Combined therapy of a DR5 agonist and an IAP antagonist

Abstract

Provided herein are methods of treating cancer using a combination of a DR5 agonist and an IAP antagonist.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 344,675, filed on May 23, 2022, and U.S. Provisional Application No. 63 / 494,276, filed on April 5, 2023, the entire contents of each of which are hereby incorporated by reference in their entirety for all purposes.

[0002] [Incorporation by Reference of Sequence Listing] This application incorporates by reference a Sequence Listing of 14,058 bytes in size, titled 01202 - 0058 - 00PCT_Sequence_Listing, created on May 5, 2023, and filed electronically with this application.

[0003] The present invention relates to the treatment of cancer using a combination of a DR5 agonist and an IAP antagonist.

Background Art

[0004] Death receptor 5 (DR5; also known as TNFRSF10B or TRAILR2) is a member of the TNF receptor superfamily (TNFRSF) and is a cell surface receptor of the TNF receptor superfamily that binds to TNF-related apoptosis-inducing ligand (TRAIL). TRAIL has evolved to play an important role in mammalian development and host defense by selectively removing unwanted cells, infected cells, and malignant cells from healthy cell populations. TRAIL induces cell death by caspase-dependent apoptosis by binding to DR4 or DR5, members of the TNF receptor family. DR5 appears to be the major receptor on tumor cells that promotes the tumor-biased activity observed in the TRAIL pathway. DR5 is activated by the natural ligand TRAIL, and by bringing three DR5 receptors into proximity, it activates intracellular caspase-8 and initiates the activation of other cell death-inducing caspases such as caspase-9 and caspase-3. Thus, clustering of DR5 receptors for efficient cell death is required for the initiation of this cell death pathway. DR5 agonists are promising therapeutic candidates for the treatment of cancer.

[0005] Apoptosis inhibitor proteins (IAPs) are a class of proteins that negatively regulate caspases and apoptosis. IAPs are frequently dysregulated in many cancers and have been suggested to contribute to apoptosis resistance in cancer cells. IAPs contribute to tumor cell survival, chemotherapy resistance, disease progression, and poor prognosis. In addition, IAPs play important roles in immune regulation. Examples of IAPs include cellular IAP1 (cIAP1), cellular IAP2 (cIAP2), and X-linked inhibitor of apoptosis protein (XIAP). IAPs have emerged as drug targets in a wide range of malignancies due to their important biological functions in cell death and immune responses. Several IAP antagonists have been developed and their clinical benefits are currently being investigated. For example, the second mitochondria-derived activator of caspase (SMAC) protein inhibits IAP function, and multiple small molecules (known as SMAC mimetics) that mimic SMAC function have been developed. Summary of the Invention

[0006] Methods for treating cancer in a subject using a death receptor 5 (DR5) agonist and an apoptosis inhibitory protein (IAP) antagonist are provided herein. In some embodiments, the method comprises administering a multivalent death receptor 5 (DR5) binding polypeptide and an apoptosis inhibitory protein (IAP) antagonist. In some embodiments, the multivalent DR5 binding polypeptide is at least tetravalent. In some embodiments, the multivalent DR5 binding polypeptide is tetravalent.

[0007] Embodiment 1. A method for treating cancer in a subject in need of treatment, the method comprising administering to the subject (a) a death receptor 5 (DR5) agonist that is tetravalent, and (b) an apoptosis inhibitory protein (IAP) antagonist.

[0008] Embodiment 2. The method according to Embodiment 1, wherein the DR5 agonist is a DR5 binding polypeptide.

[0009] Embodiment 3. The method according to Embodiment 1 or Embodiment 2, wherein the DR5 binding polypeptide comprises at least one VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3.

[0010] Embodiment 4. The method according to Embodiment 3, wherein the at least one VHH domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4.

[0011] Embodiment 5. The method according to any one of Embodiments 2 to 4, wherein the DR5 binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 4.

[0012] Embodiment 6. The method according to any one of Embodiments 2 to 5, wherein the DR5-binding polypeptide contains an Fc region.

[0013] Embodiment 7. The method according to Embodiment 6, wherein the Fc region contains the amino acid sequence of SEQ ID NO: 6.

