Chimeric antigen receptors targeting CD123 and uses thereof

Abstract

The present invention provides a single-chain antibody scFv that comprises an amino acid sequence selected from SEQ ID NOs: 56, 57, 58, 59, 60, and 61 and specifically binds to CD123; a chimeric antigen receptor that comprises an amino acid sequence selected from SEQ ID NOs: 65, 66, 67, 68, 69, and 70 and targets CD123; nucleic acid molecules encoding the scFv or chimeric antigen receptor; vectors and cells containing the nucleic acid molecules; and use of the antibody, chimeric antigen receptor, nucleic acid molecule, vector, or cell in the preparation of a therapeutic agent for a tumor that expresses CD123.

Description

[Technical field]

[0001] The present invention relates to scFv sequences bearing different antigen recognition domains and targeting CD123, chimeric antigen receptors (CARs) and CAR-T cells designed based on these scFv sequences, and their use in the treatment of tumors expressing CD123, in particular acute myeloid leukemia. [Background technology]

[0002] Acute myeloid leukemia (AML) is a malignant tumor with an increasing incidence rate, affecting mostly elderly people, with a median age of 66 years. According to estimates by the National Cancer Institute, the number of new cases of the disease in 2018 will be 19,520, including 10,670 deaths (Non-Patent Document 1). Although the 12-month survival rate for patients aged 65-74 years has increased from 20% to 30% over the past 30 years, current AML treatment efficacy remains insufficient, with a 5-year survival rate of less than 5% for these patients (Non-Patent Document 2). The main treatment for younger AML patients is intensive chemotherapy, and for patients at high risk of relapse (cure rate 35-40%), allogeneic hematopoietic stem cell transplantation (AHCT) is performed, but the prognosis for elderly patients remains unimproved (Non-Patent Document 2). Patients with relapsed AML have less than a 50% chance of recovery, and only 16% of patients successfully suppress the disease after a second salvage chemotherapy (Non-Patent Document 3). Since the introduction of combination chemotherapy with anthracycline and cytarabine in the 1970s, it has become the main treatment (Non-Patent Documents 4, 5). Therefore, new therapeutic strategies are needed to treat this disease.

[0003] Chimeric antigen receptor T cells (CAR-T cells) are a concept that combines antibody specificity and the killing effect of T cells to form an adoptive immune effect, and were first proposed in the late 1980s (Non-Patent Documents 6, 7). The molecular structure of chimeric antigen receptor (CAR) mainly includes a single-chain variable fragment (scFv) antibody, an intracellular signal domain, and a hinge region and a transmembrane region that connect the scFv and the intracellular signal domain. In recent years, anti-CD19 and anti-CD20 chimeric antigen receptor T cells (CAR-T cells) have been used to treat lymphatic tumors (B-cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma) with good results (Non-Patent Document 8). Currently, the targets of CAR-T for AML treatment include CD123, CD33, CLL1, FLT3, etc., and among them, the most research is on CAR-T targeting CD123.

[0004] CD123 is the α chain of the IL-3 receptor (IL-3Rα). Together with CD131, CD123 forms the high-affinity IL-3 receptor, which promotes cell proliferation and differentiation and inhibits apoptosis of hematopoietic cells when bound to IL-3 (Non-Patent Documents 9 and 10). High expression of CD123 is associated with activation of the signal transduction and activator of transcription 5 (STAT5) signaling pathway. The STAT5 activation pathway is involved in various malignant hematologic diseases (Non-Patent Document 11). CD123 is mainly expressed in the hematopoietic system and is associated with normal CD34 + CD38 - In cells, CD123 is expressed at low levels or not at all (Non-Patent Document 12). In addition, under normal conditions, CD123 is highly expressed in myeloid progenitor cells and B lymphoid progenitor cells, and is expressed at low levels or not at all in erythroid progenitor cells and multi-directional differentiation potential progenitor cells (Non-Patent Documents 13 and 14). CD123 is highly expressed in more than 60 to 90% of primary AML cells (Non-Patent Documents 15 to 18). Bras AE et al. (Non-Patent Document 19) conducted a test on 455 AML patients and found that CD123 was expressed in the AML cells of approximately 55% of the patients, with a positive rate of more than 80%.

