 11);

[0254] (v) CDR1-GRTSSSST (SEQ ID No. 12); CDR2-ITLSGGST (SEQ ID No. 13); CDR3-AARRWSSNNRGGYDRAGYDY (SEQ ID No. 14); or

[0255] (vi) CDR1-GRTFSSYA (SEQ ID No. 15); CDR2-ITWSGGST (SEQ ID No. 16); CDR3-AMLLRGGLYDYTDYILYNY (SEQ ID No. 17).

[0256] The anti-CD33 dAb can include one of the sequences shown in SEQ ID No.18, 19, 20, 21, 22 or 23.

[0257] SEQ ID No.18 (CD33 dAb P1.E4-44738)

[0258] QVQLESGGGLVQAGGSLRLSCAASGRTFSMHSMGWFRQAPGKEREFVAAVTWSGDTFAYADFVKGRFTISRGIAKNTLYLQMNSLKPEDTAVYYCAAKDDPYRPAYDYWGQGTQVTVSS

[0259] SEQ ID No.19 (CD33 dAb P1.H3-44739)

[0260] QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVAAISWSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAMELRGGSYNYASSRQYDYWGQGTQVTVSS

[0261] SEQ ID No.20 (CD33 dAb P1.G8–44742)

[0262] QVQLQESGGGLVQTGGSLTLSCAASEIAFSNFNMGWYRQGSGKQRTLVAQISSHGDTNYLDSMKGRFTISRDNNKKTVYLQMNALKPEDTAVYYCNANDPFLSVSDFWGQGTQVTVSS

[0263] SEQ ID No.21 (CD33 dAb P2.A7–46173)

[0264] QVQLQQSGGGLVQAGGSLRLSCAASGSIFSINAMGWFRQAPGKEREFVAAISWSGGSTYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAISGWGRSIRVGERYEYDYWGQGTQVTVSS

[0265] SEQ ID No.22 (CD33 dAb P2.B12–46174)

[0266] QVQLQESGGGLVQAGGSLRLSCAASGRTSSSSTMAWFRQAPGKEREFVAAITLSGGSTHYADSAKGRFTISRESAKNTVYLQMNSLKPEDTADYYCAARRWSNNRGGYDRAGYDYWGQGTQVTVSS

[0267] SEQ ID No.23(CD33 dAb P2.F2–46176)

[0268] QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAMLLRGGLYDYTDYILYNYWGQGTQVTVSS

[0269] The present invention also provides:

[0270] a CAR comprising such a CD33 dAb as an antigen binding domain;

[0271] Nucleic acid sequences encoding such dAbs or CARs.

[0272] CD123

[0273] CD123 is a transmembrane subunit of the interleukin 3 receptor (IL-3Ra), which together with CD131 forms a high affinity IL-3R. After binding to IL-3, IL-3R promotes cell proliferation and survival. CD123 is usually expressed at high levels on plasmacytoid dendritic cells and basophils. It is expressed at low levels on monocytes, eosinophils, and myeloid dendritic cells. The amino acid sequence of human CD123 can be obtained from the NCBI reference sequence NP_002174.1.

[0274] Several commercially available antibodies against CD123 are known, such as 6H6 and 5B11 (ThermoFisher).

[0275] The present invention provides domain antibodies (dAbs) that bind CD123 and comprise the following complementarity determining regions:

[0276] (i) CDR1-GRSINTYA (SEQ ID No. 24); CDR2-INYNSRYT (SEQ ID No. 25); CDR3-AATSYYPTDYDVASRVATWPS (SEQ ID No. 26);

[0277] (ii) CDR1-GISLNA (SEQ ID No. 27); CDR2-IKIGGVS (SEQ ID No. 28); CDR3-NTYPPYLNGMDY (SEQ ID No. 29);

[0278] (iii) CDR1-GRSFNTDA (SEQ ID No. 30); CDR2-ISWDGTRT (SEQ ID No. 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32);

[0279] (iv) CDR1-GSSISV (SEQ ID No. 33); CDR2-ISWSDGNT (SEQ ID No. 34); CDR3-AVEPRGWPKGHRY (SEQ ID No. 35);

[0280] (v) CDR1-GSSFSINV (SEQ ID No. 36); CDR2-ISWSDGST (SEQ ID No. 37); CDR3-AVEPRGWPKGHRY (SEQ ID No. 38); or

[0281] (vi) CDR1-GSIFRINA (SEQ ID No. 39); CDR2-VNWIGGTT (SEQ ID No. 40); CDR3-SATDKGGSSRY (SEQ ID No. 41).

[0282] The anti-CD123 dAb may comprise one of the sequences shown as SEQ ID No. 42, 43, 44, 45, 46 or 47.

[0283] SEQ ID No.42(CD123 dAb H11 45897)

[0284] QVQLQESGGGLVQAGGSLRLSCTASGRSINTYAMAWFRQAPGKEREFVASINYNSRYTHYVDSVKGRFTISRDNTKNTLFLQMDSLNREDTAVYYCAATSYYPTDYDVASRVATWPSWGQGTQVTVSS

[0285] SEQ ID No.43(CD123 dAb F8 45888)

[0286] QVQLQESGGGLVQAGESLRLTCAVSGISLNAMGWYRQAPGKQLREWVAVIKIGGVSNYAVSVKGRFTISRDNAKNTIYLQMNSLKPEDTGVYYCNTYPPYLNGMDYWGKGTLVTVSS

[0287] SEQ ID No.44(CD123 dAb A7 45865)

[0288] QVQLQQSGGGLVQAGGSLRLSCAFSGRSFNTDAVAWFRQAPGKEREFVAAISWDGTRTYYADSAKGRFTISRDNAKNTVYLQMNSLNSEDTAVYYCAAEPQKAWPIGTSAAGFRSWGQGTQVTVSS

[0289] SEQ ID No.45(CD123 dAb B4 45868)

[0290] QVQLQESGGGSVQSGGSLRLSCAASGSSISVMGWFRQAPGKEREFVAAISWSDGNTNYADSVNGRFSVSRDNTKNTVYLQMNSLKPEDTAIYYCAVEPRGWPKGHRYWGQGTQVTVSS

[0291] SEQ ID No.46(CD123 dAb A10 45866)

[0292] QVQLQESGGSSVQAGGSLRLSCAASGSSFSINVMGWFRQAPGKEREFVAAISWSDGSTNYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAIYYCAVEPRGWPKGHRYWGQGTQVTVSS

[0293] SEQ ID No.47(CD123 dAb C11 45874)

[0294] QVQLQESGGGLVQAGGSLRLSCAASGSIFRINAMGWFRQAPGKEREFVTAVNWIGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYFCSATDKGGSSRYWGQGTQVTVSS

[0295] The present invention also provides:

[0296] A CAR comprising such a CD123 dAb as an antigen binding domain;

[0297] Nucleic acid sequences encoding such dAbs or CARs.

[0298] FLT3

[0299] FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase (RTK), a membrane-bound receptor with an intrinsic tyrosine kinase domain. FLT3 consists of an immunoglobulin-like extracellular ligand binding domain, a transmembrane domain, a near-membrane dimerization domain, and a highly conserved intracellular kinase domain interrupted by a kinase insert. FLT3 belongs to the class III subfamily of RTK, which includes structurally similar members such as c-FMS, c-KIT, and PDGF receptors. FLT3 is primarily expressed in stereotyped myeloid and lymphoid progenitors, with variable expression in more mature monocytic lineages.

[0300] FLT3 expression has been described in lymphoid hematopoietic organs such as liver, spleen, thymus and placenta.Under unstimulated state, FLT3 receptor exists in the form of monomer, unphosphorylated with inactive kinase part.After receptor interacts with FLT ligand (FL), receptor undergoes conformational change, causing receptor unfolding and dimerization domain exposure, thereby receptor-receptor dimerization occurs.This receptor dimerization is the prelude to the activation of tyrosine kinase, causing phosphorylation of various sites in the intracellular domain.The amino acid sequence of people FLT3 can be obtained from NCBI reference sequence: NP_004110.2.

[0301] FLT3 is important for the biology of some AML cases, with mutations in FLT3 being one of the most common mutations in the disease. These mutations often lead to constitutive activation. Some AML cases respond to small molecule inhibition of FLT3.

[0302] Several commercially available antibodies against FLT3 are known, such as A2F10 and BV10A4H2 (ThermoFisher).

[0303] The present invention provides domain antibodies (dAbs) that bind to FLT3 and comprise the following complementarity determining regions:

[0304] (i) CDR1-GIFKTNY (SEQ ID No. 72); CDR2-FTNDGST (SEQ ID No. 73); CDR3-YGLGH (SEQ ID No. 74);

[0305] (ii) CDR1 - GTISSIRY (SEQ ID No.75); CDR2 - ITSSGNT (SEQ ID No.76); CDR3 - YTMGY (SEQ ID No.77);

[0306] (iii) CDR1 - GIFSTNY (SEQ ID No.78); CDR2 - FTNDGGT (SEQ ID No.79); CDR3 - CGLGH (SEQ ID No.80);

[0307] (iv) CDR1 - GSISSIRY (SEQ ID No.81); CDR2 - ITSSGST (SEQ ID No.82); CDR3 - YTMGY (SEQ ID No.83); or

[0308] (v) CDR1 - GIFSTNH (SEQ ID No.84); CDR2 - FTNDGST (SEQ ID No.85); CDR3 - YGLGH (SEQ ID No.86).

[0309] The anti - FLT3 dAb can comprise one of the sequences as shown in SEQ ID No.87, 88, 89, 90 or 91.

[0310] SEQ ID No.87 (FLT3 dAb B5)

[0311] QVQLQQSGGGLVQAGGSLRLSCAASGIFKTNYMAWYRQAPGKQRELVAAFTNDGSTLYGDSVKGRFTISRDDAKYTVSLQMNSLKPEDTAVYYCYGLGHWGQGTQVIVSSEPKTPKPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0312] SEQ ID No.88 (FLT3 dAb G3)

[0313] QVQLQESGGGLVQAGGSLRLSCAASGTISSIRYMNWYRQAPGKQREVVAYITSSGNTNYADSVKGRFTISRDNAKNTVYLQMDNLKPEDTAAYYCYTMGYWGQGTQVTVSSEPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHH

[0314] GAA

[0315] SEQ ID No.89 (FLT3 dAb H5)

[0316] QVQLQESGGGLVQAGGSLRLSCAASGIFSTNYMVWCRQAPGKQRELVAAFTNDGGTLYADSLKGRFSISQDNAKNTVLLLMNSLKPEDTAVYYCCGLGHWGRGTKVTVSSEPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0317] SEQ ID No.90 (FLT3 dAb D12)

[0318] QVQLQESGGGLVQAGGSLRLSCAASGSISSIRYMNWYRQAPGKQRESVAWITSSGSTNYADSVQGRFTISSRDNAKNTVYLQMDNLKPEDTAVYYCYTMGYWGQGTQVTVSSEPKIPQPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0319] SEQ ID No.91 (FLT3 dAb F10)

[0320] QAQVQLQESGGGLVQAGGSLRLSCAASGIFSTNHMAWYRQAPGKQRELVAAFTNDGSTLYGDSVKGRFVISRDNAKYTVFLQMNSLKPEDTAVYYCYGLGHWGQGTQVTVSSEPKTPKPQPAAADDDDKEQKLISEEDLNGAAHHHHHHGAA

[0321] The present invention also provides:

[0322] a CAR comprising such a FLT3 dAb as an antigen binding domain;

[0323] Nucleic acid sequences encoding such dAbs or CARs.

