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WO2025027529A1 - Conjugués médicament-anticorps anti-il-1rap et leurs procédés d'utilisation - Google Patents

Conjugués médicament-anticorps anti-il-1rap et leurs procédés d'utilisation Download PDF

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WO2025027529A1
WO2025027529A1 PCT/IB2024/057373 IB2024057373W WO2025027529A1 WO 2025027529 A1 WO2025027529 A1 WO 2025027529A1 IB 2024057373 W IB2024057373 W IB 2024057373W WO 2025027529 A1 WO2025027529 A1 WO 2025027529A1
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cancer
antibody
1rap
linker
inhibitor
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PCT/IB2024/057373
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Richard Charles Alfred SAINSON
Edouard AUPEPIN DE LAMOTHE-DREUZY
Olivier Favre-Bulle
Alexis Collette
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Advesya
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • interleukin-1 (IL-1) family of cytokine ligands and receptors is associated with inflammation, autoimmunity, immune regulation, cell proliferation, and host defence 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).
  • 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.
  • a co-receptor 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.
  • IL-1RAP (Wesche, H., J. Biol. Chem.272: 7727-7731, 1997; Genbank Accession No.
  • AAB4059 serves as the common cellular membrane co-receptor (common node) 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.
  • 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-1 ⁇ , IL-1 ⁇ , IL-33, IL-36 ⁇ , IL-36 ⁇ , and IL-36 ⁇ .
  • 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 Jär ⁇ s et al., 2010, Proc. Natl. Acad. Sci.
  • 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).
  • PDAC pancreatic ductal adenocarcinoma
  • Ewing sarcoma non-small cell lung cancer
  • gliomas gliomas
  • triple-negative breast cancer and stomach adenocarcinoma
  • IL-1RAP is a thus a promising tumor- associated antigen for therapeutic targeting.
  • 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.
  • 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.
  • cytotoxic molecules such as bacteria and plant toxins, radionuclides, and certain chemotherapeutic drugs have been chemically linked to monoclonal antibodies that bind tumor- specific 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 A1, 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.
  • a humanized antibody or antigen binding fragment thereof comprising the heavy chain variable region of any one of SEQ ID NOs: 8-11 and the light chain variable region of any one of SEQ ID NOs:14-17.
  • the humanized 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.
  • the antibody or antigen binding fragment thereof comprises the heavy chain of SEQ ID NO:8 and the light chain of SEQ ID NO:14.
  • the humanized antibody is a humanized ADV58 antibody.
  • 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 humanized antibody or antigen binding fragment thereof as described herein.
  • the cancer is a bladder cancer, kidney cancer, esophageal cancer, liver cancer, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, pancreatic cancer, stomach cancer, Ewing sarcoma, or acute myeloid leukemia.
  • the cancer is a squamous cancer, an esophageal cancer, esophageal squamous cell carcinoma, or cancer of the esophagogastric junction.
  • the cancer is an Ewing sarcoma comprising a EWSR1:FLI1 mutation.
  • provided herein is a use of a humanized antibody or antigen binding fragment thereof as described herein to treat cancer in a subject in need thereof.
  • a use of a humanized antibody or antigen binding fragment thereof as described herein as described herein in the manufacture of a medicament is provided herein.
  • 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, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs:4-6, 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).
  • the antibody or antigen binding fragment thereof comprises the heavy chain variable region of any one of SEQ ID NOs:8-11 and the light chain variable region of any one of SEQ ID NOs:14-17.
  • the antibody comprises the heavy chain of SEQ ID NO:8 and the light chain of SEQ ID NO:14.
  • the humanized antibody is a humanized ADV58 antibody.
  • 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.
  • the linker is a cleavable linker.
  • the cleavable linker is cleavable under intracellular conditions.
  • the cleavable linker is cleavable under extracellular conditions (e.g., cleavable by proteases present in the tumor microenvironment).
  • the cleavable linker comprises a hydrazine 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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 or/and 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,
  • the drug moiety is a topoisomerase I inhibitor.
  • the topoisomerase I inhibitor is selected from the group consisting of deruxtecan, camptothecins, topotecan, irinotecan, belotecan, exatecan, Exatecan mesylate, DXd, indenoisoquinolines, indotecan, indimitecan, SN-38, and lamellarin D, or their derivatives.
  • a pharmaceutical composition comprising an antibody drug conjugate as described herein and a pharmaceutically acceptable carrier.
  • an anti-IL-1RAP antibody drug conjugate comprising: (a) chemically linking a linker as described herein to the drug moiety deruxtecan to form a linker-drug; (b) conjugating the linker-drug to an anti-IL-1RAP antibody described herein; and (c) purifying the antibody drug conjugate.
  • 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.
  • DAR drug to antibody ratio
  • the cancer is a bladder cancer, kidney cancer, esophageal cancer, liver cancer, lung cancer, melanoma, head and neck cancer, ovarian cancer, glioblastoma, pancreatic cancer, stomach cancer, Ewing sarcoma, or acute myeloid leukemia.
  • the cancer is a squamous cancer, an esophageal cancer, esophageal squamous cell carcinoma, or cancer of the esophagogastric junction.
  • the cancer is an Ewing sarcoma comprising a EWSR1:FLI1 mutation.
  • the antibody drug conjugate induces cell death of IL-1RAP+ tumor cells.
  • the antibody drug conjugate induces bystander killing of tumor cells which do not express IL-1RAP.
  • the antibody drug conjugate induces death of immune system inhibitory IL-1RAP+ cells (e.g., IL-1RAP+ inhibitory immune cells of the tumor microenvironment).
  • 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).
  • the antibody drug conjugate or pharmaceutical composition are administered to the subject in combination with one or more additional therapeutic compounds.
  • the one or more additional therapeutic compounds is a standard of care chemotherapeutic agent or immune checkpoint inhibitor.
  • the subject is a human.
  • FIG.1 shows a graph of the expression of membranous IL-1RAP in the The Cancer Genome Atlas (TCGA) dataset based on the primary diagnosis for the 15 highest IL-1RAP- expressing indications.
  • FIG.1 shows a graph of the expression of membranous IL-1RAP in the The Cancer Genome Atlas (TCGA) dataset based on the primary diagnosis for the 15 highest IL-1RAP- expressing indications.
  • FIG. 2 shows a graph of IL-1RAP mRNA expression (log2-transformed, using a pseudo-count of 1) in cell line models from the Dependency Map (DepMap) Public 24Q2 dataset (depmap.org/portal/).
  • Cell lines are grouped by cancer types defined as Oncotree Subtype. Cancer subtypes with more than 10 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.
  • FIG.3 shows a graph the levels of IL-1RAP expression vs the number of samples (sample density) in healthy and tumor tissues based on the TCGA dataset separated between squamous and non-squamous tumors, and in healthy cells as reported in the genotype-tissue expression (GTEx) dataset.
  • FIG.4 shows a Joyplot comparing the levels of IL-1RAP expression and the density of samples in squamous tumors (by tissue) and non-squamous tumors (TCGA data), with that of matching healthy tissues (GTEx dataset).
  • FIG. 5 shows a graph comparing the expression of membranous IL-1RAP expression in tumor samples vs the matching healthy tissue.
  • the y-axis indicates the difference in mean expression of IL-1RAP (in TPM) between the tumor and the matching healthy tissue (as the mean expression in TCGA versus the mean expression in the matching healthy GTEx tissue) and the x-axis indicates the baseline expression in healthy tissue (mean expression in healthy tissue in Log2(TPM+1)).
  • the size of the dots indicates the number of samples and the colour intensity the fold change between TCGA and GTex TPM.
  • FIGs. 7A-7C show results of IL-1RAP expression analysis in several cancer cell lines from solid tumors.
  • FIG.7A reports IL-1RAP expression as antibody binding capacity (ABC) as detected by flow cytometry across several cancer cell lines as described in Example 2 and Table 2, including Capan-1, Capan-2, DoTc24510, HS 746.T, OVCAR-5, SiHa, and SNU-601.
  • FIG. 7B-7C show surface expression of IL-1RAP in SK-MEL-28 cells (FIG.7B) and DoTc24510 cells (FIG.7C).
  • FIG.8 shows the results of plasma membrane IL-1RAP protein expression analysis across several cancer cell lines from both solid tumors and AML. IL-1RAP expression is reported in terms of antibody binding capacity (ABC derived from MedFI). Raji are used as a negative control (IL-1RAP negative).
  • FIGs. 9A-9G show the results of immunohistological analysis of a pan-cancer, multi-tumor FFPE tissue microarray (TMA) for IL-1RAP expression.
  • TMA multi-tumor FFPE tissue microarray
  • FIG.9A shows a summary of IL-1RAP positivity measured as a percent of each cancer type screened in the TMA, where n indicates the number of cores representing a given cancer. Individual IL-1RAP staining scores are shown for melanoma (FIG. 9B), ovarian cancer (FIG. 9C), pancreatic cancer (FIG.
  • a score of 0 indicates no staining
  • a score of 1 indicates weak staining in 80% of tumor cells or marked staining in less than 10% of cells
  • a score of 2 indicates slight staining in 80% of tumor cells or moderate staining in 10-50 % of cells
  • a score of 3 indicates moderate staining in 80% of tumor cells or marked staining in more than 50% of cells
  • a score of 4 indicates marked to strong staining in 80% of tumor cells
  • a score of 5 indicates marked to strong and uniform staining in 100% of tumor cells.
  • FIG.10 shows the results of immunohistological analysis of an oesophagus TMA (squamous) for IL-1RAP protein expression, wherein the scores shown for individual IL-1RAP staining are as described in FIGs.9A-9G.
  • FIGs.11A-11B show the results of immunohistological analysis of a head and neck TMA.
  • FIG.11A shows the percentage of IL-1RAP positive cores across three tumor subtypes highlighting the hiher positivity in squamous cancers vs adenocarcinoma
  • FIG.11B shows the scores for individual IL-1RAP staining across all the cores (all tumor subtypes) analyzed, which are as described in FIGs.9A-9G.
  • 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.
  • FIG. 12A shows the results of Renal Papillary Cell Carcinoma (FIG. 12A), Hepatocellular Carcinoma (FIG. 12B), Stomach Adenocarcinoma (FIG. 12C), and Pancreatic Ductal Adenocarcinoma (FIG.12D).
  • FIG.14 shows IL-1RAP mRNA expression across various cancer indications from The Cancer Genome Atlas (TCGA) database.
  • TCGA Cancer Genome Atlas
  • SPR surface plasmon resonance
  • FIGs.16A-16F show the interaction analysis of a titration from 1.17 nM to 300 nM of the anti-IL-1RAP antibodies: chimeric (hIgG1) ADV58 (ADV580) (FIG.16A), ADV581 (FIG. 16B), ADV582 (FIG.16C), ADV583 (FIG.16D), mIgG1 ADV58-Vedotin (FIG.16E), and mIgG1 ADV58-DXd (FIG.16F) with reversibly immobilized human IL-1RAP.
  • 17A-17D show the binding of anti-human IL-1RAP (ADV58; mIgG1) and chimeric ADV58 (ADV580; hIgG1) to recombinant IL-1RAP protein. Binding to human IL-1RAP (hIL-1RAP; FIG. 17A), cynomolgus IL-1RAP (cIL-1RAP; FIG. 17B), rhesus IL-1RAP (rheIL- 1RAP; FIG.17C), and rat IL-1RAP (rIL-1RAP; FIG.17D) was measured by ELISA (OD 450 ) across a titration of antibody concentrations.
  • ADV580 (hIgG1) was compared to a goat anti-human IL-1 RAcP/IL-1 R3 biotinylated polyclonal antibody (BAF676, gIgG).
  • BAF676 gIgG, biotin
  • 18A-18D show the binding of anti-IL-1RAP antibodies: chimeric (hIgG1) ADV58 (ADV580), ADV581, ADV582, and ADV583 to recombinant IL-1RAP protein. Binding to human IL-1RAP (hIL-1RAP; FIG.18A), cynomolgus IL-1RAP (cIL-1RAP; FIG.18B), rhesus IL-1RAP (rheIL-1RAP; FIG. 18C), and rat IL-1RAP (rIL-1RAP; FIG. 18D) was measured by ELISA (OD450) across a titration of antibody concentrations.
  • ELISA OD450
  • FIG. 19 shows the results of an ELISA experiment to compare the binding of parental mIgG1 ADV58 antibody, vcMMAE-conjugated ADV58 antibody, and vcMMAE- conjugated isotype control B-D38 antibody to human IL-1 RAcP / IL-1 R3.
  • FIG.20 shows the binding of ADV58, deruxtecan (DXd)-conjugated ADV58, and DXd-conjugated isotype control B-D38 (3-fold serial dilutions starting from 20 nM) to human IL- 1RAP protein (1 ⁇ g/mL).
  • FIGs. 21A-21C show the binding of MMAE-, DXd-, and Tesirine-conjugated ADV581 (FIG.21A), ADV582 (FIG.21B), and ADV583 (FIG.21C) to human IL-1RAP protein (1 ⁇ g/mL).
  • FIGs.22A-22B show the results of a flow cytometry experiment to determine the expression of IL-1RAP on the surface of SK-MEL-28 cells using the anti-IL-1RAP antibody mIgG1 ADV58 or the isotype control antibody B-D38. Results are shown as either percent positive cells (FIG.2A2) or Mean Fluorescence Intensity (MFI) (FIG.22B).
  • FIGs. 23A-23B show the results of a flow cytometry experiment to compare the binding of several humanized ADV58 antibodies, including ADV581, ADV582, ADV583, and the parental murine chimeric ADV580 antibody to the surface of SK-MEL-28 cells.
  • FIG. 24 shows the results of an antibody titration experiment to determine the binding of the anti-IL-1RAP antibody ADV582 (as determined by Median Fluorescence Intensity (MFI)) to the IL-1RAP positive cell lines TC71 (Ewing Sarcoma), SK-MEL-28 (Melanoma), Kyse-270 (Esophageal squamous cell carcinoma), Hs-746T (Stomach adenocarcinoma), DETROIT-562 (Head and Neck squamous cell carcinoma), and OVCAR5 (Ovarian serous adenocarcinoma).
  • MFI Median Fluorescence Intensity
  • FIG. 25 shows the results of an anti-IL-1RAP antibody (ADV58) internalization experiment using pHrodo-labeled antibodies incubated with SK-MEL-28 cells at 37°C and monitored for 2 days.
  • FIGs. 26A-26D show the results of an anti-IL1-RAP antibody (ADV582) internalization experiment.
  • FIGs. 26A and 26B show the results of an anti-IL-1RAP antibody (ADV582) internalization experiment using FabFluor-labeled antibodies incubated at different concentrations with SK-MEL-28 cells (FIG. 26A) and TC-71 cells (FIG. 26B) at 37°C and monitored for 24 hours.
  • FIGs. 26C and 26D show the results of an anti-IL-1RAP antibody (ADV582) internalization experiment using FabFluor-labeled antibodies incubated with SK-MEL- 28 cells at 4nM (FIG. 26C) and TC-71 cells at 2nM (FIG. 26D) at 37°C and monitored for 24 hours.
  • FIGs.27A-27B show the results of show the results of two anti-IL-1RAP antibody (ADV580 and ADV582) signalling blocking experiments for either IL-1 ⁇ (FIG. 27A) or IL-33 (FIG. 27B).
  • IL-1RA was included as a positive control for IL-1 ⁇ blocking
  • astegolimab was included as a positive control for IL-33 blocking.
  • FIGs.28A-28C show the results of an in vivo mouse study on the effects of an anti- IL-1RAP antibody conjugated to vcMMAE (ADV58-vedotin) on SK-MEL-28 tumor growth.
  • FIG. 28A shows the mean tumor volume and standard deviation (STDEV) plots comparing the anti- tumor efficacy of vehicle, isotype control-vedotin, and ADV58-vedotin treated mice (dosed intravenously (IV) 3 times on day 0 [day of randomization], day 5, and day 10).
  • FIG.28B shows the mean body weight and standard deviation (STDEV) of the mice in the 3 groups treated with vehicle, isotype-vedotin and ADV58-vedotin.
  • FIG.28C shows a comparison of tumor volumes on day 36.
  • the tumor volume (in mm 3 ) from the mice in the 7 groups is indicated.
  • the statistical analysis (One-Way ANOVA with a Dunnett’s multiple comparison) compared the mean tumor size of the 7 groups on day 38. * adj. p ⁇ 0.05, ** adj. p ⁇ 0.01 and *** adj. p ⁇ 0.001.
  • FIGs.29A-29C show the results of an in vivo mouse study on the effects of an anti- IL-1RAP antibody conjugated to DXd (ADV58 DXd) on SK-MEL-28 tumor growth.
  • FIG. 29A shows the mean tumor volume and standard deviation (STDEV) plots comparing the anti-tumor efficacy of vehicle, isotype control-DXd, and ADV58-DXd treated mice (dosed intravenously (IV) 3 times on day 0 [day of randomization], day 5, and day 10).
