Anti-il-1RAP binding domains and antibody-drug conjugates thereof

Abstract

The present disclosure is directed to antibody drug conjugates (ADCs) comprising an antibody or antigen binding fragment thereof that binds IL-1RAP. The ADCs of the present disclosure are useful for, among other things, treating diseases with upregulated IL-1RAP expression.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Application No. 63 / 610,670, filed December 15, 2023, U.S. Provisional Application No. 63 / 641,123, filed May 1, 2024, and U.S. Provisional Application No. 63 / 706,373, filed October 11, 2024, each of which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 5041_010PC03_SequenceListing_ST26.xml; Size: 95,406 bytes; and Date of Creation: December 1, 2024), filed with the application, is incorporated herein by reference in its entirety. FIELD OF DISCLOSURE

[0003] The present disclosure pertains to the medical field and cancer therapy, including antibodies and antibody drug conjugates for treating cancers with upregulated IL-1RAP expression. BACKGROUND

[0004] Cancer is one of the leading causes of death in the developed world, with over one million people diagnosed with cancer and 500,000 deaths per year in the United States alone. Overall, it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime.

[0005] The interleukin-1 (IL-1) family of cytokine ligands and receptors is associated with inflammation, autoimmunity, immune regulation, cell proliferation, and host defense and contributes to the pathology of inflammatory, autoimmune, immune regulatory, degenerative, and cell proliferative (e.g., cancer) diseases and disorders and its cytokines and receptors serve as pathogenic mediators of such diseases and disorders. See, e.g., Garlanda et al., Immunity, 39:1003-1018(2013).

[0006] The IL-1 family of cytokines includes interleukin-1 alpha, interleukin-1 beta, interleukin-33, interleukin-36 alpha, interleukin-36 beta and interleukin-36 gamma. Each of these cytokines serves as a ligand capable of binding a specific IL-1 family cell membrane receptor expressed on the surface of certain cells. Upon binding of an IL-1 family cytokine to its cognate receptor, a co-receptor is recruited to form a ternary complex comprising the cytokine, its cognate membrane receptor, and its co-receptor. The resulting complex facilitates intracellular signal transduction and activation of a set of transcription factors, including NF-kB and AP-1 and mitogen-activated protein kinases, which triggers a cascade of inflammatory and immune responses, including the production of numerous cytokines, chemokines, enzymes, and adhesion molecules.

[0007] IL-1RAP (Wesche, H., J. Biol. Chern. 272: 7727-7731, 1997; Genbank Accession No. AAB4059) serves as the common cellular membrane co-receptor for several receptors in the IL-1 family, including interleukin-1 receptor 1, ST2 also known as interleukin-1 receptor-like 1 and interleukin-1 receptor-like 2 (IL1RL2). IL-1RAP is a necessary component of the ternary signaling complex formed by one of the IL-1 family cytokines noted above, the cytokine's specific cognate receptor, and the IL-1RAP co-receptor. Thus, IL-1RAP serves an important function in the IL-1 family signal transduction pathways, since it is required to facilitate particular downstream signaling pathways stimulated by the IL-1 family cytokines IL-la, IL-ip, IL-33, IL-36a, IL-36P, and IL-36y.

[0008] IL-1RAP has previously been identified as a cell-surface biomarker associated with hematological neoplastic disorders such as chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and myelodysplatic syndromes (MDS) (for example, see Jaras et al., 2010, Proc. Natl. Acad. Sci. USA 107(37):16280-5, Askmyret al., 2013, Blood. 121(18):3709-13 andBarreyro et al., 2012, Blood 120(6):1290-8, each of which are incorporated herein by reference). IL-1RAP has also been described as overexpressed in several solid cancers, including pancreatic ductal adenocarcinoma (PDAC), Ewing sarcoma, non-small cell lung cancer, gliomas, triple-negative breast cancer, and stomach adenocarcinoma (for example, see Frenay J et al. Int. J. Mol. Sci. 2022; 23(23):14918, which is incorporated herein by reference). IL-1RAP is a thus a promising tumor-associated antigen for therapeutic targeting.

[0009] The treatment of cancer has progressed significantly with the development of pharmaceuticals that more efficiently target and kill cancer cells or key supporting tumor stromal cells. To this end, researchers have taken advantage of cell-surface receptors and antigens selectively expressed by cancer cells or immunosuppressive cells in the tumor microenvironment to develop drugs based on antibodies that bind the tumor-specific or tumor-associated antigens. In this regard, cytotoxic molecules such as bacteria and plant toxins, radionuclides, and certain chemotherapeutic drugs have been chemically linked to monoclonal antibodies that bind tumorspecific or tumor-associated cell surface antigens (see, e.g., International Patent Applications WO 00 / 02587, WO 02 / 060955, and WO 02 / 092127, U.S. Pat. Nos. 5,475,092, 6,340,701, and 6,171,586, U.S. Patent Application Publication No. 2003 / 0004210 Al, and Ghetie et al., J. Immunol. Methods, 112: 267-277 (1988)). Such compounds are typically referred to as toxin, radionuclide, and drug “conjugates,” respectively. Often they also are referred to as immunoconjugates, radioimmunoconjugates, and immunotoxins. Tumor cell or supporting stromal cells killing occurs upon binding of the drug conjugate to a tumor cell and release or / and activation of the cytotoxic activity of the drug. The selectivity afforded by antibody drug conjugates lowers the minimum effective dose of the drug in the patient.

[0010] Accordingly, there remains a need in the art for anti-IL-lRAP antibodies and antibody drug conjugates (ADCs) that can be used for therapeutic purposes in the treatment of cancer. BRIEF SUMMARY

[0011] Illustrative, non-limiting embodiments of the present invention described below overcome the above disadvantages and other disadvantages not described above. Also, the present invention is not required to overcome the disadvantages described above, and an illustrative, nonlimiting embodiment of the present invention described below may not overcome any of the problems described above.

[0012] In one aspect of the present invention is provided an antibody or antigen binding fragment thereof comprising the heavy chain variable region of any one of SEQ ID NOs: 36-40 or 73-76 and the light chain variable region of any one of SEQ ID NOs: 31-35 or 77-79.

[0013] In another aspect, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3,7-9, 13-15, 19-21, or 25-27, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, or 28-30, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 4-6, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 10-12, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 13-15, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 16-18, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 19-21, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 22-24, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 25-27, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 28-30, respectively.

[0014] In some aspects, the antibody is a humanized antibody. In some aspects, the humanized antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 10-12, respectively. In some aspects, the humanized antibody or antigen binding fragment thereof comprises the heavy chain and the light chain of any one of: a) SEQ ID NO: 73 and SEQ ID NO: 77, respectively; b) SEQ ID NO: 74 and SEQ ID NO: 77, respectively; c) SEQ ID NO: 73 and SEQ ID NO: 78, respectively; d) SEQ ID NO: 73 and SEQ ID NO: 79, respectively; e) SEQ ID NO: 75 and SEQ ID NO: 77, respectively; f) SEQ ID NO: 75 and SEQ ID NO: 78, respectively;g) SEQ ID NO: 76 and SEQ ID NO: 77, respectively; or h) SEQ ID NO: 76 and SEQ ID NO: 78. In some aspects, the humanized antibody or antigen binding fragment thereof comprises the heavy chain of SEQ ID NO: 76 and the light chain of SEQ ID NO: 78.

[0015] In some aspects, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen binding fragment thereof as described herein (e.g., a humanized anti-IL-lRAP antibody or antigen binding fragment as described herein). In aspects, the cancer is an esophageal cancer, liver cancer, bile duct cancer, kidney cancer, thyroid cancer, bladder cancer, mesothelioma, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, malignant peripheral nerve sheath tumor, astrocytoma, pancreatic cancer, cholangiocarcinoma, stomach cancer, ALK-positive anaplastic large cell lymphoma, or acute myeloid leukemia. In some aspects, the cancer is a squamous cancer. In some aspects, the squamous cancer is an esophageal cancer, esophageal squamous cell carcinoma, cancer of the esophagogastric junction, squamous head and neck cancer, squamous NSCLC or cervical cancer. In some aspects, the cancer is an esophageal cancer, lung cancer, melanoma, head and neck cancer, glioblastoma, pancreatic cancer, acute myeloid leukemia, squamous cancer, esophageal squamous cell carcinoma, squamous head and neck cancer, squamous non-small cell lung cancer, cervical cancer, or Ewing sarcoma. In some aspects, the cancer is a Ewing sarcoma. In some aspects, the cancer is a Ewing sarcoma comprising a EWSR1:FLI1 mutation.

[0016] In an aspect, provided herein is a use of an antibody or antigen binding fragment thereof as described herein (e.g., a humanized anti-IL-lRAP antibody or antigen binding fragment as described herein) to treat cancer in a subject in need thereof.

[0017] In another aspect, provided herein is a use of an antibody or antigen binding fragment thereof as described herein (e.g., a humanized anti-IL-lRAP antibody or antigen binding fragment as described herein) as described herein in the manufacture of a medicament.

[0018] In an aspect, provided herein is an anti-IL-lRAP antibody or antigen binding fragment thereof as described herein (e.g., a humanized anti-IL-lRAP antibody or antigen binding fragment as described herein) for use to treat cancer in a subject in need thereof. In some aspects, the cancer is an esophageal cancer, liver cancer, bile duct cancer, kidney cancer, thyroid cancer, bladder cancer, mesothelioma, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, malignant peripheral nerve sheath tumor, astrocytoma pancreatic cancer, cholangiocarcinoma, stomach cancer, ALK-positive anaplastic large cell lymphoma, or acute myeloid leukemia. In some aspects, the cancer is an esophageal cancer, lung cancer, melanoma, head and neck cancer, glioblastoma, pancreatic cancer, acute myeloid leukemia, squamous cancer, esophageal squamous cell carcinoma, squamous head and neck cancer, squamous non-small cell lung cancer, cervical cancer, or Ewing sarcoma. In some aspects, the cancer is a Ewing sarcoma. In some aspects, the cancer is a Ewing sarcoma comprising a EWSR1:FLI1 mutation. In some aspects, the subject is a human.

[0019] In another aspect, provided herein is an antibody or antigen binding fragment thereof as described herein (e.g., a humanized anti-IL-lRAP antibody or antigen binding fragment as described herein) for use in the manufacture of a medicament.

[0020] In one aspect of the present invention is provided an antibody drug conjugate comprising the formula (Ab) - [(L) - (D)m]n, or a pharmaceutically acceptable salt thereof; wherein: (Ab) is an antibody or antigen binding fragment thereof that binds IL-1RAP, wherein the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3, 7-9, 13-15, 19-21, or 25-27, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, or 28-30, respectively; (L) is a linker; (D) is a drug moiety; m is an integer from 1 to 8; and n is an integer from 1 to 12, wherein the linker (L) links (Ab) to (D). In some aspects, the antibody or antigen binding fragment thereof comprises the heavy chain variable region of any one of SEQ ID NOs: 36-40 or 73-76 and the light chain variable region of any one of SEQ ID NOs: 31-35 or 77-79.

[0021] In some aspects, the the antibody comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3, 7-9, 13-15, 19-21, or 25-27, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, or 28-30, respectively.

[0022] In some aspects, the antibody is a humanized antibody. In some aspects, the antibody comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 10-12, respectively, and the heavy chain of SEQ ID NO:37 and the light chain of SEQ ID NO:32.

[0023] In some aspects, the humanized antibody or antigen binding fragment thereof comprises the heavy chain and the light chain of any one of: a) SEQ ID NO: 73 and SEQ ID NO: 77, respectively; b) SEQ ID NO: 74 and SEQ ID NO: 77, respectively; c) SEQ ID NO: 73 and SEQ ID NO: 78, respectively; d) SEQ ID NO: 73 and SEQ ID NO: 79, respectively; e) SEQ ID NO: 75 and SEQ ID NO: 77, respectively; f) SEQ ID NO: 75 and SEQ ID NO: 78, respectively; g) SEQ ID NO: 76 and SEQ ID NO: 77, respectively; or h) SEQ ID NO: 76 and SEQ ID NO: 78, respectively. In some aspects, the humanized antibody or antigen binding fragment thereof comprises the heavy chain of SEQ ID NO: 75 and the light chain of SEQ ID NO: 78. In some aspects, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker, and a dicarboxylic acid based linker.

[0024] In some aspects, the linker is a cleavable linker. In further embodiments, the cleavable linker is cleavable under intracellular conditions. In other embodiments, the cleavable linker is cleavable under extracellular conditions (e.g., cleavable by proteases present in the tumor microenvironment). In some embodiments, the cleavable linker comprises a hydrazine group, a maleimide group, a disulfide group, a cis-aconityl group, a peptide comprising 1 to 10 amino acid residues, a para-aminobenzyl alcohol group, a photolabile group, a dimethyl group, a glucuronic acid group, or a combination thereof. In some aspects, the cleavable linker comprises a maleimide group. In some embodiments, the cleavable linker is a peptide linker cleavable by an intracellular protease. In some embodiments, the cleavable linker is a peptide linker cleavable by an extracellular protease (e.g., cleavable by an extracellular protease present in the tumor microenvironment).

[0025] In some aspects, the cleavable linker is a peptide linker comprising a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide. In some aspects, the dipeptide is alanine-alanine (alaala), valine-alanine (val-ala), valine-glycine (val-gly), glycine-glycine (gly-gly), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), phenylalanine-lysine (phe-lys), or N-methyl-valine-citrulline (Me-val -cit). In some aspects, the tripeptide is alanine-alanine-asparagine (ala-ala-asn), glutamic acid-valine-citrulline (glu-val-cit), glycine-valine-citrulline (glv-val-cit), or glycine-glycine-glycine (gly-gly-gly). In some aspects, the tetrapeptide is glycine-phenylalanine-leucine-glycine (gly-phe-leu-gly), glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly), or alanine-leucine-alanine-leucine (ala-leu-ala-leu). In some aspects, the tetrapeptide is glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly, also referred to herein as GGFG).

[0026] In some aspects, the drug moiety is selected from a group consisting of a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, a topoisomerase I and / or II inhibitor, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an auristatin, an amanitin, a pyrrolobenzodiazepine, an RNA polymerase inhibitor, an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a TLR agonist, a STING agonist, a DNA minor groove binder, and a DHFR inhibitor.

[0027] In specific aspects, the drug moiety is a topoisomerase I inhibitor. In some aspects,, the topoisomerase I inhibitor is selected from the group consisting of CHEM008, deruxtecan, camptothecins, topotecan, irinotecan, belotecan, exatecan, Exatecan mesylate, DXd, indenoisoquinolines, indotecan, indimitecan, SN-38, and lamellarin D, or their derivatives. In some aspects, the topoisomerase I inhibitor is CHEM008.

[0028] In another aspect, provided herein is an antibody drug conjugate comprising the formula: , wherein Ab comprises a humanized antibody or antigen binding fragment thereof comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 10-12, respectively, and wherein n is an integer between 1 to 10. In some aspects, the antibody drug conjugate is also referred to as Mc-GGFG-CHEM008. In some aspects, the humanized antibody or antigen binding fragment thereof comprises the heavy chain and the light chain of SEQ ID NO: 75 and SEQ ID NO: 78, respectively. In some aspects, n is 8. In some aspects, the linker of the antibody drug conjugate is Mc-GGFG.

[0029] In another aspect, provided herein is an antibody drug conjugate comprising the formula (Ab) - [(L) - (D)m]n, or a pharmaceutically acceptable salt thereof; wherein: (Ab) is an antibody or antigen binding fragment thereof that binds IL-1 RAP, wherein the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 10-12, respectively; (L) is -GGFG; (D) is CHEM008; m is 1; and n is 8, wherein the linker (L) links (Ab) to (D). In some aspects, the linker (L) is Mc-GGFG.

[0030] In another aspect, provided herein is the antibody drug conjugate ADC019.

[0031] In another aspect, provided herein is a pharmaceutical composition comprising an antibody drug conjugate as described herein and a pharmaceutically acceptable carrier.

[0032] In an aspect, provided herein is a method for producing an anti-IL-lRAP antibody drug conjugate comprising: (a) chemically linking a linker as described herein to the drug moiety CHEM008 to form a linker-drug; (b) conjugating the linker-drug to an anti-IL-lRAP antibody described herein; and (c) purifying the antibody drug conjugate. In some embodiments, the anti-IL-1RAP antibody drug conjugates produced herein have a drug to antibody ratio (DAR) of about 8 as measured by UV spectrophotometry, mass spectrometry, hydrophobic interaction chromatography, reverse-phase HPLC, and / or capillary electrophoresis.

[0033] In another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-IL-1RAP antibody drug conjugate and / or pharmaceutical composition as described herein. In embodiments, the cancer is an esophageal cancer, liver cancer, bile duct cancer, kidney cancer, thyroid cancer, bladder cancer, mesothelioma, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, malignant peripheral nerve sheath tumor, astrocytoma, pancreatic cancer, cholangiocarcinoma, stomach cancer, ALK-positive anaplastic large cell lymphoma, or acute myeloid leukemia. In some aspects, the cancer is a squamous cancer. In some aspects, the squamous cancer is an esophageal cancer, esophageal squamous cell carcinoma, cancer of the esophagogastric junction, squamous head and neck cancer, squamous non-small cell lung cancer, or cervical cancer. In some aspects, the cancer is an esophageal cancer, lung cancer, melanoma, head and neck cancer, glioblastoma, pancreatic cancer, acute myeloid leukemia, squamous cancer, esophageal squamous cell carcinoma, squamous head and neck cancer, squamous non-small cell lung cancer, cervical cancer, or Ewing sarcoma. In some aspects, the cancer is an Ewing sarcoma. In some aspects, the cancer is an Ewing sarcoma comprising a EWSR1:FLI1 mutation.

[0034] In some aspects, the antibody drug conjugate induces cell death of IL-1RAP+ tumor cells and / or IL-1RAP+ tumor stromal cells. In some aspects, the antibody drug conjugate induces cell death of IL-1RAP+ tumor cells and IL-1RAP+ tumor stromal cells. In some aspects, the antibody drug conjugate induces cell death of IL-1RAP+ tumor stromal cells. In some aspects, the IL-1RAP+ tumor stromal cells comprise tumor associated myeloid cells and cancer associated fibroblasts.

[0035] In other aspects, the antibody drug conjugate induces bystander killing of tumor cells which do not express IL-1RAP. In some aspects, the antibody drug conjugate induces death of immune system inhibitory IL-1RAP+ cells (e.g., IL-1RAP+ inhibitory immune cells of the tumor microenvironment). In some aspects, the humanized antibody or antigen binding fragment thereof induces cell death of IL-1RAP+ tumor cells. In some aspects, the humanized antibody or antigen binding fragment thereof induces death of immune system inhibitory IL-1RAP+ cells (e.g., IL-1RAP+ inhibitory immune cells of the tumor microenvironment).

[0036] In aspects, the antibody drug conjugate or pharmaceutical composition are administered to the subject in combination with one or more additional therapeutic compounds. In some aspects, the subject (e.g., the subject with Ewing Sarcoma,has been pretreated with a treatment regimen comprising topoisomerase-1 and / or topisomerase 2 inhibitors. In some aspects, the subject is a topoisomerase-1 inhibitor refractory patient. In embodiments, the one or more additional therapeutic compounds is a standard of care chemotherapeutic agent or immune checkpoint inhibitor. In embodiments, the subject is a human.

[0037] In an aspect, provided herein is a use of an anti-IL-lRAP antibody drug conjugate and / or pharmaceutical composition as described herein to treat cancer in a subject in need thereof.

[0038] In another aspect, provided herein is a use of an anti-IL-lRAP antibody drug conjugate and / or pharmaceutical composition as described herein in the manufacture of a medicament.

[0039] In some aspects of the antibody drug conjugate as described here or the pharmaceutical composition as decribed herein, the drug moiety is an MCL-1 inhibitor. In some aspects, the MCL-1 inhibitor is selected from the group consisting of: AZD5991, S63845, AMG176, AMG397, dMCLl-2, or compound C3.

[0040] In another aspect, provided herein is an antibody drug conjugate which is ADC019.

[0041] In another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ADC019. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIGs. 1A-1B show IL-1RAP expression in tumors and cell lines. FIG. 1A shows the expression of membranous IL-1RAP (mbIL-lRAP) in the 15 highest expressing indications in cancer indications from The Cancer Genome Atlas (TCGA) dataset. FIG. IB shows total IL-1RAP mRNA expression (log2-transformed, using a pseudo-count of 1) in established cell line models from the Dependency Map (DepMap) Public 24Q2 dataset (depmap.org / portal / ). Cell lines are grouped by cancer subtypes. Only cancer subtypes with more than 5 cell line models are shown. Box and whiskers plot, horizontal line indicates the median value, box shows the interquartile range, and whiskers indicate the range up to 1.5 times the interquartile range. TPM=Transcript per Million.

[0043] FIG. 2 shows the expression of the main IL-1RAP isoforms (mbIL-lRAP, solIL-1RAP, ncIL-lRAP, stopIL-lRAP, and cnsIL-lRAP) from the GTEx database in transcripts per million (TPM). Tissues were ranked from high expression to low expression for mbIL-lRAP.

[0044] FIG. 3 shows the distribution of mbIL-lRAP expression in the TCGA and GTEx databases discriminated between the non-squamous cell tumors, the squamous cell tumor, and the healthy tissues.

[0045] FIG. 4 shows the increased mbILlRAP in cancers with squamous histology. The graphs show the density of mbIL-lRAP expression in the TCGA dataset in solid tumors (squamous and non-squamous) and in the GTEx data, split by tumour indications. SCC=squamous cell carcinoma, NSCLC=non-small cell lung cancer, and TPM=transcript per million.

[0046] FIG. 5 shows membranous IL-1RAP over-expression in tumor tissue compared to healthy tissue expression. The x-axis is the mean expression of the membranous IL-1RAP in healthy tissues and the y-axis is the difference between the mean expression in tumor and matching healthy tissues. The area of the circle is proportional to the number of samples in TCGA and the colour is the fold-change between tumor and healthy tissue. The top 15 indications (based on the highest fold change) are labelled. MPNST= Malignant Peripheral Nerve Sheath Tumors. ESCC=Oesophageal Squamous-Cell Carcinoma; GBM=Glioblastoma Multiforme; SCC=Squamous Cell Carcinoma (cervical); PTC= Papillary Thyroid Carcinoma; CC=Renal Cell Carcinoma; PDAC=Pancreatic Ductal Adenocarcinoma; AML=Acute Myeloid Leukaemia; NSCLC=Non-Small Cell Lung Cancer; and HNSCC=Head and Neck Squamous Cell Carcinoma.

[0047] FIGs. 6A-6H show the expression of IL-1RAP by tissue type in cancer indications compared to normal tissue in TCGA and GTEx, including AML / blood (FIG. 6A), oesophageal cancer (FIG. 6B), lung cancer (FIG. 6C), glioblastoma / brain (FIG. 6D), ovarian cancer (FIG. 6E), pancreatic cancer (FIG. 6F), cervical cancer (FIG. 6G), and skin cancer (FIG. 6H).

[0048] FIGs. 7A-7B are from single cell datasets showing expression of IL-1RAP in tumour cells and in stroma cells (such as tumor-associated myeloid cells and / or cancer-associated fibroblasts) in glioblastoma (UMAP in FIG. 7A) and esophageal squamous cell carcinoma (ESCC) (Box plot in FIG. 7B).

[0049] FIG. 8 shows membrane IL-1RAP (mbIL-lRAP) protein expression across various cancer cell lines, reported as antibody binding capacity (ABC). Negative control cell lines were tested, as well as cell lines representative of bladder cancer, acute myeloid leukemia (AML), head & neck cancer (H&N), ovarian cancer, stomach cancer, oesophageal cancer, Ewing sarcoma, and melanoma.

[0050] FIG. 9 shows immunohistochemical scoring of IL-1RAP protein expression in a multi-tumor (n=10 indications) microarray (TMA). 120 cores (10 per indications) from FFPE tumour tissues were stained with a polyclonal anti-IL-lRAP antibody. IL-1RAP expression was scored as either negative, minimal (1), slight (2), moderate (3), or marked (4). The percentages of IL-1RAP positive tissues for each indication are indicated.

[0051] FIGs. 10A-10D shows the percentage of IL-1RAP positive cores in a TMA with different subtypes of head and neck cancer. 102 cores from FFPE tissues were stained with anti-IL-1RAP antibody. IL-1RAP positivity is shown (FIG. 10A) segregated by tissue origin including pharynx, larynx, oral cavity, mandibular, cutaneous, and salivary gland as % of cores analyzed). FIGs. 10B-10C show the percentage of IL-1RAP positive cores in a TMA with different histology subtypes of head and neck cancer. IL-1RAP positivity is shown segregated by mucoepidermoid and adenosquamous carcinoma, adenocarcinoma and adenoid cystic carcinoma, and squamous cell carcinoma (FIG. 10B). FIG. 10C shows IL-1RAP expression scoring for H&N squamous cell carcinoma samples. FIG. 10D shows that IL-1RAP protein expression is significantly higher in H&N cancer pateints below the age of 60 years. IL-1 RAP staining scores in a head and neck cancer TMA. IL-1RAP positivity is shown segregated by age for head and neck squamous cell carcinoma samples.

[0052] FIG. 11 shows the percentage of IL-1RAP positive cores in a TMA from patients suffering from different esophageal squamous cancers. 72 cores from FFPE tissues were stained with anti-IL-lRAP antibody, including 3 non-malignant cores and 67 malignant cores.