[0324] CLL1

[0325] Human C-type lectin-like molecule-1 (CLL-1, MICL or CLEC12A) is a type II transmembrane glycoprotein and a member of the large family of C-type lectin-like receptors involved in immunomodulation. The intracellular domain of CLL-1 contains an ITIM motif and a binding site for PI-3 kinase. The expression pattern of CLL-1 in hematopoietic cells is restricted; it is particularly found in myeloid cells derived from peripheral blood and bone marrow. The amino acid sequence of human CLL1 can be obtained from Uniprot accession number Q5QGZ9.

[0326] Several antibodies against CLL-1 have been described in, for example, WO2009051974, WO2013169625, WO2016205200, and WO2016040868.

[0327] The present invention provides a domain antibody (dAb) that binds to CLL1 and comprises the following complementarity determining regions:

[0328] (i) CDR1-GFTFGNHD (SEQ ID No. 48); CDR2-IDSGGNVI (SEQ ID No. 49); CDR3-ATDLDSGAESLESVY (SEQ ID No. 50);

[0329] (ii) CDR1-GFAFGSAD (SEQ ID No. 51); CDR2-IDSGGNTQ (SEQ ID No. 52); CDR3-TDLDPTTDSLENVY (SEQ ID No. 53);

[0330] (iii) CDR1-GRTFSAYF (SEQ ID No. 54); CDR2-INWNGDSS (SEQ ID No. 55); CDR3-AADTHGAVGLGSERLYDY (SEQ ID No. 56);

[0331] (iv) CDR1-GIGVSSTG (SEQ ID No. 57); CDR2-IDRDGTT (SEQ ID No. 58); CDR3-TVVGDYY (SEQ ID No. 59);

[0332] (v) CDR1-GFIFGNYD (SEQ ID No. 60); CDR2-ISSGGNDI (SEQ ID No. 61); CDR3-AADLDPGTDSLDNIH (SEQ ID No. 62); or

[0333] (vi) CDR1 - GFTLDYYA (SEQ ID No.63); CDR2 - ISSSDGST (SEQ ID No.64); CDR3 - AEAVYYAGVCVAMYDS (SEQ ID No.65).

[0334] The anti - CLL1 dAb can include one of the sequences shown in SEQ ID No.66, 67, 68, 69, 70 or 71.

[0335] SEQ ID No.66 (CLL - 1dAb 44548)

[0336] QVQLQQSGGGLVQPGGSLRLSCVGSGFTFGNHDMSWVRQAPGKEVEFVAGIDSGGNVIVYEEVVKGRFTISRDNAKNTLYLQMDGLKPEDAGMYFCATD

[0337] LDSGAESLESVYHGQGTQVTVSS

[0338] SEQ ID No.67 (CLL - 1dAb 44544)

[0339] QVQLQESGGGLVESGGSLRISCTGFGFAFGSADMSWVRQAPGKEVEFVAGIDSGGNTQTYEDTVKGRFTISRDNAKNTLYLQMNSLQSEDAGVYFCATDLDPTTDSLENVYHGQGTQVIVSS

[0340] SEQ ID No.68 (CLL - 1dAb 44538)

[0341] QVQLQESGGGLVQTGDSLRLSCVASGRTFSAYFMGWFRQAPGKEREFVSAINWNGDSSWYRDSVKGRFTVSRDNAKNTVYLQMNSLEPEDTAVYYCAADTHGAVGLGSERLYDYWGQGTQVTVSS

[0342] SEQ ID No.69 (CLL - 1dAb 44546)

[0343] QVQLQESGGGVVQAGGSLRLSCAVSGIGVSSTGMGWSRQTPGKQVELVALIDRDGTTNYADTVKGRFTISKDNSKNMVYLQMNSLKPEDTALYHCTVVGDYYWGQGTQVTVSS

[0344] SEQ ID No.70 (CLL-1dAb 44545)

[0345] QVQLQQSGGGLVQPGGSLRLSCVGSGFIFGNYDMSWVRQAPGKEVEFVAGISSGGNDIVYEDAVKGRFSISRDNARNTVYLDMASVKPEDAGVYYCAADLDPGTDSLDNIHHGQGTQVFVSS

[0346] SEQ ID No.71 (CLL-1dAb 44536)

[0347] QVQLQESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTAYADSVKGRFTISRDNAKNSVYLQMNSLKPEDTAVYYCAEAVYYAGVCVAMYDSWGQGTQVTVSS

[0348] The present invention also provides:

[0349] A CAR comprising such a CD123 dAb as an antigen binding domain;

[0350] Nucleic acid sequences encoding such dAbs or CARs.

[0351] Spacer

[0352] Classical CARs contain a spacer sequence to connect the antigen binding domain and the transmembrane domain and to spatially separate the antigen binding domain from the intracellular domain. The flexible spacer allows the antigen binding domain to be oriented in different directions to facilitate binding.

[0353] The spacer can cause dimerization of the two polypeptide chains forming the CAR. The two polypeptide chains can, for example, contain one or more suitable cysteine ​​residues to form a disulfide bridge. Commonly used spacers include IgG1 Fc regions, IgG1 hinges, or human CD8 stems. The hinge spacer can include a sequence as shown in SEQ ID No.92.

[0354] SEQ ID No.92 (hinge spacer)

[0355] EPKSCDKTHTCPPCP

[0356] The spacer can be selected to suit the target antigen, i.e., the location and orientation of the epitope on the target antigen and the distance of the target epitope from the target cell membrane. In the OR gate, different spacers can be used to suit different relative positions of the target epitope and also to prevent cross-pairing between the two CARs.

[0357] Transmembrane domain

[0358] The transmembrane domain is the part of the CAR that spans the membrane. The transmembrane domain can be any protein structure that is thermodynamically stable in the membrane. This is usually an alpha helix containing several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to supply the transmembrane portion of the chimeric receptor. The presence and span of the transmembrane domain of the protein can be determined by a person skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Alternatively, an artificially designed TM domain can be used.

[0359] Intracellular domain

[0360] The intracellular domain is the signal transmission part of CAR. It can be a part of the intracellular domain of CAR or associated with it. After antigen recognition, receptor clusters, natural CD45 and CD148 are excluded from the synapse, and the signal is transmitted to the cell. The most commonly used intracellular domain component is the intracellular domain component of CD3-zeta containing 3 ITAMs. This transmits activation signals to T cells after antigen binding. CD3-zeta may not provide fully capable activation signals, and may require additional costimulatory signaling. Costimulatory signals promote T cell proliferation and survival. There are two main types of costimulatory signals: costimulatory signals (CD28, ICOS) belonging to the Ig family and costimulatory signals (OX40, 41BB, CD27, GITR, etc.) belonging to the TNF family. For example, chimeric CD28 and OX40 can be used together with CD3-Zeta to transmit proliferation / survival signals, or all three can be used together.

[0361] The intracellular domain may contain:

[0362] (i) an ITAM-containing intracellular domain, such as the intracellular domain from CD3 zeta; and / or

[0363] (ii) a co-stimulatory domain, such as the intracellular domain from CD28 or ICOS; and / or

[0364] (iii) Domains that transmit survival signals, such as intracellular domains of the TNF receptor family, such as OX-40, 4-1BB, CD27 or GITR.

[0365] Many systems have been described in which the antigen recognition portion is on a molecule separate from the signaling portion, such as those described in WO015 / 150771; WO2016 / 124930 and WO2016 / 030691. Thus, the cells of the present invention may express a CAR system comprising an antigen binding component comprising an antigen binding domain and a transmembrane domain; which is capable of interacting with a separate intracellular signaling component comprising a signaling domain.

[0366] The CAR may comprise a signal peptide so that when it is expressed intracellularly, the nascent protein is directed to the endoplasmic reticulum and subsequently to the surface of the cell expressing it. The signal peptide may be at the amino terminus of the molecule.

[0367] Suicide gene

[0368] The cells of the present invention may also express a suicide gene.

[0369] Suicide genes are genetically encoded mechanisms that allow for the selective destruction of adoptively transferred cells (e.g., T cells) in the face of unacceptable toxicity (e.g., off-target tumor toxicity), cytokine release syndrome (CRS), or neurotoxicity.

[0370] When the cells of the present invention are used to treat acute myeloid leukemia (AML), treatment may cause myeloid dysplasia in patients, which may be long-term or permanent. Allogeneic transplants, such as allogeneic hematopoietic stem cell transplantation (alloHSCT) can be used to save patients from this state. If a transplant is used, the incorporation of a suicide gene in cells expressing CAR can also enable cells expressing CAR to be deleted to prevent CAR-mediated graft rejection.

[0371] The cells of the invention may comprise one of the suicide genes previously tested in clinical studies, such as herpes simplex virus thymidine kinase (HSV-TK) or inducible caspase 9 (iCasp9).

[0372] WO2013 / 153391 describes a compact suicide gene comprising a CD20 epitope, which enables the selective killing of cells expressing the polypeptide using Rituximab. The cell of the present invention may express a suicide gene having a sequence as shown in SEQ ID No.93.

[0373] SEQ ID No.93

[0374] CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGG

[0375] GSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG

[0376] TCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

[0377] WO2016 / 135470 describes suicide genes that dimerize in the presence of a chemical inducer of dimerization (CID), such as rapamycin or a rapamycin analog, which causes caspase-mediated apoptosis.

[0378] Suicide genes can have the following structures:

[0379] Ht1-HT2-Casp

[0380] in:

[0381] Ht1 and Ht2 are heterodimerization domains, one of which contains FK506 binding protein (FKBP) and the other contains the FRB domain of mTOR; and

[0382] Casp is the caspase 9 domain.

[0383] The suicide gene may have the sequence shown in SEQ ID No. 94 or a variant thereof having 90, 95 or 99% sequence identity.