  • FIGs.30A-30C show the results of an in vivo mouse study on the effects of an anti- IL-1RAP antibody conjugated to DXd (ADV581 DXd) or MMAE DXd (ADV581 MMAE) on Kyse-270 tumor growth.
  • FIG.30A shows the mean tumor volume (with last observations carried forward) and standard deviation (STDEV) plots comparing the anti-tumor efficacy of vehicle, isotype control-MMAE, ADV581 MMAE, isotype control DXd, and ADV581 DXd treated mice (dosed intravenously (IV) 3 times on day 0 [day of randomization based on tumor volume around 100mm 3 ], day 5, and day 10).
  • IV intravenously
  • FIG.30C shows a Kaplan-Meier analysis of median survival probability based on the time for the mean tumor volume to reach 1500 mm 3 .
  • FIGs.31A-31C show the results of an in vivo mouse study on the effects of an anti- IL-1RAP antibody conjugated to DXd (ADV581 DXd) or MMAE DXd (ADV581 MMAE) on Hs- 746T tumor growth.
  • FIG. 31A shows the mean tumor volume (with last observations carried forward) and standard deviation (STDEV) plots comparing the anti-tumor efficacy of vehicle, isotype control-MMAE, ADV581 MMAE, isotype control DXd, and ADV581 DXd treated mice (dosed intravenously (IV) 3 times on day 0 [day of randomization based on tumor volume around 100mm 3 ], day 5, and day 10).
  • IV intravenously
  • FIG.31C shows a Kaplan-Meier analysis of median survival probability based on the time for the mean tumor volume to reach 1500 mm 3 .
  • FIGs.32A-32C show the results of an in vivo mouse study on the effects of an anti- IL-1RAP antibody conjugated to DXd (ADV581 DXd) or MMAE DXd (ADV581 MMAE) on RD-ES tumor growth.
  • FIG.32A shows the results of an in vivo mouse study on the effects of the anti-IL-1RAP antibody ADV581 conjugated to DXd (ADV581 DXd) or conjugated to MMAE on RD-ES Ewind Sarcoma tumor growth.
  • the figure shows the mean tumor volume (with last observations carried forward) and standard deviation (STDEV) plots comparing the anti-tumor efficacy of vehicle, or the isotype control HEL conjugated to MMAE (isotype HEL MMAE) or DXd (isotype HEL DXd) treated mice (dosed intravenously (IV) 3 times on day 0 [day of randomization based on tumor volume around 80-120 mm3], day 5, and day 10).
  • FIG.32C shows a Kaplan-Meier analysis of median survival probability based on the time for the mean tumor volume to reach 1500 mm 3 .
  • FIGs.33A-33C show the results of an in vivo mouse study on the effects of an anti- IL-1RAP antibody conjugated to DXd (ADV581 DXd) or MMAE DXd (ADV581 MMAE) on DETROIT-562 tumor growth.
  • FIG. 33A shows the mean tumor volume (with last observations carried forward) and standard deviation (STDEV) plots comparing the anti-tumor efficacy of vehicle, isotype control-MMAE, ADV581 MMAE, isotype control DXd, and ADV581 DXd treated mice (dosed intravenously (IV) 3 times on day 0 [day of randomization based on tumor volume around 100mm 3 ], day 5, and day 10).
  • IV intravenously
  • FIG. 33C shows a Kaplan-Meier analysis of median survival probability based on the time for the mean tumor volume to reach 1500 mm 3 .
  • FIGs.34A-34C show the results of an in vivo mouse study on the effects of an anti- IL-1RAP antibody conjugated to DXd (ADV581 DXd) or MMAE DXd (ADV581 MMAE) on TOV21G tumor growth.
  • FIG.34A shows the mean tumor volume (with last observations carried forward) and standard deviation (STDEV) plots comparing the anti-tumor efficacy of vehicle, isotype control-MMAE, ADV581 MMAE, isotype control DXd, and ADV581 DXd treated mice (dosed intravenously (IV) 3 times on day 0 [day of randomization based on tumor volume around 100mm 3 ], day 5, and day 10).
  • FIG.34C shows a Kaplan-Meier analysis of median survival probability based on the time for the mean tumor volume to reach 600 mm 3 .
  • ADCs antibody-drug conjugates
  • ADC derivatives that bind to IL-1RAP.
  • a drug conjugated to the antibody exerts a cytotoxic, cytostatic, or immunostimulatory effect on IL-1RAP-expressing cells to treat IL-1RAP-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 (e.g., a humanized or chimeric form of ADV58), and such antibodies conjugated to cytotoxic or immunostimulatory agents such as, for example, chemotherapeutic drugs or activator of the innate immune system.
  • 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, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs: 4-6, 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).
  • 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.
  • 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.
  • 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.
  • m is about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8.
  • 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.
  • 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.
  • 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).
  • “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.
  • 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.
  • “about” or “comprising essentially of” can mean within 1 or more than 1 standard deviation per the practice in the art.
  • “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.
  • the terms can mean up to an order of magnitude or up to 5-fold of a value.
  • the meaning of “about” or “comprising essentially of” should be assumed to be within an acceptable error range for that particular value or composition.
  • the term “approximately,” as applied to one or more values of interest refers to a value that is similar to a stated reference value.
  • 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).
  • 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.
  • 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 IgG1 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
  • 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)
  • disease microenvironment e.g., cleavage by a protease enriched or specific in a disease microenvironment
  • chimeric antibodies e.g., cleavage by a protease enriched or specific in a disease microenvironment
  • chimeric antibodies e.g., 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. IgG1, IgG2, IgG3, IgG4, IgA1 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.
  • 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.
  • CDR refers to the complementarity determining region within antibody variable sequences.
  • CDR1 CDR1
  • CDR2 CDR2
  • CDR3 CDR3
  • HC CDR1 HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3
  • CDR set 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.
  • CDR-grafted antibody 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.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • 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.
  • labeled antibody 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.
  • 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).
  • marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides 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.
  • fluorescent labels e.g., FITC, rhodamine, lanthanide phosphors
  • enzymatic labels e.g., horseradish peroxidase, luciferase, alkaline phosphatase
  • biotinyl groups e.g., predetermined polypeptide epitop
  • an analog is meant a molecule that is not identical, but has analogous functional or structural features.
  • 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.
  • aryl generally refers to residues having an aromatic ring derived by removing one hydrogen atom.
  • aromatic ring may refer to a 6- to 14-membered all- carbon 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.
  • heteroaryl generally refers to a residue having a hydrogen atom removed from a carbon atom of a heteroaromatic ring.
  • heteromatic 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.
  • 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.
  • 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.
  • 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, heterocycloalkylthio and one or more substituents in oxo, such as hydrogen, protium, deuterium, tritium, halogen, -NO 2 , - CN, -OH, -
  • alkenyl generally refers to a straight or branched chain hydrocarbon group containing one or more double bonds.
  • alkenyl groups include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl.
  • 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.
  • 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.
  • 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.
  • 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 Contains 3 to 8 carbon atoms.
  • Non- limiting 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.
  • the term "carbocyclyl" generally refers to a residue derived from a carbon atom having a carbocyclic ring by removing one hydrogen atom.
  • carbocycle generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, wherein the carbocycle may contain 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, or may contain 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.
  • 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.
  • 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.
  • antigens are tumor antigens.
  • 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).
  • 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.
  • 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.
  • inhibition means negatively affecting (e.g., decreasing proliferation) or killing the cell.
  • inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation).
  • 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.
  • 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).
  • 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.
  • DAR drug-to-antibody ratio
  • 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.
  • the DAR is between 1 and 12.
  • the DAR is between 2 and 10.
  • the DAR is between 4 and 8.
  • the DAR is between 1 and 2.
  • the DAR is between 2 and 3. In some aspects, the DAR is between 3 and 4. In some aspects, the DAR is between 4 and 5. In some aspects, the DAR is between 5 and 6. 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 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.
  • IL-1RAP also referred to herein as IL1RAP, IL-1RAcP, C3orf13, 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).
  • 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.
  • 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: 39.
  • the extracellular domain of IL-1RAP spans residues 21 to 359, and is provided below as SEQ ID NO: 40.
  • IL-1RAP expressing tumor 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).
  • 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.
  • 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.
  • IL-1RAP positive expression is determined based on membrane staining as determined by, e.g., immunohistochemistry (IHC) or immunofluorescence (IF) analysis.
  • 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.
  • an “elevated level of IL-1RAP” is one in which 5% or more of the cells in a tumor sample have membrane staining.
  • 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.
  • the protein expression levels can be measured by IHC and/or IF analysis.
  • 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.
  • 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.
  • the protein expression levels can be measured by IHC and/or IF analysis.
  • a cell that expresses no IL-1RAP can also be described as expressing a “low level of IL-1RAP”.
  • the phrase “expresses a low level of IL-1RAP” encompasses no IL-1RAP expression.
  • a low level of IL-1RAP is within the background staining levels.
  • a sample that is IL-1RAP “negative” has no IL-1RAP expression or a low level of IL-1RAP.
  • 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.
  • NK cells include 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.
  • T-cells 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
  • 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.
  • APCs antigen-presenting cells
  • immature B-cells are formed in the bone marrow, where its name is derived from.
  • Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (KD) of 10 -5 to 10 -11 M or less. Any KD greater than about 10 -4 M is generally considered to indicate nonspecific binding.
  • 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 10 -10 M or less, but does not bind with high affinity to unrelated antigens.
  • 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.
  • K D 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 OctetTM system (ForteBio, Pall Corp. Fremont, Calif.).
  • OctetTM system FormeBio, Pall Corp. Fremont, Calif.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL-1RAP). 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.
  • an antigen e.g., IL-1RAP
  • 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 CH1 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 CH1 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 A1 herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • 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).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antigen binding portion” of an antibody.
  • scFv molecules may be incorporated into a fusion protein.
  • 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. J., et al. (1994) Structure 2:1121-1123).
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody includes: (i) a fragment or linked fragments or a similar monovalent fragment consisting of the HCDR1, HCDR2, and HCDR3 of SEQ ID NOs:1-3 and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs:4-6; or (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:7-11, represented by any one of DNA SEQ ID NOs: 8-16, respectively, and the light chain (VL) variable region of any one of SEQ ID NO
  • IL-1RAP Humanized Antibody Sequences SEQ ID NO: Name Encoded Sequence 1 HCDR1 GYTFTSYW 2 HCDR2 IYPSDSYT 3 HCDR3 KRGYYGDYFDY 4 LCDR1 QDINSY 5 LCDR2 RAN LCDR3 LQYDEFPYT VH QVQLQQPGAELVRPGASVNLACKASGYTFTSYWIN WVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKAT LTVDKSSSTAYMQLSSPTSEDSAVYYCKRGYYGDY FDYWGQGTTLTVSS VH V1 QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWIN WVKQAPGQGLEWMGNIYPSDSYTNY
  • human monoclonal antibody 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.
  • 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.
  • 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.
  • antibody construct 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.
  • recombinant human antibody 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.
  • variable 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.
  • 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.
  • 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).
  • a "human” antibody 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).
  • 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.
  • human antibodies and “fully human” antibodies are used synonymously.
  • 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.
  • 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).
  • CDR complementary determining region
  • 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.
  • 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.
  • Fc immunoglobulin constant region or domain
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG1, IgG2, IgG3 and IgG4.
  • the humanized antibody comprises a mutant version of the Fc domain, for example, a mutant IgG1 Fc domain.
  • mutant human Fc domains are provided of all the other wild-type IgG subclasses (human IgG2, IgG3, and IgG4).
  • the mutant Fc domains may exhibit improved half-life or reduced degradation after administration to a mammalian subject in vivo.
  • the humanized antibody comprises a mutant IgG1 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).
  • 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.
  • 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.
  • Humanized antibodies may be produced using several technologies, such as resurfacing and CDR grafting.
  • 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.
  • 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.
  • (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.
  • Antibodies can be humanized using a variety of other techniques including CDR- grafting (EP 0239400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and 5,585,089), veneering or resurfacing (EP 0592106; EP 0519596; 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).
  • 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.).
  • a chimeric antibody comprises at least one mouse variable region and at least one human constant region.
  • a chimeric antibody comprises at least one cynomolgus variable region and at least one human constant region.
  • 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.
  • isotype refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant region genes.
  • 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.”
  • An “isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other proteins and cellular material.
  • isolated refers 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.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • 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.
  • 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
  • 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.
  • 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.
  • topoisomerase I inhibitors include, for example, deruxtecan, camptothecins, topotecan, irinotecan, belotecan, exatecan, Exatecan mesylate, DXd, indenoisoquinolines, indotecan, indimitecan, SN-38, and lamellarin D, or their derivatives.
  • 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.
  • 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.
  • the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker.
  • 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 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).
  • SPDP N- succinimidyl 3-(2-pyridyldithio) propionate
  • SPDB N-succinimidyl 4-(2-pyridyldithio)butanoate
  • SPP disulfide N- succinimidyl 4-(2-pyridyldithio)pentanoate
  • 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.
  • the cleavable linker may be a peptide linker comprising a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide.
  • dipeptides examples include 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), and N-methyl-valine-citrulline (Me-val -cit).
  • tripeptides examples include 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).
  • tetrapeptides examples 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).
  • 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.
  • the cleaved moieties of the subject compositions are thus intracellular metabolites (e.g., Ab, Ab-L fragment, D-L fragment, D).
  • 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.
  • 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.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • diseases include neoplasias and cancers to be treated with a composition of the invention.
  • 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.
  • 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.
  • 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 urethr
  • 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.
  • 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
  • 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, Abemethy'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 sar
  • 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.
  • 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
  • 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.
  • the metastatic tumor and its cells are presumed to be similar to those of the original tumor.
  • 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.
  • metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors.
  • 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.
  • 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.
  • a disease e.g., cancer (e.g. leukemia, acute myeloid leukemia)
  • cancer e.g. leukemia, acute myeloid leukemia
  • a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.
  • the substance may be an indicator of the disease (e.g., cancer (e.g. leukemia, acute myeloid leukemia)).
  • 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)).
  • 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.
  • IL interleukin
  • 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.
  • IFN interferon
  • 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).
  • IL-la tumor necrosis factor
  • FGF fibroblast growth factor
  • FGF fibroblast growth factor
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • sICAM-1 soluble intercellular adhesion molecule 1
  • sVCAM-1 soluble vascular adhesion molecule 1
  • VEGF vascular endothelial growth factor
  • effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin.
  • acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).
  • CRP C-reactive protein
  • SAA serum amyloid A
  • 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), MIP-Ib (MIP-lb), gamma- induced protein 10 (IP- 10), and thymus and activation regulated chemokine (TARC or CCL17).
  • MDC macrophage-derived chemokine
  • MCP-1 or CCL2 monocyte chemotactic protein 1
  • MCP-4 macrophage inflammatory protein la
  • MIP-la MIP-la
  • MIP-Ib MIP-Ib
  • IP- 10 gamma- induced protein 10
  • TARC or CCL17 thymus and activation regulated chemokine
  • 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- IBB ligand (4-1BBL), a proliferation- inducing ligand (APRIL), CD70, CD153, CD178, glucocorticoid-induced TNFR-related ligand (GITRL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L, TNF- and ApoL- related leukocyte-
  • CCL chem
  • 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.
  • a T lymphocyte, B lymphocyte, natural killer (NK) cell for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil
  • soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results
  • 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.
  • an immune response refers to NK cell-mediated killing of a foreign cell, e.g., an allogeneic T cell therapy.
  • “Immune cell” as used herein means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., an autoantigen).
  • an immune cell is a T lymphocyte, a B lymphocyte, or a dendritic cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.
  • 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.
  • aqueous solvents e.g., water, alcoholic/aque
  • compositions of the present invention 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.
  • “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.
  • 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.
  • 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.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents
  • 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.
  • 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.
  • 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-1RAP antibody, or anti-IL-1RAP antibody-drug conjugate described herein for use in the treatment and/or prevention of at least one of a cancer.
  • active agent(s) e. g. anti-IL-1RAP antibody, or anti-IL-1RAP 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • at least one antibody of the present invention at least one antibody of the present invention.
  • 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.
  • Parenteral 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • compositions of the present invention can focus the delivery of the compositions of the present invention into the target cells in vivo.
  • the compositions of the present invention can also be delivered as nanoparticles.
  • a pharmaceutical composition of the present invention can be a pharmaceutical composition which comprises an anti-IL-1RAP antibody-drug conjugate, or pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable carrier.
  • doses of anti-IL-1RAP 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 short- acting, fast-releasing, long-acting, or sustained-releasing.
  • a pharmaceutical composition of the present invention can comprise further biologically active agents, depending on the intended use of the pharmaceutical composition.
  • 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.
  • a patient is human.
  • 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.
  • terapéuticaally effective dose or amount 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).
  • a drug e.g., an ADC
  • 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)).
  • 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.
  • 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.
  • DFS disease free survival
  • OS overall survival
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • 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.
