Anti-il-1RAP car-t cells for the treatment of acute myeloid leukemia

Abstract

The disclosure provides methods of treating a refractory or relapsed acute myeloid leukemia (AML) in a subject comprising administration of a cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antigen-binding fragment thereof that binds IL-1RAP, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, wherein prior to administering the cell, the subject is preconditioned with a lymphodepleting chemotherapy (LDC).

Description

ANTI-IL-1RAP CAR-T CELLS FOR THE TREATMENT OF ACUTE MYELOID LEUKEMIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No.63 / 599,180, filed November 15, 2023, and U.S. Provisional Application No. 63 / 693,501, filed September 11, 2024, each of which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 5041_015PC02_SequenceListing_ST26; Size: 21,525 bytes; and Date of Creation: November 12, 2024), filed with the application, is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] The disclosure provided herein relates to methods of treating acute myeloid leukemia (AML) (e.g., refractory or relapsed (r / r) AML) in a subject comprising administration of a cell expressing a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antigen-binding fragment thereof that specifically binds IL-1RAP, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, wherein prior to the administering the cell, the subject is preconditioned with a lymphodepleting chemotherapy (LDC).BACKGROUND OF THE DISCLOSURE

[0004] AML is a form of haematological cancer characterized by clonal expansion of immature blasts of myeloid origin in the bone marrow (BM), resulting in impaired haematopoiesis and BM failure. While AML is the most frequent acute leukaemia, it is a rare disease. In the United States (US), the age-adjusted incidence of AML is 4.3 per 100,000 annually and there were an estimated 73,168 people living with AML in2020, and 20,050 estimated new cases and 11,540 estimated deaths were reported for 2022. In Europe, the prevalence was reported to be 13.7 per 100,000 inhabitants (males 13.2 and females 13.9 per 100,000). The incidence of AML is age-dependent, rising markedly in patients aged >60 years. The median age at diagnosis is ~70 years.

[0005] For patients with primary refractory or relapsed (r / r) AML, the prognosis remains poor and treatment is challenging. With the ever-growing knowledge of the molecular mutation landscape of AML leukemogenesis, other targeted therapy options with lower toxicity compared to the conventional treatments and stem cell transplantation have emerged. Although the treatment paradigm for r / r AML is moving toward more targeted agents, cytotoxic chemotherapy continues to have a role for young / fit patients as a bridge to haematopoietic stem cell transplantation (HSCT), especially in those with a prolonged initial complete response duration who are expected to have chemotherapy-sensitive disease.

[0006] Based on the success and subsequent approval of cellular CD19-targeted immunotherapies and the development of B cell maturation antigen-targeting CARs in multiple myeloma, CAR T-cells are being developed as anti-AML therapies for r / r patients. Several AML cell surface targets have been explored, e.g., CD33, CD 123, CD44v6, CLL-1, and B7H6. However, all of these targets have potential toxicities due to their expression on healthy haematopoietic stem cells or progenitor cells (HSPCs), and can lead to ablation of all myeloid progeny. Thus, more selective AML cell surface targets need to be explored.

[0007] IL-1RAP is overexpressed on the cell surface of multiple solid cancer types and has been identified as potential therapeutic target in AML, chronic myeloid leukaemia (CML) and myelodysplastic syndromes (MDS) as well as various other indications, including Ewing sarcoma. IL-1RAP potentiates multiple oncogenic signalling pathways in AML and promotes leukaemia cell proliferation and survival via the FLT3 and c-Kit pathways, representing a potentially promising target to treat AML. Accordingly, there exists a therapeutic opportunity for treating AML by targeting IL-1RAP with CAR T-cell-based therapies.SUMMARY OF THE DISCLOSURE

[0008] In one aspect of the present disclosure is provided a method of inhibiting the growth of a tumor cell in a subject in need thereof comprising administering to thesubject a therapeutically effective amount of T cells expressing a chimeric antigen receptor (CAR) at their surface, wherein the CAR comprises an antibody or antigenbinding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 comprising the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14, wherein prior to administering the T cells expressing the CAR, the subject is preconditioned with a lymphodepleting chemotherapy (LDC).

[0009] In one aspect of the present disclosure is provided a method of treating acute myeloid leukemia (AML) (e.g., a refractory or relapsed AML) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of T cells expressing a chimeric antigen receptor (CAR) at their surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an antiinterleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 comprising the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14, wherein prior to administering the T cells expressing the CAR, the subject is preconditioned with a lymphodepleting chemotherapy (LDC).

[0010] In some aspects, the CAR comprises the amino acid sequence of SEQ ID NO: 19.

[0011] In some aspects, the T cells comprise CD4+ and CD8+ T cells. In some aspects, the T cells are autologous.

[0012] In some aspects, the subject has increased IL-1RAP expression compared to a normal subject.

[0013] In some aspects, the T cells expressing the CAR are administered intravenously.

[0014] In some aspects, the LDC comprises administering radiotherapy, fludarabine, or cyclophosphamide.

[0015] In some aspects, the LDC is completed at least 48 hours prior to administering the T cells expressing the CAR. In some aspects, the LDC is initiated at 6 to 10 days prior to administering the T cells expressing the CAR.

[0016] In some aspects, the T cells expressing the CAR are administered at a dose of 0.1 x 106cells / kg, 0.5 x 106cells / kg, 1 x 106cells / kg, 5 x 106cells / kg, or 10 x 106cells / kg.

[0017] In some aspects, the T cells expressing the CAR are administered as a single dose.

[0018] In some aspects, IL-1RAP expression levels are determined by immunohistochemistry (IHC), flow cytometry, or quantitave polymerase chain reaction (qPCR).

[0019] In some aspects, the subject is a human.

[0020] In some aspects, the tumor cell is a hematologic cancer tumor cell, or a tumor cell resulting from a hematologic cancer.

[0021] In some aspects, the AML (e.g., the refractory or relapsed AML) is a minimal residual disease (MRD).

[0022] In some aspects, the AML is an IL-lRAP-expressing AML.

[0023] In some aspects, the method further comprises further comprising detectingIL-1RAP in a sample obtained from the AML (e.g., the refractory or relapsed AML) prior to, during, or after administering the T cells.

[0024] In some aspects, the subject received at least one prior line of therapy, at least two prior lines of therapy, or at least three prior lines of therapy.

[0025] In an aspect, provided herein is a use of the T cells as described herein for inhibiting the growth of an AML (e.g., a refractory or relapsed AML) in a subject in need thereof.BRIEF DESCRIPTION OF THE FIGURES

[0026] FIG. 1 shows a schematic of the CCTx-001 CAR.

[0027] FIG. 2 shows the in vitro cytotoxic capacity of toward IL-1RAP+ expressing cells of CAR T-cells derived from B-L43 / A3C3 mAh. Effector cells (NTD or CAR T- cells) were labelled with Cell Proliferation Dye eFluor™ 450 and co-cultured with IL- 1RAP+ Molm-13 target cell line at the indicated E:T ratios. Target cells without effector cells were also included as negative control. The cell suspensions were incubated for twenty-four hours at 37°C, 5% CO2 prior to staining with a viability marker and analysis by flow cytometry. The cytotoxicity was calculated based on the percentage of viable target cells, normalized by the percentage of viable target cells in the target only condition (target cells without effector cells).

[0028] FIGs. 3A-3F shows the in vitro efficacy and specificity of CCTx-001 (using batches representative of the clinical process, also referred to herein as TR batches) in a co-culture cytotoxicity assay. MOLM-13 (IL-1RAP positive) and Raji (IL-1RAP negative) target cells were co-cultured with CCTx-001 cell products from 3 independent healthy donors (or their donor-matched NTD counterparts) and subsequently analysed by flow cytometry (FC) for the quantification of target cell viability, to confirm the functionality of the CCTx-001 CAR T-cells. Data show viable MOLM-13 (IL- 1RAP -positive) (FIGs. 3A-3C) or Raji (IL-1RAP negative) (FIGs. 3D-3F) target cells as percentage of the target cell only control (in the absence of T-cells, 100% viability). Target cells were co-cultured for 24 hours with nontransduced (NTD) T-cells (dotted curves) or CAR T (solid curves) of donors TR3 (FIGs. 3A and 3D), TR4 (FIGs. 3B and 3E) and TR5 (FIGs. 3C and 3F) at the indicated E:T ratios (blue curves) or were treated with 0.025% Triton X-100 as a toxicity control (red data point). Data represent mean ± SD of 3 technical replicates. Statistical differences between log transformed values of CAR T-cell and NTD T-cell co-cultures (within the same E:T ratio and same T-cell donor) were determined by an unpaired multiple t-test with Welch correction and the Holm-Sidak method to correct for multiple comparisons. *** p < 0.001; ** p < 0.01; *p < 0.05; ‘ns’ (not significant) p > 0.05. CAR T = Chimeric Antigen Receptor T-cells, NTD T = non transduced T- cells. SD = standard deviation.

[0029] FIGs. 4A-4I shows the concentration of IFNy (in pg / mL) secreted from NTD T-cells (dotted curves) or CCTx-001 (C4-derived) anti-IL-lRAP CAR T (solid curves) from donors TR3 (FIGs. 4A, 4D, and 4G), TR4 (FIGs. 4B, 4E, and 4H) and TR5 (FIGs. 4C, 4F, and 41) cultured for 24 hours in the absence (T-cells only) (FIGs. 4A-4C) or presence of IL- 1 RAP -positive MOLM-13 (FIGs. 4D-4F) or IL-1RAP- negative Raji cells (FIGs. 4G-4I) at the indicated E:T ratios. The dotted line represents the LLOQ. Data represent mean ± SD of 3 technical replicates. Statistical differences between log-transformed values of CAR T-cell vs NTD T-cell co-cultures, between CAR T MOLM-13 co-cultures vs CAR T Raji co-cultures, or between CAR T MOLM-13 co-culture vs CAR T-cells alone (within the same E:T ratio and same T- cell donor) were determined by an unpaired multiple t-test with Welch correction and the Holm-Sidak method to correct for multiple comparisons. Statistical analysis for cytokine secretion results were not performed if mean values were below LLOQ (352 pg / mL for 200x diluted samples and 17,6 pg / mL for lOx diluted samples). **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ‘ns’ (not significant) p > 0.05. LLOQ=lower limit of quantification, NTD T = non-transduced T-cells, CAR T = chimeric antigen receptor T-cells.

[0030] FIG. 5 shows the in vitro cytotoxic capacity of CCTx-001 (using TR batches) toward AML cell lines with varying levels of IL-1RAP expression using a co-culture cytotoxicity assay. Effector cells (NTD or CAR T-cells) were labelled with Cell Proliferation Dye eFluor™ 450 and co-cultured with the different AML cell lines at the indicated E:T ratios. Specific cytotoxicity of CAR T-cells are reported as percentages of remaining living cells, gated in FC as eFluor- / 7-AAD-, normalized to cytotoxicity percentages obtained in the control condition using NTD T-cells. Symbols represent median value and shaded area represent the range of the values. IL-1RAP negative: Raji and K562. Three subgroups were identified based on their IL- 1RAP surface expression by FC: IL-1RAP low (ABC between 100 and 500): KG-1 and HEL cell lines; IL-1RAP medium (ABC between 500 and 5000: EOL-1, OCL AML3, HL60 and MOLM-13; and IL-1RAP high (ABC above 5000): Mono-Mac-6. ABC=antibodies bound per cell.

[0031] FIG. 6 shows Kaplan-Meier plot of survival of MOLM-13 -Luc tumourbearing NCG mice treated with vehicle (Group 1, n=8), with non transduced (NTD) human T-cells (Group 2 n=8, labelled as “untransduced T-cells” in the figure) or withdonor matched C4-derived CAR T-cells (Group 3 n=5). iv = intravenous; TTE = Median time to endpoint. Statistical Significance (Logrank test): ne = not evaluable, ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, compared to Group 1. NCG = NOD- rA:t / cem26Crf52Z / 2rgem26Crf22 / NjuCrl.

[0032] FIG. 7 shows the percentage of AML cells with all live human cells in the circulation for group 2 (NTD / C0) and group 3 (CCTx-001). Each error bar is constructed using 1 standard error of the mean. CO = non transduced (NTD) T-cells.

[0033] FIGs. 8A-8C show the kinetics of CAR T -cell (FIG. 8 A) and CAR T-cell subtypes (CD4 and CD8 CAR T-cells) expansion (FIG. 8B) and peak blood levels (FIG. 8C) are shown after adoptive cell transfer (on Day 1) until Day 30 (study endpoint). For each timepoint, the following number of independent mice were analysed: (n=4) from Days 0-14, n=3 at Day 21 and n=l at Day 30. Each error bar is constructed using 1 standard error of the mean.

[0034] FIGs. 9A-9E show the results of an in vivo study of MOLM-13-Luc tumourbearing NCG mice treated with non-transduced (NTD) human T-cells or with donor matched C4-derived CAR T-cells (CCTx-001). FIG. 9A shows an illustration of the in vivo study design. 6 mice were present in each study group. Tumours were injected subcutaneously and allowed to establish for up to 4 days before injection of untransduced or transduced CAR T-cells (IxlO7T-cells intravenously). The study was concluded by day 30 following T-cell injection, or sooner due to tumor burden and / or comorbidities. FIG. 9B shows the mean body weight of each study group across the duration of the study. FIG. 9C shows the results of bioluminescence imaging (BLI) at days 0, 7, 14, and 21 across each mouse in the study. MOLM-13-Luc signal intensity is depicted as luminescence (units in radiance; p / sec / cm2 / sr). FIG. 9D shows a longitudinal analysis of the BLI of each study group, shown as total flux (p / s) per time point. FIG. 9E shows a Kaplan-Meier plot of survival of MOLM-13-Luc tumourbearing NCG mice treated with vehicle, NTD T-cells, or CCTx-001. Probability was of survival was based on the time to reach BLI of 1E+10 p / s. NCG = NOD- Pr]((icem26Cd52Il2r^;m26Cd22 / Nj uCrl .

[0035] FIG. 10 shows a flow chart of the CCTx-001 open-label multicentre Phase 1 / 2 study to assess safety, tolerability, and clinical activity of CCTx-001 in patients with r / r AML. Abbreviations: EOS = end of study; LDC = lymphodepleting chemotherapy;PK = pharmacokinetics; PVS = persistent vector sequence; RCL = replication competent lentivirus.

[0036] FIG. 11 shows a graphical outline of the dose escalation protocol of the CCTx-001 study.

[0037] FIG. 12 shows the in vitro specific cytotoxicity of different CAR T-cells towards primary AML cells as percentages of remaining live AML blasts normalised with NTD T-cells condition. MCL004-009 and MCL004-088 are batches of healthy donor-derived CCTx-001 CAR T-cells. CL045-007 to CL045-011 are the identifiers of the co-cultured primary allogenic AML patient PBMC samples.

[0038] FIG. 13 shows the in vitro cytotoxicity of the different NTD and CAR T-cells towards primary autologous AML blasts reported as percentages of remaining live AML blasts normalised with target alone condition.

[0039] FIG. 14 shows the in vitro specific cytotoxicity of different CAR T-cells toward primary AML cells reported as percentages of remaining live AML blasts normalised with NTD T-cells condition. MCL004-009 and MCL004-088 are batches of healthy donor derived CCTxOOl CAR T-cells. CL045-007 to CEL045-011 are the identifiers of the co-cultured primary allogenic AML patient PBMC samples.

[0040] FIGs. 15A-15B show a Kaplan-Meier curve for low or high IL-1RAP mRNA expression from a publicly available data set (FIG. 15 A) and an in silico analysis of IL-1RAP mRNA in AML patients from multiple available gene set enrichment studies by AML subtype (FAB) classification (FIG. 15B).

[0041] FIGs. 16A-16B show the results of immunophenotyping of IL-1RAP and major AML targets (CD33, CD 123, and CLL1) on blood cell populations from PBMCs of health donors (FIG. 16 A) and on CD34+ hematopoietic stem and progenitor cells from health donors (FIG. 16B).

[0042] FIG. 17 shows IL-1RAP membrane expression (as antibody bound per cell) from AML patient cells in comparison to healthy donor blood and marrow cells.