[0384] SEQ ID No.94(FRB-FKBP12-L3-dCasp9)

[0385] <-----------------------FRB---------------------------------MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR

[0386] ------------------FRB-------------------> <l1-><--FKBP12------DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEGVQVETISPGDGR

[0387] ------------------FKBP12------------------------------------TFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK

[0388] ---------------------------------><------L3------><--dCasp9-LTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGSGGGGSGGGGSGVDGFGDVGA

[0389] ------------------------dCasp9------------------------------LESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMV

[0390] ------------------------dCasp9------------------------------EVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVE

[0391] ------------------------dCasp9------------------------------KIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQE

[0392] ------------------------dCasp9------------------------------GLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQ

[0393] ------------------dCasp9---------------->

[0394] SLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS

[0395] Nucleic acid construct

[0396] The nucleic acid sequence encoding CAR may have the following structure:

[0397] AgB-spacer-TM-endo

[0398] in:

[0399] AgB is the nucleic acid sequence encoding the antigen binding domain of CAR;

[0400] spacer is a nucleic acid sequence encoding the spacer region of CAR;

[0401] TM is a nucleic acid sequence encoding the transmembrane domain of CAR;

[0402] endo is a nucleic acid sequence encoding the intracellular domain of CAR.

[0403] The nucleic acid sequence encoding tanCAR may have the following structure:

[0404] AgB1-linker-AgB2-spacer-TM-endo

[0405] in:

[0406] AgB1 is the nucleic acid sequence encoding the first antigen binding domain of tanCAR;

[0407] linker is a nucleic acid sequence encoding the linker of tanCAR;

[0408] AgB2 is a nucleic acid sequence encoding the second antigen binding domain of tanCAR;

[0409] spacer is the nucleic acid sequence encoding the spacer region of tanCAR;

[0410] TM is the nucleic acid sequence encoding the transmembrane domain of tanCAR;

[0411] endo is a nucleic acid sequence encoding the intracellular domain of tanCAR.

[0412] When expressed in a cell, the nucleotide sequence encodes a polypeptide that expresses the tandem first and second antigen binding domains at the cell surface.

[0413] The linker may be or comprise a Gly-Ser flexible linker.

[0414] The antigen binding domain of a CAR or tanCAR can be, for example, a scFv or a dAb.

[0415] The present invention provides a nucleic acid construct encoding a triple OR gate comprising three CARs.

[0416] The nucleic acid construct encoding the triple OR gate may have the following structure:

[0417] AgBD1-spacer1-TM1-endo1-coexpr1-AgBD2-spacer2-TM2-endo2-coexpr2-AgBD3-spacer3-TM3-endo3

[0418] in:

[0419] AgBD1 is a nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0420] Spacer1 is a nucleic acid sequence encoding the spacer region of the first CAR;

[0421] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0422] Endo1 is a nucleic acid sequence encoding the intracellular domain of the first CAR;

[0423] coexpr1 and coexpr2, which may be the same or different, are nucleic acid sequences that enable the co-expression of the first, second, and third CARs;

[0424] AgBD2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0425] Spacer2 is a nucleic acid sequence encoding the spacer region of the second CAR;

[0426] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

[0427] Endo2 is a nucleic acid sequence encoding the intracellular domain of the second CAR;

[0428] AgBD3 is a nucleic acid sequence encoding the antigen binding domain of the third CAR;

[0429] Spacer3 is a nucleic acid sequence encoding the spacer region of the third CAR;

[0430] TM3 is a nucleic acid sequence encoding the transmembrane domain of the third CAR; and

[0431] Endo3 is a nucleic acid sequence encoding the intracellular domain of the third CAR.

[0432] The antigen binding domains of the first, second and third CARs can be, for example, scFv or dAb. In particular, all three CARs can have dAb antigen binding domains.

[0433] The present invention provides a nucleic acid construct encoding a quadruple OR gate comprising four CARs.

[0434] A nucleic acid construct encoding a quadruple OR gate may have the following structure:

[0435] AgBD1-spacer1-TM1-endo1-coexpr1-AgBD2-spacer2-TM2-endo2-coexpr2-AgBD3-spacer3-TM3-endo3-coexpr3-AgBD4-spacer4-TM4-endo4

[0436] in:

[0437] AgBD1 is a nucleic acid sequence encoding the antigen binding domain of the first CAR;

[0438] Spacer1 is a nucleic acid sequence encoding the spacer region of the first CAR;

[0439] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

[0440] Endo1 is a nucleic acid sequence encoding the intracellular domain of the first CAR;

[0441] coexpr1, coexpr2, and coexpr3, which may be the same or different, are nucleic acid sequences that enable the co-expression of the first, second, third, and fourth CARs;

[0442] AgBD2 is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

[0443] spacer2 is a nucleic acid sequence encoding the spacer region of the second CAR;

[0444] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

[0445] endo2 is a nucleic acid sequence encoding the intracellular domain of the second CAR;

[0446] AgBD3 is a nucleic acid sequence encoding the antigen binding domain of the third CAR;

[0447] spacer3 is a nucleic acid sequence encoding the spacer region of the third CAR;

[0448] TM3 is a nucleic acid sequence encoding the transmembrane domain of the third CAR; and

[0449] endo3 is a nucleic acid sequence encoding the intracellular domain of the third CAR;

[0450] AgBD4 is a nucleic acid sequence encoding the antigen binding domain of the fourth CAR;

[0451] spacer4 is a nucleic acid sequence encoding the spacer region of the fourth CAR;

[0452] TM4 is a nucleic acid sequence encoding the transmembrane domain of the fourth CAR; and

[0453] endo4 is a nucleic acid sequence encoding the intracellular domain of the fourth CAR.

[0454] The antigen binding domains of the first, second, third and fourth CARs can be, for example, scFv or dAb. In particular, all four CARs can have dAb antigen binding domains.

[0455] The present invention also provides nucleic acid constructs encoding scFv / dAb CAR and tanCAR. In this embodiment, the nucleic acid construct may have the following structure:

[0456] AgB1-linker-AgB2-spacer1-TM1-endo1-coexpr-AgB3-spacer2-TM2-endo2

[0457] in:

[0458] AgB1 is the nucleic acid sequence encoding the first antigen binding domain of tanCAR;

[0459] linker is a nucleic acid sequence encoding the linker of tanCAR;

[0460] AgB2 is a nucleic acid sequence encoding the second antigen binding domain of tanCAR;

[0461] spacer1 is the nucleic acid sequence encoding the spacer region of tanCAR;

[0462] TM1 is the nucleic acid sequence encoding the transmembrane domain of tanCAR;

[0463] endo1 is a nucleic acid sequence encoding the intracellular domain of tanCAR;

[0464] coexpr is a nucleic acid sequence that enables co-expression of CAR and tanCAR;

[0465] AgB3 is a nucleic acid sequence encoding the antigen binding domain of CAR;

[0466] spacer2 is the nucleic acid sequence encoding the spacer region of CAR;

[0467] TM2 is a nucleic acid sequence encoding the transmembrane domain of CAR;

[0468] endo2 is a nucleic acid sequence encoding the intracellular domain of CAR;

[0469] or the following structure:

[0470] AgB1-spacer1-TM1-endo1-coexpr-AgB2-linker-AgB3-spacer2-TM2-endo2

[0471] in:

[0472] AgB1 is the nucleic acid sequence encoding the antigen binding domain of CAR;

[0473] spacer1 is the nucleic acid sequence encoding the spacer region of CAR;

[0474] TM1 is a nucleic acid sequence encoding the transmembrane domain of CAR;

[0475] endo1 is a nucleic acid sequence encoding the intracellular domain of CAR;

[0476] coexpr is a nucleic acid sequence that enables co-expression of CAR and tanCAR;

[0477] AgB2 is a nucleic acid sequence encoding the first antigen binding domain of tanCAR;

[0478] linker is a nucleic acid sequence encoding the linker of tanCAR;

[0479] AgB3 is a nucleic acid sequence encoding the second antigen binding domain of tanCAR;

[0480] spacer2 is the nucleic acid sequence encoding the spacer region of tanCAR;

[0481] TM2 is a nucleic acid sequence encoding the transmembrane domain of tanCAR;

[0482] endo2 is a nucleic acid sequence encoding the intracellular domain of tanCAR.

[0483] The present invention also provides a nucleic acid construct encoding two tanCARs. In this embodiment, the nucleic acid construct may have the following structure:

[0484] AgB1-linker1-AgB2-spacer1-TM1-endo1-coexpr-AgB3-linker2-AgB4-spacer2-TM2-endo2

[0485] in:

[0486] AgB1 is a nucleic acid sequence encoding the first antigen binding domain of the first tanCAR;

[0487] linker1 is a nucleic acid sequence encoding the linker of the first tanCAR;

[0488] AgB2 is a nucleic acid sequence encoding the second antigen binding domain of the first tanCAR;

[0489] spacer1 is the nucleic acid sequence encoding the spacer region of the first tanCAR;

[0490] TM1 is a nucleic acid sequence encoding the transmembrane domain of the first tanCAR;

[0491] endo1 is a nucleic acid sequence encoding the intracellular domain of the first tanCAR;

[0492] coexpr is a nucleic acid sequence that enables co-expression of the first and second tanCARs;

[0493] AgB3 is a nucleic acid sequence encoding the first antigen binding domain of the second tanCAR;

[0494] linker2 is a nucleic acid sequence encoding a linker of a second tanCAR;

[0495] AgB4 is a nucleic acid sequence encoding the second antigen binding domain of the second tanCAR;

[0496] spacer2 is the nucleic acid sequence encoding the spacer region of the second tanCAR;

[0497] TM2 is a nucleic acid sequence encoding the transmembrane domain of the second tanCAR;

[0498] endo2 is a nucleic acid sequence encoding the intracellular domain of the second tanCAR.

[0499] The nucleic acid construct of the present invention may further comprise a nucleic acid sequence encoding a suicide gene.

[0500] As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

[0501] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it should be understood that the skilled person can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any specific host organism in which the polypeptide is to be expressed.

[0502] Nucleic acids according to the present invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides comprising synthetic or modified nucleotides. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, with acridine or polylysine chains added at the 3' and / or 5' ends of the molecule. For the purposes of the uses described herein, it will be appreciated that polynucleotides may be modified by any method available in the art. Such modifications may be performed to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0503] The terms "variant", "homologue" or "derivative" with respect to a nucleotide sequence include any substitution, change, modification, replacement, deletion or addition to the sequence or to one (or more) nucleic acids of the sequence.

[0504] In the above structure, "coexpr" is a nucleic acid sequence that enables co-expression of two polypeptides as independent entities. It may be a sequence encoding a cleavage site, such that the nucleic acid construct produces two polypeptides joined by the cleavage site. The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into separate peptides without the need for any external cleavage activity.

[0505] The cleavage site may be any sequence capable of separating the two polypeptides.

[0506] The term "cleavage" is used herein for convenience, but the cleavage site can separate the peptide into separate entities by mechanisms other than classical cleavage. For example, for the foot-and-mouth disease virus (FMDV) 2A self-cleavage peptide (see below), various models have been proposed to explain the "cleavage" activity: proteolysis by host-cell proteases, autoproteolysis or translation effects (Donnelly et al (2001) J. Gen. Virol. 82: 1027-1041). As long as the cleavage site causes the protein to be expressed as a separate entity when located between nucleic acid sequences encoding the protein, the exact mechanism of such "cleavage" is not important for the purposes of the present invention.

