Anti-IL-1RAP CAR-T cells for the treatment of acute myeloid leukemia

Abstract

The present disclosure provides a method for treating refractory or relapsed acute myeloid leukemia (AML) in a subject, the method comprising administering cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody that binds to IL-1RAP or an antigen-binding fragment thereof, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulation domain, wherein prior to administering the cells, the subject is pre-conditioned with lymphocyte-depleting chemotherapy (LDC).

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This 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.

[0003] Reference to the electronically submitted sequence list

[0004] The contents of the electronically submitted sequence list submitted with the application (Name: 5041_015PC02_SequenceListing_ST26; Size: 21,525 bytes; and Date of creation: November 12, 2024) are incorporated herein by reference in their entirety.

[0005] Initiation field

[0006] The disclosure provided herein relates to a method for treating acute myeloid leukemia (AML) (e.g., refractory or relapsed (r / r) AML) in a subject, comprising administering cells expressing a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or an antigen-binding fragment thereof that specifically binds to IL-1RAP, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulation domain, wherein prior to administering the cells, the subject is pre-conditioned with lymphocyte-depleting chemotherapy (LDC). Background Technology

[0007] AML is a form of blood cancer characterized by the clonal expansion of immature blasts of bone marrow origin in the bone marrow (BM), resulting in impaired hematopoiesis and BM failure. Although AML is the most frequent acute leukemia, it is a rare disease. In the United States (US), the age-adjusted incidence of AML is 4.3 per 100,000 people per year; in 2020, there were an estimated 73,168 people living with AML, and in 2022, 20,050 estimated new cases and 11,540 estimated deaths were reported. In Europe, the prevalence was reported to be 13.7 per 100,000 residents (13.2 for men and 13.9 for women per 100,000). The incidence of AML is age-dependent and increases significantly in patients aged ≥60 years. The median age at diagnosis is approximately 70 years.

[0008] For patients with primary refractory or relapsed (r / r) AML, the prognosis remains poor and treatment is difficult. As knowledge of the molecular mutational patterns of AML leukemia continues to increase, other targeted therapy options with lower toxicity compared to conventional treatments and stem cell transplantation have emerged. While the treatment paradigm for r / r AML is shifting toward more targeted agents, cytotoxic chemotherapy continues to play a role for young / healthy patients as a bridge to hematopoietic stem cell transplantation (HSCT), particularly in those with extended durations of initial complete response who are expected to develop chemotherapy-sensitive disease.

[0009] Based on the success and subsequent approval of cellular CD19-targeted immunotherapy and the development of B-cell maturation antigen-targeted CARs in multiple myeloma, CAR T-cells are being developed as anti-AML therapies for r / r patients. Several AML cell surface targets, e.g., CD33, CD123, CD44v6, CLL-1, and B7H6, have been explored. However, all of these targets have potential toxicity due to their expression in healthy hematopoietic stem cells or progenitor cells (HSPCs), which can lead to the elimination of all myeloid progeny. Therefore, there is a need to explore more selective AML cell surface targets.

[0010] IL-1RAP is overexpressed on the surface of cells in multiple solid tumor types and has been identified as a potential therapeutic target in AML, chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS), as well as for various other indications including Ewing sarcoma. IL-1RAP enhances multiple oncogenic signaling pathways in AML and promotes leukemia cell proliferation and survival through the FLT3 and c-Kit pathways, representing a potentially promising target for treating AML. Therefore, there is a therapeutic opportunity to treat AML by targeting IL-1RAP with CAR T-cell-based therapies.

[0011] In one aspect of the present disclosure, a method for inhibiting the growth of tumor cells in a subject requiring inhibition of tumor cell growth is provided, comprising the step of administering a therapeutically effective amount of T cells expressing a chimeric antigen receptor (CAR) on its surface to the subject, wherein the CAR comprises an antibody or an antigen-binding fragment thereof comprising an anti-interleukin 1 receptor helper protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulation domain, wherein the anti-IL-1RAP binding domain comprises (i) a light chain comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO. 6, a CDR2 having the amino acid sequence of SEQ ID NO. 7, and a CDR3 having the amino acid sequence of SEQ ID NO. 8, and (ii) a heavy chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 12, a CDR2 having the amino acid sequence of SEQ ID NO. 13, and a CDR3 having the amino acid sequence of SEQ ID NO. 14, and before administering the T cells expressing the CAR, the subject It is pre-conditioned with lymphocyte depletion chemotherapy (LDC).

[0012] In one aspect of the present disclosure, a method for treating a subject requiring treatment for acute myeloid leukemia (AML) (e.g., refractory or relapsed AML) is provided, comprising the step of administering to the subject a therapeutically effective amount of T cells expressing a chimeric antigen receptor (CAR) on its surface, wherein the CAR comprises an antibody or an antigen-binding fragment thereof comprising an anti-interleukin 1 receptor helper protein (IL-IRAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulation domain, wherein the anti-IL-IRAP binding domain comprises: (i) a light chain comprising a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO. 6, a CDR2 comprising the amino acid sequence of SEQ ID NO. 7, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 8; and (ii) a heavy chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO. 12, a CDR2 comprising the amino acid sequence of SEQ ID NO. 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 14. Before administering T cells expressing CAR, the subject is pre-conditioned with lymphocyte depletion chemotherapy (LDC).

[0013] In some embodiments, CAR comprises the amino acid sequence of SEQ ID NO. 19.

[0014] In some embodiments, T cells include CD4+ and CD8+ T cells. In some embodiments, T cells are autologous.

[0015] In some embodiments, the subject has increased IL-1RAP expression compared to normal subjects.

[0016] In some embodiments, T cells expressing CAR are administered intravenously.

[0017] In some embodiments, LDC includes administering radiation therapy, fludarabine, or cyclophosphamide.

[0018] In some embodiments, the LDC is completed at least 48 hours prior to administering the CAR-expressing T cells. In some embodiments, the LDC is initiated 6 to 10 days prior to administering the CAR-expressing T cells.

[0019] In some embodiments, CAR-expressing T cells are 0.1 x 10 6 cells / kg, 0.5 x 10⁶ 6 cells / kg, 1 x 10⁶ 6 cells / kg, 5 x 10 6 cells / kg, or 10 x 10⁶ 6 It is administered at a dose of cells / kg.

[0020] In some embodiments, CAR-expressing T cells are administered as a single dose.

[0021] In some embodiments, IL-1RAP expression levels are determined by immunohistochemistry (IHC), flow cytometry, or quantitative polymerase chain reaction (qPCR).

[0022] In some modes, the object is a human.

[0023] In some forms, the tumor cells are blood cancer tumor cells, or tumor cells originating from blood cancer.

[0024] In some forms, AML (e.g., refractory or relapsed AML) is minimal residual disease (MRD).

[0025] In some aspects, AML is IL-1RAP-expressing AML.

[0026] In some embodiments, the method further comprises the step of detecting IL-1RAP in a sample obtained from AML (e.g., refractory or relapsed AML) before, during, or after administering T cells.

[0027] In some embodiments, the subject was provided with at least one previous treatment course, at least two previous treatment courses, or at least three previous treatment courses.

[0028] In one embodiment, the present invention provides a use of T cells as described herein for inhibiting the growth of AML (e.g., refractory or relapsed AML) in subjects requiring inhibition of such growth. Brief explanation of the drawing

[0029] Fig. 1 This shows a schematic diagram of the CCTx-001 CAR. Fig. 2 This demonstrates the in vitro cytotoxic ability of CAR T-cells derived from B-L43 / A3C3 mAb against IL-1RAP+ expressing cells. Effector cells (NTD or CAR T-cells) were labeled with the cell proliferation dye eFluor™ 450 and co-cultured with IL-1RAP+ Molm-13 target cell lines at the indicated E:T ratio. Target cells without effector cells were also included as a negative control. After incubating the cell suspension at 37°C and 5% CO2 for 24 hours, the cells were stained with viability markers and analyzed by flow cytometry. Cytotoxicity was calculated based on the percentage of viable target cells, normalized to the percentage of viable target cells under the target-only condition (target cells without effector cells). Figures 3a-3fThis demonstrates the in vitro efficacy and specificity of CCTx-001 (using a batch representing the clinical process, also referred to herein as the TR batch) 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 three independent healthy donors (or their donor-matched NTD counterparts) and subsequently analyzed by flow cytometry (FC) to quantify target cell viability in order to confirm the functionality of CCTx-001 CAR T-cells. The data show viable MOLM-13 (IL-1RAP-positive) (Figs. 3a-3c) or Raji (IL-1RAP negative) (Figs. 3d-3f) target cells as a percentage of the target cell-only control (100% viability in the absence of T-cells). Target cells were co-cultured for 24 hours with non-transformed (NTD) T-cells (dotted curves) or CAR T-cells (solid curves) from 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 treated with 0.025% Triton X-100 as a toxicity control (red data points). Data represent the mean ± SD of three descriptive replicates. Statistical differences between the 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 calculated using Holm- to correct for non-corresponding multiple t-tests and multiple comparisons using Welch correction. Determined by the method. *** p ≤ 0.001; ** p ≤ 0.01; *p ≤ 0.05; 'ns' (not significant) p > 0.05. CAR T = Chimeric antigen receptor T-cell, NTD T = Untransduced T-cell. SD = Standard deviation. Figs. 4a-4iFigures show the concentration (in pg / mL) of IFNγ secreted from NTD T-cells (dashed curves) or CCTx-001 (C4-derived) anti-IL-1RAP CAR T-cells (solid curves) from donor TR3 (Figs. 4a, 4d, and 4g), TR4 (Figs. 4b, 4e, and 4h), and TR5 (Figs. 4c, 4f, and 4i) cultured for 24 hours in the absence of the indicated E:T ratio (T-cells alone) (Figs. 4a-4c) or in the presence of IL-1RAP-positive MOLM-13 (Figs. 4d-4f) or IL-1RAP-negative Raji cells (Figs. 4g-4i). The dashed line indicates the LLOQ. Data represent the mean ± SD of three technical replicates. Statistical differences between the log-transformed values ​​of CAR T-cells versus NTD T-cells co-culture, CAR T MOLM-13 co-culture versus CAR T Raji co-culture, or CAR T MOLM-13 co-culture versus CAR T-cells alone (within the same E:T ratio and the same T-cell donor) are Holm- to correct for non-corresponding multiple t-tests and multiple comparisons using Welch correction. It was determined by the method. Statistical analysis of cytokine secretion results was not performed if the mean value was less than the LLOQ (352 pg / mL for 200-fold diluted samples and 17.6 pg / mL for 10-fold 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 = untransduced T-cells, CAR T = chimeric antigen receptor T-cells. Fig. 5This demonstrates the in vitro cytotoxic ability of CCTx-001 (using TR batches) against AML cell lines with varying levels of IL-1RAP expression using a co-culture cytotoxicity assay. Effector cells (NTD or CAR T-cells) were labeled with the cell proliferation dye eFluor™ 450 and co-cultured with different AML cell lines at the indicated E:T ratios. The specific cytotoxicity of CAR T-cells is reported as the percentage of remaining living cells gated as eFluor- / 7-AAD- in the FC, normalized to the percentage of cytotoxicity obtained under control conditions using NTD T-cells. Symbols represent the median, and shaded areas represent the range of values. IL-1RAP negative: Raji and K562. Three subgroups were identified based on IL-1RAP surface expression by the FC: IL-1RAP low (100 to 500 ABC): KG-1 and HEL cell lines; IL-1RAP Intermediate (500 to 5000 ABC: EOL-1, OCI-AML3, HL60, and MOLM-13; and IL-1RAP High (greater than 5000 ABC): Mono-Mac-6. ABC = antibody bound per cell. Fig. 6 <> shows Kaplan-Meier plots of survival for MOLM-13-Luc tumor-bearing NCG mice treated with vehicle (Group 1, n=8), untransduced (NTD) human T-cells (Group 2, n=8, labeled as "untransduced T-cells" in the figure), or donor-matched C4-derived CAR T-cells (Group 3, n=5). iv = intravenously; TTE = median time to end. Statistical significance (Logrank test): ne = unevaluable, ns = not significant, * = P < 0.05, ** = P < 0.01, *** = P < 0.001, compared to Group 1. NCG = NOD- Prkdc em26Cd52 Il2rg em26Cd22 / NjuCrl. Fig. 7 Figure 2 shows the percentage of AML cells relative to all living human cells in the circulatory system for Group 2 (NTD / C0) and Group 3 (CCTx-001). Each error bar is constructed using one standard error of the mean. C0 = Untransduced (NTD) T-cells. Figs. 8a-8c Figure 8a shows the kinetics of CAR T-cell (Fig. 8a) and CAR T-cell subtype (CD4 and CD8 CAR T-cells) expansion (Fig. 8b) and peak blood levels (Fig. 8c) occurring from adoptive cell delivery (at Day 1) to Day 30 (end point of the study). For each time point, the following number of independent mice were analyzed: Days 0–14 (n=4), Day 21 (n=3), and Day 30 (n=1). Each error bar is constructed using one standard error of the mean. Figures 9a-9e Figure 9a shows the results of an in vivo study of MOLM-13-Luc tumor-bearing NCG mice treated with non-transgenic (NTD) human T-cells or donor-matched C4-derived CAR T-cells (CCTx-001). Figure 9a shows an example of the in vivo study design. Six mice were present in each study group. Tumors were injected subcutaneously, and injections of non-transgenic or transgenic CAR T-cells (intravenously 1 x 10⁶ 7 Canine T-cells were allowed to settle for up to 4 days prior. The study was terminated by day 30 after T-cell injection, or earlier due to tumor burden and / or comorbidities. Figure 9b shows the mean body weight of each study group throughout the duration of the study. Figure 9c shows the results of bioluminescence imaging (BLI) at days 0, 7, 14, and 21 across each mouse within the study. MOLM-13-Luc signal intensity was measured in luminescence (in units of radiant luminance; p / sec / cm²). 2It is described as / sr). Figure 9d shows a longitudinal analysis of BLI for each study group, expressed as total flux per time point (p / s). Figure 9e shows Kaplan-Meier plots of survival for MOLM-13-Luc tumor-bearing NCG mice treated with vehicle, NTD T-cells, or CCTx-001. The probability of survival was based on the time to reach a BLI of 1E+10 p / s. NCG = NOD- Prkdc em26Cd52 Il2rg em26Cd22 / NjuCrl. Fig. 10 Figure [] shows the flowchart of the CCTx-001 open-label multicenter Phase 1 / 2 study to evaluate the safety, tolerability, and clinical activity of CCTx-001 in patients with r / r AML. Abbreviations: EOS = End of study; LDC = Lymphocyte depletion chemotherapy; PK = Pharmacokinetics; PVS = Durability vector sequence; RCL = Replication eligible lentivirus. Fig. 11 This shows a graphical overview of the dose escalation protocol of the CCTx-001 study. Fig. 12 is the percentage of remaining living AML blasts normalized to NTD T-cell conditions of different CAR T-cells relative to primary AML cells. In vitro It exhibits specific cytotoxicity. MCL004-009 and MCL004-088 are batches of healthy donor-derived CCTx-001 CAR T-cells. CL045-007 to CL045-011 are identifiers of co-cultured primary allogeneic AML patient PBMC samples. Fig. 13 It shows the in vitro cytotoxicity of different NTD and CAR T-cells against primary autologous AML blasts, reported as the percentage of remaining living AML blasts normalized to target-only conditions. Fig. 14shows the in vitro specific cytotoxicity of different CAR T-cells against primary AML cells reported as the percentage of remaining living AML blasts normalized to NTD T-cell conditions. MCL004-009 and MCL004-088 are batches of CCTx001 CAR T-cells derived from healthy donors. CL045-007 to CEL045-011 are identifiers of co-cultured primary allogeneic AML patient PBMC samples. Figs. 15a-15b It shows Kaplan-Meier curves for low or high IL-1RAP mRNA expression from publicly available datasets (Fig. 15a) and in silico analysis of IL-1RAP mRNA in AML patients from multiple gene set enrichment studies available by AML subtype (FAB) classification (Fig. 15b). Figs. 16a-16b Figure 16 shows the results of immunophenotyping analysis of IL-1RAP and major AML targets (CD33, CD123, and CLL1) in blood cell populations from PBMCs from healthy donors (Figure 16a) and CD34+ hematopoietic stem and progenitor cells from healthy donors (Figure 16b). Fig. 17 It shows IL-1RAP membrane expression (as a bound antibody per cell) from AML patient cells compared to healthy donor blood and bone marrow cells. Figs. 18a-18b Figure 18 shows the functional results of AML patient-derived CCTx-001 CAR-T cells. Figure 18a shows the results of an IFNγ release assay by four different patient-derived CCTx-001 CAR-T cell products before and after IL-1RAP-mediated stimulation. Figure 18b shows the results of an in vitro cytotoxicity assay of patient-derived CCTx-001 CAR-T cell products against IL-1RAP-expressing MOLM-13 AML cell lines at an effector:target ratio of 1:1. Specific details for implementing the invention

[0030] The present disclosure relates to a method for inhibiting the growth of a blood malignancy / treating cancer, comprising administering cells containing a nucleic acid molecule encoding an IL-1RAP-specific CAR. In an embodiment, lymphocyte-depleting chemotherapy (LDC) is performed prior to administering the cells.

[0031] 1. Definition

[0032] To facilitate a better understanding of the present disclosure, specific terms are defined first. As used in this application, each of the following terms will have the meanings set forth below unless otherwise explicitly provided herein. Additional definitions are provided throughout the application.

[0033] Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to any specific composition or process step, and that such may vary. As used in this specification and the appended claims, the singular forms “one (a),” “an,” and “the” include multiple references unless the context clearly indicates otherwise. The terms “a (or an),” as well as “one or more” and “at least one,” may be used interchangeably herein.

[0034] Additionally, as used herein, "and / or" shall be deemed to specifically disclose each of the two designated features or components, either together with or without the other. Accordingly, the term "and / or," as used herein in phrases such as "A and / or B," is intended to include "A and B," "A or B," "A alone," and "B alone." Likewise, the term "and / or," as used herein in phrases such as "A, B, and / or C," is intended to encompass each of the following modes: 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).

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure relates. 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 a general dictionary of many terms used in this disclosure to those skilled in the art.

[0036] Units, prefixes, and symbols are indicated in their International System of Units (SI) approved forms. Numerical ranges include the number defining the range. Unless otherwise indicated, amino acid sequences are written from left to right with an amino-to-carboxy orientation. The subject matter provided herein is not limiting in various aspects, which may be obtained by referring to the specification in its entirety. Accordingly, terms defined immediately below are more fully defined by referring to the specification in its entirety.

[0037] Wherever an aspect is described in this document with the language of “comprising,” it is understood that other similar aspects described in terms of “comprising” and / or “essentially comprising” are also provided. As used herein, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “including”) are to be understood as indicating the inclusion of a group of the mentioned components, features, elements, or steps or components, features, elements, or steps, but not the exclusion of any other components, features, elements, or steps or groups of components, features, elements, or steps. Any of the terms “comprising,” “essentially comprising,” and “comprising” may be replaced by either of the other two terms while retaining their ordinary meanings.

