IL-1RAP-Targeting CAR-T Cells and Uses Thereof

Abstract

To provide CAR-T cells targeting IL-1RAP and uses thereof.SOLUTION: The present invention pertains to: an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) where the CAR has an antibody or antibody fragment having an anti-IL-1RAP binding region; a polynucleotide encoding the nucleic acid molecule; the isolated chimeric antigen receptor (CAR) molecule having the antibody or antibody fragment; a vector having a nucleic acid molecule encoding CAR; and a T-cell having the vector. The present invention also pertains to the use of the T-cell expressing CAR molecules (autologous or allogeneic) for treating proliferative diseases in mammals.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to isolated nucleic acid molecules encoding chimeric antigen receptors (CARs), the CARs having an antibody or antibody fragment having a humanized anti-IL-1RAP binding region, a transmembrane region, and an intracellular signaling region that includes at least a stimulatory region, polypeptides encoded by the nucleic acid molecules, and isolated chimeric antigen receptor (CAR) molecules having the antibody or antibody fragment.

[0002] The present invention also relates to a vector carrying a nucleic acid molecule encoding a CAR, and a T cell carrying said vector.

[0003] The present invention also relates to the use of the above-mentioned T cells expressing a CAR molecule for treating a proliferative disease in a mammal. [Background technology]

[0004] Chronic myelogenous leukemia (CML, also known as chronic myeloid leukemia) is a myeloproliferative disorder characterized by increased proliferation of granulocytic cell lines without loss of differentiation potential.

[0005] CML is a disease of hematopoietic stem cells that arises from a t(9;22)(q34;q11) translocation involving a shortened chromosome 22, termed the Philadelphia chromosome 22q-. The translocation juxtaposes the ABL1 gene on chromosome 9 with the BCR gene on chromosome 22, resulting in the BCR-ABL1 transcript and the BCR-ABL1 fusion gene, which encodes a fusion protein with high tyrosine kinase activity. As the molecular cause of CML is well understood, the mechanism by which the genetic translocation occurs is unknown.

[0006] The incidence of CML is 10-15 cases / 10, with no significant geographic or racial differences. 6 / year. The median age at diagnosis is 60-65 years in Europe, but is significantly lower in countries with younger populations. CML is rare in children.

[0007] The diagnosis of CML is generally straightforward. In most cases, it can be diagnosed based on characteristic blood counts. The diagnosis can be confirmed by identifying Philadelphia chromosome 22q- or BCR-ABL1 transcripts, or both, in peripheral blood or bone marrow cells.

[0008] Before the early 2000s, interferon alpha (IFNα) and hematopoietic stem cell transplantation were the only effective treatments in CML. Allogeneic transplantation of hematopoietic stem cells was considered the only potentially curative treatment for eligible patients, if an HLA-matched donor was available. Such allogeneic transplantation is a technique called adoptive immunotherapy that is employed in the treatment of the majority of aggressive hematological diseases. Its principle is immune transfer that relies on the activity of cytotoxic T effectors via specific T receptors. However, the cytotoxic activity specific to these lymphocytes is limited by the presentation of tumor antigens using molecules of the human leukocyte antigen (HLA) system. The mortality of such transplantation procedures and the risk of relapse after allogeneic transplantation remain a major gamble for such immunotherapy.

[0009] Despite transplant-related toxicity and mortality, it is well-known that the graft-versus-leukemia immunological effects of allogeneic stem cell transplantation, as well as the efficacy of donor lymphocyte infusion (DLI), remain the only treatments capable of achieving long-term remission of the disease, if not a cure.

[0010] Since the early 2000s, the discovery and widespread use of tyrosine kinase inhibitors (TKIs) for the treatment of chronic phase CML has significantly changed the prognosis of this hematological disease, achieving a survival rate of over 90%. Currently, the indications for allogeneic hematopoietic stem cell transplantation in CML are limited to patients who are intolerant / resistant to TKIs and those in the advanced stage (accelerated or blast crisis) of CML.

[0011] In 2017, the use of tyrosine kinase inhibitors (TKIs) remains the treatment of choice for first-line treatment, although other alternatives are available. With TKI therapy, most patients can recover normal hematopoiesis. However, although TKIs such as imatinib, dasatinib, nilotinib, bosutinib, or ponatinib have provided CML patients with much in terms of overall survival and quality of life, the ability of these drugs to cure CML is limited.

[0012] Discontinuation of a TKI may also be considered due to intolerance and toxicity, potential risks to pregnancy, or for financial reasons of funding agencies.

[0013] In a multicenter trial of imatinib, imatinib treatment was discontinued (for more than 2 years) in CML patients with submilligram disease. Of 69 patients enrolled, 42 (61%) of these patients relapsed. At 12 months, the probability of persistent submilligram disease in these 69 patients was 41%. This failure was due to the inability of TKIs to eradicate quiescent CML stem cells.

[0014] Recently, the French study STIM1 (n=100 patients) investigating the attempt to discontinue imatinib in patients with complete molecular response was updated in 2017. The molecular relapse rate after discontinuation of TKI was 61% with a median time of 2.5 months, and these relapsed patients were found to have persistent medullary accumulation of disease.

[0015] In fact, current TKIs are suppressive rather than curative, and therefore require long-term administration, which may lead to unexpected and unknown adverse events. Furthermore, long-term administration of TKIs to young CML patients may be a future challenge.

[0016] Therefore, there is a need to eliminate persistent TKI-resistant CML quiescent precursors. Genetic approaches offer promising avenues to improve immune recognition and elimination of cancer cells. One promising strategy involves genetically engineering immune effector cells to express chimeric antigen receptors that redirect cytotoxicity to tumor cells.

[0017] In recent years, thanks to advances in cell engineering that allow to bypass tumor escape mechanisms, the latest generation of CAR (chimeric antigen receptor)-T cells has emerged. CAR-T cells are T lymphocytes expressing a chimeric TCR composed of the constant part of a TCR fused to an immunoglobulin variable fragment. Target recognition is not HLA restricted, so they can target any kind of tumor marker.