  • treatment refers to slowing, stopping, or reversing the progression of an IL-1RAP-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-1RAP-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.
  • a subject in need thereof may be treated for a disease or for alleviating symptoms associated with a cancer.
  • 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.
  • a heterologous polypeptide is from a different species.
  • the term “functional,” in the context of an anti-IL-1RAP 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-1RAP-expressing cells when conjugated to a cytotoxic agent, or has an immunostimulatory effect on an immune cell when conjugated to an immunostimulatory agent such as, for example, a chemotherapeutic drug or an activator of the innate immune system.
  • an anti-IL1RAP antibody or derivative thereof is (1) capable of binding to IL- 1RAP and (2) modulating (e.g., inhibiting) IL-1, e.g., IL-1 ⁇ and/or IL-1 ⁇ , signaling; inducing cell death in cells expressing IL-1RAP, 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.
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • NSCLC non-small cell lung cancer
  • 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.
  • 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.
  • 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.
  • standard controls can be designed for assessment of any number of parameters (e.g.
  • 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.
  • Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections. [0185] Various aspects described herein are described in further detail in the following subsections. II. Compositions of the Disclosure A. Anti-IL-1RAP Antibodies [0186] Aspects disclosed herein provide humanized anti-IL-1RAP antibodies, or antigen binding portions thereof. Another aspect disclosed herein provides human anti-IL-1RAP antibodies, or antigen binding portions thereof.
  • 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). [0187] In aspects, disclosed herein are antibody drug conjugates (ADCs) comprising an anti-IL-1RAP antibody described herein and at least one drug(s) (i.e., at least one drug moiety conjugated to an anti-IL-1RAP antibody).
  • ADCs antibody drug conjugates
  • 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 have characteristics including, but not limited to, inducing cell death in cells expressing IL-1RAP, e.g., cancer cells expressing IL-1RAP.
  • an anti-IL-1RAP ADC as disclosed herein is capable of being internalized into a cell expressing IL-1RAP.
  • anti-IL-1RAP 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.”
  • the anti-IL-1RAP 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.
  • a sample that is IL-1RAP “negative” has no IL-1RAP expression or a low level of IL-1RAP.
  • 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.
  • the protein expression levels can be measured by IHC or IF analysis.
  • 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.
  • the anti-IL-1RAP antibodies, ADCs, or antigen binding fragments thereof bind IL-1RAP on cells expressing IL-1RAP.
  • the disclosure includes anti-IL-1RAP antibodies, ADCs, or antigen binding fragments thereof, that are effective at inhibiting or decreasing tumor growth.
  • 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-1 ⁇ signaling through IL-1RAP; IL-1 ⁇ , IL-1 ⁇ , and IL-38 signaling through the IL-1R; IL-33 signaling through the IL-33R, and IL-36Ra, IL-36 ⁇ , and IL-36 ⁇ signaling through the IL-36R.
  • the anti-IL-1RAP antibodies, antigen-binding portions thereof, and ADCs described herein are useful for downstream inhibition of IL-1 ⁇ pathways for the treatment of cancers, e.g., lung cancer.
  • the anti-IL-1RAP 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-1RAP antibodies, ADCs, and antigen-binding portions thereof can be used for the treatment of bladder cancer, kidney cancer, 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.
  • a cancer sample e.g., a sample representative of a cancer described herein
  • a tumor sample has a high level of IL-1RAP expression.
  • at least 5% or more of the cells in a leukemia or tumor sample have membrane staining.
  • a tumor sample obtained from a subject displays a low level of expression of IL-1RAP.
  • the expression level of IL-1RAP can be determined by any method known in the art.
  • the expression level of IL-1RAP can be determined via immunohistochemical (IHC) analysis and/or immunofluorescence (IF) analysis.
  • the cancer has been previously treated with another anti-cancer agent or anti-cancer therapy, e.g., a chemotherapy.
  • the cancer is resistant to chemotherapy.
  • 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.
  • anti-human IL-1RAP anti-hIL-1RAP
  • ADC antibody drug conjugate
  • anti-IL-1RAP antibodies and sequences thereof that can be used in the ADCs are described herein.
  • the anti-IL-1RAP antibodies described herein provide the ADCs with the ability to bind to IL-1RAP such that the cytotoxic molecule attached to the antibody may be delivered to the IL-1RAP-expressing cell, particularly an IL-1RAP expressing cancer cell.
  • an anti-IL-1RAP antibody fragment may be conjugated to the drugs, as described herein.
  • an anti-IL-1RAP 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.
  • the disclosure provides human anti-IL-1RAP 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: 8-11; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14- 17.
  • the humanized (i.e., human) anti-IL-1RAP antibodies, or antigen binding portions thereof are derived from a murine anti-IL-1RAP antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 7; and a light chain variable region comprising an amino acid sequence consisting of SEQ ID NO: 13.
  • the anti-IL-1RAP antibodies, or angen binding portions thereof comprise a heavy chain constant region comprising an amino acid sequence consisting of SEQ ID NO: 19 or SEQ ID NO: 21; and a light chain constant region comprising an amino acid sequence consisting of any one of SEQ ID NO: 23-30.
  • the humanized (i.e., human) anti-IL-1RAP antibodies, or antigen binding portions thereof are derived from a murine anti-IL-1RAP antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising a nucleic acid sequence consisting of SEQ ID NO: 12; and a light chain variable region comprising a nucleic acid sequence consisting of SEQ ID NO: 18.
  • the anti-IL-1RAP antibodies, or antigen binding portions thereof comprise a heavy chain constant region comprising a nucleic acid sequence consisting of SEQ ID NO: 20 or SEQ ID NO: 22; and a light chain constant region comprising a nucleic acid sequence consisting of any one of SEQ ID NO: 31-38.
  • an anti-IL- 1RAP antibody or antigen-binding portion thereof, comprises a heavy chain constant region comprising a nucleic acid sequence set forth in SEQ ID NO: 20 or SEQ ID NO: 22, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20 or SEQ ID NO: 22, and/or a light chain constant region comprising a nucleic acid sequence set forth in any one of SEQ ID NO: 31-38, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 31-38.
  • the disclosure includes a human anti-IL-1RAP 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.
  • an anti-IL-1RAP antibody, or antigen binding portion thereof is an ADV58 antibody.
  • the ADV58 antibody 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.
  • an anti-IL-1RAP antibody (e.g., ADV58 antibody), or antigen- binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 7, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7, and/or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13.
  • an anti-IL-1RAP antibody, or antigen binding portion thereof is a humanized ADV58 antibody (e.g., ADV50).
  • the ADV50 antibody 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.
  • the anti-IL-1RAP antibody (e.g., ADV50), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 8, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8, and/or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 14, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14.
  • an anti-IL-1RAP antibody, or antigen binding portion thereof is a humanized ADV58 antibody (e.g., ADV51).
  • the ADV51 antibody 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.
  • the anti-IL-1RAP antibody (e.g., ADV51), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 9, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9, and/or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15.
  • an anti-IL-1RAP antibody, or antigen binding portion thereof is a humanized ADV58 antibody (e.g., ADV52).
  • the ADV52 antibody 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.
  • the anti-IL-1RAP antibody (e.g., ADV52), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 10, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10, and/or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16.
  • an anti-IL-1RAP antibody, or antigen binding portion thereof is a humanized ADV58 antibody (e.g., ADV53).
  • the ADV53 antibody 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.
  • the anti-IL-1RAP antibody (e.g., ADV53), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11, and/or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17.
  • an anti-IL-1RAP antibody, or antigen binding portion thereof is a humanized ADV58 antibody (e.g., ADV581).
  • the ADV581 antibody 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.
  • the anti-IL-1RAP antibody (e.g., ADV581 ), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 8, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8, and/or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15.
  • an anti-IL-1RAP antibody, or antigen binding portion thereof is a humanized ADV58 antibody (e.g., ADV583).
  • the ADV583 antibody 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.
  • the anti-IL-1RAP antibody (e.g., ADV583 ), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 8, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8, and/or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16.
  • an anti-IL-1RAP antibody, or antigen binding portion thereof is a humanized ADV58 antibody (e.g., ADV582).
  • the ADV582 antibody 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.
  • the anti-IL-1RAP antibody (e.g., ADV582 ), or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 9, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9, and/or a light chain comprising an amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15.
  • the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region.
  • 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.
  • the antibody, or antigen binding portion thereof has an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 constant region, or an IgG4 heavy chain constant region.
  • the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region.
  • the antibody, or antigen binding portion thereof is an IgG4 isotype.
  • the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region.
  • the antibody comprises a kappa light chain constant region.
  • the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.
  • 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 V L or V H domain, or fragments produced by a Fab expression library, or an IL-1RAP-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, CH1, CH2, CH3 and CL domains.
  • antigen-binding fragments can comprise any combination of variable region(s) with a hinge region, CH1, CH2, CH3 and C L domains.
  • the antibodies are human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken.
  • “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.
  • the anti-IL-1RAP antibody is agonistic, non- agonistic or antagonistic with respective to IL-1RAP.
  • the anti-IL- 1RAP antibody does not block binding of IL-1RAP ligand to IL-1RAP.
  • 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 or IL-1RAP coreceptors 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 IgG1 and IgG3, mediate ADCC and CDC via binding to Fc ⁇ Rs and complement C1q, respectively.
  • Neonatal Fc receptors (FcRn) are the critical components determining the circulating half-life of antibodies.
  • 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.
  • Another embodiment of the disclosure provides a glycosylated binding protein wherein the anti-IL-1RAP 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).
  • 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.
  • 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.
  • the glycosylation of the anti-IL-1RAP antibody or antigen binding portion is modified.
  • 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.
  • 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.
  • a modified anti-IL-1RAP 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.
  • 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.
  • 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.
  • glycosylation enzymes e.g., glycosyltransferases and glycosidases
  • glycosylation pattern 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.
  • the glycosylated binding protein comprises glycosyl residues such that the glycosylation pattern is human.
  • Differing protein glycosylation may result in differing protein characteristics.
  • the efficacy of a therapeutic protein produced in a microorganism host such as yeast
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • electroporation e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • 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.
  • Preferred mammalian host cells for expressing the recombinant antibodies disclosed herein include Chinese Hamster Ovary (CHO or CHO K1 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.
  • Chinese Hamster Ovary CHO or CHO K1 cells
  • dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220
  • a DHFR selectable marker e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621
  • the antibodies 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.
  • 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.
  • 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.
  • ADC anti-IL-1RAP antibody- drug conjugate
  • Anti-IL-1RAP ADCs comprise an anti-IL-1RAP antibody, i.e., an antibody that specifically binds to IL-1RAP, 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-1RAP.
  • an anti-IL-1RAP 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.
  • cytotoxic drug(s) i.e., cytotoxic drug moieties
  • linkers that may be used to conjugate the antibody and the one or more drug(s).
  • drug drug
  • agent agent
  • 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.
  • 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, respectively and the LCDR1, LCDR2, and LCDR3 or SEQ ID NOs:4-6, 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).
  • an anti-IL-1RAP 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) 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.
  • a linker as described herein e.g., a cleavable linker described herein such as a cleavable peptide linker
  • a drug moiety described herein e.g., a topoisomerase I inhibitor described herein such as DXd
  • 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 A1.
  • 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 SephadexTM G25 chromatography columns.
  • the anti-IL-1RAP ADC or ADC derivative may be internalized within an IL-1RAP-expressing cell, where the ADC or ADC derivative exerts a therapeutic effect (e.g., a cytotoxic, cytostatic, or immunostimulatory effect).
  • a therapeutic effect e.g., a cytotoxic, cytostatic, or immunostimulatory 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.
  • the payload released when the payload released is permeable or transmembrane, it may also induce bystander effect to enhance the efficacy of ADC.
  • ADCs 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.
  • the anti-IL-1RAP ADC or ADC derivative is not internalized, and is effective to deplete or inhibit IL-1RAP-expressing cells by direct binding to the IL-1RAP receptor and blocking its function. Blocking IL-1RAP function has been reported, for example, to lead to an increase in apoptosis and differentiation of THP-1 cells (AML cells), alongside a reduction in cell growth (Mitchell K et al. J. Exp. Med. 2018; 215(6):1709-1727).
  • the anti-IL-1RAP ADC or ADC derivative is effective to deplete or inhibit IL- 1RAP-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.
  • 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).
  • 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-1RAP-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-1RAP-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).
  • a cleavable linker that is sensitive to cleavage in the intracellular environment of the activated immune cell or IL-1RAP-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-1RAP-expressing cancer cell (e.g., in the endosomal or, for example by virtue
  • 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-1RAP-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-1RAP-expressing cancer cell.
  • 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.
  • 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.
  • 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).
  • the anti-IL-1RAP ADC or ADC derivative is 40% pure, more typically about 50% pure, and most typically about 60% pure.
  • an anti-IL-1RAP ADC comprises an anti-IL-1RAP antibody 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.
  • the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment.
  • 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.
  • 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.
  • 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.
  • 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, ⁇ -haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.
  • 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.
  • 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.
  • 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., peptidase- sensitive) 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.
  • the linker can be a dipeptide linker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine (phe-lys) linker.
  • 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.
  • 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.
  • linkers are cleaved extracellularly in the mielieu surrounding a target cell (e.g., cleaved by an extracellular protease present in the extracellular tumor microenvironment surround an IL-1RAP-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 surround 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).
  • extracellular cleavable linkers include disulfide based linkers and dipeptide valine- citrulline (Val-Cit) linkers (see, e.g., Bahou C et al. Chem. Commun.2019; 55(98):14829-14832).
  • 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.
  • the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.
  • the pH-sensitive linker is hydrolyzable under acidic conditions.
  • 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
  • a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like can be used.
  • a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like can be used.
  • 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).
  • 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).
  • a cleaving agent e.g., an enzyme
  • 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.
  • 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-1RAP-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.
  • 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.
  • the linker is a malonate linker (Johnson et al., 1995, Anticancer Res.
  • An ADC (e.g., anti-IL-1RAP 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.
  • 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.
  • the linker is selected from the following group: independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, -NO2 , -CN, -OH, -SH, -NH 2 , -C(O)H, -CO 2 H, -C(O)C(O)H, -C(O)CH 2 C(O)H, - S(O)H, -S(O) 2 H, -C(O)NH 2 , -SO 2 NH 2 , -OC(O)H, -N(H)SO 2 H, alkyl, alkenyl, alkynyl, alicyclic, heterocyclic, aryl and heteroaryl.
  • the linker is selected from the [0247]
  • 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.
  • 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 O,O- acetal or O,N-acetal to release eventually an aliphatic alcohol or amino group, which is part of the payload.
  • cleavage of the linker liberates a hydroxy-acylated version of the original payload with enhanced bystander killing function.
  • the linker comprises a group according to the following formula or a salt thereof: wherein: a is 0 or 1; and R 1 is selected from the group consisting of hydrogen, C1-C24 alkyl groups, C3-C24 cycloalkyl groups, C2-C24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, the C 1 -C 24 alkyl groups, C 3 - C24 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 NR 3 wherein R 3 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 1 is an additional target molecule D, wherein the target
  • 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; l is 0 or 1; Sp 1 is a spacer moiety; Sp 2 is a spacer moiety; Sp 3 is a spacer moiety; Sp 4 is a spacer moiety; Sp 5 is a spacer moiety; Sp 6 is a spacer moiety; Z 1 is a connecting group; Z 2 is a connecting group, wherein one of the bonds labelled with * is connected to reactive group Q 1 and one of the bonds labelled with * is connected to target molecule D, and wherein the sulfamide group SG is
  • 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.
  • 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; Sp 1 and Sp 2 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 R 17 is individually an amino acid side chain, and R 21 is selected from H, R 22 , C(O)OH and C(O)R 22 , wherein R 22 is Ci - C24 (hetero)alkyl groups, C3 - Cw (hetero)cycloalkyl groups, C2 - Cw (hetero)aryl groups, C3 - C10 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 NR 23 wherein R 23 is independently selected from
  • 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.
  • the linker comprises a spacer unit selected from the group consisting of (C1-C10)alkylene-C(O), (C3-C8)carbocyclo-C(O), arylene-C(O), (C1-C10)alkylene-arylene-C(O), arylene-(C1-C10)alkylene-C(O), (C1-C10)alkylene-(C3- C8)carbocyclo-C(O), (C3-C8)carbocyclo-(C1-C10)alkylene-C(O), (C3-C8)heterocyclo-C(O), (C1-C10)alkylene-(C3-C8)heterocyclo-C(O), and (C3-C8)heterocyclo-(C1-C10)alkylene-C(O).
  • the linker comprises the structure: wherein # indicated the attachment point to the antibody moiety and * indicates the attachment point to the drug moiety.
  • the linker comprises a spacer comprising the structure: wherein L p is a parallel connector unit; R s is, each independently, a polyalkylene glycol unit; M is, each independently, a bond or a moiety that binds R s with L p ; 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.