[0005] Although CAR-T cells have been successful in tumor cell therapy, the efficacy and safety of CAR-T remain a major problem, mainly due to off-target effects, cytokine storm, and neurotoxicity (Non-Patent Document 20). The optimal efficacy and safety of CAR-T cells depend on many factors, among which effective activation of CAR-T cells plays an important role. The degree of activation of CAR-T cells is influenced by many factors, such as the affinity of the CAR molecule scFv, the length of the hinge region, the costimulatory signal domain, and the antigen density on the surface of the target cell. The scFv of the CAR molecule is used as a region that directly binds to the antigen on the surface of the target cell, and its affinity is very important in the activation of the entire CAR-T cell. If the affinity of the CAR molecule scFv is low, the CAR-T cell cannot obtain sufficient antigen recognition and cannot obtain an optimal activation state. If the affinity of the CAR molecule scFv is high, the CAR-T cell can recognize tumor cells, but may also recognize normal cells with relatively low antigen density, which may cause serious off-target effects.

[0006] Therefore, to further improve the efficacy and safety of CAR-T cells, ensure the effective activation of CAR-T cells, and avoid off-target effects, it is necessary to screen for scFvs with suitable affinity for the target. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Surveillance, Epidemiology, and End Results (SEER) Program. Research Data (1973-2015), National Cancer Institute, Dccps, Surveillance Research Program, Released April 2018, Based on the November 2017 Submission. 2019. (www.seer.cancer.gov). [Non-Patent Document 2] Thein MS, Ershler WB, Jemal A, Yates JW, Baer MR. Outcome of older patients with acute myeloid leukemia: an analysis of SEER data over 3 decades. Cancer 2013;119(15):2720-7. [Non-Patent Document 3] Kantarjian HM, DiNardo CD, Nogueras-Gonzalez GM, Kadia TM, Jabbour E, Bueso-Ramos CE, et al. Results of second salvage therapy in 673 adults with acute myelogenous leukemia treated at a single institution since 2000. Cancer 2018;124(12):2534-40. [Non-Patent Document 4] Jang J, Lee J, Jang JH, Jung CW, Park S. Anti-leukemic effects of simvastatin on NRAS(G12D) mutant acute myeloid leukemia cells. Mol Biol Rep. 2019;46:5859-66. [Non-Patent Document 5] Roboz GJ. Novel approaches to the treatment of acute myeloid leukemia. Hematology Am Soc Hematol Educ Program. 2011;2011:43-50. [Non-Patent Document 6] Benmebarek, MR; Karches, CH; Cadilha, BL; Lesch, S.; Endres, S.; Kobold, S. Killing mechanisms of chimeric antigen receptor (CAR) T cells. Int. J. Mol. Sci. 2019, 20, 1283. [Non-Patent Document 7] Gross, G.; Waks, T.; Eshhar, Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc. Natl. Acad. Sci. USA 1989, 86, 10024-10028. [Non-Patent Document 8] Khalil DN, Smith EL, Brentjens RJ, Wolchok JD. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol 2016;13(6):394. [Non-Patent Document 9] Munoz L, Nomdedeu JF, Lopez O, et al. Interleukin-3 receptor α chain (CD123) is widely expressed in hematologic malignancies. Haematologica. 2001;86:1261-9. [Non-Patent Document 10] Blalock WL, Weinstein-Oppenheimer C, Chang F, et al. Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs. Leukemia Aug. 1999;13:1109–6