[0018] Among novel immunotherapies, these CAR-T cells against cell surface tumor-associated antigens have shown unexpected success not only in patients with refractory / relapsed ALL (CD19) or CLL (CD19), but also in preclinical studies in solid tumors and hematology, mainly MM (CD38, BCMA (B cell maturation antigen), CD44v6, or CS1), AML (CD33 or CD123), T-cell malignancies (CD5), or lymphomas (CD30).

[0019] In CML, gene expression profiling studies have shown that normal CD34 + / CD38 - We have identified cell surface biomarkers (IL-1RAP or IL-1R3) that are expressed in leukemic but not hematopoietic stem cells, and IL-1RAP expression correlates with tumor burden and clinical phase of CML disease.

[0020] 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 signaling, and activates various signaling pathways, including MAP kinase, p38, and NF-·B genes involved in inflammation and proliferation. This protein is expressed on the surface of tumor cells. Therefore, IL-1RAP is a promising tumor-associated antigen. Summary of the Invention [Means for solving the problem]

[0021] The applicant has discovered that this IL-1RAP antigen can be used to generate genetically modified CAR T cells that are administered to patients with cancer or tumors, particularly CML.

[0022] Developing cellular CAR-T targeting the hematopoietic stem cells Phi+ responsible for CML using targeted IL-1RAP could provide a means to eradicate the cause of hematologic diseases in addition to, or instead of, TKIs that essentially target the precursors of hematologic diseases.

[0023] Such novel therapeutic approaches are applicable to: Patients who relapse after discontinuing TKI Patients who relapsed after allogeneic transplants (graft-versus-leukemia, allogeneic stem cell transplants, donor lymphocyte infusions (DLI)) Ineligible patients with suboptimal response to TKI Accelerated / blast phase CML patients at high risk of relapse Young or pediatric CML patients

[0024] In one embodiment, a polynucleotide is provided that encodes a CAR, the CAR having an extracellular domain that binds to a target antigen, a transmembrane domain, and one or more intracellular signaling domains. In one embodiment, a T cell genetically modified with a vector carrying a CAR is contemplated herein. A T cell expressing a CAR is referred to herein as a CAR T cell or a CAR modified T cell.

[0025] The present invention, in certain embodiments, contemplates cells genetically modified to express a CAR as contemplated herein for use in the treatment of cancer. 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.

[0026] The words "#E3C3" and "#A3C3" are understood to be identical, and #E3C3 can be freely used to represent #A3C3 and vice versa.

[0027] Other objects, features, aspects, and advantages of the present invention will become more apparent with reference to the following description, drawings, and examples. [Brief description of the drawings]

[0028] [Figure 1] Use of #E3C3mAb in Western blot. Leukemia cell lines KU812, KG-1, Nalm-20, Jurkat, and Raji were used. HT1080 cell line transfected with IL-1RAP cDNA mutant was used as control. Actin was detected as a protein loading control. Line a: detection of IL-1RAP (72 kDA), line b: detection of control actin (43 kDA), *: weak signal. [Diagram 2] Recognition of IL-1RAP recombinant protein by #E3C3mAb by ELISA (b). BSA is used as a negative control (a). [Diagram 3] Immunophenotyping of peripheral blood (PM) or bone marrow (BM) from two CML positive patients at diagnosis (Diag) or after imatinib (IM) treatment. IL-1RAP (#E3C3) was used in combination with CD34+ and CD38- fluorescent staining. Fluorochrome-conjugated isotype control mAbs for the various mAbs were systematically used. [Figure 4](a) KU812 cell line and (b) Raji cell line stained with fluorescent mAb (left panel: anti-mouse Fc-IgG, center panel: IL-1RAP (#E3C3)). Counterstained with nuclear stain DAPI and overlaid with fluorescent stain (right panel, merge). [Diagram 5] 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. [Figure 6] Design of SIN lentiviral construct carrying the safety cassette iCASP9, single chain variable fragment (scFv) of #E3C3mAb, and cell surface expression marker CD19. The three transgenes are separated by a 2A peptide cleavage sequence and are controlled by the EF1 promoter + SP163 enhancer sequence. [Figure 7] Western blotting of IL-1RAP-transduced T cell subcellular fractions: a: total lysate, b: membrane, c: cytoplasm, d: nucleus. (1) CAR-associated CD3ζ (55 kDa), (2) endogenous CD3ζ (16 kDa), (3) CD45 (147 kDa), (4) lamin (68 kDa), (5) GAPDH (35 kDa). [Figure 8] 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 9] 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 CAR293T cells. [Figure 10] 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 11]Flow cytometry (11A) and Western blot (11B) of isoform 1 (v1) and isoform 3 (v5), (a) actin, (b) IL-IRAP-v1 or v5 (72 kDa), (1) total cellular K562 protein, (2) medium supernatant of K562 cultures. [Figure 12] (a) Proliferative potential of IL-1RAP CART cells induced by IL-1RAP target expressing cells by co-culture of CFSE stained C0, Mock, or IL-1RAP CART cells in the presence of K562, (b) K562-v1, (c) K562-v5, or (d) KU812 cell lines (p<0.001, n=4). [Figure 13] Measurement of Th1 / Th2 / Th17 cytokines in the supernatants after co-culture with (a) C0, (b) Mock T cells, or (c) CART cells. [Figure 14] CD107a&b degranulation assay applied to co-cultured IL-1RAP CART cells against IL-1RAP+ (K532-V1, KU812) expressing target cells. Effectors were treated with monensin and stained with CD107a and CD107b mAbs for 1 h at 37°C. After 5 h, CD3+ / CD19+ / CD8+ cells were analyzed by flow cytometry for CD107a and CD107b staining. PMA / Iono activation was used as a control (p<0.001, n=4). [Figure 15] Fluorescence (eFluor) and 7-AAD staining show the IL-1RAP-dependent cytolytic capacity of IL-1RAP CAR-expressing T cells in vitro. Non-transduced or mock-transduced T cells were used as controls (p<0.001, n=4). [Figure 16] Mouse study depicted. A / NSG mouse K562 xenograft model. B / BLI analysis of different mouse groups from day 2 to day 28. [ka] Left: untreated, middle: Mock T cells, right: IL1-RAP CART cells. [Figure 17]In vitro toxicity on primary IL1-RAP+ circulating cells of CML patients. Left: Kinetic quantification of BCR-ABL1 transcript ratio (% on international scale) according to the Europe Against Cancer (EAC) method and recommended methods. RM3.0, RM4.0, RM4.5, and RM5.0 represent molecular response levels corresponding to 3, 4, 4.5, and 5 Log reductions, respectively. IM400: Imatinib 400 mg / day, DAS100: Dasatinib 100 mg / day, BOS400: Bosutinib 400 mg / day, NIL600: Nilotinib 600 mg / day. Right: CD3+ / CD19+ staining and flow cytometry analysis of transduction efficiency of PBMCs of CML patients. [Figure 18] Graph of persistent KU812 viable cells in FSC+ / 7-AAD-gate after co-culture of eFluor-labeled effector C0, Mock-T, or CAR T cells with KU812 cells at various E:T ratios. Graph of persistent KU812 viable cells in FSC+ / 7-AAD-gate. [Figure 19] Figure 1 shows in vitro toxicity against primary IL1-RAP+ circulating cells from CML patients. The percentage of total targets killed was calculated in duplicate. Results are expressed as mean ± SD. [Figure 20] Cytotoxicity of IL-1RAP CAR T cells or Mock T cells against CML autografts at various effector:target (E:T) ratios. Results are compiled from three independent studies performed on three different CML patients. The remaining percentage of CD34+ / IL-1RAP+ viable cells calculated from control cells (C0) is shown. **p<0.01. [Figure 21] Autologous IL-1RAP CART cells obtained from PBMCs of CML patients (n=3) who were alive and had been or had not been treated with various TKIs for more than 20 years (min. 16 years, max. 21 years) were co-cultured in vitro with frozen stocks of autologous peripheral blood stem cell grafts (PBSCs, obtained at diagnosis, more than 20 years ago). CML patients, peripheral blood stem cell autograft characteristics. φ: untreated, IFNγ: interferon gamma; IM: imatinib; DAS: dasatinib; NIL: nilotinib; PON: ponatinib; BOS: bosutinib. [Figure 22A] Tissue microarrays are shown. Representative #A3C3 staining of a FDA standard frozen tissue array containing 90 tissue cores (30 organs) from 3 individual donors per organ (US Biomax, Rockville, MD, USA). Immunostaining was detected using the 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 1 of 3 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 22B] 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 23A] Effect of IL-1RAP CAR T cells on healthy hematopoietic cells and efficiency of suicide gene iCASP9 safety cassette. IL-1RAP cell surface expression on peripheral blood cells (left) or bone marrow cells (right) of healthy donors (n=5). SSC-A / CD45+ allowed 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 23B-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 23B-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 23C]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 23D] 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 autologous non-transduced T cells (C0) co-cultured for 24 h with autologous Mock T cells (dashed lines) or IL-1RAP CAR T cells (solid lines). [Figure 23E] 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 23F] 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 23G]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 24] This represents an immune safety experiment 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 (days 5, 8, and 15) after infusion, 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) on days 3, 8, and 15 were compared to the time of peripheral blood collection (day -9). Cell numbers were counted in peripheral blood collected from retro-orbital samples, and fold changes were calculated relative to day -7. ns: not significant. [Diagram 25] Colony forming unit (CFU-GM) assays of CD34+ HSCs taken from three different cord blood samples and cultured alone (white bars) or co-cultured with respective autologous non-transduced (C0, grey bars) or IL-1RAP CART cells (black bars) (Giavridis, 2018, #1861) are shown. [Figure 26]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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The sequence identifiers are summarized in the table below.