[0507] The cleavage site can be, for example, a furin cleavage site, a tobacco etch virus (TEV) cleavage site, or encode a self-cleaving peptide.

[0508] "Self-cleaving peptide" refers to a peptide that functions such that when a polypeptide comprising a protein and the self-cleaving peptide is produced, it is immediately "cleaved" or separated into different and discrete first and second polypeptides without the need for any external cleavage activity.

[0509] The self-cleaving peptide can be a 2A self-cleaving peptide from foot-and-mouth virus or cardiovirus. The major 2A / 2B cleavage of foot-and-mouth virus and cardiovirus is mediated by 2A that "cleaves" at its own C-terminus. In foot-and-mouth viruses such as foot-and-mouth disease virus (FMDV) and equine rhinitis type A virus, the 2A region is a short segment of about 18 amino acids, which together with the N-terminal residue (conserved proline residue) of protein 2B represents an autonomous element that can mediate "cleavage" at its own C-terminus (Donelly et al (2001), as above).

[0510] "2A-like" sequences have been found in picornaviruses other than foot-and-mouth virus or cardioviruses, "picornavirus-like" insect viruses, type C rotaviruses, repetitive sequences in Trypanosoma species, and bacterial sequences (Donelly et al (2001), supra).

[0511] The cleavage site may comprise a 2A-like sequence as shown in SEQ ID No.95.

[0512] SEQ ID No.95:

[0513] RAEGRGSLLTCGDVEENPGP

[0514] Carrier

[0515] The present invention also provides a vector or a kit of vectors, which comprises one or more nucleic acid sequences encoding one or more chimeric antigen receptors of the cells of the present invention. Such vectors can be used to introduce nucleic acid sequences into host cells so that they express the CAR or each CAR.

[0516] The vector may be, for example, a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or a synthetic mRNA.

[0517] The vector may be capable of transfecting or transducing a cell, such as a T cell or a NK cell.

[0518] cell

[0519] The present invention provides cells comprising a plurality of chimeric antigen receptors.

[0520] The cell may be a cytolytic immune cell, such as a T cell or a NK cell.

[0521] T cells or T lymphocytes are lymphocyte types that play a central role in cell-mediated 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 the cell surface. There are many types of T cells, as summarized below.

[0522] Helper T helper cells (TH cells) assist other white blood cells 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 are activated when they present peptide antigens through MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to promote different types of immune responses.

[0523] Cytolytic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I present on the surface of all nucleated cells. IL-10, adenosine and other molecules secreted by regulatory T cells can inactivate CD8+ cells to an incapable state, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0524] Memory T cells are a subset of antigen-specific T cells that persist long after an infection has resolved. They rapidly expand into large numbers of effector T cells upon re-exposure to their cognate antigens, thereby providing the immune system with "memory" of past infections. Memory T cells contain 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 CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0525] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are essential for maintaining immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of the immune response and to suppress autoreactive T cells that escape the negative selection process in the thymus.

[0526] Two major types of CD4+ Treg cells have been described, namely naturally occurring Treg cells and adaptive Treg cells.

[0527] Naturally occurring Treg cells (also called CD4+CD25+FoxP3+Treg cells) appear in the thymus and are associated with interactions between developing T cells and myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent regulatory T cell development, leading to the fatal autoimmune disease IPEX.

[0528] Adaptive Treg cells (also called Tr1 cells or Th3 cells) can be originated during a normal immune response.

[0529] The cell may be a natural killer cell (or NK cell). NK cells form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0530] NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third type of cell differentiated from a common lymphoid progenitor that generates B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they enter the circulation.

[0531] The cells of the present invention may be any of the cell types described above.

[0532] T or NK cells according to the first aspect of the invention may be created ex vivo from the patient's own peripheral blood (first party), or in the context of a hematopoietic stem cell transplant from donor peripheral blood (second party), or from the peripheral blood of an unrelated donor (third party).

[0533] Alternatively, the T or NK cells according to the first aspect of the invention may be derived from induced progenitor cells or embryonic progenitor cells differentiated ex vivo into T or NK cells. Alternatively, immortalized T cell lines may be used which retain their lytic function and can act as therapeutic agents.

[0534] In all of these embodiments, cells expressing the chimeric polypeptide are generated by introducing DNA or RNA encoding the chimeric polypeptide by one of a number of means including transduction with a viral vector, transfection with DNA or RNA.

[0535] The cells of the present invention may be ex vivo T or NK cells from a subject. The T or NK cells may be from a peripheral blood mononuclear cell (PBMC) sample. The T or NK cells may be activated and / or expanded prior to transduction with a nucleic acid encoding a molecule providing a chimeric polypeptide according to the first aspect of the present invention, for example by treatment with an anti-CD3 monoclonal antibody.

[0536] The T or NK cells of the present invention can be prepared by:

[0537] (i) isolating a sample containing T or NK cells from a subject or other sources listed above; and

[0538] (ii) transducing or transfecting T or NK cells using the nucleic acid construct, vector or vector kit of the present invention.

[0539] The T or NK cells may then be purified, for example by selection based on expression of the antigen binding domain of the antigen binding polypeptide.

[0540] Pharmaceutical composition

[0541] The invention also relates to a pharmaceutical composition comprising a plurality of cells according to the invention.

[0542] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds. Such formulations may be, for example, in a form suitable for intravenous infusion.

[0543] Treatment

[0544] The present invention provides a method for treating a disease, comprising the step of administering the cells of the present invention (eg, in a pharmaceutical composition as described above) to a subject.

[0545] Methods for treating disease involve therapeutic uses of the cells of the invention. In this context, cells may be administered to subjects with an existing disease or condition to alleviate, reduce or ameliorate at least one symptom associated with the disease and / or to slow, reduce or block the progression of the disease.

[0546] The method may include the following steps:

[0547] (i) isolating a sample containing T or NK cells;

[0548] (ii) transducing or transfecting such cells with the nucleic acid sequence or vector provided by the present invention;

[0549] (iii) administering the cells from (ii) to a subject.

[0550] A sample containing T or NK cells can be isolated from a subject or from other sources, for example as described above. T or NK cells can be isolated from the patient's own peripheral blood (first party), or in the context of a hematopoietic stem cell transplant from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0551] The method may involve the following steps:

[0552] (i) administering the cell of the first aspect of the present invention to a subject;

[0553] (ii) administering an allogeneic hematopoietic stem cell transplant (alloHSCT) to the subject.

[0554] For example, the method may involve the following steps:

[0555] (i) administering the cell of the first aspect of the present invention to a subject;

[0556] (ii) monitoring subjects for myeloid dysplasia;

[0557] (ii) If myeloid dysplasia is detected, an allogeneic hematopoietic stem cell transplant (alloHSCT) is administered to the patient.

[0558] Myeloid metaplasia is a clinical and pathological syndrome characterized by persistent extramedullary hematopoiesis in the spleen and almost always in the liver, splenomegaly and usually hepatomegaly, and anemia with immature erythrocytes and leukocytes in the peripheral blood.

[0559] The present invention provides the cell of the present invention for use in treating and / or preventing a disease.

[0560] The present invention also relates to the use of the cells of the present invention in preparing a drug for treating and / or preventing a disease.

[0561] The disease treated by the method of the present invention may be a cancerous disease. In particular, the disease may be acute myeloid leukemia (AML).

[0562] Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells characterized by the rapid growth of abnormal cells that accumulate in the bone marrow and blood and interfere with normal blood cells. Symptoms may include feeling tired, shortness of breath, easy bruising and bleeding, and increased risk of infection. Diagnosis is usually based on a bone marrow aspirate and blood tests. As an acute leukemia, AML progresses quickly and is usually fatal within weeks or months if untreated.

[0563] The cells of the present invention may be capable of killing target cells, such as cancer cells. The target cells may 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.

[0564] The cells and pharmaceutical compositions of the present invention can be used to treat and / or prevent the above-mentioned diseases.

[0565] The present invention will now be further described by way of examples, which are intended to help those of ordinary skill in the art to practice the present invention and are not intended to limit the scope of the present invention in any way. Example

[0566] Example 1 - Design and generation of cells expressing a single CAR and a triple OR gate

[0567] A set of nucleic acid constructs encoding CARs were generated as follows:

[0568] RQR8-2A-V5CD123CAR - Single CAR construct expressing CD123 CAR with V5 tag

[0569] RQR8-2A-V5FLT3CAR - Single CAR construct expressing FLT3 CAR with a V5 tag

[0570] RQR8-2A-HACD33CAR - Single CAR construct expressing CD33 CAR with HA tag

[0571] RQR8-2A-FLAGCLL1CAR - Single CAR construct expressing CLL1 CAR with FLAG tag

[0572] RQR8-2A-V5CD123CAR-2A-HACD33CAR-2A-FLAGCLL1CAR - triple CAR construct expressing CD123CAR with V5 tag; CD33 CAR with HA tag; and CLL1 CAR with FLAG tag

[0573] RQR8-2A-V5FLT3CAR-2A-HACD33CAR-2A-FLAGCLL1CAR - triple CAR construct expressing FLT3CAR with V5 tag; CD33 CAR with HA tag; and CLL1CAR with FLAG tag

[0574] All constructs co-expressed the sorting suicide gene RQR8, which is described in WO2013 / 153391.

[0575] All CARs are dAb CARs with a second generation intracellular domain containing CD3ζ and 4-1BB co-stimulatory domains.

[0576] Peripheral blood-derived CD4+ and CD8+ T cells were transduced with the constructs. Expression of individual CARs was detected by co-staining T cells with CAR-specific tag antibodies (against V5, HA, or FLAG) and QBEND10 (to detect expression of RQR8). Figure 3 The expression of aCD33 CAR is shown. Figure 4 The expression of aCD123CAR is shown. Figure 5 Expression of aCLL1 CAR is shown.

[0577] The expression of triple CAR was detected by co-staining T cells with all three CAR-specific tag antibodies (against V5, HA, and FLAG) and QBEND10 (to detect the expression of RQR8).

[0578] Example 2 - FACS-based killing assay (FBK)

[0579] The ability of cells expressing individual CARs to kill target cells expressing individual antigens CD123, CLL1, and CD33 was investigated using a FACS-based killing assay. The assay was performed using the SupT1 cell line engineered to express the desired antigens as well as human patient-derived cell lines from the literature (Molm-14, KG1α, HL-60, K562, and THP-1) that were studied for uniform antigen expression prior to use ( Figure 6 and 7 ).

[0580] T cells were co-cultured with target cells at a 1:1 ratio. The assay was performed in a 96-well plate in a total volume of 0.2 ml using 5x10 4 Transduced T cells and target cells at a ratio of 1:1, 1:2, 1:4 or 1:8. Co-cultures were established after normalization for transduction efficiency. FBK was performed after 24 or 48 hours of incubation.