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[0008] The antibody according to the first aspect of the present invention is an antibody that specifically binds to CD123 and comprises a heavy chain variable domain VH and a light chain variable domain VL. The heavy chain variable domain comprises an amino acid sequence VH-CDR1 (GYX1X2X3X4(D)YX5X6X7) shown in SEQ ID NO: 1 and an amino acid sequence VH-CDR2 (X8X9X 10 (X 11 )X 12 X 13 X 14 X 15 X 16 X 17 X 18 NX 19 X 20 X 21 KX 22 X 23 X 24 ) and the amino acid sequence VH-CDR3 (AX 25 X 26 X 27 X 28 X 29 X 30 X 31 (YD)X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X1 is selected from S and T, X2 is selected from F and I, X3 is selected from T, S and M, X4 is selected from S, D, T and G, X5 is selected from W, Y, V, A and N, X6 is selected from M, V, I and W, X7 is selected from N and H, X8 is selected from Y and R, X9 is selected from I and C, and X 10 is selected from D, N, Y and S; X 11 is selected from P and C; X 12 is selected from Y and G; X 13 is selected from D, N and S; X 14 is selected from S, N, D and G; X 15 is selected from E, A, G and S; X 16 is selected from T, I and S; X 17is selected from H, S and N; X 18 is selected from Y and S; X 19 is selected from Q, E and P; X 20 is selected from K, N and S; X 21 is selected from F and L; X 22 is selected from D, G and S; X 23 is selected from K and R; X 24 is selected from A and I; X 25 is selected from R and G; X 26 is selected from G, S and E; X 27 is selected from E, P and R; X 28 is selected from G and S; X 29 is selected from N, Y, L and W; X 30 is selected from W, Y and L; X 31 is selected from G and D; X 32 is selected from R and E; X 33 is selected from Y, S and T; X 34 is selected from D, Y and G; X 35 is selected from G and Y; X 36 is selected from Y and L; X 37 is selected from A, G and P; X 38 is selected from M and L; X 39 is selected from D and A; X 40 is selected from Y and C.

[0009] The light chain variable domain has the amino acid sequence VL-CDR1(X 41 SX 42 X 43 X 44 X 45 X 46 X 47 X 48 X 49 (QK)X 50 X 51 X 52 X 53 and the amino acid sequence VL-CDR2 (X 54 SX 55 X 56 X57 X 58 X 59 ) and the amino acid sequence VL-CDR3 (X 60 X 61 X 62 X 63 X 64 (P)X 65 TF) and X 41 is selected from A and S; X 42 is selected from Q, K and S; X 43 is selected from S and D; X 44 is selected from I, V and L; X 45 is selected from S, N, D and L; X 46 is selected from K, Y, S, D and N; X 47 is selected from D and S; X 48 is selected from G and S; X 49 is selected from D and N; X 50 is selected from S and N, X 51 is selected from D and Y; X 52 is selected from L, I and M; X 53 is selected from A, N and H; X 54 is selected from G, T and A; X 55 is selected from T, N and Q; X 56 is selected from L, S and R; X 57 is selected from Q, E, A, I and D; X 58 is selected from S and P; X 59 is selected from G and E; X 60 is selected from Q and N; X 61 is selected from H, Y, S, F and G; X 62 is selected from N, D, H and Y; X 63 is selected from K, L, E, R and S; X 64 is selected from Y, L, D, S, F and T; X 65 is selected from Y, R and W, and preferably said antibody is an scFv.

[0010] A chimeric antigen receptor targeting CD123 according to a second aspect of the present invention comprises the antibody according to the first aspect, a hinge region, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular signaling domain.

[0011] A nucleic acid molecule according to a third aspect of the invention comprises a nucleotide sequence encoding the antibody of the first aspect or the chimeric antigen receptor of the second aspect.

[0012] A vector according to the fourth aspect of the invention comprises the nucleic acid molecule of the third aspect.

[0013] The cell according to the fifth aspect of the invention comprises the nucleic acid molecule of the third aspect or the vector of the fourth aspect by being transfected with the vector of the fourth aspect.

[0014] A pharmaceutical composition according to a sixth aspect of the present invention comprises the antibody of the first aspect, the chimeric antigen receptor of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect or the cell of the fifth aspect, and a pharma- ceutically acceptable carrier.