[0043] FIGs. 18A-18B show functional results of AML patient-derived CCTx-001 CAR-T cells. FIG. 18A shows the results of an IFN y release assay by 4 different patient-derived CCTx-001 CAR-T cell products before and after IL-lRAP-mediated stimulation. FIG. 18B shows the results of an in vitro cytotoxicity assay of patient- derived CCTx-001 CAR-T cell products towards the IL-1RAP expressing MOLM-13 AML cell line at an effectortarget ratio of 1 : 1.DETAILED DESCRIPTION OF THE DISCLOSURE

[0044] The present disclosure is directed to methods of inhibiting growth of a hematologic malignancy / treating cancer comprising administration of a cell comprising a nucleic acid molecule encoding an IL-lRAP-specific CAR. In aspects, prior to administering the cell, a lymphodepleting chemotherapy (LDC) is administered.1. Definitions

[0045] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0046] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to specific compositions or process steps, as such can vary. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein.

[0047] Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0048] Unless defined otherwise, 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 disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0049] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0050] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. As used herein, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms, while retaining their ordinary meanings.

[0051] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

[0052] As used herein, the term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term “approximately” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0053] Amino acids are referred to herein by either their commonly known three letter symbols or by the one -letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single -letter codes.

[0054] As used herein, the terms “ug” and “uM” are used interchangeably with “pg” and “pM,” respectively.

[0055] The term “polypeptide,” as used herein, is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and comprises any chain or chains of two or more amino acids. Thus, as used herein, a “peptide,” a “peptide subunit,” a “protein,” an “amino acid chain,” an “amino acid sequence,” or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of a “polypeptide,” even though each of these terms can have a more specific meaning. The term “polypeptide” can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone post-translational or post-synthesis modifications, for example, conjugation of a palmitoyl group, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, disulfide bond formation, proteolytic cleavage, or modification by non-naturally occurring amino acids. The term “peptide,” as used herein, encompasses full-length peptides and fragments, variants or derivatives thereof. A “peptide” as used herein can be part of a fusion polypeptide comprising additional components such as, e.g., an albumin or PEG moiety, to increase half-life. A peptide as used herein can also be derivatized in a number of different ways. A peptide can comprise modifications including e.g., conjugation of a palmitoyl group. The term “nucleic acid molecule,” as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single- stranded or double- stranded, and can be cDNA.

[0056] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0057] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identitybetween two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0058] The nucleic acid and protein sequences described herein can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res . 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0059] The terms “operatively linked,” “operatively inserted,” “operatively positioned,” “under control” or “under transcriptional control,” as used herein, mean that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The term “operably linked” means that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). The term “operably inserted” means that the DNA of interest introduced into a cell is positioned adjacent a DNA sequence which directs transcription and translation of the introduced DNA (i.e., facilitates the production of, e.g., a polypeptide encoded by a DNA of interest).

[0060] The terms “#E3C3” and “#A3C3” are understood to be identical: #E3C3 being able to be freely used to refer to #A3C3 and vice versa. B-L43 is also identical to #E3C3 and #A3C3, and each refer to hybridoma clones that share identical nucleotide sequences for the heavy and light chains. #E3C3 and #A3C3 are described further in PCT Publication Nos. W02019101604A1 and W02020239801 Al, each of which is incorporated herein by reference in its entirety.

[0061] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an engineered antigen-binding polypeptide, comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Expression of a CAR on the surface of a cell, e.g., an immune cell, allows the cell to target and bind a particular antigen. In some aspects, the CAR is expressed by an immune cell, e.g., a T cell. In some aspects, the antigen binding domain comprises an Fab, Fab’, F(ab’)2, Fd, Fv, single-chain fragment variable (scFv), single chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof. In some aspects, the transmembrane domain comprises a transmembrane domain selected from the transmembrane domain of CD4, CD8a, or CD28. In some aspects, the intracellular domain comprises a costimulatory domain or a portion thereof. In some aspects, the intracellular domain comprises a costimulatory domain selected from the group consisting of the intracellular domain of CD3z, a CD28 co-stimulatory domain, a CD27 co-stimulatory domain, a 4- IBB co-stimulatory domain, an ICOS costimulatory domain, an OX-40 co-stimulatory domain, a GITR co-stimulatory domain, a CD2 co-stimulatory domain, an IL-2RP co-stimulatory domain, an MyD88 / CD40a CD28 co-stimulatory domain, and any combination thereof. A CAR can further comprise a “hinge” or “spacer” domain. Non-limiting examples of hinge / spacer domains include immunoglobulin hinge / spacer domains, such as an IgGl hinge domain, and IgG2 hinge domain, an IgG3 hinge domain, or an IgG4 hinge domain. In some aspects, the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some aspects, the domains in the CAR polypeptide construct are not contiguous with each other, e.g., are in different polypeptide chains.

[0062] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., aCAR T cell. Examples of immune effector function, e.g., in a CAR T cell, include cytolytic activity and helper activity, including the secretion of cytokines. In embodiments, the intracellular signal domain is the portion of the protein which transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0063] In an aspect, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an aspect, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CAR T, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

[0064] A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or IT AM. Examples of IT AM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 (“ICOS”), FcsRI, CD66d, CD32, DAPlO and DAP12.

[0065] The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as the protein provided as GenBank Acc. No. BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that aresufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof.

[0066] The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signalling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDl la / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83.

[0067] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.

[0068] The term “4- IBB” refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like; and a “4-1BB costimulatory domain” is defined as amino acid residues 214-255 ofGenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0069] An “antibody” (Ab) shall include, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as Vzz) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CHI, CHI and CH3. Each light chain comprises a light chain variable region (abbreviated herein as Vz) and a light chain constant region. The light chain constant region is comprises one constant domain, CL. The Vzz and Vz regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each Vzz and Vz comprises three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. A heavy chain may have the C-terminal lysine or not. Unless specified otherwise herein, the amino acids in the variable regions are numbered using the Kabat numbering system and those in the constant regions are numbered using the EU system.

[0070] An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. “Isotype” refers to the antibody class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The term “antibody” includes, by way of example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man. Where not expressly stated, and unless the context indicates otherwise, the term “antibody” includes monospecific,bispecific, or multi-specific antibodies, as well as a single chain antibody. In aspects, the antibody is a bispecific antibody. In other aspects, the antibody is a monospecific antibody.

[0071] As used herein, an “IgG antibody” has the structure of a naturally occurring IgG antibody, z.e., it has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an anti-ICOS IgGl, IgG2, IgG3 or IgG4 antibody consists of two heavy chains (HCs) and two light chains (LCs), wherein the two heavy chains and light chains are linked by the same number and location of disulfide bridges that occur in naturally occurring IgGl, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bonds)

[0072] An “isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds specifically to PD-L1 is substantially free of antibodies that bind specifically to antigens other than PD-1). An isolated antibody that binds specifically to PD-L1 may, however, have cross-reactivity to other antigens, such as PD-L1 molecules from different species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0073] The antibody may be an antibody that has been altered (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, an antibody may include one or more variant amino acids (compared to a naturally occurring antibody) which change a property (e.g., a functional property) of the antibody. For example, numerous such alterations are known in the art which affect, e.g., half-life, effector function, and / or immune responses to the antibody in a patient. The term antibody also includes artificial polypeptide constructs which comprise at least one antibody-derived antigen binding site.

[0074] The term “monoclonal antibody” (“mAb”) refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules whose primary sequences are essentially identical, and which exhibits a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody. MAbs may be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art.

[0075] A “human” antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms “human” antibodies and “fully human” antibodies and are used synonymously.

[0076] A “humanized antibody” refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one aspect of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A “humanized” antibody retains an antigenic specificity similar to that of the original antibody.

[0077] A “chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

[0078] An “anti-antigen” antibody refers to an antibody that binds specifically to the antigen. For example, an anti-IL-lRAP antibody binds specifically to IL-1RAP.

[0079] An “antigen-binding portion” of an antibody (also called an “antigen-binding fragment”) refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody. It has been shown that the antigen-binding function of an antibody can be performed by fragments or portions of a full-length antibody. Examples of binding fragments encompassedwithin the term “antigen-binding portion” or “antigen-binding fragment” of an antibody, e.g., an anti-IL-lRAP antibody described herein, include:(1) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC and CHI domains;(2) a F(ab’)2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;(3) a Fd fragment consisting of the VH and CHI domains;(4) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody,(5) a single domain antibody (dAb) fragment (Ward et al., (1989) Nature 341 :544-46), which consists of a VH domain;(6) a bi-single domain antibody which consists of two VH domains linked by a. hinge (dual-affinity re-targeting antibodies (DARTs));(7) a dual variable domain immunoglobulin;(8) an isolated complementarity determining region (CDR); and(9) a combination of two or more isolated CDRs, which can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigenbinding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0080] The term “IL1RAP,” as used herein, refers to the interleukin-1 receptor accessory protein that is the cellular membrane co-receptor for several receptors in the IL-1 family, including interleukin-1 receptor 1 (IL1R1), ST2 (also known as interleukin-1 receptor-like 1 or IL-IRLI), and interleukin- 1 receptor-like protein 2(IL-1RL2). It is noted that the interleukin-1 receptor accessory protein, or IL1RAP, is sometimes referred to in the art as “IL-1RAP,” “IL-lRAcP,” “ILlRAcP” or “IL- 1R3.” The terms “IL 1 RAP,” “IL- 1 Rap” and “IL 1 RAP protein” are used herein interchangeably.

[0081] A “cancer-associated fibroblast” (CAF) or “tumor-associated fibroblast” or “activated fibroblast” is a cell type within the tumor microenvironment that promotes tumorigenic features by initiating the remodelling of the extracellular matrix or by secreting cytokines.

[0082] An “immune checkpoint inhibitor” refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In particular the immune checkpoint protein is a human immune checkpoint protein. Thus the immune checkpoint protein inhibitor in particular is an inhibitor of a human immune checkpoint protein.

[0083] “Immunotherapy” refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying the immune system or an immune response.

[0084] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0085] The term “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0086] The term “xenogeneic” refers to a graft derived from an animal of a different species.

[0087] As used herein, the term “linked” refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage also can be genetic (i.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.

[0088] As used herein, the terms “subject,” “individual,” or “patient,” refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapyis desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0089] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules, or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In some aspects contacting includes allowing a compound described herein to interact with a protein or enzyme.

[0090] “Administering” refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some aspects, the formulation is administered via a non-parenteral route, in some aspects, orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0091] Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down orpreventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.

[0092] As used herein, “effective treatment” refers to treatment producing a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method. A beneficial effect can also take the form of arresting, slowing, retarding, or stabilizing of a deleterious progression of a marker of a hematologic malignancy. Effective treatment may refer to alleviation of at least one symptom of a hematologic malignancy. Such effective treatment may, e.g., reduce patient pain, reduce the size and / or number of lesions, may reduce or prevent metastasis of a tumor, and / or may slow tumor growth.

[0093] The term “effective amount” refers to an amount of an agent that provides the desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to hematologic malignancies, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. In some aspects, an effective amount is an amount sufficient to completely eradicate the tumor. In some aspects, an effective amount is an amount sufficient to completely eradicate the tumor cells. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and may stop tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer. In one example, an “effective amount” is the amount of IL-1RAP CAR-T clinically proven to affect a significant decrease in cancer orslowing of progression of cancer, such as an advanced hematologic malignancy (e.g., AML).

[0094] The term “progression-free survival,” which can be abbreviated as PFS, as used herein refers to the length of time during and after the treatment of a hematologic malignancy that a patient lives with the disease but it does not get worse. The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a standard control. A therapeutically effective dose or amount may ameliorate one or more symptoms of a disease. A therapeutically effective dose or amount may prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is being administered is to treat a person who is at risk of developing the disease.

[0095] The term “combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present disclosure and a combination partner (e.g. another drug as explained below, also referred to as “therapeutic agent” or “co-agenf ’) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. “Concurrently,”as used herein to, refers to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

[0096] A “cancer” refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor.

[0097] The term “leukemia” refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or nonincrease in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic). Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute myeloid leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross’ leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling’s leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

[0098] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and / or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.

[0099] A “refractory” cancer is one that progresses even though an anti-tumor treatment, such as a chemotherapy, is administered to the cancer patient. An example of a refractory cancer is one which is platinum refractory.

[0100] A “relapsed” cancer is one in which the cancer or the signs and symptoms of a cancer returns after a period of improvement.

[0101] A “complete response” or “complete remission” or “CR” indicates the disappearance of all signs of tumor or cancer in response to treatment. This does not always mean the cancer has been cured. A “CRi” refers to a morphologically complete remissions with an incomplete hematological (blood count) recovery. A “CRMRD-” refers to a complete recovery without measurable residual disease.

[0102] The term “objective response rate” or “ORR” refers to the percentage of patients who achieve a response, which can either be a complete response, or a partial response, after receiving a treatment.

[0103] The term “duration of response” or “DoR” refers to the time from randomization to disease progression or death in patients who achieve complete or partial response. DoR measures how long a patient will respond to treatment without tumor growth or metastasis.

[0104] The term “overall survival” or “OS” refers to the time from randomization to death. With regards to OS, any patients lost to follow up or still alive at the time of evaluation are censored.

[0105] A “CRc” or “complete remission clinical” indicates no evidence of disease with some skin changes not indicative of active disease. A “CR with partial hematologic recovery” or “CRh” refers to a hematologic recovery which is defined as a patient having no signs of leukemia, but one or more blood counts (e.g., platelets and neutrophils) have not returned to normal levels (e.g., absolute neutrophil count (ANC) of over 500 / pl and platelet count over 50,000 / pl) (see, Dohner et al, 2022). A “CR with incomplete hematologic recovery” or “CRi” refers to a hematologic recovery which is defined as a patient having no signs of leukemia, but where an ANC of less than 1 x 109 / L or a platelet count of < 100 x 109 / L (i.e., thrombocytopenia) are present.

[0106] A “morphologic leukaemia-free state” or “MLFS” refers to a patient having less than 5% bone marrow blasts, no circulating blasts, no extramedullary disease, and wherein no hematologic recovery is required. In MLFS, marrow should not merely be “aplastic”; bone marrow spicules should be present, and at least 200 cells should be enumerated in the aspirate or cellularity should be at least 10% in the biopsy.

[0107] A “partial response” or “PR” refers to a decrease in the size or volume of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment.

[0108] “Progressive disease” refers to the appearance of one more new lesions or tumors and / or the unequivocal progression of existing non-target lesions. Progressive disease can also refer to a tumor growth of more than 20% since treatment began, either due to an increases in mass or in spread of the tumor.

[0109] The term “tumor” as used herein refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions, independent of cell of origin.

[0110] An “immune response” refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from a vertebrate’s body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4+cell, a CD8+T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell.[OHl] The term “lymphocyte” as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a major component of the inherent immune system. NK cells reject tumors and cells infected by viruses by inducing apoptosis or programmed cell death in the target cell. They were termed “natural killers” because NK cells do not require activation in order to kill a target cell. T-cells play a major role in cell-mediated- immunity. T-cell receptors (TCR) expressed on the surface of T cells differentiate T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for T cell maturation. There are six types of T-cells, namely: Helper T-cells (e.g. CD4+ cells); Cytotoxic T-cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cells or killer T cell); Memory T-cells ((i) stem memory TSCM cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+, but they also express large amounts of CD95, IL-2R.p, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory TCM cells express L-selectin and the CCR7, they secrete IL-2, but not IFNy or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFNy and IL-4); Regulatory T-cells (Tregs, suppressorT cells, or CD4+CD25+ regulatory T cells); Natural Killer T-cells (NKT); and Gamma Delta T-cells.

[0112] As used herein, “survival” refers to the patient remaining alive, and includes overall survival as well as progression free survival. 1-year survival rate and 2-year survival rate refers to the K-M estimate of the proportion of subjects alive at 12 month or 24 months.

[0113] By “extending survival” is meant increasing overall survival and / or progression free survival in a treated patient relative to a control treatment protocol, such as treatment with the antibody drug conjugates described herein. Survival is monitored for at least about one month, two months, four months, six months, nine months, or at least about 1 year, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc., following the initiation of treatment or following the initial diagnosis.