  • the linker comprises the structure: wherein R s is a polyalkylene glycol unit; and * indicates the attachment point to the antibody moiety; and ## indicates the attachment point to the drug moiety.
  • R s each independently, comprises 1 to 100 subunits having the structure:
  • M is each independently selected from the group consisting of -NH-, -O-, -S-, - C(O)-O-, -C(O)-NH- and -(C1-C10)alkylene.
  • the linker comprises at least one spacer unit joining the antibody moiety to the cleavable moiety.
  • 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.
  • the spacer unit in the linker comprises (PEG)2.
  • an ADC that comprises a shorter spacer unit e.g., (PEG)2
  • the spacer unit in the linker attaches to the antibody moiety of the ADC via a maleimide moiety (Mal).
  • 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.
  • the Mal in the linker is reactive with a cysteine residue on the antibody moiety.
  • the Mal in the linker is joined to the antibody moiety via a cysteine residue.
  • the Mal-spacer unit comprises a PEG moiety.
  • 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.
  • a spacer unit also attaches the cleavable moiety in the linker to the drug moiety.
  • the spacer unit that attaches the cleavable moiety in the linker to the drug moiety is self-immolative.
  • the self-immolative spacer is capable of releasing the drug moiety in a target cell.
  • the self-immolative spacer unit comprises a p-aminobenzyl alcohol.
  • the self-immolative spacer unit comprises p-aminobenzyloxycarbonyl (pAB).
  • pAB p-aminobenzyloxycarbonyl
  • the pAB in the linker attaches the cleavable moiety to the drug moiety.
  • the cleavable moiety is a cleavable peptide moiety, e.g., an amino acid unit.
  • the linker comprises Val-Cit-pAB.
  • the linker comprises Val-Cit-pAB and a PEG spacer unit joining the linker to the antibody moiety through a Mal.
  • 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.
  • 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. Chem.
  • 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.
  • the presence of a sterically hindered disulfide can increase the stability of a particular disulfide bond, enhancing the potency of the ADC.
  • 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.
  • 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.
  • the aforementioned linker types are not mutually exclusive.
  • the linker used in the anti-IL-1RAP 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.
  • the linker is cleavable under reducing conditions.
  • the linker is cleavable in the presence of a reducing agent, such as glutathione or dithiothreitol.
  • the linker is a cleavable disulfide linker or a cleavable sulfonamide linker. [0260] In some embodiments, the linker is a cleavable disulfide linker.
  • 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.
  • SATA N-succinimidyl-5-acetylthioacetate
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • SPDB N-succinimidyl-3-(2-pyridyldithio)butyrate
  • SMPT N-succinimid
  • 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 ⁇ M (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.
  • 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.
  • the cleavable disulfide moiety is disulfidyl-dimethyl.
  • the linker is a cleavable sulfonamide linker.
  • 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.
  • 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. Chem. Lett.
  • Dendritic linkers can increase the molar ratio of drug to antibody, i.e., drug loading, which is related to the potency of the ADC.
  • drug loading i.e., drug loading
  • a multitude of drug moieties may be attached through a dendritic linker.
  • 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., Intl. Publ. Nos.
  • the linker is a cleavable peptide linker.
  • a cleavable peptide linker refers to any linker that comprises a cleavable peptide moiety.
  • 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.
  • 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.
  • the linker is an enzyme-cleavable linker and a cleavable peptide moiety in the linker is cleavable by the enzyme.
  • the cleavable peptide moiety is cleavable by a lysosomal enzyme, e.g., cathepsin.
  • the linker is a cathepsin-cleavable linker.
  • 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.
  • the cleavable peptide moiety is cleavable by cathepsin B.
  • An exemplary dipeptide that may be cleaved by cathepsin B is valine- citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69).
  • 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).
  • an alternate cleavable moiety e.g., a cleavable disulfide moiety or a cleavable sulfonamide moiety.
  • the linker or the cleavable peptide moiety in the linker comprises an amino acid unit.
  • 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).
  • 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-Val), valine-alanine (Val-Ala), valine-lysine (Val- Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit), tryptophan-citrulline (Trp
  • 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).
  • Al-Ala- Asn alanine-alanine-asparagine
  • Gly-Val-Cit glycine-glycine-glycine
  • Phe-Phe-Lys phenylalanine-phenylalanine-lysine
  • Gly-Phe- Lys glycine-phenylalanine-lysine
  • amino acid units include, but are not limited to, Gly-Phe-Leu-Gly, Ala-Leu- Ala-Leu, Phe-N 9 -tosyl-Arg, and Phe-N 9 -Nitro-Arg, as described in, e.g., U.S. Pat. No.6,214,345.
  • the amino acid unit in the linker comprises Val-Cit.
  • the amino acid unit in the linker comprises Ala-Ala-Asn.
  • 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.
  • the linker is a selectively cleavable peptide linker.
  • the selectively cleavable peptide linker comprises a tripeptide.
  • the tripeptide has the sequence -P3-P2-P1-, wherein P1, P2, and P3 are each an amino acid, wherein a first one of the amino acids P1, P2, or P3 is negatively charged; a second one of the amino acids P1, P2, or P3 has an aliphatic side chain with hydrophobicity no greater than that of leucine; and a third one of the amino acids P1, P2, or P3 has hydrophobicity lower than that of leucine, wherein the first one of the amino acids P1, P2, or P3 corresponds to any one of P1, P2, or P3, the second one of the amino acids P1, P2, or P3 corresponds to one of the two remaining amino acids P1, P2, or P3, and the third one of the amino acids P1, P2, or P3 corresponds to the
  • 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).
  • the linker is not substantially sensitive to the extracellular environment.
  • “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.
  • a predetermined time period e.g., 2, 4, 8, 16, or 24 hours
  • 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).
  • serine proteases e.g., granzyme B and kallikreins
  • cysteine proteases e.g., cathepsins, caspases, and calpains
  • aspartic proteases e.g., renin, cathepsins D and E, pepsin C, and napsin A
  • the linker promotes cellular internalization.
  • 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-1RAP antibody or derivative thereof (i.e., in the milieu of the ADC or ADC derivative as described herein).
  • the amino acid unit is valine-citrulline (vc or val-cit). In another aspect, the amino acid unit is phenylalanine-lysine (i.e., fk).
  • the amino acid unit is N-methylvaline-citrulline.
  • 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.
  • Another approach for the generation of ADCs involves the use of heterobifunctional cross-linkers which link the anti-IL-1RAP antibody to the drug moiety.
  • cross-linkers examples 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-pyri
  • 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).
  • charged linkers are used to conjugate anti-IL-1RAP 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.
  • 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.
  • 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-1-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, WO1994/016729A1, WO1995/015770A1, WO1997/031655A2, WO1998/035704A1, WO1999/019500A1, WO2001/09785A2, WO2001/090198A1, WO2003/093793A2, WO2004/050016A2, WO2005/081898A2, WO2006/083562A2, WO2006/089668A1, WO2007/150020A1, WO2008/135237A1, WO2010/111198A1, WO2011/057216A1, WO2011/058321A1, 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • bifunctional agents such as dialdehydes or imidoesters have been used to link the amino group of a compound to amino groups of an antibody.
  • the Schiff base reaction also available for attachment of drugs to antibodies. 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.
  • an intermediate which is the precursor of the linker
  • 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-1RAP 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.
  • Anti-IL-1RAP 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-1RAP 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.
  • 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
  • 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, morpholino- doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide, eleutherobin, and mitoxantrone.
  • dolastatins e.g., auristatin E
  • 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-1RAP 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
  • anti-IL-1RAP 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.
  • Mitotic Inhibitors [0286]
  • anti-IL-1RAP antibodies may be conjugated to one or more mitotic inhibitor(s) to form an ADC for the treatment of cancer.
  • mitotic inhibitor 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).
  • an anti-IL-1RAP antibody of the invention is conjugated to one or more mitotic inhibitor(s) that disrupts microtubule formation by inhibiting tubulin polymerization.
  • an anti-IL-1RAP antibody of the invention is conjugated to one or more mitotic inhibitor(s) that stabilizes the microtubule cytoskeleton from deploymerization.
  • 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-1RAP ADCs of the invention are provided below. Included in the genus of mitotic inhibitors are auristatins, described below. Dolastatins [0287]
  • the anti-IL-1RAP 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. Chem. 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.
  • the anti-IL-1RAP ADC of the invention comprises an anti- IL-1RAP antibody, as described herein, and at least one dolastatin.
  • Auristatins are synthetic derivatives of dolastatin 10.
  • Auristatins [0288] Anti-IL-1RAP 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.
  • Auristatin E U.S. Pat. No. 5,635,483
  • Auristatin E 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. Chem. Org. Nat.
  • 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).
  • MMAD or auristatin D derivative monomethyl auristatin E
  • MMAF or auristatin F derivative monomethyl auristatin F
  • AFP auristatin EB
  • AEFP 5-benzoylvale
  • anti-IL-1RAP antibodies are conjugated to at least one MMAE (monomethyl auristatin E).
  • MMAE Monomethyl auristatin E
  • mAb monoclonal antibody
  • the linker linking MMAE to the anti-IL-1RAP 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.
  • MMAE The structure of MMAE is provided below.
  • the antibody is coupled to a single drug and, therefore, has a DAR of 1.
  • the ADC will have a DAR of 2 to 8, or, alternatively, 2 to 4.
  • Maytansinoids [0292]
  • the anti-IL-1RAP 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. Chem.
  • 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).
  • conventional chemotherapeutic agents such as, for example, methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Pat. No.5,208,020).
  • 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.
  • 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.
  • 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.
  • DM1 maytansinoid
  • maytansinoids include, but are not limited, to DM1 (N 2 ′-deacetyl-N 2 ′-(3-mercapto-1-oxopropyl)-maytansine; also referred to as mertansine, drug maytansinoid 1; ImmunoGen, Inc.; see also Chari et al.
  • an anti-IL-1RAP antibody is conjugated to at least one DM1. In one embodiment, an anti-IL-1RAP antibody is conjugated to at least one DM2. In one embodiment, an anti-IL-1RAP antibody is conjugated to at least one DM3. In one embodiment, an anti-IL-1RAP antibody is conjugated to at least one DM4. Antitumor Antibiotics [0298] Anti-IL-1RAP antibodies may be conjugated to one or more antitumor antibiotic(s) for the treatment of cancer.
  • 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-1RAP ADCs include, but are not limited to, actinomycines (e.g., pyrrolo[2,1-c][1,4]benzodiazepines), anthracyclines, calicheamicins, and duocarmycins.
  • anti-IL-1RAP antibodies may be conjugated to at least one immunomodulating agent.
  • immunomodulating agent refers to an agent that can stimulate or modify an immune response.
  • an immunomodulating agent is an immunostimulator which enhances a subject's immune response.
  • 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.
  • myeloid cells monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes
  • lymphoid cells T cells, B cells and natural killer (NK) cells
  • Representative examples include, but are not limited to, bacillus calmette-guerin (BCG) and levamisole (Ergamisol).
  • BCG bacillus calmette-guerin
  • Ergamisol levamisole
  • Other examples of immunomodulating agents that may be used in the ADCs include, but are not limited to, cancer vaccines, and cytokines.
  • immunostimulatory 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 I:C), 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).
  • TLR3 agonists e.g., polyribosinic-polyribocytidic acid (Poly I:C)
  • TLR4 agonists e.g., monophosphoryl lipid A
  • TLR7/8 agonists e.g., imidazoquinolines such as imiquimod and re
  • 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-1RAP antibody and a cancer vaccine.
  • the immune response results in the eradication of tumor cells in the body (e.g., primary or metastatic tumor cells).
  • 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.
  • 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-1RAP 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).
  • the anti- IL-1RAP antibody is conjugated to at least one cancer vaccine that is either an immunostimulator or is an immunosuppressant.
  • the anti-IL-1RAP antibodies may be conjugated to at least one cytokine.
  • 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).
  • cytokines examples 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 ⁇ , ⁇ , and ⁇ , colony stimulating factors (CSFs);
  • CSFs colony stimulating factors
  • the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
  • the disclosure provides an ADC comprising an anti-IL-1RAP antibody described herein and a cytokine.
  • the anti-IL-1RAP antibodies may be conjugated to at least one colony stimulating factor (CSF).
  • CSFs 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 (granulocyte- macrophage colony-stimulating factor and GM-CSF); Genzyme Corporation), promegapoietin, and Oprelvekin (recombinant IL-11; Pfizer, Inc.).
  • Epoetin erythropoietin
  • filgrastim also known as granulocyte colony-stimulating factor (G-CSF); Amgen, Inc.
  • sargramostim leukine (granulocyte- macrophage colony
  • an ADC may comprise an anti-IL-1RAP antibody described herein and a CSF.
  • Alkylating Agents [0304] The anti-IL-1RAP antibodies may be conjugated to one or more alkylating agent(s).
  • 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.
  • DNA damaging agent 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).
  • DNA damaging agents include DNA alkylating agents.
  • DNA alkylating agents are a class of antineoplastic compounds that attaches an alkyl group (C n H 2n+1 ) to DNA at a guanine base of DNA.
  • DNA alkylating agents examples 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.
  • alkyl sulfonates e.g., busulfan
  • ethylenimimes e.g., altretamine and thiotepa
  • methylamine derivatives epoxides
  • nitrogen mustards e.g., benda
  • DNA damaging agents also include indolino-benzodiazepines (IGNs).
  • IGNs represent a chemical class of cytotoxic molecules with high in vitro potency (IC 50 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.
  • 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].
  • PBD pyrrolobenzodiazepine
  • PPDD pyridinobenzodiazepine
  • the DNA damaging agent is a PARP inhibitor, e.g., olaparib, rucaparib, niraparib, or iniparib.
  • the PARP inhibitor is olaparib.
  • the PARP inhibitor is rucaparib.
  • the PARP inhibitor is niraparib.
  • the PARP inhibitor is iniparib.
  • the agent is a saporin toxin.
  • Antiangiogenic Agents [0311]
  • the anti-IL-1RAP 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.
  • VEGF vascular endothelial growth factor
  • certain antiangiogenic agents that prevent the interaction of VEGF with its cognate receptor, prevent VEGF from initiating angiogenesis.
  • 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.
  • 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.
  • these agents interfere with intercellular signaling cascades.
  • these agents disable specific targets that activate and promote cell growth or by directly interfering with the growth of blood vessel cells.
  • antiangiogenic agents include, but are not limited to, angiostatin, ABX EGF, C1-1033, 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, RhuMa
  • the anti-IL-1RAP 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.
  • antimetabolies examples 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.
  • 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
  • an adenosine deaminase inhibitor e.g.
  • 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).
  • Chemoprotective Agents [0315] The anti-IL-1RAP 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.
  • 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.
  • amifostine Ethyol, Medimmune, Inc.
  • Dexrazoxane Totect, Apricus Pharma; Zinecard
  • Zinecard antitumor antibiotic doxorubicin
  • mesna Mesnex, Bristol-Myers Squibb
  • the anti-IL-1RAP 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.
  • 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.
  • Radionuclide Agents Radioactive Isotopes
  • the anti-IL-1RAP 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.
  • the therapeutic agent is a radionuclide selected from the group consisting of 111 In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 475 c, 111 Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 186 Re, 188 Re, 189 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, and 211 Pb.
  • a radionuclide selected from the group consisting of 111 In, 177 Lu, 212 Bi, 213 Bi, 211 At, 62 Cu, 64 Cu, 67 Cu, 90 Y, 125 I, 131 I, 32 P, 33 P, 475
  • radionuclides that substantially decay with Auger-emitting particles.
  • Decay energies of useful beta-particle-emitting nuclides are preferably Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255.
  • Decay energies of useful alpha-particle- emitting 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 11 C, 13 N, 15 O, 75 Br, 198 Au, 224 Ac, 126 I, 133 I, 77 Br, 113m In, 95 Ru, 97 Ru, 103 Ru, 105 Ru, 107 Hg, 203 Hg, 121m Te, 122m Te, 125m Te, 165 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 105 Rh, 142 Pr, 143 Pr, 161 Tb, 161 Tb, 166 H o, 199 Au, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 201 Tl, 225 Ac, 76 Br, 169 Yb, and the like.
  • Radiosensitizers [0318] The anti-IL-1RAP 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).
  • Radiosensitizers include gemcitabine, 5- fluorouracil, taxane, and cisplatin.
  • Radiosensitizers may be activated by the electromagnetic radiation of X-rays.
  • Representative examples of X-ray activated radiosensitizers 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 (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same.
  • metronidazole misonidazole
  • desmethylmisonidazole pimonidazo
  • radiosensitizers may be activated using photodynamic therapy (PDT).
  • PDT photodynamic therapy
  • 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.
  • Topoisomerase Inhibitors [0320]
  • the anti-IL-1RAP 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.
  • topoisomerase I inhibitors include, but are not limited to, camptothecins and its derivatives irinotecan (CPT-11, Camptosar, Pfizer, Inc.) and topotecan (Hycamtin, GlaxoSmithKline Pharmaceuticals).