[0015] A seventh aspect of the invention is the use of an antibody according to the first aspect, a chimeric antigen receptor according to the second aspect, a nucleic acid molecule according to the third aspect, a vector according to the fourth aspect or a cell according to the fifth aspect in the preparation of a medicament for treating a tumour expressing CD123. Effect of the Invention

[0016] The present invention screens the affinity of the scFv of the CAR molecule to the target CD123 and compares the killing effect of CAR-T cells to obtain the best scFv sequence of the target CD123, and the CAR-T cells have higher safety and efficacy.Compared with the prior art, the present invention can effectively improve the killing efficiency of CAR-T cells targeting CD123 against tumor cell lines and primary tumor cells, and can also improve the cell proliferation ability. [Brief description of the drawings]

[0017] [Figure 1]FIG. 1 shows the transduction efficiency of different scFv CD123 CAR molecules in T cells. [Diagram 2] Figure 2 shows the total cell expansion of CD123 CAR-T in culture. [Diagram 3] Figure 3 shows the effect of target cells, primary AML cells and THP-1 cells, on the differentiation of CD123 CAR-T cells under stimulation. [Figure 4] Figure 4 shows the killing efficiency of CD123 CAR-T against CD123 positive cell line (THP-1) and CD123 negative cell line (K562). [Diagram 5] Figure 5 shows the killing efficiency of CD123 CAR-T against primary AML cells. [Figure 6] Figure 6 shows cytokine expression of CD123 CAR-T after stimulation of AML cells for 5 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The antibody of the present invention specifically binds to CD123 and comprises a heavy chain variable domain VH and a light chain variable domain VL, the heavy chain variable domain comprising VH-CDR1, VH-CDR2, and VH-CDR3, and the light chain variable domain comprising VL-CDR1, VL-CDR2, and VL-CDR3.

[0019] In a specific embodiment, the VH-CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11 and 12, the VH-CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17 and 18, the VH-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 21, 22, 23 and 24, the VL-CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29 and 30, the VL-CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 33, 34, 35 and 36, and the VL-CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, 41 and 42.

[0020] In particular, antibodies that specifically bind to CD123 of the present invention include the following: (1) A heavy chain variable domain comprising VH-CDR1 shown in SEQ ID NO: 8, VH-CDR2 shown in SEQ ID NO: 14, and VH-CDR3 shown in SEQ ID NO: 20, and a light chain variable domain comprising VL-CDR1 shown in SEQ ID NO: 26, VL-CDR2 shown in SEQ ID NO: 32, and VL-CDR3 shown in SEQ ID NO: 38, and / or (2) A heavy chain variable domain comprising VH-CDR1 shown in SEQ ID NO: 9, VH-CDR2 shown in SEQ ID NO: 15, and VH-CDR3 shown in SEQ ID NO: 21, and a light chain variable domain comprising VL-CDR1 shown in SEQ ID NO: 27, VL-CDR2 shown in SEQ ID NO: 33, and VL-CDR3 shown in SEQ ID NO: 39, and / or (3) A heavy chain variable domain comprising VH-CDR1 shown in SEQ ID NO: 10, VH-CDR2 shown in SEQ ID NO: 16, and VH-CDR3 shown in SEQ ID NO: 22, and a light chain variable domain comprising VL-CDR1 shown in SEQ ID NO: 28, VL-CDR2 shown in SEQ ID NO: 34, and VL-CDR3 shown in SEQ ID NO: 40, and / or (4) A heavy chain variable domain comprising VH-CDR1 shown in SEQ ID NO: 11, VH-CDR2 shown in SEQ ID NO: 17, and VH-CDR3 shown in SEQ ID NO: 23, and a light chain variable domain comprising VL-CDR1 shown in SEQ ID NO: 29, VL-CDR2 shown in SEQ ID NO: 35, and VL-CDR3 shown in SEQ ID NO: 41, and / or (5) A heavy chain variable domain comprising VH-CDR1 shown in SEQ ID NO: 12, VH-CDR2 shown in SEQ ID NO: 18, and VH-CDR3 shown in SEQ ID NO: 24, and a light chain variable domain comprising VL-CDR1 shown in SEQ ID NO: 30, VL-CDR2 shown in SEQ ID NO: 36, and VL-CDR3 shown in SEQ ID NO: 42.