[0038] The term “about” is used herein to mean approximately, generally, about, or in the vicinity. When the term “about” is used with a numerical range, it modifies the range by extending the boundaries above and below the presented numerical value. Generally, the term “about” may modify an upward or downward (higher or lower) numerical value above or below the specified value, for example, by a variation of 10 percent.

[0039] As used herein, the term “approximately,” as applied to one or more values ​​of interest, refers to a value similar to the mentioned reference value. In certain modalities, the term “approximately,” unless otherwise specified or otherwise evident from the context, refers to a range of values ​​falling within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less in either direction (greater or less than) the mentioned reference value (except where such a number exceeds 100% of possible values).

[0040] Amino acids are referred to by the three-letter symbols commonly known herein or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Nucleotides are likewise referred to by their commonly accepted single-letter codes.

[0041] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM", respectively.

[0042] As used herein, the term “polypeptide” is intended to encompass both singular “polypeptide” and plural “polypeptide” and comprises any chain or chain of two or more amino acids. Accordingly, as used herein, any other term used to refer to “peptide,” “peptide subunit,” “protein,” “amino acid chain,” “amino acid sequence,” or a chain or chain of two or more amino acids is included in the definition of “polypeptide,” even though each of these terms may have a more specific meaning. The term “polypeptide” may be used in place of or interchangeably with any of these terms. The term further comprises polypeptides that have undergone post-translational or post-synthetic modification, e.g., conjugation of palmitoyl groups, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, disulfide bond formation, proteolytic cleavage, or modification by non-naturally occurring amino acids. As used herein, the term “peptide” encompasses full-length peptides and their fragments, variants, or derivatives. As used herein, “peptide” may be part of a fusion polypeptide that includes additional components, such as albumin or PEG moiety, to increase the half-life. As used herein, peptides may also be derivatized in a number of different ways. Peptides may include modifications, for example, including the conjugation of palmitoyl groups. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded and may be cDNA.

[0043] The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., homology % = number of identical positions / total number of positions x 100), the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap. The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm, as described in the non-limiting examples below.

[0044] The percentage of identity between two nucleotide sequences can be determined using the NWSgapdna.CMP matrix and the GAP program of the GCG software package (available at worldwideweb.gcg.com), using gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4, as described by E. Meyers and W. Miller (integrated into the ALIGN program (version 2.0)). CABIOS The percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch ( , 4: 11-17 (1989)). Additionally, using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6, integrated into the GAP program of the GCG software package (available at http: / / www.gcg.com) J. Mol. Biol It can be determined using the algorithm. (48):444-453 (1970))

[0045] The nucleic acid and protein sequences described herein may be further used, for example, as "query sequences" to perform a search against public databases to identify related sequences. Such a search is Altschul, et al. (1990) J. Mol. Biol This can be performed using the NBLAST and XBLAST programs of .215:403-10 (version 2.0). A BLAST nucleotide search can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search 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 gap alignment for comparison purposes, Altschul et al. , (1997) Nucleic Acids Res Gapped BLAST may be utilized as described in . 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used. worldwideweb.ncbi.nlm.nih.gov reference .

[0046] As used herein, the terms “operationally linked,” “operationally inserted,” “operationally located,” “under control,” or “under transcriptional control” mean that a promoter is located at an accurate position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression. The term “operationally linked” means that the DNA sequence and the regulatory sequence(s) are linked in such a way that gene expression is allowed when an appropriate molecule (e.g., a transcription activator protein) binds to the regulatory sequence(s). The term “operationally inserted” means that DNA of interest introduced into the cell is located adjacent to a DNA sequence that directs the transcription and translation of the introduced DNA (i.e., promotes the production of a polypeptide encoded by the DNA of interest).

[0047] The terms "#E3C3" and "#A3C3" are understood to be synonymous: #E3C3 may be freely used to refer to #A3C3 and vice versa. B-L43 is also synonymous with #E3C3 and #A3C3, each referring to a hybridoma clone that shares the same nucleotide sequence for its heavy and light chains. #E3C3 and #A3C3 are further described in PCT publications No. WO2019101604A1 and WO2020239801A1, each incorporated herein by reference in its entirety.

[0048] As used herein, the term “chimeric antigen receptor” or “CAR” refers to an engineered antigen-binding polypeptide comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Expression of the CAR on the surface of a cell, e.g., an immune cell, enables the cell to target and bind to a specific antigen. In some embodiments, the CAR is expressed by immune cells, e.g., T cells. In some embodiments, the antigen-binding domain comprises Fab, Fab', F(ab')2, Fd, Fv, single-chain fragment variable portion (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD4, CD8α, or CD28. In some embodiments, the intracellular domain comprises a co-stimulatory domain or a part thereof. In some embodiments, the intracellular domain comprises a co-stimulatory domain selected from the group consisting of the intracellular domain of CD3z, the CD28 co-stimulatory domain, the CD27 co-stimulatory domain, the 4-1BB co-stimulatory domain, the ICOS co-stimulatory domain, the OX-40 co-stimulatory domain, the GITR co-stimulatory domain, the CD2 co-stimulatory domain, the IL-2Rβ co-stimulatory domain, the MyD88 / CD40a CD28 co-stimulatory domain, and any combination thereof. The CAR may further comprise a "hinge" or "spacer" domain. Non-limiting examples of the hinge / spacer domain include an IgG1 hinge domain, and immunoglobulin hinge / spacer domains such as an IgG2 hinge domain, an IgG3 hinge domain, or an IgG4 hinge domain. In some embodiments, the domains within the CAR polypeptide construct are located within the same polypeptide chain and comprise, for example, a chimeric fusion protein. In some embodiments, the domains within the CAR polypeptide construct are not continuous with each other and, for example, are located within different polypeptide chains.

[0049] As used herein, "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate signals that promote immune effector functions in CAR-containing cells, e.g., CAR T cells. Examples of immune effector functions include cytolytic activity and helper activity, such as the secretion of cytokines, in CAR T cells, for example. In the embodiments, the intracellular signaling domain is a portion of a protein that conveys effector function signals and directs the cell to perform a specialized function. The entire intracellular signaling domain may be used, but 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 instead of the whole chain, provided that it conveys an effector function signal. Thus, the term intracellular signaling domain means comprising any truncated portion of the intracellular signaling domain sufficient to convey an effector function signal.

[0050] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. An exemplary primary intracellular signaling domain includes those derived from a molecule responsible for a primary stimulus or antigen-dependent simulation. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. An exemplary co-stimulatory intracellular signaling domain includes those derived from a molecule responsible for a co-stimulatory signal or antigen-independent stimulus. For example, in the case of CAR T, the primary intracellular signaling domain may include a cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may include a cytoplasmic sequence from a co-receptor or a co-stimulatory molecule.

[0051] The primary intracellular signaling domain may include a signaling motif known as an immune receptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon rib (Rib)), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, CD32, DAP10, and DAP12.

[0052] The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as a protein provided as GenBank accession number BAG36664.1, or an equivalent residue from non-human species, e.g., mice, rodents, monkeys, and apes, etc., and “zeta stimulating domain” or alternatively “CD3-zeta stimulating domain” or “TCR-zeta stimulating domain” is defined as an amino acid residue from the cytoplasmic domain of the zeta chain sufficient to functionally deliver an initial signal required for T cell activation, or a functional derivative thereof. In one embodiment, the cytoplasmic domain of zeta comprises residues 52 to 164 of GenBank accession number BAG36664.1 or an equivalent residue from non-human species, e.g., mice, rodents, monkeys, and apes, etc., which are functional orthologs thereof.

[0053] The term "co-stimulator" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand and thereby mediates a co-stimulatory response by the T cell, such as, but not limited to, proliferation. A co-stimulator is a cell surface molecule other than an antigen receptor or their ligand that contributes to an efficient immune response.Co-stimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte-activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / 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, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1. CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, 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, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), Ligands that specifically bind to BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 are included, but not limited to.

[0054] A co-stimulatory intracellular signaling domain refers to the intracellular portion of a co-stimulatory molecule. The intracellular signaling domain may include the entire intracellular portion of the molecule from which it is derived, the entire natural intracellular signaling domain, or a functional fragment or derivative thereof.

[0055] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank accession number AAA62478.2, or an equivalent residue from non-human species, e.g., mice, rodents, monkeys and apes, etc.; "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or an equivalent residue from non-human species, e.g., mice, rodents, monkeys and apes, etc.

[0056] "Antibodies (Ab) comprise, without limitation, a glycoprotein immunoglobulin that specifically binds to an antigen, or an antigen-binding portion thereof, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (V in this application) H It includes the heavy chain invariant region (abbreviated as) and the heavy chain invariant region. The heavy chain invariant region consists of three invariant domains, C H1 , C H 2 and C H 3. Each light chain includes a light chain variable region (V in this application). L It includes (abbreviated as) and a light chain invariant region. The light chain invariant region is a single invariant domain, C L Includes. V H and V L The region can be further subdivided into hypervariable regions named Complementarity Determination Regions (CDR), which are interspersed among more conserved regions named Framework Regions (FR). Each V H and V L It comprises three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have C-terminal lysine. Unless otherwise specified herein, amino acids within the variable region are numbered using the Kabat numbering system, and amino acids within the constant region are numbered using the EU system.

[0057] Immunoglobulins may be derived from any of the commonly known isotypes, including but not limited to IgA, secreted IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to a class or subclass of antibodies (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term "antibody" includes, for example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and unless the context otherwise indicates, the term "antibody" includes not only single-chain antibodies but also monospecific, bispecific, or multispecific antibodies. In some embodiments, the antibody is a bispecific antibody. In another aspect, the antibody is a monospecific antibody.

[0058] As used herein, "IgG antibody" has the structure of a naturally occurring IgG antibody, that is, it has the same number of heavy chains, light chains, and disulfide bonds as naturally occurring IgG antibodies of the same subclass. For example, anti-ICOS IgG1, IgG2, IgG3, or IgG4 antibodies consist of two heavy chains (HC) and two light chains (LC), wherein the two heavy chains and light chains are each connected by the same number and positions of disulfide crosslinks that occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies (unless the antibody is mutated to modify the disulfide bonds).

[0059] "Isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to PD-L1 is substantially free of antibodies that specifically bind to antigens other than PD-1). However, an isolated antibody that specifically binds to PD-L1 may have cross-reactivity to other antigens, such as PD-L1 molecules from different species. Additionally, the isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0060] Antibodies may be modified antibodies (e.g., by mutation, deletion, substitution, or conjugation to non-antibody moiety). For example, antibodies may contain one or more variant amino acids that alter the properties (e.g., functional properties) of the antibody (compared to naturally occurring antibodies). For example, numerous such modifications are known in the art and affect, for example, the half-life, effector function, and / or immune response to the antibody in patients. The term antibody also includes artificial polypeptide constructs comprising at least one antibody-derived antigen binding site.

[0061] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of a single molecular composition, that is, having essentially identical primary sequences and exhibiting single-binding specificity and affinity for a specific epitope. An 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.

[0062] "Human" antibody (HuMAb) refers to an antibody having a variable region in which both the framework and the CDR region are derived from a human germline immunoglobulin sequence. Additionally, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibody of the disclosure may contain amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is transplanted into a human framework sequence. The terms "human" antibody and "fully human" antibody are used synonymously.

[0063] "Humanized antibody" refers to an antibody in which some, most, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulin. In one embodiment of the humanized form of an antibody, some, most, or all of the amino acids outside the CDR domain are replaced with amino acids from human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not eliminate the antibody's ability to bind to a specific antigen. "Humanized" antibodies retain antigen specificity similar to that of the original antibody.

[0064] "Chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody.

[0065] "Anti-antigen" antibodies refer to antibodies that specifically bind to an antigen. For example, anti-IL-1RAP antibodies specifically bind to IL-1RAP.

[0066] The “antigen-binding portion” (also referred to as an “antigen-binding fragment”) of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen bound by the whole antibody. It has been demonstrated that the antigen-binding function of an antibody can be performed by a fragment or portion of a full-length antibody. Examples of binding fragments encompassed within the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, for example, the anti-IL-1RAP antibody described herein, include the following:

[0067] (1) Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of VL, VH, LC and CH1 domains;

[0068] (2) A similar divalent fragment comprising an F(ab')2 fragment (a fragment from pepsin cleavage) or two Fab fragments connected by a disulfide crosslink at the hinge region;

[0069] (3) Fd fragment consisting of VH and CH1 domains;

[0070] (4) Fv fragment consisting of the VL and VH domains of a single arm of the antibody,

[0071] (5) A single-domain antibody (dAb) fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-46);

[0072] (6) A double-single-domain antibody consisting of two VH domains connected by a hinge (double-affinity re-targeting antibody (DART));

[0073] (7) Bivariable domain immunoglobulin;

[0074] (8) Isolated complementarity determination region (CDR); and

[0075] (9) A combination of two or more isolated CDRs that can be combined by any synthetic linker. Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined by a synthetic linker using a recombination method so that the VL and VH regions can be paired to form a single protein chain that forms a monovalent molecule (known as single-strand 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-strand antibodies are also intended to be encompassed within the terms “antigen-binding portion” or “antigen-binding fragment” of the antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same way as intact antibodies. The antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulin.

[0076] As used herein, the term "IL1RAP" refers to an interleukin-1 receptor helper protein that is a cell membrane co-receptor for several receptors of the IL-1 family, including interleukin-1 receptor 1 (IL1R1), ST2 (also known as interleukin-1 receptor-like 1 or IL-1RL1), and interleukin-1 receptor-like protein 2 (IL-1RL2). Note that the interleukin-1 receptor helper protein, or IL1RAP, is sometimes referred to as "IL-1RAP," "IL-1RAcP," "IL1RAcP," or "IL-1R3" in the art. The terms "IL1RAP," "IL-1Rap," and "IL1RAP protein" are used interchangeably herein.

[0077] Cancer-associated fibroblasts (CAFs), tumor-associated fibroblasts, or activated fibroblasts are cell types within the tumor microenvironment that promote tumorigenic characteristics by initiating the remodeling of the extracellular matrix or by secreting cytokines.

[0078] "Immune checkpoint inhibitor" refers to any compound that inhibits the function of immune checkpoint proteins. Inhibition includes a reduction in function and complete blockade. In particular, immune checkpoint proteins are human immune checkpoint proteins. Therefore, immune checkpoint protein inhibitors are specifically inhibitors of human immune checkpoint proteins.

[0079] "Immunotherapy" refers to the treatment of subjects who have a disease, are at risk of developing a disease, or are at risk of recurrence, by means of a method that includes steps of inducing, enhancing, inhibiting, or otherwise modifying the immune system or immune response.

[0080] The term "autodermium" refers to any substance derived from the same individual that will later be reintroduced into the same individual.

[0081] The term “homologous” refers to any substance derived from a different animal of the same species as the individual into which the substance is introduced. Two or more individuals are referred to as homologous to each other if the genes at one or more loci are not identical. In some modalities, homologous substances from individuals of the same species may be genetically sufficiently different to interact antigenically.

[0082] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0083] As used herein, the term “linked” refers to the combination of two or more molecules. The linkage may be covalent or non-covalent. The linkage may also be genetic (i.e., recombinantly fused). Such linkage may be achieved using various techniques recognized in the art, such as chemical conjugation and recombinant protein production.

[0084] As used herein, the terms “subject,” “individual,” or “patient” refer to any subject requiring diagnosis, prognosis, or therapy, in particular mammalian subjects. Mammal subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears.

[0085] "To bring into contact" is used in its obvious ordinary sense and refers to a process that allows at least two distinct species (e.g., chemical compounds containing biomolecules, or cells) to come into sufficient proximity to react, interact, or come into physical contact. However, it should be understood that the resulting reaction product may be produced directly from the reaction between the added reagents or from an intermediate from one or more of the added reagents that may be produced within the reaction mixture. The term "to bring into contact" may include allowing two species to react, interact, or come into physical contact, wherein the two species may be compounds and proteins or enzymes as described herein. In some embodiments, bringing into contact includes allowing a compound described herein to interact with a protein or enzyme.

[0086] “Administering” refers to the physical introduction of a composition containing a therapeutic agent into a target 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 administration routes, e.g., by injection or infusion. As used herein, the term “parenteral administration” means a mode of administration other than intestinal and local administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intra-articular, intra-articular, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, and in some embodiments, is administered orally. Other parenteral routes include local, epidermal, or mucosal administration routes, e.g., intranasal, vaginal, rectal, sublingual, or topical. Administration may also be performed, e.g., once, multiple times, and / or over one or more extended periods.

[0087] "Treatment" or "therapy" of the subject refers to any type of intervention or process performed on the subject, or the administration of an activator to the subject, for the purpose of reversing, alleviating, improving, suppressing, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, or conditions associated with the disease, or biochemical indicators.

[0088] As used herein, "effective treatment" refers to a treatment that produces a beneficial effect, for example, an improvement in at least one symptom of the disease or disorder. The beneficial effect may take the form of improvement compared to baseline, that is, improvement compared to measurements or observations made prior to the initiation of the therapy according to the method. The beneficial effect may also take the form of inhibiting, slowing, delaying, or stabilizing the detrimental progression of markers of the hematological malignancy. Effective treatment may refer to the alleviation of at least one symptom of the hematological malignancy. Such effective treatment may, for example, reduce patient pain and / or reduce the size and / or number of lesions, reduce or prevent tumor metastasis and / or slow tumor growth.

[0089] The term "effective dose" refers to an amount of a drug that provides a desired biological, therapeutic, and / or prophylactic result. The result may be a reduction, improvement, alleviation, reduction, delay, and / or remission of one or more signs, symptoms, or causes of a disease, or any other desired alteration of the biological system. In relation to hematological malignancies, the effective dose comprises an amount sufficient to shrink the tumor and / or reduce the tumor growth rate (e.g., an amount to inhibit tumor growth) or an amount sufficient to prevent or delay other unwanted cell proliferation. In some embodiments, the effective dose is an amount sufficient to delay tumor development. In some embodiments, the effective dose is an amount sufficient to prevent or delay tumor recurrence. In some embodiments, the effective dose is an amount sufficient to completely eradicate the tumor. In some embodiments, the effective dose is an amount sufficient to completely eradicate tumor cells. The effective dose may be administered in a single or more doses. The effective dose of a drug or composition may (i) reduce the number of cancer cells and / or; (ii) reduce the size of the tumor and / or; (iii) inhibit, delay, slow down, and stop cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis (i.e., slow down and stop); (v) inhibit tumor growth; (vi) prevent or delay the onset and / or recurrence of tumors; and (vii) alleviate one or more of the symptoms associated with cancer to some degree. In one example, the “effective dose” is the amount of IL-1RAP CAR-T that has been clinically proven to have a significant reduction in cancer, such as advanced hematological malignancies (e.g., AML), or to slow the progression of cancer.

[0090] The term “progression-free survival,” which may be abbreviated as PFS as used herein, refers to the period during and after treatment of a hematological malignancy during which a patient survives with the disease but the disease does not worsen. The precise dosage and formulation will depend on the purpose of treatment and will be identifiable by those 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 a given parameter, the therapeutically effective dose will represent an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy may also be expressed as a "multiple" increase or decrease. For example, the therapeutically effective dose may have an effect of at least 1.2 times, 1.5 times, 2 times, or 5 times or more compared to a standard control group. The therapeutically effective dose or amount may improve one or more symptoms of the disease. The therapeutically effective dose or amount may prevent or delay the onset of the disease or one or more symptoms of the disease, provided that the administered effect is intended to treat a person at risk of developing the disease.