[0053] FIGs. 12A-12D show the results of a Kaplan-Meier survival analysis performed using the KMplot tool for high IL-1RAP and low IL-1RAP patient groups with gene expressions (separated by median expression) and clinical data available in the pan-cancer datasets GEO, EGA and TCGA. Survival analysis was performed for Kidney Renal Papillary Cell Carcinoma (FIG. 12A), Liver Hepatocellular Carcinoma (FIG. 12B), Stomach Adenocarcinoma (FIG. 12C), and Pancreatic Ductal Adenocarcinoma (FIG. 12D).

[0054] FIG. 13 shows an analysis of the median IL-1RAP expression level across a panel of cancer cell lines derived from several indications from the Depmap database (depmap.org) plotted against the median IC50 concentration for the SN-38 topo-I inhibitor in each cell line. The diameter of the circle (n) is representative of the number of cells lines for each indication is also shown. The indications with higher I1-1RAP expression (Log2(TPM=l)>2) located in the top left rectangle are more sensitive to the topoisomerase I inhibitor (IC50<0.1uM).

[0055] FIG. 14 shows the interaction analysis (in triplicate) of a titration from 1.95 nM to 500 nM of the anti-IL-lRAP chimeric antibody ADVH60 with reversibly immobilized IL-1RAP (also referred to herein as IL-1 RAcP).

[0056] FIG. 15 shows the results of chimeric antibodies titration experiment to determine the binding of anti-IL-lRAP antibodies (as determined by MFI) for the chimeric clones ADVH40, ADVH60, ADVA70, and ADVA12 to IL-1RAP positive cells SK-MEL-28 cells. A human IgGl antibody was used as a negative control.

[0057] FIGs. 16A-16E shows the results of an internalization experiment assessing the anti-IL-lRAP antibody chimeric clones ADVH70 (FIG. 16A), ADVH40 (FIG. 16B), ADVA12 (FIG. 16C), and ADVH60 (FIG. 16D), or isotype control (FIG. 16E) at various concentrations using pHrodo-labeled secondary antibodies incubated with IL-1RAP positive SK-MEL-28 cells at 37°C and monitored for 28 hours.

[0058] FIGs. 17A-17B shows the results of an antibody titration experiment to determine the binding of the humanized anti-IL-lRAP antibody ADVH66 (as determined by Median Fluorescence Intensity (MedFI)) to the IL-1RAP positive cell lines A673 and TC71 (Ewing Sarcoma), SK-MEL-28 (Melanoma), Kyse-270 (Esophageal squamous cell carcinoma), Hs-746T (Stomach adenocarcinoma), FaDu, DETROIT-562, and Cal27 (Head and Neck squamous cell carcinoma), BFTC-905 and TCCSUP (bladder carcinoma), and TOV-21G and OVCAR5 (Ovarian serous adenocarcinoma), Mono-Mac-6, Molm-13, OCLAML-2, SKNO-1, and AML-11 (Acute myeloid leukemia), A-204 (Rhabdomyosarcoma), and SH-SY5Y (Neuroblastoma) (FIG. 17A), and a comparison of ADVH66 binding percentages on Molm-13 and Molm-13 IL-1 RAP KO cell lines (FIG. 17B). Normalized background-subtracted data (to the highest observed fluorescence intensity) was analyzed using a non-linear regression curve, 4 parameters model. Ploted data points are the results of one representative experiment.

[0059] FIG. 18 shows a plot comparing the ABC values plotted against the EC50 (nM) values for ADVH66 binding to the various cell lines assayed in FIGs. 17A.

[0060] FIG. 19 shows the quantitative pharmacological analysis of IncuCyte® FabFluor labeled ADVH66, or a HuIgGl control, or anti-TfR (transferrin) control, in the MonoMac-6, AML-11, SK-MEL-28, TC-71, or Raji (negative control) cell lines. Datapoints are the mean of 2 technical replicates. Error bars indicate the standard deviation. Internalization percent corresponds to the counts of fluorescent positive cells / total cells at 24h.

[0061] FIG. 20 shows the percent of internalization at 24 hours of either ADVH66 or HuIgGl isotype control in IL-1RAP negative and positive cell lines. Cell lines are ordered by their IL-1RAP expression as ABC values. Internalization percent is shown after 24 hours incubation with ADVH66 and isotype control mAb at 2 nM. Datapoints are the mean of 2 technical replicates. Error bars show standard deviation. Raji (IL-1RAP ABC=0) was used as a negative control cell line.

[0062] FIG. 21 Quantitative pharmacological analysis of IncuCyte® FabFluor labeled ADVH66. Kyse-270 IL-1 RAP-positive cells were treated with increasing concentrations (0 nM, 0.5 nM, 1 nM, 2 nM, and 4 nM) of FabFluor labeled ADVH66. The time course graph displays an increase in normalized red area over time with increasing ADVH66 concentrations .

[0063] FIG. 22 shows the results of two anti-IL-lRAP antibody (ADVH60 and ADVH66) signaling blocking experiments in HEK-Blue IL-ip, pre-incubated 45 minutes with antibodies, and subsequentlty incubated overnight with the receptor agonist (IL-1P), and revealed with QuantiBlue reagent after incubation with culture supernatant. Datapoints are the mean of 2 technical replicates.

[0064] FIG. 23 shows the results of two anti-IL-lRAP antibody (ADVH60 and ADVH66) signaling blocking experiment in HEK-Blue IL-33, pre-incubated 45 minutes with antibodies, and subsequentlty incubated overnight with the receptor agonist (IL-33), and revealed with QuantiBlue reagent after incubation with culture supernatant.

[0065] FIG. 24 shows the results of two anti-IL-lRAP antibody (ADVH60 and ADVH66) signaling blocking experiment in HEK-Blue IL-36, pre-incubated 45 minutes with antibodies, and subsequentlty incubated overnight with the receptor agonist (IL-36P), and revealed with QuantiBlue reagent after incubation with culture supernatant.

[0066] FIGs. 25A-25B compare the binding ability by ELISA of ADVH66 unconjugated antibody and ADC019 (ADVH66-mc-GGFG-CHEM008) to recombinant human IL-1RAP (FIG. 25A) and to recombinant cynomolgus IL-1RAP (FIG. 25B).

[0067] FIG. 26 compares the binding kinetics of ADVH66 and ADC019 (ADVH66-mc-GGFG-CHEM008) to recombinant human IL-1RAP as assessed by SPR.

[0068] FIGs. 27A-27B show cell binding percentage of ADVH66, AB017 (isotype control), ADC019, and ADC020 (AB017-mc-GGFG-CHEM008), on the IL-1RAP positive TC71 cells (FIG. 27A) and A673 cells (FIG. 27B). Datapoints are simplicate. Data are background-subtracted based on secondary only condition. Data were analyzed with [Agonist] vs. response (variable slope), and binding % were obtained by normalizing background-subtracted values based on regression top and bottom values.

[0069] FIGs. 28A-28B show average drug-antibody ratios (DAR) for ADC019 (ADVH66-mc-GGFG-CHEM008) and ADC015 (ADVH66-mc-GGFG-DXd) over time when incubated in vitro in human plasma (FIG. 28A), or cynomolgus plasma (FIG. 28B).

[0070] FIGs. 29 shows the ratio of ADC to total monoclonal antibody in human and cynomolgus plasma over time.

[0071] FIG. 30 shows the low percentage of free payload (CHEM008) released in vitro in human and cynomolgus plasma over time for ADC019. The percent free payload (%) was calculated as the ratio of the free CHEM008 determined by LC / MS-MS to the hypothetical total CHEM008 conjugated to ADC019.

[0072] FIG. 31 shows the results of an in vitro cytotoxicity potential of ADC019, ADVH66 (naked antibody), or payload (CHEM008) on various cancer cell lines treated with varying concentrations. For Mono-Mac-6, AML-11, and SKNO-1 cells, cells were treated for 6 days, and datapoints are the mean of 2 technical replicates. For TC-71 and KYSE-270 cells, cells were treated for 5 days, datapoints are the mean of 3 technical replicates for ADC, and 2 technical replicates for payload and mAb. Error bars indicate the standard deviation. Regression was performed using a 4-parameter analysis in Graphpad Prism.

[0073] FIGs. 32A-32C show the results of an in vivo mouse study on the effects of the anti-IL-1RAP antibody ADV582 conjugated (DAR 8) to DXd (ADV582 DXd) or the anti-IL-lRAP antibody ADVH66 conjugated (DAR 8) to DXd (ADVH66 DXd) on the growth of established Kyse-270 tumors implanted sub-cutaniously. FIG. 32A shows the mean tumor volume (n=8 with last observations carried forward) and standard error of the mean (SEM) plots comparing the antitumor efficacy of vehicle, naked ADV582, isotype HEL control DXd (isotype HEL DXd), ADV582 DXd, and ADVH66 DXd treated mice (dosed intravenously (IV) 3 times on day 0 [day of randomization based on tumor volume around 100mm3], day 5, and day 10). The naked ADV582, isotype HEL control DXd, ADV582 DXd, and ADVH66 DXd were each dosed at 5mg / kg. Tumors were measured 3 times a week. N=8 animals per group. FIG. 32B shows the mean body weight and standard error of the mean (SEM) of the mice in the 5 groups treated with vehicle, naked ADV582, isotype control DXd (isotype HEL DXd), ADV582 DXd, and ADVH66 DXd. Mice (n=8 animals per group) were weighed 3 times a week until the end of the study. FIG. 32C shows a Kaplan-Meier analysis of median survival probability based on the time for the tumor volume to reach 1500 mm3.

[0074] FIGs. 33A-33D show the results of an in vivo mouse study assessing the effects of ADC019 on the growth of human CDX implanted in immunocompromised mice. The data shows the efficiency of ADC019 in inducing an anti-tumour reponse. SK-ES-1 200 mm3 tumors (FIG. 33A), TOV21G 200 mm3 tumors (FIG. 33B), Kyse-270 200 mm3 tumors (FIG. 33C), and Kyse-270 400 mm3 tumors (FIG. 33D) were used as cancer models (n=8 animals per treatment), and tumor growth and survival curves are shown for each. The figures show the mean tumor volume (with last observations carried forward) and standard error of the mean (SEM) plots comparing the anti-tumor efficacy of ADC019 with ADC020 (Isotype ADC) and a vehicle control group (dosed intravenously (IV) 3 times on day 0 [day of randomization based on tumor volume], day 5, and day 10). In addition, a Kaplan-Meier analysis of median survival probability based on the time for the mean tumor volume to reach 1500 mm3 (SK-ES-1 and Kyse-270) and 600 mm3 for TOV-21G. Median survival for the 3 groups is indicated on the figures.

[0075] FIG. 34A-34C show the results of an in vivo mouse study assessing the effects of ADC019 on the growth of Ewing sarcoma patient-derived xenograft (PDX) models implanted on the flank of immunocompromised mice. Vehicle treatment and ADC020 isotype control were included as controls (n=3 animals per group / model). Body weight (FIG. 34A) and mean tumor volume (FIG. 34B) are shown for each PDX model. Datapoints are mean + standard error of the mean of 3 individual animals (dosed intravenously (IV) 3 times on day 0 [day of randomization based on tumor volume], day 5, and day 10). FIG 34C shows a Waterfall plot showing the antitumor activity of ADC019 EWS PDX models (n=6), measured as percentage of tumor volume change compared with the tumor volume on day 0 (day of randomization). Each bar indicates the mean of three biological replicates. The level of IL-1 RAP expression and the information on the origin of the tumours and response to relevant clinical treatments are also indicated.

[0076] FIG. 35 shows animal body weight over time of female cynomolgus monkeys (n=3 / group) dosed twice I.V. (Q3W) with 3 different concentrations of ADC019 on day 1 and day 22.

[0077] FIG. 36 Pharmacokinetics of ADC019 ADCs in NHP. Concentration vs time profiles following two doses (dosed IV Q3W) administered in female NHP (n=3 / group). Concentrations of the ADC (mAb conjugated to 1 or more CHEM008 drug molecules) were determined using a ligand binding assay. DETAILED DESCRIPTION

[0078] Provided are methods and compositions relating to the use of antibody-drug conjugates (ADCs) and ADC derivatives that bind to IL-1RAP. A drug conjugated to the antibody exerts a cytotoxic, cytostatic, or immunostimulatory effect on IL-lRAP-expressing cells to treat IL-lRAP-expressing cancers. Antibodies to IL-1RAP which can be used in accordance with the methods and compositions described herein include monoclonal antibodies as well as chimeric, humanized, or human antibodies, and such antibodies conjugated to cytotoxic or immuno stimulatory agents such as, for example, chemotherapeutic drugs or activators of the innate immune system.

[0079] Some aspects of the present disclosure are directed to an antibody drug conjugate comprising the formula (Ab) - [(L) - (D)m]n, or a pharmaceutically acceptable salt thereof; wherein: (Ab) is a humanized antibody or antigen binding fragment thereof that binds IL-1RAP, wherein the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3, 7-9, 13-15, 19-21, or 25-27, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, or 28-30, respectively; (L) is a linker; (D) is a drug moiety; m is an integer from 1 to 8; and n is an integer from 1 to 12, wherein the linker (L) links (Ab) to (D).

[0080] In some aspects, n is between 1 and 12. In some aspects, n is between 2 and 10. In some aspects, n is between 4 and 8. In some aspects, n is between 1 and 2. In some aspects, n is between 2 and 3. In some aspects, n is between 3 and 4. In some aspects, n is between 4 and 5. In some aspects, n is between 5 and 6. In some aspects, n is between 6 and 7. In some aspects, n is between 7 and 8. In some aspects, n is between 7 and 8. In some aspects, n is between 8 and 9. In some aspects, n is between 9 and 10. In some aspects, n is between 10 and 11. In some aspects, n is between 11 and 12. In some aspects, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some aspects, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12.

[0081] In some aspects, m is between 1 and 8. In some aspects, m is between 2 and 6. In some aspects, m is between 4 and 8. In some aspects, m is between 1 and 2. In some aspects, m is between 2 and 3. In some aspects, m is between 3 and 4. In some aspects, m is between 4 and 5. In some aspects, m is between 5 and 6. In some aspects, m is between 6 and 7. In some aspects, m is between 7 and 8. In some aspects, m is between 7 and 8. In some aspects, m is 1, 2, 3, 4, 5, 6, 7, or 8. In some aspects, m is about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.

[0082] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to the particular compositions or process steps described, as such can, of course, vary. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0083] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. I. Definitions

[0084] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0085] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range, including the endpoints, and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0086] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a chimeric polypeptide," is understood to represent one or more chimeric polypeptides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0087] 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). In addition, "or" is used mean an open list of the components in the list. For example, "wherein X comprises A or B" means X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other components.

[0088] The terms "about" or "comprising essentially of" refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "comprising essentially of" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "comprising essentially of" can mean a range of up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0089] As used herein, the term "approximately," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term "approximately" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0090] The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, epitope binding antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, immunoglobulin new antigen receptor antibodies (IgNARs), which comprise single variable new antigen receptor domain antibody fragments (VNARs, or VNAR domains), unibodies, in which the hinge region has been removed, nanobodies, antibody fragments consisting of a single monomeric variable antibody domain (Ablynx), minibodies, which are engineered antibody fragments comprising an scFv linked to a CH domain (Hu et al., Cancer Res. 56:3055-3061, 1996), DuoBodies®, which are bispecific modified IgGl antibodies that include (i) a stable hinge region that is non-permissive for Fab arm exchange in vivo and (ii) an IgG4-like CH3 domain modified to be permissive for Fab arm exchange in vivo. (See, for example, WO2008 / 119353 and WO2011 / 131746), multispecific antibodies, such as bispecific antibodies generated from at least two intact antibodies, probodies, which are recombinant, masked monoclonal antibodies that remain inert in healthy tissue, but are activated specifically in the disease microenvironment (e.g., cleavage by a protease enriched or specific in a disease microenvironment) (See Desnoyers et al., Sci Transl Med 5:207ral44, 2013), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0091] The terms “Kabat numbering,” “Kabat definitions,” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0092] As used herein, the term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain (HC) and the light chain (LC), which are designated CDR1, CDR2 and CDR3 (or specifically HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3), for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These subportions were designated as LI, L2 and L3 or Hl, H2 and H3 where the “L” and the “H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs.

[0093] A “CDR-grafted antibody” as used herein refers to a humanized antibody in which the complementarity determining regions (CDRs) of a first (non-human) species have been grafted onto the framework regions (FRs) of a second (human) species.

[0094] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

[0095] The term “labeled antibody” as used herein, refers to an antibody, or an antigen binding portion thereof, with a label incorporated that provides for the identification of the binding protein, e.g., an antibody. Preferably, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following:      radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, "TC, inIn, 125I, 1311,177Lu 166Ho, or 153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.

[0096] The term "aryl" generally refers to residues having an aromatic ring derived by removing one hydrogen atom. The term "aromatic ring" may refer to a 6- to 14-membered allcarbon monocyclic or fused polycyclic ring (ie, rings that share adjacent pairs of carbon atoms) having a conjugated pi-electron system, and may be 6 to 10 membered, such as benzene and Naphthalene. The aromatic ring can be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring linked to the parent structure is an aryl ring. Aryl may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Aryl groups can be substituted or unsubstituted.

[0097] The term "heteroaryl" generally refers to a residue having a hydrogen atom removed from a carbon atom of a hetero aromatic ring. The term "heteroaromatic ring" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms may be selected from the group consisting of oxygen, sulfur and nitrogen. Heteroaryl can be 5 to 10 membered, 5 membered or 6 membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazole Base et al. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring. Heteroaryl groups can be optionally substituted or unsubstituted, and when substituted, the substituents can be one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy group, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, ring Alkylthio, and heterocycloalkylthio. Heteroaryl groups can be substituted or unsubstituted.

[0098] The term "alkyl" generally refers to a residue derived from an alkane by removal of a hydrogen atom. Alkyl groups can be substituted or unsubstituted, substituted or unsubstituted. The term "alkyl" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having a residue derived by removing a hydrogen atom from the same carbon atom or two different carbon atoms of the parent alkane, which may be a group containing 1 to A straight or branched chain group of 20 carbon atoms, eg 1 to 12 carbon atoms, eg, an alkane alkyl group containing 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, and the like. Alkyl groups may be substituted or unsubstituted, substituted or non-substituted, for example when substituted, substituents may be substituted at any available point of attachment, and the substituents may be independently optionally selected from alkyl groups , alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy , heterocycloalkoxy, cycloalkylthio, heterocyclo alkyl thio and one or more substituents in oxo, such as hydrogen, protium, deuterium, tritium, halogen, -NO 2 , - CN, -OH, -SH, -nh2 , -C(O)H, -co2H , -C(O)C(O)H, -C(O) ch2c (O)H, -S (O)H, -S(O)2H, -c (0) nh2 , -so2nh2 , -OC ( O)H, -N(H) SO2H or Cl-6 aliphatic group.

[0099] The term "alkenyl" generally refers to a straight or branched chain hydrocarbon group containing one or more double bonds. Illustrative examples of alkenyl groups include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl. Illustrative examples of C2-6 alkenyl groups having more than one double bond include butadienyl, pentadienyl, hexadienyl, and hexatrienyl and branched forms thereof. The position of the unsaturated bond (double bond) can be anywhere in the carbon chain. Alkenyl groups can be substituted or unsubstituted.

[0100] The term "alkynyl" generally refers to unsaturated straight or branched chain alkynyl groups such as ethynyl, 1-propynyl, propargyl, butynyl, and the like. Alkynyl groups can be substituted or unsubstituted.

[0101] The term "alicyclic group" generally refers to a residue having a hydrogen atom removed from the same carbon atom or a plurality of different carbon atoms of an alicyclic ring. The term "cycloalkane" generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, the carbocyclic ring containing 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, may contain 3 to 8 carbon atoms. Nonlimiting examples of alicyclic groups include cyclopropanyl, cyclobutanyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclopentyl Heptatrienyl, cyclooctyl, etc.; polycyclic carbocycles may include spiro, fused, and bridged carbocycles. Alicyclic groups can be substituted or unsubstituted. In this application, the term "carbocyclyl" generally refers to a residue derived from a carbon atom having a carbocyclic ring by removing one hydrogen atom. The term "carbocycle" generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, the carbocycle contains 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, may Contains 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane etc.; polycyclic carbocycles may include spiro, fused and bridged carbocycles. Carbocyclyl groups can be substituted or unsubstituted. Alicyclic and carbocyclic may be used interchangeably in some cases.

[0102] By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

[0103] An "antigen" refers to any molecule, e.g., a peptide that provokes an immune response or is capable of being bound by a TCR. The immune response may involve antibody production, the activation of specific immunologically-competent cells, or a combination thereof. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen can be endogenously expressed, i.e. expressed by genomic DNA, or can be recombinantly expressed. An antigen and / or an epitope can be specific to a certain tissue, such as a cancer cell, or it can be broadly expressed. In addition, fragments of larger molecules can act as antigens. In one aspect, antigens are tumor antigens.

[0104] An "anti-tumor effect" as used herein, refers to a biological effect that can present as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival of a patient, an increase in life expectancy of a patient, or amelioration of various physiological symptoms in a patient associated with the tumor. An anti-tumor effect can also refer to the prevention of the occurrence of a tumor, e.g., a vaccine. An anti-tumor effect may present via targeting inhibitory immune cells present in the tumor microenvironment (e.g., targeting immune system inhibitory IL-1RAP+ cells of the tumor microenvironment).

[0105] As used herein, the term "bystander killing" or "bystander effect" refers to the killing of target-negative cells (e.g., IL-1RAP negative tumor cells or tumor cells expressing a low level of IL-1RAP) in the presence of target-positive cells (e.g., IL-1RAP+ tumor cells or tumor cells expressing an elevated level of IL-1RAP), wherein killing of target-negative cells is not observed in the absence of target-positive cells. Cell-to-cell contact, or at least proximity between target-positive and target-negative cells, enables bystander killing. This type of killing is distinguishable from “off-target killing,” which refers to the indiscriminate killing of targetnegative cells. “Off-target killing” may be observed in the absence of target-positive cells.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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 embodiments, 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).

[0110] An “antibody drug conjugate” or “ADC” as used herein refers to a compound that is linked to a cell binding agent (i.e., an antibody or fragment thereof). Typically, the cell binding agent (e.g., antibody) is covalently bound to the drug by a linker.

[0111] By “drug-to-antibody ratio” or "DAR" refers to the average number of “drug” (i.e., cytotoxic agent) molecules conjugated per antibody in an ADC. The DAR for a particular batch of ADC represents an average number of drugs attached to each antibody molecule within that batch. DAR is characterized using any method known in the art including, but not limited to, spectroscopy, dynamic light scattering, size exclusion chromatography (SEC), size exclusion chromatography coupled with mass spectrometry (SEC-MS) and mass spectrometry. In some aspects, the DAR is between 1 and 12. In some aspects, the DAR is between 4 and 8. In some aspects, the DAR is between 2 and 3. In some aspects, the DAR is between 4 and 5. In some aspects, the DAR is between 6 and 7. In some aspects, the DAR is between 7 and 8. In some aspects, the DAR is between 2 and 10. In some aspects, the DAR is between 1 and 2. In some aspects, the DAR is between 3 and 4. In some aspects, the DAR is between 5 and 6. In some aspects, the DAR is between 7 and 8. In some aspects, the DAR is between 8 and 9. In some aspects, the DAR is between 9 and 10. In some aspects, the DAR is between 10 and 11. In some aspects, the DAR is between 11 and 12. In some aspects, the DAR is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some aspects, the DAR is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12.

[0112] IL-1RAP (also referred to herein as IL1RAP, IL-lRAcP, C3orfl3, or IL1R3) as used herein refers to any recombinant or naturally-occurring forms of interleukin-1 receptor accessory protein (IL-1RAP) or variants or homologs thereof that maintain IL-1RAP activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to IL-1RAP). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 10, 20, 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL-1RAP polypeptide. In embodiments, IL-1RAP is substantially identical to the protein identified by the UniProt reference number Q9NPH3 or a variant or homolog having substantial identity thereto. An exemplary amino acid sequence of wild type human IL-1RAP, which contains 570 amino acids, is provided below as SEQ ID NO: 71. The extracellular domain of IL-1RAP spans residues 21 to 359, and is provided below as SEQ ID NO: 72.

[0113] MTLLWCVVSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLF EHFLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTG NYTCMLRNTTYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSS VKPTITWYMGCYKIQNFNNVIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLT VKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKP DDITIDVTINESISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQK VPAPRYTVELACGFGATVLLVVILIVVYHVYWLEMVLFYRAHFGTDETILDGKEYDIYV SYARNAEEEEFVLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLVVLSP NYVLQGTQALLELKAGLENMASRGNINVILVQYKAVKETKVKELKRAKTVLTVIKWKG EKSKYPQGRFWKQLQVAMPVKKSPRRSSSDEQGLSYSSLKNV (SEQ ID NO:71).

[0114] SERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYSTAHSAGLTLIW YWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTTYCSKVAFPL EVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNN VIPEGMNLSFLIALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPND HVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDITIDVTINESISHSRTEDET RTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVE (SEQ ID NO:72).

[0115] The term “IL-1RAP expressing tumor,” as used herein, refers to a tumor which expresses IL-1RAP protein (including a tumor comprising tumor infiltrating immunosuppressive cells such as myeloid derived suppressor cells that express IL-1RAP protein). In one embodiment, IL-1RAP expression in a tumor is determined using immunohistochemical staining of tumor cell or tumor infiltrating cell membranes, where any immunohistochemical staining above background level in a tumor sample indicates that the tumor is an IL-1RAP expressing tumor. In another embodiment, an IL-1RAP expressing tumor is identified in a patient when greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or more of the cells in a tumor sample are positive for IL-1RAP expression. In another embodiment, IL-1RAP positive expression is determined based on membrane staining as determined by, e.g., immunohistochemistry (IHC) or immunofluorescence (IF) analysis.

