WO2024150177A1

WO2024150177A1 - Treatment methods for solid tumors

Info

Publication number: WO2024150177A1
Application number: PCT/IB2024/050305
Authority: WO
Inventors: Haberbache RAFIK; Walid WARDA; Marina Deschamps; Christophe Ferrand
Original assignee: Advesya; Etablissement Français Du Sang
Priority date: 2023-01-11
Filing date: 2024-01-11
Publication date: 2024-07-18

Abstract

The disclosure provides methods of treating a solid tumor in a subject comprising administration of a cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antigen-binding fragment thereof that binds IL-1RAP, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain.

Description

TREATMENT METHODS FOR SOLID TUMORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No. 63/479,471, filed January 11, 2023, and U.S. Provisional Application No. 63/599,189, filed November 15, 2023, each of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 5041_005PC02_SequenceListing_ST26; Size: 21,477 bytes; and Date of Creation: January 4, 2024), filed with the application, is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The invention disclosed herein relates to methods of treating a solid tumor in a subject comprising administration of a cell expressing a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antigen-binding fragment thereof that specifically binds IL-1RAP, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain.

BACKGROUND OF THE INVENTION

[0004] Based on World Health Organization (WHO), approximatively 20 million new cancers are diagnosed per year and almost 10 million people die because of cancers. WHO predictions suggest an increase in cancers incidence up to 29 million and cancers caused deaths up to 16.6 million in 2040. This highlights the need for new early diagnostic tools/methods and to find new, more efficient therapies. Promising recent results concerning diagnosis and therapies have been performed and overall cancer mortality has decreased to 53%. However, despite these advances, several cancers still have very poor prognosis, including pancreatic cancers (94% mortality), hepatic cancers (93% mortality) and lung cancers (84% mortality) because of late diagnosis and poor therapeutic options. [0005] Immunotherapies, like chimeric antigen receptor (CAR)-T cells, are based on the engineered expression of T lymphocytes for targeting a cell surface tumor associated antigen. CAR-T cells have shown success in hematological malignancies with four approved products directed against CD 19 to treat several types of leukemias and two FDA approved products directed against BCMA to treat myelomas. However, CAR-T cells as a viable therapeutic for solid tumors have been much more mitigated. Indeed, solid tumors present complex challenges as the tumor microenvironment, the presence of immunosuppressive cells, and the lack of identifiable target antigens that will not contribute to off-target toxicity all negatively contribute to efficacy. Therefore, there exists a need in the art for immunotherapies that are efficacious in solid tumors.

SUMMARY OF THE INVENTION

[0006] The present disclosure is directed to a method of inhibiting growth of a solid tumor in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an antiinterleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

[0007] The present disclosure is also directed to a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

[0008] The present disclosure is also directed to a method of targeting a tumor microenvironment for treatment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

[0009] In one aspect, the CAR comprises the amino acid sequence of SEQ ID NO: 19. In another aspect, the cell is a T cell. In another aspect, the cell is an autologous cell.

[0010] In one aspect, the the subject has increased IL-1RAP expression compared to a normal subject.

[0011] In one aspect, the methods further comprise administering a therapeutically effective amount of one or more chemotherapeutic agents and/or immune checkpoint inhibitors. In one aspect, the cell expressing the CAR, and the chemotherapeutic agent and/or immune checkpoint inhibitor are administered concurrently. In another aspect, the cell expressing the CAR, and the chemotherapeutic agent and/or immune checkpoint inhibitor are administered sequentially. In another aspect, the cell expressing the CAR is administered prior to administration of the chemotherapeutic agent and/or immune checkpoint inhibitor. In another aspect, the chemotherapy is docetaxel, 5-fluorouracil, and/or oxaliplatin. In one aspect, the immune checkpoint inhibitor is an antibody that binds PD-1, PD-L1, or CTLA-4. In one aspect, the antibody is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab or tremelimumab.

[0012] In one aspect, the IL-1RAP expression levels are determined by immunohistochemistry (IHC) or quantitave polymerase chain reaction (qPCR). [0013] In one aspect, the tumor or cancer is a solid tumor or a cancer resulting from a solid tumor growth. In one aspect, the solid tumor is a non-small cell lung carcinoma (NSCLC), pancreatic carcinoma, breast carcinoma, colorectal carcinoma, liver carcinoma, esophageal carcinoma, head and neck carcinoma, or melanoma. In one aspect, the subject is a human.

[0014] The present disclosure is also directed to the use of cell described herein in any one of the methods of inhibiting the growth of a solid tumor in a subject in need thereof that are described herein.

BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1 shows the percentage of patients with IL-1RAP+ tumors. Circle size represents the number of deaths per year in the United States.

[0016] Figures 2A-2D show that IL-1RAP is overexpressed in many solid tumors at the RNA and protein levels in patients and tumor cell lines. (FIG. 2A) RT-qPCR IL-1RAP mRNA expression levels on 33 human tumoral cell lines. (FIG. 2B) Flow cytometry gating strategies of IL-1RAP labeling. RFIs were calculated by comparing the IL-1RAP and isotype signals. (FIG. 2C) Flow cytometry analysis has been performed on 3 hepatocellular carcinoma tumor cell lines, 2 pancreas carcinoma cell lines and 3 lung carcinoma cell lines looking for IL-1RAP cell surface expression. (FIG. 2D) Comparison between IL-1RAP mRNA expression in patients healthy tissues (denoted by the circle) and in patients tumoral tissues (denoted by the square), results were obtained through RNA Scope technology, y axis shows IL-1RAP scores (semi-quantitative method calculated from mRNA molecules per cell).

[0017] Figure 3 shows a schematic of a CD 107a, IFN-y, and TNFa workflow.

[0018] Figures 4A-4C show IL-1RAP CAR-T cells can efficiently degranulate, secrete IFN-y and TNFa and kill solid tumor cells cultured as monolayers in in vitro models. IL-1RAP CAR-T cells (denoted by the diamond) or untransduced T cells (denoted by the triangle) were cultured with tumor cell lines (NICH440, BXPC3 or HEPG2) at a 1 :5 E:T ratio. (FIG. 4A) A CD 107a degranulation assay was performed after 4 hours of co-culture. (FIG. 4B) IFN-y and (FIG. 4C) TNFa secretion assays were performed after 24 hours of co-culture.

[0019] Figure 5 shows a schematic of a cytotoxicity assay workflow.

[0020] Figures 6A-6D show CAR-T cell cytoxocity in 2D and 3D models. (FIG. 6A) CAR-T-cell cytotoxicity was measured through GFP intensity measurement in tumor cell lines. CAR-T cells (denoted by the triangle) or untransduced cells (denoted by the square) were co- cultured with GFP-expressing tumor cells cultured as a monolayer for 72 hours. One photo was taken each hour, and the total green object integrated intensity (CGU x pm²/image) was measured. (FIG. 6B) CAR-T cells (denoted by the triangle) or untransduced cells (denoted by the square) were co-cultured with GFP-expressing tumor cells cultured as spheroids for 72 hours. One photo was taken each hour, and the total green object integrated intensity (CGU x pm²/image) was measured. Cytotoxicity was also assessed through an LDH release assay performed either in two dimensions (FIG. 6C) or in three dimensions (spheroids) (FIG. 6D) coculture between CAR-T cells (denoted by the triangle) or untransduced cells (denoted by the square) and tumor cell lines.

[0021] Figure 7 shows a sIL-lRAP quantification workflow.

[0022] Figures 8A-8B show that solid tumor-bearing patients showed high levels of soluble IL-1RAP concentrations in their serum, but these concentrations did not impair IL-1RAP CAR-T cell cytotoxicity against tumor cell lines. (FIG. 8A) The soluble IL-1RAP concentration was measured in serum from patients bearing 5 types of solid tumors (10 patients per tumor type) and in 10 healthy donors. (FIG. 8B) CAR-T cell cytotoxicity was measured by adding 500 ng/mL, 1000 ng/mL or 2000 ng/mL soluble IL-1RAP to tumor cell lines and determining GFP intensity measurements at IncuCyte. CAR-T cells or untransduced cells were co-cultured with GFP-expressing tumor cells cultured as a monolayer for 72 hours. One photo was taken each hour, and the total green object integrated intensity (CGU x pm²/image) was measured.

[0023] Figure 9 shows an in vivo cytotoxicity assessment workflow.