[0014] Embodiment 8. The method according to any one of Embodiments 2 to 7, wherein the DR5-binding polypeptide has a structure of VHH-linker-VHH-linker-Fc.

[0015] Embodiment 9. The method according to any one of Embodiments 2 to 8, wherein each VHH domain contains CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO: 3.

[0016] Embodiment 10. The method according to any one of Embodiments 2 to 9, wherein the VHH-linker-VHH contains an amino acid sequence that is at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5.

[0017] Embodiment 11. The method according to Embodiment 10, wherein the VHH-linker-VHH contains the amino acid sequence of SEQ ID NO: 5.

[0018] Embodiment 12. The method according to any one of Embodiments 2 to 11, wherein the DR5-binding polypeptide contains an amino acid sequence that is at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7.

[0019] Embodiment 13. The method according to any one of Embodiments 2 to 12, wherein the DR5-binding polypeptide contains the amino acid sequence of SEQ ID NO: 7.

[0020] Embodiment 14. The method according to any one of Embodiments 2 to 12, wherein the DR5-binding polypeptide consists of the amino acid sequence of SEQ ID NO: 7.

[0021] Embodiment 15. The method according to Embodiment 1 or Embodiment 2, wherein the DR5 agonist is INBRX-109.

[0022] Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein the IAP antagonist is a small molecule.

[0023] Embodiment 17. The method according to any one of Embodiments 1 to 16, wherein the IAP antagonist is APG-1387 (Ascentage Pharma Group International), birinapant (IGM Biosciences, Inc.), AZD5582 (AstraZeneca), LCL161 (Novartis), Debio 1143 (Merck, Debiopharm) or ASTX660 (Astex Pharmaceuticals, Inc.).

[0024] Embodiment 18. The method according to Embodiment 17, wherein the IAP antagonist is APG-1387.

[0025] Embodiment 19. The method according to Embodiment 17, wherein the IAP antagonist is birinapant.

[0026] Embodiment 20. The method according to Embodiment 17, wherein the IAP antagonist is Debio 1143.

[0027] Embodiment 21. The method according to Embodiment 17, wherein the IAP antagonist is LCL161.

[0028] Embodiment 22. The method according to any one of Embodiments 1 to 21, wherein the DR5 agonist and the IAP antagonist are administered separately.

[0029] Embodiment 23. The method according to Embodiment 22, wherein the DR5 agonist and the IAP antagonist are administered sequentially.

[0030] Embodiment 24. The method according to embodiment 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered before the IAP antagonist.

[0031] Embodiment 25. The method according to embodiment 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered after the IAP antagonist.

[0032] Embodiment 26. The method according to any one of embodiments 1 to 21, wherein the DR5 agonist and the IAP antagonist are administered in combination.

[0033] Embodiment 27. The method according to any one of embodiments 1 to 26, wherein the DR5 agonist and the IAP antagonist act synergistically.

[0034] Embodiment 28. The method according to embodiment 27, wherein the synergistic effect is determined in an in vitro cell survival assay.

[0035] Embodiment 29. The method according to any one of embodiments 1 to 28, wherein the administration of the DR5 agonist and the IAP antagonist results in a synergistic effect as compared to the administration of each agent alone.

[0036] Embodiment 30. The method according to any one of Embodiments 1 to 29, wherein the cancer is adrenal cancer, astrocytoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing sarcoma, colorectal cancer (colon cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, melanoma, multiple myeloma, neuroblastoma, oral cancer (lip, tongue, mouth and / or pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary system cancer and vulvar cancer, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), as well as nevus, edema (such as edema related to brain tumor) and abnormal blood vessel proliferation related to Meigs syndrome.

[0037] Embodiment 31. A DR5 agonist used in a method for treating cancer, the method comprising administering the DR5 agonist in combination with an IAP antagonist.

[0038] Embodiment 32. Use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is administered together with an IAP antagonist.

Brief Description of the Drawings

[0039]

Fig. 1A-E

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Best Mode for Carrying Out the Invention

[0040] The embodiments described herein relate to a method for treating cancer using a combination of a cell death receptor 5 (DR5) agonist and an IAP (inhibitor of apoptosis protein) antagonist.