[0581] The results of FBK for aCD33 are shown in Figure 8 and 9 For aCD123, Fig.10 and 11 and for aCLL1 is shown in Fig.12 and 13 middle.

[0582] All single aCD33 dab CARs showed potent cytotoxic activity against each CD33-expressing AML cell compared to negative controls. Dose-response killing was as expected, with higher effector to target ratios (1:1) leading to higher target killing. The aCD19 FMC63 CAR showed some level of low background cytotoxic activity in some cells. No major significant differences in cytotoxicity levels were observed between the CD33 single domain CARs. Approximately 50-60% killing responses were seen in all cells, even at the lowest E:T ratios.

[0583] At 1:2 and 1:4 for SupT1 CD123, CD123-VHH-CAR-2 and CD123-VHH-CAR_1 showed significant differences (p=0.0205N=4 and p=0.0012n=4, respectively). However, only at 1:4 for SupT1 CD123, a significant difference between CD123-VHH-CAR_2 and CD123-VHH-CAR_3 can be seen (p=0.0194, n=4). For the remaining 24-hour co-cultures, all CD123 CARs had potent CD123-specific cytotoxic activity, and there were no significant differences between constructs in all ratios ( Fig.10 ). However, significant antigen-independent basal cytotoxicity was only observed for CD123- / SupT1NT cells at a 1:1 ratio. As expected, the dose-response behavior showed greater target survival with increasing E:T ratios. In the absence of basal antigen-independent cytotoxicity, all constructs showed 70% target lysis against SupT1 CD123 and KG1a cell lines (E:T, 1:2). For 48 hours of co-culture, at ratios of 1:1 and 1:2, there was significant nonspecific cytotoxicity against SupT1 NT in all constructs. At 1:8, there was no significant basal cytotoxicity against CD123- / SupT1 NT for all CAR constructs, with all CAR constructs achieving 70% target lysis against Molm14 and THP1 ( Fig.11 ).

[0584] No significant differences were observed for SupT1 CLL1, where all VHH binders showed potent CLL1-specific cytotoxicity ( Fig.12 ). This trend was shown at all E:T ratios, with no single VHH showing enhanced activity. For all CARs incubated with CLL1 / SupT1 cells at a 1:1 ratio for 48 hours, basal activity was significant, with only CLL1-VHH-CAR-3 showing significant basal activity for a 1:2 E:T ratio (p=0.0425, n=7). When incubated with THP1 and KG1a cells, all CARs showed CLL-specific cytotoxicity, while VHH-CAR 2 and 5 showed enhanced VHHCAR killing ( Fig.13 ). At a 1:2 E:T ratio, the only CAR that exhibited significant CLL1-specific cytotoxicity against THP1 was CAR-5 versus CAR-3 (p=0.0133, n=7). There was no significant difference in CLL1-specific cytotoxicity between CAR 2 and 5 at all ratios using all cell lines.

[0585] Example 3 - Cytokine Release

[0586] IL-2 secretion is an indicator of T cell activation and was assessed by using supernatants from cytotoxic co-culture assays. Cytokine secretion of individual CARs versus relevant controls was analyzed, and the production of IL-2 and IFNγ as markers of T cell activation was investigated. IL-2 and IFNγ production was measured by ELISA.

[0587] The results of the cytokine release assay are shown for aCD33. Fig.14 and 15 16 for aCD123 and in FIG17 for aCLL1.

[0588] Higher IL-2 secretion was observed when aCD33 CAR T cells were exposed to CD33-positive cells. All aCD33 sdAb CAR T cells produced relatively more IL-2 compared to non-transduced and negative controls. IL-2 secretion levels were relatively low compared to aCD33 scFv positive control. Similar IL-2 production levels were observed with the FMC63 negative control compared to the aCD33 scFv positive control. IFNγ production by aCD33 CAR T cells was significantly higher than that of the negative control. All aCD33 sdAb CARs produced similar levels of IFN-γ ( Fig.15 sdAbCD33.6 maintained high levels of IFN-γ production in all cells, whereas sdAb CD33.2 produced lower levels of IFN-γ compared to all other sdAb CD33 CARs.

[0589] When challenged with THP1 and MOLM14 cells, aCD33 scFv produced lower levels of IFNγ. Little difference in IFN-γ production was observed between engineered SupT1 and AML-derived cells. The FMC63 scFv negative control also showed the same level of IFN-γ production (approximately 10,000 pg / ml) compared to the aCD33 scFv positive control on MOLM14 cells.

[0590] When exposed to CD123+ cells, all aCD123 CAR T cells produce IL-2, but there is no significant difference in IL-2 production between VHH-CAR-T cell constructs. Using Molm14, VHH-CAR-2 and 6 produce the highest levels of IL-2 production (average of about 1.77x104 and 1.63x104pg / ml), and using THP1, VHH-CAR-4 produces the lowest IL-2 production (average of about 3.5x103pg / ml). Compared with the scFv CAR positive control, VHH-CAR-constructs showed improved IL-2 production during the 24-hour and 48-hour time course, even if no significant differences were seen in the cytotoxicity assay.

[0591] For SupT1 CD123 and Molm14, all aCD123 CARs produce similar levels of IFNγ (average of about 3.4x103 and about 4.0x104 pg / ml, respectively). For IFNγ production in all constructs, there are no significant differences between CD123 CAR constructs. As with IL-2 production, VHH-CAR-6 consistently produces higher levels of cytokines under all conditions, although there are no significant differences. The IFNγ levels produced by the scFv control CAR are similar to those of VHH CAR, which is consistent with the trend of target cleavage seen in the cytotoxicity assay. The highest IFNγ production is with Molm14's VHH-CAR-6 (average of about 4.0x104 pg / ml), while the lowest is with THP1's VHH-CAR-4 (average of about 1.45x103pg / ml).

[0592] Antigen-specific IL-2 production was observed for all CLL1-VHH-CAR constructs with SupT1 CLL1 and THP1, but no significant differences were observed between the constructs (Figure 17, a). However, even though the killing data did show antigen-specific cytotoxicity, there was no antigen-specific IL-2 production for the CAR co-cultured with KG1a. This observation may be related to the extremely low expression level of CLL1 on the cell surface of KG1a cells (638 / cell, Figure 7 ). A similar trend was observed for IFNγ production, where for SupT1 CLL1 and THP1, all CAR constructs showed antigen-specific IFNγ production with no significant differences between constructs (Figure 17, b). Similarly, relatively low levels of IFNγ were produced for KG1a, with higher levels of cytokines produced by CARs that performed well in cytotoxicity assays (VHH-CAR 2, 4, and 5).

[0593] Example 4 - Proliferation Assay (PA)

[0594] To measure proliferation, the same set of CAR-expressing T cells described in Example 1 were labeled with the dye Cell Trace Violet (CTV), a fluorescent dye that is hydrolyzed and retained inside the cell. It is excited by a 405 nm (violet) laser and fluorescence can be detected in the pacific blue channel. CTV dye was reconstituted in DMSO to 5 mM. T cells were cultured at 2x10 6 10 cells / ml were resuspended in PBS and 1 ul / ml of CTV was added. T cells were incubated with CTV for 20 minutes at 37°C. Subsequently, the cells were quenched by adding 5V of complete medium. After 5 minutes of incubation, T cells were washed and resuspended in 2 ml of complete medium. Incubated for another 10 minutes at room temperature to allow acetate to hydrolyze and retain the dye.

[0595] The labeled T cells were co-cultured with target cells for four days. The assay used 5x10 4 Transduced T cells and an equal number of target cells (ratio 1:1) were cultured in a 96-well plate in a total volume of 0.2 ml. At the fourth day time point, T cells were analyzed by flow cytometry to measure the dilution of CTV as T cells divided. The number of T cells present at the end of co-culture was calculated and expressed as the proliferation fold compared to the number of T cells input.

[0596] The results of the proliferation assay are shown for aCD33 Fig.18 For aCD123, Fig.19 and for aCLL1 is shown in Fig. 20 middle.

[0597] There were significant differences in the expansion fold of aCD33 CAR T cells observed after 6 days. A higher expansion fold was observed for aCD33 sdAb CAR T cells compared to the non-transduced control and the negative control (FMC63 scFv). It should be noted that in all AML cells, the positive control (aCD33 scFv) had lower proliferation levels compared to aCD33 sdAb CAR T cells. The expansion fold of CAR T cells was significantly higher in SupT1 artificially transduced with CD33 compared to the other cells.

[0598] No significant differences were observed between all CD123-VHH-CAR constructs under all conditions.

[0599] Using CLL1+ target cell lines, antigen-specific proliferation was observed for all CLL1-VHH-CAR constructs, however, using SupT1 CLL1 and THP1, no significant differences in proliferation capacity were observed. For KG1a co-cultures, at all ratios, CLL1-VHH-CAR 2 and 5 had significant proliferation advantages over CLL1-VHH-CAR 1, 2, and 3 (p values ​​ranged from <0.05 to <0.01, n=7). Using KG1a, CLL1-VHH-CAR-5 showed a significant proliferation advantage over CLL1-VHH-CAR-2 (p=0.0445, n=7).

[0600] All six aCD33 single dab CARs exhibited similar levels of response to target cells in all proliferation, cytotoxicity, and cytokine production assays compared to a negative CAR control (FMC63 scFv). The sdAb CD123 CAR showed efficacy against engineered SupT1 CD123 cells and other AML-derived cells (MOLM14, THP1, KG1a) in a dose-response manner. The sdAb CLL1 CAR was also effective against CLL1+ cells. The results obtained from the first phase of the study indicate that each single domain binder (CD123, CD33, and CLL1) can be effective as a second-generation CAR against AML.

