IL-1RAP-Targeted CAR-T Cells and Their Use in Acute Myeloid Leukemia (AML)

Abstract

Provided are CAR-T cells targeting IL-1RAP and uses thereof in acute myeloid leukemia (AML). [Solution] The present invention relates to a cell containing a nucleic acid molecule encoding a chimeric antigen receptor (CAR) for use in the treatment of acute myeloid leukemia (AML), wherein the CAR has an antibody or antibody fragment having an anti-IL-1RAP binding domain, a transmembrane domain, and an intracellular signaling domain having at least a stimulatory domain.

Description

[Technical field]

[0001] The present invention relates to a cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), said CAR comprising an antibody or antibody fragment having an anti-IL-1RAP binding domain, a transmembrane domain and an intracellular signaling domain comprising at least a stimulatory domain, for use in the treatment of acute myeloid leukemia (AML). [Background technology]

[0002] Acute myeloid leukemia (AML, also known as “acute myeloid leukemia”, “acute myelocytic leukemia”, “acute myeloblastic leukemia”, “acute granulocytic leukemia”, and “acute nonlymphocytic leukemia”) is a devastating clonal hematopoietic stem cell neoplasm characterized by the unregulated proliferation and accumulation of leukemic blast cells in the bone marrow, peripheral blood, and sometimes other tissues. These cells inhibit normal hematopoiesis and rapidly cause bone marrow failure and death. AML is the most common type of acute leukemia in adults and is the leading cause of leukemia deaths annually. AML progresses rapidly and is typically fatal within weeks to months if left untreated. In the United States, there were approximately 20,830 new cases of AML in 2015, and 10,460 patients died from the disease. The median age at diagnosis is approximately 70 years. The overall five-year survival rate for patients under 60 years is approximately 30-40%, while for those over 60 years it remains less than 10%.

[0003] Currently, the standard treatment for AML is induction with high-dose chemotherapy or high-dose radiation therapy, followed by consolidation consisting of allogeneic stem cell transplantation and, if necessary, additional courses of chemotherapy. Most patients treated in this manner can achieve a transient, but complete, remission. Upon relapse, the disease becomes increasingly resistant to further treatment.

[0004] Over the past 30 years, the outcomes of younger patients have improved somewhat, but the outcomes of older patients have remained essentially unchanged. Unfortunately, the majority of patients with AML, including those who achieve an initial complete remission, will experience disease relapse. Allogeneic hematopoietic stem cell transplantation during second remission offers the only chance of long-term survival, but this option is unavailable to most patients with relapsed AML, either because they are unable to achieve a second remission or because they are unable to tolerate transplantation.

[0005] For relapsed AML, there are several combination chemotherapy salvage regimens, including MEC (mitoxantrone, etoposide, cytarabine) and FLAG-IDA (fludarabine, cytarabine, idarubicin, granulocyte colony-stimulating factor). However, none of these regimens are superior to the others and do not provide long-term survival rates, so they are not accepted standard of care. Clinical trials are recommended in guidelines such as the NCCN (National Comprehensive Cancer Network), and relapsed AML is widely recognized as an urgent unmet medical need.

[0006] Currently, many novel approaches, such as antibody-drug bispecific T cell-receptor antibody (AMG330) and CAR-T cells (CART-33 cells or CART-123 cells), are being investigated to treat AML, especially relapsed AML. However, some new approaches are on hold due to clinical toxicity and / or lack of efficacy. Therefore, efficient new therapies with acceptable toxicity profiles are needed.

[0007] In AML, gene expression profiling studies, cell surface staining studies, and Western blotting studies showed normal CD34 + / CD38 - Cell surface biomarkers (IL-1RAP, IL-1R3, C3orf13, or IL-1RAcP) that are not expressed in hematopoietic stem cells but are expressed in leukemic ones have been identified, and IL-1RAP expression correlates with tumor burden and clinical phase of AML disease. Summary of the Invention [Means for solving the problem]

[0008] IL-1RAP (Interleukin-1 Receptor Accessory Protein, Genbank Accession No. AAB4059) is a co-receptor for IL-1 and IL33 receptors involved in IL-1 signal transduction and activates various signal transduction pathways, including genes involved in inflammation and proliferation, such as MAP kinase, p38, and NF-κB. This protein is expressed on the surface of tumor cells. Therefore, IL-1RAP is a promising tumor-associated antigen.

[0009] The applicant has discovered that AML patients can be treated using these CART-IL-1RAP cells.

[0010] Accordingly, the present invention provides a cell expressing at its membrane a nucleic acid molecule encoding a chimeric antigen receptor (CAR) for use in the treatment of acute myeloid leukemia (AML), the CAR comprising an antibody or antibody fragment having an anti-IL-1RAP binding domain, a transmembrane domain and an intracellular signalling domain having at least a stimulatory domain, the anti-IL-1RAP binding domain comprising: (i) a light chain having a complementarity determining region 1 (CDR1) that has at least 80% identity to the amino acid sequence of SEQ ID NO:6, a complementarity determining region 2 (CDR2) that has at least 80% identity to the amino acid sequence of SEQ ID NO:7, and a complementarity determining region 3 (CDR3) that has at least 80% identity to the amino acid sequence of SEQ ID NO:8; (ii) a heavy chain having a complementarity determining region 1 (CDR1) that has at least 80% identity to the amino acid sequence of SEQ ID NO: 12, a complementarity determining region 2 (CDR2) that has at least 80% identity to the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) that has at least 80% identity to the amino acid sequence of SEQ ID NO: 14; The present invention relates to a cell having

[0011] Herein, a T cell expressing a CAR is referred to as a "CAR T cell" or a "CAR modified T cell." Herein, a T cell expressing a CAR that targets IL1-RAP is referred to as a "CART-IL1-RAP cell" or a "CART-IL1-RAP modified T cell."

[0012] The cells used in the present invention are genetically modified to express a CAR as described herein for use in treating AML. As used herein, the terms "genetically engineered" or "genetically modified" refer to the addition of additional genetic material, either DNA or RNA, to the total genetic material in a cell. [Brief description of the drawings]