[0024] Figures 10A-10B show IL-1RAP CAR-T cells are efficient in vivo, decreasing tumor volumes for NCI-H460- and HEPG2 -bearing mice and increasing overall mouse lifespan. NSG mice aged 6-8 weeks were injected subcutaneously with IxlO⁶ tumor cells three days prior to the first 10xl0⁶ intravenous injection of IL-1RAP CAR-T cells or untransduced T cells (tail vein). The second same injection was performed three days after the first injection. (FIG. 10A) The radiance of the in vivo bioluminescent signal (radiance p/s/cm²/sr) collected using the IVIS Illumina III (Perkin Elmer) is shown. (FIG. 10B) Tumor volumes were calculated by measuring tumor length and width with a digital caliper.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present disclosure is directed to methods of inhibiting growth of a solid tumor/treating cancer comprising administration of a cell comprising a nucleic acid molecule encoding an IL-lRAP-specific CAR. In some aspects, the cell is administered in combination with chemotherapy, and/or an immune checkpoint inhibitor.

1. Definitions

[0026] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0027] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to specific compositions or process steps, as such can vary. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein.

[0028] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0030] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. [0031] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and/or "consisting essentially of are also provided.

[0032] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower).

[0033] Amino acids are referred to herein by either their commonly known three letter symbols or by the one -letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single -letter codes.

[0034] The term “polypeptide,” as used herein, is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and comprises any chain or chains of two or more amino acids. Thus, as used herein, a “peptide,” a “peptide subunit,” a “protein,” an “amino acid chain,” an “amino acid sequence,” or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of a “polypeptide,” even though each of these terms can have a more specific meaning. The term “polypeptide” can be used instead of, or interchangeably with, any of these terms. The term further includes polypeptides that have undergone post-translational or post-synthesis modifications, for example, conjugation of a palmitoyl group, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, disulfide bond formation, proteolytic cleavage, or modification by non-naturally occurring amino acids. The term “peptide,” as used herein, encompasses full-length peptides and fragments, variants or derivatives thereof. A “peptide” as used herein can be part of a fusion polypeptide comprising additional components such as, e.g., an albumin or PEG moiety, to increase half-life. A peptide as used herein can also be derivatized in a number of different ways. A peptide can comprise modifications including e.g., conjugation of a palmitoyl group. The term "nucleic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single- stranded or double- stranded, and can be cDNA.

[0035] As used herein, the term "linked" refers to the association of two or more molecules. The linkage can be covalent or non-covalent. The linkage also can be genetic (i.e., recombinantly fused). Such linkages can be achieved using a wide variety of art recognized techniques, such as chemical conjugation and recombinant protein production.

[0036] The term “chimeric antigen receptor” or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some aspects, the domains in the CAR polypeptide construct are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some aspects, the domains in the CAR polypeptide construct are not contiguous with each other, e.g., are in different polypeptide chains.

[0037] An "antibody" (Ab) shall include, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as Vzz) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CHI, CHI and CH3. Each light chain comprises a light chain variable region (abbreviated herein as Vz) and a light chain constant region. The light chain constant region is comprises one constant domain, CL. The Vzz and Vz regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each Vzz and Vz comprises three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. A heavy chain may have the C-terminal lysine or not. Unless specified otherwise herein, the amino acids in the variable regions are numbered using the Kabat numbering system and those in the constant regions are numbered using the EU system.

[0038] An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man. Where not expressly stated, and unless the context indicates otherwise, the term "antibody" includes monospecific, bispecific, or multi-specific antibodies, as well as a single chain antibody. In aspects, the antibody is a bispecific antibody. In other aspects, the antibody is a monospecific antibody.

[0039] As used herein, an “IgG antibody” has the structure of a naturally occurring IgG antibody, /.< ., it has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an anti-ICOS IgGl, IgG2, IgG3 or IgG4 antibody consists of two heavy chains (HCs) and two light chains (LCs), wherein the two heavy chains and light chains are linked by the same number and location of disulfide bridges that occur in naturally occurring IgGl, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bonds)

[0040] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds specifically to PD-L1 is substantially free of antibodies that bind specifically to antigens other than PD-1). An isolated antibody that binds specifically to PD-L1 may, however, have crossreactivity to other antigens, such as PD-L1 molecules from different species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

[0041] The antibody may be an antibody that has been altered (e.g., by mutation, deletion, substitution, conjugation to a non-antibody moiety). For example, an antibody may include one or more variant amino acids (compared to a naturally occurring antibody) which change a property (e.g., a functional property) of the antibody. For example, numerous such alterations are known in the art which affect, e.g., half-life, effector function, and/or immune responses to the antibody in a patient. The term antibody also includes artificial polypeptide constructs which comprise at least one antibody-derived antigen binding site.

[0042] The term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, /.< ., antibody molecules whose primary sequences are essentially identical, and which exhibits a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody. MAbs may be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art. [0043] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" antibodies and "fully human" antibodies and are used synonymously.

[0044] A "humanized antibody" refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one aspect of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains an antigenic specificity similar to that of the original antibody.

[0045] A "chimeric antibody" refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

[0046] An "anti-antigen" antibody refers to an antibody that binds specifically to the antigen. For example, an anti-IL-lRAP antibody binds specifically to IL-1RAP.

[0047] An "antigen-binding portion" of an antibody (also called an "antigen-binding fragment") refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody. It has been shown that the antigenbinding function of an antibody can be performed by fragments or portions of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” or “antigen-binding fragment” of an antibody, e.g., an anti-IL-lRAP antibody described herein, include: (1) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC and CHI domains;

(2) a F(ab’)2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

(3) a Fd fragment consisting of the VH and CHI domains;

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

(5) a single domain antibody (dAb) fragment (Ward et al., (1989) Nature 341 :544-46), which consists of a VH domain;

(6) a bi-single domain antibody which consists of two VH domains linked by a hinge (dual-affinity re-targeting antibodies (DARTs));

(7) a dual variable domain immunoglobulin;

(8) an isolated complementarity determining region (CDR); and

(9) a combination of two or more isolated CDRs, which can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” or “antigen-binding fragment” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0048] The term “IL1RAP,” as used herein, refers to the interleukin-1 receptor accessory protein that is the cellular membrane co-receptor for several receptors in the IL-1 family, including interleukin-1 receptor 1 (IL1R1), ST2 (also known as interleukin-1 receptor-like 1 or IL-1RL1), and interleukin-1 receptor-like protein 2 (IL-1RL2). It is noted that the interleukin-1 receptor accessory protein, or ILlRAP, is sometimes referred to in the art as “IL-1RAP,” “IL- IRAcP,” “ILlRAcP” or “IL-1R3.” The terms “IL1RAP,” “IL-lRap” and “IL1RAP protein” are used herein interchangeably. [0049] A “cancer-associated fibroblast” (CAF) or “tumor-associated fibroblast” or “activated fibroblast” is a cell type within the tumor microenvironment that promotes tumorigenic features by initiating the remodelling of the extracellular matrix or by secreting cytokines.

[0050] An “immune checkpoint inhibitor” refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In particular the immune checkpoint protein is a human immune checkpoint protein. Thus the immune checkpoint protein inhibitor in particular is an inhibitor of a human immune checkpoint protein.

[0051] A "patient" as used herein includes any patient who is afflicted with a solid tumor. The terms "subject" and "patient" are used interchangeably herein.

[0052] "Administering" refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some aspects, the formulation is administered via a non-parenteral route, in some aspects, orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

[0053] Treatment" or "therapy" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.

[0054] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual. [0055] The term “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0056] The term “xenogeneic” refers to a graft derived from an animal of a different species.

[0057] As used herein, "effective treatment" refers to treatment producing a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder. A beneficial effect can take the form of an improvement over baseline, i.e., an improvement over a measurement or observation made prior to initiation of therapy according to the method. A beneficial effect can also take the form of arresting, slowing, retarding, or stabilizing of a deleterious progression of a marker of solid tumor. Effective treatment may refer to alleviation of at least one symptom of a solid tumor. Such effective treatment may, e.g., reduce patient pain, reduce the size and/or number of lesions, may reduce or prevent metastasis of a tumor, and/or may slow tumor growth.

[0058] The term "effective amount" refers to an amount of an agent that provides the desired biological, therapeutic, and/or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and/or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to solid tumors, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. In some aspects, an effective amount is an amount sufficient to completely eradicate the tumor. In some aspects, an effective amount is an amount sufficient to completely eradicate the tumor cells. An effective amount can be administered in one or more administrations. The effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and may stop tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer. In one example, an "effective amount" is the amount of IL- 1RAP CAR-T clinically proven to affect a significant decrease in cancer or slowing of progression of cancer, such as an advanced solid tumor.

