CAR-T cells targeting IL-1RAP and their use in acute myeloid leukemia (AML)

Abstract

The present invention relates to a cell for treating acute myeloid leukemia (AML), wherein the cell comprises a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or an antibody fragment, the CAR comprises an anti-IL-1RAP binding domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain comprises at least a stimulatory domain.

Description

Technical Field

[0001] The present invention relates to a cell for treating acute myeloid leukemia (AML), wherein the cell comprises a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or an antibody fragment, the CAR comprises an anti-IL-1RAP binding domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain comprises at least a stimulatory domain. Background Art

[0002] Acute myeloid leukemia (AML; also known as "acute myeloid leukemia," "acute myeloid leukemia," "acute myeloblastic leukemia," "acute granulocytic leukemia," and "acute non-lymphocytic leukemia") is a devastating clonal hematopoietic stem cell neoplasm characterized by the uncontrolled proliferation and accumulation of leukemic blasts in the bone marrow, peripheral blood, and occasionally in other tissues. These cells disrupt normal hematopoiesis and rapidly lead to bone marrow failure and death. AML is the most common type of acute leukemia in adults and accounts for the largest number of leukemia deaths each year. AML progresses rapidly and is usually fatal within weeks or months if not treated. In 2015, there were approximately 20,830 new cases of AML in the United States, and 10,460 patients died from the disease. The median age of diagnosis is approximately 70 years. The overall 5-year survival rate is only about 30%-40% in patients under 60 years of age, and for patients over 60 years of age, the overall 5-year survival rate is less than 10%.

[0003] The current standard of care for AML involves remission induction therapy with high-dose chemotherapy or radiation therapy, followed by consolidation therapy, including allogeneic stem cell transplantation and additional chemotherapy courses as needed. Most patients treated in this way will achieve a complete but short-lived remission. Once relapse occurs, the disease becomes increasingly resistant to further treatment.

[0004] Although outcomes for younger patients have improved over the past 30 years, the dismal outcomes for older patients have remained essentially unchanged. Unfortunately, the majority of patients with AML experience disease relapse, including those who achieve an initial complete remission. Allogeneic hematopoietic stem cell transplantation in a second remission offers the only chance for long-term survival, but this option is not available for most patients with relapsed AML because they either do not achieve a second remission or are unable to tolerate the procedure.

[0005] There are multiple multi-agent chemotherapy salvage regimens for relapsed AML (such as MEC (mitoxantrone, etoposide, and cytarabine) and FLAG-IDA (fludarabine, cytarabine, idarubicin, granulocyte colony-stimulating factor)). However, there is no recognized standard of care because none of these regimens is superior to the others, and none of these regimens achieve long-term survival. NCCN (National Comprehensive Cancer Network) and other guidelines recommend clinical trials, and relapsed AML is widely recognized as an urgent unmet medical need.

[0006] A variety of new approaches to treat AML (particularly relapsed AML) are currently being studied, including antibody-drug bispecific T-cell-engaging antibodies (AMG330) and CAR-T cells (CART-33 cells or CART-123 cells). However, several new approaches have stalled due to clinical toxicity and / or lack of efficacy. Therefore, there is a clear need for new effective therapies with acceptable toxicity profiles.

[0007] In AML, gene expression profiling, cell surface staining, and Western blot studies have revealed that leukemic, but not normal, CD34 + / CD38 - Cell surface biomarkers expressed by hematopoietic stem cells (IL-1RAP, IL-1R3, C3orf13 or IL-1RAcP). In addition, IL-1RAP expression is associated with the clinical stage of AML disease as well as tumor burden. Summary of the invention

[0008] IL-1RAP (interleukin 1 receptor accessory protein, Genbank accession number AAB4059) is a co-receptor for IL-1 and IL33 receptors and is involved in IL-1 signal transduction, which activates different signal transduction pathways, including MAP kinase, p38, NF-κB, and other genes involved in inflammation and proliferation. The protein is expressed on the surface of tumor cells. Therefore, IL-1RAP is a promising tumor-associated antigen.

[0009] Applicants have discovered that by using such CART-IL-1RAP cells, it is possible to treat patients with AML.

[0010] Therefore, the present invention relates to a cell for use in treating acute myeloid leukemia (AML), wherein the cell expresses a nucleic acid molecule encoding a chimeric antigen receptor (CAR) on its membrane, wherein the CAR comprises an antibody or an antibody fragment, wherein the CAR comprises an anti-IL-1RAP binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain comprises at least a stimulatory domain, and wherein the anti-IL-1RAP binding domain comprises:

[0011] (i) a light chain comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80% identical to the amino acid sequence of SEQ ID NO: 6, the complementarity determining region 2 (CDR2) is at least 80% identical to the amino acid sequence of SEQ ID NO: 7, and the complementarity determining region 3 (CDR3) is at least 80% identical to the amino acid sequence of SEQ ID NO: 8; and

[0012] (ii) a heavy chain, comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80% identical to the amino acid sequence of SEQ ID NO: 12, the complementarity determining region 2 (CDR2) is at least 80% identical to the amino acid sequence of SEQ ID NO: 13, and the complementarity determining region 3 (CDR3) is at least 80% identical to the amino acid sequence of SEQ ID NO: 14.

[0013] T cells expressing CAR are referred to herein as "CAR T cells" or "CAR-modified T cells". T cells expressing CAR targeting IL-1RAP are referred to herein as "CART-IL-1RAP cells" or "CART-IL-1RAP modified T cells".

[0014] Cells for use according to the present invention are genetically modified to express CAR as described herein, so as to be used for the treatment of AML. As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Western blot of different hematopoietic AML cell lines and CD14+ cell-sorted monocytes.

[0016] Figure 2: ELISA technique was used to recognize IL-1RAP recombinant protein using #E3C3 mAb (b). BSA was used as a negative control (a).

[0017] Figure 3 : Flow cytometry gating strategy on primary samples of AML patients. RFI of blasts and monocyte subsets is provided (left). Percentage of IL-1RAP+ in AML blasts and monocytes and percentage of CD123+ and IL-1RAP+ / CD123+ cells in AML blasts in the same cohort (n=30) of conventional AML (all subtypes) patients (right, top). RFI is also provided (right, bottom).

[0018] Figure 4 : Specific tissue binding using frozen tissue arrays. High IL-1RAP expressing cell line (KU812) (a) or IL-1RAP negative expressing cell line (Raji) (b) were used as positive or negative controls, respectively. The following tissues were tested: a: lymph node; b: colon; c: small intestine; d: placenta; e: stomach; f: lung; g: spleen; and h: prostate.

[0019] Figure 5 : Design of SIN lentiviral construct carrying the safety cassette iCASP9, the single chain fragment variable (scFv) of #E3C3 mAb, and the cell surface expressed marker ΔCD19. The three transgenes are separated by a 2A peptide cleavage sequence and are under the control of the EF1 promoter plus the SP163 enhancer sequence.

[0020] Figure 6 : CD3 Western blot of subcellular fractions of IL-1RAP-transduced T cells. a: total lysate; b: membrane; c: cytoplasm; d: nuclear; (1) CAR-associated CD3ζ (55 kDa); (2) endogenous CD3ζ (16 kDa); (3) CD45 (147 kDa); (4) lamin (68 kDa); (5) GAPDH (35 kDa).

[0021] Figure 7 : FACS analysis of IL-1RAP CAR transduced CEM T cell lines or primary T cells. The percentage of biotin+ / CD19+ CEM cells or T cells (a) is plotted against the amount of biotinylated recombinant protein (b).

[0022] Figure 8: Safety switch of iCASP9 / AP1903 suicide system cassette after exposure to chemical inducer dimer (10 nM CID). (a) 293T cells; (b) IL-1RAP CAR 293T cells.

[0023] Fig. 9 : Clearance of IL-1RAP CART cells after 24h or 48h CID exposure compared with untransduced T cells (C0) (***p<0.001, n=3).

[0024] Figure 10: (A) Gating strategy for flow cytometry CFSE dye dilution analysis, representative experiment. (B) Percentage of total dividing CFSE-positive cells. Mean ± SD of 3 independent experiments. ***p < 0.001 (Student's test).

[0025] Figure 11: (A) Gating strategy for intracellular IFNγ cytokine detection, representative experiment. (B) Percentage of total cells producing intracellular IFNγ. Mean ± SD of 3 independent experiments for CD8+ and CD8- (mainly CD4+) cells. ***p<0.001. **p<0.01 (Student's test)

[0026] Figure 12: (A) Gating strategy for efficacy studies of IL-1RAP CAR T cells in lysing cells expressing cell surface IL-1RAP. (B) Percentage of total live target cells. Mean ± SD of 3 independent experiments.

[0027] Figure 13: (A) Tissue microarray. Representative #A3C3 staining of a US Food and Drug Administration standard frozen tissue array, including 90 tissue cores (30 organs) from 3 different donors for each organ (US Biomax, Rockville, MD, USA). Immunostaining was detected using the UltraView Universal DAB Detection Kit (Ventana, USA). Images were acquired and analyzed using NDP.view 2.0 software. Tissues that showed some degree of #A3C3 mAb staining in at least 1 of the 3 individuals analyzed are shown (scale bar 100 μm). High IL-1RAP expressing cell lines (KU812) or IL-1RAP negative expressing cell lines (Raji) were used as positive or negative controls, respectively. (B) IL-1RAP R&D (red) staining or #A3C3 (blue) staining of the HMEC-1 dermal endothelial cell line. Isotype IgG1 (grey) is depicted as an overlay. RFI of both stainings is provided.