[0013] [Figure 1] Western blotting of various hematopoietic AML cell lines and monocyte sorted CD14+ cells. [Diagram 2] Recognition of IL-1RAP recombinant protein by ELISA using #E3C3mAb (b). BSA is used as a negative control (a). [Diagram 3] Flow cytometry gating strategy for patient AML primary samples. RFI is shown for blast and monocyte subpopulations (left). Percentage of IL-1RAP+ in AML blasts and monocytes, percentage of CD123+ cells and IL-1RAP+ / CD123+ cells in AML blasts (top right) in a group of routine AML (all subtypes) patients (n=30). RFI is also shown (bottom right). [Figure 4] Specific tissue binding using frozen tissue arrays. (a) IL-1RAP high expressing (KU812) or (b) negative expressing (Raji) cell lines 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, h: prostate. [Diagram 5]Figure 1 shows the design of a SIN lentiviral construct carrying the safety cassette iCASP9, the single chain variable fragment (scFv) of #E3C3mAb, and the cell surface expression marker ΔCD19. The three transgenes are separated by a 2A peptide cleavage sequence and are under the control of the EF1 promoter + SP163 enhancer sequence. [Figure 6] Western blotting of CD3 on intracellular fractions of IL-1RAP-transduced T cells: a: total lysate, b: membrane, c: cytoplasm, d: nucleus. (1) CAR bound to CD3ζ (55 kDa), (2) endogenous CD3ζ (16 kDa), (3) CD45 (147 kDa), (4) lamin (68 kDa), (5) GAPDH (35 kDa). [Figure 7] Figure 1 depicts FACS analysis detection of either IL-1RAP CAR-transduced CEM T cell lines or primary T cells. The percentage of biotin+ / CD19+ CEM or T cells (a) is plotted against the amount of labeled biotin recombinant protein (b). [Figure 8] Safety switch of the iCASP9 / AP1903 suicide cassette following exposure to chemically induced dimerizers (10 nM CID) in (a) 293T cells and (b) IL-IRAP CAR 293T cells. [Figure 9] Depletion of IL-1RAP CART cells after 24 or 48 h CID exposure compared to non-transduced T cells (C0) is shown (*** p<0.001, n=3). [Figure 10A] Representative experimental example showing the gating strategy for flow cytometry CFSE dye dilution analysis. [Figure 10B] The total percentage of dividing CFSE positive cells is represented. Mean ± SD of three independent experiments. *** p<0.001 (Student's test). [Figure 11A] Representative experimental example showing the gating strategy for intracellular IFNγ cytokine detection. [Figure 11B]Percentage of total intracellular IFNγ-producing cells. Mean ± SD of three independent experiments for CD8+ and CD8- (mainly CD4+) cells. *** p<0.001. ** p<0.01 (Student's test). [Figure 12A] 1 depicts the gating strategy for the efficacy study of IL-1RAP CAR T cells to lyse cell surface IL-1RAP expressing cells. [Figure 12B] The percentage of total viable target cells is expressed as the mean ± SD of three independent experiments. [Figure 13A] Tissue microarrays are shown. Representative #A3C3 staining of a US Food and Drug Administration standard frozen tissue array containing 90 tissue cores (30 organs) from three individual donors per organ (US Biomax, Rockville, MD, USA). Immunostaining was detected using UltraView Universal DAB detection kit (Ventana, USA). Images were acquired and analyzed with NDP.view 2.0 software. Tissues that showed some staining with #A3C3mAb in at least one of the three individuals analyzed are shown (scale bar: 100 μm). IL-1RAP high-expressing (KU812) or negative-expressing (Raji) cell lines were used as positive or negative controls, respectively. [Figure 13B] Figure 1 shows IL-1RAP R&D (red) or #A3C3 (blue) staining of HMEC-1 dermal endothelial cell line. Isotype IgG1 (grey) is overlaid. RFI is shown for both stainings. [Figure 14A] Figure 14 shows the effect of IL-1RAP CAR T cells on healthy hematopoietic cells and the efficiency of the suicide gene iCASP9 safety cassette. Figure 14A shows IL-1RAP cell surface expression on peripheral blood cells (left) or bone marrow cells (right) of healthy donors (n=5). SSC-A / CD45+ allowed the identification of subpopulations as lymphocytes (low SSC-A), monocytes (CD33+), granulocytes (high SSC-A), or HSCs (CD33- / CD34+). RFI was calculated from isotype staining and shown in each window. [Figure 14B-1]Representative (1 of 3) IL-1RAP staining of total human cord blood cells is shown. IL-1RAP staining is shown for total CD34+, CD34+ / CD38-, and CD34+ / CD38+ HSC cord blood subpopulations. [Figure 14B-2] Representative (1 of 3) IL-1RAP staining of total human cord blood cells is shown. IL-1RAP staining is shown for total CD34+, CD34+ / CD38-, and CD34+ / CD38+ HSC cord blood subpopulations. [Figure 14C] IL-1RAP positive cells among CD34+ cells in umbilical cord blood (CB, n=5) or bone marrow (BM) of healthy donors (n=5) are shown compared to CD34+ cells obtained from BM (n=10) or peripheral blood (PB, n=10) of CML patients. [Figure 14D] Left: Dot plots of SSC-A / CD45+ granulocyte (G), monocyte (M), and lymphocyte (L) subpopulations cultured in the presence of autologous non-transduced T cells or Mock or IL-1RAP CAR T cells at various effector:target (E:T) ratios. Right: Relative percentages of viable cells among lymphocytes (squares), monocytes (circles), and granulocytes (triangles) normalized to non-transduced autologous T cells (C0) co-cultured for 24 h with autologous Mock T cells (dashed lines) or IL-1RAP CAR T cells (solid lines). [Figure 14E] The relative percentage of viable cells in monocyte (squares), KU812 (circles), or K562 (triangles) subpopulations in the presence of Mock (black, dashed line) or IL-1RAP CAR T cells (white, solid line) at various E:T ratios are shown. Percentages were calculated by absolute cell counts measured using Trucount tubes based on 5000 fluorescent bead cytometry acquisition. [Figure 14F]Left: Gating strategy and analysis for absolute number of CID AP1903-induced cell death. Non-transduced (C0) or IL-1RAP CAR T cells were exposed to medium only or medium + CID (20 nM, 24 h). Quantification was performed after acquisition of 5000 fluorescent beads. 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)] x 100. (D) Absolute percentage of mortality. CID exposure of C0 or IL-1RAP CAR (gated on CD3+ / CD19+) T cells for 24 or 48 h. Right: Results are the average of three independent experiments. **p<0.001. [Figure 14G] Absolute quantification of IL-1RAP CAR T cells injected into tumor (CML KU812, iv) xenograft NSG model 24 hours after intraperitoneal AP1903 (white bars) treatment (n=3 mice / group). Mice injected with control T cells (C0) were used as controls (n=2 mice / group). **p<0.01. Cell counts are shown per ml of peripheral blood. [Figure 15]This represents an immune safety experiment human CD34+ engrafted NOG mouse model to investigate the specific toxicity of autologous IL-1RAP CART cells to HSCs and / or immune cells in the human-CD34+ cord blood cell engrafted / NOG mouse model (hu-NOG). Briefly, 10x10E6 autologous CART cells or control T cells (C0) (obtained from mouse PBMCs, spleen, or bone marrow cell-sorted human CD45+) were infused. At each time point after infusion (days 5, 8, and 15), monitoring of mature immune cells (hCD3+, hCD19+, hCD56+, hCD14+, hCD11b+) was assessed by cytometry. Fold changes were calculated from baseline immunophenotypes obtained on day -7 before CART cell infusion and compared to the time of peripheral blood collection (day -9). Fold changes in various immunocompetent cell subpopulations in non-transduced (C0, open bars) or IL-1RAP CART cells (closed bars) were compared to the time of peripheral blood collection (day -9) for days 3, 8, and 15. Cell numbers were counted in peripheral blood collected from retro-orbital samples, and fold changes were calculated relative to day -7. ns: not significant. [Figure 16] Colony forming unit (CFU-GM) assay of CD34+ HSCs taken from three different cord blood cells and cultured alone (white bars) or co-cultured with respective autologous non-transduced (C0, grey bars) or IL-1RAP CART cells (black bars) is shown. [Figure 17] Top panel: Light microscopic evaluation of CID effect on transduced 293T cells. Bottom panel: Flow cytometric analysis of IL-1RAP CART cells after CID AP1930 exposure. Flow cytometric analysis of GMTC mixtures expressing or not IL-1RAP CAR and non-transduced T cells (C0) after CID exposure (20nM, 24h, dark grey) or without exposure (light grey). GMTC expressing CAR could be identified by CD3+ / CD19+ staining. [Figure 18] FIG. 1 shows the expression of IL-1RAP mRNA and the absolute location of the cell surface IL-1RAP antigen in primary samples of AML patients according to the European LeukemiaNet (ELN) prognostic classification. [Figure 19A]Figure 19 shows the cytotoxicity of IL-1RAP CART cells against AML blasts. Gating strategy is shown. Targeted AML blasts were identified using CD34 antibody (Figure 19A). The number of events of AML blasts showing cytotoxicity of IL-1RAP CART cells is also shown (Figure 19A). [Figure 19B] The results of three independent experiments are shown in Figure 19B. Legends for Figures 19A and 19B: FSC: Forward Scatter, indicating cell size. 7-AAD: 7-aminoactinimycin D, a marker for cell viability. E-Fluor: a dye that stains cell membranes. #1 and #2 experiments were performed with allogeneic IL-1RAP CART cells made from healthy donors. #3 experiment was performed with allogeneic IL-1RAP CART cells made from T cells of an AML patient. Viable cells refer to viable AML blasts. [Figure 20A] Figure 20 depicts the 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 at a later date (day 3) with 1x106 luciferase expressing HL-60 or Molm-13 or Mono-Mac-6 AML cells. Figure 20A: On day 0 after AML cell injection, mice were left untreated or treated with control T cells, i.e. MockT or UNT cells (10x106 cells in 300 μL PBS) or IL-1RAP CAR-T cells (10x106 cells in 300 μL PBS) and monitored for leukemia development using bioluminescence imaging (BLI) at the indicated days. Bioluminescence imaging analysis of mice was performed for different groups from day 0 to day 21. Legend: (x): dead mice. ▲: injection of UNT or IL-1RAP CAR T cells. [Figure 20B] Brightness of in vivo bioluminescence signal collected using bioluminescence imaging (BLI) (brightness p / s / cm2 / sr). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention provides a cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) for use in the treatment of acute myeloid leukemia (AML), said CAR comprising an antibody or antibody fragment having an anti-IL-1RAP binding domain, a transmembrane domain, and an intracellular signaling domain having at least a stimulatory domain, said anti-IL-1RAP binding domain comprising: (i) a light chain having a complementarity determining region 1 (CDR1) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:6, a complementarity determining region 2 (CDR2) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:7, and a complementarity determining region 3 (CDR3) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:8; (ii) a heavy chain having a complementarity determining region 1 (CDR1) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 12, a complementarity determining region 2 (CDR2) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 14. The present invention relates to a cell having the

[0015] The sequence identifiers are summarized in the table below.

[0016] [Table 1-1]

[0017] [Table 1-2]

[0018] [Table 1-3]

[0019] The terms "#E3C3" and "#A3C3" are understood to be identical. "#E3C3" can be freely used to mean "#A3C3" and vice versa.

[0020] The sequences of the hinge regions of the IgG1, IgG4, CD8α, 4-1BB, CD3ζ, CD28, and ICasp9 genes are available in Genbank.

[0021] The practice of the present invention will employ, unless otherwise indicated, 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 by way of illustration, and which are fully explained in the literature.For example, refer to the papers in academic journals such as Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 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, N.Y., 1998); Current Protocols in Immunology, Q.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and Advances in Immunology, etc.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those 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.