[0116] An IL-1RAP expressing tumor is identified as having an “elevated level of IL-1RAP” or “expressing IL-1RAP at an elevated level” when the level of IL-1RAP is higher than in tissue surrounding the cancer. In some embodiments, an “elevated level of IL-1RAP” is one in which 5% or more of the cells in a tumor sample have membrane staining. In some embodiments a “high level” in regard to IL-1RAP is 5% or more staining, for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the cells in the tumor sample are stained. In some embodiments, the protein expression levels can be measured by IHC and / or IF analysis.

[0117] An IL-1RAP expressing tumor is identified as having a “low level of IL-1RAP” or “expressing IL-1RAP at a low level” is one in which 5% or less of the cells in a tumor sample have membrane staining. In some embodiments a “low level” in regard to IL-1RAP is 5% or less staining, for example, 4.9, 4.5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1% or less of the cells in the tumor sample are stained. In some embodiments, the protein expression levels can be measured by IHC and / or IF analysis.

[0118] A cell that expresses no IL-1RAP can also be described as expressing a “low level of IL-1RAP”. Thus, the phrase “expresses a low level of IL-1RAP” encompasses no IL-1RAP expression. In some embodiments, a low level of IL- 1RAP is within the background staining levels. In some embodiments, a sample that is IL-1RAP “negative” has no IL-1RAP expression or a low level of IL-1RAP. In some embodiments, IL-1RAP staining is negative when no or less than 5%, 4%, 3%, 2%, or 1% of the cells have membrane staining for IL-1RAP.

[0119] 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.

[0120] B-cells play a principal role in humoral immunity (with antibody involvement). A B cell makes antibodies and antigens and performs the role of antigen-presenting cells (APCs) and turns into memory B-cells after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow, where its name is derived from.

[0121] The term “MCL-1 inhibitor,” as used herein, refers to an agent capable of reducing the expression and / or activity of MCL-1 and / or one or more upstream modulators or downstream targets thereof. As used herein, the terms “derivative” and “analog” when referring to an Mcl-1 inhibitor, or the like, means any such compound that retains essentially the same, similar, or enhanced biological function or activity as compared to the original compound but has an altered chemical or biological structure.

[0122] Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (Kd) of 10-5 to 1011 M or less. Any Kd greater than about 10-4 M is generally considered to indicate nonspecific binding. As used herein, an antibody that "binds specifically" to an antigen refers to an antibody that binds to the antigen and substantially identical antigens with high affinity, which means having a Kd of 10-7 M or less, 10-8 M or less, 5 x 10-9 M or less, or between 10-8 M and 1010 M or less, but does not bind with high affinity to unrelated antigens. By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of interest, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention. In one embodiment, Kd is determined by surface plasmon resonance or Bio-Layer Interferometry, or by any other method known in the art. Bio-Layer Interferometry refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by measuring the interference patterns of reflected white light, for example using the Octet™ system (ForteBio, Pall Corp. Fremont, Calif.). For further description of the Octet™ system, see Li, B et al. (2011) J. Pharm. Biomed. Anal. 54(2):286-294 and Abdiche, Y. N., et al. (2009) Anal. Biochem. 386(2):172-180, the contents of which are incorporated herein by reference.

[0123] The term “antigen binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL-1 RAP). It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats; specifically binding to two or more different antigens. 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 VL, VH, CL and CHI 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 VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90 / 05144 Al herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). 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” of an antibody. In certain embodiments, scFv molecules may be incorporated into a fusion protein. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. L, et al. (1994) Structure 2:1121-1123). Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-54041354-5).

[0124] Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody, e.g., a IL-1RAP -binding antibody or “binding portion” described herein, includes: (i) a fragment or linked fragments or a similar monovalent fragment consisting of the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs:l-3, 7-9, 13-15, 19-21, or 25-27 and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs:4-6, 10-12, 16-18, 22-24, or 28-30; (ii) a fragment or linked fragments or a similar bivalent fragment comprising two fragments comprising the heavy chain variable (VH) region of any one of SEQ ID NOs:36-40, represented by any one of DNA SEQ ID NOs: 46-50, respectively, and the light chain (VL) variable region of any one of SEQ ID NOs: 3135, represented by any one of DNA SEQ ID NOs: 41-45; or (iii) a fragment or linked fragments or a similar bivalent fragment comprising two fragments comprising the heavy chain variable (VH) region of any one of SEQ ID NOs:73-76, and the light chain (VL) variable region of any one of SEQ ID NOs:77-79. Any of a combination of the six CDRs, VH and VL, or HC and LC, can optionally be joined by natural or synthetic linkers. Sequences contained herein are listed below in Table 1. Table 1: IL-1RAP Antibody Sequences (Kabat Numbering) SEQ ID NO: Name Encoded Sequence 1 ADVH40-HCDR1 SYWIN 2 ADVH40-HCDR2 NIYPSDSYTNYNQKFKD 3 ADVH40-HCDR3 GWTHYDYFDF 4 ADVH40-LCDR1 KASQNVGTNVA 5 ADVH40-LCDR2 SASYRYS 6 ADVH40-LCDR3 QQYNSYPLT 7 ADVH60-HCDR1 SYWMN 8 ADVH60-HCDR2 MIHPSDSETRLNQKFKD 9 ADVH60-HCDR3 LYYDNFPFDY 10 ADVH60-LCDR1 KVSQNVGTNVA 11 ADVH60-LCDR2 SASYRNS 12 ADVH60-LCDR3 QQYNSYPYT 13 ADVA70-HCDR1 DYSMH 14 ADVA70-HCDR2 WINTYTGEPTYVDDFKG 15 ADVA70-HCDR3 YYGYFDV 16 ADVA70-LCDR1 KASQNVGTNVA 17 ADVA70-LCDR2 SASYRYS 18 ADVA70-LCDR3 QQYSGYPLT 19 ADVE10-HCDR1 NYGMN 20 ADVE10-HCDR2 WINTNTGEPTYAEEFKG 21 ADVE10-HCDR3 RDVGFAY 22 ADVE10-LCDR1 KASQDVSTAVA 23 ADVE10-LCDR2 WASTRHT 24 ADVE10-LCDR3 QQHYSTPYT 25 ADVA12-HCDR1 NYGMN 26 ADVA12-HCDR2 WINTYTGEPTYADDFKG 27 ADVA12-HCDR3 RGGSSSFDY 28 ADVA12-LCDR1 RASGNIHNYLA 29 ADVA12-LCDR2 NAKTLAD 30 ADVA12-LCDR3 QHFWSTPYT 31 ADVH40-VL-AA DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQ QKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISN VQSEDLAEYFCQQYNSYPLTFGAGTKLELK 32 ADVH60-VL-AA DIQMNQSQKFMSTSVGDRVSVTCKVSQNVGTNVAWY QQKPGQSPKALIYSASYRNSGVPDRFTGSGSGTDFTLTIS NVQSEDLAEYFCQQYNSYPYTFGGGTKLEIK 33 ADVA70-VL-AA DIQMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQ QKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISN VQSEDLADYFCQQYSGYPLTFGGGTKLEIK 34 ADVE10-VL-AA DIQMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQ QKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTIS SVQAEDLALYYCQQHYSTPYTFGGGTKLEIK 35 ADVA12-VL-AA DIVITQSPASLSASVGETVTITCRASGNIHNYLAWYQQK QGKSPQVLVYNAKTLADGVPSRFSGSGSGTQYSLKINS LQPEDFGSYYCQHFWSTPYTFGGGTKLEIK 36 ADVH40-VH-AA QIQLQQSGAEVARPGASVKMSCKASGYTFTSYWINWM KQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKS SSTAYMQLSSPTSEDSAVYYCARGWTHYDYFDFWGQG TTLTVSS 37 ADVH60-VH-AA QVQLQQPGAELVKPGASVKLSCKASGYSFTSYWMNW VKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDK SSSTAYMQLSSPTSEDSAVYYCARLYYDNFPFDYWGQG TLVTVSS 38 ADVA70-VH-AA QIQFVQSGPELKKPGETVKISCKASGYTFTDYSMHWVK QAPGKGLKWMGWINTYTGEPTYVDDFKGRFAFSLETS ASTAYLQINNLKNEDTATYFCARYYGYFDVWGAGTTV TVSA 39 ADVE10-VH-AA QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVK QAPGQGLKWMGWINTNTGEPTYAEEFKGRFVFSLETSA STAYLQINNLKNEDTATYFCTRRDVGFAYWGQGTLVT VSS 40 ADVA12-VH-AA QIQFVQSGPELKKPGETVKISCKASGYTFTNYGMNWVK QAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETS ASTAYLQISNLKNEDTATYFCARRGGSSSFDYWGQGTT LTVSR 41 ADVH40-VL-DNA GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCA CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGG CCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCA ACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTA CTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGC TTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGT ATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTT CGGTGCTGGGACCAAGCTGGAGCTCAAA 42 ADVH60-VL-DNA GACATCCAGATGAACCAGTCTCAAAAATTCATGTCCA CATCAGTAGGAGACAGGGTAAGCGTCACCTGCAAGG TCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCA ACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTA CTCGGCATCTTACCGGAACAGTGGAGTCCCTGATCGC TTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAATGTGCAGTCTGAAGACTTGGCTGAGTA TTTCTGTCAGCAATATAATAGCTATCCGTACACGTTC GGAGGGGGCACCAAGCTGGAAATCAAA 43 ADVA70-VL-DNA GACATCCAGATGACACAGTCTCAAAAATTCATGTCCA CATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGG CCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCA ACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTA CTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGC TTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCA CCATCAGCAATGTCCAGTCTGAAGACCTGGCAGATTA TTTCTGCCAGCAATATAGCGGCTATCCTCTCACGTTC GGAGGGGGGACCAAGCTGGAAATAAAA 44 ADVE10-VL-DNA GACATCCAGATGACCCAGTCTCACAAATTCATGTCCA CATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGG CCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCA ACAAAAACCAGGGCAATCTCCTAAACTACTGATTTAC TGGGCATCCACCCGGCACACTGGAGTCCCTGATCGCT TCACAGGCAGTGGATCTGGGACAGATTATACTCTCAC CATCAGCAGTGTGCAGGCTGAAGACCTGGCACTTTAT TACTGTCAGCAACATTATAGCACTCCGTACACGTTCG GAGGGGGGACCAAGCTGGAAATAAAA 45 ADVA12-VL-DNA GATATTGTGATAACCCAGTCTCCAGCCTCCCTATCTG CATCTGTGGGAGAAACTGTCACCATCACATGTCGAGC AAGTGGGAATATTCACAATTATTTAGCATGGTATCAG CAGAAACAGGGAAAATCTCCTCAGGTCCTGGTCTATA ATGCAAAAACCTTAGCAGATGGTGTGCCATCTAGGTT CAGTGGCAGTGGCTCAGGAACACAATATTCTCTCAAG ATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATT ACTGTCAACATTTTTGGAGTACTCCGTACACGTTCGG AGGGGGCACCAAGCTGGAAATCAAA 46 ADVH40-VH-DNA CAGATCCAACTGCAGCAGTCTGGGGCTGAAGTGGCA AGACCTGGGGCCTCAGTCAAGATGTCCTGCAAGGCTT CTGGCTACACCTTCACCAGCTACTGGATAAACTGGAT GAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGG AAATATTTATCCTTCTGATAGTTATACTAACTACAAT CAAAAGTTCAAGGACAAGGCCACATTGACTGTAGAC AAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCC CGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAG AGGGTGGACTCACTACGACTACTTTGACTTCTGGGGC CAAGGCACCACTCTCACAGTCTCCTCG 47 ADVH60-VH- CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTG DNA AAGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTT CTGGCTACTCCTTCACCAGCTACTGGATGAACTGGGT GAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGG CATGATTCATCCTTCCGATAGTGAAACTAGATTAAAT CAGAAGTTCAAGGACAAGGCCACATTGACTGTAGAC AAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCC CGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAG GCTCTACTATGATAACTTCCCCTTTGACTACTGGGGC CAAGGGACTCTGGTCACAGTCTCCTCG 48 ADVA70-VH-DNA CAGATCCAGTTCGTGCAGTCTGGACCTGAGCTGAAGA AGCCTGGAGAGACTGTCAAGATCTCCTGCAAGGCTTC TGGTTATACCTTCACAGACTATTCAATGCACTGGGTG AAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGC TGGATAAACACCTACACTGGAGAGCCAACATATGTT GATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAA CCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCT CAAAAATGAGGACACGGCTACATATTTCTGTGCACGT TACTACGGGTACTTCGATGTCTGGGGCGCAGGGACCA CGGTCACTGTCTCTGCG 49 ADVE10-VH-DNA CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAG AAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCT TCTGGGTATACCTTCACAAACTATGGAATGAACTGGG TGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGG GCTGGATAAACACCAACACTGGAGAGCCAACATATG CTGAAGAGTTCAAGGGACGGTTTGTCTTCTCTTTGGA AACCTCTGCCAGCACTGCCTATTTGCAGATCAACAAC CTCAAAAATGAGGACACGGCTACATATTTCTGTACAA GAAGGGACGTGGGGTTTGCTTACTGGGGCCAAGGGA CTCTGGTCACAGTCTCCTCG 50 ADVA12-VH-DNA CAGATCCAGTTCGTGCAGTCTGGACCTGAGCTGAAGA AGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTT CTGGGTATACCTTCACAAACTATGGAATGAACTGGGT GAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGG CTGGATAAACACCTACACTGGAGAGCCAACATATGC TGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAA ACCTCTGCCAGCACTGCCTATTTGCAGATCAGCAACC TCAAAAATGAGGACACGGCGACATATTTCTGTGCAA GGAGGGGCGGTAGTAGCTCCTTTGACTACTGGGGCC AAGGCACCACTCTCACAGTCTCTCGG 51 Murine IGHG1 AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVT VTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPS ETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSS VFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGK EFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNT QPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGL HNHHTEKSLSHSPGK 52 Murine IGHG1 DNA GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCC CTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCT GGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTG ACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTG TGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTA CACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACC TGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACC CGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGC CCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGT CCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAG CCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGG TCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCC CGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAG GTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAG TTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCA TCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCA AATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCAT CGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAA GGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAG CAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATG ATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGT GGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGA ACACTCAGCCCATCATGGACACAGATGGCTCTTACTT CGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTG GGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACAT GAGGGCCTGCACAACCACCATACTGAGAAGAGCCTC TCCCACTCTCCTGGTAAA 53 Human IGHG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 54 Human IGHG1 DNA GCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCG CCGAGCAGCAAAAGCACCAGCGGCGGCACCGCGGCG CTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGG TGACCGTGAGCTGGAACAGCGGCGCGCTGACCAGCG GCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGG CCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGC AGCAGCCTGGGCACCCAGACCTATATTTGCAACGTGA ACCATAAACCGAGCAACACCAAAGTGGATAAAAAAG TGGAACCGAAAAGCTGCGATAAAACCCATACCTGCC CGCCGTGCCCGGCGCCGGAACTGCTGGGCGGCCCGA GCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCT GATGATTAGCCGCACCCCGGAAGTGACCTGCGTGGT GGTGGATGTGAGCCATGAAGATCCGGAAGTGAAATT TAACTGGTATGTGGATGGCGTGGAAGTGCATAACGC GAAAACCAAACCGCGCGAAGAACAGTATAACAGCAC CTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAG GATTGGCTGAACGGCAAAGAATATAAATGCAAAGTG AGCAACAAAGCGCTGCCGGCGCCGATTGAAAAAACC ATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAG GTGTATACCCTGCCGCCGAGCCGCGATGAACTGACCA AAAACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCT TTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCA ACGGCCAGCCGGAAAACAACTATAAAACCACCCCGC CGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAG CAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGG CAACGTGTTTAGCTGCAGCGTGATGCATGAAGCGCTG CATAACCATTATACCCAGAAAAGCCTGAGCCTGAGC CCGGGCAAA 55 Murine IGKC*01 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV KWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK DEYERHNSYTCEATHKTSTSPIVKSFNRNEC 56 Murine IGKC*02 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINV KWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK DEYERHNSYTCEATHKTSTSPIVKSFNRNEC 57 Murine IGKC*03 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPRDINV KWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTK DEYERHNSYTCEATHKTSTSPIVKSFNRNEC 58 Human IGKC*01 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 59 Human IGKC*02 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQESKDSTYSLSSTLTLSKA DYEKHKVYAGEVTHQGLSSPVTKSFNRGEC 60 Human IGKC*03 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QRKVDNALQSGNSQESVTEQESKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 61 Human IGKC*04 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKLYACEVTHQGLSSPVTKSFNRGEC 62 Human IGKC*05 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 63 Murine IGKC*01 DNA CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCAC CATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGT CGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATC AATGTCAAGTGGAAGATTGATGGCAGTGAACGACAA AATGGCGTCCTGAACAGTTGGACTGATCAGGACAGC AAAGACAGCACCTACAGCATGAGCAGCACCCTCACG TTGACCAAGGACGAGTATGAACGACATAACAGCTAT ACCTGTGAGGCCACTCACAAGACATCAACTTCACCCA TTGTCAAGAGCTTCAACAGGAATGAGTGT 64 Murine IGKC*02 DNA CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCAC CATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGT CGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATC AATGTCAAGTGGAAGATTGATGGCAGTGAACGACAA AATGGCGTCCTGAACAGTTGGACTGATCAGGACAGC AAAGACAGCACCTACAGCATGAGCAGCACCCTCACG TTGACCAAGGACGAGTATGAACGACATAACAGCTAT ACCTGTGAGGCCACTCACAAGACATCAACTTCACCCA TCGTCAAGAGCTTCAACAGGAATGAGTGT 65 Murine IGKC*03 DNA CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCAC CATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGT CGTGTGCTTCTTGAACAACTTCTACCCCAGAGACATC AATGTCAAGTGGAAGATTGATGGCAGTGAACGACAA AATGGTGTCCTGAACAGTTGGACTGATCAGGACAGC AAAGACAGCACCTACAGCATGAGCAGCACCCTCACA TTGACCAAGGACGAGTATGAACGACATAACAGCTAT ACCTGTGAGGCCACTCACAAGACATCAACTTCACCCA TCGTCAAGAGCTTCAACAGGAATGAGTGT 66 Human IGKC*01 DNA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGT 67 Human IGKC*02 DNA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAGGAGAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCGGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC GTCACAAAGAGCTTCAACAGGGGAGAGTGT 68 Human IGKC*03 DNA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGCGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAGGAGAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGT 69 Human IGKC*04 DNA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG CTGAGCAAAGCAGACTACGAGAAACACAAACTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGT 70 Human IGKC*05 DNA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGC CATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGT TGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC AAGGACAGCACCTACAGCCTCAGCAACACCCTGACG CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGGGGAGAGTGC 73 ADVH60-H146-V1 QVQLVQPGAEVVKPGASVKVSCKASGYSFTSYWMNW VRQAPGQGLEWMGMIHPSDSETRLNQKFKDRVTLTVD KSTSTVYMELSSLRSEDTAVYYCARLYYDNFPFDYWG QGTLVTVSS 74 ADVH60-H146-V2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNW VRQAPGQGLEWMGMIHPSDGETRYNQKFKGRVTMTV DTSTSTVYMELSSLRSEDTAVYYCARLYYDNFPFDYWG QGTLVTVSS 75 ADVH60-H169-V1 QVQLVQPGAEVVKPGSSVKVSCKASGYSFTSYWMNW VRQAPGQGLEWMGMIHPSDSETRLNQKFKDRVTLTAD KSTSTAYMELSSLRSEDTAVYYCARLYYDNFPFDYWG QGTLVTVSS 76 ADVH60-H551-V1 EVQLVQPGAEVVKPGESLKISCKGSGYSFTSYWMNWV RQRPGKGLEWMGMIHPSDSETRLNQKFKDQVTLSADK SISTA YMQLSSLKASDTAVYYCARLYYDNFPFDYWGQ GTLVTVSS 77 ADVH60-L116-V1 DIQMTQSPSSLSASVGDRVTITCKVSQNVGTNVAWYQQ KPGKAPKALIYSASYRNSGVPSRFSGSGSGTDFTLTISSL QPEDFATYFCQQYNSYPYTFGQGTKLEIK 78 ADVH60-L315-V1 DIVMTQSPATLSVSPGERATLSCKVSQNVGTNVAWYQ QKPGQAPRALIYSASYRNSGIPARFSGSGSGTEFTLTISSL QSEDFAVYFCQQYNSYPYTFGQGTKLEIK 79 ADVH60-L116-V2 DIQMTQSPSSLSASVGDRVTITCRASQNIGTNLAWYQQ KPGKAPK ALIYS AS YLNSG VPS RFSGSGSGTDFTLTISSL QPEDFATYYCQQYNSYPYTFGQGTKLEIK

[0125] The term "monoclonal antibody," as used herein, refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are substantially similar and bind the same epitope(s) (e.g., the antibodies display a single binding specificity and affinity), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The term "human monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies that display(s) a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In some aspects, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0126] The term “antibody construct” as used herein refers to a polypeptide comprising one or more the antigen binding portions disclosed herein linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Antibody portions, such as Fab and F(ab)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0127] The term "recombinant human antibody," as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant regions that utilize particular human germline immunoglobulin sequences encoded by the germline genes, but include subsequent rearrangements and mutations which occur, for example, during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9): 1117- 1125), the variable region contains the antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen. The constant region will change in further response to an antigen (i.e., isotype switch). Therefore, the rearranged and somatically mutated nucleic acid molecules that encode the light chain and heavy chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar (i.e., have at least 80% identity).

[0128] 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 also is derived from human germline immunoglobulin sequences. The anti-C3 antibodies described herein can 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 are used synonymously.

[0129] The term “humanized antibody” refers to forms of non-human (e.g. murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539. The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgGl, IgG2, IgG3 and IgG4. In embodiments, the humanized antibody comprises a mutant version of the Fc domain, for example, a mutant IgGl Fc domain. In further embodiments, mutant human Fc domains are provided of all the other wild-type IgG subclasses (human IgG2, IgG3, and IgG4). In some aspects, the mutant Fc domains may exhibit improved half-life or reduced degradation after administration to a mammalian subject in vivo. In some aspects, the humanized antibody comprises a mutant IgGl comprising the L234A / L235A (LALA) substitution mutations (see, e.g., Wilkinson I et al. PLoS One. 2021; 16(12):e0260954, which is incorporated herein by reference). These substitutions reduce binding to the IgG Fc receptors FcyRI, FcyRII and FcyRIII as well as to complement component Clq. Such antibodies may be useful where binding and activation of Fc receptors is undesirable, for example when the product is being used as an antagonist of a cytokine or similar. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art.

[0130] The goal of humanization is a reduction in the immunogenicity of a xenogenic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and specificity of the antibody.

[0131] Humanized antibodies may be produced using several technologies, such as resurfacing and CDR grafting. As used herein, the resurfacing technology uses a combination of molecular modeling, statistical analysis and mutagenesis to alter the non-CDR surfaces of antibody variable regions to resemble the surfaces of known antibodies of the target host.

[0132] Strategies and methods for the resurfacing of antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are disclosed in U.S. Pat. No. 5,639,641 (Pedersen et al.), which is hereby incorporated in its entirety by reference. Briefly, in a preferred method, (1) position alignments of a pool of antibody heavy and light chain variable regions are generated to give a set of heavy and light chain variable region framework surface exposed positions wherein the alignment positions for all variable regions are at least about 98% identical; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues is defined for a rodent antibody (or fragment thereof); (3) a set of heavy and light chain variable region framework surface exposed amino acid residues that is most closely identical to the set of rodent surface exposed amino acid residues is identified; (4) the set of heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) is substituted with the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3), except for those amino acid residues that are within 5 angstroms of any atom of any residue of the complementarity-determining regions of the rodent antibody; and (5) the humanized rodent antibody having binding specificity is produced.

[0133] Antibodies can be humanized using a variety of other techniques including CDR-grafting (EP 0 239 400; WO 91 / 09967; U.S. Pat. Nos. 5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka G. M. et al., 1994, Protein Engineering 7(6):805-814; Roguska M. A. et al., 1994, PNAS 91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety of methods known in the art including phage display methods. See also U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and International Pat. Appl. Publication Nos.: WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741 (said references incorporated by reference in their entireties).

[0134] A “chimeric antibody” as used herein refers to an antibody comprising at least one variable region from a first species (such as mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (such as human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region. In some embodiments, all of the variable regions of a chimeric antibody are from a first species and all of the constant regions of the chimeric antibody are from a second species.

[0135] As used herein, "isotype" refers to the antibody class (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant region genes.

[0136] The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen."

[0137] An "isolated antibody," as used herein, is intended to refer to an antibody which is substantially free of other proteins and cellular material.