[0028] Figure 14: Effects of IL-1RAP CAR T cells on healthy hematopoietic cells and the efficiency of the safe suicide gene iCASP9 box. (A) IL-1RAP cell surface expression on peripheral blood (left) or bone marrow (right) cells from healthy donors (n=5). SSC-A / CD45+ enables the distinction of subpopulations into lymphocytes (SSC-A low), monocytes (CD33+), granulocytes (SSC-A high) or HSC (CD33- / CD34+). RFI is calculated from isotype staining and provided in each window. (B) Representative IL-1RAP staining of whole human umbilical cord blood cells (1 of 3). IL-1RAP staining of overall CD34+, CD34+ / CD38- and CD34+ / CD38+HSC umbilical cord blood subsets is provided. (C) IL-1RAP positive cells in CD34+ cells from cord blood (CB, n=5) or BM from healthy donors (n=5) compared to CD34+ cells from bone marrow (BM, n=10) or peripheral blood (PB, n=10) of CML patients. (D) Left: Dot plots of SSC-A / CD45+ granulocyte (G), monocyte (M) and lymphocyte (L) subsets cultured in the presence of autologous untransduced T cells or Mock T cells or IL-1RAP CAR T cells at different effector: target (E:T) ratios. Right: Relative percentage of live cells in lymphocytes (squares), monocytes (circles) and granulocytes (triangles) co-cultured for 24 h with autologous Mock T cells (dashed line) or IL-1RAP CAR T cells (solid line), normalized to untransduced autologous T cells (C0). (E) Relative percentage of live cells in monocytes (squares), KU812 (circles) or K562 (triangles) subsets in the presence of different E:T ratios of Mock T cells (black, dotted lines) or IL-1RAP CAR T cells (white, solid lines). The percentage was calculated using the absolute number of cells determined using Trucount tubes based on 5000 fluorescent bead cell counting acquisition (cytometry acquisition). (F) Left: Gating strategy and analysis of absolute counts of cell death induced by CID AP1903. Untransduced T cells (C0) or IL-1RAP CAR T cells were exposed to medium alone or medium + CID (20nM, 24h). Quantification was performed after collecting 5000 fluorescent beads. The killing efficiency was normalized to control cells (untreated cells). Cell killing was calculated as follows: % dead cells = [1-(absolute number of live cells in AP1903-treated cells / absolute number of live cells in untreated cells)] × 100. (D) Absolute percentage of mortality. C0 or IL-1RAP CAR (gated on CD3+ / CD19+) T cells were exposed to CID for 24h or 48h. Right: Results are the average from 3 independent experiments.**p<0.001. (G) Absolute quantification of IL-1RAP CAR T cells injected into tumor (CML KU812, iv) xenograft NSG model 24 hours after ipAP1903 (white bars) treatment (n=3 mice / group). Mice infused with control T cells (C0) were used as controls (n=2 mice / group). **p<0.01. The number of cells per mL of peripheral blood is provided.

[0029] Fig.15 :Experimental immune safety human CD34+ transplanted NOG mouse model, used to study the specific toxicity of autologous IL-1RAP CART cells to HSC and / or immune cells of human CD34+ umbilical cord blood cell transplantation / NOG mouse model (hu-NOG). In short, 10.10E6 autologous CART cells or control T cells (C0) (produced by human CD45+ cells, selected from mouse PBMC, spleen or bone marrow) were infused. Monitoring of mature immune cells (hCD3+, hCD19+, hCD56+, hCD14+, hCD11b+) was evaluated by cell counting at different times after infusion (day 5, day 8 and day 15). The immunophenotyping reference obtained on day -7 before CART cell infusion calculated the fold change. Compared with the peripheral blood collection time (day -9). Fold changes of different immunoreactive cell subsets at days 3, 8, and 15 after infusion of untransduced cells (C0, white bars) or IL-1RAP CART cells (black bars) compared to the time of peripheral blood collection (day -9). Cell counts were performed on peripheral blood harvested from retroorbital samples, and fold changes were calculated against the day -7 reference. ns: not significant.

[0030] Fig.16 : Colony forming unit (CFU-GM) experiment: This experiment was derived from CD34+ HSCs obtained from 3 different umbilical cord blood, which were cultured alone (white bars) or co-cultured with their respective autologous non-transduced T cells (C0, gray bars) or IL-1RAP CART cells (black bars).

[0031] Fig.17 :Top: The effect of CID on transduced 293T cells was evaluated by optical microscopy. Bottom: Flow cytometry analysis of IL-1RAP CART cells after CID AP1903 exposure. Flow cytometry analysis after CID exposure (20nM, 24 hours, dark gray) or no exposure (light gray) was performed on untransduced T cells (C0) and GMTC mixtures expressing or not expressing IL-1RAP CAR. CD3+ / CD19+ staining enables the distinction of GMTCs expressing CAR.

[0032] Fig.18: Absolute cell surface IL-1RAP antigenic sites and IL-1RAP mRNA expression on primary samples of AML patients according to the European Leukemia Net (ELN) prognostic classification.

[0033] Figure 19: AML blast cell toxicity of IL-1RAP CART cells. The gating strategy is shown. CD34 antibody was used to identify target AML blasts ( Fig.19A ). The number of AML blast events showing cytotoxicity of IL-1RAP CART cells is also shown ( Fig.19A The results of three independent experiments are shown in Fig.19B . Fig.19A and Fig.19B Legend: FSC: forward scatter, indicating the size of cells. 7-AAD: 7-aminoactinomycin D, a cell viability marker. E-Fluor: a dye that stains cell membranes. Experiments #1 and #2 were performed with allogeneic IL-1RAP CART cells generated from healthy donors. Experiment #3 was performed with allogeneic IL-1RAP CART cells generated from T cells of AML patients. Viable cells mean viable AML blasts.

[0034] Figure 20: Efficacy of IL-1RAP CAR-T cells in a human AML mouse xenograft model. Immunodeficient NSG-S mice were irradiated (2.5 Gy) and injected with 1×10 6 HL-60, Molm-13, or Mono-Mac-6 AML cells expressing luciferase. Fig. 20A On day 0, after AML cell injection, mice were either left untreated or treated with control T cells (i.e., MockT or UNT cells, 10 × 10 6 cells) or IL-1RAP CAR-T cells (10×10 in 300 μL PBS) 6 Cells) were treated and leukemia development was monitored using bioluminescence imaging (BLI) on the mentioned days. Bioluminescence imaging analysis was performed on different groups of mice from day 0 to day 21. Legend: (x): dead mice. UNT or IL-1RAP CAR T cell injection. Fig. 20B : Radiance of in vivo bioluminescent signal collected using bioluminescent imaging (BLI) (radiance p / s / cm 2 / sr). DETAILED DESCRIPTION

[0035] Therefore, the present invention relates to a cell for use in treating acute myeloid leukemia (AML), the cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or an antibody fragment, the CAR comprises an anti-IL-1RAP binding domain, a transmembrane domain and an intracellular signal transduction domain, the intracellular signal transduction domain comprises at least a stimulatory domain, and wherein the anti-IL-1RAP binding domain comprises:

[0036] (i) a light chain comprising a complementary determining region 1 (CDR1), a complementary determining region 2 (CDR2), and a complementary determining region 3 (CDR3), wherein the complementary determining region 1 (CDR1) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:6, the complementary determining region 2 (CDR2) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and the complementary determining region 3 (CDR3) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8; and

[0037] (ii) a heavy chain comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12, the complementarity determining region 2 (CDR2) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and the complementarity determining region 3 (CDR3) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0038] The following table summarizes the sequence identifiers

[0039]

[0040]

[0041]

[0042] Table 1: Sequence Listing

[0043] The terms "#E3C3" and "#A3C3" should be understood to be equivalent: #E3C3 can be used freely to refer to #A3C3 and vice versa.

[0044] The sequences of the hinge regions of IgG1, IgG4, CD8α, 4-1BB, CD3ζ, CD28, and ICasp9 genes can be found on Genbank.

[0045] Unless otherwise indicated, the practice of the present invention will employ conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology, which are within the skill of the art, many of which are described below for illustrative purposes. Such techniques are fully explained in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Current Protocols in Immunology, QE Coligan, A.M. Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991; Annual Review of Immunology; and monographs in journals such as Advances in Immunology.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, preferred embodiments of the compositions, methods and materials are described herein.

[0047] As will be understood by those skilled in the art and as described elsewhere herein, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first constant region, a second constant region, and a third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are divided into α, δ, ε, γ, and μ, and mammalian light chains are divided into λ or κ. Immunoglobulins comprising α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y consists of the second constant region and the third constant region (the fourth constant region for IgE and IgM) of the two heavy chains combined together, and a disulfide bond (interchain) is formed in the hinge. Heavy chains γ, α, and δ have a constant region consisting of 3 Ig domains in series (on a line) and a hinge region for increasing flexibility; heavy chains μ and ε have a constant region consisting of 4 immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and "CH3 domain," respectively. Each arm of the Y contains the first constant region and variable region of one heavy chain combined with the variable region and constant region of one light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0048] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also called "complementarity determining regions" or "CDRs"). The CDRs can be defined or identified by conventional methods, for example, by sequences according to Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132(2): 211-50, (1970); Borden, P. and Kabat EA, PNAS, 84: 2440-2443 (1987) (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991); or by structures according to Chothia et al. (Choithia, C. and Lesk, AM, J Mol. Biol., 196(4): 901-917 (1987); Choithia, C. et al., Nature, 342: 877-883 (1989)).

[0049] The sequences of the framework regions of different light chains or heavy chains are relatively conservative within species (e.g., humans). The framework region of an antibody (which is the combined framework region of the light chain and heavy chain of the composition) is used to locate and arrange CDRs in three-dimensional space. CDRs are primarily responsible for binding antigenic epitopes. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3, which are numbered in sequence from the N-terminus, and are usually also identified by the chain where a specific CDR is located. Therefore, the CDRs located in the variable domains of the heavy chain of an antibody are referred to as CDRH1, CDRH2, and CDRH3, and the CDRs located in the variable domains of the light chain of an antibody are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs.

[0050] Mentions of "VH" or "V H ” refers to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody, Fv, scFv, Fab or other antibody fragments as disclosed herein.

[0051] Mentions of "VL" or "V L ” refers to the variable region of an immunoglobulin light chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab or other antibody fragments as disclosed herein.

[0052] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by making hybrid cells that form antibodies by fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include chimeric monoclonal antibodies and humanized monoclonal antibodies.

[0053] The articles “a”, “an” and “the” are used herein to refer to one or to more than one (ie to at least one) of the grammatical object of the article.

[0054] As used herein, the term "about" refers to a quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length that differs from a reference quantity, level, value, number, frequency, percentage, size, size, amount, weight, or length by up to 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In a specific embodiment, the term "about" when preceding a numerical value indicates a range of ±15%, ±10%, ±5% or ±1% of that value.