[0114] By “reduce or inhibit” is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

[0115] The term “heterologous,” in the context of a polypeptide, means from a different source (e.g., a cell, tissue, organism, or species) as compared with another polypeptide, so that the two polypeptides are different. Typically, a heterologous polypeptide is from a different species.

[0116] As used herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to cell proliferation (e.g., cancer cell proliferation) means negatively affecting (e.g., decreasing proliferation) or killing the cell. In some aspects, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. Similarly an “inhibitor” is a compound or protein that inhibits a receptor or another protein, e.g., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity (e.g., a receptor activity or a protein activity).

[0117] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophage-like synoviocytes, etc).

[0118] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0119] Various aspects of the disclosure are described in further detail in the following subsections.2. Methods of the Disclosure

[0120] IL-1RAP is a part of a protein receptor complex and assembled with IL-la, IL-ip or IL-33 receptors on several cells, particularly hematopoietic cells. IL-1RAP has been shown to be overexpressed at cell surface of leukemic stem cells in acute myeloid leukemia (AML), myelodysplastic syndrome and chronic myeloid leukemia without being expressed on normal HSCs. IL-1RAP is known to play a key role intumor microenvironment inflammation through p38 MAPK and NF-kp signaling pathways (De Boer et al. 2020).

[0121] Currently IL-1RAP is targeted by two monoclonal antibodies through the CAN-04 and CAN-10 projects of Cantargia. CAN-04 targets IL-1RAP in solid tumors, mainly lung and pancreas, and CAN-10 targets IL-1RAP in autoimmune and inflammatory diseases. Nidalinimab (CAN-04) is under phase I / IIa clinical investigations (ClinicalTrials.gov: NCT03267316 and NCT04452214) in lung and pancreas cancers, used in association with chemotherapies. However, monoclonal antibodies have shown critical drawbacks in this context compared to adoptive cell therapies such as CAR-T cells, the main being the absence of long-term immune memory establishment.

[0122] The present disclosure relates to a cell comprising a nucleic acid molecule encoding a CAR for use in the treatment of hematologic malignancies (e.g., acute myeloid leukemia (AML), such as refractory or relapsed AML), wherein the CAR comprises an antibody or antigen-binding fragment thereof which includes an anti-IL- 1RAP binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain. Exemplary CARs and their sequences are disclosed in PCT Publication Nos. WO 2019 / 101604 and WO 2020 / 239801, which are herein incorporated by reference in their entirety.

[0123] In one aspect, the anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 6, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 7 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 12, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 13 and a complementary determining region 3 (CDR3) having at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 14.

[0124] CARs contemplated herein, may comprise one, two, three, four, or five or more linkers. In particular aspects, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some aspects, 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 long.

[0125] Illustrative examples of linkers include glycine polymers (G)n; glycine-serine polymers (Gi_sSi_5)n, where n is an integer of at least one, two, three, four, or five; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the CARs described herein. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains {see Scheraga, Rev. Computational Chem. 1 1173-142 (1992)). In one aspect, the design of a CAR can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired CAR structure.

[0126] In a particular aspect, the linker is between the VH and VL domains.

[0127] In a particular aspect, the linker comprises or consists in the amino acid sequence of SEQ ID NO: 5.

[0128] In one aspect, the IL-1RAP binding domain is a scFv comprising a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable regions of SEQ ID NO: 4 and a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region of SEQ ID NO: 2.

[0129] In one aspect, the IL-1 RAP binding domain is a scFv comprising (i) a light chain variable region comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 6, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100%identity with the amino acid sequence SEQ ID NO: 7 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain variable region comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 12, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 13 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 14.

[0130] The binding domain of the CAR is generally followed by one or more “hinge regions”, which play a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. A CAR generally comprises one or more hinge regions between the binding domain and the transmembrane domain. The hinge region may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.

[0131] In one aspect, the anti-IL-lRAP binding domain is connected to the transmembrane domain by a hinge region.

[0132] In one aspect, the hinge region comprises the hinge sequence of IgGl or a sequence with 95-99% identity thereof. IgG hinges are encoded by single exons. Thus, the term “hinge sequence of IgGl” as used herein has the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs, i .e. the 15 amino acid residues encoded by the IgGl exon for the hinge (Fundamental Immunology, Fifth edition, Chapter 3, Immunoglobulins: Structure and Function - The immunoglobulin Flinge).

[0133] In further aspects, the hinge region comprises the hinge sequence of IgG4 or a sequence with 95-99% identity thereof. In further aspects, the hinge region may also comprise the CH2-CH3 region of IgGl or IgG4 or a sequence with 95-99% identity thereof.

[0134] In further aspects, the hinge region comprises CD8alpha or a sequence with 95-99% identity thereof.

[0135] The “transmembrane domain” is the portion of the CAR that fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain can be derived either from a natural, synthetic, semi -synthetic, or recombinant source.

[0136] In one aspect, the encoded CAR includes a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154, more preferably CD28.

[0137] In certain aspects, the CARs contemplated herein comprise an intracellular signaling domain. An “intracellular signaling domain,” refers to the part of a CAR that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain.

[0138] The term “effector function” refers to a specialized function of the cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term “intracellular signaling domain” is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal.

[0139] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigendependent primary activation through the TCR (e.g. a TCR / CD3 complex) and costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or co- stimulatory signal. In some aspects, a CAR contemplated hereincomprises an intracellular signaling domain that comprises one or more “costimulatory signaling domain.”

[0140] In one aspect, the isolated nucleic acid molecule may encode an intracellular signaling domain comprising at least one costimulatory domain. In this aspect, the intracellular signaling domain therefore comprises at least one costimulatory domain.

[0141] As used herein, the term “co-stimulatory signaling domain,” or “co stimulatory domain”, refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen.

[0142] Preferably, the at least one costimulatory domain of the functional intracellular signaling domain is obtained from one or more protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, CD3 zeta, ICAM-1, LFA-1 (CD1 la / CD18), ICOS (CD278), and 4-1BB (CD137).

[0143] In one aspect, the costimulatory domain obtained from 4-1BB (CD137) has a sequence having 95-99% identity with the amino acid sequence of the costimulatory domain of 4-1BB. In one aspect, the costimulatory domain obtained from CD3 zeta has a sequence having 95-99% identity with the amino acid sequence of the costimulatory domain of CD3 zeta. In another aspect, the intracellular signaling domain comprises a costimulatory domain obtained from 4- IBB and / or a costimulatory domain obtained from CD3 zeta.

[0144] In particular aspects, a CAR comprises a CD3z primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling and co-stimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.

[0145] In one aspect, the CAR comprises the amino acid sequence of SEQ ID NO: 19.

[0146] A cell that can be used according to the disclosure may be isolated. An “isolated cell” refers to a cell that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix. The cell that is used for treating a hematologic malignancy according to the disclosure can be prepared by inserting; within the genome of a host cell, the nucleic acid molecule encoding the chimeric antigen receptor (CAR) using a vector.

[0147] The term “vector” is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.

[0148] The cell that is used for treating a hematologic malignancy according to the disclosure may be prepared using a vector comprising a nucleic acid molecule encoding the CAR, said vector is selected from a DNA, a RNA, a plasmid, a lentivirus vector, an adenoviral vector, or a retrovirus vector, preferably a lentivirus vector. In some aspects, the vector comprises a promoter, preferably an EF-1 alpha promoter.

[0149] Retroviruses are a common tool for gene delivery. In particular aspects, a retrovirus is used to deliver a polynucleotide encoding a chimeric antigen receptor (CAR) to a cell. 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 subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.

[0150] Thus, the T cells transduced with the vector can elicit a stable, long-term, and persistent CAR-mediated T-cell response. In particular aspects, the T cell is transduced with a retroviral vector, e.g., a lentiviral vector, encoding the CAR.

[0151] As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0152] The term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus. “Self-inactivating” (SIN) vectors refers to replication-defective vectors, e.g., retroviral or lentiviral vectors, 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. In one aspect, SIN vector backbones are preferred. In one aspect, the vector used further comprises a promoter, e.g. an EF-1 alpha short promoter and spl63 enhancer and / or a CMV promoter.

[0153] The term “promoter” as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an R A polymerase binds. An R A polymerase initiates and transcribes polynucleotides operably linked to the promoter. In a particular aspect, it may be desirable to express a polynucleotide comprising a CAR from a promoter that provides stable and long-term CAR expression in T cells and at sufficient levels to redirect the T cells to cells expressing the target antigen.

[0154] The cell for use according to the disclosure can be a T cell, e.g., human T cell. In one aspect, the cell is a CD8+ T cell, e.g., human CD8+ T cell. In one aspect, the cell for use according to the disclosure (e.g. T cell) expresses the CAR (e.g., SEQ ID NO: 19) at its membrane. The term “at its membrane” as used herein has the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs, i .e. “at the cell surface membrane”.

[0155] In particular aspects, prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells is obtained from a subject. In particular aspects, the cells for use according to the disclosure encompass T cells. T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g ., FICOLL™ separation. Cells from the circulating blood of an individual may be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing.

[0156] T cells may be isolated from peripheral blood mononuclear cells by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through aPERCOLL™ gradient. A specific subpopulation of T cells, expressing one or several markers like CD4 or CD8 can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0157] Thus, disclosed herein is cellular therapy where T cells are genetically modified ex vivo to express a CAR and the CAR-T cell is infused to a recipient in need thereof. The infused cell is able to inhibit the growth of hematologic malignancy cells (e.g., AML cells) in the recipient, preferably a human. Unlike antibody therapies, CAR-T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. In aspects, prior to infusing the CAR-T cell to a recipient in need thereof, the recipient is preconditioned with a lymphodepleting chemotherapy (LDC).

[0158] Moreover, CARs allow for the redirection and activation of effector T cells towards any cell surface molecule upon binding by the antibody derived receptor, and are independent of MHC restriction.

[0159] The genetically-modified cells, e.g. T cells, for use according to the disclosure can be constructed starting from the cells of the patient (autologous), but they can also originate from other allogenic donors to provide allogenic genetically-modified cells in bone marrow or peripheral hematopoietic stem cell allograft context (Donor lymphocytes infusion). These cells expressing the IL-1RAP CAR molecule are useful to treat cancer in a mammal, preferably a human.

[0160] These cells, e.g. T cells, express a CAR molecule comprising an antigen binding domain that is an anti-IL-lRAP scFv comprising an anti-IL-lRAP binding domain, a transmembrane domain of the CD28 protein, a costimulatory 4-1BB signaling domain, and a CD3(^ signaling domain, wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 6, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 7 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ IDNO: 8, and (ii) a heavy chain comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 12, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 13 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 14. In one aspect, the CAR molecule comprises the amino acid sequence of SEQ ID NO: 19. In some aspects, the CAR molecule further comprises an IgGl hinge sequence between the scFv and transmembrane domain. In some aspects, the CAR molecule comprises an immunoglobulin G (IgG) hinge region, a CD28 transmembrane and intracellular signaling domain, a 4- IBB signaling domain, and the CD3(^ chain signaling domain.

[0161] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g ., cancer. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0162] Thus, the present disclosure provides a method for the treatment of acute myeloid leukemia (AML) (e.g., refractory or relapsed (r / r) AML) comprising administering to a subject in need thereof, a therapeutically effective amount of a cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment which includes an anti- IL-1RAP binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) having at least 80% identity with the amino acid sequence SEQ ID NO: 6, a complementary determining region 2 (CDR2) having at least 80% identity with the amino acid sequence SEQ ID NO: 7 and a complementary determining region 3 (CDR3) having at least 80% identity with the amino acid sequence SEQ IDNO: 8, and (ii) a heavy chain comprising a complementary determining region 1 (CDR1) having at least 80% identity with the amino acid sequence SEQ ID NO: 12, a complementary determining region 2 (CDR2) having at least 80% identity with the amino acid sequence SEQ ID NO: 13 and a complementary determining region 3 (CDR3) having at least 80% identity with the amino acid sequence SEQ ID NO: 14, wherein prior to administering the cell, the subject is preconditioned with a lymphodepleting chemotherapy (LDC). In one aspect, the CAR molecule comprises the amino acid sequence of SEQ ID NO: 19.

[0163] The cells, e.g. T cell, may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as chemokines, cytokines (e.g., IL-2), or cell populations. Briefly, pharmaceutical compositions may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0164] In one aspect, the CAR-modified cells or the compositions are administered to a subject by direct injection into a tumor. In one aspect, the CAR-modified cell, e.g. CAR-modified T cell, are useful to treat a subject diagnosed with cancer, by removing immune effector cells from the subject, genetically modifying said immune effector cells with a vector comprising a nucleic acid encoding a CAR as described herein, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In one aspect, the immune effector cells comprise T cells.

[0165] A “therapeutically effective amount” of a genetically modified therapeutic cell may vary according to factors such as the disease state, age, sex, and weight of theindividual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The quantity, frequency of administration and the sequence of the possible association with conventional cancer treatments will be determined by such factors as the condition of the patient, and the type and severity of the cancer.

[0166] In some aspects, wherein the T cells expressing the CAR are administered at a dose of 0.1 x 106cells / kg, 0.5 x 106cells / kg, 1 x 106cells / kg, 5 x 106cells / kg, or 10 x 106cells / kg. In some aspects, wherein the T cells expressing the CAR are administered at a dose of about 0.1 x 106cells / kg. In some aspects, wherein the T cells expressing the CAR are administered at a dose of about 0.5 x 106cells / kg. In some aspects, wherein the T cells expressing the CAR are administered at a dose of about 1 x 106cells / kg. In some aspects, wherein the T cells expressing the CAR are administered at a dose of about 5 x 106cells / kg. In some aspects, wherein the T cells expressing the CAR are administered at a dose of about 10 x 106cells / kg. In some aspects, the T cells expressing the CAR are administered as a single dose. In some aspects, the T cells expressing the CAR are administered intravenously.Lymphodepleting chemotherapy (LDC)

[0167] In one aspect, the T cell expressing the CAR molecule specific for IL-1RAP may be used in a method to inhibit growth of a refractory or relapsed AML in a subject. In one aspect, the subject has already been treated by at least one therapy line, such as chemotherapy. In some aspects, the subject has already been treated with one or more of cytarabine, anthracycline, daunorubicin, venetoclax, decitabine, idarubicin, cladribine, G-CSF, and / or azacytidine. In some aspects, the subject has received an allogeneic haematopoietic stem cell transplantation (HSCT). In some aspects, the subject has already been treated with one or more of a conjugated antibody (CD33- GO, gemtuzumab ozogamicin, and antibodies targeting CD44, CD 123 or CD47), bispecific T-cell engager (BiTE) antibodies (targeting CD3 / CD33 or CD3 / CD123), and immune checkpoint inhibitors (targeting PD-l / PDL-1, anti-TIM-3, and anti- CTLA4).

[0168] In one aspect, the cell expressing the CAR molecule specific for IL-1RAP may be used in a method to treat cancer in a subject. In one aspect, the subject has already been treated by at least one therapy line, such as chemotherapy. In another aspect, the subject is treated with a combination of the T cell and chemotherapy, either simultaneously or sequentially, in any order.

[0169] In some aspects, prior to administering the cell (e.g., the T cell) expressing the CAR molecule specific for IL-1RAP in a subject, the subject is preconditioned with a lymphodepleting chemotherapy (LDC). In some aspects, the LDC comprises administering radiotherapy, fludarabine, or cyclophosphamide. In some aspects, the LDC comprises administering fludarabine and cyclophosphamide. In some aspects, the fludarabine is administered at a dose of about 20 to about 40 mg / m2 / day. In some aspects, the fludarabine is administered at a dose of about 20 mg / m2 / day. In some aspects, the fludarabine is administered at a dose of about 30 mg / m2 / day. In some aspects, the fludarabine is administered at a dose of about 40 mg / m2 / day. In some aspects, the cyclophosphamide is administered at a dose of about 200 to about 400 mg / m2 / day. In some aspects, the cyclophosphamide is administered at a dose of about 200 mg / m2 / day. In some aspects, the cyclophosphamide is administered at a dose of about 300 mg / m2 / day. In some aspects, the cyclophosphamide is administered at a dose of about 400 mg / m2 / day.