[0023] As will be understood by those skilled in the art and as described elsewhere herein, a complete antibody has two heavy chains and two light chains. Each heavy chain is composed of a variable region and a first, second, and third constant region, and each light chain is composed of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. Immunoglobulins with α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y is composed of the second and third constant regions of the two heavy chains (and in the case of IgE and IgM, a fourth constant region) bound together, forming (interchain) disulfide bonds at the hinge. The γ, α, and δ heavy chains have a constant region composed of three tandem (tandem) Ig regions and a hinge region that confers flexibility. The μ and ε heavy chains have a constant region made up of four immunoglobulin domains. The second and third constant domains are also called "CH2 domain" and "CH3 domain", respectively. Each arm of the Y has a variable domain, the first constant domain of a single heavy chain bound to the variable domain, and the constant domain of a single light chain. The variable domains of the light and heavy chains are responsible for antigen binding.

[0024] The light and heavy chain variable regions have 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, such as the sequences of 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, USDepartment of Health and Human Services, 1991) or the structures of 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)).

[0025] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans. The framework regions of an antibody are the combined framework regions of the constituent light and heavy chains, and function to position and arrange the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically numbered sequentially from the N-terminus and are referred to as CDR1, CDR2, and CDR3, and are typically identified by the chain in which the individual CDRs are located. Thus, the CDRs located in the variable region of the heavy chain of an antibody are referred to as CDRH1, CDRH2, and CDRH3, and the CDRs located in the variable region of the light chain of an antibody are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., having different binding sites for different antigens) have different CDRs.

[0026] "VH" or "V H " refers to the variable region of an immunoglobulin heavy chain, including that of an antibody, Fv, scFv, Fab, or other antibody fragment disclosed herein.

[0027] "VL" or "V L " refers to the variable region of an immunoglobulin light chain, including that of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein.

[0028] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies can be obtained by methods known to those skilled in the art, for example, fusing myeloma cells with immune spleen cells to produce hybrid antibody-forming cells. Monoclonal antibodies include chimeric and humanized monoclonal antibodies.

[0029] As used herein, the articles "a", "an" and "the" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0030] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the terms "about" or "approximately" when used before a numerical value indicates a range of ±15%, 10%, 5%, or 1% of that value.

[0031] Throughout this specification, unless the context otherwise requires, the words "comprise", "comprises" and "comprising" are to be interpreted as meaning the inclusion of a stated step or element or group of steps or elements and not the exclusion of any other step or element or group of steps or elements.

[0032] Throughout this specification, the term "one embodiment," "an embodiment," "a particular embodiment," "an embodiment," "an additional embodiment," "a further embodiment," or any combination 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.

[0033] For the purposes of the present invention, "identity" or "homology" is calculated by comparing two aligned sequences in a comparison window. By aligning the sequences, the number of positions (nucleotides or amino acids) common to the two sequences in the comparison window can be determined. The number of common positions is then divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage of homology. The determination of the percentage of sequence identity can be performed manually or using well-known computer programs.

[0034] The present invention provides immune effector cells engineered with vectors designed to express an engineered receptor that redirects cytotoxicity to tumor cells for use in the treatment of acute myeloid leukemia (AML), the engineered receptor being referred to herein as a chimeric antigen receptor (CAR).

[0035] CAR is a molecule that combines antibody-based specificity for a target antigen (such as a tumor antigen) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-tumor cell-mediated immune activity. In this specification, the term "chimera" refers to a molecule that is composed of various proteins or DNA fragments of different origins.

[0036] The main property of CARs is that they can redirect immune effector cell specificity, leading to proliferation, cytokine production, phagocytosis, or production of molecules that can mediate cell death of target antigen-expressing cells independent of major histocompatibility (MHC), exploiting the cell-specific targeting potential of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0037] As used herein, the terms "binding region," "extracellular binding region," "antigen-specific binding region," and "extracellular antigen-specific binding region" are used interchangeably and confer to the CAR the ability to specifically bind to a target antigen of interest. A binding region may comprise any protein, polypeptide, oligopeptide, or peptide capable of specifically recognizing and binding to a biological molecule (such as a cell surface receptor, a tumor protein, a cell surface target molecule such as a lipid, a polysaccharide, or a component thereof). A binding region includes any natural, synthetic, semi-synthetic, or recombinantly produced binding partner to a biological molecule of interest. As used herein, the terms "specific binding affinity," "specifically binds," "specifically bound," "specific binding," or "specifically targets" refer to binding between molecules with a higher binding affinity than background binding. A binding region (or a CAR having a binding region or a fusion protein comprising a binding region) has an affinity or Ka (i.e., the equilibrium association constant of a particular binding interaction in units of 1 / M) of, for example, about 10 5 M -1 When the above binds to or associates with a target molecule, it "specifically binds" to the target molecule. The affinity of the binding domain polypeptides and CAR proteins of the present disclosure can be readily measured by conventional methods such as competitive ELISA (enzyme-linked immunosorbent assay).

[0038] The antibody is a human antibody, a murine antibody, a chimeric antibody, or a humanized antibody.

[0039] In a preferred embodiment, the antibody is a chimeric antibody (such as a chimeric monoclonal antibody) that specifically binds to a surface protein on a tumor cell. A "chimeric" antibody is an immunoglobulin that has a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. Thus, all parts of a chimeric immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. Chimeric or other monoclonal antibodies can additionally have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Chimeric antibodies can be constructed by genetic engineering (see, e.g., U.S. Pat. No. 5,585,089).

[0040] 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 a full-length antibody that is 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.

[0041] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, which are present in a single polypeptide chain in either orientation (such as VL-VH or VH-VL).

[0042] Single chain antibodies may be cloned from the V region genes of hybridomas specific for the desired target. The production of such hybridomas is routine. The methods used to clone the variable heavy (VH) and variable light (VL) chains are described, for example, in Orlandi et al, PNAS, 1989;86:3833-3837.

[0043] 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.

[0044] CARs contemplated herein may have one, two, three, four, or five or more linkers. In certain embodiments, the linker is about 1 to about 25 amino acids in length, about 5 to about 20 amino acids in length, about 10 to about 20 amino acids in length, or any intervening amino acid length. In some embodiments, the linker 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 in length.

[0045] Specific examples of linkers include glycine polymers (G), glycine-serine polymers (Gi_sSi_5) (where n is an integer of 1, 2, 3, 4, or 5 or more), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively amorphous and can function as neutral tethers between regions of fusion proteins such as the CARs described herein. Glycine has access to a great deal of phi-psi space compared to alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 1 1173-142 (1992)). As will be appreciated by those of skill in the art, the design of CARs in certain embodiments may include flexible linkers or linkers that are fully or partially flexible, such that the linker includes one or more moieties that provide a less flexible structure than the desired CAR structure.

[0046] In a specific embodiment, the linker is between the VH and VL domains.

[0047] In a particular embodiment, the linker has or consists of the amino acid sequence of SEQ ID NO:5.

[0048] In one embodiment, the IL-1RAP binding region is an scFv having a light chain variable region having an amino acid sequence with at least 1, 2, or 3 and at most 30, 20, or 10 modifications in the amino acid sequence of the light chain variable region of SEQ ID NO:4, and a heavy chain variable region having an amino acid sequence with at least 1, 2, or 3 and at most 30, 20, or 10 modifications in the amino acid sequence of the heavy chain variable region of SEQ ID NO:2.

[0049] The IL-1RAP binding region comprises (i) a light chain variable region having a complementarity determining region 1 (CDR1) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:6, a complementarity determining region 2 (CDR2) that 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 a complementarity determining region 3 (CDR3) that 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 (ii) ) a heavy chain variable region having a complementarity determining region 1 (CDR1) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12, a complementarity determining region 2 (CDR2) that 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 a complementarity determining region 3 (CDR3) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0050] Generally, the binding region of a CAR is followed by one or more "hinge regions" that serve to position the antigen binding region away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation. CARs generally have one or more hinge regions between the binding region and the transmembrane region. The hinge region may be from either natural, synthetic, semi-synthetic, or recombinant sources.

[0051] The anti-IL-1RAP binding region is preferably connected to the transmembrane region by a hinge region.

[0052] In one embodiment, the hinge region has an IgG1 hinge sequence or a sequence having 95-99% identity thereto. The IgG hinge is encoded by a single exon. Thus, the term "IgG1 hinge sequence" as used herein has the same meaning as commonly understood by those skilled in the art to which the present invention pertains, i.e., the 15 amino acid residues encoded by the IgG1 exon for the hinge (Fundamental Immunology, 5th Edition, Chapter 3, Immunoglobulins: Structure and Function-The Immunoglobulin Hinge).

[0053] In a further embodiment, the hinge region has an IgG4 hinge sequence or a sequence having 95 to 99% identity thereto. In a further embodiment, the hinge region may have an IgG1 or IgG4 CH2-CH3 region or a sequence having 95 to 99% identity thereto.

[0054] In a further embodiment, the hinge region has CD8α or a sequence having 95 to 99% identity thereto.

[0055] The "transmembrane region" is the part of the CAR that fuses the extracellular binding portion with the intracellular signaling region and anchors the CAR to the cell membrane of the immune effector cell. The transmembrane region may be from either natural, synthetic, semi-synthetic, or recombinant sources.