[0091] The term “combination” refers to a fixed combination within a single dosage unit, or a combination of the compounds of this disclosure and combination partners (e.g., other drugs as described below, also referred to as “therapeutics” or “co-therapeutics”) that may be administered simultaneously, independently, or separately within a time interval, in particular where such time intervals allow the combination partners to exhibit cooperative, e.g., synergistic effects. A single component may be packaged within a kit or separately. One or both of the components (e.g., powder or liquid) may be reconstituted or diluted to a desired dose prior to administration. As used herein, the terms “co-administration” or “combined administration,” etc., are intended to encompass the administration of a selected combination partner to a single subject (e.g., a patient) requiring it, and are intended to include a therapeutic regimen in which the agents are not necessarily administered via the same route of administration or simultaneously. As used herein, “simultaneously” refers to the administration of two or more therapeutic agents, wherein at least a portion of the administrations overlap in time. Accordingly, simultaneous administration includes a method of medication administration in which the administration of one or more drug(s) continues even after the administration of one or more other drug(s) has been discontinued.

[0092] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells within the body. Uncontrolled cell division and growth lead to the formation of malignant tumors that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0093] The term "leukemia" broadly refers to a progressive malignant disease of the blood-forming organs, generally characterized by the distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified based on (1) the duration and nature of the disease—acute or chronic; (2) the type of cells involved—myeloid, lymphoid, or monocytic; and (3) an increase or non-increase in the number of abnormal cells within the blood—leukemic or aleukemic. Exemplary leukemias that can be treated by the compounds or methods provided herein include, for example, acute myeloid leukemia, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, leukemia-achymatic leukemia, monocytic leukemia, basophilic leukemia, blastocyst leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, Gross' leukemia, hair-cell leukemia, hemoblastic leukemia, hemocytic leukemia, histocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphogenic leukemia, lymphoid leukemia, lymphosarcoma cells Includes leukemia, mast cell leukemia, meganucleic leukemia, micromyeloid leukemia, monocytic leukemia, myeloid leukemia, myeloid 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.

[0094] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” may 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 part of the body. Cancer originates at a site of origin, e.g., the breast, which is referred to as the primary tumor, e.g., primary breast cancer. Some cancer cells from the primary tumor or the site of origin acquire the ability to infiltrate and spread surrounding normal tissue within a local area and / or to penetrate the walls of the lymphatic or vascular system and circulate through the system to other parts and tissues within the body. A clinically detectable secondary tumor formed from cancer cells of the 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, in the case of lung cancer metastasizing to the breast, the secondary tumor in the breast area consists of abnormal lung cells rather than abnormal breast cells. The secondary tumor within the breast is referred to as metastatic lung cancer. Therefore, the phrase metastatic cancer refers to a disease in which the subject has or has a primary tumor and has one or more secondary tumors. The phrase non-metastatic cancer, or a subject with non-metastatic cancer, refers to a disease in which the subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject who has or has a history of a primary lung tumor and has one or more secondary tumors in a second or multiple locations, e.g., the breast.

[0095] "Refractory" cancer is cancer that progresses despite anti-tumor treatments, such as chemotherapy, being administered to the patient. An example of refractory cancer is platinum-refractory cancer.

[0096] "Recurrent" cancer is when cancer or the signs and symptoms of cancer return after a period of improvement.

[0097] "Complete Response," "Complete Remission," or "CR" indicates that all signs of a tumor or cancer disappear in response to treatment. This does not always mean that the cancer has been cured. "CRi" refers to a morphologically complete remission accompanied by incomplete hematological (blood cell count) recovery. "CRMRD-" refers to a complete recovery with no measurable residual disease.

[0098] The term "Objective Response Rate" or "ORR" refers to the percentage of patients who achieve a response, which may be either a complete response or a partial response, after receiving treatment.

[0099] The term "Duration of Response" or "DoR" refers to the time from randomization to disease progression or death in patients who have achieved a complete or partial response. DoR measures how long a patient will respond to treatment without tumor growth or metastasis.

[0100] The term "Total Survival" or "OS" refers to the time from randomization to death. With respect to OS, any patient who is lost to follow-up or still alive at the time of evaluation is censored.

[0101] "CRc" or "clinical complete remission" indicates the absence of evidence of disease, accompanied by some skin changes that do not indicate active disease. "CR with partial hematological recovery" or "CRh" refers to hematological recovery defined as a condition where the patient does not show signs of leukemia, but one or more blood cell counts (e.g., platelets and neutrophils) have not recovered to normal levels (e.g., an absolute neutrophil count (ANC) greater than 500 / μl and a platelet count greater than 50,000 / μl) (see Dohner et al, 2022). "CR with incomplete hematological recovery" or "CRi" refers to a condition where the patient does not show signs of leukemia, but 1 x 10 9 ANC less than / L or < 100 x 10 9It refers to hematological recovery defined by the presence of a platelet count of / L (i.e., thrombocytopenia).

[0102] "Morphological leukemia-free state" or "MLFS" refers to a patient with less than 5% myeloblasts, no circulating blasts, and no extramedullary disease, where hematological recovery is not required. In MLFS, the bone marrow must not be merely "aplastic"; bone marrow fragments must be present, and at least 200 cells must be listed in the aspirate or the cellularity must be at least 10% in the biopsy.

[0103] "Partial response" or "PR" refers to a reduction in the size or volume of one or more tumors or lesions, or the extent of cancer within the body, in response to treatment.

[0104] "Advanced disease" refers to the appearance of one or more new lesions or tumors and / or the apparent progression of existing non-target lesions. Advanced disease may also refer to tumor growth exceeding 20% ​​due to an increase in tumor mass or tumor spread since the start of treatment.

[0105] As used herein, the term “tumor” refers to any mass of tissue resulting from excessive cell growth or proliferation of either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions, regardless of the originating cell.

[0106] "Immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or by the liver, resulting in the selective targeting, binding to, damage to, destruction of, and / or removal of invading pathogens, cells or tissues infected by pathogens, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues from the vertebrate body. Immune response involves, for example, T cells, e.g., effector T cells, Th cells, CD4 + Cell, CD8 + This includes the activation or inhibition of T cells, or Treg cells, or any other cells of the immune system, such as NK cells.

[0107] As used herein, the term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that represents a major component of the innate immune system. NK cells reject virus-infected cells and tumors by inducing apoptosis or programmed cell death in target cells. They are named "natural killers" because NK cells do not require activation to kill target cells. T cells play a major role in cell-mediated immunity. T-cell receptors (TCRs) expressed on the surface of T cells distinguish them 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: 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 cell, or killer T cell); There are memory T-cells ((i) stem memory TSCM cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Ra+ like naive cells, but also express large amounts of CD95, IL-2R.p, CXCR3, and LFA-1 and exhibit many functional properties characteristic of memory cells); (ii) central memory TCM cells express L-selectin and CCR7 and secrete IL-2 but do not secrete IFNy or IL-4, and (iii) however, effector memory TEM cells do not express L-selectin or CCR7 but produce effector cytokines such as IFNy and IL-4); regulatory T-cells (Treg, repressor T cells, or CD4+CD25+ regulatory T cells); natural killer T-cells (NKT); and gamma delta T-cells.

[0108] As used herein, “survival” refers to a patient remaining alive and includes overall survival as well as progression-free survival. 1-year survival and 2-year survival rates refer to KM estimates of the proportion of subjects alive at 12 or 24 months.

[0109] “Prolonging survival” means increasing overall survival and / or progression-free survival in treated patients compared to a control treatment protocol, such as treatment with the antibody-drug conjugate described herein. Survival is monitored for at least about 1 month, 2 months, 4 months, 6 months, 9 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., after the initiation of treatment or initial diagnosis.

[0110] "Reduce or inhibit" means the ability to cause an overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more. "Reduce or inhibit" may refer to the symptoms of the disorder to be treated, the presence or size of metastases, or the size of the primary tumor.

[0111] In the context of polypeptides, the term "heterogeneous" means that two polypeptides are different from each other, derived from a different source (e.g., cell, tissue, organism, or species) compared to other polypeptides. Typically, heterogeneous polypeptides are derived from different species.

[0112] As used herein, in relation to cell proliferation (e.g., cancer cell proliferation), terms such as “inhibition,” “to inhibit,” and “inhibiting” mean to negatively affect a cell (e.g., by reducing proliferation) or to kill the cell. In some embodiments, inhibition refers to the reduction of a disease or symptoms of a disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes at least partially, partially or entirely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. Similarly, “inhibitor” is a compound or protein that inhibits a receptor or other protein by, for example, blocking, reducing, preventing, delaying, inactivating, desensitizing, or down-regulating binding, partial or entirely, or activity (e.g., receptor activity or protein activity).

[0113] A "control group" or "standard control group" refers to a sample, measurement, or value that serves as a reference—commonly known reference—for comparison with a test sample, measurement, or value. For example, a test sample may 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 group may also represent an average measurement or value collected from a population of healthy individuals (e.g., a standard control group) who do not have the given disease (e.g., a standard control subject), having a similar medical background, age, weight, etc. Standard control values ​​may also be obtained from the same individual, for example, from a sample previously obtained from the patient prior to the onset of the disease. For example, a control group may be designed to compare therapeutic benefits based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Control groups are also useful for determining the significance of data. For example, if the value for a given parameter varies widely in the control group, the variation in the test sample will not be considered significant. Those skilled in the art will recognize that standard control groups may be designed for the evaluation of any number of parameters (e.g., RNA levels, protein levels, specific cell types, specific body fluids, specific tissues, synovial cells, synovial fluid, synovial tissue, fibroblast-like synovial cells, macrophage-like synovial cells, etc.).

[0114] The ranges provided herein are understood to be abbreviations for all values ​​within the range. For example, the range from 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 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.

[0115] Various aspects of the present disclosure are described in more detail in the following subsections.

[0116] 2. Method of disclosure

[0117] IL-1RAP is part of a protein receptor complex and assembles with IL-1α, IL-1β, or IL-33 receptors on various cells, particularly hematopoietic cells. IL-1RAP is not expressed in normal HSCs, but has been shown to be overexpressed on the cell surface of leukemia stem cells in acute myeloid leukemia (AML), myelodysplastic syndrome, and chronic myeloid leukemia. IL-1RAP is known to play a key role in inflammation in the tumor microenvironment through p38 MAPK and NF-kβ signaling pathways (De Boer et al. 2020).

[0118] Currently, IL-1RAP is being targeted by two monoclonal antibodies through Cantargia's CAN-04 and CAN-10 projects. CAN-04 targets IL-1RAP in solid tumors, primarily lung and pancreatic, while CAN-10 targets IL-1RAP in autoimmune and inflammatory diseases. Nidalinimab (CAN-04) is used in combination with chemotherapy and is currently undergoing Phase I / IIa clinical investigations in lung and pancreatic cancers (ClinicalTrials.gov: NCT03267316 and NCT04452214). However, monoclonal antibodies have shown significant drawbacks in this context compared to adoptive cell therapies such as CAR-T cells, the main disadvantage being the absence of long-term immune memory establishment.

[0119] The present disclosure relates to cells comprising a nucleic acid molecule encoding a CAR for use in the treatment of hematological malignancies (e.g., acute myeloid leukemia (AML), such as refractory or relapsed AML), wherein the CAR comprises an antibody or an antigen-binding fragment thereof comprising an anti-IL-IRAP binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulation domain. Exemplary CARs and their sequences are disclosed in PCT publications No. WO 2019 / 101604 and WO 2020 / 239801, the entirety of which is incorporated herein by reference.

[0120] In one embodiment, the anti-IL-IRAP 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 of 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 of 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 of SEQ ID NO. 8, and (ii) the amino acid of SEQ ID NO. 12 It includes a heavy chain comprising a complementary determination region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence, a complementary determination region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO. 13, and a complementary determination region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO. 14.

[0121] The CAR considered herein may comprise 1, 2, 3, 4, or 5 or more linkers. In certain embodiments, the length of the 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 embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more amino acid lengths.

[0122] Exemplary examples of linkers include glycine polymers (G)n; glycine-serine polymers (Gi_sSi_5)n, where n is at least an integer of 1, 2, 3, 4, or 5; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and thus can serve as neutral tethers between domains of fusion proteins such as the CARs described herein. Glycine has significantly more access to the phi-psi space than alanine and is much less restricted than residues with longer side chains {cf. Scheraga, Rev. Computational Chem. 1 1173-142 (1992)). In one embodiment, the design of the CAR may include a linker that is wholly or partially flexible, and accordingly, the linker may include one or more parts that impart a less flexible structure as well as a flexible linker to provide a desired CAR structure.

[0123] In a specific embodiment, the linker is between the VH and VL domains.

[0124] In a specific embodiment, the linker comprises or consists of the amino acid sequence of SEQ ID NO. 5.

[0125] In one embodiment, the IL-1RAP binding domain is a scFv comprising a light chain variable region having at least 1, 2, or 3 variations of the amino acid sequence of the light chain variable region of SEQ ID NO. 4, but having 30, 20, or 10 or fewer variations, and a heavy chain variable region having at least 1, 2, or 3 variations of the amino acid sequence of the heavy chain variable region of SEQ ID NO. 2, but having 30, 20, or 10 or fewer variations.

[0126] In one embodiment, the IL-1 RAP binding domain comprises (i) a light chain variable region comprising a complementation determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO. 6, a complementation determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO. 7, and a complementation determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO. 8, and (ii) SEQ ID NO It is a single scFv comprising a heavy chain variable region including a complementary determination region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of 12, a complementary determination region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO. 13, and a complementary determination region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO. 14.

[0127] The binding domain of a CAR is typically followed by one or more "hinge regions," which serve to position the antigen-binding domain away from the surface of the effector cell to enable proper cell / cell contact, antigen binding, and activation. CARs generally contain one or more hinge regions between the binding domain and the transmembrane domain. The hinge regions may be derived from any one of natural, synthetic, semi-synthetic, or recombinant sources.

[0128] In one embodiment, the anti-IL-IRAP binding domain is connected to the transmembrane domain by a hinge region.

[0129] In one embodiment, the hinge region comprises the hinge sequence of IgGl or a sequence having 95-99% identity therewith. The IgG hinge is coded by a single exon. Accordingly, the term “hinge sequence of IgGl” as used herein has the same meaning as generally understood by those skilled in the art to which the disclosure pertains, namely, having 15 amino acid residues coded by the IgGl exon for the hinge (Fundamental Immunology, Fifth edition, Chapter 3, Immunoglobulins: Structure and Function - The immunoglobulin Flinge).

[0130] In a further embodiment, the hinge region comprises the hinge sequence of IgG4 or a sequence having 95-99% identity therewith. In a further embodiment, the hinge region may also comprise the CH2-CH3 region of IgG1 or IgG4 or a sequence having 95-99% identity therewith.

[0131] In an additional embodiment, the hinge region comprises CD8alpha or a sequence having 95-99% identity with it.

[0132] The "transmembrane domain" is a part of the CAR that fuses the extracellular binding portion and the intracellular signaling domain and anchors the CAR to the plasma membrane of an immune effector cell. The transmembrane domain may be derived from any one of natural, synthetic, semi-synthetic, or recombinant sources.

[0133] In one embodiment, the encoded CAR comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, more preferably CD28.

[0134] In certain embodiments, the CAR considered herein comprises an intracellular signaling domain. The “intracellular signaling domain” refers to a part of the CAR that participates in inducing effector cell function, e.g., cytotoxic activity including activation, cytokine production, proliferation, and release of cytotoxic factors to CAR-bound target cells, by transmitting the message of effective CAR binding to a target antigen into the immune effector cell, or other cellular responses induced by antigen binding to an extracellular CAR domain.

[0135] The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be auxiliary or active, including cytolytic activity or the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transmits effector function signals and directs the cell to perform a specialized function. Typically, the entire intracellular signaling domain may be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used instead of the entire domain, provided that it transmits an effector function signal. The term "intracellular signaling domain" means containing any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0136] It is known that signals generated by TCRs alone are insufficient for the complete activation of T cells and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation via a TCR (e.g., TCR / CD3 complex), and a co-stimulatory signaling domain that acts in an antigen-independent manner to provide secondary or co-stimulatory signals. In some embodiments, the CAR considered herein comprises an intracellular signaling domain comprising one or more "co-stimulatory signaling domains."

[0137] In one embodiment, the isolated nucleic acid molecule may encode an intracellular signaling domain comprising at least one co-stimulatory domain. In this embodiment, the intracellular signaling domain thus comprises at least one co-stimulatory domain.

[0138] As used herein, the terms “co-stimulatory signaling domain” or “co-stimulatory domain” refer to the intracellular signaling domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides a secondary signal necessary for the efficient activation and function of T lymphocytes upon binding to an antigen.

[0139] Preferably, at least one co-stimulatory domain of the functional intracellular signaling domain is obtained from one or more proteins selected from the group consisting of OX40, CD2, CD27, CD28, CDS, CD3 zeta, ICAM-1, LFA-1 (CD1 la / CD18), ICOS (CD278) and 4-1BB (CD137).

[0140] In one embodiment, the co-stimulation domain obtained from 4-1BB (CD137) has a sequence having 95-99% identity with the amino acid sequence of the co-stimulation domain of 4-1BB. In one embodiment, the co-stimulation domain obtained from CD3 zeta has a sequence having 95-99% identity with the amino acid sequence of the co-stimulation domain of CD3 zeta. In another embodiment, the intracellular signaling domain comprises the co-stimulation domain obtained from 4-1BB and / or the co-stimulation domain obtained from CD3 zeta.

[0141] In a specific embodiment, a CAR comprises one CD3z primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling and co-stimulatory signaling domains may be connected in series in any order to the carboxyl terminus of the transmembrane domain.

[0142] In one embodiment, CAR comprises the amino acid sequence of SEQ ID NO. 19.

[0143] Cells that may be used according to the disclosure may be isolated. "Isolated cells" refers to cells obtained from in vivo tissues or organs that are substantially devoid of extracellular matrix. Cells used to treat hematological malignancies according to the disclosure may be produced by using a vector to insert a nucleic acid molecule encoding a chimeric antigen receptor (CAR) into the genome of a host cell.

[0144] The term "vector" is used herein to refer to a nucleic acid molecule capable of delivering or transporting other nucleic acid molecules. The delivered nucleic acid is generally linked to the vector nucleic acid molecule, for example, inserted therein. The vector may contain a sequence directing autonomous replication within a cell, or may contain a sequence sufficient to allow integration into host cell DNA.

[0145] According to the disclosure, cells used to treat blood malignancies may be prepared using a vector comprising a nucleic acid molecule encoding a CAR, said vector being selected from DNA, RNA, plasmid, lentiviral vector, adenoviral vector, or retroviral vector, preferably a lentiviral vector. In some embodiments, the vector comprises a promoter, preferably an EF-1 alpha promoter.