[0059] The term "progression-free survival," which can be abbreviated as PFS, as used herein refers to the length of time during and after the treatment of a solid tumor that a patient lives with the disease but it does not get worse. The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a standard control. A therapeutically effective dose or amount may ameliorate one or more symptoms of a disease. A therapeutically effective dose or amount may prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is being administered is to treat a person who is at risk of developing the disease.

[0060] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.

[0061] The term “combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present invention and a combination partner (e.g. another drug as explained below, also referred to as “therapeutic agent” or “co-agent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. “Concurrently,” as used herein to, refers to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).

[0062] A "cancer" refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A "cancer" or "cancer tissue" can include a tumor.

[0063] The term "tumor" as used herein refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions, independent of cell of origin.

[0064] A “relapsed” cancer is one in which the cancer or the signs and symptoms of a cancer returns after a period of improvement.

[0065] An "immune response" refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0066] A "complete response" or "complete remission" or "CR" indicates the disappearance of all signs of tumor or cancer in response to treatment. This does not always mean the cancer has been cured.

[0067] The term "lymphocyte" as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a major component of the inherent immune system. NK cells reject tumors and cells infected by viruses by inducing apoptosis or programmed cell death in the target cell. They were termed "natural killers" because NK cells do not require activation in order to kill a target cell. T-cells play a major role in cell-mediated-immunity. T-cell receptors (TCR) expressed on the surface of T cells differentiate T cells from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for T cell maturation. There are six types of T-cells, namely: Helper T-cells (e.g. CD4+ cells); Cytotoxic T-cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cells or killer T cell); Memory T-cells ((i) stem memory TSCM cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L- selectin), CD27+, CD28+ and IL-7Ra+, but they also express large amounts of CD95, IL-2R.p, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory TCM cells express L-selectin and the CCR7, they secrete IL-2, but not IFNy or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFNy and IL-4); Regulatory T-cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells); Natural Killer T-cells (NKT); and Gamma Delta T-cells.

[0068] As used herein, “survival” refers to the patient remaining alive, and includes overall survival as well as progression free survival. 1-year survival rate and 2-year survival rate refers to the K-M estimate of the proportion of subjects alive at 12 month or 24 months.

[0069] By “extending survival” is meant increasing overall survival and/or progression free survival in a treated patient relative to a control treatment protocol, such as treatment with the antibody drug conjugates described herein. Survival is monitored for at least about one month, two months, four months, six months, nine months, or at least about 1 year, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc., following the initiation of treatment or following the initial diagnosis.

[0070] By “reduce or inhibit” is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

[0071] As used herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to cell proliferation (e.g., cancer cell proliferation) means negatively affecting (e.g., decreasing proliferation) or killing the cell. In some aspects, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. Similarly an “inhibitor” is a compound or protein that inhibits a receptor or another protein, e.g., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity (e.g., a receptor activity or a protein activity).

[0072] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophagelike synoviocytes, etc).

[0073] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0074] Various aspects of the disclosure are described in further detail in the following subsections.

2. Methods of the Disclosure

[0075] IL-1RAP is a part of a protein receptor complex and assembled with IL-la, IL-ip or IL-33 receptors on several cells, particularly hematopoietic cells. IL-1RAP has been shown to be overexpressed at cell surface of leukemic stem cells in acute myeloid leukemia (AML), myelodysplastic syndrome and chronic myeloid leukemia without being expressed on normal HSCs. IL-1RAP is known to play a key role in tumor microenvironment inflammation through p38 MAPK and NF-kp signaling pathways (De Boer et al. 2020). Recently, several studies suggested that IL-1RAP could be a potential target in solid tumors. IL-1RAP has been shown to be overexpressed in papillary thyroid carcinoma cells (Smallridge RC, et al. J Clin Endocrinol Metab. 2014 Feb; 99(2): E338-47), in stomach cancer cells (LvQ et al Cancer Res Treat. 2021), in Ewing sarcoma cells (Zhang HF et al Cancer Discov. 2021 Nov; 11(11):2884-2903) and in pancreas cancer cells (Herremans K et al Neoplasia. 2022 Jun; 28: 100789). In these studies, it was also shown that the overexpression of IL-1RAP of cancer cell surface is associated with poor prognosis. More interesting however, it was found that impairment of IL-1RAP axis on in vivo models decreased all the tumoral characteristics of cancer cells including proliferation, migration and invasion. These studies suggest that IL-1RAP could be a potential optimal target for solid tumors treatment but also that and the impairment of IL-1RAP axis could lead to better prognosis for patients.

[0076] Currently IL-1RAP is targeted by two monoclonal antibodies trough CAN-04 and CAN- 10 projects of Cantargia, CAN-04 targets IL-1RAP in solid tumors, mainly lung and pancreas, and CAN-10 targets IL-1RAP in autoimmune and inflammatory diseases. Nidalinimab (CAN-04) is under phase I/IIa clinical investigations (ClinicalTrials.gov: NCT03267316 and NCT04452214) in lung and pancreas cancers, used in association with chemotherapies. However, monoclonal antibodies have shown critical drawbacks in this context compared to adoptive cell therapies such as CAR-T cells, the main being the absence of long-term immune memory establishment.

[0077] The present disclosure relates to a cell comprising a nucleic acid molecule encoding a CAR for use in the treatment of solid tumors, wherein the CAR comprises an antibody or antigen-binding fragment thereof which includes an anti-IL-lRAP binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain. Exemplary CARs are disclosed in PCT Publication Nos. WO 2019/101604 and WO 2020/239801, which are herein incorporated by reference in their entirety.

[0078] In one aspect, the anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 6, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 7 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 12, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 13 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence SEQ ID NO: 14.

[0079] CARs contemplated herein, may comprise one, two, three, four, or five or more linkers. In particular aspects, the length of a linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some aspects, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids long.

[0080] Illustrative examples of linkers include glycine polymers (G)n; glycine-serine polymers (Gi_sSi_5)n, where n is an integer of at least one, two, three, four, or five; glycinealanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between domains of fusion proteins such as the CARs described herein. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains {see Scheraga, Rev. Computational Chem. 1 1173-142 (1992)). In one aspect, the design of a CAR can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired CAR structure.

[0081] In a particular aspect, the linker is between the VH and VL domains. [0082] In a particular aspect, the linker comprises or consists in the amino acid sequence of SEQ ID NO: 5.

[0083] In one aspect, the IL-1RAP binding domain is a scFv comprising a light chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a light chain variable regions of SEQ ID NO: 4 and a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications of an amino acid sequence of a heavy chain variable region of SEQ ID NO: 2.

[0084] In one aspect, the IL-1 RAP binding domain is a scFv comprising (i) a light chain variable region comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 6, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 7 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain variable region comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 12, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 13 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 14.

[0085] The binding domain of the CAR is generally followed by one or more "hinge regions", which play a role in positioning the antigen binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding and activation. A CAR generally comprises one or more hinge regions between the binding domain and the transmembrane domain. The hinge region may be derived either from a natural, synthetic, semi -synthetic, or recombinant source.

[0086] In one aspect, the anti-IL-lRAP binding domain is connected to the transmembrane domain by a hinge region.

[0087] In one aspect, the hinge region comprises the hinge sequence of IgGl or a sequence with 95-99% identity thereof. IgG hinges are encoded by single exons. Thus, the term "hinge sequence of IgGl" as used herein has the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs, i .e. the 15 amino acid residues encoded by the IgGl exon for the hinge (Fundamental Immunology, Fifth edition, Chapter 3, Immunoglobulins: Structure and Function - The immunoglobulin Flinge).

[0088] In further aspects, the hinge region comprises the hinge sequence of IgG4 or a sequence with 95-99% identity thereof. In further aspects, the hinge region may also comprise the CH2-CH3 region of IgGl or IgG4 or a sequence with 95-99% identity thereof.

[0089] In further aspects, the hinge region comprises CD8alpha or a sequence with 95- 99% identity thereof.

[0090] The "transmembrane domain" is the portion of the CAR that fuses the extracellular binding portion and intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain can be derived either from a natural, synthetic, semi-synthetic, or recombinant source.

[0091] In one aspect, the encoded CAR includes a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154, more preferably CD28.

[0092] In certain aspects, the CARs contemplated herein comprise an intracellular signaling domain. An "intracellular signaling domain," refers to the part of a CAR that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain.

[0093] The term "effector function" refers to a specialized function of the cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term "intracellular signaling domain" is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal. [0094] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g. a TCR/CD3 complex) and co-stimulatory signaling domains that act in an antigenindependent manner to provide a secondary or co- stimulatory signal. In some aspects, a CAR contemplated herein comprises an intracellular signaling domain that comprises one or more "costimulatory signaling domain".