[0138] The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0139] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0140] A “drug moiety” as used herein may be for instance any drug, biological agent, compound, or molecule. The drug moiety may be a cytotoxic agent. The drug moiety may be a V-ATPase inhibitor. The drug moiety may be a pro-apoptotic agent. The drug moiety may be a Bcl2 inhibitor. The drug moiety may be an MCL1 inhibitor. The drug moiety may be a HSP90 inhibitor. The drug moiety may be an IAP inhibitor. The drug moiety may be an mTor inhibitor. The drug moiety may be a microtubule stabilizer. The drug moiety may be a microtubule destabilizer. The drug moiety may be an auristatin. The drug moiety may be a dolastatin. The drug moiety may be a maytansinoid. The drug moiety may be a MetAP (methionine aminopeptidase). The drug moiety may be an auristatin. The drug moiety may be an amanitin. The drug moiety may be a pyrrolobenzodiazepine. The drug moiety may be an RNA polymerase inhibitor. The drug moiety may be an inhibitor of nuclear export of proteins CRM1. The drug moiety may be a DPPIV inhibitor. The drug moiety may be a proteasome inhibitor. The drug moiety may be an inhibitor of phosphoryl transfer reactions in mitochondria. The drug moiety may be a protein synthesis inhibitor. The drug moiety may be a kinase inhibitor. The drug moiety may be a CDK2 inhibitor. The drug moiety may be a CDK9 inhibitor. The drug moiety may be a kinesin inhibitor. The drug moiety may be an HDAC inhibitor. The drug moiety may be a DNA damaging agent. The drug moiety may be a DNA alkylating agent. The drug moiety may be a DNA intercalator. The drug moiety may be a DNA minor groove binder. The drug moiety may be a DHFR inhibitor. The drug moiety may be an immunomodulatory. The drug moiety may be a STING agonist. The drug moiety may be a TLR agonist. The drug moiety may be a topoisomerase I and / or II inhibitor. The drug moiety may be a topoisomerase I inhibitor. Examples of topoisomerase I inhibitors include, for example, CHEM008, deruxtecan, camptothecins, topotecan, irinotecan, belotecan, exatecan, Exatecan mesylate, DXd, indenoisoquinolines, indotecan, indimitecan, SN-38, and lamellarin D, or their derivatives.

[0141] A “linker” as used herein may be a molecule or molecular compound, compound, or structure, that is capable of linking a compound to a protein. In one embodiment, a linker links a drug, such as a topoisomerase I inhibitor, to a cell-binding agent, such as an antibody or a fragment thereof in a stable, covalent manner. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound and / or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and known in the art. The linker may be a cleavable linker. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker. Other suitable linkers include linkers hydrolyzable at a pH of less than 5.5, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers. The linker may be cleavable under intracellular conditions (e.g., cleavable by an intracellular protease, such as a lysosomal protease or an endosomal protease). The linker may be cleavable under extracellular conditions (e.g., cleavable by an extracellular protease present in the tumor microenvironment). The linker may include a hydrazine group, a maleimide group (Me), a disulfide group, a cis-aconityl group, a peptide comprising 1 to 10 amino acid residues, a para-aminobenzyl alcohol group, a photolabile group, a dimethyl group, a glucuronic acid group, or a combination thereof. The linker may be a non-cleavable linker. The linker may be a hydrophilic linker. The linker may be a procharged linker. The linker may be a dicarboxylic acid based linker. Exemplary cleavable linkers include, but are not limited to: N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), and disulfide N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP). Exemplary non-cleavable linkers include, but are not limited to: 2-iminothiolane, acetylsuccinic anhydride, and succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). The generic linkers 2-iminothiolane and acetylsuccinic anhydride can be used as cleavable or non-cleavable linkers. The cleavable linker may be a peptide linker cleavable by an intracellular protease. The cleavable linker may be a peptide linker cleavable by an extracellular protease. Additional linkers contemplated for use with the antibody-drug conjugate described herein include those disclosed in U.S. Pat. Pub. US2023 / 0086097, and PCT Pubs. WO2023 / 025248, WO2022 / 171101, WO2022 / 135332, and WO2023040793, each of which is incorporated herein by reference in its entirety. Examples of cleavable and non-cleavable linkers are additionally described in, e.g., U.S. Pat. Nos. 9,504,756, 10,442,790, 10,808,039, 11,510,993, 11,312,769, 11,020,489, 11,116,847, 10,933,112, 10,383,948, and 10,322,192, each of which is incorporated herein by reference in its entirety.

[0142] The cleavable linker may be a peptide linker comprising a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide. Examples of dipeptides include alanine-alanine (ala-ala), valinealanine (val-ala), valine-glycine (val-gly), glycine-glycine (gly-gly), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), phenylalanine-lysine (phe-lys), and N-methyl-valine-citrulline (Me-val -cit). Examples of tripeptides include alanine-alanineasparagine (ala-ala-asn), glutamic acid-valine-citrulline (glu-val-cit), glycine-valine-citrulline (glv-val-cit), or glycine-glycine-glycine (gly-gly-gly). Examples of tetrapeptides include glycine-phenylalanine-leucine-glycine (gly-phe-leu-gly), glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly), or alanine-leucine-alanine-leucine (ala-leu-ala-leu).

[0143] The terms “intracellularly cleaved” and “intracellular cleavage” refer to metabolic processes or reactions inside a cell on a composition of the invention whereby the covalent attachment, e.g., the linker (L), between the drug moiety (D) and the antibody (Ab) is broken, resulting in the free drug dissociated from (Ab) inside the cell. In one embodiment, the cleaved moieties of the subject compositions are thus intracellular metabolites (e.g., Ab, Ab-L fragment, D-L fragment, D).

[0144] The term “extracellular cleavage” refers a metabolic process or reaction outside a cell on a composition of the invention whereby the covalent attachment, e.g., the linker (L), between the drug moiety (D) and the antibody (Ab) is broken, resulting in the free drug dissociated from (Ab) outside the cell. In one embodiment, the cleaved moieties of the subject compositions are thus initially extracellular metabolites (e.g., Ab, Ab-L fragment, D-L fragment, D), which may move intracellularly by diffusion and cell permability or transport.

[0145] By ‘‘disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasias and cancers to be treated with a composition of the invention.

[0146] A "cancer" refers to 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. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system including lymphoma, leukemia, and other leukocyte malignancies. In some embodiments, the methods of the present invention can be used to reduce the tumor size of a tumor derived from, for example, the cancer comprises bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian 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, breast cancer, prostate cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC)), Hodgkin's Disease, non-Hodgkin'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, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, 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, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, such as mesotherlioma, pleural mesothelioma, or malignant pleural mesotherlioma (MPM), or any combination thereof. The particular cancer can be responsive to chemo- or radiation therapy or the cancer can be refractory. A refractory cancer refers to a cancer that is not amendable to surgical intervention, and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time.

[0147] The term “leukemia” refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic). Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute myeloid leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

[0148] The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.

[0149] The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.

[0150] The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatinifomi carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hairmatrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signetring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma villosum.

[0151] 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.

[0152] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g., cancer (e.g. leukemia, acute myeloid leukemia)) means that the disease (e.g., cancer (e.g. leukemia, acute myeloid leukemia)) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. Alternatively, the substance (e.g., IL-1RAP) may be an indicator of the disease (e.g., cancer (e.g. leukemia, acute myeloid leukemia)). Thus, an associated substance may serve as a means of targeting disease tissue (e.g., cancer cells (e.g., leukemia stem cells, acute myeloid leukemia cells)).

[0153] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.A "cytokine," as used herein, refers to a non-antibody protein that is released by one cell in response to contact with a specific antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. A cytokine can be endogenously expressed by a cell, added to a cell in culture, administered to a subject, or any combination thereof. Cytokines may be released by immune cells, including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines can induce various responses in the recipient cell. Cytokines can include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and pro-inflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, IL-21, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include, but are not limited to, IL-la, IL-lb, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0154] "Chemokines" are a type of cytokine that mediates cell chemotaxis, or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL- 16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein la (MIP-la, MIP-la), MlP-Ib (MIP-lb), gammainduced protein 10 (IP- 10), and thymus and activation regulated chemokine (TARC or CCL17).

[0155] Other examples of cytokines include, but are not limited to chemokine (C-C motif) ligand (CCL) 1, CCL5, monocyte-specific chemokine 3 (MCP3 or CCL7), monocyte chemoattractant protein 2 (MCP-2 or CCL8), CCL13, IL-1, IL-3, IL-9, IL-11, IL-12, IL-14, IL-17, IL-20, IL-21, granulocyte colony- stimulating factor (G-CSF), leukemia inhibitory factor (LIF), oncostatin M (OSM), CD 154, lymphotoxin (LT) beta, 4-1BB ligand (4-1BBL), a proliferationinducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL-related leukocyte-expressed ligand 1 (TALL-1), or TNF-related apoptosis-inducing ligand (TRAIL).

[0156] An "immune response" is as understood in the art, and generally refers to a biological response within a vertebrate against foreign agents or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) 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 the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell, a Th cell, a CD4+ cell, a CD8+ T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g., NK cell. In some aspects, an immune response refers to NK cell-mediated killing of a foreign cell, e.g., an allogeneic T cell therapy.

[0157] “Immune cell” as used herein means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., an autoantigen). In typical embodiments, an immune cell is a T lymphocyte, a B lymphocyte, or a dendritic cell.

[0158] "Immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying the immune system or an immune response.

[0159] “Cytotoxic effect,” in reference to the effect of an agent on a cell, means killing of the cell. “Cytostatic effect” means an inhibition of cell proliferation. A “cytotoxic agent” means an agent that has a cytotoxic or cytostatic effect on a cell, thereby depleting or inhibiting the growth of, respectively, cells within a cell population.

[0160] The term “deplete,” in the context of the effect of an IL-lRAP-targeting moiety-drug conjugate on IL-lRAP-expressing cells, refers to a reduction or elimination of the IL-1RAP-expressing cells.

[0161] As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function. Numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD1 with its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer 12: 252-264, 2012). These proteins are responsible for costimulatory or inhibitory interactions of T-cell responses Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

[0162] As used herein, the term "inactivating" or "inactivation," e.g., in reference to a gene or protein, refers to a measure that can induce the reduced activity of the protein or antigen target. In some aspects, inactivation can be achieved by a binding of an antibody to the antigen target, such as IL-1 RAP.

[0163] Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of interest or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.

[0164] By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0165] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0166] As used herein, the term “active pharmaceutical ingredient” or “therapeutic agent” (“API”) refers to a biologically active compound.

[0167] As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, polymers, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0168] One of ordinary skill in the art would recognize that, when an amount of “a compound or a pharmaceutically acceptable salt thereof’ is disclosed, the amount of the pharmaceutically acceptable salt form of the compound is the amount equivalent to the concentration of the free base of the compound. It is noted that the disclosed amounts of the compounds or their pharmaceutically acceptable salts thereof herein are based upon their free base form.

[0169] The term “pharmaceutically acceptable” as used herein can in particular indicate that the “pharmaceutically acceptable” compound or “pharmaceutically acceptable” composition is suitable for administration to a subject to achieve a treatment and / or prevention of a disease, of a disorder or of a condition, in particular of at least one of a cancer such as a solid tumor cancer.

[0170] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0171] The term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0172] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

[0173] A pharmaceutical composition of the present invention can be in solid or liquid form and can be, inter alia, in a form of one or more powder(s), one or more tablet(s), one or more fluids, in particular one or more solution(s), or one or more aerosol(s). A pharmaceutical composition of the invention can also comprise one or more further biologically active agent(s), such as for example active agent(s), e. g. anti-IL-lRAP antibody, or anti-IL-lRAP antibody-drug conjugate described herein for use in the treatment and / or prevention of at least one of a cancer. The administration of a pharmaceutical composition of the present invention can be for example an administration selected from the group consisting of intraperitoneal, intravenous, parenteral, intrarenal, subcutaneous, topical, intrabronchial, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration. The compositions of the invention can also be administered directly to the target site, e.g., by biolistic delivery to the target site, like a specific organ afflicted with a disease, disorder or condition.

[0174] In particular, said administration can be carried out by injection and / or infusion and / or delivery, such as e.g. intravenous or intraperitoneal injection or infusion. The pharmaceutical composition can be present in the form of an injectable dosage form or a dosage form for administration by infusion, in particular in the form of an injectable dosage form for intravenous or intraperitoneal injection or an infusion dosage form for intravenous or intraperitoneal administration.

[0175] “Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids. Polypeptides are not limited to a specific length, e.g., they may comprise a full length protein sequence or a fragment of a full length protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0176] A pharmaceutical composition according to the present invention can be administered to the subject at a suitable dose. The dosage regimen can be for example determined by an attending physician. As well known in the art, dosages for a patient can depend upon many factors, such as the patient's size, body surface area, age, weight, administration for prevention or treatment purposes, target indication, the particular compound to be administered, general health, and other drugs being administered concurrently. According to one embodiment, at least one antibody of the present invention.

[0177] As used herein, the term “administering” means administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By “co-administer” it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be co-administered to the patient. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present invention can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0178] The compositions of the present invention may additionally include components to provide sustained release and / or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; or as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995). In embodiments, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.

[0179] According to one aspect, a pharmaceutical composition of the present invention can be a pharmaceutical composition which comprises an anti-IL-lRAP antibody-drug conjugate, or pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable carrier.

[0180] Moreover, doses of anti-IL-lRAP antibody-drug conjugate and optionally at least one pharmaceutically acceptable carrier of the present invention below or above the above indicated exemplary ranges can be administered, e.g. for treating and / or preventing at least one of a cancer. A pharmaceutical composition of the present invention can be formulated to be shortacting, fast-releasing, long-acting, or sustained-releasing.

[0181] Furthermore, a pharmaceutical composition of the present invention can comprise further biologically active agents, depending on the intended use of the pharmaceutical composition.

[0182] As used herein, the terms “patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0183] The term "therapeutic benefit" or "therapeutically effective" as used herein, refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.

[0184] By “therapeutically effective dose or amount” as used herein is meant the amount of a drug, e.g., an ADC, that produces effects for which it is administered (e.g. treating or preventing a disease). 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. The effective amount of an ADC may, for example, inhibit tumor growth (e.g., inhibit an increase in tumor volume), decrease tumor growth (e.g., decrease tumor volume), reduce the number of cancer cells, and / or relieve to some extent one or more of the symptoms associated with the cancer. The effective amount may, for example, improve disease free survival (DFS), improve overall survival (OS), or decrease likelihood of recurrence.

[0185] 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.

[0186] As used herein, the terms “treatment” or “treat” refer to slowing, stopping, or reversing the progression of an IL-lRAP-expressing cancer in a subject, as evidenced by a decrease or elimination of a clinical or diagnostic symptom of the disease, by administration of an anti-IL-1RAP ADC or ADC derivative to the subject after the onset of the clinical or diagnostic symptom of the IL-lRAP-expressing cancer at any clinical stage. Treatment can include, for example, a decrease in the severity of a symptom, the number of symptoms, or frequency of relapse. In various aspects, a subject in need thereof may be treated for a disease or for alleviating symptoms associated with a cancer.

[0187] The term “heterologous,” in the context of a polypeptide, means from a different source (e.g., a cell, tissue, organism, or species) as compared with another polypeptide, so that the two polypeptides are different. Typically, a heterologous polypeptide is from a different species.

[0188] As used herein, the term “functional,” in the context of an anti-IL-lRAP antibody or derivative thereof to be used in accordance with the methods described herein, indicates that the antibody or derivative thereof is (1) capable of binding to IL-1RAP and (2) depletes or inhibits the proliferation of IL-lRAP-expressing cells when conjugated to a cytotoxic agent, or has an immuno stimulatory effect on an immune cell when conjugated to an immuno stimulatory agent such as, for example, a chemotherapeutic drug or an activator of the innate immune system. In some embodiments, an anti-ILlRAP antibody or derivative thereof is (1) capable of binding to IL-1RAP and (2) modulating (e.g., inhibiting) IL-1, e.g., IL-ip and / or IL-la, signaling; inducing cell death in cells expressing IL1RAP, including leukemia cells; inhibiting cancer cell invasion and metastasis; decreasing or inhibiting cancer, e.g., acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), lung cancer, including non-small cell lung cancer (NSCLC) and ovarian cancer; and decreasing or inhibiting tumor cellular proliferation or tumor growth in vivo.

[0189] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0190] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophage-like synoviocytes, etc).

[0191] 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.

[0192] 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. As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0193] Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0194] Various aspects described herein are described in further detail in the following subsections. II. Compositions of the Disclosure A. Anti-IL-IRAP Antibodies

[0195] Aspects disclosed herein provide anti-IL-lRAP antibodies, or antigen binding portions thereof. Another aspect disclosed herein provides humanized anti-IL-lRAP antibodies, or antigen binding portions thereof. In some aspects, the anti-IL-lRAP antibodies are chimeric (e.g., anti-IL-lRAP antibodies comprising a mouse ScFv and a human Fc). In aspects, the antibodies disclosed herein bind human IL-1RAP. In some aspects, the antibodies disclosed herein bind human IL-1RAP expressed on tumor cells (i.e., human IL-1RAP expressed on the surface of tumor cells).

[0196] In aspects, disclosed herein are antibody drug conjugates (ADCs) comprising an anti-IL-lRAP antibody described herein and at least one drug(s) (i.e., at least one drug moiety conjugated to an anti-IL-lRAP antibody). The ADCs disclosed herein have characteristics including, but not limited to, binding to human IL-1RAP in vitro, modulating, e.g., inhibiting IL- 1 signaling, inducing cell death in cells expressing IL- 1RAP, including, but not limited to, leukemia cells, melanoma cells, Ewing sarcoma cells, and decreasing or inhibiting cancer, tumor cellular proliferation or tumor growth, or tumor invasion and metastasis. The ADCs disclosed herein have characteristics including, but not limited to, inducing bystander killing of low-expressing IL- 1RAP tumor cells. Bystander killing (the killing of neighboring cells) may be facilitated through diffusion of the linker-drug and / or the drug alone to neighboring cells. ADCs disclosed herein, in particular, have characteristics including, but not limited to, inducing cell death in cells expressing IL-1RAP, e.g., cancer cells expressing IL-1RAP. In aspects, an anti-IL-lRAP ADC as disclosed herein is capable of being internalized into a cell expressing IL-1RAP.

[0197] In aspects, anti-IL-lRAP antibodies are disclosed which have the ability to bind to IL-1RAP, as described in the Examples below. Collectively, the novel antibodies are referred to herein as “IL-1RAP antibodies.” In embodiments, the anti-IL-lRAP antibodies, ADCs, or antigen binding fragments thereof, are able to inhibit or decrease tumor growth in vivo. The tumor can be a tumor expressing a low level of IL-1RAP or a tumor expressing an elevated level of IL-1RAP. In some embodiments, a sample that is IL-1RAP “negative” has no IL-1RAP expression or a low level of IL-1RAP. In some embodiments, IL-1RAP staining is negative when no or less than 5%, 4%, 3%, 2%, or 1% of the cells have membrane staining for IL-1RAP. In some embodiments, the protein expression levels can be measured by IHC or IF analysis. In various embodiments, anti-IL-1RAP antibodies, ADCs, or antigen binding fragments thereof, are capable of modulating a biological function of IL-1RAP and its co-receptors signaling. In other embodiments of the foregoing aspects, the anti-IL-lRAP antibodies, ADCs, or antigen binding fragments thereof, bind IL-1RAP on cells expressing IL-1RAP. Thus, the disclosure includes anti-IL-lRAP antibodies, ADCs, or antigen binding fragments thereof, that are effective at inhibiting or decreasing tumor growth. Without wishing to be bound by any particular theory, in one embodiment, the anti-IL-1RAP antibodies, antigen-binding portions thereof, and ADCs are capable of inhibiting multiple IL-1RAP activities including, but not limited to, IL-ip signaling through IL-1RAP; IL-la, IL-ip, and IL-38 signaling through the IL-1R; IL-33 signaling through the IL-33R, and IL-36Ra, IL-36P, and IL-36y signaling through the IL-36R. It is known in the art that blocking IL-ip signaling is effective for treating lung cancer (see, for example, Ridker et al., Anti-inflammatory Therapy with Canakinumab for Atherosclerotic Disease, New England J. Med., 2017; and Ridker et al., The Lancet, 390:1833-1842, 2017, the entire contents of each of which are expressly incorporated herein by reference). Therefore, in one embodiment, the anti-IL-lRAP antibodies, antigen-binding portions thereof, and ADCs described herein are useful for downstream inhibition of IL-ip pathways for the treatment of cancers, e.g., lung cancer.

[0198] In embodiments, the anti-IL-lRAP antibodies, ADCs, and antigen-binding portions thereof, can be used for the treatment of melanoma in a subject, e.g., malignant melanoma. The anti-IL-lRAP antibodies, ADCs, and antigen-binding portions thereof, can be used for the treatment of esophageal cancer, head and neck cancer, glioblastoma, stomach cancer, acute myeloid leukemia (AML), ovarian cancer, pancreatic cancer, melanoma, Ewing sarcoma, liver cancer, lung cancer, lung squamous cell carcinoma, and non-small cell lung cancer. In one aspect, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or more of the cells in a cancer sample (e.g., a sample representative of a cancer described herein) is positive for IL-1RAP expression. In another embodiment, a tumor sample has a high level of IL-1RAP expression. For example, in one embodiment, at least 5% or more of the cells in a leukemia or tumor sample have membrane staining. In another aspect, a tumor sample obtained from a subject displays a low level of expression of IL- 1RAP. The expression level of IL-1 RAP can be determined by any method known in the art. For example, the expression level of IL-1 RAP can be determined via immunohistochemical (IHC) analysis and / or immunofluorescence (IF) analysis. In another embodiment, the cancer has been previously treated with another anti-cancer agent or anti-cancer therapy, e.g., a chemotherapy. In one embodiment, the cancer is resistant to chemotherapy.

[0199] Antibodies having combinations of any of the aforementioned characteristics are contemplated as aspects of the disclosure. ADCs, described in more detail below, may also have any of the foregoing characteristics.

[0200] One aspect of the disclosure features an anti-human IL-1RAP (anti-hIL-lRAP) antibody drug conjugate (ADC) comprising an anti-hIL-lRAP antibody conjugated to a drug via a linker. Exemplary anti-IL-lRAP antibodies (and sequences thereof) that can be used in the ADCs are described herein.

[0201] The anti-IL-lRAP antibodies described herein provide the ADCs with the ability to bind to IL-1 RAP such that the cytotoxic molecule attached to the antibody may be delivered to the IL-lRAP-expressing cell, particularly an IL-1RAP expressing cancer cell.

[0202] While the term “antibody” is used throughout, it should be noted that antibody fragments (i.e., antigen-binding portions of an anti-IL-lRAP antibody) are also included in the disclosure and may be included in the embodiments (methods and compositions) described throughout. For example, an anti-IL-lRAP antibody fragment may be conjugated to the drugs, as described herein. In certain embodiments, an anti-IL-lRAP antibody binding portion is a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, an scFv, a single domain antibody, or a diabody.

[0203] In aspects, the disclosure provides anti-IL-lRAP antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-40 or 73-76; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3135 or 77-79. In embodiments, the anti-IL-lRAP antibodies, or antigen binding portions thereof, comprise a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 76; and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 78.

[0204] In aspects, the disclosure provides anti-IL-lRAP antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 46-50; and a light chain variable region comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 41-45. In embodiments, the anti-IL-lRAP antibodies, or antigen binding portions thereof, comprise a heavy chain variable region comprising a nucleic acid sequence consisting of SEQ ID NO: 47; and a light chain variable region comprising a nucleic acid sequence consisting of SEQ ID NO: 42.

[0205] In aspects, the disclosure includes an anti-IL-lRAP antibody, or antigen binding portion thereof, comprising an HC CDR set (CDR1, CDR2, and CDR3) and an LC CDR set (CDR1, CDR2, and CDR3) selected from those set forth in Table 1.

[0206] In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 4-6, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 10-12, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 13-15, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 16-18, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 19-21, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 22-24, respectively. In some aspects, the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 25-27, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 28-30, respectively.

[0207] In some aspects, the antibody or antigen binding fragment thereof comprises the heavy chain of SEQ ID NO: 36 and the light chain of SEQ ID NO: 31. In some aspects, the antibody or antigen binding fragment thereof comprises the heavy chain of SEQ ID NO: 37 and the light chain of SEQ ID NO: 32. In some aspects, the antibody or antigen binding fragment thereof comprises the heavy chain of SEQ ID NO: 38 and the light chain of SEQ ID NO: 33. In some aspects, the antibody or antigen binding fragment thereof comprises the heavy chain of SEQ ID NO: 39 and the light chain of SEQ ID NO: 34. In some aspects, the antibody or antigen binding fragment thereof comprises the heavy chain of SEQ ID NO: 40 and the light chain of SEQ ID NO: 35.

[0208] In some aspects, an anti-IL-lRAP antibody, or antigen binding portion thereof, comprises a heavy chain constant region comprising a nucleic acid sequence consisting of SEQ ID NO: 52 or SEQ ID NO: 54; and / or a light chain constant region comprising a nucleic acid sequence consisting of any one of SEQ ID NO: 63-70. In some aspects, an anti-IL-lRAP antibody, or antigen-binding portion thereof, comprises a heavy chain constant region comprising a nucleic acid sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 54, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 52 or SEQ ID NO: 54, and / or a light chain constant region comprising a nucleic acid sequence set forth in any one of SEQ ID NO: 63-70, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 63-70.

[0209] In an aspect, an anti-IL-lRAP antibody (e.g., ADVH40), or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4.

[0210] In an aspect, an anti-IL-lRAP antibody (e.g., ADVH60), or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0211] In an aspect, an anti-IL-lRAP antibody (e.g., ADVA70), or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 14, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 17, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16.

[0212] In an aspect, an anti-IL-lRAP antibody (e.g., ADVE10), or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 21, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 23, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 22.

[0213] In an aspect, an anti-IL-lRAP antibody (e.g., ADVA12), or antigen binding portion thereof, comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 27, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 26, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 25, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 30, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 28.

[0214] In some aspects, an anti-IL-lRAP antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 36, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 36, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 31, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 31. In an aspect, the anti-IL-1RAP antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 2, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 1, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 4.

[0215] In some aspects, an anti-IL-lRAP antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 37, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 37, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 32, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 32. In an aspect, the anti-IL-1RAP antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10.

[0216] In some aspects, an anti-IL-lRAP antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 38, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 38, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 33, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 33. In an aspect, the anti-IL-1RAP antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 14, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 13, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 17, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 16.