[0041] Definitions and Various Embodiments The section headings used herein are for the purpose of organization only and are not to be construed as limiting the subject matter described.

[0042] All references cited herein, including patent applications, patent publications, and Genbank accession numbers, are incorporated herein by reference as if each individual reference were specifically and individually indicated as being incorporated by reference in its entirety.

[0043] The techniques and procedures described or referenced herein are generally well understood and commonly practiced, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3 rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)), the series METHODS IN ENZYMOLOGY (Academic Press, Inc.), PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)), Oligonucleotide Synthesis (M. J. Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press, Animal Cell Culture (R. I. Freshney, ed., 1987), Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell eds., 1993 - 8) J. Wiley and Sons, Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (J. E. Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C. A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 1993) and the conventional methodologies by those skilled in the art, such as the widely used methodologies described in their latest editions, are used.

[0044] Unless otherwise specified, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those skilled in the art. Further, unless the context requires otherwise or specifically indicates otherwise, singular nouns shall include the plural and plural nouns shall include the singular. In case of any conflict in definitions between various sources or references, the definitions set forth herein shall prevail.

[0045] Generally, the numbering of residues in immunoglobulin heavy chains is according to the EU index numbering as in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibody.

[0046] The embodiments of the invention described herein are understood to include embodiments "consisting of" and / or embodiments "consisting essentially of". As used herein, the singular forms ("a", "an", and "the") include plural references unless specifically indicated otherwise. The use of the term "or" herein is not to be construed as meaning that the alternatives are mutually exclusive.

[0047] In this application, the use of "or" means "and / or" unless specifically specified otherwise or not understood by those skilled in the art. In the context of multiple dependent claims, the use of "or" refers to citing two or more preceding independent or dependent claims.

[0048] The terms "reference sample", "reference cell", or "reference tissue" refer to a sample having at least one known characteristic that can be used for comparison with a sample having at least one unknown characteristic. In some embodiments, the reference sample can be used as a positive or negative indicator. By using a reference sample, it is possible to establish, for example, the levels of proteins and / or mRNAs present in a sample having unknown characteristics relative to the levels of proteins and / or mRNAs present in healthy tissue. In some embodiments, the reference sample is a sample that is derived from the same subject but from a part of the subject that is different from the part being tested. In some embodiments, the reference sample is a sample that is derived from a tissue region surrounding or adjacent to a cancer. In some embodiments, the reference sample is not derived from the subject being tested but is a sample that is derived from a subject known to have or not have a target disorder (e.g., a specific cancer or a DR5-related disorder). In some embodiments, the reference sample is derived from the same subject but is a sample at a time point before the subject develops cancer. In some embodiments, the reference sample is a sample that is derived from a benign cancer sample from the same or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the target molecule in the negative reference sample indicates a level at which one of ordinary skill in the art, considering the present disclosure, would recognize that the molecule is absent and / or that a low level of the molecule is present. When a positive reference sample is used for comparison, the expression level or amount of the target molecule in the positive reference sample indicates a level at which one of ordinary skill in the art, considering the present disclosure, would recognize that a certain level of the molecule is present.

[0049] As used herein in the context of a subject receiving a benefit from or responding to administration of a therapeutic agent, the terms "benefit", "clinical benefit", "responsiveness", and "therapeutic responsiveness" can be gauged by assessing various endpoints, such as inhibition of disease progression, including deceleration and complete cessation; reduction in the number of disease episodes and / or symptoms; reduction in lesion size; inhibition (i.e., reduction, deceleration, or complete cessation) of infiltration of diseased cells into adjacent peripheral organs and / or peripheral tissue; inhibition (i.e., reduction, deceleration, or complete cessation) of disease spread; some alleviation of one or more symptoms associated with the disorder; disease-free presentation after treatment, such as an increase in the length of the progression-free survival period, an extension of the overall survival period, a higher response rate, and / or a decrease in the mortality rate at a given point in time after treatment. A "non-responsive" or "non-responding" subject or cancer is one that does not meet the above conditions for "responding".