[0095] In one aspect, the isolated nucleic acid molecule may encode an intracellular signaling domain comprising at least one costimulatory domain. In this aspect, the intracellular signaling domain therefore comprises at least one costimulatory domain.

[0096] As used herein, the term "co-stimulatory signaling domain," or "co stimulatory domain", refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen.

[0097] Preferably, the at least one costimulatory domain of the functional intracellular signaling domain is obtained from one or more protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, CD3 zeta, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137).

[0098] In one aspect, the costimulatory domain obtained from 4-1BB (CD137) has a sequence having 95-99% identity with the amino acid sequence of the costimulatory domain of 4-1BB. In one aspect, the costimulatory domain obtained from CD3 zeta has a sequence having 95-99% identity with the amino acid sequence of the costimulatory domain of CD3 zeta. In another aspect, the intracellular signaling domain comprises a costimulatory domain obtained from 4- IBB and/or a costimulatory domain obtained from CD3 zeta.

[0099] In particular aspects, a CAR comprises a CD3z primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling and co-stimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.

[0100] In one aspect, the CAR comprises the amino acid sequence of SEQ ID NO: 19.

[0101] A cell that can be used according to the invention may be isolated. An "isolated cell" refers to a cell that has been obtained from an in vivo tissue or organ and is substantially free of extracellular matrix. The cell that is used for treating a solid tumor according to the invention can be prepared by inserting; within the genome of a host cell, the nucleic acid molecule encoding the chimeric antigen receptor (CAR) using a vector.

[0102] The term "vector" is used herein to refer to a nucleic acid molecule capable transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA.

[0103] The cell that is used for treating a solid tumor according to the invention may be prepared using a vector comprising a nucleic acid molecule encoding the CAR, said vector is selected from a DNA, a RNA, a plasmid, a lentivirus vector, an adenoviral vector, or a retrovirus vector, preferably a lentivirus vector. In some aspects, the vector comprises a promoter, preferably an EF-1 alpha promoter.

[0104] Retroviruses are a common tool for gene delivery. In particular aspects, a retrovirus is used to deliver a polynucleotide encoding a chimeric antigen receptor (CAR) to a cell. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus." The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.

[0105] Thus, the T cells transduced with the vector can elicit a stable, long-term, and persistent CAR-mediated T-cell response. In particular aspects, the T cell is transduced with a retroviral vector, e.g., a lentiviral vector, encoding the CAR.

[0106] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0107] The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus. "Self-inactivating" (SIN) vectors refers to replication-defective vectors, e.g., retroviral or lentiviral vectors, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. In one aspect, SIN vector backbones are preferred. In one aspect, the vector used further comprises a promoter, e.g. an EF-1 alpha promoter.

[0108] The term "promoter" as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an R A polymerase binds. An R A polymerase initiates and transcribes polynucleotides operably linked to the promoter. In a particular aspect, it may be desirable to express a polynucleotide comprising a CAR from a promoter that provides stable and long-term CAR expression in T cells and at sufficient levels to redirect the T cells to cells expressing the target antigen.

[0109] The cell for use according to the disclosure can be a T cell, e.g., human T cell. In one aspect, the cell is a CD8+ T cell, e.g., human CD8+ T cell. In one aspect, the cell for use according to the disclosure (e.g. T cell) expresses the CAR (e.g., SEQ ID NO: 19) at its membrane. The term "at its membrane" as used herein has the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs, i .e. "at the cell surface membrane".

[0110] In particular aspects, prior to in vitro manipulation or genetic modification of the immune effector cells described herein, the source of cells is obtained from a subject. In particular aspects, the cells for use according to the disclosure encompass T cells. T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g ., FICOLL™ separation. Cells from the circulating blood of an individual may be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocyte, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing.

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

[0112] In some aspects of the invention, the cell harboring the nucleic acid molecule encoding a CAR utilizes a suicide gene, including an inducible suicide gene to reduce the risk of direct toxicity and/or uncontrolled proliferation of gene modified cells. In specific aspects, the suicide gene is not immunogenic to the host harboring the polynucleotide or cell. A certain example of a suicide gene that may be used is inducible caspase-9 (iCASP9), thymidine kinase of Herpes simplex (HSV-tk), CD20, truncated EGFR, caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID). Others systems may be activated by metabolizing prodrugs (Ganciclovir), or by binding antibodies (Rituximab, Cituximab)

[0113] Thus, disclosed herein is cellular therapy where T cells are genetically modified ex vivo to express a CAR and the CAR-T cell is infused to a recipient in need thereof. The infused cell is able to inhibit the growth of solid tumor cells in the recipient, preferably a human. Unlike antibody therapies, CAR-T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

[0114] Moreover, CARs allow for the redirection and activation of effector T cells towards any cell surface molecule upon binding by the antibody derived receptor, and are independent of MHC restriction.

[0115] The genetically-modified cells, e.g. T cells, for use according to the disclosure can be constructed starting from the cells of the patient (autologous), but they can also originate from other allogenic donors to provide allogenic genetically-modified cells in bone marrow or peripheral hematopoietic stem cell allograft context (Donor lymphocytes infusion). These cells expressing the IL-1RAP CAR molecule are useful to treat cancer in a mammal, preferably a human.

[0116] These cells, e.g. T cells, express a CAR molecule comprising an antigen binding domain that is an anti-IL-lRAP scFv comprising an anti-IL-lRAP binding domain, a transmembrane domain of the CD28 protein, a costimulatory 4- IBB signaling domain, and a CD3 zeta signaling domain, wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 6, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 7 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a complementary determining region 1 (CDR1) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 12, a complementary determining region 2 (CDR2) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 13 and a complementary determining region 3 (CDR3) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or having 100% identity with the amino acid sequence SEQ ID NO: 14. In one aspect, the CAR molecule comprises the amino acid sequence of SEQ ID NO: 19.

[0117] As used herein "treatment" or "treating," includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g ., cancer. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0118] Thus, the present disclosure provides a method for the treatment of cancer comprising administering to a subject in need thereof, a therapeutically effective amount of a cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody or antibody fragment which includes an anti-IL-lRAP binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) having at least 80% identity with the amino acid sequence SEQ ID NO: 6, a complementary determining region 2 (CDR2) having at least 80% identity with the amino acid sequence SEQ ID NO: 7 and a complementary determining region 3 (CDR3) having at least 80% identity with the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a complementary determining region 1 (CDR1) having at least 80% identity with the amino acid sequence SEQ ID NO: 12, a complementary determining region 2 (CDR2) having at least 80% identity with the amino acid sequence SEQ ID NO: 13 and a complementary determining region 3 (CDR3) having at least 80% identity with the amino acid sequence SEQ ID NO: 14. In one aspect, the CAR molecule comprises the amino acid sequence of SEQ ID NO: 19. [0119] The cells, e.g. T cell, may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0120] In one aspect, the CAR-modified cells or the compositions are administered to a subject by direct injection into a tumor. In one aspect, the CAR-modified cell, e.g. CAR- modified T cell, are useful to treat a subject diagnosed with cancer, by removing immune effector cells from the subject, genetically modifying said immune effector cells with a vector comprising a nucleic acid encoding a CAR as described herein, thereby producing a population of modified immune effector cells, and administering the population of modified immune effector cells to the same subject. In one aspect, the immune effector cells comprise T cells.

[0121] A "therapeutically effective amount" of a genetically modified therapeutic cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The quantity, frequency of administration and the sequence of the possible association with conventional cancer treatments will be determined by such factors as the condition of the patient, and the type and severity of the cancer.

Combination therapy - chemotherapy

[0122] In one aspect, the T cell expressing the CAR molecule specific of IL-1RAP may be used in a method to inhibit growth of a solid tumor in a subject. In one aspect, the subject has already been treated by at least one therapy line, such as chemotherapy. In another aspect, the subject is treated with a combination of the T cell and chemotherapy, either simultaneously or sequentially, in any order.

[0123] In one aspect, the cell expressing the CAR molecule specific of IL-1RAP may be used in a method to treat cancer in a subject. In one aspect, the subject has already been treated by at least one therapy line, such as chemotherapy. In another aspect, the subject is treated with a combination of the T cell and chemotherapy, either simultaneously or sequentially, in any order.