[0217] In some aspects, an anti-IL-lRAP antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 39, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 39, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 34, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 34. In an aspect, the anti-IL-1RAP antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 21, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 20, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 23, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 22.

[0218] In some aspects, an anti-IL-lRAP antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 40, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 40, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 35, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 35. In an aspect, the anti-IL-1RAP antibody, or antigen-binding portion thereof comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 27, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 26, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 25, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 30, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 28.

[0219] In an aspect, an anti-IL-lRAP antibody, or antigen binding portion thereof, is a humanized ADVH60 antibody (e.g., ADVH61). The humanized ADVH61 antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-lRAP antibody (e.g., ADVH61), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 73, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 73, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 77, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 77.

[0220] In an aspect, an anti-IL-lRAP antibody, or antigen binding portion thereof, is a humanized ADVH60 antibody (e.g., ADVH62). The humanized ADVH62 antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-lRAP antibody (e.g., ADVH62), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 74, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 74, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 77, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 77.

[0221] In an aspect, an anti-IL-lRAP antibody, or antigen binding portion thereof, is a humanized ADVH60 antibody (e.g., ADVH63). The humanized ADVH63 antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-lRAP antibody (e.g., ADVH63), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 73, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 73, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 78, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 78.

[0222] In an aspect, an anti-IL-lRAP antibody, or antigen binding portion thereof, is a humanized ADVH60 antibody (e.g., ADVH64). The humanized ADVH64 antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-lRAP antibody (e.g., ADVH64), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 73, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 73, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 79, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 79.

[0223] In an aspect, an anti-IL-lRAP antibody, or antigen binding portion thereof, is a humanized ADVH60 antibody (e.g., ADVH65). The humanized ADVH65 antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-lRAP antibody (e.g., ADVH65), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 75, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 75, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 77, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 77.

[0224] In an aspect, an anti-IL-lRAP antibody, or antigen binding portion thereof, is a humanized ADVH60 antibody (e.g., ADVH66). The humanized ADVH66 antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-lRAP antibody (e.g., ADVH66), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 75, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 75, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 78, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 78.

[0225] In an aspect, an anti-IL-lRAP antibody, or antigen binding portion thereof, is a humanized ADVH60 antibody (e.g., ADVH67). The humanized ADVH67 antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-lRAP antibody (e.g., ADVH67), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 76, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 76, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 77, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 77.

[0226] In an aspect, an anti-IL-lRAP antibody, or antigen binding portion thereof, is a humanized ADVH60 antibody (e.g., ADVH68). The humanized ADVH68 antibody comprises a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-lRAP antibody (e.g., ADVH68), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 76, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 76, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 78, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 78.

[0227] To generate and select CDRs having preferred IL- 1RAP binding and / or neutralizing activity with respect to hIL-lRAP, standard methods known in the art for generating antibodies, or antigen binding portions thereof, and assessing the IL-1 RAP binding and / or neutralizing characteristics of those antibodies, or antigen binding portions thereof, may be used, including but not limited to those specifically described herein.

[0228] In certain aspects, the antibody comprises a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In certain embodiments, the anti-IL-1RAP antibody, or antigen-binding portion thereof, comprises a heavy chain immunoglobulin constant domain selected from the group consisting of a human IgG constant domain, a human IgM constant domain, a human IgE constant domain, and a human IgA constant domain. In further embodiments, the antibody, or antigen-binding portion thereof, has an IgGl heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 constant region, or an IgG4 heavy chain constant region. Preferably, the heavy chain constant region is an IgGl heavy chain constant region or an IgG4 heavy chain constant region. In one embodiment, the antibody, or antigen-binding portion thereof, is an IgG4 isotype.

[0229] Furthermore, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.

[0230] In certain embodiments, the antibody is an antigen-binding antibody fragment such as, for example, a Fab, a F(ab'), a F(ab')2, a Fd chain, a single-chain Fv (scFv), a single-chain antibody, a disulfide-linked Fv (sdFv), a fragment comprising either a Vl or Vh domain, or fragments produced by a Fab expression library, or an IL-1 RAP-binding fragments of any of the above antibodies described supra. Antigen-binding antibody fragments, including single-chain antibodies, can comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, ChI, Ch2, Ch3 and Cl domains. Also, antigen-binding fragments can comprise any combination of variable region(s) with a hinge region, ChI, Ch2, Ch3 and Cl domains. Typically, the antibodies are human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries, from human B cells, or from animals transgenic for one or more human immunoglobulin, as described infra and, for example in U.S. Pat. Nos. 5,939,598 and 6,111,166.

[0231] In certain specific embodiments, the anti-IL-lRAP antibody is agonistic, non-agonistic or antagonistic with respective to IL-1RAP. In another specific embodiment, the anti-IL-1RAP antibody does not block binding of IL-1RAP ligand or IL-1RAP coreceptors to IL-1RAP. In yet another embodiment, the anti-IL- 1RAP antibody or derivative thereof is a blocking antibody (i.e., an antibody that blocks the binding of an IL-1RAP ligand to IL-1RAP). Replacements of amino acid residues in the Fc portion to alter antibody effector function are have been described (Winter, et al. U.S. Pat. Nos. 5,648,260 and 5,624,821, incorporated by reference herein). The Fc portion of an antibody mediates several important effector functions e.g. cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life / clearance rate of antibody and antigen-antibody complexes. In some cases these effector functions are desirable for therapeutic antibody but in other cases might be unnecessary or even deleterious, depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgGl and IgG3, mediate ADCC and CDC via binding to FcyRs and complement Clq, respectively. Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies. In still another embodiment at least one amino acid residue is replaced in the constant region of the antibody, for example the Fc region of the antibody, such that effector functions of the antibody are altered.

[0232] Another embodiment of the disclosure provides a glycosylated binding protein wherein the anti-IL-1 RAP antibody or antigen-binding portion thereof comprises one or more carbohydrate residues. Nascent in vivo protein production may undergo further processing, known as post-translational modification. In particular, sugar (glycosyl) residues may be added enzymatically, a process known as glycosylation. The resulting proteins bearing covalently linked oligosaccharide side chains are known as glycosylated proteins or glycoproteins. Antibodies are glycoproteins with one or more carbohydrate residues in the Fc domain, as well as the variable domain Carbohydrate residues in the Fc domain have important effect on the effector function of the Fc domain, with minimal effect on antigen binding or half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast, glycosylation of the variable domain may have an effect on the antigen binding activity of the antibody. Glycosylation in the variable domain may have a negative effect on antibody binding affinity, likely due to steric hindrance (Co, M. S., et al., Mol. Immunol. (1993) 30:1361-1367), or result in increased affinity for the antigen (Wallick, S. C., et al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717-2723).

[0233] One aspect of the disclosure is directed to generating glycosylation site mutants in which the O- or N-linked glycosylation site of the binding protein has been mutated. One skilled in the art can generate such mutants using standard well-known technologies. Glycosylation site mutants that retain the biological activity, but have increased or decreased binding activity, are another object of the disclosure.

[0234] In still another embodiment, the glycosylation of the anti-IL-lRAP antibody or antigen-binding portion is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos. 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety.

[0235] Additionally or alternatively, a modified anti-IL-lRAP antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC / ADCP ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. See, for example, Shields, R. L. et al. (2002) J. Biol. Chern. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP 1,176,195; PCT Publications WO 03 / 035835; WO 99 / 54342 80, each of which is incorporated herein by reference in its entirety.

[0236] Protein glycosylation depends on the amino acid sequence of the protein of interest, as well as the host cell in which the protein is expressed. Different organisms may produce different glycosylation enzymes (e.g., glycosyltransferases and glycosidases), and have different substrates (nucleotide sugars) available. Due to such factors, protein glycosylation pattern, and composition of glycosyl residues, may differ depending on the host system in which the particular protein is expressed. Glycosyl residues useful may include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Preferably the glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human.

[0237] Differing protein glycosylation may result in differing protein characteristics. For instance, the efficacy of a therapeutic protein produced in a microorganism host, such as yeast, and glycosylated utilizing the yeast endogenous pathway may be reduced compared to that of the same protein expressed in a mammalian cell, such as a CHO cell line. Such glycoproteins may also be immunogenic in humans and show reduced half-life in vivo after administration. Specific receptors in humans and other animals may recognize specific glycosyl residues and promote the rapid clearance of the protein from the bloodstream. Other adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, a practitioner may prefer a therapeutic protein with a specific composition and pattern of glycosylation, for example glycosylation composition and pattern identical, or at least similar, to that produced in human cells or in the species-specific cells of the intended subject animal.

[0238] Expressing glycosylated proteins different from that of a host cell may be achieved by genetically modifying the host cell to express heterologous glycosylation enzymes. Using recombinant techniques, a practitioner may generate antibodies or antigen binding portions thereof exhibiting human protein glycosylation. For example, yeast strains have been genetically modified to express non-naturally occurring glycosylation enzymes such that glycosylated proteins (glycoproteins) produced in these yeast strains exhibit protein glycosylation identical to that of animal cells, especially human cells (U.S. patent Publication Nos. 20040018590 and 20020137134 and PCT publication WO2005100584 A2).

[0239] Antibodies may be produced by any of a number of techniques. For example, expression from host cells, wherein expression vector(s) encoding the heavy and light chains is (are) transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express antibodies in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferable, and most preferable in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[0240] Preferred mammalian host cells for expressing the recombinant antibodies disclosed herein include Chinese Hamster Ovary (CHO or CHO KI cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0241] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and / or the heavy chain of an antibody. Recombinant DNA technology may also be used to remove some, or all, of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the disclosure. In addition, bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the disclosure and the other heavy and light chain are specific for an antigen other than the antigens of interest by crosslinking an antibody of the disclosure to a second antibody by standard chemical crosslinking methods. B. Antibody-Drug Conjugates

[0242] Some aspects of the present disclosure are directed to an anti-IL-lRAP antibodydrug conjugate (ADC). Some aspects disclosed herein provide antibody-drug conjugates comprising anti-IL-lRAP antibodies (e.g., humanized anti-IL-lRAP antibodies, such as the humanized ADVH60 derivatives described herein), or antigen binding portions thereof. Other aspects of the disclosure are related to a pharmaceutical composition comprising an anti-IL-lRAP antibody-drug conjugate and a pharmaceutically acceptable carrier. In an embodiment, the antibodies disclosed herein bind human IL-1RAP expressed on tumor cells and immunosuppressive cells such as MDSC cells or others.

[0243] Anti-IL-lRAP ADCs comprise an anti-IL-lRAP antibody, i.e., an antibody that specifically binds to IL-1 RAP, linked to one or more drug moieties via a linker. The specificity of the ADC is defined by the specificity of the antibody, i.e., anti-IL-lRAP. In one embodiment, an anti-IL-lRAP antibody is linked to one or more cytotoxic drug(s) (i.e., cytotoxic drug moieties) which is delivered internally to a cancer cell or immunosuppressive cells expressing IL-1RAP.

[0244] Examples of drugs that may be used in the anti-IL- 1RAP ADCs are provided below, as are linkers that may be used to conjugate the antibody and the one or more drug(s). The terms “drug,” “agent,” and “drug moiety” are used interchangeably herein. The terms “linked” and “conjugated” are also used interchangeably herein and indicate that the antibody and moiety are covalently linked.

[0245] In an aspect, an antibody drug conjugate (ADC) is provided comprising the formula (Ab) - [(L) - (D)m]n, or a pharmaceutically acceptable salt thereof; wherein: (Ab) is an antibody or antigen binding fragment thereof that binds IL-1 RAP, wherein the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3, 7-9, 13-15, 19-21, or 25-27, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, or 28-30, respectively; (L) is a linker; (D) is a drug moiety; m is an integer from 1 to 8; and n is an integer from 1 to 12, wherein the linker (L) links (Ab) to (D).

[0246] In some aspects, an ADC is provided comprising a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 8, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-1RAP antibody (e.g., ADVH66), or antigenbinding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 75, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 75, and / or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 78, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 78. In some aspects, the linker comprises a mc-GGFG linker. In some aspects, the drug moiety comprises CHEM008.

[0247] In another aspect, provided herein is a method for producing an anti-IL-1 RAP antibody drug conjugate comprising: (a) chemically linking a linker as described herein (e.g., a cleavable linker described herein such as a cleavable peptide linker) to a drug moiety described herein (e.g., a topoisomerase I inhibitor described herein such as DXd or CHEM008) to form a linker-drug; (b) conjugating the linker-drug to an antibody as described herein (a humanized anti-IL-1RAP antibody described herein); and (c) purifying the antibody drug conjugate.

[0248] Techniques for conjugating therapeutic agents to proteins, and in particular to antibodies, are well-known. (See, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO 89 / 12624.)

[0249] Processes for conjugating antibodies to sulfhydryl-containing cytotoxic agents such as maytansinoids have been described previously (see, e.g., U.S. Pat. Nos. 5,208,020, 5,416,064, and 6,441,163). For example, U.S. Pat. Nos. 5,208,020 and 5,416,064 disclose a process for manufacturing antibody-maytansinoid conjugates wherein the antibody is first modified with a heterobifunctional reagent such as described in U.S. Pat. Nos. 4,149,003, 4,563,304 and U.S. Patent Application Publication No. 2004 / 0241174 Al. U.S. Pat. Nos. 5,208,020 and 5,416,064 further describe conjugation of a modified antibody with an excess of a sulfhydryl-containing cytotoxic agent at pH 7, followed by purification on Sephadex™ G25 chromatography columns. Purification of antibody-drug conjugates by size exclusion chromatography (SEC) also has been described (see, e.g., Liu et al., Proc. Natl. Acad. Sci. (USA), 93: 8618-8623 (1996), and Chari et al., Cancer Research, 52: 127-131 (1992)). Additional methods for generating purified ADCs has been described in, e.g., U.S. Pat. No. 9,789,204, which is incorporated herein by reference in its entirety.

[0250] In accordance with the methods described herein, the anti-IL-lRAP ADC or ADC derivative may be internalized within an IL-lRAP-expressing cell, where the ADC or ADC derivative exerts a therapeutic effect (e.g., a cytotoxic, cytostatic, or immuno stimulatory effect). Methods for determining accumulation and rates of accumulation are found in, e.g., U.S. Patent Pub. No. 2005 / 0180972, the disclosure of which is incorporated by reference herein. Alternatively, or in addition to internalization of the payload, when the payload released is permeable or transmembrane, it may also induce bystander effect to enhance the efficacy of ADC. Moreover, the bystander effect of these drugs may also alter the tumor microenvironment, which in turn may further enhance the killing effect of ADCs (Staudacher AH and Brown MP. Br. J. Cancer. 2017; 117(12): 1736-42). The degree to which an ADC mediates bystander killing may depend on factors such as the extent of ADC internalisation after binding to the target antigen, the presence of a non-cleavable or cleavable linker, and the hydrophobicity of the attached drug moiety. ADCs may contain a cleavable linker, which can be cleaved at a defined pH range or by specific proteases to release the free drug. Although the resulting free drug can directly kill the target cell, it can diffuse out of the target cell to cause bystander killing depending on the drug type and its physicochemical properties.

[0251] In some embodiments, the anti-IL-lRAP ADC or ADC derivative is effective to deplete or inhibit IL-lRAP-expressing cells via ADCC, ADCP, and CDC effects from Fc binding to FCR on the surface of killers (NK cells, macrophages, etc.), thereby mediating the direct killing effects.

[0252] To minimize activity of the therapeutic agent outside the activated immune cells or IL- IRAP-expressing cancer cells, the therapeutic agent may be conjugated in a manner that reduces its activity unless it is cleaved off the antibody (e.g., by hydrolysis or by a cleaving agent). In some embodiments, the therapeutic agent is attached to the antibody or derivative thereof with a cleavable linker that is sensitive to cleavage in the intracellular environment of the activated immune cell or IL-IRAP-expressing cancer cell but is not substantially sensitive to the extracellular environment, such that the conjugate is cleaved from the antibody or derivative thereof when it is internalized by the activated immune cell or IL-IRAP-expressing cancer cell (e.g., in the endosomal or, for example by virtue of pH sensitivity or protease sensitivity, in the lysosomal environment or in a caveolea). In other embodiments, the therapeutic agent is attached to the antibody or derivative thereof with a cleavable linker that is sensitive to cleavage in the extracellular environment surrounding the target activated immune cell or IL-IRAP-expressing cancer cell (e.g., the tumor microenvironment), such that the conjugate is cleaved from the antibody or derivative thereof when it reaches the extracellular space surrounding the activated immune cell or IL-IRAP-expressing cancer cell.

[0253] Further, in certain embodiments, the ADC or ADC derivative comprises a therapeutic agent that is charged relative to the plasma membrane, thereby further minimizing the ability of the agent to cross the plasma membrane once internalized by a cell. As used herein, a “charged agent” means an agent that (a) is polarized, such that one region of the agent has a charge relative to the plasma membrane, or (b) has a net charge relative to the plasma membrane.

[0254] Typically, the anti- IL-1RAP antibody-drug conjugate (ADC) or ADC derivative is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). In certain specific embodiments, the anti-IL-lRAP ADC or ADC derivative is 40% pure, more typically about 50% pure, and most typically about 60% pure. In other specific embodiments, the anti-IL-lRAP ADC or ADC derivative is at least approximately 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-98% pure. In another specific embodiment, the anti-IL-lRAP ADC or ADC derivative is approximately 99% pure. C. Linkers

[0255] An anti-IL-lRAP ADC comprises an anti-IL-lRAP antibody (e.g., a humanized anti-ILlRAP antibody, such as a humanized ADVH60 derivative as described herein) and at least one drug(s), wherein the antibody and the at least one drug are conjugated by a linker. A linker may include one conjugating component or may include multiple components. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment. In some embodiments, the linker is cleavable under extracellular conditions (e.g., the extracellular tumor microenvironment surrounding an IL-lRAP-expressing cancer cell), such that cleavage of the linker releases the therapeutic agent from the antibody in the extracellular environment.

[0256] For example, the linker may include a spacer, which is a moiety that extends the drug linkage to avoid, for example, shielding the active site of the antibody or improving the solubility of the ADC. Other examples of components of linkers include a stretcher unit and an amino acid unit.

[0257] Two methods are commonly used for conjugating drugs to antibodies: alkylation of reduced interchain cysteine disulfides through an enzymatically non-cleavable maleimido or simple and cleavable disulfide linker, and acylation of lysines by cleavable linear amino acids. Site-specific conjugation strategies have also been developed, and include the introduction of reactive cysteine residues (see, e.g., Junutula JR et al. Nat. Biotechnol. 2008; 26, 925-932), unnatural amino acids including N-acetyl-L-phenylalanine, azido methyl-L-phenylalanine and azido lysine (see, e.g., Hallam TJ et al. Mol. Pharm. 2015; 12, 1848-1862), glycan remodeling and glycoconjugation (see, e.g., Agarwal P et al. Bioconjug. Chern. 2015; 26, 176-192), and click chemistry conjugation of azide-containing moieties to lysine residues on the antibody (see, e.g., Chio TI and Bane SL. Methods Mol. Biol. 2020; 2078:83-97).

[0258] In one aspect, a linker covalently attaches an antibody to a drug moiety. An ADC is prepared using a linker having reactive functionality for binding to the antibody and the drug. For example, a cysteine thiol, or an amine, e.g., N-terminus or amino acid side chain such as lysine, of the antibody may form a bond with a functional group of the linker.

[0259] In one aspect, a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary such reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., the conjugation method at page 766 of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773.

[0260] In some embodiments, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Non-limiting exemplary such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0261] Suitable linkers include, for example, cleavable and non-cleavable linkers. A linker may be a “cleavable linker,” facilitating release of a drug. Non-limiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), protease-sensitive (e.g., peptidasesensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020). A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker.

[0262] Linkers may be stable extracellularly in a sufficient manner to be therapeutically effective. Before transport or delivery into a cell, the ADC is preferably stable and remains intact, i.e. the antibody remains conjugated to the drug moiety. Linkers that are stable outside the target cell may be cleaved at some efficacious rate once inside the cell. Thus, an effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety; and (iii) maintain the therapeutic effect, e.g., cytotoxic effect, of a drug moiety.

[0263] In some cases, linkers are cleaved extracellularly in the milieu surrounding a target cell (e.g., cleaved by an extracellular protease present in the extracellular tumor microenvironment surrounding an IL-lRAP-expressing cancer cell). A linker may be stable outside of a cell but then be cleaved once it has reached the extracellular space surrounding the target cell, wherein, for example, the linker may be sensitive to the proteases present in the extracellular space surrounding the target cell. Linkers that are stable outside the target cell may be cleaved at some efficacious rate once in an area of extracellular space containing cleaving agents (e.g., extracellular proteases). Examples of extracellular cleavable linkers include disulfide based linkers and dipeptide valinecitrulline (Val-Cit) linkers (see, e.g., Bahou C et al. Chern. Commun. 2019; 55(98):14829-14832). Disulfide linkers release cargo through disulfide exchange in environments containing high concentrations of thiols. The Val-Cit linker undergoes enzymatic cleavage by cathepsin B, a cysteine protease that is typically overexpressed in cancers; although this protease is usually found in the lysosome it has also displayed extracellular activity in cancers. Thus, an effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow delivery, e.g., extracellular and / or intracellular delivery, of the drug moiety; and (iii) maintain the therapeutic effect, e.g., cytotoxic effect, of a drug moiety.

[0264] In one embodiment, the linker is cleavable under intracellular or extracellular conditions, such that cleavage of the linker sufficiently releases the drug from the antibody in the intracellular environment or extracellular space of a target cell to be therapeutically effective. In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chern. 264:14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome, or the pH in the tumor microenvironment surrounding a solid tumor (see, e.g., Zhang X et al. J. Nucl. Med. 2010; 51(8):1167-1170). In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0265] In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea). In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, that is present in the extracellular environment (e.g., within the tumor microenvironment of a target cancer cell).The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typical are peptidyl linkers that are cleavable by enzymes that are present in IL-lRAP-expressing cells. Examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the val-cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.

[0266] In other embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0267] In yet other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation. See, e.g., U.S. Publication No. 20050238649, which is incorporated by reference herein in its entirety. An ADC (e.g., anti-IL-lRAP ADC) comprising a non-cleavable linker may be designed such that the ADC remains substantially outside the cell and interacts with certain receptors on a target cell surface such that the binding of the ADC initiates or prevents / blocks a particular cellular signaling pathway, for example, blocking of IL-1 or IL-33 signaling downstream of the IL-1RAP receptor.

[0268] In embodiments, the linker is a linker disclosed in U.S. Pat. Pub. US2023 / 0086097, and PCT Pubs. WO2023 / 025248, WO2022 / 171101, WO2022 / 135332, and WO2023040793, each of which is incorporated herein by reference in its entirety. For example, in some embodiments, the linker is selected from the following group:

[0269] , wherein each of R I Lla , R I Llb and R I Llc is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -no2 , -CN, -OH, -SH, -NH 2 , -C(0)H, -CO 2 H, -C(O)C(O)H, -C(O)CH 2 C(O)H, -S(O)H, -S(O) 2 H, -C(0)NH 2 , -SO 2 NH 2 , -0C(0)H, -N(H)SO 2 H, alkyl, alkenyl, alkynyl, alicyclic, heterocyclic, aryl and heteroaryl. In other embodiments, the linker is selected from the following group: N3' and In some aspects, the linker comprises H. In some aspects, the linker consists of H K. In some aspects, the linker is Mc-GGFG.

[0270] In embodiments, the linker is a linker disclosed in U.S. Pat. Nos. 9,987,373, 11,547,763, and 10,072,096, U.S. Pat. Pubs. US20210030886, US20210353766, US20190262467, US20220096652, US20230226208, US20230114866, US20210163623, US20160257764, US20160235861, and US20230100074, and International Pat. Pubs. WO2022108452, WO2022167689, WO2022136705, WO2022058395, and WO2021260232, each of which is incorporated herein by reference in its entirety. In embodiments, the linker comprises a substituted benzylic O,O-acetal or O,N-acetal, that is first activated by an enzymatic hydrolysis or reduction mechanism, that induces enhanced acid-sensitivity and thus acid-mediated hydrolysis of the 0,0-acetal or O,N-acetal to release eventually an aliphatic alcohol or amino group, which is part of the payload. In embodiments, cleavage of the linker liberates a hydroxy-acylated version of the original payload with enhanced bystander killing function. In embodiments, the linker comprises a group according to the following formula or a salt thereof: , wherein: a is 0 or 1; and R1 is selected from the group consisting of hydrogen, C1-C24 alkyl groups, C3-C24 cycloalkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups, the C1-C24 alkyl groups, C3- C 24 cycloalkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3- C24 (hetero)arylalkyl groups optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S or NR3 wherein R3is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R1 is an additional target molecule D, wherein the target molecule is optionally connected to N via a spacer moiety.