[0021] In another specific embodiment, the heavy chain variable domain of an antibody of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47 and 48, or an amino acid sequence which has at least 85%, at least 90%, at least 95%, or at least 99% identity thereto, and / or the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 51, 52, 53 and 54, or an amino acid sequence which has at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0022] In particular, antibodies that specifically bind to CD123 of the present invention include the following: (1) a heavy chain variable domain as set forth in SEQ ID NO: 44 and a light chain variable domain as set forth in SEQ ID NO: 50, and / or (2) a heavy chain variable domain as set forth in SEQ ID NO: 45 and a light chain variable domain as set forth in SEQ ID NO: 51, and / or (3) a heavy chain variable domain as set forth in SEQ ID NO: 46 and a light chain variable domain as set forth in SEQ ID NO: 52, and / or (4) a heavy chain variable domain as set forth in SEQ ID NO: 47 and a light chain variable domain as set forth in SEQ ID NO: 53, and / or (5) a heavy chain variable domain shown in SEQ ID NO: 48 and a light chain variable domain shown in SEQ ID NO: 54

[0023] In another specific embodiment, an antibody of the present invention that specifically binds to CD123 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 57, 58, 59, 60 and 61, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto.

[0024] Preferably, the antibody of the present invention is a single-chain antibody scFv, and more preferably, the heavy chain variable domain and the light chain variable domain of the single-chain antibody scFv are linked by a linker peptide chain rich in glycine and serine, and the order of the heavy chain variable domain and the light chain variable domain can be interchanged.

[0025] The chimeric antigen receptor targeting CD123 of the present invention comprises an antibody of the present invention, a hinge region, a transmembrane domain, a costimulatory domain, and a CD3ζ intracellular signaling domain.

[0026] In certain embodiments, the costimulatory domain is selected from CD27, CD28, 4-1BB, OX-40, CD30, CD40, PD-1, ICOS, LFA-1, CD-2, CD7, LIGHT, NKG2C, B7-H3, or any combination thereof, preferably, the costimulatory domain is selected from 4-1BB and CD28, wherein the 4-1BB comprises the amino acid sequence set forth in SEQ ID NO:62.

[0027] In one specific embodiment, the CD3 zeta intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:63.

[0028] In a specific embodiment, said hinge region and said transmembrane domain are selected from an IgG1, IgG4, CD8α, CD28, IL-2 receptor, IL-7 receptor, IL-11 receptor, PD-1 or CD34 hinge region and transmembrane domain.

[0029] Preferably, the chimeric antigen receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 66, 67, 68, 69 and 70, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99% identity thereto.

[0030] A nucleic acid molecule of the present invention comprises a nucleotide sequence that encodes an antibody of the present invention or a chimeric antigen receptor of the present invention.

[0031] A vector of the present invention comprises a nucleic acid molecule of the present invention, preferably said vector is a viral vector, more preferably said vector is a lentiviral vector.

[0032] A cell of the invention comprises said nucleic acid molecule or said vector by being transfected with the vector of the invention, preferably said cell is a T cell.

[0033] The present invention relates to the use of said antibody, said chimeric antigen receptor, said nucleic acid molecule, said vector or said cell in the preparation of a medicament for treating a tumor expressing CD123, preferably said tumor expressing CD123 being acute myeloid leukemia (AML).

[0034] Unless otherwise specified, the technical solutions described herein may be combined in any combination.

[0035] The present invention will now be described in more detail with reference to examples. EXAMPLES

[0036] [Example 1] (Design of CD123 CAR molecule and construction of CAR-T) In this example, we designed CD123 CAR molecules with different antigen recognition domains and generated CAR-T cells.

[0037] The principles of designing the antigen recognition domain are as follows: CAR-T-1 is the control group, the scFv sequence of CD123 was reported by the University of Pennsylvania (Non-Patent Document 21), and the scFv sequence is referred to as scFv-01 in this disclosure; CAR-T-2 to CAR-T-7, which have scFv sequences derived from the monoclonal antibody sequence obtained from the CD123 extracellular fusion protein of mouse immunization, are the modified group, and these scFv sequences are referred to as scFv-02-07 in this specification.