[0124] A wide variety of chemotherapeutic agents may be used in accordance with the present aspects. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0125] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5 -fluorouracil (5- FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0126] In one aspect, the present disclosure is directed to identifying a patient as having increased IL-1RAP protein expression and treating the subject by administering a CAR-T cell of the disclosure. In another aspect, the patient is treated with a combination of a CAR-T cell of the disclosure and chemotherapy. Combination therapy - immune checkpoint inhibitors

[0127] In the tumor microenvironment, cancer cells can evade the immunosurveillance by changing their surface antigens, thus avoiding the detection and destruction by host lymphocytes. A central mechanism of tumor-induced immune suppression is the increased expression of ligands able to bind inhibitory T cell receptors. These ligands are known as T-cell or immune checkpoints and act in physiological conditions to prevent the development of autoimmunity at multiple steps during the immunological response. The main mechanisms involved in the T cell modulation are the suppression of potential autoreactive naive T cell (characterized by a TCR directed against self-antigens) at initial stages in lymph nodes, or in later phases the T cell deactivation in peripheral tissues. This process is called peripheral tolerance and is exerted mainly by the immune checkpoints cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) pathways. Tumor cells have developed ways to take advantage of peripheral tolerance by inducing a deranged immune checkpoint expression by T cell in order to avoid immune recognition.

[0128] In another aspect, the present disclosure is directed to a method for inhibiting solid tumor growth in a subject comprising administering a combination therapy of a CAR-T cell of the disclosure and an immune checkpoint inhibitor. In one aspect, the immune checkpoint inhibitor is a monoclonal antibody. In another aspect, the monoclonal antibody binds PD-1, PD- Ll, or CTL4. In another aspct, the anti-PD-1, anti-PD-Ll or anti-CTLA4 antibodies are nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab or tremelimumab.

[0129] In some aspects, the immune checkpoint inhibitor is a PD-1 pathway inhibitor, for example an anti-PD-1 antibody or a PD-L1 antibody. In some aspects, the PD-1 pathway inhibitor is a PD-L2-binding agent, for example an anti-PD-L2 antibody. In further aspects, the PD-Ll-binding agent is a soluble PD-1 polypeptide, for example, a PD-l-Fc fusion polypeptide capable of binding to PD-L1. In further aspects, the PD-L2 -binding agent is a soluble PD-1 polypeptide, for example, a PD-l-Fc fusion polypeptide capable of binding to PD-L2.

[0130] Anti-human-PD-1 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the disclosure can be generated using methods well known in the art. Alternatively, art recognized anti-PD-1 antibodies can be used. In other aspects, the anti-PD-1 antibody is Nivolumab or BMS-936558 described in WO 2006/121168. Other known PD-1 antibodies include lambrolizumab (MK-3475) described in WO 2008/156712. Further known PD-1 antibodies and other PD-1 inhibitors include those described in, for example, WO 2009/014708, WO 03/099196, WO 2009/114335 and WO 2011/161699, which are herein incorporated by reference. In one aspect, the anti-PD-1 antibody is REGN2810. In one aspect, the anti-PD-1 antibody is PDR001. Another known anti-PD-1 antibody is pidilizumab (CT-011).

[0131] In some aspects, the anti-PD-1 antibody or fragment thereof cross-competes with pembrolizumab. In some aspects, the anti-PD-1 antibody or fragment thereof binds to the same epitope as pembrolizumab. In certain aspects, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In another aspect, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "KEYTRUDA®", lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody directed against human cell surface receptor PD-1 (programmed death- 1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0132] In certain aspects, the first antibody is an anti-PD-1 antagonist. One example of the anti-PD-1 antagonist is AMP-224, which is a B7-DC Fc fusion protein. AMP-224 is discussed in U.S. Publ. No. 2013/0017199.

[0133] In other aspects, the anti-PD-1 antibody or fragment thereof cross-competes with BGB-A317. In some aspects, the anti-PD-1 antibody or fragment thereof binds the same epitope as BGB-A317. In certain aspects, the anti-PD-1 antibody has the same CDRs as BGB-A317. In certain aspects, the anti-PD-1 antibody is BGB-A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S. Publ. No. 2015/0079109.

[0134] In some aspects, the antibody is pidilizumab (CT-011), which is an antibody previously reported to bind to PD-1 but which is believed to bind to a different target, pidilizumab is described in US Pat. No. 8,686,119 B2 or WO 2013/014668 Al.

[0135] In certain aspects, the antibodies that cross-compete for binding to human PD-1 with, or bind to the same epitope region of human PD-1 as, nivolumab are mAbs. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, or humanized or human antibodies. Such chimeric, humanized or human mAbs can be prepared and isolated by methods well known in the art.

[0136] Other anti-PD-1 monoclonal antibodies have been described in, for example, U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757 and 8,354,509, US Publication No. 2016/0272708, and PCT Publication Nos. WO 2012/145493, WO 2008/156712, WO 2015/112900, WO 2012/145493, WO 2015/112800, WO 2014/206107, WO 2015/35606, WO 2015/085847, WO 2014/179664, WO 2017/020291, WO 2017/020858, WO 2016/197367, WO 2017/024515, WO 2017/025051, WO 2017/123557, WO 2016/106159, WO 2014/194302, WO 2017/040790, WO 2017/133540, WO 2017/132827, WO 2017/024465, WO 2017/025016, WO 2017/106061, WO 2017/19846, WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540 each of which is incorporated by reference in its entirety.

[0137] Anti-PD-1 antibodies useful for the compositions of the disclosure also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the Vz, Vzz, Cz and Czzz domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the Vzz and Czzz domains; and (iv) a Fv fragment consisting of the Vz and Vzz domains of a single arm of an antibody.

[0138] Anti-PD-1 antibodies usable in the disclosed methods also include isolated antibodies that bind specifically to human PD-1 and cross-compete for binding to human PD-1 with any anti-PD-1 antibody disclosed herein. In some aspects, the anti -PD-1 antibody binds the same epitope as any of the anti-PD-1 antibodies described herein. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody.

[0139] In certain aspects, the present application encompasses use of an anti-PD-Ll antibody as the T-cell checkpoint inhibitor. In one aspect, the anti-PD-Ll antibody inhibits the binding of PD-L1 receptor, i.e., PD-1 to its ligand PD-L1.

[0140] Anti-human-PD-Ll antibodies (or VH and/or VL domains derived therefrom) suitable for use in the methods of the disclosure can be generated using methods well known in the art. In certain aspects, the anti-PD-Ll antibody or antigen-binding fragment thereof is an antibody described in PCT Publication No. WO 2011/066389. In some aspects, the anti-PD-Ll antibody comprises the HC CDR1-3 and LC CDR1-3 of SEQ ID NOs: l-3 and 4-6, respectively. In some aspects, the anti-PD-Ll antibody comprises the VH and VL of SEQ ID NOs:7 and 8, respectively.

[0141] Alternatively, art recognized anti-PD-Ll antibodies can be used. For example, anti-PD-Ll antibodies useful in the claimed methods are disclosed in U.S. Pat. No. 7,943,743. Such anti-PD-Ll antibodies include 12A4 (also referred to as BMS-936559). In some aspects, the anti-PD-Ll antibody is atezolizumab (Tecentriq or RG7446) (see, e.g., Herbst et al. (2013) J Clin Oncol 31(suppl):3000. Abstract; U.S. Patent No. 8,217,149), or avelumab (Bavencio). Other art recognized anti-PD-Ll antibodies which can be used include those described in, for example, U.S. Pat. Nos. 7,635,757 and 8,217,149, U.S. Publication No. 2009/0317368, and PCT Publication Nos. WO 2011/066389 and WO 2012/145493, which are herein incorporated by reference. Antibodies that compete with any of these art-recognized antibodies or inhibitors for binding to PD-L1 also can be used.

[0142] In certain aspects, antibodies that cross-compete for binding to human PD-L1 with, or bind to the same epitope region of human PD-L1 as the above-references PD-L1 antibodies are mAbs. For administration to human subjects, these cross-competing antibodies can be chimeric antibodies, or can be humanized or human antibodies. Such chimeric, humanized or human mAbs can be prepared and isolated by methods well known in the art.

[0143] In certain aspects, the PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgGl kappa monoclonal anti-PD-Ll antibody.

[0144] In certain aspects, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgGl monoclonal anti-PD-Ll antibody.

[0145] In certain aspects, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgGl lambda monoclonal anti-PD-Ll antibody.

[0146] Anti-PD-Ll antibodies usable in the disclosed methods also include isolated antibodies that bind specifically to human PD-L1 and cross-compete for binding to human PD- L1 with any anti-PD-Ll antibody disclosed herein, e.g., durvalumab, atezolizumab, and/or avelumab. In some aspects, the anti-PD-Ll antibody binds the same epitope as any of the anti- PD-Ll antibodies described herein, e.g., durvalumab, atezolizumab, and/or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody by virtue of their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and/or avelumab in standard PD-L1 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).