[0050] The terms "nucleic acid molecule", "nucleic acid", and "polynucleotide" are used interchangeably and can refer to a polymer of nucleotides. Such a polymer of nucleotides can include natural and / or non-natural nucleotides and can include, but is not limited to, DNA, RNA, and PNA. A "nucleotide sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0051] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Such polymers of amino acid residues include natural or unnatural amino acid residues and, without limitation, may include peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, for the purposes of the present disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative with respect to properties) to the native sequence, so long as the protein maintains the desired activity. These modifications can be intentional, such as by site-directed mutagenesis, or can be accidental, such as by mutations in the host producing the protein or errors in PCR amplification.

[0052] As used herein, the terms "DR5", "death receptor 5", "TNFRSF10B", and "TRAILR2" refer to any native mature DR5 resulting from the processing of the DR5 precursor intracellularly. This term includes DR5 from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. This term also includes naturally occurring variants of DR5, such as splice variants or allelic variants. A non-limiting exemplary precursor human DR5 amino acid sequence is shown, for example, in NCBI accession number NP_003833.4. See SEQ ID NO:8. A non-limiting exemplary precursor human DR5 amino acid sequence is shown, for example, in SEQ ID NO:9.

[0053] The terms "IAP" and "apoptosis inhibitory protein" refer to any native, mature protein of the IAP protein family, which has at least eight known members. This term includes IAPs from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. This term also includes naturally occurring variants of IAPs, such as splice variants or allelic variants. IAPs function as major regulators of caspase activity and are defined by the presence of at least one baculovirus IAP repeat (BIR) domain. These approximately 70-residue zinc-binding domains enable interaction with caspases and caspase inhibition, thus promoting the inhibition of apoptosis. Most IAPs also contain a RING finger E3 ligase domain at the C-terminus, which enables these proteins to participate in diverse cellular processes, including signaling events that promote inflammation, cell cycle progression, and migration.

[0054] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" when it reacts or associates more frequently, more rapidly, for a longer duration and / or with a higher affinity with a particular cell or substance than with another cell or substance. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target when it binds with a higher affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a DR5 epitope is an sdAb or VHH-containing polypeptide that binds to this epitope with a higher affinity, avidity, more readily, and / or for a longer duration than it binds to other DR5 epitopes or non-DR5 epitopes. Also, by interpreting this definition, it is understood that, for example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, although not always, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0055] The terms "inhibit" or "inhibiting" refer to a decrease or cessation of any phenotypic feature, or a decrease or cessation in the incidence, degree, or likelihood of that feature. "Reduce" or "inhibit" means to cause a decrease, reduction, or cessation of activity, function, and / or amount, as compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 10% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 50% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall decrease of 75%, 85%, 90%, 95% or more. In some embodiments, the above amounts are inhibited or decreased over a period of time as compared to a control over the same period of time.

[0056] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or a VHH-containing polypeptide) binds. Epitopes often include chemically active surface configurations of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features and specific charge features. Epitopes can be formed from both contiguous residues and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are usually retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8 to 10 residues (e.g., amino acids or nucleotides). In some embodiments, the epitope has a length of less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope can be defined by a certain minimum distance from CDR residues on the antigen-binding molecule. In some embodiments, an epitope can be defined by such a distance and further limited to those residues involved in a bond (e.g., a hydrogen bond) between a residue of the antigen-binding molecule and an antigen residue. Epitopes can also be defined by various scans. For example, an alanine scan or an arginine scan can identify one or more residues with which the antigen-binding molecule can interact. Unless explicitly stated otherwise, a set of residues as an epitope does not exclude the possibility that other residues are part of the epitope for a particular antigen-binding molecule. Rather, the presence of such a set indicates a minimal epitope sequence (or set of types). Thus, in some embodiments, a set of residues defined as an epitope does not represent an exclusive list of residues for the epitope on the antigen, but rather indicates the minimal epitope associated with the antigen.

[0057] The term "antibody" is used in the broadest sense and is not limited, but includes conventional antibodies (typically including at least one heavy chain and at least one light chain), single domain antibodies (sdAb, including at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides containing at least one VHH domain), and antibody-like antigen-binding domains including any of the above fragments as long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody includes a dimerization domain. Such dimerization domains include, but are not limited to, heavy chain constant domains (including CH1, hinge, CH2, and CH3, where CH1 typically pairs with the light chain constant domain CL, and the hinge mediates dimerization) and Fc regions (including hinge, CH2, and CH3, and the hinge mediates dimerization).