[0030] [Table 1-1]

[0031] [Table 1-2]

[0032] The sequences of the hinge regions of the IgG1, IgG4, CD8α, 4-1BB, CD3ζ, CD28, and ICasp9 genes are available in Genbank.

[0033] 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, see 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.

[0034] 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.

[0035] 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, a first constant domain of a single heavy chain bound to the variable domain, and a constant domain of a single light chain. The variable domains of the light and heavy chains are responsible for antigen binding.

[0036] 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)).

[0037] 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.

[0038] "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.

[0039] "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.

[0040] 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 humanized monoclonal antibodies.

[0041] 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.

[0042] 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.

[0043] 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 steps or elements or groups of steps or elements.

[0044] 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.

[0045] 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.

[0046] The present invention provides immune effector cells genetically engineered with vectors designed to express chimeric antigen receptors that redirect cytotoxicity to tumor cells. Herein, the genetically engineered receptors are referred to as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for target antigens (such as tumor antigens) with T cell receptor activating intracellular domains to generate chimeric proteins that exhibit specific anti-tumor cell-mediated immune activity. Herein, the term "chimera" refers to being composed of various proteins or DNA portions of different origins.

[0047] The present invention provides an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), 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) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid SEQ ID NO: 14. The present invention relates to an isolated nucleic acid molecule having the formula:

[0048] 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.

[0049] 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).

[0050] The antibody is a human antibody, a murine antibody, or a humanized antibody.

[0051] In a preferred embodiment, the antibody is a humanized antibody (such as a humanized monoclonal antibody) that specifically binds to a surface protein on a tumor cell. A "humanized" antibody is an immunoglobulin having 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 humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized or other monoclonal antibody can additionally have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (see, e.g., U.S. Pat. No. 5,585,089).

[0052] 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 chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof.

[0053] "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).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] In a specific embodiment, the linker is between the VH and VL domains.

[0059] In a particular embodiment, the linker has or consists of the amino acid sequence of SEQ ID NO:5.

[0060] 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.

[0061] 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 amino acid 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 amino acid 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 amino acid 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 amino acid sequence 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 amino acid sequence 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 amino acid sequence SEQ ID NO:14.

[0062] 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.

[0063] The anti-IL-1RAP binding region is preferably connected to the transmembrane region by a hinge region.

[0064] In one embodiment, the hinge region has an IgG1 hinge sequence or a sequence having 95 to 99% identity thereto.

[0065] 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.

[0066] In a further embodiment, the hinge region has CD8α or a sequence having 95 to 99% identity thereto.

[0067] 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.