[0146] Retroviruses are a common tool for gene transfer. In certain embodiments, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) to cells. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates the genomic DNA into the host genome. Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus acts as a template for RNA polymerase II and directs the expression of RNA molecules encoding structural proteins and enzymes necessary to produce new viral particles.

[0147] Therefore, T cells transformed with a vector can induce a stable, long-term, and sustained CAR-mediated T-cell response. In certain embodiments, T cells are transformed with a retroviral vector encoding CAR, for example, a lentiviral vector.

[0148] As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visnar-Medii virus (VMV); goat arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and monkey immunodeficiency virus (SIV).

[0149] The term “lentiviral vector” refers to a viral vector or plasmid containing a structural and functional genetic element, or a portion thereof, comprising an LTR derived primarily from a lentivirus. “Self-inactivation (SIN) vector” refers to a replication-defective vector, e.g., a retroviral or lentiviral vector, wherein the right (3’) LTR enhancer-promoter region, known as the U3 region, is modified to prevent viral transcription beyond the first round of viral replication (e.g., by deletion or substitution). In one embodiment, a SIN vector backbone is preferred. In one embodiment, the vector used further comprises a promoter, e.g., an EF-1 alpha short promoter and an sp163 enhancer and / or CMV promoter.

[0150] As used herein, the term “promoter” refers to a recognition site of a polynucleotide (DNA or RNA) to which an RA polymerase binds. The RA polymerase initiates and transcribes a polynucleotide operatively linked to the promoter. In certain embodiments, it may be desirable to express a polynucleotide containing a CAR from a promoter that provides stable and long-term CAR expression in T cells at a level sufficient to redirect T cells to cells expressing a target antigen.

[0151] The cell for use according to the disclosure may be a T cell, e.g., a human T cell. In one embodiment, the cell is a CD8+ T cell, e.g., a human CD8+ T cell. In one embodiment, the cell for use according to the disclosure (e.g., a T cell) expresses a CAR (e.g., SEQ ID NO. 19) on its membrane. As used herein, the term “on its membrane” has the same meaning as commonly understood by those skilled in the art to which the disclosure pertains, namely, “on the cell surface membrane.”

[0152] In certain embodiments, prior to the in vitro manipulation or genetic modification of the immune effector cells described herein, the source of the cells is obtained from the subject. In certain embodiments, the cells for use according to the disclosure comprise T cells. T cells may be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic ischium, tissue from an infection site, ascites, pleural fluid, splenic tissue, and tumors. In certain embodiments, T cells may be obtained from a single blood unit collected from the subject using any number of techniques known to those skilled in the art, such as sedimentation, e.g., FICOLL™ separation. Cells from the subject's circulating blood may be obtained by apheresis. The apheresis product typically comprises lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing.

[0153] T cells can be isolated from peripheral blood monocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Specific subpopulations of T cells expressing one or more markers, such as CD4 or CD8, can be further isolated by positive or negative selection techniques. For example, enrichment of a single T cell population by negative selection can be achieved with a combination of antibodies targeting surface markers unique to the negatively selected cells.

[0154] Accordingly, a cell therapy is disclosed herein in which T cells are genetically modified in vitro to express a CAR, and the CAR-T cells are injected into a single recipient who requires them. The injected cells can inhibit the growth of blood malignant tumor cells (e.g., AML cells) in the recipient, preferably in a human. Unlike antibody therapy, CAR-T cells can replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control. In an embodiment, prior to injecting the CAR-T cells into a single recipient who requires them, the recipient is pre-conditioned with lymphocyte-depleting chemotherapy (LDC).

[0155] Furthermore, CARs allow for the redirection and activation of effector T cells toward any cell surface molecule upon binding to antibody-derived receptors, and are independent of MHC restriction.

[0156] Genetically modified cells for use according to the present disclosure, e.g., T cells, may be produced starting from the patient's cells (autologous), but may also originate from another allogeneic donor to provide allogeneic genetically modified cells in bone marrow or peripheral hematopoietic stem cell allografts (donor lymphocyte infusion). These cells expressing the IL-1RAP CAR molecule are useful for treating cancer in mammals, preferably humans.

[0157] These cells, e.g., T cells, express a CAR molecule comprising one antigen-binding domain that is an anti-IL-IRAP scFv containing an anti-IL-IRAP binding domain, a transmembrane domain of a CD28 protein, a co-stimulatory 4-1BB signaling domain, and a CD3ζ signaling domain, wherein the anti-IL-IRAP binding domain comprises: (i) a complementation determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity with the amino acid sequence of SEQ ID NO. 6, a complementation determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or 100% identity with the amino acid sequence of SEQ ID NO. 7, and at least with the amino acid sequence of SEQ ID NO. 8 A light chain comprising a complementation determining region 3 (CDR3) having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO. 12, a complementation determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO. 12, a complementation determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO. 13, and at least 80%, 85%, 90%, It comprises a heavy chain comprising a complementation determining region 3 (CDR3) having 95%, 96%, 97%, 98%, 99% identity or 100% identity. In one embodiment, the CAR molecule comprises the amino acid sequence of SEQ ID NO. 19. In some embodiments, the CAR molecule further comprises an IgG1 hinge sequence between the scFv and the transmembrane domain.In some embodiments, the CAR molecule comprises an immunoglobulin G (IgG) hinge region, a CD28 transmembrane and intracellular signaling domain, a 4-1BB signaling domain, and a CD3ζ chain signaling domain.

[0158] 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 a minimal reduction in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment may optionally involve a reduction or improvement in the symptoms of the disease or condition, or a delay in the progression of the disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of the disease or condition, or associated symptoms.

[0159] Accordingly, the present disclosure provides a method for treating acute myeloid leukemia (AML) (e.g., refractory or relapsed (r / r) AML), comprising the step of administering a therapeutically effective amount of cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) to a subject requiring treatment for acute myeloid leukemia (AML) (e.g., refractory or relapsed (r / r) AML), wherein the CAR comprises an antibody or antibody fragment comprising an anti-IL-IRAP binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulation domain, wherein the anti-IL-IRAP binding domain comprises (i) a complementation determining region 1 (CDR1) having at least 80% identity with the amino acid sequence of SEQ ID NO. 6, a complementation determining region 2 (CDR2) having at least 80% identity with the amino acid sequence of SEQ ID NO. 7, and a complementation determining region 3 (CDR3) having at least 80% identity with the amino acid sequence of SEQ ID NO. 8. It comprises a light chain, and (ii) a heavy chain comprising a complementation determining region 1 (CDR1) having at least 80% identity with the amino acid sequence of SEQ ID NO. 12, a complementation determining region 2 (CDR2) having at least 80% identity with the amino acid sequence of SEQ ID NO. 13, and a complementation determining region 3 (CDR3) having at least 80% identity with the amino acid sequence of SEQ ID NO. 14, wherein, prior to administering cells, the subject is pre-conditioned with lymphocyte depletion chemotherapy (LDC). In one embodiment, the CAR molecule comprises the amino acid sequence of SEQ ID NO. 19.

[0160] Cells, e.g., T cells, may be administered as a pharmaceutical composition alone or in combination with other components such as a diluent and / or chemokines, cytokines (e.g., IL-2), or a cell population. Briefly, the pharmaceutical composition may comprise a target cell population as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline and phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The phrase "pharmaceuticalally acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the scope of sound medical judgment and corresponding to a reasonable benefit / risk ratio.

[0161] In one embodiment, the CAR-modified cells or composition are administered to a subject by direct injection into a tumor. In one embodiment, the CAR-modified cells, e.g., CAR-modified T cells, are useful for treating a subject diagnosed with cancer by removing immune effector cells from a subject and genetically modifying said immune effector cells with a vector containing 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 embodiment, the immune effector cells include T cells.

[0162] The "therapeutic effective dose" of genetically modified therapeutic cells may vary depending on factors such as the individual's disease status, age, sex, and body weight, as well as the ability of stem and progenitor cells to induce the desired response within the individual. The therapeutic effective dose is also the amount at which any toxic or harmful effects of the virus or transduced therapeutic cells are offset by therapeutically beneficial effects. The quantity, frequency, and sequence of administration for possible association with conventional cancer treatment will be determined by factors such as the patient's pathological condition and the type and severity of the cancer.

[0163] In some embodiments, CAR-expressing T cells are 0.1 x 10 6 cells / kg, 0.5 x 10⁶ 6 cells / kg, 1 x 10⁶ 6 cells / kg, 5 x 10 6 cells / kg, or 10 x 10⁶ 6 It is administered at a dose of cells / kg. In some embodiments, CAR-expressing T cells are approximately 0.1 x 10⁶ 6 It is administered at a dose of cells / kg. In some embodiments, CAR-expressing T cells are approximately 0.5 x 10⁶ 6 It is administered at a dose of cells / kg. In some embodiments, CAR-expressing T cells are approximately 1 x 10⁶ 6 It is administered at a dose of cells / kg. In some embodiments, CAR-expressing T cells are approximately 5 x 10 6 It is administered at a dose of cells / kg. In some embodiments, CAR-expressing T cells are approximately 10 x 10 6 It is administered at a dose of cells / kg. In some embodiments, CAR-expressing T cells are administered as a single dose. In some embodiments, CAR-expressing T cells are administered intravenously.

[0164] Lymphocyte depletion chemotherapy (LDC)

[0165] In one embodiment, T cells expressing a CAR molecule specific to IL-1RAP may be used in a method to inhibit the growth of refractory or relapsed AML in a subject. In one embodiment, the subject has already been treated with at least one course of treatment, such as chemotherapy. In some embodiments, the subject has already been treated with one or more of cytarabine, anthracyclines, daunorubicin, venetoclax, decitabine, idarubicin, cladribine, G-CSF, and / or azacitidine. In some embodiments, the subject has received an allogeneic hematopoietic stem cell transplant (HSCT). In some embodiments, the subject has already been treated with one or more of conjugated antibodies (CD33-GO, gemtuzumab ozogamicin, and antibodies targeting CD44, CD123, or CD47), bispecific T-cell engager (BiTE) antibodies (targeting CD3 / CD33 or CD3 / CD123), and immune checkpoint inhibitors (targeting PD-1 / PDL-1, anti-TIM-3, and anti-CTLA4).

[0166] In one embodiment, cells expressing a CAR molecule specific to IL-1RAP can be used in a method for treating cancer in a subject. In one embodiment, the subject has already been treated with at least one course of treatment, such as chemotherapy. In another embodiment, the subject is treated with a combination of T cells and chemotherapy, either simultaneously or sequentially, in any order.

[0167] In some embodiments, prior to administering cells (e.g., T cells) expressing a CAR molecule specific to IL-1RAP to the subject, the subject is pre-conditioned with lymphocyte-depleting chemotherapy (LDC). In some embodiments, the LDC includes a step of administering radiotherapy, fludarabine, or cyclophosphamide. In some embodiments, the LDC includes a step of administering fludarabine and cyclophosphamide. In some embodiments, fludarabine is about 20 to about 40 mg / m² 2It is administered at a daily dose. In some embodiments, fludarabine is approximately 20 mg / m². 2 It is administered at a daily dose. In some embodiments, fludarabine is approximately 30 mg / m². 2 It is administered at a daily dose. In some embodiments, fludarabine is approximately 40 mg / m² 2 It is administered at a daily dose. In some embodiments, cyclophosphamide is about 200 to about 400 mg / m² 2 It is administered at a daily dose. In some embodiments, cyclophosphamide is approximately 200 mg / m² 2 It is administered at a daily dose. In some embodiments, cyclophosphamide is approximately 300 mg / m² 2 It is administered at a daily dose. In some embodiments, cyclophosphamide is approximately 400 mg / m² 2 It is administered at a daily dose.

[0168] In some embodiments, LDC is completed at least 48 hours prior to administration of CAR-expressing T cells. In some embodiments, LDC is initiated 6 to 10 days prior to administration of CAR-expressing T cells. In some embodiments, LDC is initiated about 6, 7, 8, 9, or 10 days prior to administration of CAR-expressing T cells. In some embodiments, fludarabine administration is for about 4 days. In some embodiments, fludarabine administration is for about 3 days. In some embodiments, cyclophosphamide administration is for about 3 days. In some embodiments, cyclophosphamide administration is for about 2 days.

[0169] In one embodiment, the present disclosure relates to identifying a patient having increased IL-1RAP protein expression and treating the subject by administering the CAR-T cells of the disclosure, wherein, prior to administering the CAR-T cells, the subject is pre-conditioned with an LDC.

[0170] 3. Measurement of IL-1RAP expression

[0171] To evaluate IL-1RAP expression, in one embodiment, a test sample is obtained from a patient requiring therapy. In some embodiments, the test sample includes, but is not limited to, any clinically appropriate sample such as a tumor biopsy, bone marrow biopsy, bone marrow aspiration, core biopsy tissue sample, fine needle aspirate, or a sample of body fluid such as blood, plasma, serum, lymph, ascites fluid, cystic fluid, or urine. In some embodiments, the test tissue sample is derived from the primary tumor. In some embodiments, the test sample is from a metastasis. In some embodiments, the test sample (e.g., a test sample for IL-1RAP expression) is taken from the subject at multiple times, e.g., before treatment (e.g., T cells), during treatment, and / or after treatment. In some embodiments, the test sample is taken from different locations on the subject, e.g., a sample from the primary tumor and a sample from a metastasis located at a distant location. In some embodiments, IL-1RAP is detected in a single sample obtained from a patient (e.g., an AML patient) before administering therapy (e.g., T cells).

[0172] In some embodiments, the test tissue sample is a single paraffin-embedded fixed tissue sample. In some embodiments, the test tissue sample is a single formalin-fixed paraffin-embedded (FFPE) tissue sample. In some embodiments, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is a frozen tissue sample. In some embodiments, the test tissue sample is a fresh frozen (FF) tissue (e.g., tumor) sample. In some embodiments, the test tissue sample is a single cell isolated from fluid. In some embodiments, the test tissue sample contains circulating tumor cells (CTCs). In some embodiments, the test tissue sample contains circulating lymphocytes. In some embodiments, the test tissue sample is a stored tissue sample. In some embodiments, the test tissue sample is a stored tissue sample with a known history of diagnosis, treatment, and / or outcome. In some embodiments, the sample is a tissue block. In some embodiments, the test tissue sample is dispersed cells. In some embodiments, the sample size ranges from approximately 1 cell to approximately 1 x 10⁶ 6 It is more than 10 cells. In some embodiments, the sample size is about one cell to about 1 x 10⁶ cells. 5 It is a number of cells. In some embodiments, the sample size is about 1 cell to about 10,000 cells. In some embodiments, the sample size is about 1 cell to about 1,000 cells. In some embodiments, the sample size is about 1 cell to about 100 cells. In some embodiments, the sample size is about 1 cell to about 10 cells. In some embodiments, the sample size is a single cell.

[0173] In another embodiment, the evaluation of IL-1RAP expression may be achieved without obtaining a test tissue sample. In some embodiments, selecting a suitable patient comprises (i) optionally providing a test tissue sample obtained from a patient with cancer of the tissue, wherein the test tissue sample contains tumor cells; and (ii) evaluating the proportion of cells in the test tissue sample expressing IL-1RAP on the surface of the cells based on the assessment that the proportion of cells in the test tissue sample expressing IL-1RAP on the surface of the cells is higher than a predetermined threshold level.

[0174] However, it should be understood that in any method comprising the measurement of IL-1RAP expression in a test sample, the step of providing the test sample obtained from the patient is an optional step. That is, in certain embodiments, the method includes this step, and in other embodiments, this step is not included in the method. It should also be understood that in certain embodiments, the step of "measuring" or "evaluating" to identify cells expressing IL-1RAP in the test sample or to determine their number or proportion is performed by a transformative method analyzing 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 mass spectrometry imaging (MSI) assay. In certain other embodiments, the transformative step is not involved, and IL-1RAP expression is evaluated, for example, by reviewing a test result report from a laboratory. In some embodiments, IL-1RAP expression is evaluated, for example, by reviewing immunohistochemical analysis results from a laboratory. In a specific embodiment, the step of providing test results is performed by a medical professional or a person acting under the direction of a medical professional. In another embodiment, this step is performed by an independent person, such as an independent laboratory or a laboratory technician.

[0175] In a specific embodiment of any of the methods, the proportion of cells expressing IL-1RAP is evaluated by performing a single assay to detect the presence of IL-1RAP RNA. In additional embodiments, the presence of IL-1RAP RNA is detected by RT-PCR, hybridization in situ, or RNase protection. In some embodiments, the presence of IL-1RAP RNA is detected by an RT-PCR-based assay. In some embodiments, scoring the RT-PCR-based assay involves evaluating the level of IL-1RAP RNA expression in the test tissue sample against a predetermined level.

[0176] In another embodiment, the proportion of cells expressing IL-1RAP is evaluated by performing an analysis to detect the presence of the IL-1RAP polypeptide. In additional embodiments, the presence of the IL-1RAP polypeptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some embodiments, IL-1RAP expression is analyzed by IHC, imaging mass cytometry (IMC), or mass spectroscopic imaging (MSI). In other embodiments of all these methods, cell surface expression of IL-1RAP is analyzed, for example, using IHC or in vivo imaging.

[0177] Examples

[0178] Example 1: IL-1RAP as a Probable Therapeutic Target in AML

[0179] The standard frontline (first-line) "7+3" regimen for AML consists of an induction phase using 7 days of high-dose cytarabine and 3 days of anthracycline (daunorubicin) chemotherapy, followed by a consolidation phase of chemotherapy or allogeneic HSCT for high-risk patients (Heuser 2020, NCCN 2022). For patients unsuitable for standard induction therapy, venetoclax, a bcl-2 inhibitor combined with a demethylating agent such as azacitidine, has been rapidly adopted as an active and less toxic alternative (DiNardo 2020). With the continued increase in knowledge regarding the molecular mutational patterns of AML leukemia development, other targeted therapy options with lower toxicity compared to conventional treatments have emerged, such as FMS-like tyrosine kinase (FLT3) inhibitors like midostaurine and gilteritinib, isocitrate dehydrogenase 1 / 2 mutant inhibitors like ivosidenib and enasidenib, and the B-cell lymphoma 2 inhibitor venetoclax (VEN) combined with hypomethylating agents (HMA) (DiNardo 2018, DiNardo 2020). Despite improvements in first-line therapy, the only curative options currently available are high-dose chemotherapy and allogeneic HSCT (Dohner 2022). Approximately 10% to 20% of younger AML patients and 50% of older AML patients are considered to have primary refractory disease, failing to achieve complete remission after at least two courses of intensive induction therapy; Furthermore, 50% to 70% of patients who achieve CR will relapse (Dohner 2022). These data highlight a significant issue regarding the relapse of leukemia after an initial response (Roussel 2020). For patients with primary relapsed / refractory (r / r) AML, the prognosis remains poor and treatment is difficult (Heuser 2020).

[0180] Despite all these improvements in therapy, primary resistance to initial treatment and disease recurrence remain unmet needs in the treatment of AML. Current treatment strategies are not sufficiently successful, and ultimately, most AML patients still die from the disease. Despite improvements in HSCT and several other treatments, outcomes for high-risk patients remain poor, with only approximately 40% to 45% of young patients and 10% to 20% of elderly patients being cured, indicating the existence of unmet medical needs and suggesting the need for new alternative treatments (Bose 2017).