[0147] In certain aspects, the antibodies that cross-compete for binding to human PD-L1 with, or bind to the same epitope region of human PD-L1 antibody as, durvalumab, atezolizumab, and/or avelumab, are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies. Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

Anti-PD-Ll antibodies usable in the methods of the disclosed disclosure also include antigenbinding portions of the above antibodies. It has been amply demonstrated that the antigenbinding function of an antibody can be performed by fragments of a full-length antibody.

[0148] Anti-PD-Ll antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to PD-L1 with high specificity and affinity, block the binding of PD-1, and inhibit the immunosuppressive effect of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-Ll "antibody" includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits the functional properties similar to those of whole antibodies in inhibiting receptor binding and up-regulating the immune system. In certain aspects, the anti-PD-Ll antibody or antigen-binding portion thereof cross-competes with durvalumab, atezolizumab, and/or avelumab for binding to human PD-L1.

[0149] In certain aspects, the present application encompasses use of an anti-CTLA-4 antibody. In one aspect, the anti-CTLA-4 antibody binds to and inhibits CTLA-4. In some aspects, the anti-CTLA-4 antibody is ipilimumab (YERVOY), tremelimumab (ticilimumab; CP- 675,206), AGEN-1884, or ATOR-1015.

[0150] In one aspect, the CTLA-4 antagonist is a soluble CTLA-4 polypeptide. In one aspect, the soluble CTLA-4 polypeptide is abatacept (Orencia), belatacept (Nulojix), RG2077, or RG-1046. In another aspect, the CTLA-4 antagonist is a cell based therapy. In some aspects, the CTLA-4 antagonist is an anti-CTLA-4 mAb RNA/GITRL RNA-transfected autologous dendritic cell vaccine or an anti-CTLA-4 mAb RNA-transfected autologous dendritic cell vaccine.

[0151] In one aspect, the disclosure includes a method of selecting a solid tumor in a human patient for therapy, comprising: (a) determining the level of IL-1RAP expression in a tumor sample; and (b) selecting the tumor for immunotherapy if the tumor sample displays increased IL-1RAP protein expression. In one aspect, the disclosure includes a method of identifying a tumor in a human patient that is likely to be responsive to an immunotherapy, the method comprising: (a) determining the level of IL-1RAP expression in a tumor sample; and (b) identifying the tumor as likely to be responsive to treatment if the tumor displays increased protein expression.

[0152] In still other aspects, the disclosure includes a method for reducing a solid tumor size at least by 10% in a human patient afflicted with a tumor comprising administering to the patient a therapy or combination therapy disclosed herein (e.g., an IL-1RAP CAR-T cell, an IL- 1RAP CAR-T cell in combination with chemotherapy, or an IL-1RAP CAR-T cell in combination with an immune checkpoint inhibitor). In some aspects, the patient has been identified as having increased IL-1RAP expression prior to the administration and wherein the administration reduces the tumor size at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% compared to the tumor size prior to the administration.

[0153] The disclosure can also include a method of preventing a relapse and/or inducing remission in a patient comprising administering to the patient a therapy or combination therapy disclosed herein (e.g., an IL-1RAP CAR-T cell, an IL-1RAP CAR-T cell in combination with chemotherapy, or an IL-1RAP CAR-T cell in combination with an immune checkpoint inhibitor). In some aspects, the method of the disclosure comprises (i) identifying a patient as having increased IL-1RAP expression; and (ii) administering to the patient a therapy or combination therapy disclosed herein.

[0154] The disclosure can also be used to target a therapy to the tumor microenvironment in a patient comprising administering to the patient a therapy or combination therapy disclosed herein (e.g., an IL-1RAP CAR-T cell, an IL-1RAP CAR-T cell in combination with chemotherapy, or an IL-1RAP CAR-T cell in combination with an immune checkpoint inhibitor). In some aspects, the method of the disclosure comprises (i) identifying a patient as having increased IL-1RAP expression; and (ii) administering to the patient a therapy or combination therapy disclosed herein. In one aspect, the tumor microenvirmonment is targeted because CAF cells express high levels of IL-1RAP.

[0155] The methods of the disclosure, as a result of the administration of a therapy or combination therapy disclosed herein, can treat the malignant tumor, reduce the tumor size, prevent growth of the tumor, eliminate the tumor from the patient, prevent a relapse of a tumor, induce a remission in a patient, or any combination thereof. In certain aspects, the administration of a therapy or combination therapy disclosed herein induces a complete response. In other aspects, the administration of the therapy or combination therapy disclosed herein induces a partial response. In some aspects, the therapy or combination therapy comprises administering a therapeutically effective amount of an IL-1RAP CAR-T cell, an IL-1RAP CAR-T cell in combination with chemotherapy, or an IL-1RAP CAR-T cell in combination with an immune checkpoint inhibitor.

[0156] In some aspects, IL-1RAP expression is determined by receiving the results of an assay capable of determining IL-1RAP expression. 3. Measurement of IL-1RAP expression

[0157] In order to assess the IL-1RAP expression, in one aspect, a test sample is obtained from the patient who is in need of the therapy. In some aspects, a test sample includes, but is not limited to, any clinically relevant sample, such as a tumor biopsy, a bone marrow biopsy, a bone marrow aspiration, a core biopsy tissue sample, a fine needle aspirate, or a sample of bodily fluid, such as blood, plasma, serum, lymph, ascites fluid, cystic fluid, or urine. In some aspects, the test tissue sample is from a primary tumor. In some aspects, the test sample is from a metastasis. In some aspects, test samples are taken from a subject at multiple time points, for example, before treatment, during treatment, and/or after treatment. In some aspects, test samples are taken from different locations in the subject, for example, a sample from a primary tumor and a sample from a metastasis in a distant location.

[0158] In some aspects, the test tissue sample is a paraffin-embedded fixed tissue sample. In some aspects, the test tissue sample is a formalin-fixed paraffin embedded (FFPE) tissue sample. In some aspects, the test tissue sample is a fresh tissue (e.g., tumor) sample. In some aspects, the test tissue sample is a frozen tissue sample. In some aspects, the test tissue sample is a fresh frozen (FF) tissue (e.g., tumor) sample. In some aspects, the test tissue sample is a cell isolated from a fluid. In some aspects, the test tissue sample comprises circulating tumor cells (CTCs). In some aspects, the test tissue sample comprises circulating lymphocytes. In some aspects, the test tissue sample is an archival tissue sample. In some aspects, the test tissue sample is an archival tissue sample with known diagnosis, treatment, and/or outcome history. In some aspects, the sample is a block of tissue. In some aspects, the test tissue sample is dispersed cells. In some aspects, the sample size is from about 1 cell to about 1 x 10⁶ cells or more. In some aspects, the sample size is about 1 cell to about 1 x 10⁵ cells. In some aspects, the sample size is about 1 cell to about 10,000 cells. In some aspects, the sample size is about 1 cell to about 1,000 cells. In some aspects, the sample size is about 1 cells to about 100 cells. In some aspects, the sample size is about 1 cell to about 10 cells. In some aspects, the sample size is a single cell.

[0159] In another aspect, the assessment of IL-1RAP expression can be achieved without obtaining a test tissue sample. In some aspects, selecting a suitable patient includes (i) optionally providing a test tissue sample obtained from a patient with cancer of the tissue, the test tissue sample comprising tumor cells; and (ii) assessing the proportion of cells in the test tissue sample that express IL-1RAP on the surface of the cells based on an assessment that the proportion of cells in the test tissue sample that express IL-1RAP on the cell surface is higher than a predetermined threshold level. [0160] In any of the methods comprising the measurement of IL-1RAP expression in a test sample, however, it should be understood that the step comprising the provision of a test sample obtained from a patient is an optional step. That is, in certain aspects the method includes this step, and in other aspects, this step is not included in the method. It should also be understood that in certain aspects the "measuring" or "assessing" step to identify, or determine the number or proportion of, cells in the test sample that express IL-1RAP is performed by a transformative method of assaying for IL-1RAP expression, for example by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay, IHC, flow cytometry (FC), imaging mass cytometry (IMC), or a mass spectroscopy imaging (MSI) assay. In certain other aspects, no transformative step is involved and IL-1RAP expression is assessed by, for example, reviewing a report of test results from a laboratory. In some aspects, IL-1RAP expression is assessed by reviewing the results of, for example, an immunohistochemistry assay from a laboratory. In certain aspects, the steps that provide the test result is performed by a medical practitioner or someone acting under the direction of a medical practitioner. In other aspects, these steps are performed by an independent laboratory or by an independent person such as a laboratory technician.