[0271] In some aspects, the linker is represented by the formula: wherein: BM is a branching moiety; E is a capping group; SG is a sulfamide group according to formula (1); b is independently 0 or 1; c is 0 or 1; d is 0 or 1; e is 0 or 1; f is an integer in the range of 1 to 10; g is 0 or 1; i is 0 or 1; k is 0 or 1; 1 is 0 or 1; Sp1 is a spacer moiety; Sp2is a spacer moiety; Sp3is a spacer moiety; Sp4 is a spacer moiety; Sp5 is a spacer moiety; Sp6 is a spacer moiety; Z1 is a connecting          group;           Z2is           a          connecting           group, wherein one of the bonds labelled with * is connected to reactive group Q1 and one of the bonds labelled with * is connected to target molecule D, and wherein the sulfamide group SG is (1) O ° 0 ♦ 1 ¥ ♦ H | represented by formula (1):                      R1                                 , wherein a is 0 or 1; and Rxis selected from the group consisting of hydrogen, C1-C24 alkyl groups, C3-C 24 cycloalkyl groups, C2-C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups, wherein the C1-C24 alkyl groups, C3-C24 cycloalkyl groups, C2- C24 (hetero)aryl groups, C3-C24 alkyl(hetero)aryl groups and C3-C24 (hetero)arylalkyl groups are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR3 wherein R3is independently selected from the group consisting of hydrogen and Ci-C4 alkyl groups, or R1 is a further target molecule D, wherein the target molecule is optionally connected to N via a spacer moiety, and wherein one of the bonds labelled with * is connected to the branching moiety, optionally via spacer Sp5, and the other bond labelled with * to a capping group E, optionally via spacer Sp6. A spacer-moiety is herein defined as a moiety that spaces (i.e. provides distance between) and covalently links together two (or more) parts of a linker. The linker may be part of e.g. a linker-construct, the linker-conjugate or a bioconjugate, as defined below.

[0272] In embodiments, the linker has the structure: , wherein the wavy bond labeled with * is connected to Z and the wavy bond labeled with ** is connected to NH; Sp1 and Sp2 are each individually spacer moieties; n is an integer in the range of 1 - 5, A is a 5- or 6-membered aromatic or heteroaromatic ring, each R17 is individually an amino acid side chain, and R21 is selected from H, R22, C(0)0H and C(O)R22, wherein R22 is Ci - C24 (hetero)alkyl groups, C3 -Cw (hetero)cycloalkyl groups, C2 - Cw (hetero)aryl groups, C3 - CIO alkyl(hetero)aryl groups and C3 - Cw (hetero)arylalkyl groups, which optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR23 wherein R23 is independently selected from the group consisting of hydrogen and Ci - C4 alkyl groups.

[0273] In some aspects, the linker is a linker disclosed in U.S. Pat. Pub. US20230158154 and International Pat. Pubs. WO2023105087 and WO2023083900, each of which is incorporated herein by reference in its entirety. In some aspects, the linker comprises a spacer unit selected from the group consisting of (Cl-ClO)alkylene-C(O), (C3-C8)carbocyclo-C(O), arylene-C(O), (Cl-C lO)alkylene-arylene-C(O), arylene-(C 1-C lO)alkylene-C(O), (C1-C 10)alkylene-(C3-C8)carbocyclo-C(O), (C3-C8)carbocyclo-(Cl-C10)alkylene-C(O), (C3-C8)heterocyclo-C(O), (Cl-C10)alkylene-(C3-C8)heterocyclo-C(O), and (C3-C8)heterocyclo-(Cl-C10)alkylene-C(O). In comprises the structure: embodiments, the linker some # wherein # indicated the attachment point to the antibody moiety and * indicates the attachment point to the drug moiety. In some aspects, the linker comprises a spacer comprising the structure: wherein Lp is a parallel connector unit; Rs is, each independently, a polyalkylene glycol unit; M is, each independently, a bond or a moiety that binds Rs with Lp; s* is an integer ranging from 1 to 4; preferably, s* is 1; and the wavy lines indicate the attachment point to the antibody moiety and to another part of the linker, when present, or to a drug moiety. In some embodiments, the linker comprises the structure: o ’ wherein Rs is a polyalkylene glycol unit; and * indicates the attachment point to the antibody moiety; and ## indicates the attachment point to the drug moiety. In some embodiments, Rs, each independently, comprises 1 to 100 subunits having the structure: In some embodiments, M is each independently selected from the group consisting of -NH-, -0-, -S-, -0(0)-0-, -C(0)-NH- and -(Cl-ClO)alkylene.

[0274] In some embodiments, the linker comprises at least one spacer unit joining the antibody moiety to the cleavable moiety. In some embodiments, the spacer unit in the linker may comprise at least one polyethylene glycol (PEG) moiety. The PEG moiety may, for example, comprise -(PEG)m-, wherein m is an integer from 1 to 10. In some embodiments, the spacer unit in the linker comprises (PEG)2. In some embodiments, an ADC that comprises a shorter spacer unit (e.g., (PEG)2) demonstrates lower aggregation levels and / or higher drug loading relative to an ADC that comprises a longer spacer unit (e.g., (PEG)8) despite the shorter linker length.

[0275] In some embodiments, the spacer unit in the linker attaches to the antibody moiety of the ADC via a maleimide moiety (Mal). In some embodiments, an ADC that comprises a linker attached to the antibody moiety via a Mal demonstrates higher drug loading relative to an ADC that comprises a linker attached to the antibody moiety via an alternate moiety. In some embodiments, the Mal in the linker is reactive with a cysteine residue on the antibody moiety. In some embodiments, the Mal in the linker is joined to the antibody moiety via a cysteine residue. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the linker comprises Mal-(PEG)m, e.g., Mal-(PEG)2. In some embodiments, the linker comprises Mal-(PEG)2. In some embodiments, the Mal-spacer unit attaches the antibody moiety to the cleavable moiety in the linker. In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the linker comprises Mal-(PEG)2-Val-Cit. Additional examples of pegylated linkers are described in, e.g., U.S. Patent Pub. No. 2022 / 0072146, which is incorporated herein by reference in its entirety.

[0276] In some embodiments, the cleavable moiety in the linker is directly joined to the drug moiety of the ADC, and the cleavable moiety is either directly connected to the antibody moiety or connected through a spacer unit. In some embodiments, a spacer unit also attaches the cleavable moiety in the linker to the drug moiety. In some embodiments, the spacer unit that attaches the cleavable moiety in the linker to the drug moiety is self-immolative. In some embodiments, the self-immolative spacer is capable of releasing the drug moiety in a target cell. In some embodiments, the self-immolative spacer unit comprises a p-aminobenzyl alcohol. In some embodiments, the self-immolative spacer unit comprises p-aminobenzyloxycarbonyl (pAB). The pAB in the linker, in some embodiments, attaches the cleavable moiety to the drug moiety. In some embodiments, the cleavable moiety is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and a PEG spacer unit joining the linker to the antibody moiety through a Mal.

[0277] In some embodiments, the linker is substantially hydrophilic linker (e.g., PEG4Mal and sulfo-SPDB). A hydrophilic linker may be used to reduce the extent to which the drug may be pumped out of resistant cancer cells through MDR (multiple drug resistance) or functionally similar transporters.

[0278] In some aspects, the linker comprises a legumain-cleavable linker. Legumain is a tumor-associated asparaginyl endopeptidase (S. Ishii, Methods Enzymol. 1994, 244, 604; J. M. Chen et al. J. Biol. Chern. 1997, 272, 8090) and has been utilized for processing of prodrugs of small cytotoxic molecules, for example of doxorubicin and etoposide derivatives among others (W. Wu et al. Cancer Res. 2006, 66, 970; L. Stem et al. Bioconjugate Chern. 2009, 20, 500; K. M. Bajjuri et al. ChemMedChem 2011, 6, 54). Examples of legumain-cleavable moieties are described in, e.g., U.S. Pat. No. 11,660,351, which is incorporated herein by reference.

[0279] In other embodiments, the linker is designed to facilitate bystander killing (the killing of neighboring cells) through diffusion of the linker-drug and / or the drug alone to neighboring cells. In other, embodiments, the linker promotes cellular internalization.

[0280] The presence of a sterically hindered disulfide can increase the stability of a particular disulfide bond, enhancing the potency of the ADC. Thus, in one embodiment, the linker includes a sterically hindered disulfide linkage. A sterically hindered disulfide refers to a disulfide bond present within a particular molecular environment, wherein the environment is characterized by a particular spatial arrangement or orientation of atoms, typically within the same molecule or compound, which prevents or at least partially inhibits the reduction of the disulfide bond. Thus, the presence of bulky (or sterically hindering) chemical moieties and / or bulky amino acid side chains proximal to the disulfide bond prevents or at least partially inhibits the disulfide bond from potential interactions that would result in the reduction of the disulfide bond.

[0281] Notably, the aforementioned linker types are not mutually exclusive. For example, in one embodiment, the linker used in the anti-IL-lRAP ADCs described herein is a non-cleavable linker that promotes cellular internalization followed by antibody degradation and drug release. Non-cleavable linkers rely on complete degradation of the antibody component of ADC by cytosolic and lysosomal proteases, which eventually liberates the payload linked to an amino acid residue derived from the degraded antibody.

[0282] In some embodiments, the linker is cleavable under reducing conditions. In some embodiments, the linker is cleavable in the presence of a reducing agent, such as glutathione or dithiothreitol. In some embodiments, the linker is a cleavable disulfide linker or a cleavable sulfonamide linker.

[0283] In some embodiments, the linker is a cleavable disulfide linker. A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT. See, e.g., Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935. Disulfide linkers are typically used to exploit the abundance of intracellular thiols, which can facilitate the cleavage of their disulfide bonds. The intracellular concentrations of the most abundant intracellular thiol, reduced glutathione, are generally in the range of 1-10 nM, which is about 1,000-fold higher than that of the most abundant low-molecular thiol in the blood (i.e., cysteine) at about 5 pM (Goldmacher et. al., In Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (G. L. Phillips ed., Springer, 2013)). The intracellular enzymes of the protein disulfide isomerase family may also contribute to the intracellular cleavage of a disulfide linker. Cleavage of disulfide linkers may also occur in the extracellular space by the presence of free thiols. As used herein, a cleavable disulfide linker refers to any linker that comprises a cleavable disulfide moiety. The term “cleavable disulfide moiety” refers to a disulfide bond that can be cleaved and / or reduced, e.g., by a thiol or enzyme. In some embodiments, the cleavable disulfide moiety is disulfidyl-dimethyl.

[0284] In some embodiments, the linker is a cleavable sulfonamide linker. As used herein, a cleavable sulfonamide linker refers to any linker that comprises a cleavable sulfonamide moiety. The term “cleavable sulfonamide moiety” refers to a sulfonamide group, i.e., sulfonyl group connected to an amine group, wherein the sulfur-nitrogen bond can be cleaved.

[0285] In some embodiments, the linker may be a dendritic type linker for covalent attachment of more than one drug moiety to an antibody moiety through a branching, multifunctional linker moiety. See, e.g., Sun et al. (2002) Bioorg. Med. Chern. Lett. 12:2213-5; Sun et al. (2003) Bioorg. Med. Chern. 11:1761-8. Dendritic linkers can increase the molar ratio of drug to antibody, i.e., drug loading, which is related to the potency of the ADC. Thus, where an antibody moiety bears only one reactive cysteine thiol group, for example, a multitude of drug moieties may be attached through a dendritic linker. In some embodiments, the linker moiety or linker-drug moiety may be attached to the antibody via reduced disulfide bridging chemistry or limited lysine utilization technology. See, e.g., Inti. Publ. Nos. WO2013173391 and WO2013173393, each of which is incorporated herein by reference.

[0286] In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker that comprises a cleavable peptide moiety. The term “cleavable peptide moiety” refers to any chemical bond linking amino acids (natural or synthetic amino acid derivatives) that can be cleaved by an agent (e.g., a protease) that is present in the intracellular environment or extracellular environment. For instance, a linker may comprise an alanine-alanine-asparagine (Ala-Ala-Asn) sequence or a valine-citrulline (Val-Cit) sequence that is cleavable by a peptidase such as cathepsin, e.g., cathepsin B.

[0287] In some embodiments, the linker is an enzyme-cleavable linker and a cleavable peptide moiety in the linker is cleavable by the enzyme. In some embodiments, the cleavable peptide moiety is cleavable by a lysosomal enzyme, e.g., cathepsin. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the cleavable peptide moiety in the linker is cleavable by a lysosomal cysteine cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin B. An exemplary dipeptide that may be cleaved by cathepsin B is valinecitrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chern. 13:855-69). In some embodiments, an ADC that comprises a cleavable peptide moiety demonstrates lower aggregation levels and / or higher drug loading (p) relative to an ADC that comprises an alternate cleavable moiety (e.g., a cleavable disulfide moiety or a cleavable sulfonamide moiety).

[0288] In some embodiments, the linker or the cleavable peptide moiety in the linker comprises an amino acid unit. In some embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug moiety from the ADC upon exposure to one or more intracellular proteases, such as one or more lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-84; Dubowchik and Walker (1999) Pharm. Therapeutics 83:67-123). In some embodiments, the amino acid unit allows for cleavage of the linker by an extracellular protease, thereby facilitating release of the drug moiety from the ADC upon exposure to a protease presence in an extracellular space such as a tumor microenvironment. Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), alanine-lysine (Ala-Lys), alanine-valine (Ala-Vai), valine-alanine (Vai-Ala), valine-lysine (Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit), tryptophan-citrulline (Trp-Cit), and phenylalanine-alanine (Phe-Ala). Exemplary tripeptides include, but are not limited to, alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine-citrulline (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly), phenylalanine-phenylalanine-lysine (Phe-Phe-Lys), and glycine-phenylalanine-lysine (Gly-Phe-Lys). Other exemplary amino acid units include, but are not limited to, Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu, Phe-N9-tosyl-Arg, and Phe-N9-Nitro-Arg, as described in, e.g., U.S. Pat. No. 6,214,345. In some embodiments, the amino acid unit in the linker comprises Val-Cit. In some embodiments, the amino acid unit in the linker comprises Ala-Ala-Asn. In some embodiments, an ADC that comprises Val-Cit demonstrates decreased off-target cell killing, increased on-target cell killing, lower aggregation levels, and / or higher drug loading (p) relative to an ADC that comprises an alternate amino acid unit or an alternate cleavable moiety. An amino acid unit may comprise amino acid residues that occur naturally and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, a lysosomal protease such as cathepsin B, C, D, or S, or a plasmin protease.

[0289] In some aspects, the linker is a selectively cleavable peptide linker. In some embodiments, the selectively cleavable peptide linker comprises a tripeptide. In embodiments, the tripeptide has the sequence -P3-P2-P1-, wherein Pl, P2, and P3 are each an amino acid, wherein a first one of the amino acids Pl, P2, or P3 is negatively charged; a second one of the amino acids Pl, P2, or P3 has an aliphatic side chain with hydrophobicity no greater than that of leucine; and a third one of the amino acids Pl, P2, or P3 has hydrophobicity lower than that of leucine, wherein the first one of the amino acids Pl, P2, or P3 corresponds to any one of Pl, P2, or P3, the second one of the amino acids Pl, P2, or P3 corresponds to one of the two remaining amino acids Pl, P2, or P3, and the third one of the amino acids Pl, P2, or P3 corresponds to the last remaining amino acids Pl, P2, or P3, provided that -P3-P2-P1- is not -Glu-Val-Cit- or -Asp-Val-Cit-. Specific examples of selectively cleavable peptide linker are described, e.g., in U.S. Patent Pub. No. 2021 / 0138077, which is incorporated herein by reference in its entirety.

[0290] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0291] In embodiments, the linker is not substantially sensitive to the extracellular environment. As used herein, “not substantially sensitive to the extracellular environment,” in the context of a linker, means that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of ADC or ADC derivative, are cleaved when the ADC or ADC derivative is present in an extracellular environment (e.g., in plasma). Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating independently with plasma both (a) the ADC or ADC derivative (the “ADC sample”) and (b) an equal molar amount of unconjugated antibody or therapeutic agent (the “control sample”) for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample with that present in control sample, as measured, for example, by high performance liquid chromatography.

[0292] In other embodiments, the linker is sensitive to the extracellular environment (e.g., the linker is cleaved when the ADC or ADC derivative is present in an extracellular environment, for example, a tumor microenvironment). The role of extracellular proteases in cancer progression is well-known, especially in relation to the promotion of cell invasion through extracellular matrix remodeling. Metalloproteases that are highly active in the tumor microenvironment include the matrix metalloproteases (MMPs), a disintegrin and metalloproteases (ADAMs), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs). In addition, serine proteases (e.g., granzyme B and kallikreins), cysteine proteases (e.g., cathepsins, caspases, and calpains), and aspartic proteases (e.g., renin, cathepsins D and E, pepsin C, and napsin A) are found in the tumor extracellular environment (see, e.g., Vizovisek M et al. Int. J. Mol. Sci. 2021; 22(5): 2514).

[0293] In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to the therapeutic agent (i.e., in the milieu of the linker-therapeutic agent moiety of the ADC or ADC derivate as described herein). In yet other embodiments, the linker promotes cellular internalization when conjugated to both the therapeutic agent and the anti-IL-lRAP antibody or derivative thereof (i.e., in the milieu of the ADC or ADC derivative as described herein).

[0294] In one embodiment, the amino acid unit is valine-citrulline (vc or val-cit). In another aspect, the amino acid unit is phenylalanine-lysine (i.e., fk). In yet another aspect of the amino acid unit, the amino acid unit is N-methylvaline-citrulline. In yet another aspect, the amino acid unit is 5-aminovaleric acid, homo phenylalanine lysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine, glycine serine valine glutamine and isonepecotic acid.

[0295] Another approach for the generation of ADCs involves the use of heterobifunctional cross-linkers which link the anti-IL-lRAP antibody to the drug moiety. Examples of cross-linkers that may be used include N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate or the highly water-soluble analog N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), and N-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SSNPB), N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SMNP), N-succinimidyl-4-(5-N,N-dimethylcarboxamido-2-pyridyldithio) butyrate (SCPB) or N-sulfosuccinimidyl4-(5-N,N-dimethylcarboxamido-2-pyridyldithio) butyrate (SSCPB)). The antibodies may be modified with the cross-linkers N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, SPDB, SNPB, SSNPB, SMNP, SCPB, or SSCPB can then react with a small excess of a particular drug that contains a thiol moiety to give excellent yields of an ADC (see also U.S. Pat. No. 6,913,748, incorporated by reference herein).

[0296] In one embodiment, charged linkers (also referred to as pro-charged linkers) are used to conjugate anti-IL-lRAP antibodies to drugs to form ADCs. Charged linkers include linkers that become charged after cell processing. The presence of a charged group(s) in the linker of a particular ADC or on the drug after cellular processing provides several advantages, such as (i) greater water solubility of the ADC, (ii) ability to operate at a higher concentration in aqueous solutions, (iii) ability to link a greater number of drug molecules per antibody, potentially resulting in higher potency, (iv) potential for the charged conjugate species to be retained inside the target cell, resulting in higher potency, and (v) improved sensitivity of multidrug resistant cells, which would be unable to export the charged drug species from the cell. Examples of some suitable charged or pro-charged cross-linkers and their synthesis are shown in FIGS. 1 to 10 of U.S. Pat. No. 8,236,319, and are incorporated by reference herein. Preferably, the charged or pro-charged cross-linkers are those containing sulfonate, phosphate, carboxyl or quaternary amine substituents that significantly increase the solubility of the ADCs, especially for ADCs with 2 to 20 conjugated drugs. Conjugates prepared from linkers containing a pro-charged moiety would produce one or more charged moieties after the conjugate is metabolized in a cell.

[0297] Additional examples of linkers that can be used with the compositions and methods described herein include valine-citrulline; maleimidocaproyl; amino benzoic acids; p- aminobenzylcarbamoyl (PAB); lysosomal enzyme-cleavable linkers; maleimidocaproyl-polyethylene glycol (MC(PEG)6-OH); N-methyl-valine citrulline; N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate      (SMCC); N-Succinimidyl 4-(2- pyridyldithio)butanoate (SPDB); and N-Succinimidyl 4-(2-pyridylthio)pentanoate (SPP) (See also US 2011 / 0076232). Another linker for use includes an avidin-biotin linkage to provide an avidin biotin-containing ADC (See also U.S. Pat. No. 4,676,980, PCT publication Nos WO1992 / 022332A2, WO1998 / 035704A1, WO2003 / 093793A2, WO2006 / 089668A1, WO 1994 / 016729A1, WO 1999 / 019500A1, WO2004 / 050016A2, WO2007 / 150020A1, WO 1995 / 015770A1, WO2001 / 09785A2, WO2005 / 081898A2, WO2008 / 135237A1, WO 1997 / 031655A2, WO2001 / 090198A1, WO2006 / 083562A2, WO2010 / 111198A1, WO2011 / 057216A1, WO2011 / 058321Al, WO2012 / 027494A1, and EP77671B1), wherein some such linkers are resistant to biotinidase cleavage. Additional linkers that may be used include a cohesin / dockerin pair to provide a cohesion-dockerin-containing ADC (See PCT publication Nos. WO2008 / 097866A2, WO2008 / 097870A2, WO2008 / 103947A2, and WO2008 / 103953A2).

[0298] Additional linkers may contain non-peptide polymers (examples include, but are not limited to, polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (poly(lactic acid)), PLGA (poly(lactic acid-glycolic acid)), and combinations thereof, wherein a preferred polymer is polyethylene glycol) (See also PCT publication No. WO2011 / 000370). Additional linkers are also described in WO 2004-010957, U.S. Publication No. 20060074008, U.S. Publication No. 20050238649, and U.S. Publication No. 20060024317, each of which is incorporated by reference herein in its entirety).

[0299] For an ADC comprising a maytansinoid, many positions on maytansinoids can serve as the position to chemically link the linking moiety. In one embodiment, maytansinoids comprise a linking moiety that contains a reactive chemical group are C-3 esters of maytansinol and its analogs where the linking moiety contains a disulfide bond and the chemical reactive group comprises a N-succinimidyl or N-sulfosuccinimidyl ester. For example, the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy and the C-20 position having a hydroxy group are all useful. The linking moiety most preferably is linked to the C-3 position of maytansinol.

[0300] The conjugation of the drug to the antibody via a linker can be accomplished by any technique known in the art. A number of different reactions are available for covalent attachment of drugs and linkers to antibodies. This may be accomplished by reaction of the amino acid residues of the antibody, including the amine groups of lysine, the free carboxylic acid groups of glutamic and aspartic acid, the sulfhydryl groups of cysteine and the various moieties of the aromatic amino acids. One of the most commonly used non-specific methods of covalent attachment is the carbodiimide reaction to link a carboxy (or amino) group of a compound to amino (or carboxy) groups of the antibody. Additionally, bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody. Also available for attachment of drugs to antibodies is the Schiff base reaction. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the antibody. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to antibodies. Other techniques are known to the skilled artisan and within the scope of the disclosure.

[0301] In certain embodiments, an intermediate, which is the precursor of the linker, is reacted with the drug under appropriate conditions. In certain embodiments, reactive groups are used on the drug or the intermediate. The product of the reaction between the drug and the intermediate, or the derivatized drug, is subsequently reacted with the anti-IL-lRAP antibody under appropriate conditions. The synthesis and structure of exemplary linkers, stretcher units, amino acid units, self-immolative spacer units are described in U.S. Patent Application Publication Nos. 20030083263, 20050238649 and 20050009751, each if which is incorporated herein by reference.

[0302] Stability of the ADC may be measured by standard analytical techniques such as mass spectroscopy, HPLC, and the separation / analysis technique LC / MS. D. Exemplary Drug Moieties for Conjugation

[0303] Anti-IL-lRAP antibodies may be used in ADCs to target one or more drug moieties to a cell of interest, e.g., a cell expressing IL-1RAP. The anti-IL-lRAP ADCs disclosed herein provide a targeted therapy that may, for example, reduce the side effects often seen with anti-cancer therapies, as the one or more drug moieties is delivered to a specific cell or to cells surrounding the IL-1RAP expressing cells.

[0304] Useful classes of cytotoxic or immunosuppressive agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, pre-forming compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like and their derivatives.

[0305] Individual cytotoxic or immunosuppressive agents include, for example, an androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU), mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

[0306] In some typical embodiments, the therapeutic agent is a cytotoxic agent. Suitable cytotoxic agents include, for example, dolastatins (e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholinodoxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.

[0307] Examples of drug moieties that may be used in the ADCs described herein, i.e., drug moieties that may be conjugated to the anti-IL-lRAP antibodies, are provided below, and include a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, a topoisomerase inhibitor, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an auristatin, an amanitin, a pyrrolobenzodiazepine, an RNA polymerase inhibitor, an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a TLR agonist, a STING agonist, a DNA minor groove binder, and a DHFR inhibitor.

[0308] Additional examples of drug moieties that may be included in the anti-IL-lRAP antibody drug conjugates include mitotic inhibitors, antitumor antibiotics, immunomodulating agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormone agents, glucocorticoids, photoactive therapeutic agents, oligonucleotides, radioactive isotopes, radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors, and combinations thereof.

[0309] Mitotic Inhibitors

[0310] In one aspect, anti-IL-lRAP antibodies may be conjugated to one or more mitotic inhibitor(s) to form an ADC for the treatment of cancer. The term “mitotic inhibitor”, as used herein, refers to a cytotoxic and / or therapeutic agent that blocks mitosis or cell division, a biological process particularly important to cancer cells. A mitotic inhibitor disrupts microtubules such that cell division is prevented, often by effecting microtubule polymerization (e.g., inhibiting microtubule polymerization) or microtubule depolymerization (e.g., stabilizing the microtubule cytoskeleton against depolymrization). Thus, in one embodiment, an anti-IL-lRAP antibody of the invention is conjugated to one or more mitotic inhibitor(s) that disrupts microtubule formation by inhibiting tubulin polymerization. In another embodiment, an anti-IL-1 RAP antibody of the invention is conjugated to one or more mitotic inhibitor(s) that stabilizes the microtubule cytoskeleton from deploymerization. In one embodiment, the mitotic inhibitor used in the ADCs of the invention is Ixempra (ixabepilone). Examples of mitotic inhibitors that may be used in the anti-IL-lRAP ADCs of the invention are provided below. Included in the genus of mitotic inhibitors are auristatins, described below.