[0056] The encoded CAR preferably comprises a protein selected from the group consisting of the alpha, beta, or zeta 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 the transmembrane region of CD28.

[0057] In certain embodiments, the CARs discussed herein have an intracellular signaling region. By "intracellular signaling region" is meant the portion of the CAR that is involved in transmitting the message of an active CAR bound to a target antigen to the interior of an immune effector cell to trigger effector cell functions such as activation, cytokine production, proliferation, and cytotoxic activity (including cellular responses triggered upon antigen binding to the extracellular CAR region, such as the release of cytotoxic factors to the CAR-bound target cell).

[0058] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be a cytolytic activity, or an aid or activity including the secretion of a cytokine. Thus, the term "intracellular signaling region" refers to a portion of a protein that transmits an effector function signal and instructs a cell to perform a specialized function. Usually, the entire intracellular signaling region can be used, but in many cases, it is not necessary to use the entire region. When a truncated portion of an intracellular signaling region is used, such a truncated portion may be used instead of the entire region as long as it transmits an effector function signal. The term "intracellular signaling region" refers to having a truncated portion of the intracellular signaling region sufficient to transmit an effector function signal.

[0059] It is known that signals generated only through TCR are insufficient to fully activate T cells, and secondary or costimulatory signals are also required. Thus, it can be said that T cell activation is mediated by two different classes of intracellular signaling regions, namely, primary signaling regions that initiate antigen-dependent primary activation through TCR (such as TCR / CD3 complex) and costimulatory signaling regions that act antigen-independently to provide secondary or costimulatory signals. In a preferred embodiment, the CAR discussed herein has an intracellular signaling region that has one or more "costimulatory signaling regions".

[0060] In an embodiment, the isolated nucleic acid molecule may encode an intracellular signaling region having at least one costimulatory region. Thus, in this embodiment, the intracellular signaling region has at least one costimulatory region.

[0061] As used herein, the term "costimulatory signaling region" or "costimulatory region" refers to the intracellular signaling region of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or an Fc receptor, that upon antigen binding provides a secondary signal necessary for efficient activation and function of T lymphocytes.

[0062] At least one costimulatory region of the functional intracellular signaling region is preferably obtained from one or more proteins selected from the group consisting of OX40, CD2, CD27, CD28, CDS, CD3ζ, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0063] It is more preferable that the costimulatory region obtained from 4-1BB (CD137) has a sequence that has 95 to 99% identity to the amino acid sequence of the costimulatory region of 4-1BB.

[0064] It is more preferable that the costimulatory region derived from CD3ζ has a sequence that has 95 to 99% identity to the amino acid sequence of the costimulatory region of CD3ζ.

[0065] In another embodiment, the intracellular signaling region comprises a costimulatory region derived from 4-1BB and / or a costimulatory region derived from CD3ζ.

[0066] In certain preferred embodiments, a CAR comprises a CD3ζ primary signaling region and one or more costimulatory signaling regions, wherein the intracellular primary signaling region and the costimulatory signaling region may be tandemly linked in any order to the carboxyl terminus of the transmembrane region.

[0067] Cells that can be used according to the present invention may be isolated. "Isolated cells" refers to cells obtained from an in vivo tissue or organ and that are substantially free of extracellular matrix.

[0068] The cells used to treat AML according to the present invention may be generated by inserting a nucleic acid molecule encoding a chimeric antigen receptor (CAR) into the genome of a host cell using a vector.

[0069] As used herein, the term "vector" refers to a nucleic acid molecule capable of introducing or transporting another nucleic acid molecule. The introduced nucleic acid is generally linked to (e.g., inserted into) the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in a cell or may contain sequences sufficient to permit integration into the host cell DNA.

[0070] The cells used to treat AML according to the present invention can be produced using a vector carrying a nucleic acid molecule encoding a CAR of the present invention, the vector being selected from a DNA, an RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector, preferably a lentiviral vector.

[0071] In some embodiments, the vector comprises a promoter, preferably the EF-1α promoter.

[0072] Retroviruses are common tools for gene transfer. In certain embodiments, retroviruses are used to transfer polynucleotides encoding chimeric antigen receptors (CARs) into cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once integrated into the host genome, the virus is called a "provirus." The provirus acts as a template for RNA polymerase II to direct the expression of RNA molecules that code for structural proteins and enzymes required to produce new viral molecules.

[0073] Thus, T cells transduced with the above vectors are capable of eliciting stable, long-lasting CAR-mediated T cell responses.

[0074] In certain embodiments, the T cells are transduced with a retroviral vector (such as a lentiviral vector) encoding the CAR.

[0075] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; such as 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).

[0076] The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof, such as LTRs, primarily derived from a lentivirus.

[0077] "Self-inactivating (SIN)" vector means a replication-deficient vector (e.g., a retroviral or lentiviral vector) in which the right (3') LTR enhancer / promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.

[0078] In one embodiment, the SIN vector backbone is preferred.

[0079] The vector used preferably further has a promoter such as the EF-1α promoter.

[0080] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which RA polymerase binds. RA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, it may be desirable to express a polynucleotide bearing a CAR from a promoter that allows stable long-term expression of the CAR in T cells at levels sufficient to redirect the T cells to cells expressing a target antigen.

[0081] The cell for use according to the present invention is a T cell, such as a human T cell, more preferably a CD8+ T cell, such as a human CD8+ T cell. In a preferred embodiment, the cell (such as a T cell) for use according to the present invention expresses the CAR on its membrane. The term "on its membrane" as used herein has the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs, i.e., "on the membrane of the cell surface".

[0082] In certain embodiments, a source of cells is obtained from a subject prior to the in vitro manipulation or genetic modification of immune effector cells as described herein. In certain embodiments, cells for use according to the invention include T cells. T cells can be obtained from a variety of cell sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using a variety of methods known to those skilled in the art, such as sedimentation (e.g., FICOLL™ separation). Cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes lymphocytes, such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and the cells placed in an appropriate buffer or medium for further processing.

[0083] T cells are isolated from peripheral blood mononuclear cells by lysing red blood cells and depleting monocytes, such as by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells expressing one or more markers, such as CD4 or CD8, can be further isolated by positive or negative selection methods. For example, enrichment of a T cell population by negative selection can be achieved by combining antibodies against surface markers characteristic of the negatively selected cells.

[0084] In some embodiments of the invention, cells harboring a nucleic acid molecule encoding a chimeric antigen receptor (CAR) utilize a suicide gene, such as an inducible suicide gene, to reduce the risk of direct toxicity (i.e., graft-versus-host disease in an allogeneic administration setting) and / or uncontrolled proliferation of genetically modified cells. In certain aspects, the suicide gene is not immunogenic to the host harboring the polynucleotide or cell. Examples of suicide genes that can be used include inducible caspase-9 (iCASP9), herpes simplex thymidine kinase (HSV-tk), CD20, truncated EGFR, caspase-8, or cytosine deaminase. Caspase-9 can be activated using certain chemically induced dimerizers (CIDs). Other systems can be activated by metabolic prodrugs (ganciclovir) or by conjugated antibodies (rituximab, cituximab).

[0085] This specification discloses a kind of cell therapy in which T cells are genetically modified ex vivo to express CAR, and the CAR T cells are injected into recipients in need of the therapy. The injected cells can kill tumor cells in recipients, preferably humans. Unlike antibody therapy, CAR T cells can replicate in vivo, so they can persist for a long time and control tumors persistently.

[0086] CARs can also redirect and activate effector T cells to any cell surface molecule upon antibody-derived receptor binding, independent of MHC restriction.

[0087] The genetically modified cells (such as T cells) for use according to the invention are constructed from the patient's own cells (autologous), but may also be derived from other allogeneic donors in the context of bone marrow or peripheral hematopoietic stem cell allografts (donor lymphocyte infusions) to obtain allogeneic genetically modified T cells. The above cells expressing the CAR molecule are useful for treating AML in mammals, preferably humans, a disease associated with cell surface IL-1RAP expression.

[0088] Preferably, the cell (such as a T cell) expresses a CAR molecule having an antigen binding region that is an anti-IL-1RAP scFv having an anti-IL-1RAP binding region, a transmembrane region of a CD28 protein, a costimulatory 4-1BB signaling region, and a CD3ζ signaling region, wherein the anti-IL-1RAP binding region is (i) a light chain having a complementarity determining region 1 (CDR1) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:6, a complementarity determining region 2 (CDR2) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:7, and a complementarity determining region 3 (CDR3) that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:8; (ii) a heavy chain having a complementarity determining region 1 (CDR1) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 12, a complementarity determining region 2 (CDR2) that 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 a complementarity determining region 3 (CDR3) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 14.

[0089] AML may be any AML according to the European LeukemiaNet (ELN) prognostic classification and / or any subtype of AML according to the French-American-British (FAB) classification.

[0090] In one embodiment, the AML is relapsed / refractory AML.

[0091] In another embodiment, the AML is relapsed / refractory AML with complex cytogenetic abnormalities, ie, cytogenetic abnormalities according to the WHO classification, and / or AML with TP53 mutations.