[0058] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as camel (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0059] As used herein, the term "antigen-binding domain" refers to the portion of an antibody sufficient to bind an antigen. In some embodiments, the antigen-binding domain of a conventional antibody comprises three heavy-chain CDRs and three light-chain CDRs. Thus, in some embodiments, the antigen-binding domain comprises a heavy-chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen, and a light-chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen. In some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises the three CDRs of the VHH domain. Thus, in some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen.

[0060] As used herein, the term "VHH" or "VHH domain" or "VHH antigen-binding domain" refers to the antigen-binding portion of a single-domain antibody such as a camelid antibody or shark antibody. In some embodiments, a VHH comprises three CDRs and four framework regions designated FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, a VHH may be truncated at the N-terminus and / or C-terminus such that it comprises only partial FR1 and / or FR4 or lacks one or both of those framework regions, so long as the VHH substantially maintains antigen binding and specificity.

[0061] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one monomeric domain such as a VHH domain that does not have a light chain and an Fc region. In some embodiments, the sdAb is a dimer of two polypeptides, each polypeptide comprising at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" refer to polypeptides comprising multiple VHH domains, e.g., structured VHH 1 -VHH 2 -Fc or VHH 1 -VHH 2 -VHH 3 -Fc, where VHH 1 、VHH 2 、and VHH 3 may be the same or different and includes polypeptides.

[0062] The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, the VHH polypeptide comprises two, three, or four or more VHH domains, where each VHH domain may be the same or different. In some embodiments, the VHH-containing polypeptide comprises an Fc region. In some such embodiments, the VHH-containing polypeptide may be referred to as an sdAb. Further, in some such embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of VHH-containing polypeptides, also referred to as sdAbs, include VHH 1 -Fc, VHH 1 -VHH 2 -Fc, and VHH 1 -VHH 2 -VHH 3 -Fc, where VHH 1 、VHH 2 、and VHH 3may be the same or different. In some embodiments of such a structure, a VHH may be linked to another VHH by a linker, or a VHH may be linked to an Fc by a linker. In some such embodiments, the linker comprises from 1 to 20 amino acids, preferably from 1 to 20 amino acids consisting mainly of glycine and optionally serine. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH 1 -VHH 2 -Fc is considered to be tetravalent when it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH 1 -VHH 2 -VHH 3 -Fc is considered to be hexavalent when it forms a dimer (i.e., the dimer has six VHH domains).

[0063] The term "monoclonal antibody" refers to antibodies (including sdAb or VHH-containing polypeptides) of a substantially homogeneous antibody population. That is, the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Further, typically, in contrast to polyclonal antibody preparations that include different antibodies against different determinants (epitopes), each monoclonal antibody is an antibody against a single determinant on the antigen. Thus, a sample of monoclonal antibodies can bind to the same epitope on the antigen. The modifying phrase "monoclonal" indicates the nature of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be produced by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries produced using techniques such as those described in McCafferty et al., 1990, Nature 348:552-554.

[0064] The term "CDR" refers to complementarity-determining regions defined by at least one particular scheme for one of ordinary skill in the art. In some embodiments, the CDRs can be defined according to any of the Chothia numbering scheme, Kabat numbering scheme, combination of Kabat and Chothia, AbM definition, and / or contact definition. VHH contains three CDRs designated CDR1, CDR2, and CDR3.

[0065] As used herein, the term "heavy chain constant region" refers to at least three heavy chain constant domains, namely, C H 1, hinge, C H 2, and C HRefers to the region containing 3. Of course, deletions and modifications that do not change the function within the domain are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Further, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. A particular isotype may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibody (containing γ 1 constant region), IgG2 antibody (containing γ 2 constant region), IgG3 antibody (containing γ 3 constant region), and IgG4 antibody (containing γ 4 constant region), and IgA antibodies include, but are not limited to, IgA1 antibody (containing α 1 constant region) and IgA2 antibody (containing α 2 constant region), and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0066] As used herein, "Fc region" refers to a portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0067] As used herein, "acceptor human framework" refers to a heavy chain variable domain (V H) It is a framework comprising the amino acid sequence of the framework. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can contain the same amino acid sequence or can contain changes in the amino acid sequence. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all human frameworks within a single antigen-binding domain such as a VHH.