[0055] Throughout this specification, unless the context requires otherwise, the words “comprise / comprises / comprising” will be understood to imply the inclusion of stated steps or elements or groups of steps or elements but not the exclusion of any other steps or elements or groups of steps or elements.

[0056] Reference throughout this specification to "one embodiment," "an embodiment," "a specific embodiment," "an embodiment," "an additional embodiment," or "a further embodiment" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.

[0057] For the purposes of the present invention, "identity" or "homology" is calculated by comparing two aligned sequences in a comparison window. Alignment of the sequences enables the determination of the number of positions (nucleotides or amino acids) shared by the two sequences in the comparison window. The number of shared positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the homology percentage. The determination of the sequence identity percentage can be done manually or by using a well-known computer program.

[0058] The present invention provides immune effector cells for the purposes of treating acute myeloid leukemia (AML), wherein the immune effector cells are genetically engineered with vectors, and the vectors are designed to express genetically engineered receptors that redirect cytotoxicity to tumor cells. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs).

[0059] CAR is such a molecule: combining the antibody-based specificity for a target antigen (e.g., a tumor antigen) with a T cell receptor activating intracellular domain, and generating a chimeric protein that exhibits specific anti-tumor cell immune activity. As used herein, the term "chimeric" describes a portion consisting of different proteins or DNA from different sources.

[0060] The main feature of CARs is their ability to redirect immune effector cell specificity, thereby causing proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner, exploiting the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0061] As used herein, the terms "binding domain", "extracellular binding domain", "antigen-specific binding domain" and "extracellular antigen-specific binding domain" are used interchangeably and provide the ability of CAR to specifically bind to a target antigen of interest. The binding domain may comprise any protein, polypeptide, oligopeptide or peptide that has the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide or other cell surface target molecule, or a component thereof). The binding domain includes any naturally occurring binding partner, synthetic binding partner, semisynthetic binding partner or recombinantly produced binding partner of the biological molecule of interest. As used herein, the term "specific binding affinity" or "specifically binds / specifically bound / specific binding" or "specific targeting" describes a molecule that binds to another molecule with a greater binding affinity than background binding. If the binding domain (or a CAR comprising a binding domain or a fusion protein comprising a binding domain) is, for example, greater than or equal to about 10 5 M -1 The binding domain (or a CAR comprising a binding domain or a fusion protein comprising a binding domain) "specifically binds" to the target molecule if the binding domain (or CAR comprising a binding domain) binds or associates with the target molecule with an affinity or Ka (i.e., the equilibrium association constant of a specific binding interaction, in units of 1 / M). The affinity of the binding domain polypeptides and CAR proteins according to the present disclosure can be easily determined using conventional techniques such as competitive ELISA (enzyme-linked immunosorbent assay).

[0062] The antibody is a human antibody, a murine antibody, a chimeric antibody or a humanized antibody.

[0063] In certain preferred embodiments, the antibody is a chimeric antibody (e.g., a chimeric monoclonal antibody) that specifically binds to a surface protein of a tumor cell. A "chimeric" antibody is an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. Thus, all parts of the chimeric immunoglobulin may be substantially identical to the corresponding parts of the natural human immunoglobulin sequence, except for the CDRs. Chimeric or other monoclonal antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Chimeric antibodies may be constructed with the aid of genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0064] Antibodies include antigen-binding fragments thereof, such as Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv proteins ("scFv"), and the portion of the full-length antibody responsible for antigen binding. The term also includes genetically engineered forms, such as humanized antibodies, heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof.

[0065] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain in either orientation (eg, VL-VH or VH-VL).

[0066] Single-chain antibodies can be cloned from the V region genes of hybridomas specific for the desired target. The production of such hybridomas has become routine. Techniques that can be used to clone the heavy chain variable region (VH) and the light chain variable region (VL) are described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837.

[0067] Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0068] The CAR contemplated herein may comprise 1, 2, 3, 4 or 5 or more joints. In a specific embodiment, the length of the joint is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intermediate amino acid length. In some embodiments, the length of the joint is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids.

[0069] Exemplary examples of linkers include glycine polymers (G) n; glycine-serine polymers (Gi_sSi_5) n, wherein n is an integer of at least 1, 2, 3, 4 or 5; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine polymers and glycine-serine polymers are relatively unstructured and therefore may be able to act as neutral tethers (tethers) between domains of fusion proteins (e.g., CAR described herein). Glycine is even more accessible to phi-psi space than alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). It will be appreciated by those of ordinary skill in the art that the design of CAR in a specific embodiment may include a fully or partially flexible linker, so that the linker may include a flexible linker and one or more portions that confer a less flexible structure to provide a desired CAR structure.

[0070] In a specific embodiment, the linker is between the VH domain and the VL domain.

[0071] In a specific embodiment, the linker comprises or consists of the amino acid sequence of SEQ ID NO:5.

[0072] In one embodiment, the IL-1RAP binding domain is a scFv comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least 1, 2 or 3 modifications but not more than 30, 20 or 10 modifications in the light chain variable region amino acid sequence of SEQ ID NO:4, and the heavy chain variable region comprises an amino acid sequence having at least 1, 2 or 3 modifications but not more than 30, 20 or 10 modifications in the heavy chain variable region amino acid sequence of SEQ ID NO:2.

[0073] Preferably, the IL-1RAP binding domain is a scFv, which comprises (i) a light chain variable region, wherein the light chain variable region comprises a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO: 6, the complementarity determining region 2 (CDR2) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO: 7, the complementarity determining region 3 (CDR3) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO: 8 NO:8 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical; and (ii) a heavy chain variable region comprising a complementary determining region 1 (CDR1), a complementary determining region 2 (CDR2) and a complementary determining region 3 (CDR3), wherein the complementary determining region 1 (CDR1) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO:12, the complementary determining region 2 (CDR2) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO:13, the complementary determining region 3 (CDR3) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO:14, NO: 14 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical.

[0074] The binding domain of the CAR is usually followed by one or more "hinge regions" that play a role in positioning the antigen binding domain away from the effector cell surface to enable appropriate cell / cell contact, antigen binding and activation. The CAR typically comprises one or more hinge regions between the binding domain and the transmembrane domain. The hinge region may be derived from natural, synthetic, semisynthetic or recombinant sources.

[0075] Preferably, the anti-IL-1RAP binding domain is connected to the transmembrane domain via a hinge region.

[0076] In an embodiment, the hinge region comprises a hinge sequence of IgG1 or a sequence having 95%-99% identity to the hinge sequence of IgG1. The IgG hinge is encoded by a single exon. Therefore, the term "hinge sequence of IgG1" as used herein has the same meaning as that commonly understood by a person of ordinary skill in the art to which the present invention belongs, i.e., the 15 amino acid residues of the hinge encoded by the IgG1 exon (Basic Immunology, Fifth Edition, Chapter 3, Immunoglobulins: Structure and Function - Immunoglobulin Hinge).

[0077] In a further embodiment, the hinge region comprises the hinge sequence of IgG4 or a sequence having 95%-99% identity with the hinge sequence of IgG4. In a further embodiment, the hinge region may also comprise the CH2-CH3 region of IgG1 or IgG4 or a sequence having 95%-99% identity with the CH2-CH3 region of IgG1 or IgG4.

[0078] In a further embodiment, the hinge region comprises CD8α or a sequence that is 95%-99% identical to CD8α.

[0079] The "transmembrane domain" is a part of the CAR that fuses the extracellular binding portion and the intracellular signal transduction domain and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain can be derived from natural sources, synthetic sources, semisynthetic sources, or recombinant sources.

[0080] Preferably, the encoded CAR comprises a transmembrane domain of a protein selected from the group consisting of: the α chain, β chain or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, more preferably CD28.

[0081] In a specific embodiment, the CAR contemplated herein comprises an intracellular signal transduction domain. "Intracellular signal transduction domain" refers to a part of CAR that is involved in transducing information that effectively binds CAR to a target antigen to the interior of an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to target cells bound by CAR, or other cellular responses caused by antigen binding to an extracellular CAR domain.

[0082] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, a cytolytic activity or an activity or assistance including cytokine secretion. Therefore, the term "intracellular signal transduction domain" refers to the following portion of a protein: it transduces an effector function signal and instructs the cell to perform a specialized function. Although the entire intracellular signal transduction domain can generally be used, in many cases, it is not necessary to use the entire domain. With respect to the use of a truncated portion of an intracellular signal transduction domain, such a truncated portion may be used instead of the entire domain as long as it transduces an effector function signal. The term "intracellular signal transduction domain" is intended to include a truncated portion of any intracellular signal transduction domain that is sufficient to transduce an effector function signal.

[0083] It is known that the signal generated only by TCR is not enough to fully activate T cells, and secondary signals or costimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of intracellular signal transduction domains: the main signal transduction domain activated by TCR (e.g., TCR / CD3 complex) initiating antigen-dependent main activation; and the costimulatory signal transduction domain that acts in an antigen-independent manner to provide a secondary signal or costimulatory signal. In a preferred embodiment, the CAR considered herein includes an intracellular signal transduction domain, and the intracellular signal transduction domain includes one or more "costimulatory signal transduction domains".

[0084] In an embodiment, the isolated nucleic acid molecule can encode an intracellular signal transduction domain comprising at least one costimulatory domain. Thus, in this embodiment, the intracellular signal transduction domain comprises at least one costimulatory domain.

[0085] As used herein, the term "costimulatory signal transduction domain" or "costimulatory domain" refers to the intracellular signal transduction domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes after binding to an antigen.

[0086] Preferably, the at least one co-stimulatory domain of the functional intracellular signal transduction domain is obtained from one or more proteins selected from the group consisting of the following proteins: OX40, CD2, CD27, CD28, CDS, CD3ζ, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1ВB (CD137).

[0087] More preferably, the costimulatory domain obtained from 4-1ВB (CD137) has a sequence that is 95%-99% identical to the amino acid sequence of the costimulatory domain of 4-1BB.

[0088] More preferably, the costimulatory domain obtained from CD3ζ has a sequence that is 95%-99% identical to the amino acid sequence of the costimulatory domain of CD3ζ.