[0092] TP53 is a tumor suppressor protein encoded by the TP53 gene located on the short arm of chromosome 17. TP53 plays a pivotal role in maintaining genomic stability against DNA damage, activating DNA repair programs and causing cell cycle arrest. TP53 is mutated in more than half of human cancers. In AML, mutated TP53 is found primarily in patients with therapy-related AML and complex karyotypes. Studies using mouse models of AML have shown that gain-of-function mutations in hotspot regions promote more aggressive AML. Previous studies have shown that the presence of TP53 mutations correlates with poor response to chemotherapy and poor survival in patients with or without complex karyotypes. In multivariate analyses, the presence of TP53 mutations without abnormal cytogenetic abnormalities predicted poor overall survival and poor response to therapy. In some embodiments, the AML is AML associated with IL-1RAP expression (also known as "IL1-RAP expressing AML" or "IL1-RAP+AML").

[0093] In some embodiments, AML associated with IL-1RAP expression is (i) a relapsed / refractory form of IL1-RAP+ AML, or (ii) IL1-RAP+ AML with complex cytogenetic abnormalities and / or IL1-RAP+ AML with TP53 mutations.

[0094] The cells for use according to the invention may be used to treat patients of any age, particularly patients under 60 years of age, such as under 20 years of age (ie pediatric AML), and / or patients over 60 years of age.

[0095] Cells (such as T cells) expressing a CAR molecule specific for IL-1RAP may be used in a method of treating AML in a human, where the human has already been treated with at least one line of therapy, such as chemotherapy, immunotherapy, or targeted therapy. The chemotherapeutic agent may be CPX-351, and the immunotherapy may be a monoclonal antibody, such as an Antibody Drug Conjugate (ADC) (e.g., anti-CD33 linked to a toxin (Gemtuzumab ozogamicin)), a bispecific antibody (Bites, e.g., anti-CD33 / CD3, anti-CD123 / CD3, or other AML anti-cell surface marker / CD3). The targeted therapy agent may be an anti-mutated FLT3, such as midostaurin or quazartinib, or an agent targeting mutated IDH1 / IDH2, such as an IDH1 inhibitor (ivosidenib) or an IDH2 inhibitor (enasidenib).

[0096] Thus, preferably, cells (such as T cells) expressing a CAR molecule specific for IL-1RAP are useful in methods of treating AML in a mammal in combination with at least one monoclonal antibody, such as an anti-checkpoint inhibitor (i.e., anti-PD-1, anti-PDL-1, or anti-CTL4). The anti-PD-1, anti-PDL-1, or anti-CTLA4 may be nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, or tremelimumab.

[0097] Thus, cells (such as T cells) expressing a CAR molecule specific for IL-1RAP are useful in methods of treating AML in humans who have already undergone graft-versus-leukemia, allogeneic stem cell transplantation, donor lymphocyte infusion (DLI), previous CART cell therapy that is not IL-1RAP CART cells, or allogeneic or autologous hematopoietic transplantation.

[0098] As used herein, "treatment" includes a beneficial or desirable effect on the symptoms or pathology of a disease or condition, and may be a slight decrease in one or more measurable markers of the disease or disorder being treated, such as cancer. Treatment may include either a reduction or amelioration of symptoms of the disease or disorder, as the case may be, or may include a delay in the progression of the disease or disorder. "Treatment" does not necessarily refer to a complete eradication or cure of the disease or disorder or its associated symptoms.

[0099] Accordingly, the present disclosure provides a method for the treatment of AML comprising administering to a subject in need thereof a therapeutically effective amount of a cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), said CAR comprising an antibody or antibody fragment having an anti-IL-1RAP binding region, a transmembrane region, and an intracellular signaling region having at least a stimulatory region, said anti-IL-1RAP binding region comprising: (i) a light chain having a complementarity determining region 1 (CDR1) that has at least 80% identity to the amino acid sequence of SEQ ID NO:6, a complementarity determining region 2 (CDR2) that has at least 80% identity to the amino acid sequence of SEQ ID NO:7, and a complementarity determining region 3 (CDR3) that has at least 80% identity to the amino acid sequence of SEQ ID NO:8; (ii) a heavy chain having a complementarity determining region 1 (CDR1) that is at least 80% identical to the amino acid sequence of SEQ ID NO: 12, a complementarity determining region 2 (CDR2) that is at least 80% identical to the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) that is at least 80% identical to the amino acid sequence of SEQ ID NO: 14.

[0100] The cells (e.g., T cells) may be administered alone or in combination with a diluent and / or other components (e.g., cytokines such as IL-2 and cell populations) as a pharmaceutical composition. Briefly, a pharmaceutical composition may include a target cell population as described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. The composition may include a buffer, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose, 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. As used herein, the term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, consistent with a reasonable risk-benefit ratio.

[0101] The cells for use according to the invention may be formulated into a composition.

[0102] Accordingly, the present invention also provides a composition, such as a pharmaceutical composition, comprising a cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) for use in the treatment of acute myeloid leukemia (AML), said CAR comprising an antibody or antibody fragment having an anti-IL-1RAP binding domain, a transmembrane domain and an intracellular signaling domain having at least a stimulatory domain, said anti-IL-1RAP binding domain comprising: (i) a light chain having a complementarity determining region 1 (CDR1) that has at least 80% identity to the amino acid sequence of SEQ ID NO:6, a complementarity determining region 2 (CDR2) that has at least 80% identity to the amino acid sequence of SEQ ID NO:7, and a complementarity determining region 3 (CDR3) that has at least 80% identity to the amino acid sequence of SEQ ID NO:8; (ii) a heavy chain having a complementarity determining region 1 (CDR1) that has at least 80% identity to the amino acid sequence of SEQ ID NO: 12, a complementarity determining region 2 (CDR2) that has at least 80% identity to the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) that has at least 80% identity to the amino acid sequence of SEQ ID NO: 14.

[0103] Compositions for use in accordance with the present invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0104] As used herein, "parenterally administered" means a method of administration other than enteral and topical administration (usually by injection), including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intratumor, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0105] In one embodiment, the CAR modified cells or compositions are administered to the subject by direct injection into a tumor, a lymph node, the systemic circulation, or a site of infection.

[0106] In one embodiment, the CAR modified cells (e.g., CAR modified T cells) are useful for treating a subject diagnosed with AML by removing immune effector cells from the subject, genetically modifying the immune effector cells with a vector carrying a nucleic acid encoding a CAR described herein to obtain a population of modified immune effector cells, and administering the population of modified immune effector cells to the subject. In a preferred embodiment, the immune effector cells comprise T cells.

[0107] The amount and frequency of administration, as well as possible sequences of combination with conventional AML therapies, will be determined by factors such as the condition of the patient, the type and severity of AML, and appropriate dosages may be determined in animal models and ultimately in clinical trials.

[0108] A "therapeutically effective amount" of genetically modified therapeutic cells may 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. Additionally, a therapeutically effective amount is such that any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. Generally, pharmaceutical compositions comprising the T cells described herein are administered in a manner that is consistent with the administration of the cells or the therapeutically beneficial effects. 4 ~10 9 pcs / kg body weight, preferably 10 5 ~10 6 It may be administered at a dose of 100 mg / kg body weight (including all integer values ​​within these ranges).

[0109] The present invention will now be described in more detail with reference to the following examples, which are merely illustrative and not limiting unless expressly stated. EXAMPLES

[0110] <Example 1: Preparation of monoclonal antibodies> Mouse anti-hIL-1RAP monoclonal antibodies were generated by standard hybridoma techniques.

[0111] Briefly, BALB / c mice (5 weeks old, Charles River) were immunized either in the footpad (n=3) or intraperitoneally (n=5) with a recombinant fusion protein composed of the extracellular portion of IL-1RAP (NM_002182.2, NCBI) and the Fc portion of human IgG1 (R&D Systems, Lille, France). Lymph node or spleen cells and blood samples were harvested and cells were fused with mouse myeloma cell lines and then screened by FACS analysis (Becton Dickinson) against IL-1RAP positive (KU812) and negative (Raji, KG1) cell lines.

[0112] Hybridoma screening identified IL-1RAP-positive cell lines (KU812 or KG-1, AML or Phi, respectively). + p 210CML) and negative cell lines (Tom-1, NALM-20, Jurkat, or Raji, respectively, Phi+ p190 B-ALL, Phi - Five monoclonal antibody subclones were able to be selected that discriminated between AML, B-ALL, T-ALL, or Burkitt's lymphoma.

[0113] Molecular characterization of antibodies Molecular characterization was performed by Sanger sequencing of cloned PCR amplification products obtained with degenerate primers specific for FR1 and the heavy and light chain constant regions according to the protocol of Wang.Z.,et al.(J.Immunol.Methods,2000;233,pp167-77). VDJC gene rearrangements and CDR3 regions could be identified by aligning the consensus nucleotide sequence against the IMGT® database using the V-QUEST online tool according to Brochet X.,et al.(Nucleic Acids Res.,2008,36,pp503-8). Molecular Sanger sequencing showed that all five monoclonal antibodies were identical and had the same CDR3 nucleotide sequence. The monoclonal antibody subclone (#E3C3) was selected because it had the highest relative fluorescence intensity (RFI) by cytometry.