[0038] The nucleotide sequences of CD123 scFv with different antigen recognition domains were synthesized by gene synthesis, and then the nucleotide sequences of CD123 scFv were linked with the sequences of CD8α hinge region, CD8α transmembrane region, 4-1BB and CD3ζ intracellular domain by homologous recombination to form the complete CAR molecule, i.e., CAR1-7. The CAR nucleic acid fragment was incorporated into the lentiviral transfer plasmid pLenti6.3 / V5 (Thermo Fisher, Waltham, MA, USA) by restriction endonuclease digestion and ligation with ligase. HEK293T cells were transfected with lentiviral packaging plasmids pLP / VSVG, pLP1 / MDK, and pLP2 / RSK (Thermo Fisher, Waltham, Massachusetts, USA) and the transfer plasmids obtained using Lipofectamine 3000 (Thermo Fisher, Waltham, Massachusetts, USA). After 48 hours, the culture medium was collected and centrifuged at 300 g to remove cell debris, and centrifuged at 25,000 rpm for 3 hours using an ultracentrifuge. The precipitate was then dissolved in 1 mL of saline to obtain the lentiviral vector of interest.

[0039] T cells were isolated from peripheral blood mononuclear cells of healthy volunteers using CD3 / CD28 beads (Thermo Fisher, Catalog No. 40203D). The isolated and purified T cells were cultured at 1.0 × 10 6The cells were seeded and cultured in X-VIVO 15 (Lonza, Switzerland) medium containing 500 IU / mL IL-2 (Shandong Jintai Bioengineering, China). After 48 hours of culture, the T cells were infected with a lentivirus vector having a CD123 CAR molecule at an MOI of 0.5 to obtain CAR-T-1-7. After 24 hours of lentivirus infection, the cell liquid was replaced, and the lentivirus was seeded in a new culture system containing X-VIVO 15 and 500 IU / mL IL-2, and the culture was continued. After 5 days of lentivirus infection, the cells in the culture system were repeatedly sprayed with a pipette and collected in a centrifuge tube, and the CD3 / CD28 beads were removed on a magnetic stand. The T cells were centrifuged and counted, and some cells were removed to detect the CAR molecule transduction efficiency of each cell group using flow cytometry (ACEA Biosciences, San Diego, CA, USA, product name: NovoCyte 2060R).

[0040] Figure 1 shows the CAR molecule transduction efficiency of each cell group after T cells were infected with lentivirus for 5, 7, 9, and 12 days. The CAR molecule transduction efficiency of the CAR-T-2, CAR-T-3, and CAR-T-7 groups was close to that of the control group CAR-T-1, and the CAR molecule transduction efficiency of the CAR-T-4, CAR-T-5, and CAR-T-6 groups was significantly higher than that of the control group CAR-T-1. Therefore, good CAR protein expression was observed in all CAR-T cell groups.

[0041] [Example 2] (In vitro proliferation of modified CD123 CAR-T) In this example, the proliferation ability of CD123CAR-T having different antigen recognition domains was detected.

[0042] During the culture process of CD123CAR-T cells, the day on which the lentivirus was infected into the T cells was counted as the second day of culture, and thereafter, the CAR-T cells were collected every 2 to 3 days, centrifuged, and resuspended by adding 1 to 2 ml of culture medium. 10 μl of cells were removed and diluted to a certain ratio, stained with trypan blue (Solarbio, catalog number: C0040), and the total number of live cells and dead cells were counted using an inverted microscope (Nikon Corporation, Japan, product name: Ts2-FL) to calculate the cell proliferation in each group.

[0043] As shown in Figure 2, the total cell proliferation of CAR-T-2 and CAR-T-7 with different antigen recognition domains was significantly enhanced compared with the control CAR-T-1. This result indicates that the scFv design of CAR-T-2 and CAR-T-7 can enhance the proliferation ability of CD123 CAR-T cells.

[0044] [Example 3] (Cell differentiation of modified CD123 CAR-T cells under target cell stimulation) In this example, two target cells, primary AML cells or TTP-1 cells (both of which express CD123), were stimulated, and T cell differentiation of CD123CAR-T, which has a different antigen recognition domain, was detected.