[0068] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody such as an sdAb or a VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of molecule X for its partner Y is generally represented by the dissociation constant (K D ) or K D(見かけ) . Affinity can be measured by conventional methods known in the art including the methods described herein (e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance devices, etc.). Such methods include, but are not limited to, methods requiring BIAcore®, Octet®, or flow cytometry.

[0069] As used herein, the term "K D " refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. When the term "K D " is used herein, it includes K D and K D(見かけ) .

[0070] In some embodiments, the K D of an antigen-binding molecule is measured by flow cytometry using an antigen-expressing cell line and fitting the mean fluorescence measured at each antibody concentration to a non-linear one-site binding equation (graphpad's Prism Software). In some such embodiments, KD is K D(見かけ) is.

[0071] The term "biological activity" refers to any one or more biological properties of a molecule (whether occurring naturally as seen in vivo, or provided or made possible by recombinant means). Biological properties include, but are not limited to, ligand binding, induction or increase of cell proliferation, and induction or increase of cytokine expression.

[0072] An "agonist" antibody or polypeptide or an "activating" antibody or polypeptide is an antibody or polypeptide that increases and / or activates the biological activity of a target antigen. In some embodiments, an agonist antibody or polypeptide binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0073] An "antagonist" antibody, "blocking" antibody, or "neutralizing" antibody is an antibody that inhibits, reduces and / or inactivates the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0074] An "affinity matured" sdAb or VHH-containing polypeptide refers to an sdAb or VHH-containing polypeptide having one or more such modifications in one or more CDRs compared to a parental sdAb or VHH-containing polypeptide that does not have such modifications that result in an improvement in the affinity of the sdAb or VHH-containing polypeptide for the antigen.

[0075] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions are substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Further, a humanized VHH may contain residues that are not found in the original VHH or the human framework sequence but are included to further improve and optimize the performance of the sdAb or VHH-containing polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide comprises a human Fc region. As will be appreciated, a humanized sequence can be identified by its primary sequence and does not necessarily indicate the process by which the antibody was made.

[0076] An "effector-positive Fc region" has the "effector functions" of the Fc region of the native sequence. Exemplary "effector functions" include Fc receptor binding, Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be evaluated using various assays.

[0077] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of the Fc region found in nature. Native sequence human Fc regions include the native sequence human IgG1 Fc region (non-A allotype and A allotype), the native sequence human IgG2 Fc region, the native sequence human IgG3 Fc region, and the native sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

[0078] The "mutant Fc region" includes an amino acid sequence that is different from the amino acid sequence of the Fc region of the native sequence due to at least one amino acid modification. In some embodiments, the "mutant Fc region" includes an amino acid sequence that is different from the amino acid sequence of the Fc region of the native sequence due to at least one amino acid modification, but retains at least one effector function of the Fc region of the native sequence. In some embodiments, the mutant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the Fc region of the native sequence or the Fc region of the parent polypeptide as compared to the Fc region of the native sequence or the Fc region of the parent polypeptide. In some embodiments, the mutant Fc regions herein have at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the Fc region of the native sequence and / or the Fc region of the parent polypeptide.

[0079] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds to an IgG antibody (gamma receptor) and is a receptor of the FcγRI subclass, FcγRII subclass, and FcγRIII subclass, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences but mainly differ in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. For example, the terms "Fc receptor" or "FcR" also include the neonatal receptor FcRn, which plays a role in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the regulation of immunoglobulin isotype.Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997), Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997), Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004), WO 2004 / 92219 (Hinton et al.)).

[0080] As used herein, the terms "substantially similar" or "substantially the same" indicate a sufficiently high degree of similarity between two or more numerical values such that one of ordinary skill in the art would consider the difference between the two or more values to be of little or no biological and / or statistical significance within the context of the biological characteristics measured by said values. In some embodiments, two or more substantially similar values differ by no more than an approximation of any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0081] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity to a native sequence polypeptide, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, the variant has at least about 80% amino acid sequence identity. In some embodiments, the variant has at least about 90% amino acid sequence identity. In some embodiments, the variant has at least about 95% amino acid sequence identity to a native sequence polypeptide.