[0068] 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.

[0069] 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).

[0070] 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.

[0071] 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".

[0072] 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.

[0073] 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.

[0074] 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).

[0075] 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.

[0076] 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.

[0077] In another embodiment, the intracellular signaling region comprises a costimulatory region derived from 4-1BB and / or a costimulatory region derived from CD3ζ.

[0078] In certain preferred embodiments, a CAR comprises a CD3ζ primary signaling region and one or more costimulatory signaling regions, which may be tandemly linked in any order to the carboxyl terminus of the transmembrane region.

[0079] Also contemplated are isolated polypeptide molecules encoded by the nucleic acid molecules of the invention, further comprising an isolated CAR molecule having an antibody or antibody fragment having an anti-IL-1RAP binding region, a transmembrane region, and an intracellular signaling region, wherein the anti-IL-1RAP binding region is (i) a light chain having a complementarity determining region 1 (CDR1) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid 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 amino acid 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 amino acid SEQ ID NO:8; (ii) an isolated CAR molecule having a heavy chain with a complementarity determining region 1 (CDR1) that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid 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 amino acid 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 amino acid SEQ ID NO: 14.

[0080] "Polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably unless otherwise specified and according to their conventional meaning, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length and may have, for example, a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and other modifications (both natural and non-natural) known in the art.

[0081] Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic methods. The polypeptides contemplated herein specifically include the CARs of the present disclosure, or sequences in which one or more amino acids have been deleted, added, and / or substituted in the CARs disclosed herein.

[0082] As used herein, "isolated peptide" or "isolated polypeptide" and the like refer to a peptide or polypeptide molecule that is isolated and / or purified in vitro from its cellular environment and from association with other components of a cell. Similarly, an "isolated cell" refers to a cell obtained from an in vivo tissue or organ and that is substantially free of extracellular matrix.

[0083] 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.

[0084] The present invention also provides a vector comprising 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.

[0085] In some embodiments, the vector of the invention comprises a promoter, preferably the EF-1α promoter.

[0086] 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.

[0087] Thus, T cells transduced with the vectors of the invention are capable of eliciting stable, long-lasting CAR-mediated T cell responses.

[0088] In a specific embodiment, the T cells are transduced with a retroviral vector (such as a lentiviral vector) encoding a CAR of the invention.

[0089] 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).

[0090] 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.

[0091] "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.

[0092] In one embodiment, the SIN vector backbone is preferred.

[0093] The vector used preferably further has a promoter such as the EF-1α promoter.

[0094] 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.

[0095] The invention also provides a cell comprising a nucleic acid molecule encoding a CAR of the invention or a vector of the invention, which is preferably 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 of the invention (such as a T cell) expresses the CAR of the invention in its membrane.

[0096] In certain embodiments, a cell source is obtained from a subject prior to the in vitro manipulation or genetic modification of immune effector cells described herein. In certain embodiments, immune effector cells expressing the CAR of the present invention on their membranes 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). In one embodiment, 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.

[0097] In one embodiment, 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.

[0098] In some embodiments of the invention, the polynucleotide or cells harboring the polynucleotide of the invention 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 situation) 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 (CID). Other systems can be activated by metabolic prodrugs (ganciclovir) or by conjugated antibodies (rituximab, cituximab).

[0099] 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.

[0100] CARs can also redirect and activate effector T cells to any cell surface molecule upon antibody-derived receptor binding, independent of MHC restriction.

[0101] The genetically modified T cells of the invention are constructed from the patient's own T cells (autologous), but may also be derived from other allogeneic donors in order to obtain allogeneic genetically modified T cells in the context of bone marrow or peripheral hematopoietic stem cell allografts (donor lymphocyte infusions). Said T cells expressing the CAR molecule of the invention are useful for treating proliferative disorders in mammals, preferably humans, which disorders are associated with cell surface IL-1RAP expression.

[0102] Preferably, the 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 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid 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 amino acid SEQ ID NO:14.

[0103] The present invention also provides a cell (such as a T cell) according to the present invention for use as a medicament.

[0104] The invention also provides a cell (such as a T cell) according to the invention for use in the treatment of a proliferative disorder in a mammal, preferably a human.

[0105] In some embodiments, the proliferative disease is a disease associated with IL-1RAP expression.

[0106] Preferably, said disease associated with IL-1RAP expression is selected from cancer, malignant tumors, or precancerous conditions, such as myelodysplasia, myelodysplastic syndrome, or preleukemia.

[0107] Adult tumors / cancers and pediatric tumors / cancers are also included.

[0108] More preferably, the disease is a blood cancer selected from the group consisting of one or more acute leukemias, such as B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); and one or more chronic leukemias, such as chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL).

[0109] In a more preferred embodiment, the disease is chronic myelogenous leukemia.

[0110] In the first-line treatment of CML, TKIs are used, but more than half of patients relapse when treatment is stopped, indicating that the use of TKIs does not cure the disease.

[0111] Thus, T cells expressing a CAR molecule specific for IL-1RAP are useful in methods of treating CML in humans who have already been treated with at least one tyrosine kinase inhibitor (TKI).

[0112] Thus, T cells expressing a CAR molecule specific for IL-1RAP are preferably useful in methods of treating a mammalian proliferative disease in combination with at least one tyrosine kinase inhibitor (TKI).

[0113] The TKIs used may be imatinib, dasatinib, nilotinib, bosutinib, and ponatinib.

[0114] Thus, T cells expressing a CAR molecule specific for IL-1RAP are useful in methods of treating CML in humans who have already undergone graft-versus-leukemia, allogeneic stem cell transplantation, or donor lymphocyte infusion (DLI).

[0115] 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.

[0116] Thus, the present disclosure provides a method for treating or preventing CML comprising administering to a subject in need thereof a therapeutically effective amount of the T cells of the present invention.

[0117] The T cells disclosed herein 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.

[0118] The present invention also provides compositions (such as pharmaceutical compositions) comprising the cells (such as T cells) according to the present invention.

[0119] The compositions of the present invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0120] 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.

[0121] In one embodiment, the CAR modified T cells or compositions discussed herein are administered to a subject by direct injection into a tumor, lymph node, systemic circulation, or site of infection.