  • DNA topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxotrubicin, epipodophyllotoxins, ellipticines, epirubicin, etoposide, razoxane, and teniposide.
  • the topoisomerase I inhibitor is selected from the group consisting of deruxtecan, camptothecins, topotecan, irinotecan, belotecan, exatecan, Exatecan mesylate, DXd, indenoisoquinolines, indotecan, indimitecan, SN-38, and lamellarin D, or their derivatives.
  • topoisomerase I inhibitor-containing ADC formula is depicted below, including deruxtecan (MC-GGFG-DXd), wherein Ab represents an antibody (e.g., a humanized ADV58 antibody as described herein) and n is an integer between 2 to 10: [0322]
  • Another exemplary topoisomerase I inhibitor-containing ADC formula is depicted below, including deruxtecan (MC-GGFG-DXd), wherein Ab represents an antibody (e.g., a humanized ADV58 antibody as described herein) and n is an integer between 1 to 10: Tyrosine Kinase Inhibitors [0323]
  • the anti-IL-1RAP 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.
  • 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)
  • PAPI Phytolaca americana proteins
  • PAPII Phytolaca americana proteins
  • PAP-S pokeweed antiviral protein
  • Pseudomonas endotoxin Pseudomonas exotoxin
  • 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 mocetinostat
  • panobinostat COX-2 inhibitors
  • substituted ureas e.g., heat shock protein inhibitors (e.g. Geldanamycin and its numerous analogs), adrenocortical suppressants, and the tricothecenes.
  • agents also include asparaginase (Espar, Lundbeck Inc.), hydroxyurea, levamisole, mitotane (Lysodren, Bristol-Myers Squibb), and tretinoin (Renova, Valeant Pharmaceuticals Inc.).
  • asparaginase Espar, Lundbeck Inc.
  • hydroxyurea levamisole
  • mitotane Lysodren, Bristol-Myers Squibb
  • tretinoin Renova, Valeant Pharmaceuticals Inc.
  • 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 bladder cancer, kidney cancer, 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.
  • a IL-1RAP associated disorder e.g., cancer such as bladder cancer, kidney cancer, 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.
  • the IL-1RAP is human IL-1RAP
  • the subject is a human subject.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • a disorder in which IL-1RAP activity is detrimental is a disorder in which reduction of IL-1RAP 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-1RAP antibody as described above.
  • 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.
  • 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, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin’s lymphoma, cancer of
  • PVNS acute myeloid leukemia, adrenocortico carcinoma, ladder urothelial carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, diffuse large B cell lymphoma, 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.
  • 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 induction of apoptosis (caspase activation) of the ADC.
  • the antibodies and ADCs disclosed herein are administered to a subject in need thereof in order to treat advanced cancers, including solid tumor types, likely to exhibit elevated levels of IL-1RAP.
  • the disclosure includes a method for inhibiting or decreasing solid tumor growth in a subject having a solid tumor, said method comprising administering an anti-IL-1RAP ADC described herein, to the subject having the solid tumor, such that the solid tumor growth is inhibited or decreased.
  • the solid tumor is an IL-1RAP expressing solid tumor.
  • the solid tumor is a primary tumor.
  • the anti-IL- 1RAP ADCs described herein are administered to a subject having cancer.
  • the disclosure includes a method for inhibiting or decreasing solid tumor growth in a subject having a solid tumor which was identified as a IL- 1RAP expressing or IL-1RAP positive tumor, said method comprising administering an anti-IL- 1RAP ADC described herein, to the subject having the solid tumor, such that the solid tumor growth is inhibited or decreased.
  • the solid tumor is a primary tumor.
  • the disclosure includes a method for inhibiting or decreasing solid tumor growth in a subject having a solid tumor which was identified as including tumor cells expressing an elevated level of IL-1RAP, said method comprising administering an anti-IL-1RAP ADC described herein, to the subject having the solid tumor, such that the tumor cells expressing an elevated level of IL-1RAP are directly targeted by the ADC and the solid tumor growth is inhibited or decreased.
  • the disclosure includes a method for inhibiting or decreasing solid tumor growth in a subject having a solid tumor which was identified as including tumor cells expressing a low level of IL-1RAP and tumor cells expressing an elevated level of IL-1RAP, said method comprising administering an anti-IL-1RAP ADC described herein, to the subject having the solid tumor, such that the tumor cells expressing an elevated level of IL- 1RAP are directly targeted by the ADC, and the growth of tumor cells expressing a low level of IL-1RAP is inhibited or decreased, for example, by bystander killing.
  • Bystander killing the killing of neighboring cells
  • the solid tumor is a primary tumor.
  • Methods for identifying IL-1RAP expressing tumors are known in the art, and include for example, a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry or immunofluorescence assays.
  • ELISA enzyme linked immunosorbent assays
  • RIA radioimmunoassay
  • tissue immunohistochemistry or immunofluorescence assays tissue immunohistochemistry or immunofluorescence assays.
  • Alternate assays for example PCR, RT-qPCR, and/or digital PCR, may use primers that are specific for the IL-1RAP gene and/or cDNA and result in the amplification of the IL-1RAP gene/cDNA, or a portion thereof.
  • this application features a method of treating (e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse of) or preventing an IL-1RAP-associated disorder (e.g., an IL-1RAP-associated cancer), in a subject.
  • a method of treating e.g., curing, suppressing, ameliorating, delaying or preventing the onset of, or preventing recurrence or relapse of
  • an IL-1RAP-associated disorder e.g., an IL-1RAP-associated cancer
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an antibody drug conjugate (i.e., an anti-IL-1RAP ADC) or pharmaceutical composition as described herein.
  • an antibody drug conjugate i.e., an anti-IL-1RAP ADC
  • the anti-IL-1RAP antibody or fragment thereof used in the methods of the invention is a human or humanized anti-IL-1RAP antibody or fragment thereof.
  • the pharmaceutical compositions may include a “therapeutically effective amount” of an antibody or antibody portion.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the antibody or antibody portion may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody-drug conjugate, or antibody portion, are outweighed by the therapeutically beneficial effects.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response) and exposure. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect and exposure in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • this application provides a method for detecting the presence of IL-1RAP in a sample in vitro (e.g., a biological sample, such as serum, plasma, tissue, biopsy).
  • a sample in vitro e.g., a biological sample, such as serum, plasma, tissue, biopsy.
  • the subject method can be used to diagnose a disorder, e.g., a cancer.
  • the method includes: (i) contacting the sample or a control sample with the anti-IL-1RAP antibody or fragment thereof as described herein; and (ii) detecting formation of a complex between the anti-IL-1RAP antibody or fragment thereof, and the sample or the control sample, wherein a statistically significant change in the formation of the complex in the sample relative to the control sample is indicative of the presence of IL-1RAP in the sample.
  • the anti-human IL-1RAP antibodies, or portions thereof, (as well as ADCs thereof) can be used to detect human IL-1RAP (e.g., in a biological sample, such as serum or plasma), using a conventional immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry or immunofluorescence assays.
  • a conventional immunoassay such as an enzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue immunohistochemistry or immunofluorescence assays.
  • the disclosure provides a method for detecting human IL-1RAP in a biological sample comprising contacting a biological sample with an antibody, or antibody portion, and detecting either the antibody (or antibody portion) bound to human IL-1RAP or unbound antibody (or antibody portion), to thereby detect human IL-1RAP in the biological sample.
  • the antibody is directly or indirectly labelled with a detectable substance to facilitate detection of the bound or unbound antibody.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol; and examples of suitable radioactive material include 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 SM.
  • this application provides a method for detecting the presence of IL-1RAP in vivo (e.g., in vivo imaging in a subject).
  • in vivo imaging may include immune-positron emission tomography (immunoPET), a molecular imaging modality combining the high sensitivity of PET with the specific targeting ability of monoclonal antibodies (see, e.g., Manafi-Farid R et al. Front. Med.2022; 9:916693).
  • the subject method can be used to diagnose a disorder, e.g., an IL-1RAP-associated disorder.
  • the method includes: (i) administering the anti-IL- 1RAP antibody or fragment thereof as described herein to a subject or a control subject under conditions that allow binding of the antibody or fragment to IL-1RAP; and (ii) detecting formation of a complex between the antibody or fragment and IL-1RAP, wherein a statistically significant change in the formation of the complex in the subject relative to the control subject is indicative of the presence of IL-1RAP.
  • a thymidine incorporation assay may be used for determining whether an ADC exerts a cytostatic effect.
  • cancer cells expressing a target antigen at a density of 5,000 cells/well of a 96-well plated can be cultured for a 72-hour period and exposed to 0.5 ⁇ Ci of 3 H-thymidine during the final 8 hours of the 72-hour period.
  • the incorporation of 3 H-thymidine into cells of the culture is measured in the presence and absence of the ADC.
  • necrosis or apoptosis may be measured. Necrosis is typically accompanied by increased permeability of the plasma membrane; swelling of the cell, and rupture of the plasma membrane. Apoptosis can be quantitated, for example, by measuring DNA fragmentation.
  • Apoptosis may also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (e.g., a fluorescent dye such as, for example, acridine orange or ethidium bromide).
  • a fluorescent dye such as, for example, acridine orange or ethidium bromide
  • a method for measuring apoptotic cell number has been described by Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992) pp. 3.17.1-3.17.16).
  • Cells also can be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells observed for chromatin condensation and margination along the inner nuclear membrane.
  • Apoptosis may also be determined, in some embodiments, by screening for caspase activity.
  • a Caspase-Glo® Assay can be used to measure activity of caspase-3 and caspase-7.
  • the assay provides a luminogenic caspase-3/7 substrate in a reagent optimized for caspase activity, luciferase activity, and cell lysis.
  • adding Caspase-Glo® 3/7 Reagent in an “add-mix-measure” format may result in cell lysis, followed by caspase cleavage of the substrate and generation of a “glow-type” luminescent signal, produced by luciferase.
  • luminescence may be proportional to the amount of caspase activity present, and can serve as an indicator of apoptosis.
  • morphological changes that can be measured to determine apoptosis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular shrinkage. Determination of any of these effects on cancer cells indicates that an ADC is useful in the treatment of cancers.
  • Cell viability may be measured, e.g., by determining in a cell the uptake of a dye such as neutral red, trypan blue, Crystal Violet, or ALAMARTM blue (see, e.g., Page et al. (1993) Intl J Oncology 3:473-6).
  • the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically.
  • Cell viability may also be measured, e.g., by quantifying ATP, an indicator of metabolically active cells.
  • in vitro potency and/or cell viability of prepared ADCs may be assessed using a CellTiter-Glo® Luminescent Cell Viability Assay, as described in the examples provided herein.
  • the single reagent (CellTiter-Glo® Reagent) is added directly to cells cultured in serum-supplemented medium.
  • the addition of reagent results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present.
  • the amount of ATP is directly proportional to the number of cells present in culture.
  • the protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J Natl Cancer Inst.82:1107-12).
  • SRB protein-binding dye sulforhodamine B
  • the disclosed ADCs may also be evaluated for bystander killing activity. Bystander killing activity may be determined, e.g., by an assay employing two cell lines, one positive for a target antigen and one negative for a target antigen. In certain embodiments, the design of the assay allows tracking of only target negative cells.
  • cells are plated under three conditions: (i) target negative cells alone (tagged or labeled); (ii) target positive cells alone; and (iii) co-culture of target negative cells and target positive cells.
  • Cells are then treated with an ADC followed by monitoring of cytotoxicity.
  • viability of all cell populations can be monitored.
  • plates are read with OneGlo® Reagent, only the tagged or labeled target negative cells produce a signal. Killing of the target-negative cells when mixed with target-positive cells is indicative of bystander killing, whereas killing of the target-negative cells in the absence of the target-positive cells is indicative of off-target killing.
  • the disclosed ADCs may be administered in any cell or tissue that expresses IL-1RAP, such as an IL-1RAP-expressing neoplastic cell or tissue.
  • An exemplary embodiment includes a method of inhibiting IL-1RAP-mediated cell signaling or a method of killing a cell. The method may be used with any cell or tissue that expresses IL-1RAP, such as a cancerous cell or a metastatic lesion.
  • Non-limiting examples of IL-1RAP-expressing cancers include bladder cancer, kidney cancer, 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, or ovarian cancer.
  • Exemplary methods include the steps of contacting a cell with an ADC, as described herein, in an effective amount, i.e., amount sufficient to kill the cell. The method can be used on cells in culture, e.g. in vitro, in vivo, ex vivo, or in situ.
  • cells that express IL-1RAP can be cultured in vitro in culture medium and the contacting step can be affected by adding the ADC to the culture medium.
  • the method will result in killing of cells expressing IL-1RAP, including in particular tumor cells expressing IL-1RAP.
  • the ADC can be administered to a subject by any suitable administration route (e.g., intravenous, subcutaneous, or direct contact with a tumor tissue) to have an effect in vivo.
  • suitable administration route e.g., intravenous, subcutaneous, or direct contact with a tumor tissue
  • xenogeneic cancer models can be used, wherein cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al. (1997) Nature Med.3:402-8).
  • PDX Patient-derived xenografts
  • Efficacy may be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
  • xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
  • a neoplastic disorder e.g., a cancer.
  • the ADCs disclosed herein can be administered to a non-human mammal or human subject for therapeutic purposes.
  • the therapeutic methods entail administering to a subject having or suspected of having a neoplastic disorder a therapeutically effective amount of an ADC that binds to an antigen expressed, is accessible to binding, or is localized on a cancer cell surface.
  • treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic cells which do not express a target antigen but are adjacent to neoplastic cells which express a target antigen.
  • An exemplary embodiment is a method of delivering a topoisomerase I inhibitor to a cell expressing IL-1RAP, comprising conjugating the topoisomerase I inhibitor to an antibody that immunospecifically binds to an IL-1RAP epitope and exposing the cell to the ADC.
  • bladder cancer cells kidney cancer cells, esophageal cancer cells, liver cancer cells, lung squamous cell carcinoma cells, melanoma cells, head and neck cancer cells, glioblastoma cells, pancreatic cancer cells, stomach cancer cells, acute myeloid leukemia (AML) cells, myelodysplastic syndrome (MDS) cells, NSCLC cells, Ewing sarcoma cells, or ovarian cancer cells.
  • Another exemplary embodiment is a method of reducing or inhibiting growth of a tumor (e.g., an IL-1RAP-expressing tumor), comprising administering a therapeutically effective amount of an ADC or composition comprising an ADC.
  • the treatment is sufficient to reduce or inhibit the growth of the patient's tumor, reduce the number or size of metastatic lesions, reduce tumor load, reduce primary tumor load, reduce invasiveness, prolong survival time, and/or maintain or improve the quality of life.
  • the tumor is resistant or refractory to treatment with the antibody or antigen binding fragment of the ADC (e.g., an anti-IL-1RAP antibody) when administered alone, and/or the tumor is resistant or refractory to treatment with the drug moiety when administered alone.
  • the tumor is resistant or refractory to treatment with the anti-IL-1RAP antibody nadunolimab (Cantargia AB), but is susceptible to treatment with an antibody, ADC, or composition comprising an ADC described herein.
  • antibodies of the present disclosure may be administered to a non-human mammal expressing an antigen with which the ADC is capable of binding for veterinary purposes or as an animal model of human disease.
  • an exemplary embodiment is the use of an ADC in the treatment of a neoplastic disorder (e.g., an IL-1RAP-expressing cancer).
  • a neoplastic disorder e.g., an IL-1RAP-expressing cancer
  • an ADC for use in the treatment of a neoplastic disorder e.g., an IL-1RAP-expressing cancer.
  • Another exemplary embodiment is the use of an ADC in a method of manufacturing a medicament for the treatment of a neoplastic disorder (e.g., an IL-1RAP-expressing cancer).
  • treatment involves single bolus or repeated administration of the ADC preparation via an acceptable route of administration.
  • Patients may be evaluated for the levels of target antigen in a given sample (e.g. the levels of target antigen expressing cells) in order to assist in determining the most effective dosing regimen, etc.
  • An exemplary embodiment is a method of determining whether a patient will be responsive to treatment with an ADC of the present disclosure, comprising providing a biological sample from the patient and contacting the biological sample with the ADC.
  • Exemplary biological samples include tissue, stool sample, or tumor biopsy (e.g., a tumor biopsy derived from a patient having or at risk of a target antigen-expressing cancer, e.g., an IL-1RAP-expressing cancer).
  • a sample e.g., a tissue
  • a suitable immunological method can be used to detect and/or measure protein expression of the target antigen (e.g., IL-1RAP).
  • Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters.
  • the efficacy of an ADC may be evaluated by contacting a tumor sample from a subject with the ADC and evaluating tumor growth rate or volume.