[0114] The selected antibody (clone #E3C3) was characterized by Western blotting, ELISA against recombinant IL-1RAP protein, immunohistochemistry, confocal microscopy, and tissue microarrays (TMA) of normal tissues (FDA normal human organ tissue array, 99 cores / 33 sites / 75 cases) and primary samples from CML patients.

[0115] Western blotting of subcellular fractions (Figure 1) Whole cell lysates of different AML or CML (positive control) hematopoietic cell lines were separated by polyacrylamide gel electrophoresis and electrophoresed onto polyvinylidene difluoride membranes. Membranes were probed overnight with primary IL-1RAP #A3C3 (diluted 1:20). Twenty micrograms of protein were electrophoresed on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and sonicated protein fractions were suspended in RIPA buffer supplemented with a protease inhibitor cocktail (complete Mini EDTA-free; Roche, Bale, Switzerland). Protein loading of the Western blot lanes was assessed by β-actin mAb staining (1:1000, clone AC15, #A5441, Sigma-Aldrich). Immunodetection staining was performed with a secondary polyclonal antibody sheep anti-mouse IgG (#515-035-062, Jackson). Enhanced chemiluminescence detection reagents allowed detection using a camera and Bio-1D software (Vilber-Lourmat, Collégien, France).

[0116] - Relative expression of IL-1RAP mRNA (Figure 18) Relative IL-1RAP mRNA expression was measured by RT-qPCR using the Hs_00895050_m1 Taqman qPCR Gene Expression Assay (ThermoFisher Scientific) targeting mRNA variant codons for cell surface proteins. Analyses were performed in whole blood or bone marrow samples of AML patients (n=29) at diagnosis according to the European LeukemiaNet (ELN) prognostic classification. K562, MonoMac-6, and HEL-60 mRNA were used as negative, high positive, and low positive controls, respectively.

[0117] - Absolute number of IL-1RAP antigenic sites (Figure 3) The determination of the absolute number of IL-1RAP antigen sites was based on the cytometric bead array (CBA) method. 6Different leukemia lines at 0.1% / mL were labeled with anti-IL-1RAP primary antibody (unlabeled #A3C3, Diaclone®) and revealed with FITC-IgG1 anti-mouse secondary antibody (cat. no. 55526, MP Biomedicals®). The experiment was performed using the Cell Quant Calibrator kit (Biocytex no. 7208) according to the recommendations. As a control, unlinked IgG1 isotype was used (cat. no. 857-073-020, Diaclone). Analysis was performed using flow cytometry (FACS Canto™ II, BD FACS DIVA V 7.0, BD Biosciences). KU812 and K562 were used as positive and negative controls, respectively.

[0118] In vitro detection of recombinant IL-1RAP protein by ELISA (Figure 2) Anti-human Fc antibody was coated on the bottom of a plastic ELISA plate. IL-1RAP protein loaded onto human antibodies was probed with mouse and human IL-1RAP (#E3C3) antibodies, and then detected with anti-mouse Fc antibody coated with horseradish peroxidase (HRP).

[0119] ELISA confirmed that the #E3C3 monoclonal antibody recognized the IL-1RAP recombinant protein.

[0120] Flow cytometry analysis of primary cells from AML patients IL-1RAP cell surface staining of different hematopoietic AML cell lines was performed using #A3C3 mAb and IL-1RAP mouse mAb as a staining comparison.

[0121] Relative fluorescence intensity (RFI) between IL-1RAP staining and isotype was calculated. IL-1RAP+ cell line (KU812) or IL-1RAP- cell line (Raji) were used as positive or negative controls, respectively.

[0122] For primary bone marrow or peripheral blood samples from AML patients, murine IL-1RAP mAb (#EЗCЗ) was included in a panel capable of detecting CD45, CD34, CD38, CD33, CD123, and CD14 (for monocytes). Stained cells were collected using a CANTO II cytometer (BD Biosciences, Le Pont De Clerc, France) and analyzed using DIVA 6.1 software (BD Biosciences, Le Pont De Clerc, France).

[0123] In situ detection To study specific or non-target tissue binding, FDA standard frozen tissue arrays (US Biomax, Rockville, USA) containing 90 tissue cores (30 organs) from three individual donors per organ were incubated as described above. Immunostaining was detected using the UltraView Universal DAB Detection Kit (Ventana, USA). Images were acquired and analyzed with NDP.view2 software. IL-1RAP high-expressing (KU812) or negative-expressing (Raji) cell lines 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+). IL-1RAP high-expressing (KU812) or negative-expressing (Raji) cell lines were used as positive or negative controls, respectively.

[0124] IL-1RAP expression was examined using the #E3C3 monoclonal antibody, and staining of various intensities was detected mainly in epithelial or endothelial cells in only six tissues: lymph node, colon, small intestine, placenta, stomach, and prostate (Figure 4).

[0125] ·result #A3C3 can stain various AML cell lines expressing IL-1RAP on their cell surface. Therefore, by western blotting with #A3C3, we confirmed that IL-1RAP was expressed on the cell surface of different cell lines with signals of different intensity levels. Interestingly, a subpopulation of CD14+ monocytes also expressed IL-1RAP. Analysis of a cohort of patients with AML (all subtypes, n=30) by flow cytometry confirmed that IL-1RAP was present in AML blasts at various percentages (84.27±17.4%) and cell surface expression levels, while the expression level in monocytes was stable, as shown in the prior art. This result was also confirmed by the absolute number of IL-1RAP antigen sites in AML patients classified according to the European LeukemiaNet (ELN) prognostic classification. Based on these results, we defined three cell surface expression levels: low, intermediate, and high. These expression levels were modeled in cell lines for further studies.

[0126] Example 2: Lentiviral constructs Based on molecular sequencing of the VDJ or VJ rearrangements and CDR3 nucleotide sequence determination, a synthetically generated single chain variable fragment (scFv) from the #E3C3 IL-1RAP hybridoma of Example 1 was cloned into the SIN-pSDY backbone to prepare the CAR lentiviral construct (pSDY-iC9-IL-1RAPCAR-dCD19) (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).

[0127] Briefly, we constructed a SIN lentiviral construct carrying the safety cassette iCASP9, the single chain variable region fragment of the #E3C3 monoclonal antibody, and the cell surface expression marker ΔCD19 for monitoring and selection of promising cells. All three transgenes are under the control of the EF1 promoter and SP163 enhancer sequence (part of the 5'UTR of the mouse VEGF gene, GenBank accession number #U41383), separated by a 2A peptide cleavage sequence.

[0128] As shown in Figure 5, the above construct has three different parts: suicide safety cassette iCASP9 (chemically inducible caspase 9), IL-1RAP CAR, and cell surface selection marker ΔCD19 (non-signaling intracellular cleaved CD19), which are separated by two different 2A ribosomal skip sequences (P2A and T2A) under the control of the EF1a (elongation factor 1 promoter alpha) promoter attached to the SP163 enhancer. Under the control of the EF1a promoter and SP163 enhancer, the CD28-4.1BB-CD3z signaling chain is used to clone in frame the scFv composed of the variable regions of the heavy (VH) and light (VL) chains of the #E3C3 immunoglobulin. The IL-1RAP CAR has 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 composed of two costimulatory domains (engineered transmembrane and intracellular signaling CD28 and 4-1BB) and a CD3z intracellular signaling domain. Mock T is composed of a similar construct without the IL-1RAP scFv.

[0129] <Example 3: Preparation of IL-1RAP CART cells> CD3+ T lymphocytes obtained from peripheral blood mononuclear cells of healthy donors were activated using anti-CD3 / CD28 beads (Life Technologies, France) according to the manufacturer's instructions, and then isolated on a magnetic column (MACS, Miltenyi Biotec, Paris, France). On day 2, activated T cells were contacted with the supernatant (SN) and transduced with the lentiviral vector of Example 2 by spinoculation at 2000 g and 10° C. for 90 min. Transduction efficiency was measured by flow cytometry analysis to identify ΔCD19 cell surface marker expression. Four days after transduction, CD19 positive cells labeled with CD19 microbeads (Miltenyi Biotec, Paris, France) were magnetically separated on a MACS column. Isolated CD19-expressing cells were expanded and cryopreserved in X-vivo complete medium (Lonza, Basel, Switzerland) containing 500 UI / mL rhIL-2 (Proleukin, Novartis) supplemented with 8% human serum. Experimentally, 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 were used.

[0130] Example 4: Lentiviral transduction of donor T cells Lentiviral vector supernatant stocks for Example 2 were generated by transient co-transduction of subconfluent 293T cells with a helper plasmid encoding the Vesicular Stomatitis Virus (VSV) envelope (pMDG) and a GAG / POL (psPAX2) packaging plasmid (Addgene, #12259 and #12260, respectively, Trono et al, Lausanne, Switzerland) using the CaCl2 method. Viral supernatants were harvested after 48 and 72 hours, concentrated by PEG and low-speed centrifugation (3000 g, overnight) and stored at -80°C until use. The same lentiviral construct without IL-1RAP scFv (Mock) was used as a control. Titers of lentiviral supernatants were established by transduction of permissive 293T cells with serial dilutions of SN.