[0181] In summary, patients with r / r AML have few remaining therapeutic options and are encouraged to participate in clinical studies. Therefore, there is a high medical need for effective treatments, including chimeric antigen receptor T (CAR T) cell therapy.

[0182] Interleukin (IL)-1 plays a crucial role in innate and adaptive immunity and has been associated with acute and chronic inflammation, as well as cancer progression. Two forms of IL-1, IL-1α and IL-1β, have been identified. IL-1β is considered a more potent cytokine, whereas IL-1α appears to possess broader activity (Fields 2019). Both IL-1α and IL-1β bind to the same cellular receptor to induce signaling and are considered potential therapeutic targets due to their roles during cancer progression. The interleukin-1 receptor (IL-1R) is the primary receptor for IL-1. Upon receptor engagement, IL-1R1 forms a heterodimer with the interleukin-1 receptor helper 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 signaling. IL-1RAP mediates the IL-1-dependent activation of NF-kappa-B and other signaling pathways. While IL-1RAP cannot directly bind to IL-1, this is essential for IL-1-mediated signaling. 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 apparent phenotypic changes (Cullinan 1998).

[0183] IL-1RAP is overexpressed in multiple solid tumor types and has been identified as a potential therapeutic target for 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 tumor cell surface, various direct targeting approaches involving mAbs are under development in solid tumors (Robbrecht 2022, Rydberg Millrud 2022). Nadunolimab, an anti-IL-1RAP mAb, is under clinical evaluation in solid tumors as monotherapy and in combination with chemotherapy (NCT03267316, NCT04452214, NCT05181462, NCT04990037, NCT05116891).

[0184] IL-1RAP has also been identified as a potential anticancer target in chronic myeloid leukemia, AML, and myelodysplastic syndrome (Jaras 2010, Askmyr 2013, Zhao 2014, Agerstam 2015, Landberg 2016, Shastri 2017, Blatt 2018, De Boer 2021, Eldesouki 2021). IL-1RAP is selectively expressed in the majority of AML blasts and to some extent in monocytes, but is not expressed in HSCs (Houtsma 2022, Trad 2022). Recent publications have reported that patients with AML having 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 enhances multiple oncogenic signaling pathways in AML and promotes leukemia cell proliferation and survival through the FLT3 and C-kit pathways, representing a potentially promising target for treating AML (Mitchell 2018, De Boer 2021). The need for IL-1RAP on leukemia cells suggests a low risk of antigen evasion in AML cells that downregulate IL-1RAP after being targeted by IL-1RAP CAR T-cell immunotherapy. Therefore, this may provide a unique safety profile compared to other AML targets such as CLL-1 and CD123 (Mardiana 2020).

[0185] In preclinical studies, IL-1RAP-directed CAR T-cells showed promising anti-leukemic activity in AML and CML models (Zhao 2017, Warda 2019, Warda 2021, Trad 2022, Nicod 2023), and IL-1RAP-directed CAR NK-cells showed promising anti-leukemic activity in Ewing sarcoma (Luo 2022).

[0186] Example 2: CCTx-001 Preclinical Study

[0187] Example 2.1. IL-1RAP overexpression in AML cell lines and primary samples.

[0188] Overexpression of IL-1RAP has been demonstrated in AML cells (leukemia stem cells [LSCs] and blasts) compared to normal hematopoietic progenitor cells and stem cells. High IL-1RAP mRNA expression was associated with statistically significant shorter overall survival in AML (p<0.05). In this regard, membranous IL-1RAP is overexpressed in all AML FAB subtypes (Trad 2022).

[0189] Using publicly available AML patient mRNA expression data (n=539) (see Karakaslar EO et al. NPJ Precis. Oncol. 2024; 8(1):105), Kaplan-Meier curves stratified by either low or high IL-1RAP expression were generated ( Fig. 15a This analysis indicated that high IL-1RAP mRNA expression is associated with shorter overall survival in AML. Additionally, varying levels of IL-1RAP mRNA expression were noted from a single silico analysis of IL-1RAP in AML patients (n=832), depending on AML subtypes classified by morphological differentiation stage (FAB). Fig. 15b ).

[0190] The following AML cell lines were evaluated for IL-1RAP membrane expression: MOLM-13 (M5 according to FAB classification), Mono-Mac-6 (M5), EOL-1 (M4 eosinophil), KG-1 (M0), HEL (M6 erythrocyte), HL60 (M2), and OCI-AML3 (M4). Table 1As shown in [figure], all tested AML cell lines were found to be positive for IL-1RAP expression (ranging from 100 to 8752 ABC). Overall, the cell lines were classified into three subgroups: KG-1 and HEL cell lines were classified as IL-1RAP-low expression cell lines; EOL-1, OCI-AML3, HL60, and MOLM-13 were classified as IL-1RAP-medium expression cell lines; and Mono-Mac-6 cell line was classified as IL-1RAP-high expression cell line.

[0191] [Table 1]

[0192] Table 1: IL-1RAP expression in AML cell lines

[0193]

[0194] RFI = Relative fluorescence intensity, ABC = Antibody binding capacity

[0195] Comparison of IL-1RAP membrane expression (in ABC) between AML patients (AML blasts and AML-derived monocytes) and healthy donor blood and bone marrow cells (differentiated immune cells and progenitor cells) Fig. 17 This is shown in [data]. This data further indicates that IL-1RAP is overexpressed in blast cells from AML patients, whereas IL-1RAP is not expressed in healthy peripheral blood cells and bone marrow hematopoietic cells.

[0196] The expression of IL-1RAP, CD33, CD123, and CLL1 on peripheral blood mononuclear cells (PBMCs) was also evaluated. PBMCs were isolated from five healthy blood donors. Cells were stained for each marker and detected by flow cytometry. The expression of IL-1RAP was compared to CD33, CD123, CLL1, or isotype controls. Quantibrite PE beads (BD) were used in parallel to quantify the expression levels of the above markers in each immune cell population. Marker expression was studied in the following cell types: B cells, T cells, monocytes, basophils, neutrophils, eosinophils, NK cells, conventional dendritic cells (cDCs), and plasmacytic dendritic cells (pDCs).

[0197] Prior to sample acquisition, BD Quantibrite PE beads were resuspended and fixed according to the cell assay protocol, and run on a NovoCyte Penteon flow cytometer. The gate was set around the bead singlet, and histograms were used to analyze the bead statistics. Cell assay samples were acquired using the same instrument setup. The antibody-bound (ABC) values ​​per cell were generated by subtracting the isotype geoMean from the acquired sample geoMean and then extrapolating the cell sample data to a logarithmic scale using a standard curve and the equation y = a * x + b, where a is the slope and b is the y-intercept.

[0198] The results of the normal PBMC donor analysis Fig. 16aIL-1RAP expression was detected at much lower levels than other targets, and was found at very low levels only in monocytes (ABC=719.19) and basophils (ABC=470.73) across all 5 donors. The most widely expressed marker was CLL1, which was detected in monocytes (ABC=6722.46), cDc (ABC=5618.82), neutrophils (ABC=4718.81), basophils (ABC=2606.63), and eosinophils (ABC=2005), and at lower levels in pDC (ABC=766.1). CD33 was consistently detected in monocytes (ABC=5915.37) and cDCs (ABC=3404.11), and at lower levels in basophils, neutrophils, and eosinophils (ABC=1136.5, 1024.86, and 493.7, respectively); CD123 was consistently detected across donors, primarily in basophils (ABC=7240.74), and at lower levels in cDCs (ABC=1193.96) and monocytes (ABC=307.04). CD123 expression on pDCs was significantly higher compared to all other target expressions across cell subpopulations (ABC=17342.96).

[0199] In addition, flow cytometry evaluations of IL-1RAP, CLL-1, CD33, and CD123 expression were performed in hematopoietic stem and progenitor populations of human bone marrow CD34+ cells. Four lots of bone marrow (BM) CD34+ cells from normal healthy donors were evaluated by flow cytometry, and human stem and progenitor cell subpopulations were evaluated based on the expression of surface markers CD34, CD38, CD90, and CD45RA. Expression of target surface markers CLL-1, CD123, CD33, and IL-1RAP was evaluated in stem and progenitor cell populations [lineage-determined progenitor cells (CD34+CD38+), hematopoietic stem cells (HSC), lymphocyte-primed pluripotent progenitor cells (LMPP), and pluripotent progenitor cells (MPP)] and compared with isotype-stained controls. Target expression was quantified using quantification beads, and antibody binding capacity (ABC) was calculated ( Fig. 16b While the expression of CLL-1, CD123, and CD33 varied for each evaluated donor and each progenitor cell population, IL-1RAP target expression was very low in all BM CD34+ stem and progenitor cell populations for each tested donor (ABC = ≤ 140).

[0200] In summary, analysis of AML cell lines and AML primary cells confirmed the expression of IL-1RAP on the surface of cancer cells, thus constituting a promising target for the treatment of AML.

[0201] Example 2.2 Affinity of B-L43 mAb for IL-1RAP

[0202] The affinity of B-L43 mAb for human IL-1RAP was demonstrated by ELISA, FC, and surface plasmon resonance (SPR). Table 2Recombinant scFv derived from the B-L43 / A3C3 sequence was also evaluated and showed similar binding characteristics to B-L43 mAb by ELISA. Notably, A3C3 and B-L43 are two hybridoma clones that share the same nucleotide sequences for their heavy and light chains, and thus were used interchangeably in different experiments.

[0203] [Table 2]

[0204] Table 2: Summary of B-L43 mAb binding evaluation to IL-1RAP

[0205]

[0206] Quantitative kinetic characterization of B-L43 binding to IL-1RAP by SPR analysis showed fast binding and slow dissociation rates. Data evaluation using the Langmuir 1:1 binding model revealed sub-nanomolar range KD values ​​( Table 3 ).

[0207] [Table 3]

[0208] Table 3: Quantitative kinetic characterization of B-L43 binding to IL-1RAP by SPR analysis

[0209]

[0210] SD = Standard Deviation

[0211] Example 2.3: Cytotoxic activity of CAR T-cells derived from B-L43 / A3C3 mAb against IL-1RAP+ expressing cells

[0212] Anti-IL-1RAP CAR T-cells were generated using a lentiviral vector encoding a CAR polyprotein containing a scFv derived from the heavy and light chain sequences of A3C3 / B-L43 mAb, a human immunoglobulin G (IgG) hinge domain, a CD28 transmembrane and intracellular domain, a 4-1BB signaling domain, and CD3ζ chain signaling. Briefly, CD4 and CD8-positive T-cells were isolated from PBMCs of two healthy donors by immunolabeling and magnetic separation. The isolated T cells were activated with CD3 and CD28 agonists, transfected with an anti-IL-1RAP CAR-coding lentiviral vector (or untransfected as a control [NTD]), expanded in vitro, and cryopreserved for storage. The transduction efficiency was evaluated, which was 47.4% for donor batch 1 and 49.9% for donor batch 2.

[0213] The cytotoxicity of these two batches of CAR-T cells against IL-1RAP-positive cell lines was evaluated in vitro after 24 hours of co-culture with the IL-1RAP-positive AML cell line MOLM-13 at different E:T ratios, along with untransduced (NTD) T-cell controls from the same two human donors (HD). At an E:T ratio of 1:9, a cytotoxicity rate of approximately 20% to 40% was observed, and at an E:T ratio of 1:1, the cytotoxicity rate was 70% to 80%, demonstrating the potent apoptotic potential of anti-IL-1RAP CAR T-cells against the IL-1RAP-expressing MOLM-13 AML cell line. Fig. 2 ).

[0214] Example 2.4 CCTx-001 Investigational Medicinal Product (IMP)

[0215] The final CCTx-001 investigational medicinal product (IMP) comprises a suspension of CD4+CAR+ and CD8+CAR+ frozen T-cells in a medium containing dimethyl sulfoxide (DMSO). CCTx-001 is administered by intravenous (IV) infusion after thawing. The CCTx-001 investigational product is a novel IL-1RAP-targeted, genetically modified, autologous CAR T-cell immunotherapy. Additional disclosures related to anti-IL-1RAP CAR T-cells are described, for example, in U.S. Patent Publications Nos. 2021 / 0008108 and 2022 / 0235138, each of which is incorporated herein by reference in its entirety.

[0216] CCTx-001 is manufactured from autologous PBMCs obtained through standard leukocyte apheresis collection procedures. The PBMCs undergo positive selection for T-cells and are subsequently transduced into a CAR using a self-inactivated lentivirus. The IL-1RAP-specific CAR is introduced in vitro into autologous CD8+ and CD4+ T-cells using a non-replication-agnostic, self-inactivated lentivirus vector (LV). The IL-1RAP-specific CAR comprises a scFv binding domain derived from a murine anti-IL-1RAP-specific mAb (clone A3C3), an immunoglobulin G (IgG) hinge domain, a CD28 transmembrane and intracellular signaling domain, a 4-1BB signaling domain, and a CD3ζ chain signaling domain. A schematic diagram of the CAR Fig. 1 It appears in.

[0217] CCTx-001 will be provided as a cell dispersion for IV infusion. The cells are formulated in an infusion-ready cryopreservation medium and stored frozen at temperatures below -120°C. Each infusion bag will be labeled as required by national requirements.

[0218] Example 2.5: Preclinical evaluation of CCTx-001

[0219] In vitro functionality and specificity of CCTx-001

[0220] In vitro studies were performed on a representative large-scale batch of CCTx001 (referred herein to as the TR batch, i.e., the batch representing the clinical process) as described below to evaluate the functionality and specificity of CCTx-001 CAR-T. The percentage of viable transduced cells was evaluated in this batch, which showed transduction levels of 50.00%, 60.90%, and 25.50% for TR3, TR4, and TR5, respectively. NTD T-cells intended for the negative control were prepared in parallel with the CCTx-001 batch using the same cell starting materials and processes.

[0221] The in vitro efficacy and specificity of CCTx-001 (using TR batches) were 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 three independent donors (or their donor-matched NTD counterparts), and subsequently analyzed by flow cytometry (FC) to quantify target cell viability in order to confirm the functionality of CCTx-001 CAR T-cells.

[0222] The three batches of CCTx-001 cells (TR3, TR4, and TR5) representing the clinical process, unlike their NTD counterparts, demonstrated effect-expectant target cell (E:T)-independent cytotoxicity against MOLM-13 cells, but not against Raji cells, where only allogeneic, CAR-independent death was observed. Fig. 3 ).

[0223] Furthermore, when CCTx-001 cells were co-cultured in the absence of target cells ( Figs. 4a-4c ), or when co-cultured with IL-1RAP-negative Raji cells ( Figure 4g-4iWhile only low levels of IFN-γ release were observed, co-culture with IL-1RAP-positive MOLM-13 cells induced significant, E:T-dependent IFN-γ release, which was significantly higher compared to the levels obtained from NTD T-cells co-cultured with MOLM-13 cells ( Figures 4d-4f ).

[0224] Differences across batches in the levels of IFN-γ released by CCTx-001 anti-IL-1RAP CAR T-cells were consistent with differences in transduction levels between these batches. A similar trend was observed for IL-2 and IL-8 secretion (not shown). Results for IL-1β, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α showed values ​​below the lower limit of quantification (LLOQ).

[0225] In conclusion, these results confirmed the in vitro efficacy and specificity of CCTx-001 from the TR batch on IL-1RAP-expressing MOLM-13 AML cells.

[0226] 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 consisted of MOLM-13 (M5 according to FAB classification), Mono-Mac-6 (M5), EOL-1 (M4 eosinophil), KG-1 (M0), HEL (M6 erythrocyte), HL60 (M2), and OCI-AML3 (M4). Table 1 Note). K562 and Raji cell lines were used as IL-1RAP-negative cell lines.

[0227] NTD and TR CCTx-001 cells (TR4) were co-cultured with AML cell lines for 24 hours at different E:T ratios, and target cell viability was analyzed using FC. Cytotoxicity experiments confirmed CAR-dependent cytotoxicity against all IL-1RAP-expressing AML cell lines, regardless of AML subtype and IL-1RAP expression levels. However, higher IL-1RAP expression levels were associated with increased cytotoxicity at lower E:T ratios (i.e., 1:27), indicating a relationship between IL-1RAP expression levels on target cells and specific cytotoxicity by CAR T-cells ( Fig. 5 ).

[0228] Finally, the release of CAR-mediated, IL-1RAP-specific, IFN-γ, TNF-α, and Granzyme B cytokines by CCTx-001 CAR T-cells was observed when co-cultured with AML cell lines expressing different levels of IL-1RAP. When CCTx-001 cells were co-cultured with IL-1RAP-negative cell lines K562 or Raji, no specific cytotoxicity or cytokine secretion was observed. Fig. 5 of "voice".

[0229] In vivo pharmacology of CCTx-001

[0230] The anti-tumor efficacy of CCTx-001 cells (TR3) was evaluated in vivo in female NCG mice 5 days after IV injection of the luciferase-expressing human MOLM-13-luc AML cell line. The persistence and expansion of CCTx-001 (longitudinal analysis) were also monitored in the circulatory system of treated mice. Finally, the presence of CCTx-001 cells was also analyzed in a limited number of tissues and mice at different time points.

[0231] On Day 1, Day 0 Bioluminescence Imaging (BLI) Flux (Average Flux = 1.9 x 10 per group) 6Three randomized groups (n = 5 or 8 mice per group) of MOLM-13-luc tumor-bearing female NCG mice randomized based on photons / s Table 4 Treatment was initiated at ). All treatments were administered IV once on Day 1. Control group 1 received the vehicle (phosphate-buffered saline [PBS] + 0.1% HSA). Groups 2 and 3 each received 1 x 10⁻⁶. 7 I received NTD or CAR T-cells.

[0232] Overall, treatment was well tolerated, and the maximum group mean body weight loss was similar between the control animals in Group 1 (5.8% at Day 15) and Groups 2 and 3 (9.6% at Day 15 and 6.7% at Day 28, respectively). No unscheduled treatment-related deaths were observed during this 30-day study. A whole-body BLI analysis was performed on Day 0 for randomization and then twice a week until the end of the study to monitor MOLM-13-luc AML tumor progression. Fig. 6 As shown in [figure], in this 30-day study, the median time to end (TTE) for control group 1 was 15 days; individual TTEs in group 1 ranged from 14 to 17 days, indicating moderate within-group variability. The median TTE for animals in group 2 treated with NTD was 16 days, corresponding to a non-significant 7% increase in life expectancy (ILS) (Logrank test, p > 0.05 compared to the control group). In contrast, the median TTE for CCTx-001-treated mice in group 3 was 22.0 days (47% ILS, p < 0.01 compared to groups 1 and 2; Logrank test), and one mouse survived to the end of the study (day 31). Whole-body BLI images were consistent with survival kinetics.