  • an ADC when an ADC has been determined to be effective, it may be administered to the subject. [0362] In some embodiments, cleavage of an ADC releases the topoisomerase I inhibitor from the antibody or antigen binding fragment and linker. In some embodiments, the linker and/or topoisomerase I inhibitor is designed to facilitate bystander killing (the killing of neighboring cells). In some embodiments, the linker and/or topoisomerase I inhibitor is designed to facilitate bystander killing through cleavage after cellular internalization and diffusion of the linker- topoisomerase I inhibitor moiety and/or the topoisomerase I inhibitor moiety alone to neighboring cells. In some embodiments, the linker promotes cellular internalization.
  • the linker is designed to minimize cleavage in the extracellular environment and thereby reduce toxicity to off-target tissue (e.g., non-cancerous tissue), while preserving ADC binding to target tissue and bystander killing of cancerous tissue that does not express an antigen targeted by the antibody or antigen binding fragment of an ADC, but surrounds target cancer tissue expressing that antigen.
  • off-target tissue e.g., non-cancerous tissue
  • the topoisomerase I inhibitor moiety, or the catabolite of the topoisomerase I inhibitor moiety produced by cleavage of an ADC is designed to facilitate uptake by target cells or by neighboring cells (i.e., cell permeable).
  • topoisomerase I inhibitor moieties and catabolites may be referred to herein as “bystander active,” whereas drug moieties or catabolites with reduced cell permeability may be referred to as “bystander inactive.”
  • the disclosed ADCs also demonstrate bystander killing activity, but low off-target cytotoxicity. Without being bound by theory, the bystander killing activity of an ADC may be particularly beneficial where its penetration into a solid tumor is limited and/or target antigen expression among tumor cells is heterogeneous.
  • an ADC comprising a cleavable linker is particularly effective at bystander killing and/or demonstrates improved bystander killing activity, relative to comparable treatment with an ADC comprising a non-cleavable linker.
  • the ADCs disclosed herein exhibit improved solubility and target cell penetrance over the drug moieties on their own.
  • the above-described conjugates can be used in a method for targeting a topoisomerase I inhibitor to a selected cell population, the method comprising contacting a cell population or tissue suspected of containing the selected cell population with a cell-binding agent topoisomerase I inhibitor conjugate, wherein one or more topoisomerase I inhibitors is/are covalently linked to the cell-binding agent via a cleavable linker and the cell-binding agent binds to cells of the selected cell population.
  • the above-described conjugates can also be used in a method of destroying cells, the method comprising contacting the cells with a cell-binding agent topoisomerase I inhibitor conjugate, wherein one or more topoisomerase I inhibitors are covalently linked to the cell-binding agent via a cleavable linker and the cell-binding agent binds to the cells.
  • Examples of medical conditions that can be treated according to the methods of the present invention include but are not limited to malignancy of any type including, for example, cancer of the lung, breast, colon, prostate, kidney, pancreas, ovary, lymphatic organs, and other cancers as determined by one of ordinary skill in the art.
  • compositions comprising an anti-IL- 1RAP antibody, or antigen binding portion thereof, or anti-IL-1RAP ADC and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions comprising anti-IL-1RAP ADCs are for use in, but not limited to, diagnosing, detecting, or monitoring a disorder, in preventing, treating, managing, or ameliorating of a disorder or one or more symptoms thereof, and/or in research.
  • a pharmaceutical composition comprising an anti-IL-1RAP antibody drug conjugate as described herein and a pharmaceutically acceptable carrier.
  • a composition comprises one or more anti-IL-1RAP antibodies, or one or more anti-IL-1RAP ADCs.
  • the pharmaceutical composition comprises one or more anti-IL-1RAP ADCs and one or more therapeutic agents other than anti-IL-1RAP ADCs for treating a disorder.
  • the composition may further comprise of a carrier, diluent or excipient.
  • the anti-IL-1RAP antibodies and antibody-portions or anti-IL-1RAP ADCs can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • the pharmaceutical composition comprises an antibody or antibody portion and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • compositions for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • amino acids such as methionine
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the anti-IL-1RAP antibody or antibody portion or ADC.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, intranasal, or topical administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • kits for use in the laboratory and therapeutic applications described herein are within the scope of the present disclosure.
  • Such kits may comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method disclosed herein, along with a label or insert comprising instructions for use, such as a use described herein.
  • Kits may comprise a container comprising a drug moiety.
  • the present disclosure also provides one or more of the anti-IL-1RAP ADCs, or pharmaceutical compositions thereof, packaged in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity of the agent.
  • Kits may comprise the container described above, and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
  • a label may be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic, or laboratory application.
  • a label may also indicate directions for either in vivo or in vitro use, such as those described herein.
  • Directions and or other information may also be included on an insert(s) or label(s), which is included with or on the kit.
  • the label may be on or associated with the container.
  • a label may be on a container when letters, numbers, or other characters forming the label are molded or etched into the container itself.
  • a label may be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • the label may indicate that the composition is used for diagnosing or treating a condition, such as a cancer a described herein.
  • Some of the indications with high IL-1RAP mRNA expression include esophageal squamous cell carcinoma, acute myeloid leukemia, glioblastoma and head and neck squamous cell carcinoma.
  • the hinges represent the 1st and 3rd quartiles.
  • the upper (or lower) whisker expand to extend to the maximum (minimum) between the maximum (minimum) value and 1.5 times the interquantile range from the closest hinge.
  • Expression of IL-1RAP in Human Tumor Cell Lines [0382] Expression of IL-1RAP in human tumor cell lines was analysed based on cell line expression data available in the DepMap dataset (depmap.org/portal/).
  • the cell lines included in the dataset were derived from different haematological and solid tumors.
  • the dataset includes gene expression profiles of several hundred established cell lines. The results are shown in FIG.2.
  • the cell lines represented a broad spectrum of hematological malignancies (e.g., leukemia, lymphoma) and solid tumors (e.g., Ewing sarcoma, lung cancer).
  • IL-1RAP expression was represented as a box plot for each indication, with the horizontal line indicating the median value, the box showing the interquartile range, and the whiskers indicating the range up to 1.5 times the interquartile range.
  • FIG. 2 there were differences in IL-1RAP expression across cell lines derived from different cancers.
  • Cell lines derived from Ewing sarcoma, melanoma, head and neck squamous cell carcinoma (HNSCC), and esophageal cancer showed increased IL-1RAP expression at the mRNA level compared to other cell lines.
  • TCGA squamous and non squamous tumors as well as healthy cells in the genotype- tissue expression (GTEx) dataset.
  • GTEx genotype- tissue expression
  • the level of expression of membranous IL-1RAP was higher in squamous cancer (in the TCGA dataset) as shown by the higher expression (higher compared to non-squamous histologies and healthy tissues (GTEx).
  • the level of membranous IL-1RAP expression was also specifically determined in the TCGA dataset in certain squamous (by tissue) and non-squamous tumors and in healthy cells in the GTEx dataset.
  • the levels of expression of membranous IL-1RAP squamous cancer in TCGA shows a visible increase in expression with different patterns between tissues compared to non- squamous histologies and healthy tissues (GTEx).
  • GTEx healthy tissue
  • the y-axis shows the difference in mean expression (In TPM) between the tumor against matching healthy tissue.
  • the x-axis shows the mean expression in healthy tissue (in log2 TPM +1).
  • the IL-1RAP mRNA TMP expression from the cell lines were downloaded from DepMap.
  • An approximate string matching method was used to match cell line names from the Cancerxgene and DepMap databases.538 cell lines were matched together and aggregated by cancer types, with the results shown on FIG.5.
  • a similar distribution of IL-1RAP- expressing cell lines was observed for the topoisomerase inhibitors irinotecan and campothecin (data not shown).
  • Example 2 Detection of IL-1RAP Surface Expression in Cancer Cells from Solid Tumor and AML
  • This example summarizes experiments performed to determine IL-1RAP protein expression in various cancer cell lines from so called solid tumors.
  • Methods Tissue Culture and Cell Lines [0390] For the flow cytometry experiment described in this Example using anti-IL-1RAP- PE labeling antibody, 2x10 4 cells of each of the cancer cell lines A549, SCC25, Cal27, FaDu, Raji, and MM6 were seeded on 96-well plates. Cells were maintained in MEM medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma).
  • MEM medium Invitrogen
  • FBS fetal bovine serum
  • RNA levels for each of these cell lines are shown in Table 2, and are represented in log2(FPKM) based on the RNAseq dataset.
  • Flow Cytometry [0393] Staining for the first flow cytometry experiment was performed in 1 ⁇ cold PBS with 0.5% BSA. 10, 1, and 0.1 ⁇ g/mL of anti-IL-1RAP-PE (R&D systems, Clone 89412, Cat # FAB676P) antibody was incubated with each cell line. The cells were then analyzed for PE- fluorescence by flow cytometry. Quantification calculations were performed as described in the manufacturer’s protocol for BD QuantibriteTM Beads.
  • IL-1RAP quantification was performed on Capan-1, Capan-2, DoTc24510, HS 746.T, OVCAR- 5, SiHa, SK-MEL-28, and SNU-601 cells by staining with B-R58-FITC (Diaclone, Cat # 857.111.010) at a saturation concentration of 3.63 ⁇ g/mL (12.5uL/test). The cells were then analyzed for FITC-fluorescence by flow cytometry. QuantumTM Simply Cellular® (QSC) microspheres (Bangs Laboratories) were used in the quantitative analysis of cellular antigen expression.
  • QSC QuantumTM Simply Cellular®
  • Quantification calculations were performed as described in the manufacturer’s protocol for QuantumTM Simply Cellular® (QSC) microspheres. Briefly, when stained with the same antibody that is used to label cells, they permit determination of the Antibody Binding Capacity (ABC) of the cells.
  • the FITC fluorescence intensity of the samples (i.e., the cells) with the FITC conjugated mAb was compared to the fluorescence intensity of the known number of antibody molecules on the QSC beads, and the absolute number of antibody molecules bound to the target of interest was determined (i.e., the ABC of the cells).
  • viability staining was performed using 7- aminoactinomycin D (7-AAD; BD Biosciences, Cat # 559925) for 10 min at 4 °C. Fluorescent intensity was then measured using NovoExpress on a Novocyte Quanteon flow cytometer. The cells were then analyzed for PE-fluorescence by flow cytometry.
  • BD QuantibriteTM PE Phycoerythrin Fluorescence Quantitation Kit beads (BD Biosciences) were used in the quantitative analysis of cellular antigen expression. Quantification calculations were performed as described in the manufacturer’s protocol for BD QuantibriteTM PE Phycoerythrin Fluorescence Quantitation Kit.
  • the samples i.e., the cells
  • the PE conjugated mAb was compared to the fluorescence intensity of the known number of antibody molecules on the Quantibrite beads, and the absolute number of antibody molecules bound to the target of interest was determined (i.e., the ABC of the cells).
  • tumors from broad solid tumor indications may have elevated IL-1RAP expression, at the mRNA levels and/or as surface protein expression.
  • Flow cytometry also confirmed the high surface expression of IL-1RAP on SK-MEL-28 cells (FIG.7B), and the low surface expression of IL-1RAP on DoTc24510 cells (FIG.7C).
  • Example 3 IL-1RAP Expression in a Multi-Tumor Tissue Microarray (TMA), a Head & Neck TMA, and an oesophagus TMA [0397]
  • TMA Multi-Tumor Tissue Microarray
  • AF676 human anti-IL-1RAP antibody
  • OmniMap DAB was used as the detection system.
  • the optimal concentrations (tested between 0.03 to 10 ⁇ g/mL) for staining human TMAs with the selected antibody was defined at 5 ⁇ g/mL.
  • Each slide of TMA was immunostained with the IL-1 RAcP/IL-1 R3 pAb (AF676) and negative isotype control, at the selected concentration (i.e. 5 ⁇ g/mL).
  • An oesophagus TMA with 72 cores (TissueArray LLC, Cat # BC02012a) including oesophageal squamous cell carcinomas, and a head and neck TMA with 102 cores (Pantomics Inc, Cat # HNT1021) were also stained for IL-1RAP expression.
  • results for the TMA analysis for IL-1RAP expression are summarized in Table 5 and Figure 9A. Positive staining was primarily seen in melanoma (up to 89% of positive samples), ovaries (70%) and pancreas (approximately 66%), followed by urinary bladder, head and neck, lung and colon samples. No staining was observed in prostate samples.
  • ⁇ Bladder positive staining was observed in transitional epithelial cells from approximately 42% of the samples, from minimal to moderate.
  • Figure 9E ⁇ Breast: minimal positive staining was observed in a single breast sample accounting approximately for 9% of the samples.
  • ⁇ Colon moderate positive staining was observed in two colon samples accounting approximately for 20% of the samples.
  • Head and neck minimal to moderate positive staining was observed in 4/12 samples, approximately 33 % of the samples.
  • Kidney Ring Clear Cell Carcinoma
  • ⁇ Lung minimal or marked positive staining was observed in three samples, accounting for 30% of the samples. See also Figure 9G.
  • ⁇ Melanoma Minimal to marked positive staining was observed in 89% of the samples (melanocytic cells). See also Figure 9B. ⁇ Ovarian: Minimal to marked positive staining was observed in 7/10 of the samples (70%). See also Figure 9C. ⁇ Pancreatic: Minimal to moderate positive staining was observed in 6/9 samples (approximately 66%). See also Figure 9D. ⁇ Prostate: No staining was observed in prostate samples.
  • Table 5 Summary of IL-1RAP Positivity in TMA by Cancer Type Indications Number of Number of Cores Cores Percentage cores on cores showing IL- without IL- of IL-1RAP TMA assessable 1RAP 1RAP positive expression in expression in cores in the the tumoral the tumoral tumoral compartmen compartmen compartme t t nt Bladder 12 12 5 7 42% cancer Breast 12 11 1 10 9% cancer Colon 12 10 2 8 20% cancer Head and 12 12 4 8 33% neck cancer Kidney 12 12 1 11 8% cancer (RCCC) Lung 12 10 3 7 30% cancer Melanoma 12 9 8 1 89% Ovarian 12 10 7 3 70% cancer Pancreatic 12 9 6 3 66% cancer Prostate 12 11 0 11 0% cancer [0402] In the oesophageal TMA, the vast majority of oesophageal squamous cell carcinoma tumors, 65/66, or about 98%, were positive for IL-1RAP expression, with a score of between 1-4
  • FIG.10 A summary of the IL-1RAP statining scores in the oesophageal TMA is shown in FIG.10. Similarly, the majority of squamous tumors in the head and neck TMA stained positive for IL-1RAP (50 out of 79, or about 63%), as shown in FIG.11A. Some IL-1RAP positivity was also observed in adenocarcinoma albeit lower than in the tumors with squamous histology. IL- 1RAP positivity was detected across a majority of the head and neck tumor types, as shown in FIG. 11B.
  • Example 4 Correlation Between IL-1RAP Expression and Clinical Survival in Cancer
  • This example summarizes an analysis performed to determine to investigate the prognostic significance of IL-RAP mRNA expression in pan-cancer datasets including GEO, EGA and TCGA.
  • the Kaplan-Meier plotter (KMplot) is a publicly available online tool for survival analysis based on gene expression data across various cancer types.
  • the KMplot database can be accessed at www.kmplot.com (see, Lanczky A and Gyorffy B. J. Med. Internet Res. 2021; 23(7):e27633).
  • the pan-cancer dataset includes a wide range of cancer types, allowing for comprehensive survival analysis.
  • the tool provided survival curves and corresponding p-values, indicating the significance of the difference in survival between the two groups. Survival analysis was performed for Kidney Renal Papillary Cell Carcinoma, Liver Hepatocellular Carcinoma, Stomach Adenocarcinoma, and Pancreatic Ductal Adenocarcinoma. Log-rank p-values were calculated to determine the statistical significance of the differences in survival between the High IL-1RAP and Low IL-1RAP groups. A p-value less than the chosen significance level (e.g., 0.05) was considered statistically significant. [0407] Kaplan-Meier survival curves were generated using the KMplot tool and were saved as images for further analysis and reporting.
  • FIG. 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 expression and clinical data available in the pan-cancer dataset. Survival analysis was performed for Renal Papillary Cell Carcinoma (FIG. 12A), Hepatocellular Carcinoma (FIG. 12B), Stomach Adenocarcinoma (FIG.12C), and Pancreatic Ductal Adenocarcinoma (FIG.12D). In each cancer type analyzed, high levels of IL-1RAP expression correlated with poorer clinical outcomes compared to patients with low levels of IL-1RAP expression.
  • Example 5 IL-1RAP mRNA Expression in The Cancer Genome Atlas (TCGA) Indications
  • TCGA Cancer Genome Atlas
  • FIG.14 shows IL- 1RAP mRNA expression across the various cancer indications from the TCGA database associated with high IL-1RAP expression. Data is shown as RNA-Seq by Expectation-Maximization (RSEM) normalized count values.