[0122] In one embodiment, the invention is useful for treating a subject diagnosed with cancer by removing immune effector cells from the subject, genetically modifying the immune effector cells with a vector carrying a nucleic acid encoding a CAR as discussed 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.

[0123] The dosage and frequency of administration, as well as possible sequences of combination with conventional CML therapies such as TKIs, will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, but appropriate dosages may be determined in animal models and ultimately in clinical trials.

[0124] 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 105 ~10 6 It may be administered at a dose of 100 mg / kg body weight (including all integer values ​​within these ranges).

[0125] 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.

[0126] Example 1: Patient samples, healthy donor blood samples, cell lines CML sample collections were established from patients at diagnosis and follow-up after TKI treatment. Peripheral blood mononuclear cells were isolated from anonymous blood samples of healthy donors collected at the French Hematology Center (Besançon, France) by Ficoll density gradient centrifugation using Ficoll-Paque (Velizy-Villacoublay, France). Human tumor cell lines KU812 (CRL-2099), K562 (CCL-243), or epithelial cell lines 239T (CRL-3216), HT1080 (CCL-121) were from the ATCC® collection (LGC Standards, Molsheim, France).

[0127] <Example 2: Preparation of monoclonal antibodies> Mouse anti-hIL-1RAP monoclonal antibodies were generated by standard hybridoma techniques.

[0128] 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.

[0129] Hybridoma screening identified IL-1RAP-positive cell lines (KU812 or KG-1, AML or Phi, respectively). + p 210 CML) and negative cell lines (Tom-1, NALM-20, Jurkat, or Raji, respectively, Phi+ p190 B-ALL, Phi - Five monoclonal antibody subclones were able to be selected that discriminated between AML, B-ALL, T-ALL, or Burkitt's lymphoma.

[0130] 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.

[0131] 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.

[0132] Western blotting of subcellular fractions Whole cell, subcellular, or secreted protein fractions of the cells listed in Example 1 were obtained by sonication and suspended in RIPA lysis and extraction buffer (ThermoFisher Scientific) supplemented with a protease inhibitor cocktail (complete Mini EDTA-free, Roche, Switzerland). HT1080 cell line transfected with IL-1RAP cDNA variant 1 (NM_002182.2, NCBI) was used as a control. Actin was detected as a protein loading control. Primary IL-1RAP#E3C3 (1:10) was used for IL-1RAP, CAR, or β-actin expression, respectively. 3 (1:10 dilution), CD3ζ (BD Pharmigen, clone #8D3), or β-actin (1:10 dilution) 3 Proteins transferred to PDVF membranes were probed overnight with either clone AC15, (#A5441, Sigma-Aldrich). Immunodetection staining was performed with secondary polyclonal antibody sheep anti-mouse IgG (#515-035-062, Jackson, USA). Detection was performed using a camera and Bio-1D software (Vilber-Lourmat, Collégien, France). Using #E3C3 monoclonal antibody in Western blots shows KU812 (Figure 1).

[0133] In vitro detection of recombinant IL-1RAP protein by ELISA 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).

[0134] ELISA confirmed that the #E3C3 monoclonal antibody recognized the IL-1RAP recombinant protein (Figure 2).

[0135] Flow cytometry analysis of primary cells from CML patients A panel of CD45, CD34, CD38, CD33, CD133, and CD117, including mouse Alexa Fluor488-labeled IL-1RAP antibody clone #E3C3, was used to track hematopoietic stem cells from CML patients. A panel of antibodies including CD3, CD4, CD8, and CD19 was used to stain transduced cells to identify helper or cytotoxic GMTC. A panel of CD45RA, CD62L, CD95, and CCR7 monoclonal antibodies was used to analyze naïve, central, or memory T cell subsets. Cells were harvested using a CANTO II cytometer (BD Biosciences, Le Pont De Clerc, France) and analyzed using DIVA6.1 software (BD Biosciences, Le Pont De Clerc, France).

[0136] Immunophenotyping was performed on peripheral blood or bone marrow from two CML-positive patients who had or had not received imatinib (TKI) treatment. + and CD38 - Fluorescent dye-conjugated isotype control monoclonal antibodies of various monoclonal antibodies were systematically used.

[0137] Incorporation of the #E3C3 monoclonal antibody in the antibody panel demonstrated that IL-1RAP was significantly increased in bone marrow or peripheral blood of patients at diagnosis or 12 months after imatinib. + Leukemia-initiating stem cell CD34 + CD38 + or CD34 + CD38 - Subpopulations could be distinguished (Figure 3).

[0138] Confocal microscopy Confocal microscopy was performed on KU812 and Raji cell lines collected by cytospin on slides (SuperfrostTM Plus, 4951PLUS4, ThermoFisher Scientific). Briefly, the cells were stained with the fluorescent monoclonal antibody anti-mouse Fc-IgG;IL-1RAP (#E3C3) and analyzed on an Olympus BM51 microscope equipped with a QImaging Retiga2000R camera. Digital images were acquired using a 40x objective and digitized with Image-Pro Plus (version 6.0, Media Cybernetics). Counterstaining was performed with the nuclear stain DAPI (2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride, Sigma-Aldrich, France) and overlaid with the fluorescent staining.

[0139] Confocal microscopy revealed cell membrane staining corresponding to IL-1RAP expression (FIG. 4).

[0140] 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.

[0141] 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 5).

[0142] Example 3: 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).

[0143] 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 DCD19 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.

[0144] As shown in Figure 6, the construct has three different parts: the suicide safety cassette iCASP9 (chemically inducible caspase 9), IL-1RAP CAR, and the 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.

[0145] <Example 4: 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 transduced by contacting with the supernatant (SN) and spinoculating at 2000g for 90 min at 10°C. Transduction efficiency was measured by flow cytometric 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 in X-vivo complete medium (Lonza, Basel, Switzerland) containing 500UI / mL rhIL-2 (Proleukin, Novartis) supplemented with 8% human serum and cryopreserved. Experimentally, T Cell TransAct reagent and TexMACS medium (Miltenyi Biotec, Paris, France) supplemented with human IL-2, IL-7, IL-15, or IL-7+IL-15 were used.