[0233] [Table 4]

[0234] Table 4: Summary of responses in the MOLM-13-luciferase model

[0235]

[0236] Table 4 < > indicates the treatment regimen scheduled for completion of the study. Vehicle = PBS + 0.1% BSA. The study was terminated on Day 33 based on a review of SD data and the fact that only one animal remained in the study (study duration = 30 days). TTE was defined as the time to necrotic signs due to tumor progression, and all deaths due to tumor progression were classified as deaths in the survival study. TTE (in days) was recorded for each mouse. C = Control group, Med. = Median, NTD = Untransduced, TTE = Time to end, ILS = Increase in lifespan, T / C 100% - 100%, Statistical significance (Logrank test): ns = Not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, compared to Group 1 or 2; Mean weight trough = Lowest group mean weight as % change from Day 1; NA = Not applicable, T = Treatment group, TR = Treatment-related deaths, NTR = Non-treatment-related deaths.

[0237] In human T-cell transplanted animals (Group 2 NTD(C0) and Group 3 (CCTx-001 cells), blood samples were collected at different time points and analyzed for the presence of circulating MOLM-13 AML cells. Fig. 7As shown in [figure], the percentage of AML cells in Group 3 (CCTx-001 cells) remained at a low level (initial blood AML levels averaged only 0.002% of total surviving cells on day 3; at the end of the study, circulating AML was 0.6% and 0.2% of total surviving cells on days 21 and 30, respectively), whereas Group 2 (NTD) showed an increase in circulating AML cells until day 14 (just before the animals died or were euthanized due to tumor progression), confirming that CCTx-001 inhibits the growth of IL-1RAP-positive AML cells in vivo. Similar results were observed regarding the absolute count of AML cells.

[0238] In summary, CCTx-001 demonstrated anti-tumor efficacy in vivo and significantly delayed the growth of IL-1RAP-positive MOLM-13-luc AML cell xenografts in female NCGs. No findings indicating CCTx-001-related morbidity were observed.

[0239] These findings were consistent with published in vivo data from previous results using IL-1RAP CAR T-cells containing A3C3 / B-L43 mAb-derived scFvs. Anti-IL-1RAP CAR T-cells efficiently controlled the growth of AML cell lines (HL60, MOLM-13, and Mono-Mac-6) in vivo in a NOD scid gamma (NSG) mouse model (Trad 2022) and were able to control Mono-Mac-6 tumor progression in vivo and reduce the AML tumor burden compared to untreated mice or mice treated with NTD T-cells (Nicod 2023).

[0240] Pharmacokinetics and product metabolism in animals

[0241] Whole blood samples (from CCTx-001 cell-injected (Day 0) mice harboring IL-1RAP-expressing MOLM-13 tumors) were collected on Day 3, Day 7, Day 10, Day 14, Day 21, and Day 30. The samples were used to monitor the presence, expansion, and persistence of CCTx-001 cells and T-cell subsets (CD8 and CD4 CAR-positive T-cells) during the course of the anti-tumor efficacy study.

[0242] FC analysis of blood samples demonstrated the successful adoptive delivery and persistence of CCTx-001 cells in the mouse circulatory system. More specifically, when injected into IL-1RAP-expressing MOLM-13-Luc tumor-bearing mice, both CD4+ and CD8+ CAR-positive T-cell numbers significantly increased 2 weeks after adoptive delivery ( Fig. 8c This increase in CCTx-001 cells after day 14 was associated with low levels of circulating AML cells, whereas mice injected with NTD T-cells showed an increase in the incidence of circulating AML cells.

[0243] Finally, using a limited number of mice (n=4), the distribution of CCTx-001 cells in the liver, spleen, and BM of treated mice was evaluated at day 21 (n=3) and day 30 (n=1). Overall, these analyses demonstrated that CCTx-001 cells were present in the three 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 accounted for 14% to 49% of total cells, of which 43% to 67% were CCTx-001 cells), and in the BM (3% to 6% of BM viable 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, in the spleen, 0.6 x 10 4Up to 3 x 10 4 10 CAR T-cells / mg were detected, and 0.3 x 10⁶ in the liver 4 Up to 1 x 10 4 Canine CAR T-cells / mg were detected. Femoral BM is 4 x 10 per femur. 4 Up to 10 x 10 4 It contained dogs' CAR T-cells.

[0244] A second in vivo study was also conducted on OCI-AML3-Luc tumor-bearing NCG mice treated with untransduced (NTD) human T-cells or donor-matched C4-derived CAR T-cells (CCTx-001). Figure 9a shows an example of the in vivo study design. Six mice were present in each study group. Tumors were injected subcutaneously, and injections of untransduced or transduced CAR T-cells (intravenously 1 x 10⁶ 7 Canine T-cells were allowed to settle for up to 4 days prior to injection. Animal welfare was monitored throughout the course of the study. The study was terminated by day 30 after T-cell injection, or earlier due to tumor burden and / or comorbidities. Tumor progression was regularly assessed during the study by tracking tumor cell bioluminescence (BLI).

[0245] Figure 9b shows the mean body weight of each study group throughout the duration of the study. CCTx-001 treatment was well tolerated, and no treatment-related morbidity or death / euthanasia was observed. Figure 9c shows the results of bioluminescence imaging (BLI) at days 0, 7, 14, and 21 for each mouse within the study. OCI-AML3-Luc signal intensity is luminescence (in units of radiant luminance; p / sec / cm² 2It is described as / sr). Figure 9d shows a longitudinal analysis of BLI for each study group, expressed as total flux per time point (p / s). Figure 9e shows Kaplan-Meier plots of survival for OCI-AML3-Luc tumor-bearing NCG mice treated with vehicle, NTD T-cells, or CCTx-001. The probability of survival was based on the time to reach a BLI of 1E+10 p / s. The median survival for both the vehicle and NTD treatment groups was 14 days, whereas the CCTx-001 group did not reach a median survival (hazard ratio = 0.03912; p-value < 0.0005).

[0246] In summary, the data presented in Figures 6–9 indicate that CCTx-001 treatment provides significant anti-tumor effects and inhibition of tumor progression in in vivo models, without any signs of CAR-related toxicity in all tested models. In contrast, neither the vehicle nor the NTD T-cell therapy had a positive effect on survival or tumor suppression.

[0247] Cytotoxicity of CCTx-001 against primary AML cells

[0248] A study was conducted to evaluate the cytotoxic potential of two batches of healthy donor-derived CCTx-001 CAR T-cells against primary AML cells from different donors (n = 5). Anti-IL-1RAP CCTx-001 CAR T-cells and non-transgenic T-cell controls (NTD T-cells) were previously generated from healthy donor PBMCs by clinical-scale manufacturing. CCTx-001 CAR-mediated cytotoxicity was evaluated in an in vitro co-culture assay using primary AML cells. Primary AML cells purchased from BioIVT and previously characterized for blast content / phenotype and IL-1RAP expression were Table 5It is described in [document]. To promote AML survival during the 24-hour assay duration, a defined medium containing AML-supporting cytokines (SCF, TPO, FLT-3L, IL-3, IL-6, and GM-CSF) was used, and T-cell-supporting cytokines (IL-7 and IL-15) typically included in standard cytotoxicity assays for cell lines were omitted.

[0249] [Table 5]

[0250] Table 5: Primary AML cell batch

[0251]

[0252] Note: F = Female; M = Male; Coll. = Collection

[0253] Table 6 As outlined in the study design, two independent batches of anti-IL-1RAP CCTx-001 CAR T-cells and a donor-matched NTD T-cell control were used to evaluate their functionality against primary AML cells. For each test and control product, a total of one biological replicate was performed for each E:T ratio to assess cytotoxicity.

[0254] [Table 6]

[0255] Table 6: Study Design

[0256]

[0257] NTD and CAR T-cells were thawed on Day -3 of the cytotoxicity assay. Briefly, NTD and CAR T-cells were thawed in TexMACS medium and 1 x 10⁶ cells were thawed in TexMACS medium supplemented with 5% HS (human serum), 1% PS (pen-strep), 12.5 mg / mL IL-7, and 12.5 mg / mL IL-15. 6 Cells were maintained at a cell concentration of 1 x 10⁶ cells / mL. 6 Seeded at a density of 1 x 10 cells / mL, and 1 x 10 6The medium was periodically replaced (refreshed) to maintain a cell density of 10 cells / mL. Viability and cell concentration were verified and adjusted daily for 3 days prior to the start of the cytotoxicity test.

[0258] After a 3-day resting culture following thawing, test substance effector cells were labeled with the cell proliferation dye eFluor™ 450. Then, effector cells were harvested from the culture and washed twice with PBS 1X. Subsequently, 2 x 10 effector cells were placed in PBS 1X. 6 The cells were resuspended at 1 / mL. While vortexing the T-cells, an equal volume of 20 μM dye solution was added. After incubation in the dark at 37°C for 10 minutes, labeling was stopped by adding 4-5 volumes of wash buffer (PBS 1X, 10% FBS) and incubating on ice for 5 minutes. Subsequently, the cells were washed three times with wash buffer. The labeled effector cells were cultured at 4.5 x 10⁶ in culture medium (StemSpan SFEM II supplemented with CD34+ expansion supplement and 100 UI / mL GM-CSF without IL-7 and IL-15). 6 Each ratio was prepared by adjusting the concentration to cells / mL. From these preparations, dilutions were prepared by mixing with culture medium to obtain the concentrations required for each E:T ratio (3:1, 1:1, 1:3, 1:9, and 1:27).

[0259] AML primary cells were thawed on the start date of the cytotoxicity assay (after a 3-day resting period for effector cells), washed with 1X PBS, and 0.5 x 10⁶ cells were placed in a StemSpan SFEM II supplemented with CD34+ expansion supplement and 100 UI / mL GM-CSF without IL-7 and IL-15. 6 It was resuspended at a concentration of cells / mL.

[0260] 100 μL of effector cells were added into the corresponding wells. 100 μL of target cell suspension was added to all wells. 100 μL of culture medium was added to the "target alone" and "1:0 E:T ratio" sample wells. The well plates were incubated at 37°C and 5% CO2 for 24 hours.

[0261] After 24 hours of co-culture, the cells were resuspended and stained with CD33, CD34, and CD45 antibodies at +4°C for 10 minutes. Then, 7-AAD was added, and the cells were incubated at +4°C for an additional 10 minutes prior to FC analysis. Subsequently, the cells were analyzed by flow cytometry in Novocyte Quanteon, and data analysis was performed using Novoexpress software.

[0262] Co-culture was performed at different E:T ratios using culture conditions selected to preferentially support the survival of AML primary cells in vitro (SFEM medium + SCF, TPO, FLT3L, IL-3, IL-6, and G-CSF without T-cell supporting cytokines typically used to support CCTx-001). After 24 hours, samples were analyzed by FC, and cytotoxicity (viable blast cells normalized to target-alone conditions) and specific cytotoxicity (viable blast cells normalized to NTD T-cell conditions) were measured. Both batches of CCTx-001 CAR T-cells demonstrated CAR-mediated (specific) cytotoxicity against AML blast cells (as already observed starting from an E:T ratio of 1:3) for all patient samples tested ( Fig. 12 and Fig. 14 The magnitude of cytotoxicity was generally correlated with the IL-1RAP expression level on target cells ( Table 5 The allogeneic cytotoxicity observed in the NTD control group is Fig. 13As shown in the cytotoxicity results (viable blast cells normalized to target-only conditions), it varied depending on the AML patient target cells.

[0263] Preparation and Functional Evaluation of CCTx-001 CAR-T Cells Using AML Patient T-Cells

[0264] A study was conducted 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 against the patient's own primary AML blasts was also evaluated.

[0265] Several clinical trials have confirmed that the clinical-scale manufacturing of autologous CAR T-cells from AML patients is feasible (Jin et al. 2022; Pei et al. 2023; Sallman et al. 2022; Tambaro et al. 2021; Wang et al. 2015; Zhang et al. 2020; 2021; 2022; Zhao et al. 2024). Accessing relevant quantities of AML patient material to conduct further studies on manufacturability at a clinically relevant scale during development is not possible because larger quantities of patient-derived peripheral blood are unavailable. Therefore, core pharmacological and toxicological studies are performed using CAR T-cell batches generated from a clinically representative process using healthy donor cells as starting material. However, manufacturing using AML patient cells may be performed on a small scale (R&D grade) using small amounts of cryopreserved cells from routine clinical monitoring to demonstrate the functionality of CCTx-001 CAR T-cells produced from AML patient T-cells. Additionally, to enable the evaluation of the cytotoxicity of CAR-T cells against primary AML patient-derived blasts, small amounts of primary AML cells are generally sufficient when containing a substantial proportion of malignant blasts. For this study, T-cells were isolated from AML patients 1 month after diagnosis, which may have resulted in exposure to a primary chemotherapy regimen consisting of a single "3+7" regimen of daunorubicin and cytarabine.

[0266] AML blasts are notoriously difficult to maintain in vitro (Ø. Bruserud et al. 2001; Cucchi et al. 2020), and culture conditions can affect T-cell co-stimulation signaling 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 to enable the assessment of cytotoxicity, a special medium containing several AML cell-supporting cytokines (SCF, TPO, FLT-3L, IL-3, IL-6, and GM-CSF) was used, and T-cell-supporting cytokines were omitted from the culture to mitigate potential effects on AML cell survival.

[0267] Anti-IL-1RAP CCTx-001 CAR T-cells and untransduced T-cell controls (NTD T-cells) were generated on a small scale (R&D-grade manufactured) from previously collected peripheral blood mononuclear cells (PBMCs) from four AML patients diagnosed with moderate to severe prognosis AML. Blood samples were collected one month after diagnosis, which may have resulted in exposure to first-line chemotherapy. The AML patient PBMCs used as starting material for CAR T-cell production and as target cells in cytotoxicity assays were Table 7 It is listed in. All frozen samples are EFS Bourgogne Franche-Comt It was provided by and, in compliance with local regulations, collected at the one-month post-diagnosis visit with the donor's consent and without any form of compensation.

[0268] [Table 7]

[0269] Table 7: AML patient donor cells

[0270]

[0271] Note: AML-MR = AML with myelodysplastic syndrome-associated gene mutations; AML-M1 = AML with minimal differentiation; F = female; M = male

[0272] T-cell production:

[0273] T-cell isolation : gun Thawed PBMCs were incubated with AutoMACS execution buffer, CD4 micro beads, and CD8 micro beads at +4°C for 20 minutes. Subsequently, the cells were centrifuged at 500g for 5 minutes, the supernatant was removed, AutoMACS execution buffer was added, and the cell suspension was loaded onto an LS column. After the addition of AutoMACS execution buffer and human serum-free complete medium, non-T cells were eluted from the suspension; then, the column was removed from the magnetic field, and T cells were eluted from the column using a piston. Afterward, the cells were centrifuged, and the cell pellet was stored in human serum-free complete medium at a rate of 1 x 10⁶ 6 It was resuspended at a concentration of cells / mL.

[0274] T-cell activation : The cell suspension was incubated in a plate at 37°C in a humid atmosphere supplemented with 5% CO2 with TransAct.

[0275] T-cell transduction : Viral titer (5.69 x 10⁶) to reach MOI 2 1 day after T-cell activation 7 A volume of C4 lentivirus supernatant calculated according to TU / mL was added. The next day, medium containing human serum was added, and on the day after that, twice this amount was added. On day 9, the cells were diluted to a concentration of 0.5-1.10 6 It was adjusted to cells / mL. On day 9, the cell material is referred to as "Drug Substance (DS)."

[0276] cryopreservation: On day 9, T-cells in 5% human serum albumin (HSA) and 5% DMSO (CS10) were 3 x 10 6 cells / mL (CL045-016) or 5 x 10⁶ 6 It was cryopreserved at concentrations between 1 cell / mL (CL045-017, CL045-018, CL045-019).

[0277] T-cell Characterization:

[0278] The composition and transduction of the generated CAR-T cell products were characterized using flow cytometry (FCM) and digital PCR assays. Titration evaluation was based on IFN-γ release following incubation on IL-1RAP-coated plates. Briefly, DPs were incubated in 96-well plates coated with recombinant IL-1RAP, human serum albumin (HSA, negative control), or phorbol myristate acetate (PMA) and ionomycin (positive control, non-specific activation). After overnight incubation, the supernatant was tested for interferon gamma cytokine concentrations using an automated ELISA (Ella, Bio-Techne) and compared to the negative control (HSA). The results of the IFN-γ release assay were used. Fig. 18a It appears in.

[0279] The cytotoxicity of patient-derived CAR-T cells was evaluated against IL-1RAP-expressing AML cells (MOLM-13 cells). In this assay, CAR-T cells were stained with eFluor 450 dye and co-cultured with the target cell line MOLM-13 for 24 hours in TexMACS supplemented with IL-7 and IL-15, 5% human serum (SH), and 1% penicillin streptomycin (PS) (ratio: 1:1). Subsequently, cells were harvested, stained, and analyzed by flow cytometry to assess the viability of the target cells using viability dye 7-AAD. Specific death was normalized to parallel culture in which the target cells were incubated in the absence of CAR-T cells. The results for the MOLM-13 AML cytotoxicity assay Fig. 18b It appears in.

[0280] In addition, for three patients with identifiable malignant blast populations in PBMC samples, the cytotoxicity of patient-derived CAR T-cells was evaluated against the patient's own blasts in an in vitro co-culture assay adjusted to enable 24-hour culture of primary AML cells.

[0281] Analysis of the PBMC cell composition of AML patients showed that the T-cell content ranged from 2.49 to 25.77%. In addition, clearly identified malignant blasts in 3 out of 4 patients accounted for 20.94% to 93.29% of the total PBMCs, and IL-1RAP expression ranged from 2230.7 to 2383.0 ABC. High-survival CAR T-cells were obtained for all patients, showing successful transduction ranging from 22.13% to 35.35% of total T-cells and a vector copy number (VCN) maintained at less than 5. The functionality of the CAR T-cells was demonstrated by IFN-γ release titer assays and cytotoxicity against the MOLM-13 cell line. All batches exceeded release specifications for IFN-γ titers (> 5-fold increase) and demonstrated potent cytotoxicity against MOLM-13 cell lines (ranging from 58% to 70% at a 1:1 E:T ratio). Overall, all batches met expected specifications.

[0282] Example 3: Adaptive open-label multicenter Phase 1 / 2 study

[0283] 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 leukemia cells (Frenay 2022). In preclinical studies, IL-1RAP-targeted CARs 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, CCTx-001 is expected to potentially alter the natural course of r / r AML and provide a potential novel treatment option.

[0284] Research Overview:

[0285] The study will be conducted in compliance with the International Commission for Harmonization of Technical Requirements for Human Medicinal Products (ICH) technical requirements for registration / Good Clinical Practice (GCP) and applicable regulatory requirements.

[0286] This is an adaptive, open-label, multicenter Phase 1 / 2 study aimed at determining the RP2D of CCTx-001 and evaluating the safety, tolerability, and clinical activity of CCTx-001 in patients with r / r AML. The Phase 1 portion of the study will evaluate increasing doses of CCTx-001 to identify the recommended Phase 2 dose (RP2D) or optimal biological dose. Patient safety is a primary consideration for the selection of the starting dose. Dose escalation in Phase 1 will follow a Bayesian Optimal Interval (BOIN) design based on the occurrence of dose-limiting toxicity (DLT). The Phase 2 (dose escalation) portion will include a larger cohort of patients with r / r AML and aims to evaluate the clinical activity and further assess the safety of CCTx-001 in this population at the RP2D, which is considered a safe and clinically active dose.