[0170] In some aspects, the LDC is completed at least 48 hours prior to administering the T cells expressing the CAR. In some aspects, the LDC is initiated at 6 to 10 days prior to administering the T cells expressing the CAR. In some aspects, the LDC is initiated about 6, 7, 8, 9, or 10 days prior to administering the T cells expressing the CAR. In some aspects, the fludarabine administration is for about 4 days. In some aspects, the fludarabine administration is for about 3 days. In some aspects, the cyclophosphamide administration is for about 3 days. In some aspects, the cyclophosphamide administration is for about 2 days.

[0171] In one aspect, the present disclosure is directed to identifying a patient as having increased IL-1RAP protein expression and treating the subject by administering a CAR-T cell of the disclosure, wherein prior to administering the CAR-T cell, the subject is preconditioned with a LDC.3. Measurement of IL-1RAP expression

[0172] In order to assess the IL-1RAP expression, in one aspect, a test sample is obtained from the patient who is in need of the therapy. In some aspects, a test sample includes, but is not limited to, any clinically relevant sample, such as a tumor biopsy, a bone marrow biopsy, a bone marrow aspiration, a core biopsy tissue sample, a fine needle aspirate, or a sample of bodily fluid, such as blood, plasma, serum, lymph, ascites fluid, cystic fluid, or urine. In some aspects, the test tissue sample is from a primary tumor. In some aspects, the test sample is from a metastasis. In some aspects, test samples (e.g., test sample for IL-1RAP expression) are taken from a subject at multiple time points, for example, before treatment (e.g., the T cells), during treatment, and / or after treatment. In some aspects, test samples are taken from different locations in the subject, for example, a sample from a primary tumor and a sample from a metastasis in a distant location. In some aspects, IL-1RAP is detected in a sample obtained from the patient (e.g., the AML patient) prior to administering the therapy (e.g., the T cells).

[0173] In some aspects, the test tissue sample is a paraffin-embedded fixed tissue sample. In some aspects, the test tissue sample is a formalin-fixed paraffin embedded (FFPE) tissue sample. In some aspects, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some aspects, the test tissue sample is a frozen tissue sample. In some aspects, the test tissue sample is a fresh frozen (FF) tissue (e.g., tumor) sample. In some aspects, the test tissue sample is a cell isolated from a fluid. In some aspects, the test tissue sample comprises circulating tumor cells (CTCs). In some aspects, the test tissue sample comprises circulating lymphocytes. In some aspects, the test tissue sample is an archival tissue sample. In some aspects, the test tissue sample is an archival tissue sample with known diagnosis, treatment, and / or outcome history. In some aspects, the sample is a block of tissue. In some aspects, the test tissue sample is dispersed cells. In some aspects, the sample size is from about 1 cell to about 1 x 106cells or more. In some aspects, the sample size is about 1 cell to about 1 x 105cells. In some aspects, the sample size is about 1 cell to about 10,000 cells. In some aspects, the sample size is about 1 cell to about 1,000 cells. In some aspects, the sample size is about 1 cells to about 100 cells. In some aspects, the sample size is about 1 cell to about 10 cells. In some aspects, the sample size is a single cell.

[0174] In another aspect, the assessment of IL-1RAP expression can be achieved without obtaining a test tissue sample. In some aspects, selecting a suitable patient includes (i) optionally providing a test tissue sample obtained from a patient with cancer of the tissue, the test tissue sample comprising tumor cells; and (ii) assessing the proportion of cells in the test tissue sample that express IL-1RAP on the surface of the cells based on an assessment that the proportion of cells in the test tissue sample that express IL-1RAP on the cell surface is higher than a predetermined threshold level.

[0175] In any of the methods comprising the measurement of IL-1RAP expression in a test sample, however, it should be understood that the step comprising the provision of a test sample obtained from a patient is an optional step. That is, in certain aspects the method includes this step, and in other aspects, this step is not included in the method. It should also be understood that in certain aspects the “measuring” or “assessing” step to identify, or determine the number or proportion of, cells in the test sample that express IL-1RAP is performed by a transformative method of assaying for IL-1RAP expression, for example by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay, IHC, flow cytometry (FC), imaging mass cytometry (IMC), or a mass spectroscopy imaging (MSI) assay. In certain other aspects, no transformative step is involved and IL-1RAP expression is assessed by, for example, reviewing a report of test results from a laboratory. In some aspects, IL-1RAP expression is assessed by reviewing the results of, for example, an immunohistochemistry assay from a laboratory. In certain aspects, the steps that provide the test result is performed by a medical practitioner or someone acting under the direction of a medical practitioner. In other aspects, these steps are performed by an independent laboratory or by an independent person such as a laboratory technician.

[0176] In certain aspects of any of the present methods, the proportion of cells that express IL- 1 RAP is assessed by performing an assay to detect the presence of IL- 1RAP RNA. In further aspects, the presence of IL-1RAP RNA is detected by RT- PCR, in situ hybridization or RNase protection. In some aspects, the presence of IL- 1RAP RNA is detected by an RT-PCR based assay. In some aspects, scoring the RT- PCR based assay comprises assessing the level of IL-1RAP RNA expression in the test tissue sample relative to a predetermined level.

[0177] In other aspects, the proportion of cells that express IL-1RAP is assessed by performing an assay to detect the presence of IL-1RAP polypeptide. In further aspects, the presence of IL- 1 RAP polypeptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some aspects, IL-1RAP expression is assayed by IHC, imaging mass cytometry (IMC), or mass spectroscopy imaging (MSI). In other aspects of all of these methods, cell surface expression of IL-1RAP is assayed using, e.g., IHC or in vivo imaging.EXAMPLESExample 1: IL-1RAP as a putative therapeutic target in AML

[0178] The standard frontline (first-line) “7+3” therapy for AML consists of an induction phase with 7 days of high doses of cytarabine and 3 days of anthracy cline (daunorubicin) chemotherapy followed by a consolidation phase of chemotherapy or allo-HSCT for high-risk patients (Heuser 2020, NCCN 2022). For patients unfit to undergo standard induction therapy, the bcl-2 inhibitor venetoclax in combination with demethylating agents such as azacitidine is an active regimen and has rapidly been adopted as less toxic alternative (DiNardo 2020). With the ever-growing knowledge of the molecular mutation landscape of AML leukemogenesis, other targeted therapy options with lower toxicity compared to the conventional treatments have emerged, such as FMS-like tyrosine kinase (FLT3) inhibitors like midostaurin and gilteritinib, the isocitrate dehydrogenase 1 / 2 mutant inhibitors ivosidenib and enasidenib, and the B-cell lymphoma 2 inhibitor venetoclax (VEN) in combination with hypomethylating agents (HMAs) (DiNardo 2018, DiNardo 2020). Despite improvements in first line therapies, currently the only curative option is high-dose chemotherapy and allo-HSCT (Dohner 2022). Roughly 10% to 20% of younger and 50% of older AML patients do not achieve complete remission after at least 2 courses of intensive induction therapy and are considered to have primary refractory disease; in addition, 50% to 70% of patients who obtain CR will relapse (Dohner 2022). These data highlight the critical issue pertaining to a relapse of leukaemia following an initial response (Roussel 2020). For patients with primary relapsed / refractory (r / r) AML the prognosis remains poor and treatment is challenging (Heuser 2020).

[0179] Despite all these improvements in therapy, primary resistance to initial treatment and disease relapse remain unmet needs in the treatment of AML. Current treatment strategies are not successful enough, and eventually, most AML patients still succumb to the disease. Despite improvements in HSCT and several other treatments, the outcome for high-risk patients remains poor and only approximately 40% to 45% of young patients and 10% to 20% of elderly patients can be cured, indicating that there is an unmet medical need and suggesting the necessity of new alternatives treatments (Bose 2017).

[0180] In summary, patients with r / r AML have few therapeutic options left and are recommended to join clinical studies. Accordingly, there is a high medical need for effective treatments, including chimeric antigen receptor T (CAR T) cell therapies.

[0181] Interleukin (IL)- 1 plays an important role in innate and adaptive immunity and has been associated with acute and chronic inflammation, and cancer progression. Two forms of IL-1, IL-la and IL-ip, have been identified. IL-ip is considered the more potent cytokine while IL-la seems to have the broader activity (Fields 2019). IL-la and IL-ip both bind to the same cellular receptor to induce signalling and are considered as potential therapeutic targets due to their roles during cancer progression. Interleukin- 1 receptor (IL-1R) is the main receptor for IL-1. Upon receptor engagement, IL-1R1 forms a heterodimer with Interleukin- 1 Receptor Accessory Protein (IL-1RAP), which functions as a co-receptor (Volpe 1997, Wesche 1997, Frenay 2022). IL-1RAP is a transmembrane protein that interacts with IL-1R1, IL-1R2, IL-1RL1 and IL1RL2 and is required for IL-1, IL-33, and IL-36 signal transduction. IL-1RAP mediates IL- 1 -dependent activation of NF-kappa-B and other signalling pathways. While IL-1RAP cannot bind directly to IL-1, it is essential for IL-l-mediated signalling. The IL-1RAP gene is located on chromosome 3q28 (Dale 1998). Importantly, IL-1RAP is not expressed in most healthy tissues (Zhang 2021), and IL-1RAP knockout mice are viable without overt phenotypic changes (Cullinan 1998).

[0182] IL-1RAP is overexpressed in multiple solid cancer types and has been identified as potential therapeutic target in various indications, including Ewing sarcoma (Zhang 2021), pancreatic adenocarcinoma (Herremans 2022, Zhang 2022), and gastrointestinal adenocarcinoma (Lv 2021, Rehman 2022). Due to its expression on the tumour cell surface, various direct targeting approaches are in development insolid tumours, including mAbs (Robbrecht 2022, Rydberg Millrud 2022). Nadunolimab, an anti-IL-lRAP mAb, as monotherapy and in combination with chemotherapy is under clinical evaluation in solid tumours (NCT03267316, NCT04452214, NCT05181462, NCT04990037, NCT05116891).

[0183] IL-1RAP was identified as a potential anticancer target in chronic myeloid leukaemia, AML, and myelodysplastic syndromes as well (Jaras 2010, Askmyr 2013, Zhao 2014, Agerstam 2015, Landberg 2016, Shastri 2017, Blatt 2018, De Boer 2021, Eldesouki 2021). IL-1RAP is selectively expressed on the majority of AML blasts and to some degree on monocytes, but not on HSCs (Houtsma 2022, Trad 2022). A recent publication reported that patients with AML with higher expression of IL3RA / CD123 and IL-1RAP had inferior overall survival (p< 0.01 for IL3RA; p< 0.05 for IL-1RAP) (Cheng 2023). IL-1RAP potentiates multiple oncogenic signalling pathways in AML and promotes leukaemia cell proliferation and survival via the FLT3 and C-kit pathways, representing a potentially promising target to treat AML (Mitchell 2018, De Boer 2021). The need of IL-1RAP for leukemic cells suggests a low risk of antigen escape of IL-1RAP downregulating AML cells after being targeted by IL- 1RAP CAR T-cell immunotherapy. Hence, it might offer an unique safety profile compared to other AML-targets such as CLL-1 and CD123 (Mardiana 2020).

[0184] In preclinical experiments, IL- 1 RAP-directed CAR T-cells have shown promising antileukemic activity in models of AML and CML (Zhao 2017, Warda 2019, Warda 2021, Trad 2022, Nicod 2023), and IL- 1 RAP-directed CAR NK-cells in Ewing sarcoma (Luo 2022).Example 2: CCTx-001 Pre-Clinical StudiesExample 2.1. IL-1RAP overexpression in AML cell lines and primary samples.

[0185] Overexpression of IL-1RAP was demonstrated in AML cells (leukemic stem cells [LSCs] and blasts) when compared to normal haematopoietic progenitor and stem cells. High IL-1RAP mRNA expression was associated with a statistically significant shorter overall survival in AML (p<0.05). Of relevance, membranous IL- 1RAP is overexpressed in all AML FAB subtypes (Trad 2022).

[0186] Using publically available AML patient mRNA expression data (n=539) (see, Karakaslar EO et al. NPJ Precis. Oncol. 2024; 8(1): 105), a Kaplan-Meier curve was generated, stratified by either low or high IL-1RAP expression (Figure 15A). Thisanalysis indicated that high IL-1RAP mRNA expression is associated with shorter overall survival in AML. Further, varying amounts of IL-1RAP mRNA expression levels were noted from an in silico analysis of IL-1RAP in AML patients (n=832), dependent on the AML subtype by morphological differentiation stage (FAB) classification (Figure 15B).

[0187] The following AML cells lines were assessed for IL-1RAP membranous expression: MOLM-13 (M5 as per FAB classification), Mono-Mac-6 (M5), EOL-1 (M4 eosinophil), KG-1 (MO), HEL (M6 erythrocyte), HL60 (M2), OCLAML3 (M4). As shown in Table 1, all of the AML cell lines tested were shown to be positive for IL-1RAP expression (ranging from 100 to 8752 ABC). Overall, the cell lines were classified into 3 subgroups: KG-1 and HEL cell lines were classified as IL-lRAP-low expressing cell lines, EOL-1, OCI-AML3, HL60 and MOLM-13 were classified as IL- 1 RAP-medium expressing cell lines; and Mono-Mac-6 cell lines was classified as IL-lRAP-high expressing cell line.Table 1: IL-1RAP Expression on AML Cell Lines<FI= Relative fluorescence intensity, ABC= Antibody binding capacity

[0188] A comparison of IL-1RAP membrane expression (in ABC) between AML patients (AML blasts and AML-derived monocytes) and healthy donor blood and marrow cells (differentiated immune cells and progenitor cells) is shown in Figure 17. This data further indicates that IL-1RAP is overexpressed in blasts from AML patients, while IL-1RAP is not expressed in healthy peripheral blood cells and bone marrow hematopoietic cells.

[0189] Expression of IL-1RAP, CD33, CD123, and CLL1 on peripheral blood mononuclear cells (PBMC) was also assessed. PBMCs were isolated from 5 healthy blood donors. Cells were stained for each marker, and detected by flow cytometry.Expression of IL-1RAP was compared to CD33, CD123, CLL1 or Isotype control. Quantibrite PE beads (BD) were run in parallel to quantify levels of the above markers expression on each immune cell population. Marker expression was studied on the following cell types: B cells, T cells, Monocytes, Basophils, Neutrophils, Eosinophils, NK cells, conventional dendritic cells (eDC), and plasmacytoid dendritic cells (pDC).

[0190] Ahead of sample acquisition, BD Quantibrite PE beads were resuspended, fixed as per the cellular assay protocol, and run on the NovoCyte Penteon Flow Cytometer. The gate was set around the bead singlets and analysed using a histogram to view the bead statistics. Using the same instrument settings, the cellular assay samples were acquired. Antibody bound per cell (ABC) values were generated by subtracting isotype geoMean from acquired sample geoMean then extrapolating in logarithmic scale, cellular sample data using the standard curve and the equation y = a * x + b where a = slope and b is Y-intercept.

[0191] Results of the normal PBMC donor analysis are shown in Figure 16A. IL- 1RAP expression was detected at much lower levels than the other targets and was only found at very low levels on monocytes (ABC=719.19) and basophils (ABC=470.73) across the five donors. The most widely expressed marker was CLL1 which was detected on monocytes (ABC=6722.46), cDc (ABC=5618.82), neutrophils (ABC=4718.81), basophils (ABC=2606.63), , eosinophils (ABC=2005) and less so on pDC (ABC= 766.1). CD33 was consistently detected on monocytes (ABC=5915.37) and eDC (ABC=3404.11) and to a lower extent on basophils, neutrophils and eosinophils (ABC=1136.5, 1024.86 and 493.7 respectively); CD123 was consistently detected across the donors, predominately on basophils (ABC=7240.74) and to a lower extent on eDC (AB C=1193.96), and monocytes (ABC=307.04). CD123 expression on pDC was notably high (ABC= 17342.96) compared to all other target expression across cell subpopulations.