[0045] When CAR-T cells were cultured for 5 days, 5 × 10 5 Two of the cells were taken out and added to a 12-well plate, and then the target cells, primary AML cells or THP-1 cells, were added to the 12-well plate at an effector / target ratio (E:T) of 5:1. After 6 days of target cell stimulation, that is, when the CAR-T cells were cultured until day 11, 1 × 10 cells were obtained from each group. 6 The cells were removed, centrifuged, and resuspended in 100 μl of DPBS (brand name: HyClone, catalog number: SH30028.02), and each CAR-T cell group was labeled with a T cell differentiation-associated fluorescent antibody and detected using a full spectrum flow cytometer (Fremont, Cytek Biosciences, product name: N7-00008-0A).

[0046] Figure 3 shows the results. + CD62L + The cell population represents a combination of early and dried memory T cell populations and is CD45RA + CD62L - represented the effector T cell population. CD45RA in CAR-T-2 and CAR-T-7 with different antigen recognition domains + CD62L + The percentage of the cell population was higher than that in the control CAR-T-1 group, and CD45RA + CD62L - The percentage of the cell population was lower than that of the control CAR-T-1. This result demonstrated that the modified CD123 antigen recognition domain was superior in the persistence of CAR-T cells.

[0047] [Example 4] (In vitro killing efficiency of engineered CD123 CAR-T against cell lines) In this example, we detected the killing efficiency of CD123CAR-T cells, which have different antigen recognition domains, against cell lines expressing CD123 and cell lines not expressing CD123.

[0048] The cell line THP-1 expressing CD123 and the cell line K562 not expressing CD123 were collected, centrifuged, and then resuspended in 1 ml of normal saline (Hebei Tiancheng Pharmaceutical, China), and 5 μl of live cell staining dye Calcein-AM (concentration 1 μg / μL, Thermo Fisher, USA, catalog number: C3100MP) was added, mixed gently, and incubated in a 37°C incubator for 30 minutes to label the target cells. After the incubation, the cells were washed twice with normal saline, resuspended in X-VIVO 15, and counted. 1 × 10 cells per well were counted. 5The labeled target cells were added to a 48-well cell culture plate (Corning, Corning, NY, USA), and each CAR-T cell group was added to the 48-well cell culture plate at an E:T ratio of 10:1. The 48-well cell culture plate was placed in a 37°C, 5% CO2 incubator and incubated for 5 hours. After the incubation, 2% Triton-X-100 (Sigma, Germany, Catalog No.: T8787-100ML) was added to the positive control group, i.e., 1×10 labeled target cells. 5 100 μl of the killing system supernatant was taken out from each well and placed on a microplate, and the fluorescence value (excitation wavelength: 496 nm, emission wavelength: 515 nm) was detected using a multifunctional microplate reader (ThermoFisher, product name: Varioscan Lux).

[0049] As shown in Figure 4, the killing efficiency of CD123 CAR-T with different antigen recognition domains, CAR-T-3 and CAR-T-5, against the CD123-positive cell line THP-1 was significantly higher than that of the control group CAR-T-1. Meanwhile, the killing efficiency of each group of modified CD123 CAR-T and control group CAR-T-1 against the cell line K562, which does not express CD123, was consistent with the killing efficiency of the T cell group. This result showed that each CD123 CAR-T cell group had a certain killing efficiency and excellent specificity against target cells, and the scFv design of CAR-T-3 and CAR-T-5 promoted the killing of tumor cells by CAR-T.

[0050] [Example 5] (In vitro killing efficiency of modified CD123 CAR-T cells against primary AML cells) In this example, the killing efficiency of CD123 CAR-T with different antigen recognition domains against primary AML cells was detected.