[0161] In certain aspects of any of the present methods, the proportion of cells that express IL-1RAP is assessed by performing an assay to detect the presence of IL-1RAP RNA. In further aspects, the presence of IL-1RAP RNA is detected by RT-PCR, in situ hybridization or RNase protection. In some aspects, the presence of IL-1RAP RNA is detected by an RT-PCR based assay. In some aspects, scoring the RT-PCR based assay comprises assessing the level of IL-1RAP RNA expression in the test tissue sample relative to a predetermined level.

[0162] In other aspects, the proportion of cells that express IL-1RAP is assessed by performing an assay to detect the presence of IL-1RAP polypeptide. In further aspects, the presence of IL-1RAP polypeptide is detected by IHC, enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In some aspects, IL-1RAP expression is assayed by IHC, imaging mass cytometry (IMC), or mass spectroscopy imaging (MSI). In other aspects of all of these methods, cell surface expression of IL-1RAP is assayed using, e.g., IHC or in vivo imaging.

4. Patient Populations

[0163] Provided herein are clinical methods for treating tumors in subjects, e.g., human patients, using a therapy disclosed herein, for example, an IL-1RAP CAR-T cell, an IL-1RAP CAR-T cell in combination with chemotherapy, or an IL-1RAP CAR-T cell in combination with an immune checkpoint inhibitor.

[0164] Examples of cancers and/or malignant tumors that may be treated using the methods of the disclosure, include liver cancer, hepatocellular carcinoma (HCC), bone cancer, pancreatic cancer, skin cancer, oral cancer, cancer of the head or neck, breast cancer, lung cancer, small cell lung cancer, non small cell lung cancer (NSCLC), cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, squamous cell carcinoma of the head and neck (SCCHN), nonHodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, and any combinations of said cancers. The present disclosure is also applicable to treatment of metastatic cancers. In certain aspects, the cancer is NSCLC, pancreatic carcinoma, breast carcinoma, colorectal carcinoma, liver carcinoma, esophageal carcinoma, head and neck carcinoma, or melanoma.

Exemplary Aspects Provided Herein

[0165] In one aspect (Aspect 1; Al), provided herein is a method of inhibiting growth of a solid tumor in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

[0166] In one aspect, i.e., A2, provided herein is a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

[0167] In one aspect, i.e., A3, provided herein is a method of targeting a tumor microenvironment for treatment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

[0168] In one aspect of any one of A1-A3, i.e., A4, the CAR comprises the amino acid sequence of SEQ ID NO: 19.

[0169] In one aspect of any one of A1-A4, i.e., A5, the cell is a T cell.

[0170] In one aspect of any one of A4 or A5, i.e., A6, the cell is an autologous cell.

[0171] In one aspect of any one of A1-A6, i.e., A7, the subject has increased IL-1RAP expression compared to a normal subject. [0172] In one aspect of any one of A1-A7, i.e., A8, the method further comprises administering a therapeutically effective amount of one or more chemotherapeutic agents and/or immune checkpoint inhibitors.

[0173] In one aspect of A8, i.e., A9, the cell expressing the CAR, and the chemotherapeutic agent and/or immune checkpoint inhibitor are administered concurrently.

[0174] In one aspect of A8 or A9, i.e., A10, the cell expressing the CAR, and the chemotherapeutic agent and/or immune checkpoint inhibitor are administered sequentially.

[0175] In one aspect of any one of A8-A10, i.e., Al l, the cell expressing the CAR is administered prior to administration of the chemotherapeutic agent and/or immune checkpoint inhibitor.

[0176] In one aspect of any one of A8-A11, i.e., A12, the chemotherapy is docetaxel, 5- fluorouracil, and/or oxaliplatin.

[0177] In one aspect of any one of A8-A12, i.e., A13, the immune checkpoint inhibitor is an antibody that binds PD-1, PD-L1, or CTLA-4.

[0178] In one aspect of A13, i.e., A14, the antibody is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab or tremelimumab.

[0179] In one aspect of any one of A7-A14, i.e., A15, IL-1RAP expression levels are determined by immunohistochemistry (IHC) or quantitave polymerase chain reaction (qPCR).

[0180] In one aspect of any one of A1-A15, i.e., A16, the tumor or cancer is a solid tumor or a cancer resulting from a solid tumor growth.

[0181] In one aspect of A16, i.e., A17, the solid tumor is a non-small cell lung carcinoma (NSCLC), pancreatic carcinoma, breast carcinoma, colorectal carcinoma, liver carcinoma, esophageal carcinoma, head and neck carcinoma, or melanoma.

[0182] In one aspect of any one of A1-A17, i.e., A18, the subject is a human.

[0183] In one aspect, i.e., A19, the cell of any one of A1-A7 for use in inhibiting the growth of a solid tumor in a subject in need thereof.

EXAMPLES

Example 1 : IL-1RAP is overexpressed in solid tumors

[0184] IL-1RAP mRNA expression was analyzed by RT-qPCR on 33 tumor cell lines as shown in Table 1. [0185] Table 1 : IL-RAP mRNA expression in tumor cell lines

[0186] RNA was extracted and reverse transcribed into cDNA. RT-qPCR was performed on the target (IL-1RAP) and GAPDH as a housekeeping gene. The K562 cell line was used as a reference. RT-qPCR showed that among the 33 cell lines, 26 cell lines overexpressed IL-1RAP mRNA (Figure 2A).

[0187] Second, flow cytometry was used to show that IL-1RAP is overexpressed in several cell lines derived from breast carcinoma, lung carcinoma, pancreatic carcinoma and hepatocellular carcinoma. Three cell lines for liver, lung and breast carcinomas and two cell lines for pancreatic carcinoma were cultured in RPMI/DMEM, 10% FBS, and 1% PS. Cell lines were trypsinized, and 100,000 cells were labeled with 5 pL of BR58 anti-IL-lRAP antibody #857.113.020 (Diaclone) and incubated for 10 minutes at room temperature. Based on these results, IL-1RAP appears to be highly expressed in almost all cell lines. As shown in Figure 2B and 2C, HEPG2, BXPC3 and NCIH460 cells were selected for further investigation.

[0188] Finally, RNAscope™ 2.5 LSxRed ISH assays were performed to evaluate the expression of human IL 1 -RAP in one ACD prequalified human multinormal TMA, one ACD prequalified human multitumor TMA, and one ACD prequalified human HCC liver sample. RNAscope™ quantification is based on the number of dots per cell and not on intensity. The dots correlate to the number of individual RNA molecules, whereas dot intensity reflects the number of probe pairs bound to each molecule. The percentage of positive cells was scored visually based on the number of cells with >1 dot/cell and binned into categories (i.e., 0%, 1-25%, 26- 50%, 51-75%, 76-99%, 100%) (Figure 2D).

[0189] Overall, the results showed that IL-1RAP mRNA is absent or expressed at very low levels in healthy tissues. However, IL-1RAP is highly expressed in most solid tumors, such as liver hepatocarcinoma, melanomas, and ovarian cancers. Moreover, tumor tissues significantly overexpressed IL-1RAP compared to healthy tissues, especially melanoma versus skin, ovarian, and liver tissues.

Example 2: IL-1RAP CAR-T cells are stimulated against solid tumor cell lines

[0190] Functional validation of IL-1RAP CAR-T cells, whether CAR-T cells are able to secrete high levels of CD 107a, interferon gamma and TNFa was then performed.

[0191] For analysis of CD107a degranulation, 100,000 anti-IL-lRAP CAR-T cells were co-cultured with 500,000 target tumor cells (effector target ratio = 1 :5) in 96-well flat-bottom plates (150 pL per well) for 1 hour at 37°C. Fifty microliters of GolgiSTOP mix was added to each well, and the plates were incubated for 4 hours at 37°C. At the end of the incubation, all cells were transferred to FCM tubes and labeled with 1 pL of anti-CD19 antibody #130-113-165 (Miltenyi Biotec clone LT19), 1 pL of anti-CD3 antibody #130-113-129 (Miltenyi Biotec clone BW264/56) and 5 pL of anti-CD8 antibody #854.961.010 (Diaclone clone B-Z31) (Figure 3).

[0192] For the IFN-y and TNFa assays, the same cell mix and ratios were used, except that the co-culture lasted 24 hours before supernatant harvest, 1/200 dilution was used and Human TNF-a ELISA Set #851.570.010 (Diaclone) and Human IFN-y ELISA Set #851.560.010 (Diaclone) were used as shown in Figure 3.