[0311] Dolastatins

[0312] The anti-IL-lRAP antibodies of the invention may be conjugated to at least one dolastatin to form an ADC. Dolastatins are short peptidic compounds isolated from the Indian Ocean sea hare Dolabella auricularia (see Pettit et al., J. Am. Chern. Soc., 1976, 98, 4677). Examples of dolastatins include dolastatin 10 and dolastatin 15. Dolastatin 15, a seven-subunit depsipeptide derived from Dolabella auricularia, and is a potent antimitotic agent structurally related to the antitubulin agent dolastatin 10, a five-subunit peptide obtained from the same organism. Thus, in one embodiment, the anti-IL-lRAP ADC of the invention comprises an antiIL-1 RAP antibody, as described herein, and at least one dolastatin. Auristatins are synthetic derivatives of dolastatin 10.

[0313] Auristatins

[0314] Anti-IL-lRAP antibodies may be conjugated to at least one auristatin. Auristatins represent a group of dolastatin analogs that have generally been shown to possess anticancer activity by interfering with microtubule dynamics and GTP hydrolysis, thereby inhibiting cellular division. For example, Auristatin E (U.S. Pat. No. 5,635,483) is a synthetic analogue of the marine natural product dolastatin 10, a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (G. R. Pettit, Prog. Chern. Org. Nat. Prod, 70: 1-79 (1997)). Dolastatin 10, auristatin PE, and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds. Exemplary embodiments of the auristatin subclass of mitotic inhibitors include, but are not limited to, monomethyl auristatin D (MMAD or auristatin D derivative), monomethyl auristatin E (MMAE or auristatin E derivative), monomethyl auristatin F (MMAF or auristatin F derivative), auristatin F phenylenediamine (AFP), auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AE ester (AEVB). The synthesis and structure of auristatin derivatives are described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649 and 2005-0009751; International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO 02 / 088172, and U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is incorporated by reference herein.

[0315] In one embodiment, anti-IL-lRAP antibodies are conjugated to at least one MMAE (monomethyl auristatin E). Monomethyl auristatin E (MMAE, vedotin) inhibits cell division by blocking the polymerization of tubulin. Because of its super toxicity, it also cannot be used as a drug itself. In recent cancer therapy developments, it is linked to a monoclonal antibody (mAb) that recognizes a specific marker expression in cancer cells and directs MMAE to the cancer cells. In one embodiment, the linker linking MMAE to the anti-IL-1 RAP antibody is stable in extracellular fluid (i.e., the medium or environment that is external to cells), but is cleaved by cathepsin once the ADC has bound to the specific cancer cell antigen and entered the cancer cell, thus releasing the toxic MMAE and activating the potent anti-mitotic mechanism.

[0316] The structure of MMAE is provided below.

[0317]

[0318] Monomethyl Auristatin E (MMAE) In one embodiment, the antibody is coupled to a single drug and, therefore, has a DAR of 1. In certain embodiments, the ADC will have a DAR of 2 to 8, or, alternatively, 2 to 4.

[0319] Maytansinoids

[0320] The anti-IL-lRAP antibodies of the invention may be conjugated to at least one maytansinoid to form an ADC. Maytansinoids are potent antitumor agents that were originally isolated from members of the higher plant families Celastraceae, Rhamnaceae, and Euphorbiaceae, as well as some species of mosses (Kupchan et al, J. Am. Chern. Soc. 94:1354-1356

[1972] ; Wani et al, J. Chern. Soc. Chern. Commun. 390:

[1973] ; Powell et al, J. Nat. Prod. 46:660-666

[1983] ; Sakai et al, J. Nat. Prod. 51:845-850

[1988] ; and Suwanborirux et al, Experientia 46:117-120

[1990] ). Evidence suggests that maytansinoids inhibit mitosis by inhibiting polymerization of the microtubule protein tubulin, thereby preventing formation of microtubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al., Science, 189, 1002-1005 (1975)). Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models, and in vivo using laboratory animal systems. Moreover, the cytotoxicity of maytansinoids is 1,000-fold greater than conventional chemotherapeutic agents, such as, for example, methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Pat. No. 5,208,020).

[0321] Maytansinoids to include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogues and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated by reference herein). C-3 esters of maytansinol can be naturally occurring or synthetically derived. Moreover, both naturally occurring and synthetic C-3 maytansinol esters can be classified as a C-3 ester with simple carboxylic acids, or a C-3 ester with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic maytansinoid analogues are described in, for example, Kupchan et al., J. Med. Chern., 21, 31-37 (1978).

[0322] Suitable maytansinoids for use in ADCs of the invention can be isolated from natural sources, synthetically produced, or semi-synthetically produced. Moreover, the maytansinoid can be modified in any suitable manner, so long as sufficient cytotoxicity is preserved in the ultimate conjugate molecule. In this regard, maytansinoids lack suitable functional groups to which antibodies can be linked. A linking moiety desirably is utilized to link the maytansinoid to the antibody to form the conjugate, and is described in more detail in the linker section below. The structure of an exemplary maytansinoid, mertansine (DM1), is provided below.

[0323] Mertansine (DM1)

[0324] Representative examples of maytansinoids include, but are not limited, to DM1 (N2'-deacetyl-N2'-(3-mercapto-l-oxopropyl)-maytansine; also referred to as mertansine, drug maytansinoid 1; ImmunoGen, Inc.; see also Chari et al. (1992) Cancer Res 52:127), DM2, DM3 (N2'-deacetyl-N2'-(4-mercapto- l-oxopentyl)-maytansine), DM4 (4-methyl-4-mercapto-1-oxopentyl)-maytansine), and maytansinol (a synthetic maytansinoid analog). Other examples of maytansinoids are described in U.S. Pat. No. 8,142,784, incorporated by reference herein.

[0325] Ansamitocins are a group of maytansinoid antibiotics that have been isolated from various bacterial sources. These compounds have potent antitumor activities. Representative examples include, but are not limited to ansamitocin Pl, ansamitocin P2, ansamitocin P3, and ansamitocin P4.

[0326] In one embodiment of the invention, an anti-IL-lRAP antibody is conjugated to at least one DM1. In one embodiment, an anti-IL-lRAP antibody is conjugated to at least one DM2. In one embodiment, an anti-IL-1 RAP antibody is conjugated to at least one DM3. In one embodiment, an anti-IL-lRAP antibody is conjugated to at least one DM4.

[0327] Antitumor Antibiotics

[0328] Anti-IL-lRAP antibodies may be conjugated to one or more antitumor antibiotic(s) for the treatment of cancer. As used herein, the term “antitumor antibiotic” means an antineoplastic drug that blocks cell growth by interfering with DNA and is synthesized by a microorganism. Often, antitumor antibiotics either break up DNA strands or slow down or stop DNA synthesis. Examples of antitumor antibiotics that may be included in the anti-IL-lRAP ADCs include, but are not limited to, actinomycines (e.g., pyrrolo[2,l-c][l,4]benzodiazepines), anthracyclines, calicheamicins, and duocarmycins. In addition to the foregoing, additional antitumor antibiotics that may be used in the anti-IL-1 RAP ADCs include bleomycin (Blenoxane, Bristol-Myers Squibb), mitomycin, and plicamycin (also known as mithramycin).

[0329] Immunomodulating Agents

[0330] In one aspect, anti-IL-lRAP antibodies may be conjugated to at least one immunomodulating agent. As used herein, the term “immunomodulating agent” refers to an agent that can stimulate or modify an immune response. In one embodiment, an immunomodulating agent is an immuno stimulator which enhances a subject's immune response. In another embodiment, an immunomodulating agent is an immunosuppressant which prevents or decreases a subject's immune response. An immunomodulating agent may modulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) or lymphoid cells (T cells, B cells and natural killer (NK) cells) and any further differentiated cell thereof. Representative examples include, but are not limited to, bacillus calmette-guerin (BCG) and levamisole (Ergamisol). Other examples of immunomodulating agents that may be used in the ADCs include, but are not limited to, cancer vaccines, and cytokines. Representative examples of immuno stimulatory agents include, but are not limited to, STING agonists and TLR agonists. Representative STING agonists include, for example, DMXAA, c-di-AMP, c-di-GMP, diABZIs, 3'3'-cGAMP, and 2'3'-cGAMP (see, e.g., Su T et al. Theranostics. 2019; 9(25):7759). TLR agonists may include, for example, TLR3 agonists (e.g., polyribosinic-polyribocytidic acid (Poly EC), TLR4 agonists (e.g., monophosphoryl lipid A), TLR7 / 8 agonists (e.g., imidazoquinolines such as imiquimod and resiquimod), and TLR9 agonists (e.g., D-, K or C-type oligodeoxynucleotide (ODN)) (described in, e.g., Li K et al. Int. J. Mol. Sci. 2017; 18(2):404).

[0331] As used herein, the term “cancer vaccine” refers to a composition (e.g., a tumor antigen and a cytokine) that elicits a tumor-specific immune response. The response is elicited from the subject's own immune system by administering the cancer vaccine, or, in the case of the instant disclosure, administering an ADC comprising an anti-IL-lRAP antibody and a cancer vaccine. In preferred embodiments, the immune response results in the eradication of tumor cells in the body (e.g., primary or metastatic tumor cells). The use of cancer vaccines generally involves the administration of a particular antigen or group of antigens that are, for example, present on the surface a particular cancer cell, or present on the surface of a particular infectious agent shown to facilitate cancer formation. In some embodiments, the use of cancer vaccines is for prophylactic purposes, while in other embodiments, the use is for therapeutic purposes. Non-limiting examples of cancer vaccines that may be used in the anti-IL-lRAP ADCs include, recombinant bivalent human papillomavirus (HPV) vaccine types 16 and 18 vaccine (Cervarix, GlaxoSmithKline), recombinant quadrivalent human papillomavirus (HPV) types 6, 11, 16, and 18 vaccine (Gardasil, Merck & Company), and sipuleucel-T (Provenge, Dendreon). Thus, in one embodiment, the anti-IL-1RAP antibody is conjugated to at least one cancer vaccine that is either an immunostimulator or is an immunosuppressant.

[0332] The anti-IL-lRAP antibodies may be conjugated to at least one cytokine. The term “cytokine” generally refers to proteins released by one cell population which act on another cell as intercellular mediators. Cytokines directly stimulate immune effector cells and stromal cells at the tumor site and enhance tumor cell recognition by cytotoxic effector cells (Lee and Margolin (2011) Cancers 3:3856). Numerous animal tumor model studies have demonstrated that cytokines have broad anti-tumor activity and this has been translated into a number of cytokine-based approaches for cancer therapy (Lee and Margoli, supra). Recent years have seen a number of cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18 and IL-21, enter clinical trials for patients with advanced cancer (Lee and Margoli, supra).

[0333] Examples of cytokines that may be used in the ADCs include, but are not limited to, parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF; platelet-growth factor; transforming growth factors (TGFs); insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon a, p, and y, colony stimulating factors (CSFs); granulocyte-macrophage-C-SF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factor; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. Thus, in one embodiment, the disclosure provides an ADC comprising an anti-IL-lRAP antibody described herein and a cytokine.

[0334] The anti-IL-lRAP antibodies may be conjugated to at least one colony stimulating factor (CSF). Colony stimulating factors (CSFs) are growth factors that assist the bone marrow in making red blood cells. Because some cancer treatments (e.g., chemotherapy) can affect white blood cells (which help fight infection), colony-stimulating factors may be introduced to help support white blood cell levels and strengthen the immune system. Colony-stimulating factors may also be used following a bone marrow transplant to help the new marrow start producing white blood cells. Representative examples of CSFs that may be used in the anti-IL- 1RAP ADCs include, but are not limited to erythropoietin (Epoetin), filgrastim (Neopogen (also known as granulocyte colony-stimulating factor (G-CSF); Amgen, Inc.), sargramostim (leukine (granulocytemacrophage colony-stimulating factor and GM-CSF); Genzyme Corporation), promegapoietin, and Oprelvekin (recombinant IL-11; Pfizer, Inc.). Thus, in one embodiment, an ADC may comprise an anti-IL-1RAP antibody described herein and a CSF.

[0335] Alkylating Agents

[0336] The anti-IL- 1RAP antibodies may be conjugated to one or more alkylating agent(s).

[0337] Alkylating agents are a class of antineoplastic compounds that attaches an alkyl group to DNA. Examples of alkylating agents that may be used in the ADCs include, but are not limited to, alkyl sulfonates, ethylenimimes, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines and hydrazines.

[0338] DNA Damaging Agents

[0339] In one embodiment, the antibodies and antigen-binding portions thereof described herein may be conjugated to one or more DNA damaging agents. The term “DNA damaging agent”, as used herein, refers to an agent which is capable of damaging DNA and are well known to those of ordinary skill in the art (see, for example, Cheung-Ong et al., Cell Chemical Biology, 20(5): 648-659, 2013).

[0340] DNA damaging agents include DNA alkylating agents. DNA alkylating agents are a class of antineoplastic compounds that attaches an alkyl group (CnH2n+i) to DNA at a guanine base of DNA. Examples of DNA alkylating agents that may be used in the ADCs include, but are not limited to, alkyl sulfonates (e.g., busulfan), ethylenimimes (e.g., altretamine and thiotepa), methylamine derivatives, epoxides, nitrogen mustards (e.g., bendamustine, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan), nitrosoureas (e.g., carmustine, lomustine, and streptozocin), triazines (e.g., dacarbazine and temozolomide), and hydrazines.

[0341] DNA damaging agents also include indolino-benzodiazepines (IGNs). IGNs represent a chemical class of cytotoxic molecules with high in vitro potency (IC50 values in the low pmol / L range) toward cancer cells. Examples of IGN DNA alkylating agents that can be used as a cytotoxic payload in an ADC are described in Miller et al. (2016) Molecular Cancer Therapeutics, 15(8)). The IGN compounds described in Miller et al. bind to the minor groove of DNA followed by covalent reaction of guanine residues with the two imine functionalities in the molecule resulting in crosslinking of DNA. The structure of an exemplary IGN is provided below.

[0342]                                                 -

[0343] In one embodiment, a DNA damaging agent may also include a pyrrolobenzodiazepine (PBD) or pyridinobenzodiazepine (PDD) (see, e.g., N. Veillard et al. “Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity” [abstract]. In: Proceedings of the 109th Annual Meeting of the American Association for Cancer Research; 2018 Apr. 14-18; Chicago, Hl. Philadelphia (Pa.): AACR; 2018. Abstract no 736 / 3 and Stefano J. E., et al. (2013) Micro- and Mid-Scale Maleimide-Based Conjugation of Cytotoxic Drugs to Antibody Hinge Region Thiols for Tumor Targeting. In: Ducry L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology (Methods and Protocols), vol 1045. Humana Press, Totowa, N.J.)).

[0344] In another embodiment, the DNA damaging agent is a PARP inhibitor, e.g., olaparib, rucaparib, niraparib, or iniparib. In one embodiment, the PARP inhibitor is olaparib. In one embodiment, the PARP inhibitor is rucaparib. In one embodiment, the PARP inhibitor is niraparib. In one embodiment, the PARP inhibitor is iniparib. In one embodiment, the agent is a saporin toxin.

[0345] Antiangiogenic Agents

[0346] In one aspect, the anti-IL-lRAP antibodies described herein are conjugated to at least one antiangiogenic agent. Antiangiogenic agents inhibit the growth of new blood vessels. Antiangiogenic agents exert their effects in a variety of ways. In some embodiments, these agents interfere with the ability of a growth factor to reach its target. For example, vascular endothelial growth factor (VEGF) is one of the primary proteins involved in initiating angiogenesis by binding to particular receptors on a cell surface. Thus, certain antiangiogenic agents, that prevent the interaction of VEGF with its cognate receptor, prevent VEGF from initiating angiogenesis. In other embodiments, these agents interfere with intracellular signaling cascades. For example, once a particular receptor on a cell surface has been triggered, a cascade of other chemical signals is initiated to promote the growth of blood vessels. Thus, certain enzymes, for example, some tyrosine kinases, that are known to facilitate intracellular signaling cascades that contribute to, for example, cell proliferation, are targets for cancer treatment. In other embodiments, these agents interfere with intercellular signaling cascades. Yet, in other embodiments, these agents disable specific targets that activate and promote cell growth or by directly interfering with the growth of blood vessel cells. Angiogenesis inhibitory properties have been discovered in more than 300 substances with numerous direct and indirect inhibitory effects.

[0347] Representative examples of antiangiogenic agents that may be used in the ADCs include, but are not limited to, angiostatin, ABX EGF, Cl-1033, PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900, CP358, PD153035, AG1478, IMC-C225 (Erbitux, ZD1839 (Iressa), OSI-774, Erlotinib (tarceva), angiostatin, arrestin, endostatin, BAY 12-9566 and w / fluorouracil or doxorubicin, canstatin, carboxyamidotriozole and with paclitaxel, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid, IM862, Interleukin-12, Interleukin-2, NM-3, HuMV833, PTK787, RhuMab, angiozyme (ribozyme), IMC-1C11, Neovastat, marimstat, prinomastat, BMS-275291, COL-3, MM1270, SU101, SU6668, SUI 1248, SU5416, with paclitaxel, with gemcitabine and cisplatin, and with irinotecan and cisplatin and with radiation, tecogalan, temozolomide and PEG interferon a2b, tetrathiomolybdate, TNP-470, thalidomide, CC-5013 and with taxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors (deforolimus, everolimus (Afinitor, Novartis Pharmaceutical Corporation), and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinase inhibitors (e.g., erlotinib (Tarceva, Genentech, Inc.), imatinib (Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa, AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb), sunitinib (Sutent, Pfizer, Inc.), nilotinib (Tasigna, Novartis Pharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKline Pharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), phosphoinositide 3-kinases (PI3K).

[0348] Antimetabolites

[0349] The anti-IL-1 RAP antibodies may be conjugated to at least one antimetabolite. Antimetabolites are types of chemotherapy treatments that are very similar to normal substances within the cell. When the cells incorporate an antimetabolite into the cellular metabolism, the result is negative for the cell, e.g., the cell is unable to divide. Antimetabolites are classified according to the substances with which they interfere. Examples of antimetabolies that may be used in the ADCs include, but are not limited to, a folic acid antagonist (e.g., methotrexate), a pyrimidine antagonist (e.g., 5-Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine), a purine antagonist (e.g., 6-Mercaptopurine and 6-Thioguanine) and an adenosine deaminase inhibitor (e.g., Cladribine, Fludarabine, Nelarabine and Pentostatin), as described in more detail below.

[0350] Boron-Containing Agents

[0351] The anti-IL-lRAP antibody may be conjugated to at least one boron containing agent. Boron-containing agents comprise a class of cancer therapeutic compounds which interfere with cell proliferation. Representative examples of boron containing agents include, but are not limited, to borophycin and bortezomib (Velcade, Millenium Pharmaceuticals).

[0352] Chemoprotective Agents

[0353] The anti-IL-lRAP antibodies may be conjugated to at least one chemoprotective agent. Chemoprotective drugs are a class of compounds, which help protect the body against specific toxic effects of chemotherapy. Chemoprotective agents may be administered with various chemotherapies in order to protect healthy cells from the toxic effects of chemotherapy drugs, while simultaneously allowing the cancer cells to be treated with the administered chemotherapeutic. Representative chemoprotective agents include, but are not limited to amifostine (Ethyol, Medimmune, Inc.), which is used to reduce renal toxicity associated with cumulative doses of cisplatin, dexrazoxane (Totect, Apricus Pharma; Zinecard), for the treatment of extravasation caused by the administration of anthracycline (Totect), and for the treatment of cardiac-related complications caused by the administration of the antitumor antibiotic doxorubicin (Zinecard), and mesna (Mesnex, Bristol-Myers Squibb), which is used to prevent hemorrhagic cystitis during chemotherapy treatment with ifocfamide.

[0354] Photoactive Therapeutic Agents

[0355] The anti-IL-1 RAP antibodies may be conjugated to at least one photoactive therapeutic agent. Photoactive therapeutic agents include compounds that can be deployed to kill treated cells upon exposure to electromagnetic radiation of a particular wavelength. Therapeutically relevant compounds absorb electromagnetic radiation at wavelengths which penetrate tissue. In preferred embodiments, the compound is administered in a non-toxic form that is capable of producing a photochemical effect that is toxic to cells or tissue upon sufficient activation. In other preferred embodiments, these compounds are retained by cancerous tissue and are readily cleared from normal tissues. Non-limiting examples include various chromagens and dyes.

[0356] Radionuclide Agents (Radioactive Isotopes)

[0357] The anti-IL-lRAP antibodies may be conjugated to at least one radionuclide agent. Radionuclide agents comprise agents that are characterized by an unstable nucleus that is capable of undergoing radioactive decay. The basis for successful radionuclide treatment depends on sufficient concentration and prolonged retention of the radionuclide by the cancer cell. Other factors to consider include the radionuclide half-life, the energy of the emitted particles, and the maximum range that the emitted particle can travel. In preferred embodiments, the therapeutic agent is a radionuclide selected from the group consisting of niIn, 177Lu, 212Bi, 213Bi, 211At, 62Cu, 64Cu, 67Cu, 90Y, 125I, 131I, 32P, 33P, 475c, mAg, 67Ga, 142Pr, 153Sm, 161Tb, 166Dy, 166Ho, 186Re, 188Re, 189Re, 212Pb, 223Ra, 225Ac, 59Fe, 75Se, 77As, 89Sr, "Mo, 105 Rh, 109Pd, 143Pr, 149Pm, 169Er, 194Ir, 198Au, 199Au, and 211Pb. Also preferred are radionuclides that substantially decay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111 1, Sb-119,1-125, Ho-161, Os-189m and Ir-192. Decay energies of useful beta-particle-emitting nuclides are preferably Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of useful alpha-particleemitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV. Additional potential radioisotopes of use include nC, 13N, 15O, 75Br, 198Au, 224Ac, 126I, 133I, 77Br, 113mIn, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203 Hg 121niTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197pt, 109pd, 105Rh, I42p); 143pr, 161Tb, 161Tb 166H o, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 2O1T1, 225Ac, 76Br, 169Yb, and the like.

[0358] Radiosensitizers

[0359] The anti-IL-lRAP antibodies may be conjugated to at least one radiosensitizer. The term “radiosensitizer,” as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of the cells to be radiosensitized to electromagnetic radiation and / or to promote the treatment of diseases that are treatable with electromagnetic radiation. Radiosensitizers are agents that make cancer cells more sensitive to radiation therapy, while typically having much less of an effect on normal cells. Radiosensitizers are described in D. M. Goldberg (ed.), Cancer Therapy with Radiolabeled Antibodies, CRC Press (1995). Examples of radiosensitizers include gemcitabine, 5-fluorouracil, taxane, and cisplatin.

[0360] Radiosensitizers may be activated by the electromagnetic radiation of X-rays. Representative examples of X-ray activated radio sensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (lUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same. Alternatively, radiosensitizers may be activated using photodynamic therapy (PDT). Representative examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, Photofrin®, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.

[0361] Topoisomerase Inhibitors

[0362] The anti-IL-1 RAP antibodies may be conjugated to at least one topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy agents designed to interfere with the action of topoisomerase enzymes (topoisomerase I and II), which are enzymes that control the changes in DNA structure by catalyzing then breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. Representative examples of DNA topoisomerase I inhibitors include, but are not limited to, camptothecins and its derivatives irinotecan (CPT-11, Camptosar, Pfizer, Inc.) and topotecan (Hycamtin, GlaxoSmithKline Pharmaceuticals). Representative examples of DNA topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxotrubicin, epipodophyllotoxins, ellipticines, epirubicin, etoposide, razoxane, and tempo side.

[0363] In aspects, the topoisomerase I inhibitor is selected from the group consisting of CHEM008, deruxtecan, camptothecins, topotecan, irinotecan, belotecan, exatecan, Exatecan mesylate, DXd, indenoisoquinolines, indotecan, indimitecan, SN-38, and lamellarin D, or their derivatives. In some aspects, the topoisomerase I inhibitor is CHEM008. The structure of CHEM008 is shown in the formula below:

[0364] An exemplary topoisomerase I inhibitor-contianing ADC formula is depicted below, including deruxtecan (Mc-GGFG-DXd), wherein Ab represents an antibody (e.g., an anti-IL-1RAP antibody as described herein) and n is an integer between 2 to 10: Deruxtecan (MC-GGFG-Dxd)

[0365]

[0366] Another exemplary topoisomerase I inhibitor-containing ADC formula is depicted below, including CHEM008 (Mc-GGFG-CHEM008; wherein the linker is Mc-GGFG), wherein Ab represents an antibody (e.g., a humanized ADVH60 antibody disclosed herein) and n is an integer between 1 to 10 (e.g., n is 8): i                                                                                                                        n

[0367] Tyrosine Kinase Inhibitors

[0368] The anti-IL-lRAP antibodies may be conjugated to at least one tyrosine kinase inhibitor. Tyrosine kinases are enzymes within the cell that function to attach phosphate groups to the amino acid tyrosine. By blocking the ability of protein tyrosine kinases to function, tumor growth may be inhibited. Examples of tyrosine kinases that may be used on the ADCs include, but are not limited to, Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sunitinib, and Vandetanib.

[0369] MCL-1 Inhibitors

[0370] Myeloid cell leukemia sequence 1 (MCL-1) is an antiapoptotic protein that plays a key role in promoting cell survival in multiple myeloma (MM), acute myeloid leukemia (AML), and non-Hodgkin lymphoma (NHL) (see, e.g., Wei AH et al. Blood Rev. 2020; 44:100672, which is incorporated herein by reference in its entirety). Overexpression of MCL-1 is associated with treatment resistance and poor prognosis; thus, MCL-1 inhibitors are rational therapeutic options for malignancies depending on MCL-1. Examples of MCL-1 inhibitors that may be used in the ADCs described herein, i.e., MCL-1 inhibitor moieties that may be conjugated to the anti-IL- 1RAP antibodies, are provided below.