[0089] In another embodiment, the intracellular signaling domain comprises a costimulatory domain obtained from 4-1BB and / or a costimulatory domain obtained from CD3ζ.

[0090] In a particularly preferred embodiment, CAR comprises a CD3ζ primary signal transduction domain and one or more costimulatory signal transduction domains. The intracellular primary signal transduction domain and the costimulatory signal transduction domain can be connected in series to the carboxyl terminus of the transmembrane domain in any order.

[0091] The cells that can be used according to the present invention can be isolated. "Isolated cells" refer to cells obtained from tissues or organs in vivo and are substantially free of extracellular matrix.

[0092] The cell for treating AML according to the present invention can be prepared by inserting a nucleic acid molecule encoding a chimeric antigen receptor (CAR) into the genome of the host cell using a vector.

[0093] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically connected to (e.g., inserted into) a carrier nucleic acid molecule. The vector may contain sequences that direct autonomous replication in the cell, or may contain sequences sufficient to allow integration into the host cell DNA.

[0094] The cells for treating AML according to the present invention can be prepared using a vector comprising a nucleic acid molecule encoding CAR, wherein the vector is selected from DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector, preferably a lentiviral vector.

[0095] In some embodiments, the vector comprises a promoter, preferably an EF-1α promoter.

[0096] Retrovirus is a common tool for gene delivery. In a specific embodiment, retrovirus is used to deliver polynucleotides encoding chimeric antigen receptor (CAR) to cells. As used herein, the term "retrovirus" refers to an RNA virus: the RNA virus reversely transcribes its genomic RNA into a linear double-stranded DNA copy, and then covalently integrates its genomic DNA into the host genome. Once the virus is integrated into the host genome, it is referred to as "provirus". Provirus acts as a template for RNA polymerase II and guides the expression of RNA molecules encoding the structural proteins and enzymes required for the production of new virus particles.

[0097] Therefore, T cells transduced with the vector are able to elicit stable, long-term, and durable CAR-mediated T cell responses.

[0098] In a specific embodiment, T cells are transduced with a retroviral vector (e.g., a lentiviral vector) encoding a CAR.

[0099] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0100] The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements primarily derived from a lentivirus, or portions thereof, including LTRs.

[0101] A "self-inactivating" (SIN) vector refers to a replication-defective vector, such as a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region (referred to as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.

[0102] In one embodiment, a SIN vector backbone is preferred.

[0103] Preferably, the vector used further comprises a promoter, such as the EF-1α promoter.

[0104] As used herein, the term "promoter" refers to the recognition site of the polynucleotide (DNA or RNA) bound by RNA polymerase. RNA polymerase initiates and transcribes the polynucleotide operably connected to the promoter. In a specific embodiment, it is desirable to express the polynucleotides comprising CAR by a promoter providing stable and long-term CAR expression in T cells, and to express at a level sufficient to redirect T cells to cells expressing the target antigen.

[0105] The cells used for use according to the present invention are preferably T cells (eg, human T cells), more preferably CD8+T cells (eg, human CD8+T cells). In a preferred embodiment, cells (eg, T cells) used for use according to the present invention express CAR on their membranes. As used herein, the term "on its membrane" has the same meaning as that generally understood by those of ordinary skill in the art to which the present invention belongs, i.e., "on the cell surface membrane".

[0106] In a specific embodiment, before the in vitro manipulation or genetic modification of the immune effector cells described herein, a cell source is obtained from a subject. In a specific embodiment, the cells for use according to the present invention encompass T cells. T cells can be obtained from a variety of sources, including but not limited to: peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments, any number of techniques known to those skilled in the art (e.g., precipitation, such as FICOLL TM T cells are obtained from a unit of blood collected from a subject by separation. Cells from individual circulating blood can be obtained by apheresis. The product of apheresis generally comprises lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction, and the cells are placed in a suitable buffer or medium for subsequent processing.

[0107] This can be accomplished by lysing red blood cells and depleting monocytes (e.g., by PERCOLL TM Gradient centrifugation) is used to separate T cells from peripheral blood mononuclear cells. Specific T cell subsets expressing one or more markers (e.g., CD4 or CD8) can be further separated by positive selection or negative selection techniques. For example, the enrichment of T cell populations by negative selection can be accomplished by using a combination of antibodies against surface markers specific to negatively selected cells.

[0108] In some embodiments of the present invention, cells with nucleic acid molecules encoding chimeric antigen receptors (CAR) use suicide genes (including inducible suicide genes) to reduce the risk of direct toxicity (i.e., graft-versus-host disease in an allogeneic administration environment) and / or uncontrolled proliferation of genetically modified cells. In a specific aspect, the suicide gene is not immunogenic to a host with the polynucleotide or cell. Some examples of usable suicide genes are inducible caspase-9 (iCASP9), herpes simplex virus thymidine kinase (HSV-tk), CD20, truncated EGFR, caspase 8, or cytosine deaminase. Caspase 9 can be activated using a specific dimerization chemical inducer (CID). Other systems can be activated by metabolizing prodrugs (ganciclovir) or by binding antibodies (rituximab, cetuximab (Cituximab)).

[0109] Disclosed herein is a class of cell therapy in which T cells are genetically modified in vitro to express CAR, and CART cells are infused into recipients in need thereof. The infused cells are capable of killing tumor cells in the recipient (preferably human). Unlike antibody therapy, CAR T cells are able to replicate in vivo, resulting in long-term persistence that can cause sustained tumor control.

[0110] Furthermore, CARs allow the redirection and activation of effector T cells to any cell surface molecule following binding via antibody-derived receptors and are independent of MHC restriction.

[0111] Genetically modified cells (e.g., T cells) for use according to the present invention are constructed from the patient's own cells (autologous), but they may also originate from other allogeneic donors, to provide allogeneic genetically modified T cells (donor lymphocyte infusion) in a bone marrow or peripheral hematopoietic stem cell allograft environment. These cells expressing CAR molecules can be used to treat AML in mammals (preferably humans), and the disease is associated with cell surface IL-1RAP expression.

[0112] Preferably, these cells (e.g., T cells) express a CAR molecule comprising an antigen binding domain (which is an anti-IL-1RAP scFv), the CAR molecule comprising an anti-IL-1RAP binding domain, a transmembrane domain of a CD28 protein, a co-stimulatory 4-1BB signaling domain, and a CD3ζ signaling domain, wherein the anti-IL-1RAP binding domain comprises:

[0113] (i) a light chain comprising a complementary determining region 1 (CDR1), a complementary determining region 2 (CDR2), and a complementary determining region 3 (CDR3), wherein the complementary determining region 1 (CDR1) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO:6, the complementary determining region 2 (CDR2) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO:7, and the complementary determining region 3 (CDR3) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical or 100% identical to the amino acid sequence of SEQ ID NO:8; and

[0114] (ii) a heavy chain comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12, the complementarity determining region 2 (CDR2) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 13, and the complementarity determining region 3 (CDR3) is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0115] AML may be any AML according to the European Leukemia Net (ELN) prognostic classification and / or any subtype of AML according to the French American British (FAB) classification.

[0116] In one embodiment, the AML is relapsed / refractory AML.

[0117] In another embodiment, AML is relapsed / refractory AML with complex cytogenetic abnormalities (ie, cytogenetic abnormalities according to the WHO classification) and / or AML with TP53 mutations.

[0118] TP53 is a tumor suppressor protein encoded by the TP53 gene located on the short arm of chromosome 17. TP53 plays a key role in maintaining genomic stability in response to DNA damage; it activates DNA repair programs and triggers cell cycle arrest. TP53 is mutated in more than half of human cancers. In AML, mutated TP53 is mainly observed in treatment-related AML and / or in patients with complex chromosomal karyotypes. Studies from AML mouse models have shown that gain-of-function mutations in hotspot regions can promote more aggressive AML. Previous studies have shown that the presence of TP53 mutations is associated with poor survival and poor response to chemotherapy in patients with or without complex chromosomal karyotypes. In multivariate analysis, the presence of TP53 mutations in the absence of abnormal cytogenetic abnormalities predicts poor overall survival and poor treatment response. In some embodiments, AML is AML associated with IL-1RAP expression (also referred to as "AML expressing IL-1RAP" or "IL-1RAP+AML").

[0119] In some embodiments, the AML associated with IL-1RAP expression is (i) a relapsed / refractory form of IL-1RAP+ AML or (ii) IL-1RAP+ AML with complex cytogenetic abnormalities and / or IL-1RAP+ AML with TP53 mutations.

[0120] The cells for use according to the invention may be used to treat patients of all ages, in particular patients younger than 60 years old (eg patients younger than 20 years old, ie pediatric AML) and / or patients older than 60 years old.

[0121] Cells (e.g., T cells) expressing IL-1RAP-specific CAR molecules can be used in methods for treating AML in humans, wherein the humans have been treated with at least one therapy line (e.g., chemotherapy, immunotherapy, or targeted therapy). The chemotherapeutic agent may be CPX-351; immunotherapy may be a monoclonal antibody, such as an antibody drug conjugate (ADC) (e.g., anti-CD33 (Gemtuzumab ozogamicin) linked to a toxin); bispecific antibodies (Bites, such as anti-CD33 / CD3, anti-CD123 / CD3 or other AML anti-cell surface markers / CD3). Targeted therapy agents may be anti-mutated FLT3, such as Midostaurin or Quazartinib; or agents targeting mutated IDH1 / IDH2, such as IDH1 inhibitors (ivosidenib) or IDH2 inhibitors (enasidenib).

[0122] Therefore, preferably, cells (e.g., T cells) expressing IL-1RAP-specific CAR molecules can be used in combination with at least one monoclonal antibody for use in a method for treating AML in a mammal, wherein the monoclonal antibody is, for example, an anti-checkpoint inhibitor (i.e., anti-PD-1, anti-PDL-1, or anti-CTLA4). Anti-PD-1, anti-PDL-1, or anti-CTLA4 can be nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, or tremelimumab.

[0123] Therefore, cells (e.g., T cells) expressing IL-1RAP-specific CAR molecules can be used in methods for treating AML in humans who have received graft-versus-leukemia, allogeneic stem cell transplantation, donor lymphocyte infusion (DLI), a previous CART cell therapy that was not IL-1RAP CART cells, or allogeneic or autologous hematopoietic transplantation.