[0131] Transduction efficiency was measured by flow cytometry and the multiplicity of infection (MOI) was subtracted from the supernatant titer depending on the starting cell number.

[0132] Primary T cells can be transduced with Mock or CAR IL-1RAP supernatants at an MOI of 2, respectively, by an in vitro production process using lentiviral supernatants.

[0133] Western blot analysis of IL-1RAP CAR expression Proteins extracted from membrane or cytoplasmic subfractions (obtained after ultracentrifugation) or total protein lysates of IL-1RAP-transduced T cells were probed with mouse anti-human CD3z antibody. Western blotting on subcellular fractions revealed that IL-1RAP CAR associates with CD3z signaling (55KDa signal compared to the predicted endogenous CD3z signal of 16KDa) (Figure 6).

[0134] Flow cytometry analysis CAR expression on the T cell surface was analyzed using recombinant IL-1RAP biotinylated protein and detected by flow cytometry using a secondary anti-biotin antibody (Miltenyi Biotec, clone #Bio3-18E7). CEM cell lines or primary T cells were transduced with either Mock or CAR IL-1RAP. Cells were then incubated in the presence of increasing amounts of recombinant IL-1RAP labeled with biotin. Staining was performed with anti-biotin fluorescent antibody and analyzed by flow cytometry. The percentage of biotin+ / CD19+ CEM or T cells was plotted against the amount of labeled biotin recombinant protein. Dot plots of cytometric analysis are shown for representative staining including the maximum value. Non-transduced cells (C0) or Mock T cells were used as controls.

[0135] Additional analysis with serial dilutions of biotinylated IL-1RAP protein (20 ng to 2.4 pg / ml) and FACS analysis allows the detection of either IL-1RAP CAR-transduced CEM T cell lines or primary T cells. From one experiment it can be shown that different amounts of recombinant protein, 1.25 ng and 0.15 ng, respectively, are required to mobilize maximum CEM (85.8%) or primary (68.5%) GMTC (Figure 7).

[0136] CEM express more CAR on their cell surface than primary T cells, and addition of high amounts (1000-fold > plasma concentration) of unlabeled recombinant IL-1RAP protein to E:T cocultures significantly inhibits effector cytotoxicity.

[0137] These experiments confirm that CAR is addressed to the cell surface and that CAR-specific recognition and binding of the IL-1RAP protein occurs.

[0138] <Example 5: Efficiency of suicide gene iCASP9 cassette> Transduced (IL-1RAP CAR293T) or non-transduced (293T) cells were cultured for 24 hours in medium alone (-chemically induced dimerizer (CID)) or medium containing 20 nM CID AP1903. The presence and structure of live or dead cells in the cultures can be visualized by light microscopy (×40).

[0139] Light microscopy reveals that 293T cell cultures transduced with IL-1RAP CAR are susceptible to CID (Figure 8).

[0140] Flow cytometry analysis of GMTC cell mixtures expressing or not expressing IL-1RAP CAR and non-transduced T cells (C0) after CID exposure (20 nM, 24 h) or without exposure (light grey). CD3 + / CD19 + Staining allows differentiation of GMTCs expressing CAR from others.

[0141] Either non-transduced T cells (C0) or IL-1RAP CART cells were exposed to medium alone or medium + CID (20 nM, 24 h).

[0142] Accurate cell death was first assessed by flow cytometry after Annexin-V / 7-AAD gating according to the manufacturer's instructions (Beckman Coulter, IM3614). Fluorescence analysis was performed on CD3 + / CD19 + Gating was done on positive cells. 5000 fluorescent beads were acquired and then quantified. 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)] x 100. CID exposure of CO or IL-1RAP CART (gated on CD3+ / CD19+) cells for 24 or 48 h. Results are expressed as mean ± SD of three independent experiments. *** :p<0.001 (Figure 9).

[0143] Cytometric analysis revealed that IL-1RAP CAR-expressing (CD19 + ) or not (CD19 - ) After 24-hour CID exposure of a mixed population of T cells, CD19 - It can be seen that only CD3+ cells survived.26 More precisely, a quantitative AnnV / 7AAD assay of apoptosis revealed that 84.11% and 88.93% of IL-1RAP CART cells were eliminated after 24 or 48 h CID exposure, respectively, compared to non-transduced T cells (C0) (1.28% and 6.13% at 24 or 48 h, respectively) (p<0.001, n=3).

[0144] Example 6: Proliferative ability of IL-1RAP CART cells Materials and Methods Non-transduced T cells (C0), Mock T cells (MockT) or IL-1RAP CAR (IL-1RAP CART) were cultured for 3 days in either medium alone (no target), in contact with IL-1RAP negative cells (K562), or in contact with cell surface expressing target cells (KU812, CML, positive control) and AML cell lines with different levels of cell surface expression of IL-1RAP (low: HL-60, medium: MOLM-13, high: EOL-1) at an effector:target (E:T) ratio of 1:3. Effectors were labeled with 0.5 M CFSE without prior addition of IL-2. After 72 h of coculture without the addition of IL-2, division of live CD3+ / CD19- (C0) and CD3+ / CD19+ gated cells (MOCKT- or IL-1 RAP CART cells) was assessed by flow cytometry and measured by CFSE dye dilution.

[0145] result In contrast to non-transduced 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, whatever the IL-1RAP cell surface expression of the target cells (Figures 10A and 10B).

[0146] Example 7: Expression of IFNγ in cells Materials and Methods Non-transduced T cells (C0), mock-transduced T cells (MockT) or IL-1RAP CAR (IL-1RAP CART) were cultured for 6 h at an effector:target (E:T) ratio of 1:5 in contact with medium alone (no target), IL-1RAP negative cells (K562), or cell surface-expressing target cells (KU812, CML, positive control) and AML cell lines with different levels of IL-1RAP expressed on the cell surface (low: HL-60, medium: MOLM-13, high: EOL-1). As a positive control for cytokine production, cells stimulated with 10 ng / mL phorbol myristate acetate (PMA) and 1 μg / mL ionomycin (Sigma-Aldrich) were used. IFNγ labeling was performed after Brefeldin A treatment and cell permeabilization. Fluorescent IFNγ signals were detected after gating on CD3+ / CD19+ (CAR positive cells) in addition to CD8 staining to distinguish CD8+ and CD4+ (formerly CD8-) cells.

[0147] result In contrast to non-transduced (C0) or MOCK-transduced (MockT) T cells, CD8+ or CD4+ IL-1RAP CART cells can specifically express intracellular IFNγ in the presence of AML cell lines, whatever the IL-1RAP cell surface (Figures 11A and 11B).

[0148] Example 8: Cytotoxicity of IL-1RAP CART cells against AML cell lines Materials and Methods Effector cells were labeled with e-Fluor before co-culture. Non-transduced T cells (C0), Mock-transduced T cells (MockT) or IL-1RAP CAR (IL-1RAP CART) were cultured for 24 hours at various effector:target (E:T) ratios in medium alone (no target), in contact with IL-1RAP negative cells (K562), or in contact with cell surface expressing target cells (KU812, CML, positive control) and AML cell lines with different levels of cell surface expression of IL-1RAP (low: HL-60, medium: MOLM-13, high: EOL-1). Gating of cells on FSC and 7-AAD labeling allowed discrimination between target cells, effector and live cells. Gating on FSC+ / 7-AAD- measured the percentage of target cells that persisted in the presence or absence of effector cells. As controls, non-transduced T cells (C0) or mock-transduced T cells (MockT) were used.

[0149] result In contrast to MOCK-transduced T cells (MockT), effector CART cells co-cultured with IL-1RAP positive targets (AML) at various E:T ratios were able to kill target AML cell lines expressing IL-1RAP with equal efficiency, regardless of the expression level of the tumor antigen (Figures 12A and 12B).

[0150] Example 9: IL-1RAP-CART cells protected with the iCASP9 safety switch do not show significant adverse effects on healthy hematopoietic cells To predict off-target toxicity, IL-1RAP expression was examined by tissue macro assay (TMA) of 30 normal human tissues using #A3C3mAb. Staining was detected at various intensity levels, excluding inflammatory or necrotic elements, in only six tissues: lymph node, prostate, skeletal muscle, stomach, colon and small intestine, and pancreas (Figure 13A and Table 2). Interestingly, the microvascular HMEC-1 endothelial cell line was not recognized by our #A3C3 IL-1RAP mAb (Figure 13B), whereas cell surface expression was clearly detected by R&D IL-1RAP mAb (R&D Systems, Ref#89412), suggesting that a different epitope is recognized.

[0151] Regarding targeting of healthy hematopoiesis, mAb#A3C3 did not detect HSCs (RFI<1.2, n=5) in bone marrow (Fig. 14A,C) or normal umbilical cord blood (Fig. 14B,C) of healthy donors, but weak staining (RFI<2) of monocyte subpopulations was confirmed in 2 / 5 peripheral blood and 3 / 5 bone marrow of healthy donors (Fig. 14A). The in vitro sensitivity of monocytes was then examined by co-culturing PBMC and autologous CART cells at various E:T ratios. At E:T ratio=1:1, only a portion of monocytes was targeted, with 41.45% of monocytes remaining viable (Fig. 14D, right, Table 3), whereas lymphocytes, granulocytes, and K562 IL-1RAP negative cell line were unaffected, even at high E:T ratios (Fig. 13D). Interestingly, at the above E:T ratio, 94.77% of leukemic cells were killed (Figure 14E).