[0287] The study includes four periods ( Fig. 10 Note): (1) The pre-treatment period will consist of screening for eligibility, leukocyte apheresis, and pre-treatment evaluation (before lymphocyte-depleting chemotherapy (LDC)); (2) The treatment period will begin with LDC, followed by CCTx-001 infusion 2 to 7 days after the completion of LDC. The first response evaluation will be performed approximately 28 days after CCTx-001 infusion; (3) The post-treatment period will consist of additional clinical activity and safety follow-up visits at regular time points after CCTx-001 infusion, starting after the 3-month visit and up to the 24-month visit; (4) The long-term follow-up period will begin after the 24-month visit after CCTx-001 infusion and up to 15 years.

[0288] Pre-treatment period:

[0289] Prior to the initiation of any study procedure, the patient must provide informed consent. Once enrolled and during the pre-treatment period, the patient will undergo leukocyte apheresis to obtain sufficient peripheral blood mononuclear cells (PBMCs) to produce the CCTx-001 cell product.

[0290] If necessary, anticancer therapy or disease control (bridging therapy) is permitted while CCTx-001 is being manufactured (i.e., after leukocyte apheresis and before the start of LDC). All patients receiving bridging therapy must repeat all disease evaluations before the start of LDC. All patients must continue to have detectable AML and meet eligibility criteria related to adequate organ function, active infection, pregnancy, and discontinuation of previous therapy before the start of LDC.

[0291] Treatment period:

[0292] If the production of the CCTx-001 product is successful, the patient will enter the treatment period and, after receiving the LDC, will be provided with a single dose of autologous CCTx-001 T-cells.

[0293] The patient received 4 days of fludarabine IV (30 mg / m²) for LDC. 2 / day) and cyclophosphamide IV for 3 days (300 mg / m² 2 You will be provided with ( / day). The composition or dosage of this lymphocyte depletion regimen may be changed during the study depending on observations of safety, biological and / or clinical activity.

[0294] From 2 to 7 days after the completion of LDC, CCTx-001 will be administered by IV infusion as a single dose on day 1.

[0295] A review of CAR T-cell studies registered on ClinicalTrials.gov through the end of 2016 found that typical dose escalation studies involve a 2-log (100-fold) dose range and generally a total of 10 CAR T-cells per patient 6 -10 9 It is within the range of, and 1x10 6 Dogs were found to be a typical starting dose (Hartmann 2017).

[0296] The dosages of commercially available T-cell products for hematological malignancies are within the following ranges: brexucabtagene autoleucel (Tecartus™) and axicabtagene ciloleucel (Yescarta ® In the case of ), 2x10 per kg 6 Up to 1x10 8 2x10 with dogs' cells 6 Up to 6.0x10 8 Dog cells; tisagenlecleucel (Kymriah ® In the case of ), 0.6 to 6.0x10 8 Dog cells; lysocaptagene maralucel (Breyanzi ® In the case of ), 0.5 to 1.1x10 8 Dog cells; Idecaptagene bicleucel (Abecma ® In the case of ), 3.0 to 4.6x10 8 Dog cells (van der Walle 2021). Recently, CD123-oriented CAR T-cells were 100 x 10⁶ as a fixed dose. 6 and 250x10 6 Evaluated in two separate Phase 1 studies starting with 10 UniCAR-T-cells (Wermke 2021), or 2.5 x 10 5 cells / kg, 6.25x10 5 cells / kg, 1.5x10 6 1 cell / kg, or 3.03 x 10⁶ 6 It was evaluated at a dose of UCART123 of 10 cells / kg (DL3) (Sallman 2022).

[0297] Considering that IL-1RAP is a novel, yet-to-be-explored target for CAR T-cell therapy, 5x10, which is approximately twice the typical starting dose (i.e., one dose escalation step), 5 A starting dose of 10 cells / kg was selected as an appropriate starting dose. This dose is 500 times lower than the dose used in animal studies. As recommended by FDA guidelines on the development of CAR T-cells, the calculation of cell dose will be based on body weight rather than using a fixed dose (FDA 2022).

[0298] The first three patients per DL will be treated with a minimum interval of 14 days between CCTx-001 infusions to allow sufficient time to observe for acute toxicity. At the end of each dose cohort, all available safety data will be reviewed and discussed with the SRC prior to the initiation of dosing in the next cohort. By the end of the dose exploration part, a dose level (DL) for the dose expansion part of the study will be selected. For a DL in which only three patients were treated, additional patients may be enrolled to support the selection of the DL for the expansion cohort. All patients are expected to be hospitalized for at least 14 days from the infusion for Phase 1.

[0299] We will continuously review safety data. We will perform an early safety assessment 28 days after administering CCTx-001 to the 12th patient. Enrollment will be discontinued if the observed treatment-related mortality rate is > 30%, or if > 50% of patients who underwent leukocyte apheresis do not have a satisfactory cell product available for infusion.

[0300] Phase 1 (Capacity Exploration) will include capacity escalation using the BOIN capacity escalation design (Yuan 2016, Lin 2017). This is a design that is easy to implement, flexible to cohort sizes, and allows cessation rules to be predefined. A total of five different DLs of CCTx-001 can be evaluated ( Table 8 ). The study is DL1(0.5x10 6 It will start at cells / kg). If declared safe based on evaluation, the next DL can be tested (DL2: 1x10 6 cells / kg; DL3: 5x10 6 cells / kg; DL4: 10x10 6 (Cells / kg). If DL1 is not tolerated according to evaluation, DL is DL-1 (0.1 x 10⁶). 6 It will be reduced to cells / kg). All DLs are capped at a maximum patient body weight of 80 kg. If a patient does not develop toxicity to CCTx-001 and does not respond to the first dose of CCTx-001, they may be given an additional dose of CCTx-001 at the next higher DL to enable potential anti-leukemic activity (intra-patient dose escalation).

[0301] [Table 8]

[0302] Table 8: CCTx-001 Capacity Levels

[0303]

[0304] At least 3 patients per dose will be treated in up to 5 planned DL cohorts. Additional patients (up to 3 additional patients per cohort) will be added based on DLT observation, and the total number of patients eligible for DLT evaluation under the BOIN design will be up to 30. All patients are expected to be hospitalized for at least 14 days from the time of infusion.

[0305] The Phase 2 (dose expansion) part is a single-arm part that primarily evaluates clinical activity, measured by the composite complete response rate (cCRR: CR, CRh, or CRi), as assessed by an Independent Review Committee (IRC) based on the European Leukemia Network (ELN) 2022 criteria (Dohner 2022). This will include approximately 72 patients with r / r AML who are evaluable for the primary endpoint analysis. Two interim analyses are expected. The first interim analysis will evaluate the futility of treatment with CCTx-001 in r / r AML patients compared to the prior control group. The second interim analysis will evaluate only the clinical activity of treatment with CCTx-001 in r / r AML patients compared to the prior control group. The primary analysis will be performed when the patient reaches the earliest of the following points in time during the last study in which CCTx-001 was infused: a visit 90 days after CCTx-001 infusion, relapse according to ELN 2022 criteria, initiation of new AML treatment, loss of follow-up, withdrawal of consent, or death from any cause. The dose escalation part will be enrolled over approximately 18 months, and the dose expansion part will be enrolled over approximately 18 months.

[0306] Post-treatment period:

[0307] Patients will be followed up for 2 years after CCTx-001 infusion, receiving more frequent follow-up for AEs related to CCTx-001 and / or LDC, and associated concomitant medicines and procedures.

[0308] Long-term follow-up period:

[0309] Patients will be followed up for up to 15 years after CCTx-001 infusion in accordance with current health authority guidelines. This long-term follow-up serves to collect any suspected AEs associated with CAR T-cell therapy.

[0310] Delayed potential toxicity may include RCL expression, autonomous proliferation of injected CAR T-cells, and interstitial oncogenesis due to the integration of the lentiviral construct. None of these potential cases have been observed to date.

[0311] Definition of Dose-Limited Toxicity (DLT) :

[0312] Severe cytokine release syndrome (CRS) and neurotoxicity (NT) are the most significant toxicities observed in CAR T-cell therapy. While the pattern of elevated cytokines varies among patients, significant increases in several cytokines, such as IL-6, IL-5, IL-10, and interferon-γ, have been observed (Davila 2014, Gust 2017). There is some correlation between the development of CRS and efficacy. The severity of CRS does not predict response but is associated with disease burden. Preliminary observations show correlations not only with the aforementioned subsets of cytokines that can predict the severity of CRS and NT, but also with additional cytokines such as IL-8, IL-15, and transforming growth factor-α. Furthermore, C-reactive protein (CRP) and ferritin, clinical markers of inflammation, are at elevated levels in patients presenting with CRS. To understand the pathophysiology of CAR T-cell-mediated toxicity and potential changes arising from combinations, a broad panel of available factors will be tested on a multiplex assay platform.

[0313] DLT refers to an adverse event or abnormal laboratory finding assessed as unrelated to leukemia, comorbidity, or concomitant medicine. The DLT observation period will be from Day 1 (day of CCTx-001 infusion) to Day 28. The period for DLT evaluation may be extended based on newly emerging data and for the evaluation of hematological toxicity and its outcomes. AEs will be evaluated and graded according to the National Cancer Institute (NCI) Common Terminology for Adverse Events Version 5.0 (CTCAE v.5.0). CRS and neurotoxicity will be evaluated according to the American Society for Transplant and Cell Therapy (ASTCT) consensus grading (Lee 2019).

[0314] DLT is defined as the following CCTx-001-related cases: death (except due to disease progression); cytokine release syndrome (CRS) (grade 3 CRS occurring during any treatment that does not improve to grade ≤ 2 within 72 hours despite appropriate therapy; grade 4 CRS occurring during any treatment of any duration); neurotoxicity (grade 3 neurological toxicity occurring during any treatment that does not resolve to grade ≤ 2 within 72 hours of onset despite appropriate therapy; grade 4 neurological toxicity occurring during any treatment of any duration); Cytopenia (any grade 4 leukopenia not improving to grade ≤ 3 within 28 days and not considered by the investigator to be due to other clearly identifiable causes (e.g., disease progression, concomitant medication, pre-existing medical conditions); any grade 4 thrombocytopenia not improving to grade ≤ 3 within 28 days and not considered by the investigator to be due to other clearly identifiable causes (e.g., disease progression, concomitant medication, pre-existing medical conditions); grade 4 febrile neutropenia accompanied by clinically significant bleeding and not considered by the investigator to be due to other clearly identifiable causes (e.g., disease progression, concomitant medication, pre-existing medical conditions) and grade ≥ 3 thrombocytopenia); grade 4 tumor lytic syndrome (Cairo and Bishop 2004); any grade 4 allergic reaction; any grade ≥ 3 autoimmune toxicity occurring during treatment; Any grade ≥ 3 elevated liver enzymes occurring during treatment unrelated to CRS; any other grade ≥ 3 toxicity to major organs that is not pre-existing or due to underlying disease. An exception may be made for grade 3 or 4 renal function tests that improve to grade 2 or lower within 7 days.

[0315] Research period:

[0316] End of study (EOS) for each individual patient will be 15 years from the patient's last CCTx-001 infusion date, withdrawal of consent, loss of follow-up, or death, whichever comes first.

[0317] EOS is defined as the later of the last visit date of the last patient to complete the EOS visit or the date of receipt of the last data point from the last patient required for primary, secondary, and / or exploratory analysis.

[0318] Study Group and Inclusion Criteria:

[0319] Adult patients (≥ 18 years of age) with relapsed or refractory AML to at least two cycles of standard induction therapy. The investigator or designator must ensure that only patients who meet all eligibility criteria are enrolled in the study.

[0320] All patients to be enrolled in the study must meet the following inclusion criteria:

[0321] 1. Patients with active r / r AML (WHO 2022) defined by any of the following (>5% of blasts in the bone marrow):

[0322] i. Primary refractory:

[0323] 1. Patients who failed after two cycles of intensive induction including high-dose and / or standard-dose cytarabine (including liposomal formulations), + / - anthracyclines, + / - antimetabolites, or + / - targeted therapy, or

[0324] 2. Elderly patients or patients who are unsuitable for intensive induction treatment and have failed after 2 cycles of venetoclax + azacitidine or 4 cycles of azacitidine.

[0325] ii. Recurrent:

[0326] 1. Patients who experienced an early relapse after CR with first-line therapy (within ≤6 months after CR1) or

[0327] 2. Patients who experience a recurrence after a subsequent treatment cycle (recurrence after CR≥2).

[0328] iii. Patients who relapsed after allogeneic hematopoietic stem cell transplantation:

[0329] 1. At the time of consent, at least 3 months must have passed since the HSCT, and

[0330] 2. At the time of consent, immunosuppressants must have been discontinued for at least one month, and

[0331] 3. There must be no active graft-versus-host disease (GvHD).

[0332] 2. Circulating blast count is 20,000 / mm³ 3 Less than (control using hydroxyurea is permitted).

[0333] 3. Absolute lymphocyte count >200 / mm 3 lim.

[0334] 4. Eastern Collaborative Oncology Group (ECOG) performance status ≤ 1.

[0335] 5. Life expectancy exceeding 3 months.

[0336] 6. The patient is ≥ 18 years of age at the time of prior consent.

[0337] 7. Read, understand, and sign the Informed Consent Form (ICF) before any research procedure.

[0338] 8. The patient is willing and able to comply with the study visit schedule and other protocol requirements.

[0339] 9. Suitable for leukocyte apheresis.

[0340] 10. Treatment-related toxicity of the previous therapy has been completely resolved.

[0341] 11. Adequate organ function confirmed by clinical laboratory values, defined as follows:

[0342] a. Bone marrow function adequate for receiving LDC as assessed by the investigator.

[0343] b. Serum creatinine [< 1.5 x Upper Limit of Normal (ULN) or Creatinine Clearance (CrCl) > 45 mL / min] (estimated by Cockcroft Gault or Dietary Modification in Renal Disease (MDRD).

[0344] 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].

[0345] d. Adequate lung function defined as [grade 1 dyspnea according to CTCAE and oxygen saturation (SaO2) in room air ≥ 92% and forced expiratory volume within the first second ≥ 50%].

[0346] e. Ejection fraction > 40% as assessed by echocardiography (ECHO) or multigate acquisition (MUGA) scan performed within 1 month prior to CCTx-001 injection.

[0347] 12. Women of childbearing potential (WOCBP) must be negative for a serum pregnancy test performed at the time of screening and within 7 days prior to enrollment.

[0348] 13. Men who are WOCBP or whose sexual partner is WOCBP must be able to and willing to use at least one highly effective contraceptive method during the study and during the 12 months following the last dose of LDC. WOCBP refers to female patients who: 1) have reached menarche at some point; 2) have not undergone a hysterectomy or bilateral oophorectomy; or 3) have not been naturally postmenopausal for at least 12 consecutive months (amenorrhea following cancer therapy does not exclude the possibility of fertility) (i.e., have had a menstruation at any time within the preceding 12 consecutive months).

[0349] Exclusion Criteria:

[0350] The presence of any of the following criteria will exclude the patient from participation in the study:

[0351] 1. Acute promyelocytic leukemia: t(15;17)(q22;q12);(promyelocytic leukemia / retinoic acid receptor alpha) and patients with variants.

[0352] 2. Patients with active CNS leukemia involvement. If the patient has a history of CNS leukemia, they must have a negative cerebrospinal fluid (CSF) evaluation and magnetic resonance imaging (MRI) or computed tomography of the brain (if MRI is not feasible) to demonstrate the absence of evidence of CNS disease.

[0353] 3. Patients with isolated extramedullary AML.

[0354] 4. Patients who have received prior treatment or prior gene therapy targeting IL-1RAP.

[0355] 5. Patients who have undergone allogeneic HSCT within 90 days prior to leukocyte apheresis.

[0356] 6. Patients who received donor lymphocyte infusion within 60 days prior to leukocyte apheresis.

[0357] 7. Patients with active GvHD.

[0358] 8. Patients with a history of a primary malignancy other than the disease under study, excluding the following non-invasive malignancies, provided the patient has not been disease-free for ≥ 2 years:

[0359] a. Basal cell carcinoma of the skin.

[0360] b. Squamous cell carcinoma of the skin.

[0361] c. Carcinoma in situ of the cervix.

[0362] d. Carcinoma in situ of the breast.

[0363] e. Incidental histological findings of prostate cancer (T1a or T1b) or curative prostate cancer.

[0364] f. Other completely resected stage 1 solid tumors with a low risk of recurrence.

[0365] 9. Presence of systemic fungal, bacterial, viral, or other infections (including tuberculosis) that are not controlled despite appropriate antibiotics or other treatments.

[0366] 10. Active or prior history of hepatitis B or hepatitis C infection.

[0367] 11. History of active human immunodeficiency virus (HIV) infection.

[0368] 12. Activated macrophage activation syndrome (MAS) as evidenced by laboratory abnormalities (e.g., elevated ferritin, elevated triglycerides, hemophagocytosis in bone marrow samples) and / or clinical signs.

[0369] 13. History or presence of active and clinically relevant CNS disorders, such as epilepsy, generalized seizure disorder, incomplete paralysis, aphasia, stroke, cerebral edema, severe brain injury, dementia, multiple sclerosis, Parkinson's disease, cerebellar disease, organic brain syndrome, or occipital reversible encephalopathy syndrome, or any autoimmune disease with CNS involvement.

[0370] 14. Patients with active autoimmune disorders or active neurological or inflammatory disorders (e.g., Guillain-Barré syndrome, amyotrophic lateral sclerosis) requiring immunosuppressive therapy or corticosteroid therapy (defined as > 20 mg / day prednisone or equivalent). Physiological replacement, topical, and inhaled steroids are permitted.

[0371] 15. Use of the following:

[0372] a. Therapeutic dose of corticosteroid within 7 days prior to leukocyte apheresis or within 72 hours prior to CCTx-001 infusion (defined as > 20 mg / day prednisone or equivalent). Physiological replacement, topical, and inhaled steroids are permitted.

[0373] b. Immunosuppressive therapy within 4 weeks prior to signing the ICF (e.g., calcineurin inhibitors, methotrexate or other chemotherapy agents, mycophenolate, rapamycin, thalidomide, anti-tumor necrosis factor [TNF], immunosuppressive antibodies such as anti-IL-6 or anti-IL-6 receptor [IL-6R]).

[0374] c. Cytotoxic chemotherapy agents (including intrathecal) within 14 days prior to leukocyte apheresis.

[0375] d. Treatment with alemtuzumab within 6 months prior to leukocyte apheresis, or treatment with fludarabine or cladribine within 3 months prior to leukocyte apheresis.

[0376] e. Experimental agents within 4 weeks prior to the ICF signing, provided that no response to the experimental therapy or progressive disease (PD) is documented and at least 3 half-lives have elapsed prior to the ICF signing.

[0377] f. Therapeutic anticoagulation.

[0378] 16. History of any of the following cardiovascular conditions within the past 6 months prior to signing the ICF:

[0379] g. Class III or IV heart failure as defined by the New York College of Cardiology.

[0380] h. Cardiovascular angioplasty or stent placement.

[0381] i. Myocardial infarction.

[0382] j. Unstable angina.

[0383] k. Other clinically significant heart disease.

[0384] 17. Known hypersensitivity to DMSO or other excipients.