[0124] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction in one or more measurable markers of the disease or condition (e.g., cancer) being treated. Treatment may optionally include alleviation or relief of the symptoms of a disease or condition, or a delay in the progression of a disease or condition. "Treatment" does not necessarily refer to complete eradication or cure of a disease or condition or its associated symptoms.

[0125] Therefore, the present disclosure provides a method for treating AML, the method comprising administering to a subject in need thereof a therapeutically effective amount of cells, the cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or an antibody fragment, the CAR comprises an anti-IL-1RAP binding domain, a transmembrane domain, and an intracellular signaling domain, the intracellular signaling domain comprises at least a stimulatory domain, and wherein the anti-IL-1RAP binding domain comprises:

[0126] (i) a light chain comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80% identical to the amino acid sequence of SEQ ID NO: 6, the complementarity determining region 2 (CDR2) is at least 80% identical to the amino acid sequence of SEQ ID NO: 7, and the complementarity determining region 3 (CDR3) is at least 80% identical to the amino acid sequence of SEQ ID NO: 8; and

[0127] (ii) a heavy chain, comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80% identical to the amino acid sequence of SEQ ID NO: 12, the complementarity determining region 2 (CDR2) is at least 80% identical to the amino acid sequence of SEQ ID NO: 13, and the complementarity determining region 3 (CDR3) is at least 80% identical to the amino acid sequence of SEQ ID NO: 14.

[0128] The cells (e.g., T cells) may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other ingredients (e.g., IL-2 or other cytokines or cell populations). In short, a pharmaceutical composition may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications within the scope of reasonable medical judgment, and have a reasonable benefit / risk ratio.

[0129] The cells for use according to the invention may be formulated into a composition.

[0130] Therefore, the present invention also relates to a composition (e.g., a pharmaceutical composition) for treating acute myeloid leukemia (AML), the composition comprising a cell, the cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or an antibody fragment, the CAR comprises an anti-IL-1RAP binding domain, a transmembrane domain, and an intracellular signal transduction domain, the intracellular signal transduction domain comprises at least a stimulatory domain, and wherein the anti-IL-1RAP binding domain comprises:

[0131] (i) a light chain comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80% identical to the amino acid sequence of SEQ ID NO: 6, the complementarity determining region 2 (CDR2) is at least 80% identical to the amino acid sequence of SEQ ID NO: 7, and the complementarity determining region 3 (CDR3) is at least 80% identical to the amino acid sequence of SEQ ID NO: 8; and

[0132] (ii) a heavy chain, comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is at least 80% identical to the amino acid sequence of SEQ ID NO: 12, the complementarity determining region 2 (CDR2) is at least 80% identical to the amino acid sequence of SEQ ID NO: 13, and the complementarity determining region 3 (CDR3) is at least 80% identical to the amino acid sequence of SEQ ID NO: 14.

[0133] The composition for use according to the invention is preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

[0134] As used herein, "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0135] In one embodiment, the CAR-modified cells or compositions are administered to a subject by direct injection into a tumor, lymph node, systemic circulation, or site of infection.

[0136] In one embodiment, CAR-modified cells (e.g., CAR-modified T cells) can be used to treat subjects diagnosed as AML in the following manner: immune effector cells are removed from the subject; the immune effector cells are genetically modified with a carrier comprising a nucleic acid encoding CAR as described herein, so as to produce a modified immune effector cell group; and the modified immune effector cell group is administered to the same subject. In a preferred embodiment, the immune effector cell includes a T cell.

[0137] Although appropriate dosages can be determined by animal models and ultimately by clinical trials, the amount, frequency, and possible sequence of administration with conventional AML treatments will depend on factors such as the patient's condition and the type and severity of AML.

[0138] A "therapeutically effective amount" of genetically modified therapeutic cells can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutic beneficial effects. It can be generally stated that a pharmaceutical composition comprising T cells described herein can be administered in an amount of 10 4 -10 9 cells / kg body weight, preferably 10 5 -10 6 The dosage is given at 10 cells / kg body weight (including all integer values ​​within these ranges).

[0139] The present invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified.

[0140] Example

[0141] Example 1: Monoclonal Antibody Production

[0142] Mouse anti-hIL-1RAP monoclonal antibodies were generated by standard hybridoma technology.

[0143] Briefly, BALB / c mice (5 weeks, Charles River) were immunized by footpad (n=3) or intraperitoneal (n=5) with a recombinant fusion protein consisting of the extracellular portion of IL-1RAP (NM_002182.2, NCBI) and the Fc portion of human IgG1 (R&D Systems, Lille, France). Lymph nodes or spleen cells and blood samples were harvested and the cells were fused with mouse myeloma cell lines and then screened against IL-1RAP positive cell lines (KU812) and IL-1RAP negative cell lines (Raji, KG1) by FACS analysis (Becton Dickinson).

[0144] Screening of hybridomas allowed the selection of IL-1RAP-positive cell lines (KU812 or KG-1, AML or Phi + p 210 CML) and negative cell lines (Tom-1, NALM-20, Jurkat or Raji, respectively Phi+ p190 B-ALL、Phi - Five monoclonal antibody subclones were used to distinguish B-ALL, T-ALL or Burkitt's lymphoma.

[0145] - Molecular characterization of antibodies

[0146] Molecular characterization was performed by Sanger sequencing of cloned PCR amplification products obtained with degenerate primers specific for FR1 and the constant regions of the heavy and light chains according to the protocol of Wang. Z. et al. (J. Immunol. Methods, 2000; 233, pp. 167-77). Molecular characterization was performed by Sanger sequencing of cloned PCR amplification products obtained with degenerate primers specific for FR1 and the constant regions of the heavy and light chains according to Brochet X. et al. (Nucleic Acids Res., 2008, 36, pp 503-8) using the V-QUEST online tool for After database alignment of the consensus nucleotide sequence, identification of the VDJC gene rearrangement and CDR3 region was obtained. Molecular Sanger sequencing showed that all 5 monoclonal antibodies were identical and shared the same CDR3 nucleotide sequence. The monoclonal antibody subclone (#E3C3) was selected because it emitted the highest relative fluorescence intensity (RFI) by cytometry.

[0147] The selected antibody (clone #E3C3) was characterized by ELISA, Western blot, immunohistochemistry, confocal microscopy, tissue microarrays (TMA) from normal tissues (FDA Normal Human Organ Tissue Array, 99 cores / 33 sites / 75 cases) and primary samples from CML patients against recombinant IL-1RAP protein.

[0148] - Western blot of subcellular fractions ( Figure 1 )

[0149] Whole cell lysates of different AML or CML (positive control) hematopoietic cell lines were separated by polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membranes. The membrane was probed overnight with IL-1RAP#A3C3 primary antibody (diluted at 1:20). 20 micrograms of protein were electrophoresed on sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), and the ultrasonic protein fractions were suspended in RIPA buffer supplemented with a protease inhibitor cocktail (cOmplete, Mini, EDTA-free; Roche, Bale, Switzerland). Protein loading in the western blot lanes was evaluated with β-actin mAb staining (1:1000, clone AC15, #A5441, Sigma-Aldrich). Immunodetection staining was performed with sheep anti-mouse IgG polyclonal secondary antibody (#515-035-062, Jackson). Enhanced chemiluminescence detection reagents enable detection using cameras and Bio-ID software (Vilber-Lourmat, Collegien, France).

[0150] -Relative IL-1RAP mRNA expression ( Fig.18 )

[0151] Relative IL-1RAP mRNA expression was determined by RT-qPCR using the Hs_00895050_m1 Taqman qPCR gene expression assay (ThermoFisher Scientific) targeting mRNA variant codons of cell surface proteins. Whole blood samples or bone marrow samples from AML patients (n=29) at diagnosis were analyzed according to the European Leukemia Network (ELN) prognostic classification. K562, MonoMac-6, and HEL-60 mRNA were used as negative controls, high or low positive controls, respectively.

[0152] - Absolute number of IL-1RAP antigenic sites ( Figure 3 )

[0153] The absolute number of IL-1RAP antigenic sites was determined based on the cytometric bead array (CBA) technique. 6Different leukemia cell lines with 10 cells / mL were treated with anti-IL-1RAP primary antibody (unlabeled #A3C3, ) and stained with FITC-IgG1 anti-mouse secondary antibody (Catalog No. 55526, MP ) is shown. The experiments were performed using the CellQuant Calibrator kit as recommended (Ref 7208, Biocytex). Unligated IgG1 isotype was used as a control (Cat. No. 857-073-020, Diaclone). Flow cytometry (FACS Canto TM II, results were analyzed using BD FACS DIVA V7.0, BD Biosciences). KU812 and K562 were used as positive and negative controls, respectively.

[0154] - In vitro detection of recombinant IL-1RAP protein by ELISA ( Figure 2 ).

[0155] Anti-human Fc antibody was coated on the bottom of plastic ELISA plates. IL-1RAP protein loaded on human antibody was probed with mouse and human IL-1RAP (#E3C3) antibodies and then revealed by anti-mouse Fc antibody coated with horseradish peroxidase (HRP).

[0156] ELISA confirmed that #E3C3 monoclonal antibody recognized IL-1RAP recombinant protein.

[0157] - Flow cytometric analysis of primary cells from AML patients ( Figure 3 )

[0158] IL-1RAP cell surface staining of different hematopoietic AML cell lines was performed using #A3C3 mAb and IL-1RAP murine mAb as staining comparison.

[0159] Relative fluorescence intensity (RFI) between IL-1RAP staining and isoforms was calculated. IL-1RAP+ (KU812) or IL-1RAP- (Raji) cell lines were used as positive or negative controls, respectively.

[0160] For primary samples of AML patient bone marrow or peripheral blood, mouse IL-1RAP mAb (#E3C3) was included in the panel to enable detection of CD45, CD34, CD38, CD33, CD123 and CD14 (for monocytes). Stained cells were collected by a CANTO II cell counter (BD Biosciences, Le Pont-de-Claix, France) and analyzed by DIVA 6.1 software (BD Biosciences, Le Pont-de-Claix, France).