[0371] Exemplary MCL-1 modulators (including exemplary inhibitors of MCL-1) are described in WO 2020 / 236825; WO 2015 / 097123; WO 2016 / 207216; WO 2016 / 207217; WO 2016 / 207225; WO 2016 / 207226; WO 2017 / 125224; WO 2019 / 035899, WO 2019 / 035911, WO 2019 / 035914, WO 2019 / 035927, US 2019 / 0055264, WO 2016 / 033486, WO 2017 / 147410, WO 2018 / 183418, and WO 2017 / 182625, each of which is incorporated herein by reference in its entirety as exemplary MCL-1 modulators, including exemplary MCL-1 inhibitors, that can be included as drug moieties in the disclosed anti-IL-RAP ADCs. For example, exemplary MCL-1 inhibitors that can be included as drug moieties in the disclosed anti-IL-lRAP ADCs are those of formula:

[0373] wherein each variable is defined as in WO2019 / 035911; WO 2019 / 035899; WO 2019 / 035914; or WO 2019 / 035927, wherein each compound as a drug payload can be conjugated to an antibody or a linker via the nitrogen atom of the N-methyl in piperazinyl functional group of the compound.

[0374] Several MCL-1 inhibitors have entered clinical trials, including AZD5991 (NCT03218683), S64315 (NCT02979366, NCT03672695), AMG176 (NCT03797261, NCT02675452), and AMG397 (NCT03465540) (see also, Hird, AW and Tron AE. Pharmacol. Ther. 2019; 198:59-67, which is incorporated herein by reference in its entirety). AZD5991 is a highly selective macrocyclic inhibitor of MCL-1, which induces apoptosis by binding to the BH3-binding groove of MCL-1. It has shown promising activity in preclinical studies, particularly in combination with venetoclax (a BCL-2 inhibitor). S63845 is a small-molecule inhibitor that selectively targets MCL-1 by binding to its BH3-binding groove, showing effectiveness in various cancer models, including AML and multiple myeloma. AMG176 and AMG397 are potent MCL-1 inhibitors that disrupt the interaction between MCL-1 and pro-apoptotic proteins. These inhibitors are currently in clinical trials for hematologic cancers like AML and multiple myeloma.

[0375] Additionally, proteolysis-targeting chimeras (PROTACs) have been developed to be a useful technology for targeted protein degradation, for example, targeted degradation of MCL-1 (see, e.g., Li X and Song Y. J. Hematol. Oncol. 2020; 13(1):50, which is incorporated herein by reference in its entirety). A bifunctional PROTAC molecule consists of a ligand (mostly smallmolecule inhibitor) of the protein of interest (POI) and a covalently linked ligand of an E3 ubiquitin ligase (E3). Upon binding to the POI, the PROTAC can recruit E3 for POI ubiquitination, which is subjected to proteasome-mediated degradation. Exemplary PROTACs targeting MCL-1 include, but are not limited to, dMCLl-2 (CAS No: 2351218-88-5) and C3 (CAS No: 2163793-38-0). dMCLl-2 contains thalidomide and an MCL1 inhibitor A-1210477, which can successfully degrade MCL1 at nM concentrations in multiple myeloma 0PM2 cells (Papatzimas JW et al. J. Med. Chern. 2019; 62(11):5522-40). Compound C3 with pomalidomide and an MCL1 inhibitor Nap-1 induced MCL1 degradation with a DC50 of 0.7 pM (Wang Z et al. J. Med. Chern. 2019; 62(17):8152-63). C3 exhibited more potent anti-proliferative activity than MCL1 inhibitors Nap-1 and A-1210477.

[0376] Other Agents

[0377] Examples of other agents that may be used in the ADCs include, but are not limited to, abrin (e.g. abrin A chain), alpha toxin, Aleurites fordii proteins, amatoxin, crotin, curcin, dianthin proteins, diptheria toxin (e.g. diphtheria A chain and nonbinding active fragments of diphtheria toxin), deoxyribonuclease (Dnase), gelonin, mitogellin, modeccin A chain, Momordica charantia inhibitor, neomycin, onconase, phenomycin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), pokeweed antiviral protein, Pseudomonas endotoxin, Pseudomonas exotoxin (e.g. exotoxin A chain (from Pseudomonas aeruginosa}), restrictocin, ricin A chain, ribonuclease (Rnase), Sapaonaria officinalis inhibitor, saporin, alpha-sarcin, Staphylcoccal enterotoxin-A, tetanus toxin, cisplatin, carboplatin, and oxaliplatin (Eloxatin, Sanofi Aventis), proteasome inhibitors (e.g. PS-341 [bortezomib or Velcade]), HDAC inhibitors (vorinostat (Zolinza, Merck & Company, Inc.)), belinostat, entinostat, mocetinostat, and panobinostat), COX-2 inhibitors, substituted ureas, heat shock protein inhibitors (e.g. Geldanamycin and its numerous analogs), adrenocortical suppressants, and the tricothecenes. (See, for example, WO 93 / 21232). Other agents also include asparaginase (Espar, Lundbeck Inc.), hydroxyurea, levamisole, mitotane (Lysodren, Bristol-Myers Squibb), and tretinoin (Renova, Valeant Pharmaceuticals Inc.).

[0378] It should be noted that the aforementioned groups of drug moieties that may be used in the anti-IL-lRAP ADCs are not exclusive, in that certain examples of drugs may be found in more than one category, e.g., ansamitocins are both mitotic inhibitors and antitumor antibiotics.

[0379] All stereoisomers of the above drug moieties are contemplated for use herein, i.e. any combination of R and S configurations at the chiral carbons of D. III. Methods of Treatment

[0380] Disclosed herein is a method for reducing the presence of IL-1RAP positive cells and IL-1RAP activity in a subject, advantageously from a subject suffering from a IL-1RAP associated disorder, e.g., cancer such as esophageal cancer, liver cancer, lung squamous cell carcinoma, melanoma, head and neck cancer, glioblastoma, pancreatic cancer, stomach cancer, acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), NSCLC, Ewing sarcoma, or ovarian cancer. The disclosure provides methods for reducing IL-1RAP activity in a subject suffering from such a disease or disorder, which method comprises administering to the subject an ADC, antibody, or antibody portion of the disclosure such that IL-1RAP activity in the subject is reduced. ADC treatment efficacy may be evaluated for toxicity as well as indicators of efficacy and adjusted accordingly. Efficacy measures include, but are not limited to, a cytostatic and / or cytotoxic effect observed in vitro or in vivo, reduced tumor volume, tumor growth inhibition, and / or prolonged survival.

[0381] Preferably, the IL-1RAP is human IL-1RAP, and the subject is a human subject. Alternatively, the subject can be a mammal expressing an IL-1RAP to which antibodies of the disclosure are capable of binding Antibodies of the disclosure can be administered to a human subject for therapeutic purposes. Moreover, antibodies of the disclosure can be administered to a non-human mammal expressing a IL-1RAP with which the antibody is capable of binding for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies and ADCs of the disclosure (e.g., testing of efficacy, dosages and time courses of administration).

[0382] As used herein, the term “a disorder in which IL-1RAP activity is detrimental” is intended to include diseases and other disorders in which the presence of IL-1RAP in a subject suffering from the disorder has been shown to be or is suspected of being either responsible for the pathophysiology of the disorder or a factor that contributes to a worsening of the disorder. In some instances, an elevated level of IL-1RAP is present in a subject suffering from a disorder, wherein the presence of IL-1RAP is not associated with the the disorder or a factor that contributes to a worsening of the disorder. Accordingly, a disorder in which IL-1 RAP activity is detrimental is a disorder in which reduction of IL-1 RAP activity is expected to alleviate the symptoms and / or progression of the disorder. Such disorders may be evidenced, for example, by an increase in the concentration of IL-1RAP in a biological cell or tissue of a subject suffering from the disorder (e.g., an increase in the concentration of IL- 1RAP in a tumor of the subject), which can be detected, for example, using an anti-IL-lRAP antibody as described above.

[0383] Other examples of cancers that may be treated using the compositions and methods disclosed herein include, but are not limited to carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include but are not limited to breast cancer (Luminal A, TNBC, Ductal), prostate cancer, squamous cancer, squamous cell tumors, squamous cell carcinoma (e.g., squamous cell lung cancer or squamous cell head and neck cancer), neuroendocrine tumors, urothelial cancer, vulvar cancer, mesothelioma, liver cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, lung cancer, small cell lung cancer, non-small cell lung cancer, 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 (e.g., cervical squamous cell carcinoma), carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, esophageal cancer, esophageal squamous cell carcinoma, cancer of the esophagogastric junction, cancer of the small intestine, cancer of the endocrine system, 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, malignant peripheral nerve sheath tumors (MPNST), tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, environmentally induced cancers including those induced by asbestos, hematologic malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma (e.g., ALK-positive anaplastic large cell lymphoma), T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combinations of said cancers. PVNS, adrenocortico carcinoma, ladder urothelial carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, glioblastoma multiforme, chronic lymphocytic leukemia, brain lower grade glioma, head and neck squamous cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, large squamous cell carcinoma, cutaneous melanoma, ovarial serous cystadenocarcinoma, gastric cancer, soft tissue sarcoma, testicular germ cell cancer, thymoma, thyroid carcinoma, uterine corpus endometrial carcinoma, uterine carcinosarcoma, kidney renal clear cell carcinoma, and kidney renal papillary cell carcinoma. The present invention is also applicable to treatment of metastatic cancers. Methods of determining whether an ADC exerts a cytostatic and / or cytotoxic effect on a cell are known. For example, the cytotoxic or cytostatic activity of an ADC can be measured by: exposing mammalian cells expressing a target protein of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 6 days; and measuring cell viability. Cell-based in vitro assays may also be used to measure viability (proliferation), cytotoxicity, and ...

Claims

1. An antibody or antigen binding fragment thereof comprising the heavy chain variable region of any one of SEQ ID NOs: 36-40 or 73-76 and the light chain variable region of any one of SEQ ID NOs: 31-35 or 77-79.

2. The antibody or antigen binding fragment thereof of claim 1, comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3, 7-9, 13-15, 19-21, or 25-27, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, or 28-30, respectively.

3. The antibody or antigen binding fragment thereof of claim 1 or 2, wherein the antibody is a humanized antibody.

4. The humanized antibody or antigen binding fragment thereof of claim 3, wherein the antibody is a humanized ADVH60 antibody.

5. The humanized antibody or antigen binding fragment thereof of claim 3 or 4, comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 10-12, respectively.

6. The humanized antibody or antigen binding fragment thereof of any one of claims 3-5, wherein the humanized antibody or antigen binding fragment thereof comprises the heavy chain and the light chain of any one of:a) SEQ ID NO: 73 and SEQ ID NO: 77, respectively;b) SEQ ID NO: 74 and SEQ ID NO: 77, respectively;c) SEQ ID NO: 73 and SEQ ID NO: 78, respectively;d) SEQ ID NO: 73 and SEQ ID NO: 79, respectively;e) SEQ ID NO: 75 and SEQ ID NO: 77, respectively;f) SEQ ID NO: 75 and SEQ ID NO: 78, respectively;g) SEQ ID NO: 76 and SEQ ID NO: 77, respectively; orh) SEQ ID NO: 76 and SEQ ID NO: 78, respectively.

7. A method of treating cancer in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the antibody or antigen binding fragment thereof of any one of claims 1-6.

8. The method of claim 7, wherein the cancer is an esophageal cancer, liver cancer, bile ductcancer, kidney cancer, thyroid cancer, bladder cancer, mesothelioma, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, malignant peripheral nerve sheath tumor, astrocytoma, pancreatic cancer, cholangiocarcinoma, stomach cancer, ALK-positive anaplastic large cell lymphoma, Ewing sarcoma, cervical cancer, or acute myeloid leukemia.

9. The method of claim 7, wherein the cancer is a squamous cancer.

10. The method of claim 9, wherein the squamous cancer is an esophageal cancer, esophageal squamous cell carcinoma, cancer of the esophagogastric junction, squamous head and neck cancer, squamous non-small cell lung cancer, or cervical cancer.

11. The method of claim 7, wherein the cancer is an esophageal cancer, lung cancer, melanoma, head and neck cancer, glioblastoma, pancreatic cancer, acute myeloid leukemia, squamous cancer, esophageal squamous cell carcinoma, squamous head and neck cancer, squamous non-small cell lung cancer, cervical cancer, or Ewing sarcoma.

12. The method of claim 8 or 11, wherein the cancer is an Ewing sarcoma.

13. The method of claim 8 or 11, wherein the cancer is an Ewing sarcoma comprising a EWSR1:FLI1 mutation.

14. Use of the antibody or antigen binding fragment thereof of any one of claims 1-6 to treat cancer in a subject in need thereof.

15. The use of claim 15, wherein the cancer is an esophageal cancer, liver cancer, bile duct cancer, kidney cancer, thyroid cancer, bladder cancer, mesothelioma, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, malignant peripheral nerve sheath tumor, astrocytoma, pancreatic cancer, cholangiocarcinoma, stomach cancer, ALK-positive anaplastic large cell lymphoma, Ewing sarcoma, cervical cancer, or acute myeloid leukemia.

16. The use of claim 14, wherein the cancer is a squamous cancer.

17. The use of claim 15, wherein the squamous cancer is an esophageal cancer, esophageal squamous cell carcinoma, cancer of the esophagogastric junction, squamous head and neck cancer, squamous non-small cell lung cancer, or cervical cancer.

18. The use of claim 14, wherein the cancer is an esophageal cancer, lung cancer, melanoma, head and neck cancer, glioblastoma, pancreatic cancer, acute myeloid leukemia, squamous cancer, esophageal squamous cell carcinoma, squamous head and neck cancer, squamous non-small cell lung cancer, cervical cancer, or Ewing sarcoma.

19. The use of claim 15 or 18, wherein the cancer is an Ewing sarcoma.

20. The use of claim 15 or 18, wherein the cancer is an Ewing sarcoma comprising aEWSR1:FLI1 mutation.

21. The use of any one of claims 14-20, wherein the subject is a human.

22. Use of the antibody or antigen binding fragment thereof of any one of claims 1-6 in the manufacture of a medicament.

23. An antibody drug conjugate comprising the formula (Ab) - [(L) - (D)m]n, or a pharmaceutically acceptable salt thereof; wherein:(Ab) is an antibody or antigen binding fragment thereof that binds IL-1 RAP, wherein the antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3, 7-9, 13-15, 19-21, or 25-27, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, or 28-30, respectively; (L) is a linker;(D) is a drug moiety;m is an integer from 1 to 8; andn is an integer from 1 to 12,wherein the linker (L) links (Ab) to (D).

24. The antibody drug conjugate of claim 23, wherein the antibody or antigen binding fragment thereof comprises the heavy chain variable region of any one of SEQ ID NOs: 36-40 or 73-76 and the light chain variable region of any one of SEQ ID NOs: 31-35 or 77-79.

25. The antibody drug conjugate of claims 23 or 24, wherein the antibody comprises the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 1-3,7-9, 13-15, 19-21, or 25-27, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 4-6, 10-12, 16-18, 22-24, or 28-30, respectively.

26. The antibody drug conjugate of any one of claims 17-19, wherein the antibody is a humanized antibody.

27. The antibody drug conjugate of claim 26, comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 10-12, respectively.

28. The antibody drug conjugate of claim 26 or 27, wherein the humanized antibody or antigen binding fragment thereof comprises the heavy chain and the light chain of any one of:a) SEQ ID NO: 73 and SEQ ID NO: 77, respectively;b) SEQ ID NO: 74 and SEQ ID NO: 77, respectively;c) SEQ ID NO: 73 and SEQ ID NO: 78, respectively;d) SEQ ID NO: 73 and SEQ ID NO: 79, respectively;e) SEQ ID NO: 75 and SEQ ID NO: 77, respectively;f) SEQ ID NO: 75 and SEQ ID NO: 78, respectively;g) SEQ ID NO: 76 and SEQ ID NO: 77, respectively; orh) SEQ ID NO: 76 and SEQ ID NO: 78, respectively.

29. The antibody drug conjugate of any one of claims 26-28, wherein the humanized antibody or antigen binding fragment thereof comprises the heavy chain and the light chain of SEQ ID NO: 75 and SEQ ID NO: 78, respectively.

30. The antibody drug conjugate of any one of claims 23-28, wherein the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, a procharged linker, and a dicarboxylic acid based linker.

31. The antibody drug conjugate of claim 30, wherein the linker is a cleavable linker.

32. The antibody drug conjugate of claim 31, wherein the cleavable linker is cleavable underintracellular conditions.

33. The antibody drug conjugate of claim 31 or 32, wherein the cleavable linker comprises a hydrazine group, a maleimide group, a disulfide group, a cis-aconityl group, a peptide comprising 1 to 10 amino acid residues, a para-aminobenzyl alcohol group, a photolabile group, a dimethyl group, a glucuronic acid group, or a combination thereof.

34. The antibody drug conjugate of any one of claims 31-33, wherein the cleavable linker comprises a maleimide group.

35. The antibody drug conjugate of any one of claims 32-34, wherein the cleavable linker is a peptide linker cleavable by an intracellular protease.

36. The antibody drug conjugate of any one of claims 32-35, wherein the cleavable linker is a peptide linker comprising a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide.

37. The antibody drug conjugate of claim 36, wherein the dipeptide is alanine-alanine (ala-ala), valine-alanine (val-ala), valine-glycine (val-gly), glycine-glycine (gly-gly), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), phenylalanine-lysine (phe-lys), or N-methyl-valine-citrulline (Me-val -cit).

38. The antibody drug conjugate of claim 37, wherein the tripeptide is alanine-alanine-asparagine (ala-ala-asn), glutamic acid-valine-citrulline (glu-val-cit), glycine-valine-citrulline (glv-val-cit), or glycine-glycine-glycine (gly-gly-gly).

39. The antibody drug conjugate of claim 38, wherein the tetrapeptide is glycine-phenylalanine-leucine-glycine (gly-phe-leu-gly), glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly), or alanine-leucine-alanine-leucine (ala-leu-ala-leu).

40. The antibody drug conjugate of claim 36 or 39, wherein the tetrapeptide is glycine-glycine-phenylalanine-glycine (gly-gly-phe-gly).

41. The antibody drug conjugate of any one of claims 23-40, wherein the drug moiety is selected from a group consisting of a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL-1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, a topoisomerase inhibitor, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an auristatin, an amanitin, a pyrrolobenzodiazepine, an RNA polymerase inhibitor, an inhibitor of nuclear export of proteins CRM1, a DPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a TLR agonist, a STING agonist, a DNA minor groove binder, and a DHFR inhibitor.

42. The antibody drug conjugate of claim 41, wherein the drug moiety is a topoisomerase I inhibitor.

43. The antibody drug conjugate of claim 42, wherein the topoisomerase I inhibitor is selected from the group consisting of CHEM008, deruxtecan, camptothecins, topotecan, irinotecan, belotecan, exatecan, Exatecan mesylate, DXd, indenoisoquinolines, indotecan, indimitecan, SN-38, and lamellarin D, or their derivatives.

44. The antibody drug conjugate of claim 42 or 43, wherein the topoisomerase I inhibitor is CHEM008.

45. Anthe formula:antibody drug conjugate comprisinghumanized antibody or antigen binding fragment thereof comprising the HCDR1, HCDR2, andHCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 of SEQ ID NOs:10-12, respectively, and wherein n is an integer between 1 to 10.

46. The antibody drug conjugate of claim 45, wherein the humanized antibody or antigen binding fragment thereof comprises the heavy chain and the light chain of SEQ ID NO: 75 and SEQ ID NO: 78, respectively.

47. The antibody drug conjugate of claim 45 or 46, wherein n is 8.

48. An antibody drug conjugate comprising the formula (Ab) - [(L) - (D)m]n, or a pharmaceutically acceptable salt thereof; wherein:(Ab) is an antibody or antigen binding fragment thereof that binds IL-1 RAP, wherein theantibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, andHCDR3 of SEQ ID NOs: 7-9, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 10-12, respectively;(L) is Mc-GGFG;(D) is CHEM008;m is 1; andn is 8,wherein the linker (L) links (Ab) to (D).

49. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 23-48 and a pharmaceutically acceptable carrier.

50. A method for producing an anti-IL-lRAP antibody drug conjugate comprising: (a) chemically linking the linker as claimed in any one of claims 31-40 to the drug moiety CHEM008 to form a linker-drug; (b) conjugating the linker-drug to the antibody as claimed in any one of claims 23-29; and (c) purifying the antibody drug conjugate.

51. An antibody drug conjugate produced by the method of claim 50.

52. The antibody drug conjugate of claim 51 having a drug to antibody ratio (DAR) of about 8 as measured by UV spectrophotometry, mass spectrometry, hydrophobic interaction chromatography, reverse-phase HPLC, and / or capillary electrophoresis.

53. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody drug conjugate of any one of claims 2348 or 51-52 or the pharmaceutical composition of claim 49.

54. The method of claim 53, wherein the cancer is an esophageal cancer, liver cancer, bile duct cancer, kidney cancer, thyroid cancer, bladder cancer, mesothelioma, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, malignant peripheral nerve sheath tumor, astrocytoma, pancreatic cancer, cholangiocarcinoma, stomach cancer, cervical cancer, ALK-positive anaplastic large cell lymphoma, or acute myeloid leukemia.

55. The method of claim 53, wherein the cancer is a squamous cancer.

56. The method of claim 55, wherein the squamous cancer is an esophageal cancer, esophageal squamous cell carcinoma, cancer of the esophagogastric junction, squamous head and neck cancer, squamous non-small cell lung cancer, or cervical cancer.

57. The method of claim 53, wherein the cancer is an esophageal cancer, lung cancer, melanoma, head and neck cancer, glioblastoma, pancreatic cancer, acute myeloid leukemia, squamous cancer, esophageal squamous cell carcinoma, squamous head and neck cancer, squamous non-small cell lung cancer, cervical cancer, or Ewing sarcoma.

58. The method of claim 53 or 57, wherein the cancer is an Ewing sarcoma.

59. The method of claim 53 or 57, wherein the cancer is an Ewing sarcoma comprising a EWSR1:FLI1 mutation.

60. The method of any one of claims 53-59, wherein the antibody drug conjugate induces cell death of IL-1RAP+ tumor cells and / or IL-1RAP+ tumor stromal cells.

61. The method of claim 60, wherein the antibody drug conjugate induces cell death of IL-1RAP+ tumor cells and IL-1RAP+ tumor stromal cells.

62. The method of claim 60, wherein the antibody drug conjugate induces cell death of IL-1RAP+ tumor stromal cells.

63. The method of any one of claims 60-62, wherein the IL-1RAP+ tumor stromal cells comprise tumor associated myeloid cells and / or cancer associated fibroblasts.

64. The method of any one of claims 53-63, wherein the antibody drug conjugate induces bystander killing of tumor cells which do not express IL-1RAP.

65. The method of any one claims 53-64, wherein the antibody drug conjugate or pharmaceutical composition are administered to the subject in combination with one or more additional therapeutic compounds.

66. The method of claim 65, wherein the subject has been pretreated with a treatment regimen comprising topoisomerase-1 and / or topisomerase 2 inhibitors.

67. The method of claim 65 or 66, wherein the subject is a topoisomerase-1 inhibitor refractory patient.

68. The method of any one of claims 65-67, wherein the one or more additional therapeutic compounds is a standard of care chemotherapeutic agent or immune checkpoint inhibitor.

69. The method of any one of claims 53-68, wherein the subject is a human.

70. Use of the antibody drug conjugate of any one of claims 23-48 or 51-52 or thepharmaceutical composition of claim 49 to treat cancer in a subject in need thereof.

71. The use of claim 70, wherein the cancer is an esophageal cancer, liver cancer, bile duct cancer, kidney cancer, thyroid cancer, bladder cancer, mesothelioma, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, malignant peripheral nerve sheath tumor, astrocytoma, pancreatic cancer, cholangiocarcinoma, stomach cancer, cervical cancer, ALK-positive anaplastic large cell lymphoma, or acute myeloid leukemia.

72. The use of claim 70, wherein the cancer is a squamous cancer.

73. The use of claim 72, wherein the squamous cancer is an esophageal cancer, esophageal squamous cell carcinoma, cancer of the esophagogastric junction, squamous head and neck cancer, squamous non-small cell lung cancer, or cervical cancer.

74. The use of claim 70, wherein the cancer is an esophageal cancer, lung cancer, melanoma, head and neck cancer, glioblastoma, pancreatic cancer, acute myeloid leukemia, squamous cancer, esophageal squamous cell carcinoma, squamous head and neck cancer, squamous non-small cell lung cancer, cervical cancer, or Ewing sarcoma.

75. The use of claim 71 or 74, wherein the cancer is an Ewing sarcoma.

76. The use of claim 71 or 74, wherein the cancer is an Ewing sarcoma comprising aEWSR1:FLI1 mutation.

77. The use of any one of claims 70-76, wherein the subject is a human.

78. Use of the antibody drug conjugate of any one of claims 23-48 or 51-52 or thepharmaceutical composition of claim 49 in the manufacture of a medicament.

79. The antibody drug conjugate of any one of claims 23-48 or 51-52 or the pharmaceutical composition of claim 49, wherein the drug moiety is an MCL-1 inhibitor.

80. The antibody drug conjugate or pharmaceutical composition of claim 79, wherein the MCL-1 inhibitor is selected from the group consisting of: AZD5991, S63845, AMG176, AMG397, dMCLl-2, or compound C3.

81. The antibody drug conjugate of claim 48 which is ADC019.

82. A method of treating cancer in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of the antibody drug conjugate of claim 81.

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