[0051] Three bone marrow blood samples were obtained from CD123-positive AML patients with high antigen density (i.e., AML1#, CD123 expression rate 99.87%, MFI value 13298), medium antigen density (i.e., AML2#, CD123 expression rate 75.4%, MFI value 7518), and low antigen density (i.e., AML3#, CD123 expression rate 94.58%, MFI value 5252) (antigen density levels were defined as follows: high antigen density: median fluorescence intensity (MFI) > 10,000; medium antigen density: MFI value > 6,000 and < 10,000; low antigen density: MFI value < 6,000). Mononuclear cells were isolated from bone marrow blood samples obtained from CD123-positive AML patients, and CD34-positive AML primary cells were selected using CD34 microbeads (Miltenyi, Germany, catalog number: 130-100-453), resuspended in 1 ml of normal saline, and 10 μl of Callein-AM (concentration 1 μg / μL, ThermoFisher, USA) were added, mixed gently, and placed in a 37°C incubator for 30 minutes to label the AML primary cells. After incubation, the cells were washed twice with normal saline, resuspended in X-VIVO 15, and counted. 1 × 10 cells were cultured per well in a 48-well cell culture plate (Corning, Corning, NY, USA). 5 The labeled AML primary cells were added to a 48-well cell culture plate, and CD123 CAR-T cells were added to the plate at a ratio of E:T = 10:1. The 48-well cell culture plate was placed in a 37°C, 5% CO2 incubator and incubated for 5 hours. After the incubation, a positive control group, i.e., 1 x 10 labeled AML primary cells, was added to a 48-well cell culture plate at a ratio of E:T = 10:1. 5 100 μl of the killing system supernatant was taken out per well and placed on a microplate, and the fluorescence value (excitation wavelength: 496 nm, emission wavelength: 515 nm) was detected using a microplate reader (Thermo Fisher, product name: Varioscan Lux).

[0052] As shown in Figure 5, CAR-T-3 with different antigen recognition domains had significantly higher killing efficiency against three primary cells of high antigen density (i.e., AML1#), medium antigen density (i.e., AML2#), and low antigen density (i.e., AML3#) from CD123-positive AML patients compared with the control CAR-T-1. This result suggested that CD123 CAR-T cells with CAR-T-3 scFv could effectively improve the killing efficiency against primary cells.

[0053] [Example 6] (Cytokine expression of modified CD123 CAR-T under target cell stimulation) In this example, the cytokine expression levels of CD123 CAR-T with different antigen recognition domains on primary AML cells were detected.

[0054] The target cells, primary AML cells, were cultured at 1 × 10 per well. 5 The CAR-T cells were added to a 48-well cell culture plate (Corning, NY, USA). The CD123 expression rate of the AML primary cells was 99.87%, and the MFI value was 13298. Each CAR-T cell group was added to a 48-well cell culture plate at a ratio of E:T=10:1, and the 48-well cell culture plate was placed in a 37°C, 5% CO2 incubator and incubated for 5 hours. The cell supernatant was removed, and the expression levels of various cytokines were detected using a CBA kit (BD Biosciences, San Jose, CA, USA) and flow cytometry (ACEA Biosciences, San Diego, CA, USA, product name: NovoCyte 2060R).

[0055] As shown in Figure 6, the expression levels of IL-2, TNF, and IFN-γ were significantly higher in CAR-T-6 with different antigen recognition domains under target cell stimulation compared with the control CAR-T-1. And the cytokine expression levels in the remaining CAR-T cell groups were similar to that of the control CAR-T-1. This result indicated that the scFv design of CAR-T-6 promoted the expression of CD123 CAR-T cytokines.

[0056] Details of the amino acid sequences relevant to the present invention are set forth in Tables 1 and 2 below.

[0057] [Table 1-1]

[0058] [Table 1-2]

[0059] [Table 1-3]

[0060] [Table 1-4]

[0061] [Table 1-5]

[0062] [Table 2-1]

[0063] [Table 2-2]

[0064]

Table 2-3

[0065]

Table 2-4

Claims

1. A chimeric antigen receptor that targets CD123, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 66, 67, 68, 69 and 70.

2. A nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of claim 1.

3. A vector comprising the nucleic acid molecule of claim 2.

4. A cell transfected with the vector of claim 3 and comprising the nucleic acid molecule of claim 2 or the vector of claim 3.

5. The cell described in claim 4, which is a T cell.

6. A pharmaceutical composition comprising the chimeric antigen receptor described in claim 1, the nucleic acid molecule described in claim 2, the vector described in claim 3 or the cell described in claim 4 or 5, and a pharma- ceutically acceptable carrier.

7. Use of a chimeric antigen receptor as described in claim 1, a nucleic acid molecule as described in claim 2, a vector as described in claim 3, or a cell as described in claim 4 or 5 in the preparation of a therapeutic drug for a tumor expressing CD123.

8. Use of the cells described in claim 7, wherein the tumor expressing CD123 is acute myeloid leukemia.

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