[0385] 18. Uncontrolled medical, psychological, familial, sociological, or geographical condition that does not allow for compliance with the protocol, as determined by the investigator; or lack of willingness or ability to follow the procedures required by the protocol.

[0386] 19. Abnormal findings that, in the investigator's opinion, may endanger patient safety and / or clinically significant grade ≥ 3 non-hematological toxicity and any other medical condition(s) or laboratory findings.

[0387] 20. The presence of any pathological condition that confuses the ability to interpret data from the study based on the investigator's judgment.

[0388] 21. Any planned medical or surgical treatment that may interfere with the ability to comply with study requirements.

[0389] 22. Pregnant or breastfeeding women. Note: WOCBP must be negative on a serum pregnancy test performed within 48 hours of the start of LDC.

[0390] Leukocyte apheresis:

[0391] To obtain a sufficient amount of PBMC for the production of the CCTx-001 investigational product, unstimulated leukocyte apheresis collection will be performed for each patient. If technical issues arise during the procedure or product processing that prevent the product from being used for CCTx-001 administration, the patient may undergo a second collection procedure. The patient must continue to meet eligibility requirements for repeated leukocyte apheresis.

[0392] The following activities and evaluations will be performed on the day of unstimulated leukocyte apheresis, but before leukocyte apheresis:

[0393] Eligibility for Leukocyte Apheresis: The patient must be evaluated for evidence of active infection before leukocyte apheresis is initiated. If an infection is suspected, the patient must be treated, and leukocyte apheresis should be postponed until the active infection is resolved.

[0394] ECOG performance status assessment.

[0395] Measurement of vital signs and SaO2 via pulse oximetry (before and after leukocyte apheresis).

[0396] Collection of Peripheral Blood Samples and Preparation for Leukocyte Apheresis for Clinical Laboratory Evaluation:

[0397] o Hematology Panel

[0398] o Chemical Panel

[0399] o Inflammation markers

[0400] All AE records related to the procedures, concomitant medications, and concomitant procedures required by the protocol.

[0401] The patient may start bridging therapy if necessary for disease control while CCTx-001 is being manufactured.

[0402] Lymphocyte Depletion Chemotherapy (LDC):

[0403] LDC should be initiated to be completed 2 to 7 days before CCTx-001 injection.

[0404] Patients receiving bridging therapy must have a different staging (BMA / BMB, total blood count, flow cytometry) before the start of LDC.

[0405] Patients must be evaluated prior to the initiation of LDC (pre-LDC evaluation); adequate organ function and the absence of evidence of active infection are required prior to LDC. If infection is suspected, the patient should be treated accordingly, and LDC should be postponed until the active infection is resolved. Patients with rapid deterioration or rapid disease progression should not be initiated with LDC.

[0406] The patient 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 the infusion of CCTx-001. Refer to the most recent package insert for additional details regarding the administration of these agents.

[0407] LDC may be started 6 to 10 days prior to CCTx-001 injection and must be completed at least 48 hours prior to CCTx-001 injection. If adverse effects from LDC occur, CCTx-001 injection may be delayed up to 7 days after LDC.

[0408] Day 1 (CCTx-001 Infusion):

[0409] Patients must be evaluated prior to the initiation of CCTx-001; adequate organ function and the absence of evidence of active infection are required prior to LDC. If infection is suspected, the patient should be treated accordingly, and CCTx-001 should be postponed until the active infection is resolved. Patients with rapid deterioration or rapid disease progression should not be initiated with CCTx-001.

[0410] The following activities and evaluations will be performed prior to the administration of CCTx-001 infusion:

[0411] Confirmation of treatment eligibility

[0412] ECOG performance status assessment

[0413] Perform physical examination

[0414] Performing routine neurological examinations

[0415] Measurement of vital signs and SaO2 via pulse oximetry

[0416] Peripheral blood sample collection for clinical laboratory evaluation as described:

[0417] o Hematology Panel

[0418] o Coagulation test

[0419] o Chemical Panel

[0420] o Inflammation markers

[0421] PK sample collection for droplet digital polymerase chain reaction (ddPCR) and flow cytometry analysis

[0422] Sample collection for biomarkers

[0423] Collection of peripheral blood samples (serum and PBMC) for immunogenicity testing

[0424] CCTx-001 Injection Administration

[0425] Applicable to Phase 2 only: HM-PRO questionnaire administered

[0426] Records of all AEs, concomitant medications, and concomitant procedures

[0427] Hospital resource utilization evaluation

[0428] CCTx-001 will be provided as a cell dispersion for IV infusion. The cells are formulated in an infusion-ready cryopreservation medium and stored frozen at temperatures below -120°C. Each infusion bag will be labeled as required by national requirements.

[0429] Patients should be pre-treated with 500 to 650 mg of paracetamol / acetaminophen orally (PO) and 25 to 50 mg of diphenhydramine hydrochloride (PO or IV) 30 to 60 minutes before CCTx-001 infusion. If diphenhydramine is not available in the country, the use of other H1 antihistamines is acceptable.

[0430] These medications may be repeated every 6 hours as needed, based on the investigator's assessment of symptoms. Pre-treatment with steroids should be avoided.

[0431] CCTx-001 will be injected at each DL (Phase 1) or 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).

[0432] The patient must be continuously monitored during the administration of CCTx-001. Vital signs (body temperature, respiratory rate, heart rate, blood pressure, and SaO2 by pulse oximetry) will be measured approximately every 15 minutes, starting 15 minutes before the first IV administration and continuing until 1 hour after the last IV administration, and then every hour for the next 2 hours. If the patient's vital signs are not stable 4 hours after the last administration, vital signs should be monitored as clinically indicated until they stabilize.

[0433] Post-treatment visit:

[0434] Unless otherwise specified, all patients who receive the CCTx-001 infusion must complete post-treatment period visits for disease status and survival at approximately 4, 5, 6, 9, 12, 15, 18, 21, and 24 months after the CCTx-001 infusion.

[0435] The following activities and evaluations will be performed in patients without PD / recurrence who have not received subsequent anticancer therapy following the CCTx-001 infusion:

[0436] ECOG performance status assessment at months 4, 5, 6, 9, 12, 15, 18, 21, and 24

[0437] Perform physical examinations at 4, 5, 6, 9, 12, 15, 18, 21, and 24 months.

[0438] Vital signs measurements at 4, 5, 6, 9, 12, 15, 18, 21, and 24 months

[0439] SaO2 measured via pulse oximetry at 4, 5, 6, 9, 12, 15, 18, 21, and 24 months when clinically indicated.

[0440] BMB / BMA and peripheral blood collection 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 chemotherapy; if previously non-CR; if not persistent cytopenia (> grade 2), and if not transfusion-dependent).

[0441] Peripheral blood sample collection for clinical laboratory evaluation as follows:

[0442] o Hematological panel at 4, 5, 6, 9, 12, 15, 18, 21, and 24 months

[0443] o Chemical panel at months 4, 5, 6, 9, 12, 15, 18, 21, and 24

[0444] o Serum β-HCG pregnancy test for all WOCBPs at 12 months

[0445] Collection of PK samples for ddPCR at months 4, 5, 6, 9, 12, 15, 18, 21, and 24 (refer to CCTx-001 Laboratory Manual) (only if persistence vector sequence [PVS] was detected by PCR in the previous two samples)

[0446] Sample collection for biomarkers at the 6th month

[0447] Collection of peripheral blood samples (serum and PBMC) for immunogenicity testing at 6, 12, and 24 months

[0448] Collection of peripheral blood samples for RCL testing at 6, 12, and 24 months

[0449] Collection of peripheral blood samples for integrated site testing at months 6, 12, 24, 30, 36, 42, 48, 54, and 60, and annually thereafter (only if PVS is detected, and only if ≥ 1% of peripheral T-cells are positive for PVS)

[0450] AE records related to CCTx-001 and / or LDC and associated concomitant medications and procedures

[0451] New anticancer therapy record

[0452] Check survival status

[0453] Patients who received HSCT after CCTx-001 (but did not receive other anticancer treatment) must continue to be evaluated for the disease unless they developed PD before transplantation.

[0454] Long-term follow-up visit:

[0455] Follow-up visits: M36, M48, M60

[0456] During these visits, the patient will undergo one or more of the following: blood tests including ECOG, hematology, chemistry, RCL, PK, and integrated site tests (M36 only, unless PVS is detected). The patient must undergo a physical examination including vital signs. The female reproductive status and current status of the primary malignancy will be captured. Additionally, AEs defined in the protocol, including new malignancies and selected companion drugs, will be collected, including mutagens including cytotoxic drugs, radiation therapy and antineoplastic therapy (including stem cell transplantation), immunosuppressants including steroids at doses higher than the physiological replacement dose (20 mg / day hydrocortisone or equivalent), and investigational drugs.

[0457] Follow-up visits: M30, M42, and M54

[0458] During these visits, the patient will undergo one or more of the following: blood tests including ECOG, hematology, chemistry, and PK. The patient must undergo a physical examination including vital signs. The current status of the primary malignancy will be captured. Additionally, the AEs defined in the protocol, including the novel malignancy and selected companion drugs, will be collected, which include cytotoxic drugs, mutagens including radiotherapy and antineoplastic therapy (including stem cell transplantation), immunosuppressants including steroids at doses higher than the physiological replacement dose of steroids (20 mg / day hydrocortisone or equivalent), and investigational drugs.

[0459] Follow-up visit: 6 to 15 years if CAR transgene is detected

[0460] One or more of the following data will be collected: ECOG, physical examination, blood tests including hematology and chemistry, and the following until each test status is undetectable: RCL, PK, and integrated site tests.

[0461] The female reproductive status (for patients aged < 18 years at the time of initial infusion, collection will be initiated at age ≥ 8 years) and the current status of the primary malignancy will be captured. Additionally, the AEs defined in the protocol, including new malignancies and selected concomitant drugs, will be collected, which include cytotoxic drugs, mutagens including radiotherapy and antineoplastic therapy (including stem cell transplantation), immunosuppressants including steroids at doses higher than the physiological replacement dose of steroids (20 mg / day hydrocortisone or equivalent), and investigational drugs.

[0462] Follow-up visit: 6 to 15 years if CAR transgene is not detected

[0463] For patients in whom persistent vector sequences are no longer detected, their annual visits at the intervals indicated above may be conducted via telephone, video call, or mail to collect selected essential information.

[0464] One or more of the following data will be collected: body weight and height (if applicable); female reproductive status (for patients aged < 18 years at the time of initial infusion, collection will begin at age ≥ 8 years); and the current status of the primary malignancy will be captured. Additionally, AEs defined in the protocol, including new malignancies and selected concomitant medicines, will be collected. These include cytotoxic drugs, mutagens including radiotherapy and antineoplastic therapy (including stem cell transplantation), immunosuppressants including steroids at doses higher than the physiological replacement dose (20 mg / day hydrocortisone or equivalent), and investigational drugs. Repeated attempts to contact the patient must be made.

[0465] End of Study (EOS) / End of 15-Year Long-Term Follow-up Visit:

[0466] End of follow-up (EOS) will be 15 years after the last injection date of CCTx-001.

[0467] Pharmacokinetics of CCTx-001:

[0468] Evaluation of CCTx-001 PK will be determined by ddPCR to detect vector HIV gag DNA sequences and / or by flow cytometry to count CCTx-001 cells and analyze the immunophenotype. Peripheral blood will be collected as shown in Table 3.

[0469] Biomarkers, Pharmacodynamics, Pharmacogenetics:

[0470] The immune response to CCTx-001 will be evaluated using an anti-therapeutic antibody assay to detect the presence of circulating antibodies binding to the extracellular domain of the CAR. Additionally, cellular immunogenicity can also be evaluated by testing PBMCs from patients for the presence of anti-CCTx-001 cytotoxic T-cells.

[0471] Exploratory biomarker evaluations will be collected and will include evaluation of CCTx-001 and circulating cells in peripheral blood, characterization of leukemia and leukemia microenvironment, and analysis of plasma cytokines.

[0472] BMA and BMB samples will be collected to investigate cellular factors and the tumor microenvironment regarding biomarkers associated with clinical activity and disease characteristics (e.g., cytogenetics). Additionally, these samples will be analyzed for the expression of various immunologically interesting markers. This may provide insights into pathways activated in the tumor microenvironment that could influence the fate and activity of CCTx-001 cells.

[0473] Peripheral blood and plasma will be collected to examine analyses including but not limited to the following: soluble factors from plasma will be measured as markers of immune activation and to determine cytokine production, efficacy and potential correlations between CRS and the severity of ICANS; phenotypic characterization and gene expression profiling, and various analyses at the nucleotide level may be performed on CCTx-001 cells and immune cells to identify markers or gene signatures correlated with clinical response.

[0474] Data collected from these biomarker evaluations will be used to determine CAR T-cell function, persistence, disease and tumor microenvironment characteristics, and the relationship between CCTx-001 and peripheral blood characteristics and clinical response and toxicity.

[0475] Concurrent medications and procedures

[0476] To minimize the risk of infusion reactions, all patients must be pre-treated with acetaminophen and diphenhydramine before IMP infusion.

[0477] In some cases, tocilizumab, an anti-IL-6R antibody, may be required to treat toxicities such as CRS. Refer to the currently approved Actemra® / RoActemra® package insert. Depending on the label, up to four doses of tocilizumab may be administered for the treatment of CRS. It is recommended to follow local labeled guidelines. In some cases, steroids (e.g., dexamethasone) may also be administered for the treatment of CRS or ICANS.

[0478] For MAS, current guidelines recommend treatment with Anakinra (Hayden 2022). Targeting of IL-1RAP may reduce the activity of Anakinra. Currently, there is no clinical experience regarding the use of Anakinra in patients treated with IL-1RAP-targeted agents.

[0479] References cited in the examples:

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486] Since the foregoing description of specific embodiments and embodiments is sufficient to reveal the general nature of the present disclosure, others may easily modify and / or adapt such specific embodiments and embodiments for various applications by applying knowledge within the scope of the art, without excessive experimentation, and without departing from the general concept of the present disclosure. Accordingly, such adaptation and modification are intended to be within the meaning and scope of equivalents of the disclosed embodiments and embodiments based on the teachings and guidelines set forth herein. It should be understood that the terms or phrases in this specification are for descriptive purposes only and not for limiting purposes, and that the terms or phrases in this specification should be interpreted by those skilled in the art in light of the teachings and guidelines.

[0487] The breadth and scope of the present disclosure shall not be limited by any of the exemplary embodiments and embodiments described above, but shall be defined only by the following claims and their equivalents.

[0488] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) cited throughout this application are, like the references cited therein, expressly incorporated herein by reference for any purpose as if they were described herein in their entirety. In the event of any discrepancy, the material disclosed herein literally shall prevail.

[0489] Although various specific embodiments have been exemplified and described, the foregoing specification is not limiting. It will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure(s). Many variations will become apparent to those skilled in the art upon reviewing this specification.

[0490] Sequence list

[0491]

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[0493]

Claims

Claim 1 A method for inhibiting the growth of tumor cells in a subject requiring inhibition of tumor cell growth comprises the step of administering a therapeutically effective amount of T cells expressing a chimeric antigen receptor (CAR) on its surface to said subject, wherein the CAR comprises an antibody or an antigen-binding fragment thereof comprising an anti-interleukin 1 receptor helper protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulation domain, and said anti-IL-1RAP binding domain comprises (i) a light chain comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO. 6, a CDR2 having the amino acid sequence SAS, and a CDR3 having the amino acid sequence of SEQ ID NO. 8, and (ii) a heavy chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 12, a CDR2 having the amino acid sequence of SEQ ID NO. 13, and a CDR3 having the amino acid sequence of SEQ ID NO. 14, and prior to administering said T cells expressing said CAR, said subject is pre-treated with lymphocyte-depleting chemotherapy (LDC). Conditioning method. Claim 2 A method for treating acute myeloid leukemia (AML) in a subject requiring treatment, comprising the step of administering a therapeutically effective amount of T cells expressing a chimeric antigen receptor (CAR) on its surface to said subject, wherein the CAR comprises an antibody or an antigen-binding fragment thereof comprising an anti-interleukin 1 receptor helper protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulation domain, and said anti-IL-1RAP binding domain comprises (i) a light chain comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO. 6, a CDR2 having the amino acid sequence SAS, and a CDR3 having the amino acid sequence of SEQ ID NO. 8, and (ii) a heavy chain comprising a CDR1 having the amino acid sequence of SEQ ID NO. 12, a CDR2 having the amino acid sequence of SEQ ID NO. 13, and a CDR3 having the amino acid sequence of SEQ ID NO. 14, and before administering said T cells expressing said CAR, said subject is lymphocyte depleted A method that is pre-conditioned with chemotherapy (LDC). Claim 3 A method according to claim 1 or 2, wherein the CAR comprises the amino acid sequence of SEQ ID NO.

19. Claim 4 A method according to any one of claims 1 to 3, wherein the T cells comprise CD4+ and CD8+ T cells. Claim 5 A method according to claim 3 or 4, wherein the T cells are autologous. Claim 6 A method according to any one of claims 1 to 5, wherein the subject has increased IL-1RAP expression compared to a normal subject. Claim 7 A method according to any one of claims 1 to 6, wherein the T cells expressing the CAR are administered intravenously. Claim 8 A method according to any one of claims 1 to 7, wherein the LDC comprises the step of administering radiation therapy, fludarabine, or cyclophosphamide. Claim 9 A method according to any one of claims 1 to 8, wherein the LDC is completed at least 48 hours prior to administering the T cells expressing the CAR. Claim 10 A method according to any one of claims 1 to 9, wherein the LDC is initiated 6 to 10 days prior to administering the T cells expressing the CAR. Claim 11 In any one of claims 1 to 10, the T cells expressing the CAR are 0.1 x 10 6 cells / kg, 0.5 x 10⁶ 6 cells / kg, 1 x 10⁶ 6 cells / kg, 5 x 10 6 cells / kg, or 10 x 10⁶ 6 A method administered at a dose of cells / kg. Claim 12 A method according to any one of claims 1 to 11, wherein the T cells expressing the CAR are administered as a single dose. Claim 13 A method according to any one of claims 6 to 12, wherein the IL-1RAP expression level is determined by immunohistochemistry (IHC), flow cytometry, or quantitative polymerase chain reaction (qPCR). Claim 14 A method in which, in any one of paragraphs 1 to 13, the subject is a human. Claim 15 A method according to any one of claims 1 to 14, wherein the tumor cells are blood cancer tumor cells or tumor cells originating from blood cancer. Claim 16 A method according to any one of paragraphs 2 through 16, wherein the AML is a minimal residual disease (MRD). Claim 17 A method according to any one of claims 2 to 16, wherein the AML is IL-1RAP-expressing AML. Claim 18 A method according to any one of claims 2 to 17, further comprising the step of detecting IL-1RAP in a sample obtained from the AML before, during, or after administering the T cells. Claim 19 A method according to any one of claims 1 to 18, wherein the subject has received at least one previous treatment course, at least two previous treatment courses, or at least three previous treatment courses. Claim 20 Use of T cells according to any one of claims 1 to 5 for inhibiting the growth of AML in a subject requiring inhibition of AML growth.

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