[0161] - In-situ detection

[0162] To investigate specific or non-target tissue binding, FDA standard frozen tissue arrays (90 tissue cores (30 organs) from 3 different donors per organ, US Biomax, Rockville, USA) were incubated as described above. Immunostaining was detected using the UltraView Universal DAB Detection Kit (Ventana, USA). Images were acquired and analyzed using NDP.view 2 software. High IL-1RAP expressing cell lines (KU812) or IL-1RAP negative expressing cell lines (Raji) were used as positive or negative controls, respectively. Staining intensity was graded as follows: negative (0), weak staining (1+), moderate staining (2+), or strong staining (3+). High IL-1RAP expressing cell lines (KU812) or IL-1RAP negative expressing cell lines (Raji) were used as positive or negative controls, respectively.

[0163] IL-1RAP expression was investigated using the #E3C3 monoclonal antibody. Staining of varying intensity was detected in only six tissues (lymph nodes, colon, small intestine, placenta, stomach, and prostate, mainly in epithelial or endothelial cells) ( Figure 4 ).

[0164] -result

[0165] #A3C3 is able to stain different AML cell lines that express IL-1RAP on the cell surface. Using #A3C3, by Western blotting, we thus confirmed IL-1RAP expression on the cell surface of different cell lines, which had signals of different intensity levels. Interestingly, the CD14+ monocyte subset also expressed IL-1RAP. By flow cytometric analysis of the same cohort of AML (all subtypes, n=30) patients, we confirmed (as previously shown in the prior art) that IL-1RAP was present on AML blasts at different percentages (84.27±17.4%) and had cell surface level expression, while monocyte expression levels remained stable. This was also confirmed by the absolute counts of IL-1RAP antigenic sites in AML patients classified by the European Leukemia Network (ELN) prognostic stratification. Based on these results, we defined 3 different cell surface expression levels: low, medium and high. These expression levels were modeled by cell lines for further study.

[0166] Example 2: Lentiviral constructs

[0167] Based on molecular sequencing of VDJ or VJ rearrangement and CDR3 nucleotide sequence determination, a CAR lentiviral construct (pSDY-iC9-IL-1RAPCAR-dCD19) was prepared by cloning the synthetically produced single-chain variable fragment (scFv) derived from the #E3C3 IL-1RAP hybridoma of Example 1 into the SIN-pSDY backbone (Rossolillo P, Winter F, Simon-Loriere E, Gallois-Montbrun S, Negroni M. Retrovolution: HIV-driven evolution of cellular genes and improvement of anticancer drug activation. PLoS Genet. 2012; 8(8): e1002904).

[0168] Briefly, a SIN lentiviral construct carrying the iCASP9 safety cassette, the single-chain variable fragment of the #E3C3 monoclonal antibody, and the cell surface expressed marker ΔCD19 (for monitoring and potential cell selection) was constructed. All three transgenes were separated by a 2A peptide cleavage sequence and were under the control of the EF1 promoter and the SP163 enhancer sequence (part of the 5'UTR of the mouse VEGF gene, GenBank accession number #U41383).

[0169] like Figure 5As shown, the construct carries the following 3 different parts: iCASP9 (chemically inducible caspase 9) suicide safety box, IL-1RAP CAR and cell surface selection marker ΔCD19 (CD19 with intracellular part truncated to avoid signal transduction), which are separated by 2 different 2A ribosomal skipping sequences (P2A and T2A) and are under the control of EF1α (elongation factor 1 promoter α) promoter plus SP163 enhancer. The scFv (composed of the variable regions of the heavy chain (VH) and light chain (VL) sequences of #E3C3 immunoglobulin) was cloned in frame with the CD28-4.1BB-CD3z signal transduction chain and was under the control of EF1α promoter and SP163 enhancer. The IL-1RAP CAR contains a single-chain variable fragment (scFv) bound to a leader sequence (L), tagged with human influenza hemagglutinin (HA), and connected via a hinge region to a T cell activation domain consisting of two co-stimulatory domains (modified transmembrane and intracellular signaling CD28 and 4-1BB) and a CD3z intracellular signaling domain. Mock T consists of the same construct without the IL-1RAP scFv.

[0170] Example 3: Generation of IL-1RAP CART cells

[0171] According to the manufacturer's instructions, CD3+T lymphocytes obtained from healthy donor peripheral blood mononuclear cells were activated with anti-CD3 / CD28 beads (Life Technologies, France) and then separated on magnetic columns (MACS, Miltenyi Biotec, Paris, France). On the second day, activated T cells were transduced by using the lentiviral vector of Example 2 at 10°C and 2000g spinoculation for 90min in contact with the supernatant (SN). The transduction efficiency was determined by flow cytometry analysis to identify the expression of ΔCD19 cell surface markers. Four days after transduction, CD19 positive cells labeled with CD19 microbeads (Miltenyi Biotec, Paris, France) were magnetically separated using MACS columns. The isolated CD19 expressing cells were amplified in complete X-vivo medium (Lonza, Bale, Switzerland) containing 500UI / mL rhIL-2 (Proleukin; Novartis) supplemented with 8% human serum and cryopreserved. Experimentally, we used TransAct T cell reagent and TexMACS medium (Miltenyi Biotec, Paris, France) supplemented with human IL-2, IL-7, IL-15, or IL-7+IL-15.

[0172] Example 4: Lentiviral transduction of donor T cells

[0173] The lentiviral vector supernatant stock solution of Example 2 was produced by the following method: using CaCl 2 Methods: Subconfluent 293T cells were transiently co-transfected with a helper plasmid (pMDG) encoding the vesicular stomatitis virus (VSV) envelope and a GAG / POL (psPAX2) packaging plasmid (Addgene, #12259 and #12260, Trono et al., Lausanne, Switzerland). Viral supernatants were harvested after 48 and 72 hours, concentrated using PEG and low-speed centrifugation (3000g, overnight), and then stored at -80°C until use. The same lentiviral construct without IL-1RAP scFv (Mock) was used as a control. The titer of the lentiviral supernatant was determined by transduction of 293T permissive cells using serial dilutions of SN.

[0174] Transduction efficiency was measured by flow cytometry. The multiplicity of infection (MOI) was subtracted from the supernatant titer according to the number of starting cells.

[0175] The in vitro production process using lentiviral supernatants allowed transduction of primary T cells at an MOI of 2 for either Mock or CAR IL-1RAP supernatants, respectively.

[0176] - Western blot analysis of IL-1RAP CAR expression.

[0177] Whole protein lysates or proteins extracted from membrane or cytoplasmic subfractions (obtained after ultracentrifugation) of IL-1RAP-transduced T cells were probed with mouse anti-human CD3z antibodies. Western blotting of subcellular fractions showed that IL-1RAP CAR was associated with CD3z signaling (signal at 55 KDa, compared to the expected endogenous CD3z signal at 16 KDa) ( Figure 6 ).

[0178] -Flow cytometry analysis

[0179] CAR expression on the surface of T cells was analyzed using recombinant IL-1RAP biotinylated protein and displayed by flow cytometry using anti-biotin secondary antibody (Miltenyi Biotec Clone # Bio3-18E7). CEM cell lines or primary T cells were transduced with Mock or CAR IL-1RAP. Then, the cells were incubated in the presence of recombinant IL-1RAP labeled with biotin in increasing amounts. Stained with anti-biotin fluorescent antibody and analyzed by flow cytometry. The percentage of biotin+ / CD19+CEM or T cells was plotted relative to the amount of biotin-labeled recombinant protein. A dot plot of cell counting analysis of representative staining (including maximum staining) is provided. Untransduced T cells (C0) or Mock T cells were used as controls.

[0180] Additional analysis using serial dilutions of biotinylated IL-1RAP protein (20 ng-2.4 pg / ml) and FACS analysis allowed the examination of CEM T cell lines or primary T cells transduced with IL-1RAP CAR. A single experiment allowed the demonstration that different amounts of recombinant protein (1.25 ng and 0.15 ng) were required for the greatest recruitment of CEM (85.8%) or primary (68.5%) GMTCs, respectively ( Figure 7 ).

[0181] Compared with primary T cells, CEMs expressed more CAR on their cell surface. In addition, the addition of large amounts (1000-fold > plasma concentration) of cold recombinant IL-1RAP protein to E:T co-cultures resulted in a significant inhibition of effector cytotoxicity.

[0182] These experiments confirmed that CAR was localized on the cell surface and that there was CAR-specific recognition and binding to the IL-1RAP protein.

[0183] Example 5: Efficiency of the safe suicide gene iCASP9 cassette

[0184] Transduced cells (IL-1RAP CAR 293T) or untransduced cells (293T) were cultured for 24 h in medium alone (-dimer chemical inducer (CID)) or medium containing 20 nM CID AP1903. Light microscopy enabled imaging of the presence and structure of live or dead cells in culture (×40).

[0185] By light microscopy, it was shown that 293T cell cultures transduced with IL-1RAP CAR were sensitive to CID ( Figure 8 ).

[0186] Flow cytometric analysis of non-transduced T cells (C0) and a mixture of GMTC cells expressing or not expressing IL-1RAP CAR after CID exposure (20 nM, 24 h) or without exposure (light gray). + / CD19 + Staining allows differentiation of CAR-expressing GMTCs from others.

[0187] Untransduced T cells (C0) or IL-1RAP CART cells were exposed to medium only or medium + CID (20 nM, 24 h).

[0188] Precise cell death was first assessed by flow cytometry after Annexin-V / 7-AAD gating according to the manufacturer's instructions (Beckman Coulter, IM3614). + / CD19 + Gating was performed on positive cells. Quantification was determined after acquiring 5000 fluorescent beads. The killing efficiency was normalized to control cells (untreated cells). Cell killing was calculated as follows: % dead cells = [1-(absolute number of live cells in AP1903-treated cells / absolute number of live cells in untreated cells)] × 100. C0 or IL-1RAP CART (gated on CD3+ / CD19+) cells were exposed to CID for 24h or 48h. The results are shown as mean ± SD from 3 independent experiments. ***: p<0.001 ( Fig. 9 ).