[0601] All publications mentioned in the above description are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the described method and system of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, it is apparent to those skilled in the art of molecular biology or related fields that various modifications of the described modes for implementing the present invention are intended to be within the scope of the appended claims. Sequence Listing <110> Autorus Ltd. <120> cell <130> P115644PCT <150> GB 1813178.9 <151> 2018-08-13 <160> 95 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 chimeric antigen receptor (CAR) complementarity determining region (CDR) CDR1 <400> 1 Gly Arg Thr Phe Ser Met His Ser 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR2 <400> 2 Val Thr Trp Ser Gly Asp Thr Phe 1 5 <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR3 <400> 3 Lys Asp Asp Pro Tyr Arg Pro Ala Tyr Asp Tyr 1 5 10 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR1 <400> 4 Gly Arg Thr Phe Ser Ser Tyr Val 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR2 <400> 5 Ile Ser Trp Ser Gly Gly Ser Thr 1 5 <210> 6 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR3 <400> 6 Ala Ala Met Glu Leu Arg Gly Gly Ser Tyr Asn Tyr Ala Ser Ser Arg 1 5 10 15 Gln Tyr Asp Tyr 20 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR1 <400> 7 Glu Ile Ala Phe Ser Asn Phe Asn 1 5 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR2 <400> 8 Ile Ser Ser His Gly Asp Thr Asn Tyr 1 5 <210> 9 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR3 <400> 9 Asn Ala Asn Asp Pro Phe Leu Ser Val Ser Asp Phe 1 5 10 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR1 <400> 10 Gly Ser Ile Phe Ser Ile Asn Ala 1 5 <210> 11 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR3 <400> 11 Ala Ala Ile Ser Gly Trp Gly Arg Ser Ile Arg Val Gly Glu Arg Tyr 1 5 10 15 Glu Tyr Asp Tyr 20 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR1 <400> 12 Gly Arg Thr Ser Ser Ser Ser Thr 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR2 <400> 13 Ile Thr Leu Ser Gly Gly Ser Thr 1 5 <210> 14 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR3 <400> 14 Ala Ala Arg Arg Trp Ser Asn Asn Arg Gly Gly Tyr Asp Arg Ala Gly 1 5 10 15 Tyr Asp Tyr <210> 15 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR1 <400> 15 Gly Arg Thr Phe Ser Ser Tyr Ala 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR2 <400> 16 Ile Thr Trp Ser Gly Gly Ser Thr 1 5 <210> 17 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Anti-CD33 CAR, CDR3 <400> 17 Ala Met Leu Leu Arg Gly Gly Leu Tyr Asp Tyr Thr Asp Tyr Ile Leu 1 5 10 15 Tyr Asn Tyr <210> 18 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> CD33 domain antibody (dAb) sequence, CD33 dAb P1.E4 - 44738 <400> 18 Gln Val Gln Leu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser 1 5 10 15 Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Met His Ser 20 25 30 Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 35 40 45 Ala Val Thr Trp Ser Gly Asp Thr Phe Ala Tyr Ala Asp Phe Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Gly Ile Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Lys Asp Asp Pro Tyr Arg Pro Ala Tyr Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 19 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> CD33 dAb sequence, CD33 dAb P1.H3 - 44739 <400> 19 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Met Glu Leu Arg Gly Gly Ser Tyr Asn Tyr Ala Ser Ser Arg 100 105 110 Gln Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 20 <211> 118 <212> PRT <213> Artificial sequence <220> <223> CD33 dAb sequence, CD33 dAb P1.G8 - 44742 <400> 20 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Ala Ala Ser Glu Ile Ala Phe Ser Asn Phe 20 25 30 Asn Met Gly Trp Tyr Arg Gln Gly Ser Gly Lys Gln Arg Thr Leu Val 35 40 45 Ala Gln Ile Ser Ser His Gly Asp Thr Asn Tyr Leu Asp Ser Met Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Asn Lys Lys Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ala Asn Asp Pro Phe Leu Ser Val Ser Asp Phe Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 21 <211> 127 <212> PRT <213> artificial sequence <220> <223> CD33 dAb sequence, CD33 dAb P2.A7 - 46173 <400> 21 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Phe Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Ile Ser Gly Trp Gly Arg Ser Ile Arg Val Gly Glu Arg Tyr 100 105 110 Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 22 <211> 126 <212> PRT <213> artificial sequence <220> <223> CD33 dAb sequence, CD33 dAb P2.B12 - 46174 <400> 22 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Ser Ser Ser 20 25 30 Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Thr Leu Ser Gly Gly Ser Thr His Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Glu Ser Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr Cys 85 90 95 Ala Ala Arg Arg Trp Ser Asn Asn Arg Gly Gly Tyr Asp Arg Ala Gly 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 23 <211> 127 <212> PRT <213> Artificial sequence <220> <223> CD33 dAb sequence, CD33 dAb P2.F2 - 46176 <400> 23 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Thr Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Met Leu Leu Arg Gly Gly Leu Tyr Asp Tyr Thr Asp Tyr Ile 100 105 110 Leu Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 24 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti - CD123 CAR, CDR1 <400> twenty four Gly Arg Ser Ile Asn Thr Tyr Ala 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR2 <400> 25 Ile Asn Tyr Asn Ser Arg Tyr Thr 1 5 <210> 26 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR3 <400> 26 Ala Ala Thr Ser Tyr Tyr Pro Thr Asp Tyr Asp Val Ala Ser Arg Val 1 5 10 15 Ala Thr Trp Pro Ser 20 <210> 27 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR1 <400> 27 Gly Ile Ser Leu Asn Ala 1 5 <210> 28 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR2 <400> 28 Ile Lys Ile Gly Gly Val Ser 1 5 <210> 29 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR3 <400> 29 Asn Thr Tyr Pro Pro Tyr Leu Asn Gly Met Asp Tyr 1 5 10 <210> 30 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR1 <400> 30 Gly Arg Ser Phe Asn Thr Asp Ala 1 5 <210> 31 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR2 <400> 31 Ile Ser Trp Asp Gly Thr Arg Thr 1 5 <210> 32 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR3 <400> 32 Ala Ala Glu Pro Gln Lys Ala Trp Pro Ile Gly Thr Ser Ala Ala Gly 1 5 10 15 Phe Arg Ser <210> 33 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR1 <400> 33 Gly Ser Ser Ile Ser Val 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR2 <400> 34 Ile Ser Trp Ser Asp Gly Asn Thr 1 5 <210> 35 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR3 <400> 35 Ala Val Glu Pro Arg Gly Trp Pro Lys Gly His Arg Tyr 1 5 10 <210> 36 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR1 <400> 36 Gly Ser Ser Phe Ser Ile Asn Val 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR2 <400> 37 Ile Ser Trp Ser Asp Gly Ser Thr 1 5 <210> 38 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR3 <400> 38 Ala Val Glu Pro Arg Gly Trp Pro Lys Gly His Arg Tyr 1 5 10 <210> 39 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR1 <400> 39 Gly Ser Ile Phe Arg Ile Asn Ala 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR2 <400> 40 Val Asn Trp Ile Gly Gly Thr Thr 1 5 <210> 41 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Anti-CD123 CAR, CDR3 <400> 41 Ser Ala Thr Asp Lys Gly Gly Ser Ser Arg Tyr 1 5 10 <210> 42 <211> 128 <212> PRT <213> Artificial sequence <220> <223> CD123 dAb sequence, CD123 dAb H11 45897 <400> 42 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Ser Ile Asn Thr Tyr 20 25 30 Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ser Ile Asn Tyr Asn Ser Arg Tyr Thr His Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asp Ser Leu Asn Arg Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Ser Tyr Tyr Pro Thr Asp Tyr Asp Val Ala Ser Arg Val 100 105 110 Ala Thr Trp Pro Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> CD123 dAb sequence, CD123 dAb F8 45888 <400> 43 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Glu 1 5 10 15 Ser Leu Arg Leu Thr Cys Ala Val Ser Gly Ile Ser Leu Asn Ala Met 20 25 30 Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Leu Arg Glu Trp Val Ala 35 40 45 Val Ile Lys Ile Gly Gly Val Ser Asn Tyr Ala Val Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ile Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn Thr 85 90 95 Tyr Pro Pro Tyr Leu Asn Gly Met Asp Tyr Trp Gly Lys Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 44 <211> 126 <212> PRT <213> Artificial sequence <220> <223> CD123 dAb sequence, CD123 dAb A7 45865 <400> 44 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Arg Ser Phe Asn Thr Asp 20 25 30 Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Asp Gly Thr Arg Thr Tyr Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Asn Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Glu Pro Gln Lys Ala Trp Pro Ile Gly Thr Ser Ala Ala Gly 100 105 110 Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 45 <211> 118 <212> PRT <213> Artificial sequence <220> <223> CD123 dAb sequence, CD123 dAb B4 45868 <400> 45 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ser Ile Ser Val Met 20 25 30 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala 35 40 45 Ile Ser Trp Ser Asp Gly Asn Thr Asn Tyr Ala Asp Ser Val Asn Gly 50 55 60 Arg Phe Ser Val Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Val 85 90 95 Glu Pro Arg Gly Trp Pro Lys Gly His Arg Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 46 <211> 120 <212> PRT <213> Artificial sequence <220> <223> CD123 dAb sequence, CD123 dAb A10 45866 <400> 46 Gln Val Gln Leu Gln Glu Ser Gly Gly Ser Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ser Phe Ser Ile Asn 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Asp Gly Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Val Glu Pro Arg Gly Trp Pro Lys Gly His Arg Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 47 <211> 118 <212> PRT <213> Artificial sequence <220> <223> CD123 dAb sequence, CD123 dAb C11 45874 <400> 47 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Arg Ile Asn 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Thr Ala Val Asn Trp Ile Gly Gly Thr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Phe Cys 85 90 95 Ser Ala Thr Asp Lys Gly Gly Ser Ser Arg Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Anti-CLL-1 CAR, CDR1 <400> 48 Gly Phe Thr Phe Gly Asn His Asp 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR2 <400> 49 Ile Asp Ser Gly Gly Asn Val Ile 1 5 <210> 50 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR3 <400> 50 Ala Thr Asp Leu Asp Ser Gly Ala Glu Ser Leu Glu Ser Val Tyr 1 5 10 15 <210> 51 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR1 <400> 51 Gly Phe Ala Phe Gly Ser Ala Asp 1 5 <210> 52 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR2 <400> 52 Ile Asp Ser Gly Gly Asn Thr Gln 1 5 <210> 53 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR3 <400> 53 Thr Asp Leu Asp Pro Thr Thr Asp Ser Leu Glu Asn Val Tyr 1 5 10 <210> 54 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR1 <400> 54 Gly Arg Thr Phe Ser Ala Tyr Phe 1 5 <210> 55 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR2 <400> 55 Ile Asn Trp Asn Gly Asp Ser Ser 1 5 <210> 56 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR3 <400> 56 Ala Ala Asp Thr His Gly Ala Val Gly Leu Gly Ser Glu Arg Leu Tyr 1 5 10 15 Asp Tyr <210> 57 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR1 <400> 57 Gly Ile Gly Val Ser Ser Thr Gly 1 5 <210> 58 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR2 <400> 58 Ile Asp Arg Asp Gly Thr Thr 1 5 <210> 59 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR3 <400> 59 Thr Val Val Gly Asp Tyr Tyr 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR1 <400> 60 Gly Phe Ile Phe Gly Asn Tyr Asp 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR2 <400> 61 Ile Ser Ser Gly Gly Asn Asp Ile 1 5 <210> 62 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR3 <400> 62 Ala Ala Asp Leu Asp Pro Gly Thr Asp Ser Leu Asp Asn Ile His 1 5 10 15 <210> 63 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR1 <400> 63 Gly Phe Thr Leu Asp Tyr Tyr Ala 1 5 <210> 64 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR2 <400> 64 Ile Ser Ser Ser Asp Gly Ser Thr 1 5 <210> 65 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Anti-CLL-1 CAR, CDR3 <400> 65 Ala Glu Ala Val Tyr Tyr Ala Gly Val Cys Val Ala Met Tyr Asp Ser 1 5 10 15 <210> 66 <211> 122 <212> PRT <213> Artificial sequence <220> <223> CLL-1 dAb sequence, CLL-1 dAb 44548 <400> 66 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Gly Asn His 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Val Glu Phe Val 35 40 45 Ala Gly Ile Asp Ser Gly Gly Asn Val Ile Val Tyr Glu Glu Val Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asp Gly Leu Lys Pro Glu Asp Ala Gly Met Tyr Phe Cys 85 90 95 Ala Thr Asp Leu Asp Ser Gly Ala Glu Ser Leu Glu Ser Val Tyr His 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 67 <211> 122 <212> PRT <213> Artificial sequence <220> <223> CLL-1 dAb sequence, CLL-1 dAb 44544 <400> 67 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Glu Ser Gly Gly 1 5 10 15 Ser Leu Arg Ile Ser Cys Thr Gly Phe Gly Phe Ala Phe Gly Ser Ala 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Val Glu Phe Val 35 40 45 Ala Gly Ile Asp Ser Gly Gly Asn Thr Gln Thr Tyr Glu Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Gln Ser Glu Asp Ala Gly Val Tyr Phe Cys 85 90 95 Ala Thr Asp Leu Asp Pro Thr Thr Asp Ser Leu Glu Asn Val Tyr His 100 105 110 Gly Gln Gly Thr Gln Val Ile Val Ser Ser 115 120 <210> 68 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> CLL-1 dAb Sequence, CLL-1 dAb 44538 <400> 68 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Asp 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Ala Tyr 20 25 30 Phe Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Asn Gly Asp Ser Ser Trp Tyr Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Thr His Gly Ala Val Gly Leu Gly Ser Glu Arg Leu Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 69 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> CLL-1 dAb Sequence, CLL-1 dAb 44546 <400> 69 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Gly Val Ser Ser Thr 20 25 30 Gly Met Gly Trp Ser Arg Gln Thr Pro Gly Lys Gln Val Glu Leu Val 35 40 45 Ala Leu Ile Asp Arg Asp Gly Thr Thr Asn Tyr Ala Asp Thr Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Met Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr His Cys Thr 85 90 95 Val Val Gly Asp Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 100 105 110 Ser <210> 70 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> CLL-1 dAb Sequence, CLL-1 dAb 44545 <400> 70 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Phe Ile Phe Gly Asn Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Val Glu Phe Val 35 40 45 Ala Gly Ile Ser Ser Gly Gly Asn Asp Ile Val Tyr Glu Asp Ala Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr 65 70 75 80 Leu Asp Met Ala Ser Val Lys Pro Glu Asp Ala Gly Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Leu Asp Pro Gly Thr Asp Ser Leu Asp Asn Ile His His 100 105 110 Gly Gln Gly Thr Gln Val Phe Val Ser Ser 115 120 <210> 71 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> CLL-1 dAb Sequence, CLL-1 dAb 44536 <400> 71 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Glu Ala Val Tyr Tyr Ala Gly Val Cys Val Ala Met Tyr Asp Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 72 <211> 7 <212> PRT <213> artificial sequence <220> <223> Anti-FLT3 CAR, CDR1 <400> 72 Gly Ile Phe Lys Thr Asn Tyr 1 5 <210> 73 <211> 7 <212> PRT <213> artificial sequence <220> <223> Anti-FLT3 CAR, CDR2 <400> 73 Phe Thr Asn Asp Gly Ser Thr 1 5 <210> 74 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR3 <400> 74 Tyr Gly Leu Gly His 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR1 <400> 75 Gly Thr Ile Ser Ser Ile Arg Tyr 1 5 <210> 76 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR2 <400> 76 Ile Thr Ser Ser Gly Asn Thr 1 5 <210> 77 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR3 <400> 77 Tyr Thr Met Gly Tyr 1 5 <210> 78 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR1 <400> 78 Gly Ile Phe Ser Thr Asn Tyr 1 5 <210> 79 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR2 <400> 79 Phe Thr Asn Asp Gly Gly Thr 1 5 <210> 80 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR3 <400> 80 Cys Gly Leu Gly His 1 5 <210> 81 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR1 <400> 81 Gly Ser Ile Ser Ser Ile Arg Tyr 1 5 <210> 82 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR2 <400> 82 Ile Thr Ser Ser Gly Ser Thr 1 5 <210> 83 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR3 <400> 83 Tyr Thr Met Gly Tyr 1 5 <210> 84 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR1 <400> 84 Gly Ile Phe Ser Thr Asn His 1 5 <210> 85 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR2 <400> 85 Phe Thr Asn Asp Gly Ser Thr 1 5 <210> 86 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Anti-FLT3 CAR, CDR3 <400> 86 Tyr Gly Leu Gly His 1 5 <210> 87 <211> 150 <212> PRT <213> Artificial sequence <220> <223> FLT3 dAb sequence, FLT3 dAb B5 <400> 87 Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Phe Lys Thr Asn Tyr 20 25 30 Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 35 40 45 Ala Phe Thr Asn Asp Gly Ser Thr Leu Tyr Gly Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Tyr Thr Val Ser Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Gly 85 90 95 Leu Gly His Trp Gly Gln Gly Thr Gln Val Ile Val Ser Ser Glu Pro 100 105 110 Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Asp Asp Asp Asp Lys Glu 115 120 125 Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala His His His 130 135 140 His His His Gly Ala Ala 145 150 <210> 88 <211> 151 <212> PRT <213> Artificial Sequence <220> <223> FLT3 dAb sequence, FLT3 dAb G3 <400> 88 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Ser Ser Ile Arg 20 25 30 Tyr Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Val Val 35 40 45 Ala Tyr Ile Thr Ser Ser Gly Asn Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Ala Tyr Tyr Cys Tyr 85 90 95 Thr Met Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu 100 105 110 Pro Lys Ile Pro Gln Pro Gln Pro Ala Ala Ala Asp Asp Asp Asp Lys 115 120 125 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala His His 130 135 140 His His His His Gly Ala Ala 145 150 <210> 89 <211> 150 <212> PRT <213> Artificial Sequence <220> <223> FLT3 dAb sequence, FLT3 dAb H5 <400> 89 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Phe Ser Thr Asn Tyr 20 25 30 Met Val Trp Cys Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala 35 40 45 Ala Phe Thr Asn Asp Gly Gly Thr Leu Tyr Ala Asp Ser Leu Lys Gly 50 55 60 Arg Phe Ser Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Leu Leu Leu 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Cys Gly 85 90 95 Leu Gly His Trp Gly Arg Gly Thr Lys Val Thr Val Ser Ser Glu Pro 100 105 110 Lys Ile Pro Gln Pro Gln Pro Ala Ala Ala Asp Asp Asp Asp Lys Glu 115 120 125 Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala His His His 130 135 140 His His His Gly Ala Ala 145 150 <210> 90 <211> 151 <212> PRT <213> Artificial Sequence <220> <223> FLT3 dAb Sequence, FLT3 dAb D12 <400> 90 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Ile Arg 20 25 30 Tyr Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Ser Val 35 40 45 Ala Trp Ile Thr Ser Ser Gly Ser Thr Asn Tyr Ala Asp Ser Val Gln 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95 Thr Met Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu 100 105 110 Pro Lys Ile Pro Gln Pro Gln Pro Ala Ala Ala Asp Asp Asp Asp Lys 115 120 125 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala His His 130 135 140 His His His His Gly Ala Ala 145 150 <210> 91 <211> 152 <212> PRT <213> Artificial Sequence <220> <223> FLT3 dAb Sequence, FLT3 dAb F10 <400> 91 Gln Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Phe Ser Thr 20 25 30 Asn His Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu 35 40 45 Val Ala Ala Phe Thr Asn Asp Gly Ser Thr Leu Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Val Ile Ser Arg Asp Asn Ala Lys Tyr Thr Val Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Tyr Gly Leu Gly His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Asp Asp Asp Asp 115 120 125 Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala His 130 135 140 His His His His Gly Ala Ala 145 150 <210> 92 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Hinge spacer sequence <400> 92 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 93 <211> 136 <212> PRT <213> Artificial sequence <220> <223> Suicide gene sequence <400> 93 Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Glu 1 5 10 15 Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser Pro 20 25 30 Ala Lys Pro Thr Thr Thr Ala Cys Pro Tyr Ser Asn Pro Ser Leu Cys 35 40 45 Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 50 55 60 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 65 70 75 80 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 85 90 95 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 100 105 110 Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val 115 120 125 Cys Lys Cys Pro Arg Pro Val Val 130 135 <210> 94 <211> 517 <212> PRT <213> artificial sequence <220> <223> FRB-FKBP12-L3-dCasp9 <400> 94 Met Ala Ser Arg Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu 1 5 10 15 Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe 20 25 30 Glu Val Leu Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr 35 40 45 Leu Lys Glu Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu 50 55 60 Ala Gln Glu Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp 65 70 75 80 Leu Leu Gln Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser 85 90 95 Lys Leu Glu Tyr Ser Gly Gly Gly Ser Leu Glu Gly Val Gln Val Glu 100 105 110 Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr 115 120 125 Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Phe Asp 130 135 140 Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln 145 150 155 160 Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly 165 170 175 Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr 180 185 190 Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val 195 200 205 Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Gly Gly Gly Ser Gly Val Asp Gly Phe Gly Asp Val Gly Ala 225 230 235 240 Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met 245 250 255 Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg 260 265 270 Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys 275 280 285 Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly 290 295 300 Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln 305 310 315 320 Gln Asp His Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His 325 330 335 Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr 340 345 350 Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly 355 360 365 Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln 370 375 380 Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr 385 390 395 400 Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr 405 410 415 Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser 420 425 430 Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro 435 440 445 Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu 450 455 460 Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln 465 470 475 480 Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr 485 490 495 Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe 500 505 510 Lys Thr Ser Ala Ser 515 <210> 95 <211> 20 <212> PRT <213> Artificial sequence <220> <223> 2A-like cleavage site sequence <400> 95 Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu 1 5 10 15 Asn Pro Gly Pro 20