[0192] Further, flow cytometry evaluation of IL-1RAP, CLL-1, CD33 and CD123 expression on hematopoietic stem and progenitor populations of human bone marrow CD34+ cells was performed. Four lots of bone marrow (BM) CD34+ cells from normal, healthy donors were evaluated by flow cytometry and sub-populations of human stem and progenitor cells were evaluated based on expression of surface markers CD34, CD38, CD90, and CD45RA. Expression of target surface markersCLL-1, CD123, CD33 and IL-1RAP on the stem and progenitor populations [committed progenitors (CD34+CD38+), hematopoietic stem cells (HSC), Lymphoid- primed Multipotent Progenitors (LMPP) and Multipotent Progenitors (MPP)] was evaluated and compared to isotype-stained controls. Target expression was quantified using quantification beads and the antibody binding capacity (ABC) calculated (Figure 16B). While expression of CLL-1, CD123 and CD33 varied for each donor and each progenitor population evaluated, IL- 1RAP target expression was very low (ABC = < 140) on all BM CD34+ stem and progenitor cell populations for each donor tested.

[0193] Altogether the analysis of AML cell line and AML primary cells confirmed expression of IL-1RAP on the surface of the cancer cells and therefore constitute a promising target for the treatment of AML.Example 2.2 Affinity of B-L43 mAb to IL-1RAP

[0194] Affinity of the B-L43 mAb to human IL-1RAP was demonstrated by ELISA, FC and Surface Plasmon Resonance (SPR) (Table 2). The recombinant scFv derived from the B-L43 / A3C3 sequence was also assessed, showing similar binding characteristics by ELISA to the B-L43 mAb. Of note, A3C3 and B-L43 are two hybridoma clones that share identical nucleotide sequences for the heavy and light chains, and have thus been used interchangingly in different experiments.Table 2: Summary of Binding Assessment of B-L43 mAb to IL-1RAP

[0195] Quantitative kinetic characterization of B-L43 binding to IL-1RAP by SPR analysis showed a fast association and slow dissociation rate. Data evaluation using a Langmuir 1 : 1 binding model exhibited KD values in the subnanomolar range (Table 3).Table 3: Quantitative kinetic characterization of B-L43 binding to IL-1RAP by SPR analysisSD=standard deviationExample 2.3. Cytotoxic activity toward IL-1RAP+ expressing cells of CAR T- cells derived from B-L43 / A3C3 mAb

[0196] Anti-IL-IRAP CAR T-cells were generated using a lentiviral vector encoding a CAR polyprotein, comprising, the scFv derived form the A3C3 / B-L43 mAbs heavy and light chain sequence, a human immunoglobulin G (IgG) hinge region, CD28 transmembrane and intracellular domains a 4- IBB signaling domain, and the CD3(^ chain signaling. Briefly, CD4 and CD8-positive T-cell were isolated from 2 healthy donors PBMCs by immunolabelling and magnetic separation. Isolated T cells were activated with CD3 and CD28 agonists, transduced with anti-ILlRAP CAR-encoding lentiviral vector (or Non-Transduced [NTD] as controls), expanded in vitro, and cryopreserved for storage. Transduction efficiency was assessed, indicating levels of 47.4% for the donor 1 batch and 49.9% for the donor 2 batch.

[0197] The cytotoxicity of these two batches of CAR-T cells toward an IL-1RAP positive cell line was evaluated in vitro, together with non-transduced (NTD) T-cell controls from the same 2 human donors (HD), after 24 hours of co-culture at different E:T ratios against the IL-1RAP positive AML cell line MOLM-13. At an E:T ratio of 1 :9, cytotoxicity rates of about 20% to 40% were seen and at an E:T of 1 : 1, cytotoxicity rates were 70% to 80% demonstrating the strong killing potential of anti- IL-1RAP CAR T-cells towards IL-1RAP expressing MOLM-13 AML cell line (Figure 2).Example 2.4 CCTx-001 investigational medicinal product (IMP)

[0198] The final CCTx-001 investigational medicinal product (IMP) includes CD4+CAR+ and CD8+CAR+ frozen T-cell suspensions in media containing dimethyl sulfoxide (DMSO). CCTx-001 is administered after thawing by intravenous (IV)infusion. The CCTx-001 investigational product is a novel IL- 1 RAP -targeted, genetically modified, autologous CAR T-cell immunotherapy. Additional disclosure related to anti-IL-lRAP CAR T cells is described in, e.g., U.S. Pat. Pub. Nos. 2021 / 0008108 and 2022 / 0235138, each of which is incorporated herein by reference in their entirety.

[0199] CCTx-001 is manufactured from autologous PBMCs that are obtained via standard leukapheresis collection procedures. The PBMCs undergo positive selection for T-cells and subsequently are transduced with the CAR using a self-inactivating lentivirus. The IL-lRAP-specific CAR are introduced into autologous CD8+ and CD4+ T-cells ex vivo using a replication-incompetent, self-inactivating lentiviral vector (LV). The IL-lRAP-specific CAR includes a scFv binding domain derived from a murine anti-IL-lRAP specific mAb (clone A3C3), an immunoglobulin G (IgG) hinge region, a CD28 transmembrane and intracellular signaling domain, a 4- 1BB signaling domain, and the CD3(^ chain signaling domain. A schematic of the CAR is shown in Figure 1.

[0200] CCTx-001 will be provided as cell dispersion for IV infusion. The cells are formulated in an infusible cry opreservation medium and are stored frozen below - 120°C. Each infusion bag will be labelled as required per country requirement.Example 2.5 Preclinical assessment of CCTx-001In vitro functionality and specificity of CCTx-001

[0201] In vitro studies were conducted on representative large-scale batches of CCTxOOl (referred to herein as TR batches, i.e., batches representative of the clinical process), to assess functionality and specificity of CCTx-001 CAR-T, as described below. The percentage of viable transduced cells was assessed in those batches indicating transduction levels of 50.00%, 60.90%, and 25.50% of cells, for TR3, TR4, and TR5 respectively. NTD T-cells, intended for negative controls, were produced in parallel to CCTx-001 batches using the same cell starting material and process.

[0202] The in vitro efficacy and specificity of CCTx-001 (using TR batches) was further demonstrated in a co-culture cytotoxicity assay. MOLM-13 (IL-1RAP positive) and Raji (IL-1RAP negative) target cells were co-cultured with CCTx-001 cell products from 3 independent donors (or their donor-matched NTD counterparts)and subsequently analysed by flow cytometry (FC) for the quantification of target cell viability, to confirm the functionality of the CCTx-001 CAR T-cells.

[0203] Three batches (TR3, TR4 and TR5) of CCTx-001 cells that are representative of the clinical process, but not their NTD counterparts, showed effector to target cell (E:T) ratio-dependent cytotoxicity toward MOLM-13 cells but not against Raji cells where only allogenic, CAR-independent killing was observed (Figure 3).

[0204] Furthermore, while only low levels of IFN-y release where observed when CCTx-001 cells were co-cultured in the absence of target cells (Figures 4A-4C), or when co-cultured with IL- 1 RAP-negative Raji cells (Figures 4G-4I), co-culture with IL- 1 RAP-positive MOLM-13 cells induced a marked, E:T-dependent release of IFN- y, significantly higher when compared to levels obtained for NTD T-cells co-cultured with MOLM-13 cells (Figures 4D-4F).

[0205] Differences across batches in the levels of IFN-y released by CCTx-001 anti- IL-1RAP CAR T-cells were consistent with differences in transduction levels between those batches. Similar trends were observed for IL-2 and IL-8 secretion (not shown). The results for IL-ip, IL-4, IL-6, IL-8, IL- 10, IL-12p70, IL- 13 and TNF-a showed values below the lower limit of quantification (LLOQ).

[0206] In conclusion, these results confirmed the in vitro efficacy and specificity of CCTx-001 from TR batches to IL-1RAP expressing MOLM-13 AML cells.

[0207] The cytotoxicity of TR CCTx-001 cells was further confirmed in vitro against AML cell lines with different levels of IL-1RAP expression and against different AML subtypes. The cell line panel was composed of MOLM-13 (M5 as per FAB classification), Mono-Mac-6 (M5), EOL-1 (M4 eosinophil), KG-1 (M0), HEL (M6 erythrocyte), HL60 (M2), OCI-AML3 (M4) (see Table 1). K562 and Raji cell lines were used as IL- 1 RAP -negative cell lines.

[0208] NTD and TR CCTx-001 cells (TR4) were co-cultured with the AML cell lines for 24 hours at different E:T ratios, and target cell viability was analysed by FC. The cytotoxicity experiments confirmed CAR-dependent cytotoxicity against all IL-1RAP expressing AML cell lines, regardless of their AML subtype and their IL-1RAP expression level. However, higher IL-1RAP expression levels were associated with increased cytotoxicity at lower E:T ratios (i.e. 1 :27) indicative of a relationship between IL-1RAP expression levels on the target cells and specific cytotoxicity by the CAR T-cells (Figure 5).

[0209] Finally, a CAR-mediated, IL-lRAP-specific, release of IFN-y, TNF-a and granzyme B cytokines by the CCTx-001 CAR T-cells was observed when co-cultured with AML cell lines expressing different levels of IL-1RAP. No specific cytotoxicity and cytokines secretion were observed when CCTx-001 cells were co-cultured with the IL- 1 RAP -negative cell lines K562 or Raji (“Negative” in Figure 5).In Vivo Pharmacology of CCTx-001

[0210] The anti -tumour efficacy of CCTx-001 cells (TR3) was assessed in vivo in female NCG mice, 5 days after IV injection of luciferase-expressing human MOLM- 13-luc AML cell line. The persistence and expansion (longitudinal analysis) of CCTx- 001 was also monitored in the circulation of treated mice. Finally, CCTx-001 cells presence was also analysed in a limited number of tissues and mice at different time points.

[0211] On Day 1, treatment was initiated in 3 randomized groups of M0LM-13-luc tumour-bearing female NCG mice (n=5 or 8 per group, Table 4), randomized based on Day 0 bioluminescent imaging (BLI) flux (mean flux = 1.9 x 106photons / s per group). All treatments were administered IV once on Day 1. Control Group 1 received vehicle (phosphate-buffered saline [PBS] + 0.1% HSA). Groups 2 and 3 received 1 x 107NTD or CAR T-cells, respectively.

[0212] Overall, the treatments were well tolerated, the maximum group mean body weight losses were similar between Group 1 control animals (5.8% on Day 15) and Groups 2 and 3 (9.6% on Day 15 and 6.7% on Day 28, respectively). No unscheduled treatment-related deaths were observed during this 30-day study. Whole-body BLI analysis was carried out on Day 0 for randomization and then twice weekly to the end of the study to monitor M0LM-13-luc AML tumour progression. As shown in Figure 6, the median time to endpoint (TTE) of control Group 1 was 15 days in this 30-days study; individual TTEs in Group 1 ranged from 14 to 17 days, indicating a modest intragroup variability. The median TTE in Group 2 animals treated with NTD was 16 days, corresponding to a nonsignificant 7% increased lifespan (ILS) (Logrank test, p > 0.05 vs. control). In contrast, the median TTE in Group 3 CCTx-001 -treated mice was 22.0 days (47% ILS, p<0.01 against group 1 and 2; Logrank test), with 1 survivor to the end of the study (Day 31). Whole body BLI images were consistent with survival kinetics.Table 4: Response Summary in MOLM-13-luciferase model

[0213] Table 4 displays the scheduled treatment regimen at completion of the study. Vehicle = PBS + 0.1% BSA. Study was ended on Day 33 based on SD data review and only 1 animal remaining on study (Study Duration = 30 Days). TTE was defined as time to moribund signs due to tumor progression and all deaths due to tumor progression were classified as death on survival study. TTE (in days) was recorded for each mouse. C=control group, Med.=Median, NTD=non-transduced, TTE = Time to Endpoint, ILS = Increased life span, T / C100% - 100%, Statistical significance (Logrank test): ns = not significant, *= p < 0.05, **= p < 0.01, ***= p < 0.001, compared to Group 1 or 2; Mean body weight Nadir = lowest group mean body weight, as % change from Day 1; NA=not applicable, T=treatment group, TR = treatment-related death, NTR = non-treatment-related death.

[0214] In animals implanted with human T-cells (Group 2 NTD (CO) and Group 3 (CCTx-001 cells), blood samples were collected at different timepoints and analysed for the presence of circulating MOLM-13 AML cells. As shown in Figure 7, Group 3 (CCTx-001 cells) the percentage of AML cells remained at low levels (initial blood AML levels averaged only 0.002% of total live cells on Day 3; by the end of the study, circulating AML was 0.6% and 0.2% of total live cells on Days 21 and 30, respectively), whereas Group 2 (NTD) demonstrated an increase in circulating AML cells by Day 14 (just before the animals died or were euthanized due to tumour progression) confirming that CCTx-001 inhibits the growth of IL-1RAP positive AML cell in vivo. Similar results were seen regarding the absolute count of AML cells.

[0215] In summary, CCTx-001 demonstrated anti -tumor efficacy in vivo and significantly delayed the growth of IL- 1 RAP-positive M0LM-13-luc AML cellxenografts in female NCG. No findings indicative of CCTx-001 -related morbidities were observed.

[0216] These findings were in line with published in vivo data from former results with IL-1RAP CAR T-cells comprising an A3C3 / B-L43 mAb derived scFv. Anti-IL- 1RAP CAR T-cells efficiently controlled AML cell lines growth in vivo (HL60, MOLM-13 and Mono-Mac-6) in a NOD scid gamma (NSG) mouse model (Trad 2022) and were able to control Mono-Mac-6 in vivo tumor progression and reduce the AML tumor burden compared to untreated mice or mice treated with NTD T-cells (Nicod 2023).Pharmacokinetics and Product Metabolism in Animals

[0217] Whole blood samples (from CCTx-001 cells-injected (Day 0) mice harboring IL-lRAP-expressing MOLM-13 tumors) were collected on Days 3, 7, 10, 14, 21 and 30. The samples were used to monitor the presence, the expansion and persistence of CCTx-001 cells and T-cells subsets (CD8 and CD4 CAR-positive T-cells) during the course of an anti-tumor efficacy study.

[0218] FC analysis of the blood samples demonstrated successful adoptive transfer and persistence of CCTx-001 cells in the circulation of the mice. More relevant, both CD4+ and CD8+ CAR-positive T-cell numbers increased significantly after 2 weeks of the adoptive transfer (Figure 8C) when injected in IL-lRAP-expressing MOLM- 13-Luc tumor-bearing mice. This increase in CCTx-001 cells post day 14 was associated with a low level of circulating AML cells whereas mice injected with NTD T-cells demonstrated an increase in the incidence of circulating AML cells.

[0219] Finally using a limited number of mice (n=4), distribution of CCTx-001 cells was assessed at Day 21 (n=3) and Day 30 (n=l) in the liver, spleen, and BM of treated mice. Overall, this analysis demonstrated that CCTx-001 cells were present in the 3 tissues. Across both time points, CCTx-001 cells accumulated in the liver (64% to 71% were human T-cells, of which 46 to 71% were CCTx-001 cells), in the spleen (human T-cells represented 14% to 49% of total cells, of which 43 to 67% were CCTx-001 cells) and in the BM (3% to 6% of BM live cells were human T-cells of which 42% to 66% were CCTx-001 cells). When looking at the absolute number of CCTx-001 cells per mg of tissue, 0.6 x 104to 3 x 104CAR T-cells / mg were detectedin the spleen and 0.3 x 104to 1 x 104CAR T-cells / mg in the liver. The femur BM contained between 4 x 104to 10 x 104CAR T-cells per femur.

[0220] A second in vivo study of OCI-AML3-Luc tumour-bearing NCG mice treated with non-transduced (NTD) human T-cells or with donor matched C4-derived CAR T-cells (CCTx-001) was also performed. FIG. 9A shows an illustration of the in vivo study design. 6 mice were present in each study group. Tumours were injected subcutaneously and allowed to establish for up to 4 days before injection of untransduced or transduced CAR T-cells (IxlO7T-cells intravenously). Animal welfare was monitored over the course of the study. The study was concluded by day 30 following T-cell injection, or sooner due to tumor burden and / or comorbidities. Tumour progression was assessed regularly during the studies through tracking of tumour cell bioluminescence (BLI).