[0082] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide sequence, polypeptide sequence, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide sequence or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0083] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into the antibody of interest and the product screened for retention / improvement of a desired activity, such as antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC.

[0084]

Table 1

[0085] Amino acids can be grouped according to common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues that influence chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0086] Non-conservative substitutions involve exchanging one member of these classes for another.

[0087] The term "vector" is used to describe a polynucleotide that can be engineered to contain a cloned polynucleotide(s) that can be propagated within a host cell. A vector can contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., a promoter and / or enhancer, etc.) that regulate the expression of the polypeptide of interest, and / or one or more selectable marker genes (e.g., an antibiotic resistance gene and genes that can be used in a colorimetric assay, e.g., β-galactosidase, etc.). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0088] "Host cell" refers to a cell that can be or has been a recipient of a vector or an isolated polynucleotide. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells such as primate or non-primate animal cells, fungal cells such as yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6™ cells (Crucell), as well as 293 cells and CHO cells, and their derivatives, such as 293-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. Host cells include the progeny of a single host cell, but due to natural mutations, accidental mutations, or intentional mutations, the progeny are not necessarily identical (in morphology or genomic DNA complement) to the original parent cell. Host cells also include cells transfected in vivo with the polynucleotide(s) provided herein.

[0089] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are typically found together or produced together in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least a portion of the components of the cell that produced it. When a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is "isolated" when it is not part of a larger polynucleotide (e.g., genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide) in which it is typically found in nature, or when, for example, in the case of an RNA polynucleotide, it is separated from at least a portion of the components of the cell that produced it. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0090] The terms "individual" and "subject" are used interchangeably herein to refer to an animal, such as a mammal. In some embodiments, but not limited to, methods of treating mammals including humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets are provided. In some examples, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject being treated can be a patient who has been identified as having or at sufficient risk of having a disorder related to the treatment.

[0091] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0092] The terms "tumor cell", "cancer cell", "cancer", "tumor", and / or "neoplasm" are used interchangeably herein unless otherwise specified, and refer to cells (or a plurality of cells) that exhibit uncontrolled proliferation and / or increased abnormal cell survival and / or inhibition of apoptosis that interfere with the normal functions of body organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasia, and latent tumors or micrometastases.

[0093] The terms "cancer" and "tumor" include solid cancers and hematological cancers / lymphadenocarcinomas, as well as malignant tumors such as dysplasia, premalignant tumors, and benign tumors. Exemplary cancers include, but are not limited to, adrenal cancer, astrocytoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing sarcoma, colon and rectal cancer (colorectal cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer), melanoma, multiple myeloma, neuroblastoma, oral cancer (lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorder (PTLD), and angiomatosis, edema (such as edema associated with brain tumors), and abnormal vascular proliferation associated with Meigs syndrome.

[0094] In some embodiments, "increase" or "decrease" each refers to a statistically significant increase or decrease. As will be apparent to those skilled in the art, "modulating" also refers to a change (which can be either an increase or a decrease) in the affinity, avidity, specificity, and / or selectivity for one or more of the target's or antigen's ligands, binding partners, homomultimers or heteromultimers, partners or substrates that associate thereto, compared to the same conditions except in the absence of the test agent, causing a change (which can be either an increase or a decrease) in the sensitivity of the target or antigen to one or more conditions (such as pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target or antigen is present, and / or may include cell proliferation or cytokine production. This can be determined in any suitable manner known per se or described herein and / or using any suitable assay, depending on the target involved.

[0095] As used herein, "treatment" is a procedure for obtaining a beneficial or desired clinical outcome. "Treatment" as used herein is directed to any administration or application of a therapeutic agent for a disease in a mammal, including a human. For the purposes of the present disclosure, beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, reduction in the degree of the disease, prevention or delay of the progression of the disease (e.g., metastasis, e.g., metastasis to the lung or lymph nodes), prevention or delay of recurrence of the disease, delay or deceleration of the progression of the disease, improvement of the disease state, inhibition of the disease or the progression thereof, inhibition or deceleration of the disease or its progression, prevention of its development, and remission (partial or total). "Treatment" also includes reduction of the pathological consequences of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In accordance with the foregoing, the term treatment does not require the complete elimination of all aspects of the disorder.