[0193] The results showed that the anti-IL-lRAP CAR-T cells could secrete high levels of CD 107a and IFN-y with a significant difference between the untransduced (CO) T cells and the CAR-T cells, indicating high anti-IL-lRAP specificity and negligible allogenic interference (Figure 4). These results showed that the CAR-T cells are strongly stimulated and activated when they encounter their target, IL- 1 RAP.

Example 3: IL-1RAP CAR-T cells are cytotoxic against solid tumor cell lines in 2D and 3D models

[0194] Anti-IL-lRAP CAR-T cell cytotoxicity against IL-1RAP positive target cells was also analyzed. To assess cytotoxicity, 5: 1 ratio co-cultures were performed. In two dimensions first, 30,000 GFP+ target cells were cultered in 24-well flat bottom plates for 6 hours at 37°C, 150,000 CAR-T cells were added to each well, and the plate was incubated in IncuCyte® for live cell imaging and analysis for 72 hours.

[0195] Cytotoxicity was also assessed in three-dimensional models. GFP+ target cells (5000) were cultured for five days at 37 °C in 96-well ultralow attachment plates to generate spheroids. Then, 250,000 CAR-T cells were added to each well, and the plate was incubated in IncuCyte® for live cell imaging and analysis for 72 h. (Figure 5).

[0196] During all the co-cultures, one picture was taken each hour for each well, and the total green object integrated intensity (CGU x pm2/image) was calculated. The total green object integrated intensity is defined as the total sum of the objects’ fluorescent intensity in the image. The results showed significant cytotoxicity of our CAR-T cells against all tumor cell types compared to cell lines cultured alone or compared to co-cultures with untransduced cells, highlighting the specificity of our CAR against IL-1RAP. Significant cytotoxicity was observed either in two-dimensional models or in three-dimensional (spheroid) models, which is very encouraging in the solid tumor context (Figures 6A and 6B).

[0197] CAR-T-cell cytotoxicity was confirmed by performing a CyQUANT LDH Cytotoxicity Assay Kit #C20300 (Thermo Fisher). The supernatant of previous co-cultures overnight was harvested, and LDH was quantified. Cytotoxicity was confirmed in two- dimensional and three-dimensional models using the CyQUANT LDH Cytotoxicity Assay. CAR-T cells showed significant cytotoxicity compared to untransduced cells (Figures 6C and 6D). Example 4: sIL-lRAP does not impair IL-1RAP CAR-T cell cytotoxicity in a 2D model

[0198] Given that IL-1RAP is a key factor in IL-1 intracellular signaling, which is a proinflammatory protein involved in tumor progression and tumor microenvironment establishment, whether soluble IL-1RAP would interfere with CAR-T cell cytotoxicity was analyzed. First, soluble IL-1RAP was quantified in healthy donor serum/plasma or patients with lung, breast, kidney, or colorectal carcinomas and melanomas (10 donors/patients for each cancer type) using the Human IL-1 RAcP/IL-1 R3 DuoSet® ELISA kit (Figure 7).

[0199] The results showed that patients with solid tumors had an overall significantly higher amount of sIL-lRAP, with levels two- to threefold higher than that of healthy donors (Figure 8A). Cytotoxicity assays were performed under the same conditions as previously described with the addition of sIL-lRAP at 0, 500, 1000 or 2000 ng/mL in co-culture. The results showed that CAR-T cell cytotoxicity was not impaired even with higher amounts of sIL-lRAP in co-culture (Figure 8B).

Example 5: IL-1RAP CAR-T cells are cytotoxic in vivo

[0200] Finally, the in vivo efficacy of the anti-IL-lRAP CAR-T cells was assessed in NSG mouse xenograft models. One million luciferase positive tumor cells were inoculated subcutaneously three days prior to the first CAR-T cell intravenous injection (ten million transduced T cells); a second CAR-T-cell injection was performed three days after the first injection. Tumor volumes and mouse survival were monitored, and at the end of the experiment, organs, tumors and whole blood were collected. Tumor volumes were measured with a digital caliper twice a week, and once a week, a bioluminescence measurement was performed on the IVIS® Lumina III In Vivo Imaging System (Figure 9).

[0201] For the NCI-H460 cell line (lung carcinoma model), the results showed an increase in lifespan when mice were treated with IL-1RAP CAR-T cells compared to controls. Indeed, the nontreated mice or the CO-treated mice showed a 20-day lifespan, while the IL-1RAP CAR-T cell-treated mice showed a lifespan of more than 33 days. Similar results have been observed with the pancreatic carcinoma BXPC3 cell line. The nontreated mice and the CO-treated mice showed a 30-day lifespan mean, while the CAR-T-cell-treated mice showed a 41 -day lifespan mean. Concerning HEPG2, the results have been promising; indeed, untreated mice and CO-treated mice showed a 30-day survival mean, while IL-1RAP CAR-T-cell-treated mice showed complete clearance of tumor cells at Day 29 (Figure 10A). [0202] These results were completed with tumor volume monitoring. Interestingly, the results showed significantly decreased mean tumor volumes from Day 10 over time for the IL- 1RAP CAR-T cell-treated mice previously injected with NCI-H460 and decreased tumor growth (between 250 mm³ at Day 10 and 500 mm³ at Day 18) compared to the controls (up to 1700 mm³ at Day 18). For BXPC3, even if IL-1RAP CAR-T-cell-treated mice showed better survival, tumor volumes did not decrease significantly over time (from 0 mm³ up to 400 mm³). The results obtained for HEPG2 were confirmed with tumor volumes that were significantly decreased to 0 mm³ from Days 10 to 22 for the mice treated with IL-1RAP CAR-T cells compared to the controls (Figure 10B). Together, these results show that IL-1RAP CAR T cells efficiently controlled lung and liver cell line growth in vivo and increased mouse lifespan in lung, pancreatic and liver carcinoma contexts.

Table 2: LISTING OF SEQUENCES

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting growth of a solid tumor in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigenbinding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

2. A method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

3. A method of targeting a tumor microenvironment for treatment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cell expressing a chimeric antigen receptor (CAR) at its surface, wherein the CAR comprises an antibody or antigen-binding fragment thereof that includes an anti-interleukin 1 receptor accessory protein (IL-1RAP) binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least a stimulatory domain, and wherein said anti-IL-lRAP binding domain comprises: (i) a light chain comprising a complementary determining region 1 (CDR1) comprising the amino acid sequence SEQ ID NO: 6, a CDR2 comprising the amino acid sequence SEQ ID NO: 7 and a CDR3 comprising the amino acid sequence SEQ ID NO: 8, and (ii) a heavy chain comprising a CDR1 the amino acid sequence SEQ ID NO: 12, a CDR2 comprising the amino acid sequence SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence SEQ ID NO: 14.

4. The method of any one of claims 1-3, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 19.

5. The method of any one of claims 1-4, wherein the cell is a T cell.

6. The method of claim 4 or 5, wherein the cell is an autologous cell.

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

8. The method of any one of claims 1-7, further comprising administering a therapeutically effective amount of one or more chemotherapeutic agents and/or immune checkpoint inhibitors.

9. The method of claim 8, wherein the cell expressing the CAR, and the chemotherapeutic agent and/or immune checkpoint inhibitor are administered concurrently.

10. The method of claim 8 or 9, wherein the cell expressing the CAR, and the chemotherapeutic agent and/or immune checkpoint inhibitor are administered sequentially.

11. The method of any one of claims 8-10, wherein the cell expressing the CAR is administered prior to administration of the chemotherapeutic agent and/or immune checkpoint inhibitor.

12. The method of any one of claims 8-11, wherein the chemotherapy is docetaxel, 5- fluorouracil, and/or oxaliplatin.

13. The method of any one of claims 8-12, wherein the immune checkpoint inhibitor is an antibody that binds PD-1, PD-L1, or CTLA-4.

14. The method of claim 13, wherein the antibody is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab or tremelimumab.

15. The method of any one of claims 7-14, wherein IL-1RAP expression levels are determined by immunohistochemistry (IHC) or quantitave polymerase chain reaction (qPCR).

16. The method of any one of claims 1-15, wherein the tumor or cancer is a solid tumor or a cancer resulting from a solid tumor growth.

17. The method of claim 16, wherein the solid tumor is a non-small cell lung carcinoma (NSCLC), pancreatic carcinoma, breast carcinoma, colorectal carcinoma, liver carcinoma, esophageal carcinoma, head and neck carcinoma, or melanoma.

18. The method of any one of claims 1-17, wherein the subject is a human.

19. Use of the cell of any one of claims 1-7 for inhibiting the growth of a solid tumor in a subject in need thereof.