[0221] FIG. 9B shows the mean body weight of each study group across the duration of the study. CCTx-001 treatment was well tolerated, with no observed morbidity or death / euthanasia attributed to the treatment. FIG. 9C shows the results of bioluminescence imaging (BLI) at days 0, 7, 14, and 21 across each mouse in the study. OCI-AML3-Luc signal intensity is depicted as luminescence (units in radiance; p / sec / cm2 / sr). FIG. 9D shows a longitudinal analysis of the BLI of each study group, shown as total flux (p / s) per time point. FIG. 9E shows a Kaplan-Meier plot of survival of OCI-AML3-Luc tumour-bearing NCG mice treated with vehicle, NTD T- cells, or CCTx-001. Probability was of survival was based on the time to reach BLI of 1E+10 p / s. The median survival of both vehicle and NTD treatment groups was 14 days, while the median survival was not reach in the CCTx-001 group (hazard ratio = 0.03912; p-value < 0.0005).

[0222] In summary, the data shown in FIGs 6-9 indicate that CCTx-001 treatment provides significant anti-tumor effects and inhibition of tumor progression in an in vivo model, with no signs of CAR related toxicity in all tested models. In contrast, neither vehicle nor NTD T-cell treatment had a positive effect on survival or tumor inhibition.Cytotoxicity of CCTx-001 Toward Primary AML Cells

[0223] A study was performed assessing the cytotoxicity potential of two batches of healthy donor-derived CCTx-001 CAR T-cells toward primary AML cells fromdifferent donors (n=5). Anti-IL-IRAP CCTx-001 CAR T-cells and non-transduced T cell controls (NTD T-cells) were previously generated by clinical scale manufacturing from PBMCs of healthy donors. CCTx-001 CAR-mediated cytotoxicity was assessed in in vitro co-culture assays with primary AML cells. Primary AML cells, purchased from BioIVT and previously characterized for blast content / phenotype and IL-1RAP expression are described in Table 5. To promote AML survival during the 24h assay duration, a defined media with AML supporting cytokines (SCF, TPO, FLT-3L, IL-3, IL-6 and GM-CSF) was used and T-cell supporting cytokines usually included in standard cytotoxicity assay (IL-7 and IL- 15) toward cell lines were omitted.Table 5: Primary AML Cell BatchesNotes: F = female; M = male; Coll. = collection

[0224] As described in the study design outlined in Table 6, two independent batches of anti-IL-lRAP CCTx-001 CAR T-cells and donor-matched NTD T-cell controls were used to evaluate the functionality against primary AML cells. For each test and control article a total of 1 biological replicate for each E:T ratio was performed for the cytotoxicity assays.Table 6: Study Design

[0225] NTD and CAR T-cells were thawed on Day -3 of the cytotoxicity assay.Briefly, the NTD and CAR T-cells were thawed in TexMACS medium and maintained to a cell concentration of IxlO6cells / mL in TexMACS medium complemented with 5% HS (human serum), 1% PS (pen-strep), 12.5 mg / mL of IL-7 and 12.5 mg / mL of IL-15. Cells were seeded at a density of IxlO6cells / mL, with media periodically refreshed to maintain cell density of IxlO6cells / mL. The viability and cell concentrations were verified and adapted daily during the 3 days prior to cytotoxicity assay initiation.

[0226] After 3 days of post-thaw resting culture, test article effector cells were labelled with Cell Proliferation Dye eFluor™ 450. Effector cells were then harvestedfrom culture and washed twice with PBS IX. Subsequently, effector cells were resuspended at 2xlO6 / mL in PBS IX. While vortexing the T-cells, an equal volume of 20pM dye solution was added. After incubation for 10 min at 37°C in the dark, labelling was stopped by adding 4-5 volumes of wash buffer (PBS IX, 10% FBS) and incubated on ice for 5 min. Thereafter, cells were washed 3 times with wash buffer. Labelled effector cells were adjusted to a concentration of 4.5xl06cells / mL in culture media media (StemSpan SFEM II supplemented with CD34+ Expansion supplement and 100 UI / mL of GM-CSF without IL-7 and IL- 15) and the respective ratios were prepared. From this preparation, dilutions were made by mixing with culture media to obtain the concentration necessary for the respective E:T ratios (3: 1, 1 : 1, 1 :3, 1 :9, and 1 :27).

[0227] AML primary cells were thawed on the day of cytotoxicity assay initiation (after 3 days of resting of effector cells), washed with PBS IX and resuspended in StemSpan SFEM II supplemented with CD34+ Expansion supplement and 100 UI / mL of GM-CSF without IL-7 and IL-15 at a concentration of 0.5xl06cells / mL.

[0228] lOOpL of effector cells were added into the corresponding wells. To all wells, lOOpL of target cell suspension was added. In the “target only” and “1 :0 E:T ratio” sample wells, lOOpL of culture media was added. The well plate was incubated for 24 hours at 37°C, 5% CO2.

[0229] After 24 hours of co-culture, cells were resuspended and stained with CD33, CD34 and CD45 antibodies for 10 min at +4°C. Then, 7-AAD was added, and cells were incubated for 10 additional min at +4°C prior to FC analysis. Cells were then analyzed by Flow Cytometry on Novocyte Quanteon and data analysis was performed on Novoexpress software.

[0230] Co-cultures were performed at different E:T ratios using culture conditions selected to preferentially support the survival of AML primary cells in vitro (SFEM media + SCF, TPO, FLT3L, IL-3, IL-6 and G-CSF, without T-cell supporting cytokines, normally used to support CCTx-001). After 24h, samples were analysed by FC and cytotoxicity (viable blasts cells normalized to the target alone condition) and specific cytotoxicity (viable blasts cells normalized to the NTD T-cell condition) were measured. Both batches of CCTx-001 CAR T-cells demonstrated CAR-mediated (specific) cytotoxicity toward AML blasts (already observed beginning from the E:T ratio of 1 :3) for all the patient samples tested (Figure 12 and Figure 14). Themagnitude of the cytotoxicity was generally correlated with IL-1RAP expression levels on target cells (Table 5). Allogeneic cytotoxicity observed with NTD controls varied depending on the AML patient target cells as shown in the cytotoxicity results (viable blasts cells normalized to the target alone condition) shown in Figure 13.Manufacturing and Functional Assessment of CCTx-001 CAR-T cells using AML Patient T-cells

[0231] A study was performed to demonstrate that functional C4-derived anti-IL- 1RAP (CCTx-001) CAR T-cells can be produced from AML patient T-cells. In addition, the cytotoxicity of CCTx-001 CAR T-cells towards the patient’s own primary AML blasts was also assessed.

[0232] Several clinical trials have confirmed that clinical scale manufacturing of autologous CAR T-cells from AML patients is feasible (Jin et al. 2022; Pei et al. 2023; Sailman et al. 2022; Tambaro et al. 2021; Wang et al. 2015; Zhang et al. 2020; 2021; 2022; Zhao et al. 2024). Access to relevant amounts of AML patient materials to perform further studies on manufacturability in a clinically relevant scale during development is not possible due to unavailability of patient derived larger quantities of peripheral blood. Thus, pivotal pharmacology and toxicology studies are performed using CAR T cells batches generated in a clinically representative process using healthy donor cells as starting material. However, manufacturing using AML patient cells can be performed at small scale (R&D grade) using small amount of cryopreserved residual cells from routine clinical monitoring, to demonstrate the functionality of CCTx-001 CAR T-cells produced from AML patient T-cells. In addition, small amounts of AML primary cells are generally sufficient, if comprising a substantial proportion of malignant blasts, to allow the assessment of the cytotoxicity of CAR-T cells towards primary, AML patient derived blasts. For the current experiment, T-cells were isolated from AML patients one month postdiagnosis, which may have resulted in exposure to first-line chemotherapy consisting of a “3+7” regimen of daunorubicin and cytarabine.

[0233] AML blasts are notoriously difficult to maintain in vitro (0. Bruserud et al. 2001; Cucchi et al. 2020) and culture condition can impact T-cell costimulatory signal expression from AML cells (Oystein Bruserud, Frostad, et Foss 1999; O. Bruserud, Gjertsen, et von Volkman 2000). To promote the survival of AML blasts in vitro and allow for cytotoxicity assessment, a specialized media was used, comprising severalAML cells supporting cytokines (SCF, TPO, FLT-3L, IL-3, IL-6 and GM-CSF) while T-cell supporting cytokines were omitted from the culture to mitigate on potential impact on AML cell survival.

[0234] Anti-IL-IRAP CCTx-001 CAR T-cells and non-transduced T cell controls (NTD T-cells) were generated at small scale (R&D grade manufacturing) from previously collected peripheral blood mononuclear cells (PBMCs) of 4 AML patients, diagnosed of intermediate to severe prognosis AML. The blood samples were collected one month post diagnosis, which may have resulted in exposure to first-line chemotherapy. AML patient PBMC used as starting material for CAR T-cell manufacturing and as target cells in cytotoxicity assays are described in Table 7. All the PBMC frozen samples were provided by EFS Bourgogne Franche-Comte, collected at the one-month post-diagnosis visit in compliance with local regulations, with the donors’ consent and without compensation of any form.Table 7: AML Patient Donor CellsNotes: AML-MR=AML with myelodysplasia-related gene mutations; AML-M1=AML with minimal differentiation; F=Female; M=MaleT-Cell Production:

[0235] T-cell isolation: The total thawed PBMC were incubated with AutoMACS running buffer, CD4 microbeads, and CD8 microbeads for 20min at +4°C. Thereafter, cells were centrifuged 5min at 500g, supernatant was removed, AutoMacs running buffer was added and the cell suspension was loaded onto an LS column. After further addition of AutoMACS running buffer and complete medium without human serum, non T-cells were eluted from the suspension, after which the column was removed from the magnetic field and T-cells were eluted from the column using a piston. Thereafter, cells were centrifugated and the cell pellet was resuspended at the concentration of IxlO6cells / mL in complete medium without human serum.

[0236] T-cell activation: The cell suspension together with TransAct was incubated on a plate at 37°C in humid atmosphere supplemented with 5% CO2.

[0237] T-cell transduction: One day after T-cell activation, the calculated volume of C4 lentiviral supernatant was added according to the viral titre (5.69xl07TU / mL) to reach a MOI of 2. The following day, medium with human serum was added and double this amount on the next day. On day 9, cells were diluted to adjust the concentration to 0.5-1.106cells / mL. At Day 9, the cell material is referred to as “Drug Substance” (DS).

[0238] Cryopreservation: On day 9, T-cells were cryopreserved in 5% human serum albumin (HSA), 5% DMSO (CS10), at a concentration between 3xl06cells / mL (CL045-016) or 5xl06cells / mL (CL045-017, CL045-018, CL045-019).T-Cell Characterization:

[0239] The generated CAR-T cell products were characterized for composition and transduction using flow cytometry (FCM) and digital PCR assays. Potency assessment was based on IFN-y release following culture on IL-1RAP coated plates Briefly, the DP is incubated in 96-well plates coated with recombinant IL-1RAP, coated with human serum albumin (HSA, negative control) or supplied with phorbol myristate acetate (PMA) and ionomycin (positive control, unspecific activation). After an overnight incubation, the supernatant is tested by automated ELISA (Ella, Bio-Techne) for Interferon Gamma cytokine concentration and compared to the negative control (HSA). The results of the IFN-y release assay are shown in Figure 18A

[0240] Cytotoxicity of the patient-derived CAR-T cells was assessed towards IL- 1RAP expressing AML Cells (MOLM-13 cells). In this assay, CAR-T cells are stained with eFluor 450 dye and co-cultured with the target cell line MOLM-13 (ratio: 1 : 1) during 24h in TexMACS, 5% human serum (SH), 1% penicillin streptomycin (PS), supplemented with IL-7 and IL-15. Afterwards, cells are harvested, stained, and analysed by flow cytometry to evaluate the viability of the target cells via the viability dye 7-AAD. The specific killing is normalized to a parallel culture in which the target cells are incubated in the absence of CAR-T cells. Results for the MOLM-13 AML cytotoxicity assay are shown in Figure 18B.

[0241] In addition, for 3 patients with an identifiable population of malignant blasts in PBMC samples, the cytotoxicity of patient derived CAR T-cells was assessed toward the patient’s own blasts in an in vitro co-culture assay that was adapted to allow culture of primary AML cells for 24 hrs.

[0242] Analysis of AML patient PBMC cell composition showed T-cell content ranging from 2.49 to 25.77%. and malignant blasts, unambiguously identified in three of the four patients PBMCs, represented 20.94% to 93.29% of total PBMCs, with IL- 1RAP expression ranging from 2230.7 to 2383.0 ABC. High viability CAR T-cells were obtained for all patients, with successful transduction ranging from 22.13 to 35.35% of total T-cells and Vector copy number (VCN) per transduced cells maintained below 5. Functionality of the CAR T-cells was demonstrated by IFN-y release potency assay and cytotoxicity toward the MOLM-13 cell line. All batches exceeded release specification for IFN-y potency (>5-fold increase) and demonstrated potent cytotoxicity toward the MOLM-13 cell line (ranging from 58% to 70% at 1 : 1 E:T ratio). Overall, all batches met expected specifications.Example 3: Adaptive Open-Label Multicenter Phase 1 / 2 Study

[0243] The purpose of this adaptive Phase 1 / 2 study is to evaluate the safety, tolerability, pharmacokinetics (PK), and antileukemic activity of CCTx-001 in adult patients with r / r AML. CCTx-001 targets IL-1RAP, which is specifically expressed in leukemic cells (Frenay 2022). In preclinical studies, IL- 1 RAP-targeted CARs have demonstrated encouraging activity in both in vitro and in vivo experiments in AML models (Zhao 2017, Neto Da Rocha 2019, Warda 2021, Trad 2022). Based on these promising preclinical results, it is expected that CCTx-001 could potentially alter the natural course of r / r AML and provide a potential novel treatment option.Study Overview:

[0244] The study will be conducted in compliance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use / Good Clinical Practice (GCP) and applicable regulatory requirements.

[0245] This is an adaptive open-label multicenter Phase 1 / 2 study aimed to determine the RP2D of CCTx-001, and to assess safety, tolerability, and clinical activity ofCCTx-001 in patients with r / r AML. The Phase 1 part of the study will evaluate increasing doses of CCTx-001 to identify the recommended Phase 2 dose (RP2D) or optimal biological dose. The main consideration for the starting dose selection is patient safety. The dose-escalation in Phase 1 will follow a Bayesian Optimal Interval (BOIN) design based on the occurrence of dose limiting toxicities (DLTs). The Phase 2 (dose expansion) part will comprise a larger cohort of patients with r / r AML and will aim to evaluate the clinical activity and further assess the safety of CCTx-001 in this population at RP2D, a dose considered to be safe and clinically active.

[0246] The study comprises 4 periods (see Figure 10): (1) The pre-treatment period will consist of screening for eligibility, leukapheresis and a pretreatment evaluation (prior to lymphodepletion chemotherapy (LDC)); (2) The treatment period will start with LDC, followed by CCTx-001 infusion 2 to 7 days after completion of LDC. A first response evaluation will be performed at approximately 28 days after CCTx-001 infusion; (3) The post-treatment period will consist of further clinical activity and safety follow-up visits at regular timepoints after CCTx-001 infusion, starting after the Month 3 visit up to the Month 24 visit; and (4) The long-term follow-up period will start after the Month 24 visit up to 15 years post CCTx-001 infusion.Pre-Treatment Period:

[0247] Prior to initiation of any study procedure, patients must provide informed consent. Once enrolled and during the pre-treatment period, patients will undergo a leukapheresis to obtain enough peripheral blood mononuclear cells (PBMCs) to produce the CCTx-001 cell product.

[0248] If necessary, anticancer treatment or disease control (bridging therapy) is allowed while CCTx-001 is being manufactured (i.e., after leukapheresis and prior to start of LDC). All patients receiving bridging therapy need to have all disease assessments repeated before start of LDC. All patients must continue to have detectable AML, and meet eligibility criteria pertaining to adequate organ function, active infections, pregnancy, and washout of prior therapy before initiation of LDC.Treatment Period:

[0249] Upon successful CCTx-001 product generation, patients will enter the treatment period and receive LDC, followed by a single dose of autologous CCTx- 001 T-cells.