[0146] Example 5: Lentiviral transduction of donor T cells Lentiviral vector supernatant stocks 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.

[0147] 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.

[0148] 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.

[0149] 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 7).

[0150] 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.

[0151] 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 8).

[0152] 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.

[0153] These experiments confirmed that CAR is addressed to the cell surface and that CAR-specific recognition and binding of the IL-1RAP protein occurs.

[0154] Example 6: Efficiency of suicide gene iCASP9 safety 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).

[0155] Light microscopy reveals that 293T cell cultures transduced with IL-1RAP CAR are susceptible to CID (Figure 9).

[0156] Flow cytometry analysis of GMTC cell mixtures expressing or not IL-1RAP CAR and non-transduced T cells (C0) after CID exposure (20 nM, 24 h) or without exposure (light grey). CD3+ / CD19+ staining distinguishes GMTC expressing CAR from others.

[0157] Either non-transduced cells (C0) or IL-1RAP CART cells were exposed to medium alone or medium + CID (20 nM, 24 h).

[0158] 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 10).

[0159] 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).

[0160] Example 7: IL-1RAP-dependent proliferation and cytokine secretion of IL-1RAP CAR-expressing T cells To analyze the proliferation and therefore functionality of IL-1RAP CART cells, in addition to the naturally expressing IL-1RAP cell line KU812, we generated the MHC class I-deficient cell line K562, which expresses either membrane-type (isoform 1) or soluble (isoform 3) IL-1RAP translated from variant 1 (v1) or 5 (v5) transcripts, respectively. To obtain membrane-type (mb) or soluble (s) IL-1RAP expressing cell lines, K562 cells were transfected with variant 1 (isoform 1, NM_002182.2) or variant 5 (isoform 2, NM_001167930) ORF clones (pCMV6-AC-GFP vector, Origen, or pEZ-M61, Gene Copoeia), respectively. Stably expressing mb- or s-IL-1RAP K562 cells were then transduced with pLenti CMV V5-LUC Blast (Addgene, plasmid #21474).

[0161] Western blotting analysis was performed on these cells. Briefly, whole cell (total cells) or secreted protein (culture medium supernatant) fractions were obtained from transfected K562 cell lines after sonication in RIPA buffer supplemented with a protease inhibitor cocktail (complete Mini EDTA-free; Roche, Basel, Switzerland). 20 μg of protein was subjected to SDS-PAGE electrophoresis and then electrophoretically transferred to a PVDF membrane. For IL-1RAP expression, the membrane was stained with primary IL-1RAP#E3C3 (1:10 3 As an assessment of protein loading, the cells were probed overnight with β-actin mAb staining (1:10 3 , clone AC15, #A5441, Sigma-Aldrich) was used. Immunodetection staining was performed with the secondary polyclonal antibody sheep anti-mouse IgG (#515-035-062, Jackson). Chemiluminescence detection was evaluated using a camera and Bio-1D software (Vilber-Lourmat, Collégien, France).

[0162] By flow cytometry and Western blotting, these experiments confirmed that isoform 1 (v1) was highly expressed on the cell surface and that isoform 3 (v5) was detected in the culture supernatants of K562-v5 but not -v1 (Fig. 11A and B).

[0163] K562-v1 (dark) and KU812 (light) were stained with IL-1RAP antibody (red histogram) and compared to unlabeled cells (blue histogram). Relative fluorescence intensity obtained by the software is reported.

[0164] Interestingly, IL-1RAP expression in transfected K562-v1 was higher than in the native IL-1RAP-expressing KU812 cell line (ratio of fluorescence intensity (RFI) = 10.57 vs. 33.46) (Figures 11A and B).

[0165] <Example 8: Proliferation ability of IL-1RAP CART cells> To measure the proliferation potential of IL-1RAP CART cells induced by IL-1RAP target-expressing cells, CFSE-stained C0, Mock, or IL-1RAP CART cells were co-cultured in the presence of K562, K562-v1, K562-v5, or KU812 cell lines (effector / target ratio E:T = 1:1).

[0166] Compared to C0 or Mock cells, effector IL-1RAP CART cells significantly divided only in response to the presence of IL-1RAP cell surface expressing K562-v1 (76.1% ± 10.9) and KU812 cells (81.6% ± 6.16), but to the lowest levels in response to K562-v5 (27.3% ± 9.03) or medium alone (18.8% ± 7.02) (p<0.001, n=4).

[0167] In a similar experiment, except that the E:T ratio was 1:5, we investigated how CART cells induce IL-1RAP + The ability to produce IFN· in the presence of target cells was assessed by intracellular IFN· production staining.

[0168] IL-1RAP CART CD8 cells, but not C0 or Mock cells + or CD8 - The cells were IL-1RAP-expressing target cells K562-v1 (CD8 + : 23.7±0.71%, CD8 - :14.8±3.58%) and KU812(CD8 + : 22.3±2.39%, CD8 - :13.1±2.79%) (p<0.001, n=4) (Figure 12).

[0169] Example 9: Cytokine Profile To determine the cytokine profile produced by CART cells, the Human Th1 / Th2 / Th17 Cytokines Bead Array (CBA) Kit (BD Biosciences) was used according to the manufacturer's instructions, which allows for the quantification of secretion of human IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A. Briefly, 1 × 10 cells were cultured using beads and PE-conjugated anti-cytokine antibodies in the presence (1:1 ratio) or absence (control) of target cells. 5 Supernatants from overnight cultures of CART cells were incubated for 3 h. Beads were washed and acquired by standardized flow cytometry assay. Data were analyzed using FCAP Arraya software Version 3.0 (BD Biosciences). Supernatants collected from target cells alone were used as controls. IL-1RAP expressing targets (K562-IL-1RAP + Representative capture bead fluorescence analysis of culture supernatants of non-transduced, Mock-, or CAR IL-1RAP T cells in the presence or absence (medium, PMA / Iono) of IL-1RAP-positive T cells expressing IL-1RAP-positive T cells (v1, KU812) or non-expressing target (K562). For co-culture of IL-1RAP-positive cell culture supernatants with effectors, the supernatants were diluted 1 / 3. Medium and PMA / Iono are used as negative and positive controls, respectively.