  • RSEM Expectation-Maximization
  • ESCC esophageal squamous cell carcinoma
  • HNSCC head and neck squamous cell carcinoma
  • LSCC laryngeal squamous cell carcinoma
  • CSCC cervical squamous cell carcinoma
  • OVCA ovarian carcinoma
  • PDAC pancreatic ductal adenocarcinoma
  • SPR Surface Plasmon Resonance
  • Each assay cycle included the steps of: 1. Capturing of biotinylated human IL-1RAP (Acro Biosystems, ref. ILPH82E5) via reversibly coated avidin variant; 2. Interaction analysis of antibodies with captured IL-1RAP; and 3. Regeneration by complete removal of antibodies with bound antigen from capture surfaces. Results [0414] The anti-IL-1RAP antibody ADV58 was analyzed by SPR.
  • ADV58 displayed reproducible binding, with different kinetics, to reversibly immobilized IL-1RAP ( Figure 15).
  • Figure 15 Experiments of antibody interactions with IL-1RAP:ADV58 batches were analyzed by a single cycle analysis (SCK) at 4 nM, 20 nM and 100 nM with reversibly immobilized IL- 1RAP.
  • SCK single cycle analysis
  • Biacore T200 instrument was used, with a Series S Sensor Chip C1.
  • the temperature used for the analysis was 25 °C, with a flow rate of 50 ⁇ L/min (for quantitative kinetic interaction analysis).
  • Characteristics of the analysis buffer 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20.
  • Each assay cycle included the steps of: 1. Capturing of biotinylated human IL-1RAP (Acro Biosystems, ref. ILPH82E5) via reversibly coated avidin variant; 2. Interaction analysis of antibodies with captured IL-1RAP; and 3. Regeneration by complete removal of antibodies with bound antigen from capture surfaces.
  • FIG.16A shows the interaction analysis of a titration from 1.17 nM to 300 nM of chimeric ADV58 antibody (ADV580) with reversibly immobilized IL-1RAP.
  • Three replicates were performed for kinetic analysis of the ADV580:IL-1RAP interaction.
  • KD for replicate #1 was 8.16 x 10 -9 M; the KD for replicate #2 was 8.50 x 10 -9 M; and the KD for replicate #3 was 9.02 x 10 -9 M.
  • FIG.16B shows the interaction analysis of a titration from 1.17 nM to 300 nM of ADV581 antibody with reversibly immobilized IL-1RAP.
  • FIG.16C shows the interaction analysis of a titration from 1.17 nM to 300 nM of ADV582 antibody with reversibly immobilized IL-1RAP.
  • K D for replicate #1 was 1.47 x 10 -8 M; the KD for replicate #2 was 1.29 x 10 -8 M; and the KD for replicate #3 was 1.40 x 10 -8 M.
  • FIG.16D shows the interaction analysis of a titration from 1.17 nM to 300 nM of ADV583 antibody with reversibly immobilized IL-1RAP.
  • Three replicates were performed for kinetic analysis of the ADV583:IL-1RAP interaction.
  • KD for replicate #1 was 1.84 x 10 -8 M; the KD for replicate #2 was 1.73 x 10 -8 M; and the KD for replicate #3 was 2.07 x 10 -8 M.
  • FIG.16E shows the interaction analysis of a titration from 1.17 nM to 300 nM of ADV58-Vedotin-conjugated antibody with reversibly immobilized IL-1RAP.
  • FIG.16F shows the interaction analysis of a titration from 1.17 nM to 300 nM of ADV58-DXd-conjugated antibody with reversibly immobilized IL-1RAP.
  • Three replicates were performed for kinetic analysis of the ADV58-DXd:IL-1RAP interaction.
  • K D for replicate #1 was 8.74 x 10 -9 M; the KD for replicate #2 was 9.22 x 10 -9 M; and the KD for replicate #3 was 9.11 x 10 -9 M.
  • the K D to human IL-1RAP were determined to be 8.56 x 10 -9 M for ADV580, 2.07 x 10 -8 M for ADV581, 1.39 x 10 -8 M for ADV582, 1.88 x 10 -8 M for ADV583, 7.12 x 10 -9 M for ADV58-Vedotin, and 9.02 x 10 -9 M for ADV58-DXd.
  • ADV580, the ADV58-Vedotin conjugate, and the ADV58-DXd conjugate were found to have comparable K D values, while the three humanized antibodies had slightly weaker interactions with IL-1RAP.
  • the anti-IL-1RAP antibodies ADV581, ADV582, and ADV583 had comparable binding kinetics to the ADV580 antibody.
  • Example 8 Cross-Species Binding Assessment by ELISA of Anti-IL-1RAP Antibodies
  • This example summarizes experiments performed to assess the cross-species reactivity towards several IL-1RAP orthologs with the anti-IL-1RAP antibody clones disclosed herein (i.e., anti-IL-1RAP antibodies ADV581, ADV582, and ADV583, and chimeric ADV58 (i.e., ADV580)).
  • Cross-species reactivity between the anti-human IL-1RAP antibody ADV58 (mIgG1) and the chimeric ADV580 antibody (hIgG1) was also compared.
  • a 384-well plate was coated with human (hIL-1RAP), cynomolgus (cIL-1RAP), rhesus (rheIL-1RAP), and rat (ratIL-1RAP) IL-1RAP orthologs at a concentration of 1 ⁇ g/mL, at 30 ⁇ L/well, and incubated for 20 h at 4°C.
  • the plate was then blocked (PBS, 2% BSA, 0.05% Tween20, 0.2% Proclin300; 90 ⁇ L/well; 1 h at RT) and incubated with 3-fold serial dilutions (15 wells) in duplicate, starting from 3000 ng/mL, of the various anti-IL-1RAP antibodies, and incubated for 1 h at RT.
  • the plate was then incubated with goat anti-human HRP-conjugated antibody (1:20000 dilution) and the OD was read at 450 nm.
  • the MW of each primary antibody was considered to be 150 kDa.
  • EC 50 values were calculated using GraphPad Prism 9 using a non-linear regression, 4 parameter model. Results [0429] The results for EC 50 value calculations of the ADV58 clones (i.e., mIgG1 and chimeric ADV580 (hIgG1)) are shown in Table 6. Cross-reactivity of ADV58 and ADV580 to human and cynomolgus IL-1RAP (FIGs. 17A-17B, respectively) was confirmed, and binding to rhesus macaque IL-1RAP by ADV580 was also observed (FIG.17C).
  • the ADV582 antibody showed a strong binding affinity to human IL-1RAP by ELISA (in addition to control ADV580), in addition to the ADV581 and ADV583 antibodies (FIG. 18A). Binding affinity to cynomolgus or rhesus IL1-1RAP orthologs are similar (FIGs. 18B-18C, respectively). As expected, all antibodies, except D-5, showed only background signal comparable to the blank sample signal ( ⁇ 0.1) for binding to rat IL- 1RAP (FIG.18D).
  • the anti-IL-1RAP Mouse IgG1 ADV58 and the isotype control mIgG1 B-D38 were conjugated to vcMMAE in order to generate an antibody drug conjugate (ADC).
  • ADC antibody drug conjugate
  • the binding efficacy of the ADC was assessed by ELISA.
  • IL-1RAP protein For assessing binding of IL-1RAP protein to unconjugated anti-human IL-1RAP (ADV58), vedotin-conjugated ADV58, and vedotin-conjugated isotype control antibody (B-D38) antibodies, a 96-well ELISA plate was first coated with human IL-1RAP protein (1 ⁇ g/mL).
  • the plate was then blocked, incubated with a 1:3 dilution series of anti-human IL-1RAP (parental ADV58), ADV58-vedotin and B-D38-vedotin molecules (from 3000 ng/mL to 0.051 ng/mL) and incubated with secondary antibody (goat anti-mouse IgG (H+L), HRP conjugated antibody).
  • secondary antibody goat anti-mouse IgG (H+L), HRP conjugated antibody.
  • An HRP substrate was added, and the OD reading at 450 nm was recorded using Thermo Scientific Multiskan Fc and EC50 values were calculated using GraphPad Prism 9 using a non-linear regression, 4-parameter model.
  • Example 10 In Vitro Binding of Anti-IL-1RAP Antibody-Deruxtecan (DXd) Conjugates to Recombinant Human IL-1RAP [0435] This example summarizes experiments to determine the EC 50 value for binding of mIgG1 ADV58 and deruxtecan (DXd)-conjugated ADV58 to human IL-1RAP protein. Methods [0436] A 384-well ELISA plate was coated with human IL-1RAP protein (1 ⁇ g/mL). The plate was then blocked.
  • the plate was incubated with a 1:3 dilution series of mIgG1 ADV58 (naked) and mIgG1 ADV58 DXd, and compared to the isotype control mIgG1 B-D38-DXd.
  • 3-fold serial dilutions in duplicates, starting from 20 nM were made in PBS, 1% BSA, 0.05% Tween20, and Proclin300.30 ⁇ L/well was applied, and then incubated for 1 h at RT.
  • Goat anti-mouse HRP-conjugated antibody was diluted 1:10,000 in PBS, 1% BSA, 0.05% Tween20, and 30 ⁇ L/well was added.
  • Table 8 Calculated EC 50 Values For Anti-IL-1RAP Antibody and ADC Binding to Human IL-1RAP Coating Antigen Test Antibody EC 50 (nM) H uman IL1RAP ADV58 (Naked) 0.1270 m IgG1 ADV58 DXd 0.1235
  • Example 11 In Vitro Binding of Anti-IL-1RAP Antibody-Vedotin/Dxd/Tesirin Conjugates to Recombinant Human IL-1RAP [0439] This example summarizes experiments to determine the EC50 value for binding of human IgG1 Fc ADV581, ADV582, and ADV582 conjugated to either MMAE, DXd, or Tesirine to human IL-1RAP protein.
  • Humanized ADV58 antibodies were generated using techniques known in the art and described herein. The method consisted of in silico grafting of the CDRs of the murine monoclonal antibody ADV58 into human germline antibody sequences. [0445] The CDR-grafting protocol used a modernized version of the approach pioneered by Greg Winter and colleagues at the Medical Research Council, Cambridge, UK. The murine antibody was humanized by grafting the three CDRs from the light chain variable region (VL) into a unique selected human germline genes association (IGHV1-33*01/IGKJ2*01).
  • VL light chain variable region
  • the three CDRs from the heavy chain variable region (VH) were grafted into a unique selected human germline genes association (IGHV1-46*01/IGHJ4*01).
  • IGHV1-46*01 were the closest human germline sequence to respectively light chain and heavy chain of the murine ADV58 antibody.
  • some amino acid residues in the framework regions (Frs) of the selected human germline variable regions were reverted (back-mutations) to their corresponding murine amino acid residues.
  • Tissue Culture and Cell Lines [0446] 2x10 5 cells of the SK-MEL-28 malignant melanoma cell line were seeded on a 96- well plate.
  • the ADV58-derived antibodies were serially diluted, 1:5, for a final concentration range starting at 40 ⁇ g/mL and ending at 0.00256 ⁇ g/mL and added to the SK-MEL-28 cells in duplicate. Cultures were performed at 4°C to prevent antibody internalization. After incubation, goat anti-human-Fc-Alexa fluor 488 secondary antibody was added to each well. The cells were then analyzed for Alexa fluor 488-fluorescence by flow cytometry.
  • Thermostability and Purity Studies [0449] The purpose of these studies is to test the thermostability, the aggregation propensity, and the purity of ADV58-derived antibodies (parental and humanized) were assessed.
  • the UNcle instrument from Unchained Labs
  • Maurice instrument ProteinSimple
  • DSF differential scanning fluorimetry
  • SLS static light scattering
  • CE-SDS SDS capillary electrophoresis
  • FIGs.22A-22B additionally confirm high expression of IL-1RAP on SK-MEL-28 cells detected with the ADV58 mAb.
  • the EC 50 of ADV58 was calculated to be 0.1341 ⁇ g/mL, as shown in FIG.22B.
  • IL-1RAP expression is reported as mean fluorescence intensity (MFI).
  • FIGs. 23A-23B confirm comparable binding between the humanized ADV58-derived antibodies and the parental chimeric ADV580 mAb to IL-1RAP on SK-MEL-28 cells. Binding is reported as mean fluorescence intensity (MFI). Based on the MFI, the EC50 and EMax was calculated, as shown in Table 10.
  • the three humanized ADV58 antibodies also have lower polydispersity index (PDI) values and high molecular weight (HMW) percentage compared to that of the parental chimeric antibody indicating lower aggregation propensity (Table 11).
  • PDI polydispersity index
  • HMW high molecular weight
  • the DSF and SLS results showed that all the three humanized ADV58 antibodies tested have similar Tm1, Tagg266 and Tagg473 to that of the parental chimeric antibody (ADV580), indicating their thermostability are very similar.
  • the DLS results showed that all the three humanized ADV58 antibodies (ADV581, ADV582, and ADV583) and the parental chimeric antibody (ADV580) have only one peak around the size of the monomer, indicating the 4 antibodies have no or very low aggregation propensity.
  • the PDI of each of the four antibodies is less than 0.2, indicating they all can be considered as monodisperse, however, the humanized ADV58 antibodies have lower PDI values than that of the parental ADV58 antibody suggesting they are more uniform in size and have less aggregation potential.
  • the CE-SDS results showed that all four antibodies have very high purity, with no peaks detected other than those corresponding to IgG heavy chain and light chain for the reduced samples, and only a low level HHL (3.16-4.31%) peak and a single major peak (>95.69%) corresponding to IgG in the non-reduced samples.
  • A673 and TC71 (Ewing Sarcoma), SK-MEL-28 (Melanoma), Hs-746T (Stomach adenocarcinoma), DETROIT-562 (Head and Neck squamous cell carcinoma), Kyse 270 (Esophageal squamous cell carcinoma), and OVCAR5 (Ovarian serous adenocarcinoma) cell lines were cultured under the recommended ATCC culture conditions for each cell lines.
  • Flow Cytometry [0456] Staining for flow cytometry was performed with a titration of ADV582 anti-IL-1RAP antibody.
  • a human IgG1 antibody was used as a negative control. ⁇ for each model, cells were harvested from culture and stained with a live/dead cell staining reagent for 30min at RT. After washing, the cells were seeded at 2x10 5 cells/well and pre-incubated in PBS with Fc blocking reagent (FcRBlock). The ADV582 antibody was serially diluted, 1:3, for a final concentration range starting at 103nM and ending at 0.002nM and incubated with the cells for 1h. Incubations were performed at 4°C to prevent antibody internalization. After incubation, goat anti-human-Fc PE secondary antibody was added to each well and incubated for 1h.
  • the anti-IL-1RAP antibody ADV582 is binding of the anti-IL-1RAP antibody ADV582 to IL-1RAP-positive cells TC71 (Ewing Sarcoma), SK-MEL-28 (Melanoma), Kyse-270 (Esophageal squamous cell carcinoma), Hs-746T (Stomach adenocarcinoma), DETROIT-562 (Head and Neck squamous cell carcinoma), and OVCAR5 (Ovarian serous adenocarcinoma). As shown in FIG.24 and Table 12, the anti-IL- 1RAP antibody ADV582 exhibited strong binding affinity across all of the tested IL-1RAP positive cell lines.
  • EC50 values are the results of one representative experiment (EC50 values are reported as best-fit +/- standard error). For each cell line, membrane IL-1RAP expression levels (ABC values, pre-determined by quantitative flow cytometry) are also reported. As expected, the lowest EC50 values were observed for the highest IL-1RAP-expressing cell lines (i.e., those cell lines with the highest ABC value), indicative of IL-1RAP specificity. Based on the binding data, the EC 50 of the anti-IL-1RAP antibody clone ADV582 was found to be in the nanomolar to subnanomolar range. Table 12.
  • HEK-Blue IL-1 ⁇ , HEK-Blue IL-33 cell lines were cultured under recommended invivogen culture conditions for each cell line.
  • Assay HEK-Blue IL-33 or IL-1 ⁇ cells (Invivogen, CA) were harvested and plated in technical duplicates at a density of 50,000 cells per well in a 96-well plate.
  • ADV580 and ADV582 were added to the wells in a concentration range between 10 -6 and 10 -12 M. After incubating cells with the antibodies for 45 minutes at 37°C, IL-33 or IL-1 ⁇ was added to final concentration of 4x10 -10 M and 1.5x10 -10 M respectively, and the plate was incubated overnight. The following day, supernatant was harvested, substrate was added, and samples were analyzed for absorbance at 630 nm. Results [0468] As depicted in FIGs.27A-27B, the ADV580 and ADV582 anti-IL-1RAP mAb did not inhibit IL-1 ⁇ (FIG. 27A) and IL-33 (FIG. 27B) dependent signalling in HEK-Blue cells.
  • IL- 1RA is a recombinant protein with known activity to block IL-1 ⁇ /IL-1R1/IL-1RAP-dependent.
  • Astegolimab is an anti-ST2 antibody that blocks IL-33 signaling.
  • Astegolimab and IL-1RA antagonists used as positive controls respectively for HEK-Blue IL-33 and IL-1 ⁇ cell lines, induced a pronounced inhibition, as expected.
  • Example 17 In Vitro Cytotoxicity of Anti-IL-1RAP Antibody-Drug Conjugates
  • a topoisomerase I inhibitor for example deruxtecan
  • a tubulin inhibitor for example monomethyl auristatin E (MMAE).