[0250] Patients will receive 4 days of fludarabine IV (30 mg / m2 / day) and 3 days of cyclophosphamide IV (300 mg / m2 / day) for LDC. This lymphodepleting regimen may change during the study in composition or dose upon safety, biological, and / or clinical activity observations.

[0251] Two to 7 days after completion of LDC, CCTx-001 will be administered by IV infusion as a single dose on Day 1.

[0252] A review of CAR T-cell studies registered at ClinicalTrials.gov up to the end of 2016, found that the typical dose escalation study covered a 2-log (100-fold) dose range, typically within the range 106- 109total CAR T-cells per patient, with IxlO6as typical starting dose (Hartmann 2017).

[0253] Doses of marketed T-cell products for hematological malignancies are within the following ranges: 2xl06to 6.0xl08cells with 2xl06to IxlO8per kg for brexucabtagene autoleucel (Tecartus™) and axicabtagene ciloleucel (Yescarta®); 0.6 to 6.0xl08cells for tisagenlecleucel (Kymriah®); 0.5 to L lxlO8cells for lisocabtagene maraleucel (Breyanzi®); 3.0 to 4.6xl08cells for idecabtagene vicleucel (Abecma®) (van der Walle 2021). Recently, CD 123 -directed CAR T-cells were evaluated in 2 separate Phase 1 studies starting at lOOxlO6and 250xl06UniCAR-T-cells as flat doses (Wermke 2021), or at doses of UCART123 of 2.5xl05cells / kg, 6.25xl05cells / kg, 1.5xl06cells / kg, or 3.03xl06cells / kg (DL3) (Sailman 2022).

[0254] Given that IL-1RAP is a new target which has not yet been explored for CAR T-cell therapy, a starting dose of 5xl05cells / kg, which is about 2-fold (i.e., one escalation step) lower than the typical starting dose was chosen as an adequate starting dose. This dose is 500-fold lower than the dose used in animal studies. As recommended by the FDA guidance on the development of CAR T-cells, the calculation of the cell dose will be based on weight rather than using a flat dose (FDA 2022).

[0255] The first 3 patients per DL will be treated with a minimum interval of 14 days between CCTx-001 infusions to allow for enough time to observe acute toxicities. At the end of each dose cohort, all available safety data will be reviewed and discussedwith the SRC before the initiation of dosing at the next cohort. By the end of the dose finding part, a dose level (DL) for the dose expansion part of the study is selected. For DLs with only 3 patients treated, the additional patients may be enrolled to support the DL selection for expansion cohorts. All patients are expected to be hospitalized for at least 14 days from infusion for Phase 1.

[0256] Safety data will be reviewed on an ongoing basis. An early safety assessment will be performed 28 days after CCTx-001 administration to the 12th patient. Enrolment will be halted if the observed treatment-related mortality rate is > 30%, or if > 50% of patients who undergo leukaphereses fail to have a satisfactory cell product available for infusion.

[0257] The Phase 1 (dose finding) part will comprise a dose escalation using a BOIN dose escalation design (Yuan 2016, Lin 2017). It is a design which is easy to implement, flexible in cohort size, allows for stopping rules to be predefined. A total of 5 different DLs of CCTx-001 may be evaluated (Table 8). The study will start at DL1 (0.5xl06cells / kg). If declared safe as per evaluation, the next DLs may be tested (DL2: IxlO6cells / kg; DL3 5xl06cells / kg; DL4 10xl06cells / kg). If DL1 is not tolerable as per evaluation, the DL will be de-escalated to DL-1 (0. IxlO6cells / kg). All DLs are capped at a maximum patient weight of 80 kg. If patients will not develop toxi cities to CCTx-001 and will not respond to the first dose CCTx-001, they may receive an additional dose of CCTx-001 at the next higher DL (intra patient dose escalation) to allow for potential antileukemic activity.Table 8: CCTx-001 Dose Levels

[0258] At least 3 patients per dose will be treated in up to 5 planned DL cohorts.Additional patients will be added based on the observation of DLTs (up to 3additional patients in each cohort), for a total maximum of 30 DLT evaluable patients following the BOIN design. All patients are expected to be hospitalized for at least 14 days from infusion.

[0259] The Phase 2 (dose expansion) part is a single arm part evaluating primarily the clinical activity measured by composite complete response rate (cCRR: CR, CRh, or CRi), as assessed by an Independent Review Committee (IRC) based on European LeukemiaNet (ELN) 2022 criteria (Dohner 2022). It will comprise approximately 72 patients with r / r AML evaluable for the primary endpoint analysis. Two interim analyses are anticipated. The first interim analysis will evaluate the futility of treatment of r / r AML patients with CCTx-001 against historical control. The second interim analysis will evaluate solely the clinical activity of treatment of r / r AML patients with CCTx-001 against historical control. The primary analysis will be performed when the last on-study patient infused with CCTx-001 reaches the earliest between 90 days post-CCTx-001 infusion visit, relapse as per ELN 2022 criteria, the start of a new AML treatment, loss to follow-up, withdrawal of consent, or death from any cause. The dose escalation part will enroll over approximately 18 months and the dose expansion part will enroll over approximately 18 months.Post-Treatment Period:

[0260] Patients will be followed for 2 years post CCTx-001 infusion with more frequent follow ups for AEs related to CCTx-001 and / or LDC and associated concomitant medications and procedures.Long-Term Follow-Up Period:

[0261] Patients will be followed for up to 15 years post CCTx-001 infusion as per current health authority guidance. This long-term follow-up serves to collect AEs if any, suspected to be related to the CAR T-cell therapy.

[0262] Delayed potential toxicities may include expression RCL, autonomous proliferation of infused CAR T-cells, and insertional oncogenesis by integration of the lentiviral construct. None of these potential events have been observed to date.Definitions of Dose-Limiting Toxicities (DLT):

[0263] Severe cytokine release syndrome (CRS) and neurotoxicity (NT) are the most significant toxicities observed with CAR T-cell therapy. Significant increases inseveral cytokines such as IL-6, IL-5, IL- 10, and interferon-y have been observed although the pattern of elevated cytokines varies among patients (Davila 2014, Gust 2017). Some degree of correlation exists between the development of CRS and efficacy. While the severity of CRS is not predictive of response, it is associated with disease burden. Preliminary observations show a correlation of a subset of the aforementioned cytokines as well as additional cytokines such as IL-8, IL-15, and transforming growth factor-a that may predict the severity of CRS and NT. In addition, clinical markers of inflammation, C-reactive protein (CRP) and ferritin, are at elevated levels in patients displaying CRS. To understand the pathophysiology of CAR T-cell-mediated toxicity and potential changes arising from the combination, an extensive soluble factor panel will be tested on multiplex assay platforms.

[0264] A DLT is an AE or an abnormal laboratory finding assessed as unrelated to leukemia, intercurrent illness, or concomitant medications. The DLT observation period will be from Day 1 (day of CCTx-001 infusion) until Day 28. The period of DLT evaluation may be extended based on emerging data and for evaluation of hematological toxicity and its consequences. AEs will be evaluated and graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events, Version 5.0 (CTCAE v.5.0). CRS and neurotoxicity will be evaluated according to the American Society for Transplantation and Cellular Therapy (ASTCT) Consensus Grading (Lee 2019).

[0265] A DLT is defined as the following CCTx-001 -related events: Death (except due to disease progression); cytokine release syndrome (CRS) (Any treatment- emergent grade 3 CRS that does not improve to grade < 2 in 72 hours despite adequate therapy; Any treatment-emergent grade 4 CRS of any duration); Neurotoxicity (Any treatment-emergent grade 3 neurologic toxicity that fails to resolve to grade < 2 within 72 hours of onset, despite adequate therapy; Any treatment-emergent grade 4 neurologic toxicity of any duration); Cytopenia (Any grade 4 leukopenia that does not improve to grade < 3 within 28 days, and not considered by the Investigator to be attributable to another clearly identifiable cause (e.g. disease progression, concomitant medication, pre-existing medical condition; Any grade 4 thrombocytopenia that does not improve to grade < 3 within 28 days, and not considered by the Investigator to be attributable to another clearly identifiable cause (e.g. disease progression, concomitant medication, pre-existing medicalcondition; Febrile neutropenia grade 4 and thrombocytopenia grade >3 with clinically significant bleeding, and not considered by the Investigator to be attributable to another clearly identifiable cause (e.g. disease progression, concomitant medication, pre-existing medical condition)); Tumor lysis syndrome of grade 4 (Cairo and Bishop 2004); Any grade 4 allergic reactions; Any grade > 3 treatment-emergent autoimmune toxicity; Any grade > 3 treatment-emergent liver enzyme elevation not related to CRS; Any other grade > 3 toxicity to vital organs, not pre-existing or not due to underlying disease. Exceptions may be made for grade 3 or 4 renal function tests that improve to grade 2 or less within 7 days.Study Timing:

[0266] The end-of-study (EOS) for each individual patient will be the earliest between 15 years after the date of patient’s last infusion of CCTx-001, withdrawal of consent, loss to follow-up or death.

[0267] The EOS is defined as the date of the last visit of the last patient completing its EOS visit or the date of receipt of the last data point from the last patient that is required for primary, secondary and / or exploratory analyses, whichever is the later date.Study Population and Inclusion Criteria:

[0268] Adult patients (> 18 years) with AML who have relapsed or are refractory to at least 2 cycles of standard induction treatment. The investigator or designee must ensure that only patients who meet all the eligibility criteria are enrolled in the study.

[0269] All patients must meet the following inclusion criteria to be enrolled in the study:1. Patients with active (> 5 % blasts in bone marrow) r / r AML (WHO 2022) defined as either: i. Primary refractory:1. Patients who failed after two cycles of intensive induction including high-dose and / or standard dose cytarabine (including liposomal formulation), + / - anthracycline, + / - antimetabolite, + / - targeted therapy or2. Older patients or patients unfit to receive intensive induction courses who failed after two cycles of venetoclax + azacitidine or 4 cycles of azacitidine. ii. Relapsing:1. Patients with early relapse after CR to first line therapy (within < 6 months after CR1) or2. Patients with relapse after later lines of therapy (Relapse after CR>2). iii. Patients relapsing after allogeneic hematopoietic stem cell transplant:1. Patients must be at least 3 months from HSCT at the time of consent, and2. Off immunosuppression for at least 1 month at the time of consent, and3. Have no active graft versus host disease (GvHD). Have a circulating blast count of less than 20,000 / mm3(control with hydroxyurea is allowed). Absolute Lymphocyte count of >200 / mm3. Eastern Cooperative Oncology Group (ECOG) performance status < 1. Life expectancy of more than 3 months. Patient is > 18 years of age at the time of informed consent. Read, understood, and signed the informed consent form (ICF) prior to any study procedures. Patient is willing and able to adhere to the study visit schedule and other protocol requirements. Eligible for leukapheresis. Treatment-related toxi cities of previous therapies have completely resolved. Adequate organ function as confirmed by clinical laboratory values, defined as: a. Adequate bone marrow function to receive LDC as assessed by the Investigator.Serum creatinine [< 1.5 x the upper limit of normal (ULN) or creatinine clearance (CrCl) > 45 mL / min] (estimated by Cockcroft Gault or Modification of Diet in Renal Disease (MDRD)).c. Alanine aminotransferase [< 5 x ULN and total bilirubin < 2.0 mg / dL (or < 3.0 mg / dL] for patients with Gilbert’s syndrome or leukemic infiltration of the liver. d. Adequate pulmonary function, defined as [< Grade 1 dyspnoea according to CTCAE and oxygen saturation (SaO2) > 92% on room air and forced expiratory volume in the first second > 50%]. e. Ejection fraction > 40% assessed by an echocardiogram (ECHO) or multigated acquisition (MUGA) scan performed within 1 month before CCTx-001 infusion.12. Women of childbearing potential (WOCBP) must have a negative serum pregnancy test performed at screening and within 7 days before enrolment.13. WOCBP or males whose sexual partners are WOCBP must be able and willing to use at least 1 highly effective method of contraception during the study and for 12 months after the last dose of LDC. A WOCBP is a female patient who: 1) has achieved menarche at some point, 2) has not undergone a hysterectomy or bilateral oophorectomy or 3) has not been naturally postmenopausal (amenorrhea following cancer therapy does not rule out childbearing potential) for at least 12 consecutive months (i.e., has had menses at any time in the preceding 12 consecutive months).Exclusion Criteria:

[0270] The presence of any of the following criteria will exclude a patient from study participation:1. Patients with an acute promyelocytic leukemia: t(l 5; 17)(q22;ql2); (promyelocytic leukemia / retinoic acid receptor alpha) and variants.2. Patients with active CNS leukemia involvement. If the patient has prior history of CNS leukemia, they must have a negative cerebrospinal fluid (CSF) assessment and magnetic resonance imaging (MRI) or computed tomography (if MRI is not feasible) of the brain demonstrating no evidence of CNS disease.3. Patients with isolated extramedullary AML disease.4. Patients who received previous treatment targeting IL-1RAP or previous gene therapy.Patients who underwent allo-HSCT within 90 days prior to leukapheresis. Patients who received donor lymphocyte infusion within 60 days prior to leukapheresis. Patients with active GvHD. Patients with history of another primary malignancy other than disease under study unless the patient has been free of the disease for > 2 years, except for the following non-invasive malignancies: a. Basal cell carcinoma of the skin. b. Squamous cell carcinoma of the skin. c. Carcinoma in situ of the cervix. d. Carcinoma in situ of the breast. e. Incidental histologic finding of prostate cancer (Tla or Tib) or prostate cancer that is curative. f. Other completely resected stage 1 solid tumour with low risk for recurrence. Presence of systemic fungal, bacterial, viral, or other infection (including tuberculosis) that is uncontrolled despite appropriate antibiotics or other treatments. Active or prior history of hepatitis B or hepatitis C infection. History of or active human immunodeficiency virus (HIV) infection. Active macrophage activation syndrome (MAS) as evidenced by laboratory abnormalities (e.g.: elevated ferritin, elevated triglycerides, hemophagocytosis on the bone marrow sample) and / or clinical signs. History or presence of an active and clinically relevant CNS disorder such as epilepsy, generalized seizure disorder, paresis, aphasia, stroke, cerebral oedema, severe brain injury, dementia, multiple sclerosis, Parkinson’s disease, cerebellar disease, organic brain syndrome, or posterior reversible encephalopathy syndrome, or any autoimmune disease with CNS involvement. Patients with active autoimmune disorders or active neurological or inflammatory disorders (e.g., Guillain-Barre Syndrome, Amyotrophic Lateral Sclerosis) requiring immunosuppressive therapy or corticosteroid therapy (defined as >20 mg / day prednisone or equivalent). Physiologic replacement, topical, and inhaled steroids are permitted.Use of the following: a. Therapeutic doses of corticosteroids (defined as > 20 mg / day prednisone or equivalent) within 7 days prior to leukapheresis or 72 hours prior to CCTx-001 infusion. Physiologic replacement, topical, and inhaled steroids are permitted. b. Immunosuppressive therapies within 4 weeks prior to signing the ICF (e.g., calcineurin inhibitors, methotrexate or other chemotherapeutics, mycophenolate, rapamycin, thalidomide, immunosuppressive antibodies such as anti-tumour necrosis factor [TNF], anti-IL-6, or anti-IL-6 receptor [IL-6R]). c. Cytotoxic chemotherapeutic agents (including intrathecal) within 14 days prior to leukapheresis. d. Treatment with alemtuzumab within 6 months of leukapheresis, or treatment with fludarabine or cladribine within 3 months of leukapheresis. e. Experimental agents within 4 weeks prior to signing the ICF unless no response or progressive disease (PD) is documented on the experimental therapy and at least 3 half-lives have elapsed prior to signing the ICF. f. Therapeutic anti coagulation. History of any one of the following cardiovascular conditions within the past 6 months prior to signing the ICF : g. Class III or IV heart failure as defined by the New York Heart Association. h. Cardiac angioplasty or stenting. i. Myocardial infarction. j . Unstable angina. k. Other clinically significant cardiac disease. Known hypersensitivity to DMSO or other excipients. Uncontrolled medical, psychological, familial, sociological, or geographical conditions that do not permit compliance with the protocol, as judged by the Investigator; or unwillingness or inability to follow the procedures required in the protocol.19. Abnormal findings and / or clinically significant Grade >3 non-hematological toxicity and any other medical condition(s) or laboratory findings that, in the opinion of the Investigator, might jeopardize the patient’s safety.20. Presence of any condition that confounds the ability to interpret data from the study based on Investigator's judgement.21. Any planned medical / surgical treatment that might interfere with the ability to comply with the study requirements.22. Pregnant or nursing women. NOTE: WOCBP must have a negative serum pregnancy test performed within 48 hours of starting LDC.Leukapheresis:

[0271] An unstimulated leukapheresis collection will be performed for each patient to obtain a sufficient quantity of PBMCs for the production of the CCTx-001 investigational product. Should a technical issue arise during the procedure or in the processing of the product such that it cannot be used for CCTx-001 administration, the patient may have a second collection procedure performed. Patients must continue to meet eligibility requirements for repeat leukapheresis.