[0170] Finally, in addition to confirming that stimulation of IL-1RAP CART cells resulted in secretion of a Th1-like cytokine profile, we measured Th1 / Th2 / Th17 cytokines in the supernatants following co-culture with C0, Mock, or CART cells (E:T=1:1).

[0171] Only IL-1RAP cell surface expressing K562-v1 and KU812 cells were able to induce cytokine secretion, secreting strong IFNg and IL-2, moderately TNFa, and weakly IL-4, IL-6, and IL-10, but not IL-17, somewhat oriented towards a specific Th1 profile (Figure 13).

[0172] Example 10: IL-1RAP-dependent CAR cytotoxicity and lysis of IL-1RAP-expressing tumor target cell lines T cell-mediated cytotoxicity was analyzed by CD107 degranulation assay. The cytotoxicity of CAR-T cells against live tumor cells was evaluated by incubating at various E:T cell ratios for 20–24 h. IL-1RAP + CD107a&b degranulation assays performed on IL-1RAP CART cells co-cultured with (K532-V1, KU812)-expressing target cells at an E:T ratio of 1:5 showed that IL-1RAP-specific T cells were CD8 - (Mainly CD4 + ) and CD8 + There was a marked cell surface recruitment of CD107a&b in both compartments, but not cell surface IL-1RAP - (K562, K562-v5) expressing cells, and no appreciable degranulation was observed in cell donor Mock or non-transduced (C0) cells (p<0.001, n=4) (Figure 14).

[0173] To measure the IL-1RAP-dependent cytolytic capacity of IL-1RAP CAR-expressing T cells in vitro, fluorescent (eFluor) staining and 7-AAD staining were used to identify CAR T cells and live cells, respectively.

[0174] IL-1RAP +(K562-v1 and KU812) target cells and IL-1RAP - (K562, K562-v5) 7-AAD between target cells - / eFluor - Statistically significant lytic activity (p<0.001, n=4) is seen, characterized by the loss of cells in the gate. Non-transduced or mock-transduced T cells were used as controls (Figure 15).

[0175] <Example 11: Xenograft Mouse Model (In Vivo Test) (FIG. 16)> NSG (NOD.Cg-Prkdcscid Il2rgtm1WjI / SzJ) mice (Charles River, France) were implanted with Luc+, IL-1RAP+ tumor cell lines with or without injection of effector CAR T cells. Mouse survival as well as circulating CAR T cells and tumor burden were analyzed weekly by either cytometry or bioluminescence analysis.

[0176] Briefly, mice were sublethally irradiated with a dose of 3.5 Gy for 24 h prior to transplantation (n=5 / group). 6 IL-1RAP + , Luciferase + , GFP + Expression cell line K562-v1 (K562-v1 IL-1RAP+ / GFP+ / Luc+ ) were implanted into 6- to 8-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice (The Jackson Laboratory, Bar Harbor, ME) via tail vein (i.v.) injection. Four days after tumor injection, 1 × 10 E6 ~5×10 E6 Mock T or IL-1RAP CART cells were injected intravenously once. IL-1RAP+ / GFP+ / Luc+ A group of mice injected with K562-v1 but not treated with T cells served as a control. IL-1RAP+ / GFP+ / Luc+To track tumor burden, mice were intraperitoneally injected with 50 mg / g D-luciferin (Promega, Lyon, France) every week under isoflurane anesthesia, followed by imaging with a NightOwl (Berthold Technologies, Thoiry, France) 10 min later. We assessed the ability of IL-1RAP CART cells to eliminate IL-1RAP-expressing cells in vivo.

[0177] The results showed that after tumor engraftment (D4), IL-1RAP CART cells (E:T=1:1) were cultured in K562-v1 cells until tumor size decreased (D4-D9) and was completely eliminated (>D9). IL-1RAP+ / Luc+ It is known that tumors can be targeted.

[0178] In contrast, tumor growth was observed in untreated or Mock T cell-treated mice and mice died (2 / 3 in both groups at D28), but no mice died in the CART cell-treated group. Interestingly, tumors continued to grow in the absence of CART cells in surviving untreated or Mock T cell-treated mice.

[0179] Example 12: In vitro cytotoxicity against primary IL-1RAP expressing cells from CML patients From a patient with primary TKI-resistant CML (always with BCR-ABL (IS) ratio >10%) over 4 years of 5 lines of treatment with 4 TKIs (Figure 17, top), we were able to generate CART cells with a transduction efficiency of 95.5% (Figure 17, bottom). IL-1RAP CART cells showed dose-dependent cytotoxic activity against IL-1RAP+KU812 cells with an efficiency of 95% at an E:T ratio of 3:1, whereas C0 or Mock T cells showed 18% and 21% alloreactive cytotoxicity, respectively (Figure 18). This was comparable to IL1-RAP CART cells obtained from a healthy donor. Furthermore, coculture of autologous IL-1RAP CART cells with CML patient PBMCs demonstrated specific lysis of IL-1RAP+ / CD34+ cells after 24 hours (76.65±9.2% for IL-1RAP CART cells vs. 4.16±4.3% and 2.78±1.72% for C0 or Mock T cells, respectively) (FIG. 19).

[0180] Moreover, autologous IL1-RAP CART cells (transduction efficiency: 85.33±8.8%) obtained from CML patients (n=3) on or off long-term treatment including TKI (FIG. 21), versus their respective initial long-term cryopreserved (>20 years) peripheral blood stem cell autologous transplants, killed CD34+ / IL-1RAP+ cells with an efficiency of 79.78±10.7% (FIG. 20).

[0181] Example 13: 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 22A and Table 2). Interestingly, the microvascular HMEC-1 endothelial cell line was not recognized by our #A3C3 IL-1RAP mAb (Figure 22B), 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.

[0182] Regarding targeting of healthy hematopoiesis, mAb#A3C3 did not detect HSCs (RFI<1.2, n=5) in bone marrow (Fig. 23A,C) or normal umbilical cord blood (Fig. 23B,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. 23A). We then investigated the in vitro sensitivity of monocytes 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. 23D, right, Table 3), whereas lymphocytes, granulocytes, and K562 IL-1RAP negative cell line were unaffected, even at high E:T ratios (Fig. 22D). Interestingly, at the above E:T ratio, 94.77% of the leukemic cells were killed (Figure 23E).