  • MMAE monomethyl auristatin E
  • Monoclonal ADV58 antibodies are conjugated to a deruxtecan linker-payload, MC- GGFG-DXd (PubChem CID 118305111), or a MMAE linker-payload, MC-VC-MMAE (PubChem CID 46944733).
  • each antibody is partially reduced with an appropriate molar amount of TCEP for 2 hours at 37° C. After incubation, the antibodies are cooled to room temperature and an alkylating agent (e.g.
  • Example 18 In Vivo Cytotoxicity of Anti-IL-1RAP Antibody-Vedotin Conjugate [0474] The experiments described supra, e.g., in Example 2, 14, and 15, confirmed that IL- 1RAP is expressed on the surface of SK-MEL-28 and that ADV58 is internalized in the cell line.
  • an MMAE conjugated version of the anti-IL-1RAP mAb ADV58 i.e., ADV58-vedotin
  • a cleavable linker e.g., as described in Example 9
  • an MMAE-conjugated murine IgG1 isotype control B-D38 i.e., isotype-vedotin
  • mice 8-10 week old female NCG immunocompromised mice were subcutaneously injected with 1x10 7 SK-MEL-28 cells in 0.2 mL PBS with matrigel. Upon the mean tumor size reaching approximately 200 mm 3 , animals were randomized into 7 study groups, with 8 mice per group. The date of randomization is denoted as day 0. The study design is illustrated in Table 13 below. "ROA” is an abbreviation for route of administration. "i.v.” is an abbreviation for intravenous. Table 13: Anti-IL-1RAP Vedotin ADC In Vivo Study Design Group No.
  • the body weights and tumor volumes were measured by using StudyDirector TM software (version 3.1.399.19).
  • Treatment was initiated immediately after grouping (Day 0) per study design. Additional dosing was performed on Day 5 and Day 10. Measurement of tumor size and animal weight was performed until the study endpoint on day 36.
  • ADV58-vedotin treatment at 3 mg/kg was found to block tumor growth in the SK-MEL-28 subcutaneous tumor model up to 25 days after the end of the treatment (i.e., from day 10 of dosing until the end of the study).
  • FIG. 28C shows a comparison of tumor volumes on day 36 of the study across the ADV58-vedotin and isotype-vedotin groups at each of the 1 mg/kg, 3mg/kg, and 10 mg/kg dosages.
  • Example 19 In Vivo Cytotoxicity of Anti-IL-1RAP Antibody-DXd Conjugate [0480] To determine the in vivo cytotoxicity of a second anti-IL-1RAP antibody-drug conjugate (ADC), a DXd conjugated version of the anti-IL-1RAP mAb ADV58 (i.e., ADV58- DXd) with a cleavable linker (e.g., as described in Example 10), and a DXd-conjugated murine IgG1 isotype control B-D38 (i.e., isotype-DXd), were tested in vivo in the treatment of a human melanoma SK-MEL-28 subcutaneous xenograft model in female NCG immunocompromised mice.
  • ADC anti-IL-1RAP antibody-drug conjugate
  • mice 8-10 week old female NCG immunocompromised mice were subcutaneously injected with 1x10 7 SK-MEL-28 cells in 0.2 mL PBS with matrigel. Upon the mean tumor size reaching approximately 200 mm 3 , animals were randomized into 7 study groups, with 8 mice per group. The date of randomization is denoted as day 0. The study design is illustrated in Table 14 below. "ROA” is an abbreviation for route of administration. "i.v.” is an abbreviation for intravenous. Table 14: Anti-IL-1RAP DXd ADC In Vivo Study Design Group No.
  • Dosing ROA Dosing Mice Level Solution volume Frequency (mg/kg) (mg/mL (uL/g) & ) Duration 1 8 Vehicle - - 10 i.v. D0, D5, (saline) and D10 2 8 Isotype 1 0.1 3 8 DXd 3 0.3 4 8 10 1 5 8 ADV58 1 0.1 6 8 DXd 3 0.3 7 8 10 1 [0482] After tumor cell inoculation, the mice were checked daily for morbidity and mortality.
  • the body weights and tumor volumes were measured by using StudyDirector TM software (version 3.1.399.19).
  • Treatment was initiated immediately after grouping (Day 0) per study design. Additional dosing was performed on Day 5 and Day 10. Measurement of tumor size and animal weight was performed until the study endpoint on day 35.
  • ADV58 DXd treatment at all concentrations tested (1 mg/kg, 3 mg/kg, and 10 mg/kg) were found to block tumor growth in the SK-MEL-28 subcutaneous tumor model up to 25 days after the end of the treatment (i.e., from day 10 of dosing until the end of the study).
  • Example 20 In Vivo Cytotoxicity of Anti-IL-1RAP Antibody-Drug Conjugates in Esophageal Cancer
  • ADC anti-IL-1RAP antibody-drug conjugate
  • an esophageal cancer model i.e., a model of esophageal cancer, esophageal squamous cell carcinoma, and/or cancer of the esophagastric junction
  • DXd and MMAE conjugated versions of the anti-IL-1RAP mAb ADV581 i.e., ADV581 DXd and ADV581 MMAE
  • HEL DXd- and MMAE-conjugated human IgG1 hen egg lysozyme
  • Dosing ROA Dosing Mice Level Solution volume Frequency (mg/kg) (mg/mL (uL/g) & ) Duration 1 8 Vehicle - - 10 i.v. Dosing on (saline) D0, D5, 2 8 Isotype HEL 5 0.5 and D10; MMAE studu 3 8 ADV581 5 0.5 sustained MMAE for 50 4 8 Isotype HEL 5 0.5 days DXd 5 8 ADV581 5 0.5 DXd [0488] After tumor cell inoculation, the mice were checked daily for morbidity and mortality.
  • the body weights and tumor volumes were measured by using StudyDirector TM software (version 3.1.399.19). The individual mouse was euthanized if its tumor volume exceeds 1500 mm 3 .
  • treatment was initiated immediately after grouping (Day 0) per study design. Additional dosing was performed on Day 5 and Day 10. Measurement of tumor size and animal weight was performed until the study endpoint on day 50.
  • both the ADV581 DXd and ADV581 MMAE treatments at 5mg/kg were found to induce tumor shrinkage in the subcutaneous tumor model up to 40 days after the end of the treatment (i.e., from day 10 of dosing until the end of the study).
  • the ADV581 DXd treatment led to a more significant and sustained tumor shrinkage compared to the ADV581 MMAE.
  • the isotype HEL DXd and isotype HEL MMAE treatments demonstrated a moderate decrease in tumor growth compared to the vehicle control, with the isotype HEL DXd showing a stronger effect, although both isotype controls were significantly less potent than the ADV581 MMAE treatment and much less potent than the ADV581 DXd treatment. This suggests some amount of IL-1RAP target-independent anti-tumor effect from the DXd and MMAE molecules.
  • Figure 30C shows a Kaplan-Meier analysis for each of the 5 study groups, with the probability of survival based on the time for each group’s mean tumor volume to reach 1500mm 3 .
  • the median survival for each of the vehicle, isotype HEL MMAE, ADV581 MMAE, and isotype HEL DXd were 17.5 days, 21 days, 41.5 days, and 31.5 days, respectively.
  • Example 21 In Vivo Cytotoxicity of Anti-IL-1RAP Antibody-Drug Conjugates in Stomach Cancer
  • ADC anti-IL-1RAP antibody-drug conjugate
  • a stomach cancer model i.e., a model of stomach cancer or gastric adenocarcinoma
  • DXd and MMAE conjugated versions of the anti-IL-1RAP mAb ADV581 i.e., ADV581 DXd and ADV581 MMAE
  • a cleavable linker e.g., as described in Example 10
  • HEL DXd- and MMAE-conjugated human IgG1 hen egg lysozyme
  • HEL human IgG1 hen egg lysozyme
  • Dosing ROA Dosing Mice Level Solution volume Frequency (mg/kg) (mg/mL (uL/g) & ) Duration 1 8 Vehicle - - 10 i.v. Dosing on (saline) D0, D5, 2 8 Isotype HEL 5 0.5 and D10; MMAE study 3 8 ADV581 5 0.5 sustained MMAE for 60 4 8 Isotype HEL 5 0.5 days DXd 5 8 ADV581 5 0.5 DXd [0494] After tumor cell inoculation, the mice were checked daily for morbidity and mortality.
  • the body weights and tumor volumes were measured by using StudyDirector TM software (version 3.1.399.19). The individual mouse was euthanized if its tumor volume exceeds 1500 mm 3 .
  • treatment was initiated immediately after grouping (Day 0) per study design. Additional dosing was performed on Day 5 and Day 10. Measurement of tumor size and animal weight was performed until the study endpoint on day 60.
  • both the ADV581 DXd and ADV581 MMAE treatments at 5mg/kg were found to induce tumor shrinkage in the subcutaneous tumor model up to 50 days after the end of the treatment (i.e., from day 10 of dosing until the end of the study).
  • the ADV581 DXd treatment led to a more significant and sustained tumor shrinkage compared to the ADV581 MMAE.
  • the isotype HEL DXd and isotype HEL MMAE treatments demonstrated a moderate decrease in tumor growth compared to the vehicle control, with the isotype HEL DXd showing a stronger effect, although both isotype controls were significantly less potent than the ADV581 MMAE treatment and much less potent than the ADV581 DXd treatment. This suggests some amount of IL-1RAP target-independent anti-tumor effect from the DXd and MMAE molecules.
  • Figure 31C shows a Kaplan-Meier analysis for each of the 5 study groups, with the probability of survival based on the time for each group’s mean tumor volume to reach 1500mm 3 .
  • the median survival for each of the vehicle and isotype HEL MMAE were 19 days and 32 days, respectively.
  • the median survival of the ADV581 MMAE and isotype HEL DXd groups was not reached.
  • Example 22 In Vivo Cytotoxicity of Anti-IL-1RAP Antibody-Drug Conjugates in Ewing Sarcoma [0498] To determine the in vivo cytotoxicity of an anti-IL-1RAP antibody-drug conjugate (ADC) in a Ewing sarcoma model RD-ES, DXd and MMAE conjugated versions of the anti-IL- 1RAP mAb ADV581 (i.e., ADV581 DXd and ADV581 MMAE) with a cleavable linker (e.g., as described in Example 10), and DXd- and MMAE-conjugated human IgG1 hen egg lysozyme (HEL) isotype controls (i.e., Isotype DXd and Isotype MMAE), were tested in vivo in the treatment of an established a human Ewing sarcoma RD-ES subcutaneous xenograft model in female CB.17 SCID immunocompromised mice
  • mice 10-12 week old female CB.17 SCID immunocompromised mice were subcutaneously injected with 5x10 6 RD-ES cells in 0.1 mL PBS and Matrigel (50%). Upon the mean tumor size reaching an average size of 80-120 mm 3 , animals were randomized into 5 study groups, with 8 mice per group. The date of randomization is denoted as day 0. The study design is illustrated in Table 17 below. "ROA” is an abbreviation for route of administration. "i.v.” is an abbreviation for intravenous. Table 17: Anti-IL-1RAP ADC In Vivo Study Design Group No.
  • the body weights and tumor volumes were measured by using StudyDirector TM software (version 3.1.399.19). The individual mouse was euthanized if its tumor volume exceeds 1500 mm 3 .
  • treatment was initiated immediately after grouping (Day 0) per study design. Additional dosing was performed on Day 5 and Day 10. Measurement of tumor size and animal weight was performed until the study endpoint on day 46.
  • the ADV581 DXd treatment at 5mg/kg was found to induce tumor growth stasis in the subcutaneous RD-ES tumor model up to 36 days after the end of the treatment (i.e., from day 10 of dosing until the end of the study).
  • FIG. 32C shows a Kaplan-Meier analysis for each of the 5 study groups, with the probability of survival based on the time for each group’s mean tumor volume to reach 1500mm 3 .
  • the median survival for each of the vehicle, isotype HEL MMAE, and isotype HEL DXd treatment groups were 28.5 days, 25 days, and 26 days, respectively.
  • the median survival of the ADV581 MMAE and ADV581 DXd groups was not reached.
  • Both the ADV581 DXd and ADV581 MMAE treatments were well tolerated, with a similar mean body weight change across the study compared to the isotype HEL DXd, isotype HEL MMAE, and vehicle controls, as shown in Figure 32B.
  • Example 23 In Vivo Cytotoxicity of Anti-IL-1RAP Antibody-Drug Conjugates in Head and Neck Cancer
  • ADC anti-IL-1RAP antibody-drug conjugate
  • a head and neck cancer model i.e., a model of head and neck squamous cell carcinoma
  • DXd and MMAE conjugated versions of the anti-IL-1RAP mAb ADV581 i.e., ADV581 DXd and ADV581 MMAE
  • a cleavable linker e.g., as described in Example 10
  • HEL DXd- and MMAE-conjugated human IgG1 hen egg lysozyme
  • HEL human IgG1 hen egg lysozyme
  • Dosing ROA Dosing Mice Level Solution volume Frequency (mg/kg) (mg/mL (uL/g) & ) Duration 1 8 Vehicle - - 10 i.v. Dosing on (saline) D0, D5, 2 8 Isotype HEL 5 0.5 and D10; MMAE study 3 8 ADV581 5 0.5 sustained MMAE 4 8 Isotype HEL 5 0.5 for 45 DXd days 5 8 ADV581 5 0.5 DXd [0506] After tumor cell inoculation, the mice were checked daily for morbidity and mortality.
  • the body weights and tumor volumes were measured by using StudyDirector TM software (version 3.1.399.19). The individual mouse was euthanized if its tumor volume exceeds 1500 mm 3 .
  • treatment was initiated immediately after grouping (Day 0) per study design. Additional dosing was performed on Day 5 and Day 10. Measurement of tumor size and animal weight was performed until the study endpoint on day 45.
  • the ADV581 MMAE treatment at 5mg/kg was found to induce tumor shrinkage in the subcutaneous tumor model up to 35 days after the end of the treatment (i.e., from day 10 of dosing until the end of the study).
  • Figure 33C shows a Kaplan-Meier analysis for each of the 5 study groups, with the probability of survival based on the time for each group’s mean tumor volume to reach 1500mm 3 .
  • the median survival for each of the vehicle, isotype HEL MMAE, isotype HEL DXd, and ADV581 DXd treatment groups were 14 days, 45 days, 17 days, and 31 days, respectively.
  • a significant anti-tumor response was observed in the ADV581 MMAE group which was maintained after the end of treatment.
  • Example 24 In Vivo Cytotoxicity of Anti-IL-1RAP Antibody-Drug Conjugates in Ovarian Cancer
  • ADC anti-IL-1RAP antibody-drug conjugate
  • DXd and MMAE conjugated versions of the anti-IL-1RAP mAb ADV581 i.e., ADV581 DXd and ADV581 MMAE
  • a cleavable linker e.g., as described in Example 10
  • HEL human IgG1 hen egg lysozyme
  • Dosing ROA Dosing Mice Level Solution volume Frequency (mg/kg) (mg/mL (uL/g) & ) Duration 1 8 Vehicle - - 10 i.v. Dosing on (saline) D0, D5, 2 8 Isotype HEL 5 0.5 and D10; MMAE study 3 8 ADV581 5 0.5 sustained MMAE 4 8 Isotype HEL 5 0.5 for 60 DXd days 5 8 ADV581 5 0.5 DXd [0512] After tumor cell inoculation, the mice were checked daily for morbidity and mortality.
  • the body weights and tumor volumes were measured by using StudyDirector TM software (version 3.1.399.19). The individual mouse was euthanized if its tumor volume exceeds 600 mm 3 .
  • treatment was initiated immediately after grouping (Day 0) per study design. Additional dosing was performed on Day 5 and Day 10. Measurement of tumor size and animal weight was performed until the study endpoint on day 60.
  • both the ADV581 DXd and ADV581 MMAE treatments at 5mg/kg were found to induce tumor shrinkage in the subcutaneous tumor model up to 50 days after the end of the treatment (i.e., from day 10 of dosing until the end of the study).
  • the median survival for each of the vehicle, isotype HEL MMAE, and isotype HEL DXd groups was 27 days, 49.5 days, and 53 days, respectively.
  • This data highlights the long-term anti-tumor effects of ADV581 DXd and ADV581 MMAE in this ovarian cancer model.

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Abstract

La présente divulgation concerne des anticorps humanisés et des conjugués anticorps humanisés -médicament (ADC) comprenant un anticorps humanisé ou un fragment de liaison à l'antigène de celui-ci qui se lie à IL-1RAP. Les anticorps et les ADC de la présente invention sont utiles, entre autres, pour traiter des maladies avec une expression d'IL-1RAP régulée à la hausse.
PCT/IB2024/057373 2023-07-31 2024-07-30 Conjugués médicament-anticorps anti-il-1rap et leurs procédés d'utilisation WO2025027529A1 (fr)

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