[0152] [Table 2-1]

[0153] [Table 2-2]

[0154] [Table 2-3]

[0155] [Table 3]

[0156] These results were confirmed in vivo in a hCD34 engrafted mouse model (hu-NOG), where monocytes were reduced at day 15 (41±25%, n=3, p=ns), whereas other human immunocompetent cells derived from hCD34+ cells were not affected by CART cells (Figure 15). Hematopoietic stem cell culture assays performed after in vitro co-culture of healthy CD34+ cord blood HSCs with autologous CART cells (n=3) confirmed that HSCs were not affected (Figure 16). These results are consistent with IL-1RAP CART cell immunotherapy with minimal side effects on the hematopoietic system.

[0157] To limit potential toxicity, we assessed the functionality of the safety switch of the iCASP9 / AP1903 suicide cassette after exposure to a chemically induced dimerizer (CID, 10 nM). First, we demonstrated that 293T cell cultures transduced with IL-1RAP CAR were sensitive to CID using light microscopy (Figure 17, top). Cytometric analysis showed that in a mixed population of CD19+ and CD19-IL-1RAP CART cells, only CD19-CD3+ cells persisted after 24 h of CID exposure (Figure 17, bottom). More precisely, quantitative apoptosis assays showed that 84.11% and 88.93% of IL-1RAP CART cells were eliminated after 24 or 48 h of CID exposure, respectively, compared with 1.28% and 6.13% of non-transduced T cells (C0) at 24 or 48 h, respectively (p<0.001, n=3, Figure 14F). Finally, in vivo assessment of the safety switch in the NSG mouse model showed that 87 ± 7.32% (p < 0.01, n = 3) of IL-1RAP CART cells were eliminated after intraperitoneal administration of AP1903, but were unaffected after PBS administration, and control non-transduced T cells (C0) were unaffected by either treatment (Figure S14G).

[0158] Example 10: In vitro cytotoxicity of IL-1RAP CART cells against AML blasts from AML patients Materials and Methods Untransduced T cells (UNT cells), MockT cells (i.e., cells transduced with the same vector used for CART cells, but the vector does not carry a CAR), and IL-1RAP CART cells (generated from T cells of healthy donors or AML patients) were centrifuged and resuspended in 1 ml of PBS 1X, followed by the addition of 1 ml of e-fluor-V450 solution (Cell Proliferation Dye) (eBioscience) diluted 1 / 1000. The cells were incubated in the dark at room temperature for 20 minutes. Then, 8 ml of X-Vivo 15 complete medium (Lonza), 10% fecal calf serum (GIBCO, USA), 1% penicillin-streptomycin PS (GIBCO, USA) were added, followed by incubation for 5 minutes at +4°C and centrifugation for 5 minutes at 1500 rpm. The cells were washed twice with the same medium and suspended in X-Vivo 15 complete medium containing interleukin 2, 10% human serum, and 1% PBS. These cells and 100x10 3Co-cultures with primary cells (allogeneic or autologous) were performed in 96-well plates (round bottom) at different effector:target ratios (E:T ratios). Final volume / well = 200 μl. After 24 h of co-culture, the cytotoxicity of UNT, MockT and IL-1RAP CART cells towards AML blasts was evaluated by viability labelling with 3 μl 7-AAD-PerCP-Cy5.5 (BD Bioscience) and membrane staining with anti-CD34-allophycocyanin (APC) (BD Bioscience). The following antibodies (Abs) were used to stain, detect and differentiate AML blasts from effector cells (MockT or IL-1RAP CART cells) as appropriate: 1 μl anti-CD3-phycoerythrin (PE) (Miltenyi Biotec, Germany) and 1 μl anti-CD19-allophycocyanin (APC) (Miltenyi Biotec, Germany). Effector cells were distinguished from target cells by e-fluor labeling. Results were obtained by flow cytometry and analyzed with FACS Diva software. Allogenicity killing of IL-1RAP CART cells was subtracted from the cytotoxicity of control non-transduced T cells (UNT).

[0159] In an experiment in which IL-1RAP T cells were generated from AML patients, AML blasts were labeled in advance after T cell isolation to confirm the presence or absence of T cells and B cells.

[0160] result The results are shown in Figure 19. Compared to MOCKT and UNT cells, which showed no cytotoxicity, co-culture with IL-1RAP CART cells at various E:T ratios was able to kill primary AML patient blasts (low to intermediate IL-1RAP expressers previously identified by flow cytometry (not shown)) with the same efficiency regardless of the expression level of the tumor antigen.

[0161] Interestingly, IL-1RAP CART cells generated from T cells from AML patients were also able to kill AML blasts with the same efficiency, confirming that CART cells can be generated from AML patients and used in autologous transplants.

[0162] Example 11: In vivo cytotoxicity of IL-1RAP CART cells against AML blasts from AML patients Materials and Methods HL-60, Molm-13, and Mono-Mac-6 AML cell lines with low, intermediate (int), and high IL-1RAP expression, respectively, were transduced with a luciferase lentiviral vector (pLenti CMV V5-Luc Blast vector, Addgene). Luciferase-positive cells were selected by resistance to blastidine (ThermoFisher Scientific).

[0163] Six to eight week old NSG mice (Jackson Laboratory, Sacramento, CA, USA) were irradiated intraperitoneally (25 Gy) on day 4. On day 3, each mouse was injected with 1x10 IgG suspended in 300 μL of PBS. 6 HL-60, Molm-13, or Mono-Mac-6 luciferase-expressing AML cells were injected via the tail vein. On day 0 after AML cell injection, mice were treated with either naïve (UT) or untransduced T cells (UNT) (10 × 10 in 300 μL PBS). 6 cells), or IL-1RAP CAR-T cells (10 × 10 in 300 μL PBS) 6 ) was administered via the tail vein, and leukemia development was monitored on days 3, 5, 10, 14, 17, and 21 using the IVIS (registered trademark) lumina III system (PerkinElmer).

[0164] The animal protocol was carried out under the control of the Animal Care and Use Committee of the University of Besançon. Mice were followed until untreated animals reached a state of weakened health and showed signs of leukemia (i.e., weight loss >15%, reduced activity, and / or paralysis of the hind legs). Mouse experiments were approved by the local ethical committee (CELEAG and protocol 11007R (Veterinary Services for Animal Health & Protection) for the NSG-S model).

[0165] result The results are shown in Figure 20. IL-1RAP CART cells are shown to significantly reduce leukemia burden in an in vivo AML xenograft mouse model, independent of IL-1RAP cell surface expression on leukemia cells, compared to untreated or UNT-treated T cell mouse groups.

Claims

1. 1. A pharmaceutical composition comprising a T cell expressing a nucleic acid molecule encoding a chimeric antigen receptor (CAR) for use in the treatment of acute myeloid leukemia (AML), the CAR comprising an antibody or antibody fragment having an anti-IL-1RAP binding domain, a transmembrane domain, and an intracellular signaling domain having at least a stimulatory domain, the anti-IL-1RAP binding domain comprising: (i) a light chain having a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:6, a complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO:7, and a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:8; (ii) a pharmaceutical composition having a heavy chain having a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 12, a complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 13, and a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO:

14.

2. The pharmaceutical composition of claim 1 , wherein the T cells are CD8+ T cells.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the IL-1RAP binding region is selected from the group consisting of an antibody, Fv, scFv, Fab, and other antibody fragments.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein the IL-1RAP binding region is an scFv.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the transmembrane region is a transmembrane region of a protein selected from the group consisting of the α, β, or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

6. A pharmaceutical composition described in any one of claims 1 to 5, wherein the membrane-spanning domain is the membrane-spanning domain of CD28.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the anti-IL-1RAP binding region is connected to the transmembrane region by a hinge region.

8. The pharmaceutical composition described in claim 7, wherein the hinge region has an IgG1 hinge sequence or a sequence having 95 to 99% identity thereto.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the intracellular signaling region has at least one costimulatory region, and the at least one costimulatory region of the intracellular signaling region is obtained from one or more proteins selected from the group consisting of OX40, CD2, CD27, CD28, CDS, CD3ζ, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

10. The pharmaceutical composition of claim 9, wherein at least one costimulatory region of the intracellular signaling region is obtained from 4-1BB (CD137) and / or from CD3ζ.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the AML is (i) refractory / relapsed AML, or (ii) AML with complex cytogenetic abnormalities and / or AML with TP53 mutations.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the AML is IL1-RAP-expressing AML, such as refractory / relapsed IL1-RAP-expressing AML.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the T cells are bound to at least one monoclonal antibody.

14. The pharmaceutical composition of claim 13, wherein at least one monoclonal antibody is an anti-checkpoint inhibitor.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the treatment is an autologous treatment.

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