Claims

1. A cell comprising: a chimeric antigen receptor comprising an anti-CD33 domain antibody antigen binding domain; a chimeric antigen receptor comprising an anti-CLL1 domain antibody antigen binding domain; and a chimeric antigen receptor comprising an anti-CD123 domain antibody antigen binding domain; wherein the cell is a T cell; The chimeric antigen receptor comprising an anti-CD33 domain antibody antigen binding domain has an antigen binding domain comprising the following complementarity determining regions: (i) CDR1-GRTFSMHS (SEQ ID No. 1); CDR2-VTWSGDTF (SEQ ID No. 2); CDR3-KDDPYRPAYDY (SEQ ID No. 3); (ii) CDR1-GRTFSSYV (SEQ ID No. 4); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAMELRGGSYNYASSRQYDY (SEQ ID No. 6); (iii) CDR1-EIAFSNFN (SEQ ID No. 7); CDR2-ISSHGDTNY (SEQ ID No. 8); CDR3-NANDPFLSVSDF (SEQ ID No. 9); (iv) CDR1-GSIFSINA (SEQ ID No. 10); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAISGWGRSIRVGERYEYDY (SEQ ID No. 11); (v) CDR1-GRTSSSST (SEQ ID No. 12); CDR2-ITLSGGST (SEQ ID No. 13); CDR3-AARRWSSNNRGGYDRAGYDY (SEQ ID No. 14); or (vi) CDR1-GRTFSSYA (SEQ ID No. 15); CDR2-ITWSGGST (SEQ ID No. 16); CDR3-AMLRGLYDYTDYILYNY (SEQ ID No. 17).