[0189] Cell counting analysis showed that in the expression of (CD19 + ) or no expression (CD19 - )After 24 hours of CID exposure of mixed populations of IL-1RAP CAR T cells, only CD19 - CD3 + Cells persisted. More specifically, using the apoptosis quantification AnnV / 7AAD assay, it was found that 84.11% and 88.93% of IL-1RAP CART cells were eliminated after 24h or 48h CID exposure, respectively (p<0.001, n=3), compared with untransduced T cells (C0, 1.28% and 6.13% at 24h or 48h, respectively).

[0190] Example 6: Proliferation ability of IL-1RAP CART cells

[0191] Materials and methods

[0192] Untransduced T cells (C0), Mock T cells (MockT) or IL-1RAP CAR (IL-1RAP CART) were contacted with target cells (KU812, CML, positive control) expressed on the cell surface and AML cell lines with different IL-1RAP cell surface expression levels (low: HL-60; medium: MOLM-13; high: EOL-1) in culture medium only (no target), or with IL-1RAP negative (K562) cells, and cultured for 3 days with an effector: target (E: T) ratio of 1: 3. The effector was previously labeled with 0.5M CFSE without IL-2 supplementation. After co-culture for 72 hours without IL-2 supplementation, CFSE dye dilution was measured by flow cytometry to evaluate the division of live CD3+ / CD19- (C0) and CD3+ / CD19+ gated cells (MockT cells or IL-1RAP CART cells).

[0193] result

[0194] In contrast to untransduced T cells (C0) or MOCK-transduced T cells (MockT), IL-1RAP CART cells were able to be specifically activated and divide in the presence of AML cell lines, regardless of the IL-1RAP cell surface expression of target cells ( Fig. 10A and Fig. 10B ).

[0195] Example 7: Intracellular expression of IFNγ

[0196] Materials and methods

[0197] Untransduced T cells (C0), Mock transduced T cells (MockT) or IL-1RAP CAR (IL-1RAP CART) were contacted with target cells (KU812, CML, positive control) expressed on the cell surface and AML cell lines with different IL-1RAP cell surface expression levels (low: HL-60; medium: MOLM-13; high: EOL-1) in culture medium only (no target), or with IL-1RAP negative (K562) cells, and cultured for 6 hours with an effector: target (E: T) ratio of 1: 5. As a positive control for cytokine production, cells stimulated with 10ng / mL phorbol myristate acetate (PMA) and 1μg / mL ionomycin (Sigma-Aldrich) were used as positive controls. IFNγ labeling was performed after Brefeldin A treatment and cell permeabilization. In addition to CD8 staining, fluorescent IFNγ signal was further detected after gating on CD3+ / CD19+ (CAR-positive cells) to distinguish CD8+ from CD4+ (formerly CD8-) cells.

[0198] result

[0199] In contrast to untransduced T cells (C0) or MOCK-transduced T cells (MockT), CD8+ or CD4+IL-1RAP CART cells were able to specifically express intracellular IFNγ in the presence of AML cell lines, regardless of the cell surface expression of IL-1RAP. Fig.11A and Fig. 11B ).

[0200] Example 8: Cytotoxicity of IL-1RAP CART cells against AML cell lines

[0201] Materials and methods

[0202] Before co-culture, effector cells are labeled with e-Fluor. Untransduced T cells (C0), Mock transduced T cells (MockT) or IL-1RAP CAR (IL-1RAP CART) are contacted with target cells (KU812, CML, positive control) expressed on the cell surface and AML cell lines of different IL-1RAP cell surface expression levels (low: HL-60; Medium: MOLM-13; High: EOL-1) and cultured for 24 hours with multiple effectors: target (E: T) ratios. Cell gating for FSC and 7-AAD labeling allows target cells to be distinguished from effector cells and from live cells. The percentage of target cells that persist in the presence or absence of effector cells is determined in gated FSC+ / 7-AAD-. Untransduced T cells (C0) or Mock transduced T cells (MockT) are used as controls.

[0203] result

[0204] In contrast to MOCK-transduced T cells (MockT), co-culture of effector CART cells with IL-1RAP-positive targets (AML) at various E:T ratios allowed killing of target AML cell lines expressing IL-1RAP with equal efficiency, regardless of the level of tumor antigen expression ( Fig. 12A and Fig. 12B ).

[0205] Example 9: IL-1RAP-CART cells protected by the iCASP9 safety switch have no significant harmful effects on healthy hematopoietic cells

[0206] To predict off-target toxicity, we used a tissue macroarray (TMA) of 30 normal human tissues to study IL-1RAP expression using the #A3C3 mAb. Staining at varying levels of intensity was detected in only six tissues (lymph nodes, prostate, skeletal muscle, stomach, colon, and small intestine) and in the pancreas (excluding inflammatory or necrotic factors, Fig.13A and Table 2). Interestingly, the microvascular HMEC-1 endothelial cell line was not recognized by our #A3C3 IL-1RAP mAb ( Fig. 13B ), while R&D IL-1RAP mAb (R&D Systems-Ref#89412) clearly detected cell surface expression, indicating that different epitopes were recognized.

[0207] Regarding the targeting of the healthy hematopoietic system, even though mAb#A3C3 did not detect Fig.14A , Fig. 14C ) of bone marrow (RFI < 1.2, n = 5) or normal umbilical cord blood ( Fig. 14B , Fig. 14C ), we noted weak staining (RFI < 2) of monocyte subsets in peripheral blood of 2 / 5 healthy donors and bone marrow of 3 / 5 healthy donors ( Fig.14A Next, we investigated the in vitro sensitivity of monocytes by co-culturing PBMCs and autologous CAR T cells at different E:T ratios. At an E:T ratio of 1:1, only some monocytes were targeted, resulting in 41.45% survival ( Fig.14D , right, Table 3); whereas lymphocytes, granulocytes, and the K562 IL-1RAP-negative cell line were not affected even at higher E:T ratios ( Fig.14D Interestingly, at this E:T ratio, 94.77% of leukemia cells were killed ( Fig.14E ).

[0208]

[0209]

[0210]

[0211] Table 2: Immunostaining of IL-1RAP (mAb#A3C3) in normal tissues

[0212]

[0213] Table 3: Percentage of viable cells in different subsets according to co-culture at different E (Mock T cells or IL-1RAP CART cells): T ratios.

[0214] These results were confirmed in vivo in a hCD34 transplanted mouse model (hu-NOG), where we demonstrated that, despite a decrease in monocytes at day 15 (41 ± 25%, n = 3, p = ns), other human immunocompetent cells derived from hCD34+ cells were not affected by CAR T cells ( Fig.15). The hematopoietic stem cell culture assay after in vitro co-culture of healthy CD34+ umbilical cord blood HSC with autologous CART cells (n=3) confirmed that HSC were not affected ( Fig.16 ). These results are consistent with the fact that IL-1RAP CART cell immunotherapy has almost no adverse effects on the hematopoietic system.

[0215] To limit potential toxicity, we evaluated the functionality of the safety switch of the iCASP9 / AP1903 suicide system cassette after exposure to a chemical inducer of dimerization (CID; 10 nM). First, using light microscopy, we noted that IL-1RAP CAR-transduced 293T cell cultures were sensitive to CID ( Fig.17 , top). Cytometry analysis showed that after 24 hours of CID exposure, only CD19-CD3+ cells persisted in the mixed population of CD19+ and CD19-IL-1RAP CART cells ( Fig.17 , bottom). More specifically, in the apoptosis quantification assay, 84.11% and 88.93% of IL-1RAP CART cells were eliminated after 24 or 48 h CID exposure, respectively, compared with untransduced T cells (C0) (1.28% and 6.13% at 24 or 48 h, respectively) (p < 0.001, n = 3; Fig.14F Finally, in vivo evaluation of the safety switch in the NSG mouse model showed that AP1903 ip administration eliminated 87±7.32% (p<0.01, n=3) of IL-1RAP CART cells, but was not affected after PBS administration; control non-transduced T cells (C0) were not affected by either treatment ( Figure 14G ).

[0216] Example 10: In vitro cytotoxicity of IL-1RAP CART cells against AML blasts from AML patients

[0217] Materials and methods

[0218] Untransduced T cells (UNT cells), MockT cells (i.e., cells transduced with the same vector as that used for CART cells, but the vector does not carry CAR) and IL-1RAP CART cells (generated by T cells from healthy donors or AML patients) were centrifuged and resuspended in 1 mL PBS 1×, and then 1 mL e-fluor-V450 solution (cell proliferation dye) (eBioscience) diluted 1 / 1000 was added. The cells were incubated in the dark at room temperature for 20 minutes. Then 8 mL of complete X-Vivo 15 medium (Lonza), 10% fetal bovine serum (GIBCO, USA), 1% penicillin-streptomycin PS (GIBCO, USA) were added, followed by incubation at +4°C for 5 minutes and centrifugation at 1500 rpm for 5 minutes. The cells were washed twice with the same medium and suspended in complete X-Vivo 15 medium, 10% human serum, 1% PBS (containing interleukin 2). These cells were incubated with 100 × 10 50 μL of AML cells from blood samples at different effector:target ratios (E:T ratios) in 96-well plates (round bottom). 3 Co-culture of primary cells (in allogeneic or autologous environments). Final volume / well = 200 μL. After 24 hours of co-culture, the cytotoxicity of UNT cells, MockT cells and IL-1RAP CART cells to AML blasts was evaluated by viability labeling with 3 μL 7-AAD-PerCP-Cy5.5 (BD Bioscience) and membrane staining with anti-CD34-allophycocyanin (APC) (BD Bioscience). If necessary, the following antibodies (Abs) were used to stain, detect and distinguish AML blasts from effector cells (MockT or IL-1RAP CART cells): 1 μL anti-CD3-phycoerythrin (PE) (Miltenyi Biotec, Germany) and 1 μL anti-CD19-allophycocyanin (APC) (Miltenyi Biotec, Germany). Effector cells have been identified from target cells by e-fluor labeling. The results were obtained by flow cytometry and analyzed with FACS Diva software. After subtracting the cytotoxicity of control untransduced T cells (UNT), the allogenicity killing of IL-1RAP CART cells was obtained.

[0219] For experiments in which IL-1RAP T cells were generated from AML patients, prior labeling of AML blasts was performed after isolation of T cells to examine the presence or absence of T cells and B cells.