[0272] The following activities and assessments will be performed on the day of but before the unstimulated leukapheresis:• Check leukapheresis eligibility: patients must be evaluated for evidence of active infections prior to the leukapheresis being started. In case of suspected infection, patient should be treated and leukapheresis postponed until the active infection has resolved.• Assess ECOG performance status.• Measure vital signs and SaO2 via pulse oximetry (pre and post leukapheresis)• Collection of peripheral blood samples for clinical laboratory evaluations, and prepare leukapheresis: o Hematology panel o Chemistry panel o Inflammatory markers• Record all AEs related to protocol mandated procedures, concomitant medications and concomitant procedures.

[0273] Patients may start bridging therapy if needed for disease control while CCTx- 001 is manufactured.Lymphodepleting Chemotherapy (LDC):

[0274] LDC should be initiated to be completed 2 to 7 days prior to CCTx-001 infusion.

[0275] Patients receiving bridging therapy should have another staging (BMA / BMB, complete blood count, flow cytometry) prior to start of the LDC.

[0276] Patients must be evaluated prior to start of LDC (pre-LDC evaluation); adequate organ function and no evidence of active infections prior to the LDC are required. In case of suspected infection, the patient should be treated accordingly, and LDC postponed until the active infection has resolved. Patients with rapid deterioration or rapid disease progression should not start LDC.

[0277] Patients will be treated with fludarabine IV (30 mg / m2 / day for 4 days) (Dekker 2022, Fabrizio 2022) and cyclophosphamide IV (300 mg / m2 / day for 3 days) prior to CCTx-001 infusion. Refer to the most recent package inserts for further details on administration of these agents.

[0278] LDC can start 6 to 10 days before CCTx-001 infusion and must be completed at least 48 hours before CCTx-001 infusion. If side effects from the LDC occur, CCTx-001 infusion may be delayed for up to 7 days after LDC.Day 1 (CCTx-001 Infusion):

[0279] Patients must be evaluated prior to start of CCTx-001; adequate organ function and no evidence of active infections prior to the LDC are required. In case of suspected infection, the patient should be treated accordingly, and CCTx-001 postponed until the active infection has resolved. Patients with rapid deterioration or rapid disease progression should not start CCTx-001.

[0280] The following activities and assessments will be performed before administration of CCTx-001 infusion:• Confirm treatment eligibility• Assess ECOG performance status• Perform physical examination• Perform routine neurologic examination• Measure vital signs and SaCh via pulse oximetry• Collect peripheral blood samples for clinical laboratory evaluations as described: o Hematology panel o Coagulation tests o Chemistry panel o Inflammatory markers• Collect PK samples for droplet digital polymerase chain reaction (ddPCR) and flow cytometry• Collect samples for biomarkers• Collect peripheral blood sample (serum and PBMCs) for immunogenicity test• Administer CCTx-001 infusion• Phase 2 only: Administer HM-PRO questionnaire• Record all AEs, concomitant medications, and concomitant procedures• Assess hospital resource utilization

[0281] CCTx-001 will be provided as cell dispersion for IV infusion. The cells are formulated in an infusible cry opreservation medium and are stored frozen below - 120°C. Each infusion bag will be labelled as required per country requirement.

[0282] Patients should be premedicated with 500 to 650 mg paracetamol / acetaminophen per os (PO) and 25 to 50 mg diphenhydramine hydrochloride (PO or IV) 30 to 60 minutes prior to CCTx-001 infusion. In case the diphenhydramine is not available in a country, it is acceptable to use another Hl antihistamine.

[0283] These medications may be repeated every 6 hours as needed based on the Investigator’s assessment of symptoms. Premedication with steroids must be avoided.

[0284] CCTx-001 will be infused at the respective DL (Phase 1) or at RP2D (Phase 2) on Day 1 (2 to 7 days after completion of LDC). Dose calculation is based on the number of CCTx-001 -positive viable transduced T-cells (CAR+ T-cells).

[0285] The patient must be continuously monitored during administration of CCTx- 001. Vital signs (temperature, respiratory rate, heart rate, blood pressure, and SaCh by pulse oximetry) will be measured approximately every 15 minutes, starting from 15 minutes prior to the first IV administration until one hour after the last IV administration, and hourly for the next 2 hours. If the patient's vital signs are notstable 4 hours following the final administration, vital signs should be monitored as clinically indicated until stable.Post-Treatment Visits:

[0286] All patients who received the CCTx-001 infusion should complete the posttreatment period visits at approximately 4, 5, 6, 9, 12, 15, 18, 21 and 24 months after CCTx-001 infusion for disease status and survival, unless otherwise specified.

[0287] The following activities and assessments will be performed in patients without PD / relapse and who have not received subsequent anticancer treatment following CCTx-001 infusion:• Assess ECOG performance status at Months 4, 5, 6, 9, 12, 15, 18, 21, and 24• Perform physical examination at Months 4, 5, 6, 9, 12, 15, 18, 21, and 24• Measure vital signs at Months 4, 5, 6, 9, 12, 15, 18, 21, and 24• Measure SaO2via pulse oximetry at Months 4, 5, 6, 9, 12, 15, 18, 21, and 24, if clinically indicated• Collect BMB / BMA and peripheral blood for response evaluation at Months 4, 5, 6, 9, 12, 18 and 24 (Note: For Months 6, 9, 12, 18 and 24 these are not required after PD / relapse or subsequent anticancer treatment; if not previously in CR; if not persistent cytopenia (> grade 2), and if not transfusion dependent).• Collect peripheral blood samples for clinical laboratory evaluations as follows: o Haematology panel at Months 4, 5, 6, 9, 12, 15, 18, 21, and 24 o Chemistry panel at Months 4, 5, 6, 9, 12, 15, 18, 21, and 24 o Serum B-HCG pregnancy test on all WOCBP at Month 12• Collect PK samples for ddPCR (see the CCTx-001 laboratory manual) at Month 4, 5, 6, 9, 12, 15, 18, 21, and 24 (only if persistent vector sequences [PVS] were detected by PCR in the two prior samples)• Collect samples for biomarkers at Months 6• Collect peripheral blood sample (serum and PBMC) for immunogenicity test at Months 6, 12, and 24• Collect peripheral blood sample for RCL test at Months 6, 12, and 24• Collect peripheral blood sample for integration site test at Months 6, 12, 24, 30, 36, 42, 48, 54, 60, and annually thereafter (Only if PVS detected, AND if > 1% of peripheral T-cells are positive for PVS)• Record AEs related to CCTx-001 and / or LDC and associated concomitant medications and procedures• Record new anticancer therapies• Check survival status

[0288] Patients who receive HSCT post-CCTx-001 (but no other anticancer treatment) should continue to undergo disease evaluations unless they have demonstrated PD prior to transplant.Long-Term Follow-Up Visits:Follow-up visits: M36, M48, M60

[0289] At these visits, patients will undergo one or more of the following: ECOG, blood tests including hematology, chemistry, RCL, PK and integration site testing (M36 only unless PVS detected). Patients should undergo a physical exam including vital signs. Female reproductive status and current status of primary malignancy will be captured. In addition, protocol defined AEs including new malignancies and selected concomitant medications will be collected including mutagenic agents including cytotoxic drugs, radiation therapy and antineoplastic therapy (including stem cell transplant), immunosuppressive agents including dose of steroids higher than physiologic replacement doses of steroids (20 mg / day hydrocortisone or equivalent), and investigational agents.Follow-up visits: M30, M42, and M54

[0290] At these visits, patients will undergo one or more of the following: ECOG, blood tests including hematology, chemistry, and PK. Patients should undergo a physical exam including vital signs. Current status of primary malignancy will be captured. In addition, protocol defined AEs including new malignancies and selected concomitant medications will be collected including mutagenic agents including cytotoxic drugs, radiation therapy and antineoplastic therapy (including stem cell transplant), immunosuppressive agents including dose of steroids higher than physiologic replacement doses of steroids (20 mg / day hydrocortisone or equivalent), and investigational agents.Follow-up visits: 6Y to 15Y if CAR transgene is detected

[0291] One or more of the following data will be collected: ECOG, physical examination, blood tests including hematology and chemistry and the following until respective test status is undetectable: RCL, PK and integration site testing.

[0292] Female reproductive status (for patients < 18 years of age at time of initial infusion commence collecting at > 8 years of age) and current status of primary malignancy will be captured. In addition, protocol defined AEs including new malignancies and selected concomitant medications will be collected including mutagenic agents including cytotoxic drugs, radiation therapy and antineoplastic therapy (including stem cell transplant), immunosuppressive agents including dose of steroids higher than physiologic replacement doses of steroids (20 mg / day hydrocortisone or equivalent), and investigational agents.Follow-up visits: 6Y to 15Y if no CAR transgene is detected

[0293] At the intervals indicated above for patients who no longer have the persistent vector sequences detected, their annual visits can occur by phone call, video call or through the mail to collect selected required information.

[0294] One or more of the following data will be collected: weight and height (as applicable). Female reproductive status (for patients < 18 years of age at time of initial infusion commence collecting at > 8 years of age) and status of primary malignancy will be captured. In addition, protocol defined AEs including new malignancies and selected concomitant medications will be collected including mutagenic agents including cytotoxic drugs, radiation therapy and antineoplastic therapy (including stem cell transplant), immunosuppressive agents including dose of steroids higher than physiologic replacement doses of steroids (20 mg / day hydrocortisone or equivalent), and investigational agents. Repeated attempts should be made to contact the patient.End of Study (EOS) / End of 15 years long-term follow-up visit:

[0295] The end of the follow-up (EOS) will be 15 years after the date of the last infusion of the CCTx-001.Pharmacokinetics of CCTx-001:

[0296] Assessment of CCTx-001 PK will be determined by ddPCR to detect vector HIV gag DNA sequences and / or by flow cytometry to enumerate and immunophenotype CCTx-001 cells. Peripheral blood will be collected as indicated in Table 3.Biomarkers, pharmacodynamics, pharmacogenomics:

[0297] Immune responses to CCTx-001 will be evaluated with an anti -therapeutic antibody assay to detect the presence of circulating antibodies that bind to the extracellular region of the CAR. In addition, cellular immunogenicity may also be evaluated by testing PBMCs from patients for the presence of anti-CCTx-001 cytotoxic T-cells.

[0298] Exploratory biomarker assessments will be collected and will include evaluation of CCTx-001 and circulating cells in peripheral blood, characterization of leukemia, leukemia microenvironment, and analysis of plasma cytokines.

[0299] BMA and BMB samples will be collected to investigate cellular elements and the tumur microenvironment for biomarkers related to clinical activity and association with disease features (e.g., cytogenetics). In addition, these specimens will be analyzed for expression of various markers of immunological interest. This may provide insights into pathways activated in the tumor microenvironment that may influence the fate and activity of CCTx-001 cells.

[0300] Peripheral blood and plasma will be collected to look at analyses including, but not limited to the following: Soluble factors from plasma will be measured as a marker of immune activation and to determine potential correlations between cytokine production, efficacy and severity of CRS and ICANS; and Phenotypic characterization and gene expression profiling and various analyses at the nucleotide level may be conducted on CCTx-001 cells and immune cells in order to identify markers or gene signatures correlating with clinical response.

[0301] The data collected in these biomarker assessments will be used to elucidate the relationship of CAR T-cell function, persistence, disease, and tumor microenvironment features and CCTx-001 and peripheral blood characteristics to clinical response and toxicity.Concomitant medications and procedures

[0302] To minimize the risk of infusion reactions, all patients should be premedicated with acetaminophen and diphenhydramine prior to IMP infusion.

[0303] In some cases, tocilizumab, an anti-IL-6R-antibody, may be required to treat toxi cities such as CRS. Please refer to the currently approved Actemra® / RoActemra® package insert. As per label, up to 4 doses of tocilizumab can be given for treatment of CRS. The recommendation is to follow the local labelled guidance. In some cases, steroids (e.g., dexamethasone) may also be given for the treatment of CRS or ICANS.

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[0305] The foregoing description of the specific aspects and embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects and embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects and embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0306] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects and embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0307] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout thisapplication are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

[0308] While various specific aspects have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the disclosure(s). Many variations will become apparent to those skilled in the art upon review of this specification.LISTING OF SEQUENCES

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting the growth of a tumor cell in a subject in need thereof comprising administering to the subject a therapeutically effective amount of T cells expressing a chimeric antigen receptor (CAR) at their surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an antiinterleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SAS and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 comprising the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14, wherein prior to administering the T cells expressing the CAR, the subject is preconditioned with a lymphodepleting chemotherapy (LDC).

2. A method of treating acute myeloid leukemia (AML) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of T cells expressing a chimeric antigen receptor (CAR) at their surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an antiinterleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SAS and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 comprising the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14, wherein prior to administering the T cells expressing the CAR, the subject is preconditioned with a lymphodepleting chemotherapy (LDC).

3. The method of claim 1 or 2, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 19.

4. The method of any one of claims 1-3, wherein the T cells comprise CD4+ and CD8+ T cells.

5. The method of claim 3 or 4, wherein the T cells are autologous.

6. The method of any one of claims 1-5, wherein the subject has increased IL-1RAP expression compared to a normal subject.

7. The method of any one of claims 1-6, wherein the T cells expressing the CAR are administered intravenously.

8. The method of claim any one of claims 1-7, wherein the LDC comprises administering radiotherapy, fludarabine, or cyclophosphamide.

9. The method of any one of claims 1-8, wherein the LDC is completed at least 48 hours prior to administering the T cells expressing the CAR.

10. The method of any one of claims 1-9, wherein the LDC is initiated at 6 to 10 days prior to administering the T cells expressing the CAR.

11. The method of any one of claims 1-10, wherein the T cells expressing the CAR are administered at a dose of 0.1 x 106cells / kg, 0.5 x 106cells / kg, 1 x 106cells / kg, 5 x 106cells / kg, or 10 x 106cells / kg.

12. The method of any one of claims 1-11, wherein the T cells expressing the CAR are administered as a single dose.

13. The method of any one of claims 6-12, wherein IL-1RAP expression levels are determined by immunohistochemistry (IHC), flow cytometry, or quantitave polymerase chain reaction (qPCR).

14. The method of any one of claims 1-13, wherein the subject is a human.

15. The method of any one of claims 1-14, wherein the tumor cell is a hematologic cancer tumor cell, or a tumor cell resulting from a hematologic cancer.

16. The method of any one of claims 2 to 16, wherein the AML is a minimal residual disease (MRD).

17. The method of any one of claims 2 to 16, wherein the AML is an IL-lRAP-expressing AML.

18. The method of any one of claims 2 to 17, further comprising detecting IL-1RAP in a sample obtained from the AML prior to, during, or after administering the T cells.

19. The method of any one of claims 1 to 18, wherein the subject received at least one prior line of therapy, at least two prior lines of therapy, or at least three prior lines of therapy.

20. Use of the T cells of any one of claims 1-5 for inhibiting the growth of an AML in a subject in need thereof.

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