2. The cell according to claim 1, wherein the anti-CD33 domain antibody antigen binding domain comprises one of the sequences shown in SEQ ID No. 18, 19, 20, 21, 22 or 23.

3. The cell according to claim 1 or 2, wherein the chimeric antigen receptor comprising an anti-CLL1 domain antibody antigen binding domain has an antigen binding domain comprising the following complementarity determining regions: (i) CDR1-GFTFGNHD (SEQ ID No. 48); CDR2-IDSGGNVI (SEQ ID No. 49); CDR3-ATDLDSGAESLESVY (SEQ ID No. 50); (ii) CDR1-GFAFGSAD (SEQ ID No. 51); CDR2-IDSGGNTQ (SEQ ID No. 52); CDR3-TDLDPTTDSLENVY (SEQ ID No. 53); (iii) CDR1-GRTFSAYF (SEQ ID No. 54); CDR2-INWNGDSS (SEQ ID No. 55); CDR3-AADTHGAVGLGSERLYDY (SEQ ID No. 56); (iv) CDR1-GIGVSSTG (SEQ ID No. 57); CDR2-IDRDGTT (SEQ ID No. 58); CDR3-TVVGDYY (SEQ ID No. 59); (v) CDR1-GFIFGNYD (SEQ ID No. 60); CDR2-ISSGGNDI (SEQ ID No. 61); CDR3-AADLDPGTDSLDNIH (SEQ ID No. 62); or (vi) CDR1-GFTLDYYA (SEQ ID No. 63); CDR2-ISSSDGST (SEQ ID No. 64); CDR3-AEAVYYAGVCVAMYDS (SEQ ID No. 65).

4. The cell according to claim 3, wherein the anti-CLL1 domain antibody antigen binding domain comprises one of the sequences shown in SEQ ID No. 66, 67, 68, 69, 70 or 71.

5. A cell according to claim 1 or 2, wherein the chimeric antigen receptor comprising an anti-CD123 domain antibody antigen binding domain has an antigen binding domain comprising the following complementarity determining regions: (i) CDR1-GRSINTYA (SEQ ID No. 24); CDR2-INYNSRYT (SEQ ID No. 25); CDR3-AATSYYPTDYDVASRVATWPS (SEQ ID No. 26); (ii) CDR1-GISLNA (SEQ ID No. 27); CDR2-IKIGGVS (SEQ ID No. 28); CDR3-NTYPPYLNGMDY (SEQ ID No. 29); (iii) CDR1-GRSFNTDA (SEQ ID No. 30); CDR2-ISWDGTRT (SEQ ID No. 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32); (iv) CDR1-GSSISV (SEQ ID No. 33); CDR2-ISWSDGNT (SEQ ID No. 34); CDR3-AVEPRGWPKGHRY (SEQ ID No. 35); (v) CDR1-GSSFSINV (SEQ ID No. 36); CDR2-ISWSDGST (SEQ ID No. 37); CDR3-AVEPRGWPKGHRY (SEQ ID No. 38); or (vi) CDR1-GSIFRINA (SEQ ID No. 39); CDR2-VNWIGGTT (SEQ ID No. 40); CDR3-SATDKGGSSRY (SEQ ID No. 41).

6. A cell according to claim 3, wherein the chimeric antigen receptor comprising an anti-CD123 domain antibody antigen binding domain has an antigen binding domain comprising the following complementarity determining regions: (i) CDR1-GRSINTYA (SEQ ID No. 24); CDR2-INYNSRYT (SEQ ID No. 25); CDR3-AATSYYPTDYDVASRVATWPS (SEQ ID No. 26); (ii) CDR1-GISLNA (SEQ ID No. 27); CDR2-IKIGGVS (SEQ ID No. 28); CDR3-NTYPPYLNGMDY (SEQ ID No. 29); (iii) CDR1-GRSFNTDA (SEQ ID No. 30); CDR2-ISWDGTRT (SEQ ID No. 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32); (iv) CDR1-GSSISV (SEQ ID No. 33); CDR2-ISWSDGNT (SEQ ID No. 34); CDR3-AVEPRGWPKGHRY (SEQ ID No. 35); (v) CDR1-GSSFSINV (SEQ ID No. 36); CDR2-ISWSDGST (SEQ ID No. 37); CDR3-AVEPRGWPKGHRY (SEQ ID No. 38); or (vi) CDR1-GSIFRINA (SEQ ID No. 39); CDR2-VNWIGGTT (SEQ ID No. 40); CDR3-SATDKGGSSRY (SEQ ID No. 41).

7. A cell according to claim 4, wherein the chimeric antigen receptor comprising an anti-CD123 domain antibody antigen binding domain has an antigen binding domain comprising the following complementarity determining regions: (i) CDR1-GRSINTYA (SEQ ID No. 24); CDR2-INYNSRYT (SEQ ID No. 25); CDR3-AATSYYPTDYDVASRVATWPS (SEQ ID No. 26); (ii) CDR1-GISLNA (SEQ ID No. 27); CDR2-IKIGGVS (SEQ ID No. 28); CDR3-NTYPPYLNGMDY (SEQ ID No. 29); (iii) CDR1-GRSFNTDA (SEQ ID No. 30); CDR2-ISWDGTRT (SEQ ID No. 31); CDR3-AAEPQKAWPIGTSAAGFRS (SEQ ID No. 32); (iv) CDR1-GSSISV (SEQ ID No. 33); CDR2-ISWSDGNT (SEQ ID No. 34); CDR3-AVEPRGWPKGHRY (SEQ ID No. 35); (v) CDR1-GSSFSINV (SEQ ID No. 36); CDR2-ISWSDGST (SEQ ID No. 37); CDR3-AVEPRGWPKGHRY (SEQ ID No. 38); or (vi) CDR1-GSIFRINA (SEQ ID No. 39); CDR2-VNWIGGTT (SEQ ID No. 40); CDR3-SATDKGGSSRY (SEQ ID No. 41).

8. A cell according to claim 5, wherein the anti-CD123 domain antibody antigen binding domain comprises one of the sequences shown in SEQ ID No. 42, 43, 44, 45, 46 or 47.

9. The cell according to claim 6, wherein the anti-CD123 domain antibody antigen binding domain comprises one of the sequences shown in SEQ ID No. 42, 43, 44, 45, 46 or 47.

10. The cell according to claim 7, wherein the anti-CD123 domain antibody antigen binding domain comprises one of the sequences shown in SEQ ID No. 42, 43, 44, 45, 46 or 47.

11. A nucleic acid construct encoding: a chimeric antigen receptor comprising an anti-CD33 domain antibody antigen binding domain; a chimeric antigen receptor comprising an anti-CLL1 domain antibody antigen binding domain; and a chimeric antigen receptor comprising an anti-CD123 domain antibody antigen binding domain; The chimeric antigen receptor comprising an anti-CD33 domain antibody antigen binding domain has an antigen binding domain comprising the following complementarity determining regions: (i) CDR1-GRTFSMHS (SEQ ID No. 1); CDR2-VTWSGDTF (SEQ ID No. 2); CDR3-KDDPYRPAYDY (SEQ ID No. 3); (ii) CDR1-GRTFSSYV (SEQ ID No. 4); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAMELRGGSYNYASSRQYDY (SEQ ID No. 6); (iii) CDR1-EIAFSNFN (SEQ ID No. 7); CDR2-ISSHGDTNY (SEQ ID No. 8); CDR3-NANDPFLSVSDF (SEQ ID No. 9); (iv) CDR1-GSIFSINA (SEQ ID No. 10); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAISGWGRSIRVGERYEYDY (SEQ ID No. 11); (v) CDR1-GRTSSSST (SEQ ID No. 12); CDR2-ITLSGGST (SEQ ID No. 13); CDR3-AARRWSSNNRGGYDRAGYDY (SEQ ID No. 14); or (vi) CDR1-GRTFSSYA (SEQ ID No. 15); CDR2-ITWSGGST (SEQ ID No. 16); CDR3-AMLRGLYDYTDYILYNY (SEQ ID No. 17).

12. A method for preparing a cell according to claim 1, comprising the step of transducing or transfecting the cell with a nucleic acid construct according to claim 11.

13. A vector comprising the nucleic acid construct according to claim 11.

14. A kit of vectors, comprising: (i) a first vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising an anti-CD33 domain antibody antigen binding domain; (ii) a second vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising an anti-CLL1 domain antibody antigen binding domain; and (iii) a third vector comprising a nucleic acid sequence encoding a chimeric antigen receptor comprising an anti-CD123 domain antibody antigen binding domain; The chimeric antigen receptor comprising an anti-CD33 domain antibody antigen binding domain has an antigen binding domain comprising the following complementarity determining regions: (i) CDR1-GRTFSMHS (SEQ ID No. 1); CDR2-VTWSGDTF (SEQ ID No. 2); CDR3-KDDPYRPAYDY (SEQ ID No. 3); (ii) CDR1-GRTFSSYV (SEQ ID No. 4); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAMELRGGSYNYASSRQYDY (SEQ ID No. 6); (iii) CDR1-EIAFSNFN (SEQ ID No. 7); CDR2-ISSHGDTNY (SEQ ID No. 8); CDR3-NANDPFLSVSDF (SEQ ID No. 9); (iv) CDR1-GSIFSINA (SEQ ID No. 10); CDR2-ISWSGGST (SEQ ID No. 5); CDR3-AAISGWGRSIRVGERYEYDY (SEQ ID No. 11); (v) CDR1-GRTSSSST (SEQ ID No. 12); CDR2-ITLSGGST (SEQ ID No. 13); CDR3-AARRWSSNNRGGYDRAGYDY (SEQ ID No. 14); or (vi) CDR1-GRTFSSYA (SEQ ID No. 15); CDR2-ITWSGGST (SEQ ID No. 16); CDR3-AMLLRGGLYDYTDYILYNY (SEQ ID No. 17).

15. A pharmaceutical composition comprising a plurality of cells according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier, diluent or excipient.

16. Use of a cell according to any one of claims 1 to 10 in the preparation of a pharmaceutical composition for use in a method of treating acute myeloid leukemia (AML) in a subject.

17. Use according to claim 16, wherein the method involves the subsequent step of administering an allograft to the subject.

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