[0183] [Table 2-1]

[0184] [Table 2-2]

[0185] [Table 2-3]

[0186] [Table 3]

[0187] 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 24). 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 25). These results are consistent with IL-1RAP CART cell immunotherapy with minimal side effects on the hematopoietic system.

[0188] 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, optical microscopy showed that 293T cell cultures transduced with IL-1RAP CAR were sensitive to CID (Figure 26, top). Cytometric analysis showed that in a mixed population of CD19+ and CD19-IL-1RAP CART cells, only CD19-CD3+ cells remained after 24 h of CID exposure (Figure 26, 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 23F). Finally, in vivo evaluation 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 was unaffected after PBS administration, and control T cells (C0) were unaffected by either treatment (Figure 23G).

Claims

1. Use of an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) in the manufacture of a medicament for use in the treatment of chronic leukemia, said CAR having 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) having the amino acid sequence SEQ ID NO:6, a complementarity determining region 2 (CDR2) having the amino acid sequence SEQ ID NO:7, and a complementarity determining region 3 (CDR3) having the amino acid sequence SEQ ID NO:8; (ii) a heavy chain having a complementarity determining region 1 (CDR1) having the amino acid sequence SEQ ID NO:12, a complementarity determining region 2 (CDR2) having the amino acid sequence SEQ ID NO:13, and a complementarity determining region 3 (CDR3) having the amino acid sequence SEQ ID NO:14; Have, use.

2. The use of claim 1, wherein the IL-1RAP binding region is selected from the group consisting of an antibody, Fv, scFv, Fab, or other antibody fragment.

3. The use described in claim 2, wherein the IL-1RAP binding region is an scFv.

4. The use according to any one of claims 1 to 3, wherein the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

5. The use described in claim 4, wherein the membrane-spanning domain is the membrane-spanning domain of CD28.

6. The use according to any one of claims 1 to 5, wherein the anti-IL-1RAP binding region is connected to the transmembrane region by a hinge region.

7. The use of claim 6, wherein the anti-IL-1RAP binding region is connected to the transmembrane region by a hinge region having an IgG1 hinge sequence.

8. The use according to any one of claims 1 to 7, wherein the intracellular signalling domain comprises at least one costimulatory domain.

9. The use of claim 8, wherein the 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 use described in claim 8 or 9, wherein the costimulatory region of the intracellular signaling region is obtained from CD28, 4-1BB (CD137) and / or CD3ζ.

11. Use of an isolated chimeric antigen receptor (CAR) molecule having an antibody or antibody fragment having an anti-IL-1RAP binding domain, a transmembrane domain, and an intracellular signaling domain in the manufacture of a medicament for use in the treatment of chronic leukemia, wherein the anti-IL-1RAP binding domain comprises: (i) a light chain having a complementarity determining region 1 (CDR1) having the amino acid sequence SEQ ID NO:6, a complementarity determining region 2 (CDR2) having the amino acid sequence SEQ ID NO:7, and a complementarity determining region 3 (CDR3) having the amino acid sequence SEQ ID NO:8; (ii) a heavy chain having a complementarity determining region 1 (CDR1) having the amino acid sequence SEQ ID NO:12, a complementarity determining region 2 (CDR2) having the amino acid sequence SEQ ID NO:13, and a complementarity determining region 3 (CDR3) having the amino acid sequence SEQ ID NO:14; Have, use.

12. The use according to claim 11, wherein the CAR is a CAR as defined in any one of claims 2 to 10.

13. 11. A pharmaceutical composition for treating chronic leukemia comprising a vector having a nucleic acid molecule as defined in any one of claims 1 to 10, wherein the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

14. The pharmaceutical composition described in claim 13, wherein the vector is a lentiviral vector.

15. A pharmaceutical composition for treating chronic leukemia comprising a cell carrying a nucleic acid molecule as defined in any one of claims 1 to 10 or a vector as defined in claim 13 or 14, wherein the cell is a T cell.

16. The pharmaceutical composition described in claim 15, wherein the cells are CD8+ T cells.

17. The pharmaceutical composition described in claim 15 or 16, wherein the cell expresses a CAR defined in claim 11 or 12 on its membrane.

18. The pharmaceutical composition according to any one of claims 13 to 17, for use in the treatment of chronic leukemia in a mammal.

19. The pharmaceutical composition of claim 18 for use in treating chronic leukemia in humans.

20. The pharmaceutical composition according to claim 19, wherein the chronic leukemia is a disease associated with IL-1RAP expression.

21. The pharmaceutical composition described in claim 20, wherein the chronic leukemia is chronic myeloid leukemia (CML) or chronic lymphocytic leukemia (CLL).

22. The CAR has an antigen-binding region, a transmembrane region of a CD28 protein, a costimulatory 4-1BB signaling region, and a CD3ζ signaling region, and the antigen-binding region comprises (i) a light chain having a light chain variable region having a complementarity determining region 1 (CDR1) having the amino acid sequence SEQ ID NO:6, a complementarity determining region 2 (CDR2) having the amino acid sequence SEQ ID NO:7, and a complementarity determining region 3 (CDR3) having the amino acid sequence SEQ ID NO:8; (ii) a heavy chain having a heavy chain variable region having a complementarity determining region 1 (CDR1) having the amino acid sequence SEQ ID NO:12, a complementarity determining region 2 (CDR2) having the amino acid sequence SEQ ID NO:13, and a complementarity determining region 3 (CDR3) having the amino acid sequence SEQ ID NO:14; The pharmaceutical composition according to any one of claims 18 to 21, which is an anti-IL-1RAP scFv having the following structure:

23. A pharmaceutical composition according to any one of claims 18 to 22 in combination with at least one tyrosine kinase inhibitor (TKI).

24. The pharmaceutical composition of claim 23 in combination with at least one TKI selected from imatinib, dasatinib, nilotinib, bosutinib, and ponatinib.

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