[0220] result

[0221] The results are shown in Figure 19. Co-culture with IL-1RAP CART cells at various E:T ratios enabled killing of AML blasts from AML patients (low IL-1RAP expressers and medium IL-1RAP expressers, previously identified by flow cytometry (not shown)) with the same efficiency, regardless of the level of tumor antigen expression, compared to MOCKT cells or UNT cells, in which no cytotoxicity was detected.

[0222] Interestingly, IL-1RAP CART cells generated from AML patients' T cells were also able to kill AML blasts with the same efficiency, confirming that CART cells can be generated from AML patients and used in an autologous setting.

[0223] Example 11: In vivo cytotoxicity of IL-1RAP CART cells against AML blasts from AML patients

[0224] Materials and methods

[0225] HL-60, Molm-13 and Mono-Mac-6 AML (low, medium (int) and high IL-1RAP expressing cell lines, respectively) were transduced with luciferase lentiviral vector (pLenti CMV V5-Luc Blast vector, Addgene). Luciferase positive cells were selected by resistance to blasticidin (ThermoFisher Scientific).

[0226] Six- to eight-week-old NSG mice (Jackson Laboratories, Sacramento, CA, USA) were sublethally irradiated (25 Gy) on day 4. On day 3, each mouse was injected via the tail vein with 1 × 10 6 On day 0, after AML cell injection, mice were either left untreated (UT) or treated with untransduced T cells (UNT) (10 × 10 T cells in 300 μL PBS) via the tail vein. 6 cells) or IL-1RAPCAR-T cells (10×10 in 300 μL PBS 6 cells) and used on days 3, 5, 10, 14, 17, and 21 Leukemia progression was monitored using the lumina III system (PerkinElmer).

[0227] Animal Protocol The mice were followed until the animals in the untreated group reached a moribund state of health and developed signs of leukemia (i.e., weight loss>15%, decreased activity, and / or hind limb paralysis). The mouse experiments were approved by the local ethics committee (CELEAG and protocol 11007R, Veterinary Services for Animal Health & Protection, respectively, for the NSG-S model).

[0228] result

[0229] The results are shown in Figure 20. It has been shown that IL-1RAP CART cells significantly reduced the leukemic burden in an in vivo xenograft mouse model of AML compared to groups of mice with untreated or UNT-treated T cells, regardless of IL-1RAP cell surface expression by leukemic cells. Sequence Listing <110> Francis du Sang INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM) CENTER HOSPITALIER UNIVERSITAIRE DE BESANCON UNIVERSITE DE FRANCHE COMTE <120> CAR-T cells targeting IL-1RAP and their use in acute myeloid leukemia (AML) <130> 1H318460-0005 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 423 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding the H chain (VH) of the murine anti-IL-1RAP scFv <400> 1 atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt caactcccag 60 gtccaactgc agcagcctgg ggctgagctt atgatgcctg gggcttcagt gaaagtgtcc 120 tgcgaggctt ctggctacac attcactgac tcctggatgc actgggtgaa gcagaggcct 180 ggacaaggcc ttgagtggat cggagcgatt gatccttctg atagttatac tacctataat 240 caaaaattca cgggcaaggc cacattgagt gtagacgaat cctccaacac agcctacatg 300 cagctcagca gcctgacatc tgaggactct gcggtctatt actgtgcaag gtattactcc 360 ggtagtaact acatatcgcc ctttccttac tggggccaag ggactctggt cactgtctct 420 gca 423 <210> 2 <211> 141 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the H chain (VH) of murine anti-IL-1RAP scFv <400> 2 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val Asn Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Met Met 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Ser Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Ala Ile Asp Pro Ser Asp Ser Tyr Thr Thr Tyr Asn 65 70 75 80 Gln Lys Phe Thr Gly Lys Ala Thr Leu Ser Val Asp Glu Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Tyr Tyr Ser Gly Ser Asn Tyr Ile Ser Pro Phe 115 120 125 Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 130 135 140 <210> 3 <211> 382 <212> DNA <213> Artificial sequence <220> <223> Nucleotide sequence encoding the K chain (VL) of the murine anti-IL-1RAP scFv <400> 3 atggagtcac agattcaggt ctttgtattc gtgtttctct ggttgtctgg tgttgacgga 60 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcacc 120 atcacctgca aggccagtct ggatgtgagt actgctgtgg cctggtatca acagaaacca 180 ggacaatctc ctaaactact gatttactcg gcatcctacc ggtacactgg agtccctgat 240 cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgtgcaggct 300 gaagacctgg cagtttatta ctgtcagcaa cattatagtc ctccattcac gttcggctcg 360 gggacaaact tggagataaa ac 382 <210> 4 <211> 127 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of the K chain (VL) of the mouse anti-IL-1RAP scFv <400> 4 Met Glu Ser Gln Ile Gln Val Phe Val Phe Val Phe Leu Trp Leu Ser 1 5 10 15 Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser 20 25 30 Thr Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Leu Asp 35 40 45 Val Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro 50 55 60 Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp 65 70 75 80 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser 85 90 95 Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr 100 105 110 Ser Pro Pro Phe Thr Phe Gly Ser Gly Thr Asn Leu Glu Ile Lys 115 120 125 <210> 5 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Linker between VH and VL domains <400> 5 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Asp 1 5 10 15 <210> 6 <211> 6 <212> PRT <213> Artificial sequence <220> <223> CDR1 of light chain <400> 6 Leu Asp Val Ser Thr Ala 1 5 <210> 7 <211> 3 <212> PRT <213> Artificial sequence <220> <223> CDR2 of light chain <400> 7 Ser Ala Ser 1 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CDR3 of light chain <400> 8 Gln Gln His Tyr Ser Pro Pro Phe Thr 1 5 <210> 9 <211> 18 <212> DNA <213> Artificial sequence <220> <223> CDR1 of light chain (nt) <400> 9 ctggatgtga gtactgct 18 <210> 10 <211> 9 <212> DNA <213> Artificial sequence <220> <223> CDR2 of light chain (nt) <400> 10 tcggcatcc 9 <210> 11 <211> 27 <212> DNA <213> Artificial sequence <220> <223> CDR3 of light chain (nt) <400> 11 cagcaacatt atagtcctcc attcacg 27 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR1 of the heavy chain <400> 12 Gly Tyr Thr Phe Thr Asp Ser Trp 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial sequence <220> <223> CDR2 of heavy chain <400> 13 Ile Asp Pro Ser Asp Ser Tyr Thr 1 5 <210> 14 <211> 15 <212> PRT <213> Artificial sequence <220> <223> CDR3 of heavy chain <400> 14 Ala Arg Tyr Tyr Ser Gly Ser Asn Tyr Ile Ser Pro Phe Pro Tyr 1 5 10 15 <210> 15 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> CDR1 of heavy chain (nt) <400> 15 ggctacacat tcactgactc ctgg 24 <210> 16 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> CDR2 of heavy chain (nt) <400> 16 attgatcctt ctgatagtta tact 24 <210> 17 <211> 45 <212> DNA <213> Artificial sequence <220> <223> CDR3 of heavy chain (nt) <400> 17 gcaaggtatt actccggtag taactacata tcgccctttc cttac 45 <210> 18 <211> 283 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of mouse anti-IL-1RAP scFv (i.e. #A3C3 CAR) <400> 18 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val Asn Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Met Met 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Asp Ser Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Ile Gly Ala Ile Asp Pro Ser Asp Ser Tyr Thr Thr Tyr Asn 65 70 75 80 Gln Lys Phe Thr Gly Lys Ala Thr Leu Ser Val Asp Glu Ser Ser Asn 85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Tyr Tyr Ser Gly Ser Asn Tyr Ile Ser Pro Phe 115 120 125 Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Asp Met Glu Ser Gln 145 150 155 160 Ile Gln Val Phe Val Phe Val Phe Leu Trp Leu Ser Gly Val Asp Gly 165 170 175 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 180 185 190 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Leu Asp Val Ser Thr Ala 195 200 205 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 210 215 220 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 225 230 235 240 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala 245 250 255 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Pro Pro Phe 260 265 270 Thr Phe Gly Ser Gly Thr Asn Leu Glu Ile Lys 275 280

Claims

1. Use of T cells in the preparation of a medicament for treating acute myeloid leukemia (AML), wherein the T cells express a nucleic acid molecule encoding a chimeric antigen receptor (CAR) on their membrane, in, The CAR comprises: an antibody or antigen-binding fragment comprising an anti-IL-1RAP binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain comprises at least a stimulatory domain, and wherein the anti-IL-1RAP binding domain comprises: (i) a light chain, comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is the amino acid sequence of SEQ ID NO: 6, the complementarity determining region 2 (CDR2) is the amino acid sequence of SEQ ID NO: 7, and the complementarity determining region 3 (CDR3) is the amino acid sequence of SEQ ID NO: 8; and (ii) a heavy chain, the heavy chain comprising a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2) and a complementarity determining region 3 (CDR3), wherein the complementarity determining region 1 (CDR1) is the amino acid sequence of SEQ ID NO: 12, the complementarity determining region 2 (CDR2) is the amino acid sequence of SEQ ID NO: 13, and the complementarity determining region 3 (CDR3) is the amino acid sequence of SEQ ID NO: 14, Wherein, the anti-IL-1RAP binding domain is scFV, the transmembrane domain is the transmembrane domain of CD28, the intracellular signal transduction domain is from CD28 and / or from 4-1BB and / or from CD3ζ, and the anti-IL-1RAP binding domain is connected to the transmembrane domain via a hinge region.

2. The use according to claim 1, in, The T cells are CD8+T cells.

3. The use according to claim 1, in, The hinge region comprises the hinge sequence of IgG1.

4. The use according to claim 1, in, The AML is (i) refractory / relapsed AML; or (ii) AML with complex cytogenetic abnormalities and / or AML with TP53 mutation.

5. The use according to claim 1, in, The AML is AML expressing IL-1RAP.

6. The use according to claim 5, in, The AML expressing IL-1RAP is refractory / relapsed AML expressing IL-1RAP.

7. The use according to claim 1, in, The medicament comprises at least one monoclonal antibody, and wherein the cell is used in combination with the at least one monoclonal antibody.

8. The use according to claim 7, in, The at least one monoclonal antibody comprises an anti-checkpoint inhibitor.

9. The use according to claim 1, in, The treatment is autologous treatment.

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