[0096] "Improvement" means that one or more symptoms are alleviated or improved as compared to the case where no therapeutic agent is administered. "Improvement" also includes shortening or reduction of the duration of the symptoms.

[0097] The term "anticancer agent" is used herein in its broadest sense to refer to an agent used in the treatment of one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anticancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiation therapy agents, CAR-T therapy agents, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0098] As used herein, the terms "synergistic," "synergistically," and "synergy" refer to an effect that exceeds the additive effect of two or more agents. The determination of the synergistic effect between a DR5 agonist and an IAP antagonist can be performed using the assays described herein.

[0099] The term "biological sample" means an amount of substance from something that is living or was once living. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0100] The term "control" or "reference" refers to a composition known to be free of the analyte ("negative control") or a composition known to contain the analyte ("positive control"). A positive control may contain a known concentration of the analyte.

[0101] As used herein, "delaying the onset of a disease" means delaying, preventing, slowing down, retarding, stabilizing, suppressing, and / or extending the onset of a disease (such as cancer). This delay can be of various lengths of time depending on the disease history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or substantial delay can in fact encompass prevention in the sense that the individual does not develop the disease. For example, it is possible to delay the onset of advanced cancer such as the occurrence of metastasis.

[0102] As used herein, "prevention" includes providing prophylaxis against the development or recurrence of a disease in a subject who may have a predisposition to the disease but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not indicate or require complete prevention over the entire period, but rather indicate or require prevention only over the measured period.

[0103] The "therapeutically effective amount" of a substance / molecule, agonist or antagonist can vary depending on factors such as the individual's disease state, age, gender, and weight, as well as the ability of the substance / molecule, agonist or antagonist to induce a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the substance / molecule, agonist or antagonist. A therapeutically effective amount can be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective to achieve the desired therapeutic and / or prophylactic results over the necessary dosage and time period.

[0104] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation in a form that enables the biological activity of the active ingredient(s) to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered. Such formulations can be sterilized.

[0105] "Pharmaceutically acceptable carrier" refers to a conventional non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier in the art that is used together with a therapeutic agent to comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations used, is compatible with the other ingredients of the formulation, and is appropriate for the formulation used.

[0106] Administration "in combination" with one or more additional therapeutic agents includes co-administration (simultaneous administration) and sequential administration in any order.

[0107] As used herein, the term "administered in combination" refers to the administration of two or more therapeutic agents, where at least a portion of the administrations overlap in time, or the administration of one therapeutic agent is included within a short period relative to the administration of the other therapeutic agent, or the therapeutic effects of both therapeutic agents overlap for at least some period of time.

[0108] As used herein, the term "sequentially" refers to the administration of two or more therapeutic agents where the administrations do not overlap in time or the therapeutic effects of the therapeutic agents do not overlap.

[0109] As used herein, "in combination with" refers to adding another therapy to the administration of a particular therapy. Thus, "in combination with" refers to administering a particular therapy before, during, or after another therapy is administered to an individual.

[0110] The term "package insert" is used to refer to the instructions for use customarily included in the commercial package of a therapeutic product, which includes information regarding instructions, methods of use, dosage, administration, combination therapies, contraindications and / or warnings regarding the use of such a therapeutic product.

[0111] A "manufactured article" is any manufactured item (e.g., a package or container) or kit that includes at least one reagent, such as an agent for treating a disease or disorder (e.g., cancer), or a probe that specifically detects a biomarker described herein. In some embodiments, the manufactured item or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0112] The terms "label" and "detectable label" mean, for example, a moiety that binds to an antibody or antigen and enables detection of a reaction (e.g., binding) between members of a specific binding pair. A labeled member of a specific binding pair is referred to as "detectably labeled". Thus, the term "labeled binding protein" refers to a protein into which a label that provides for the identification of the binding protein is incorporated. In some embodiments, the label is a detectable marker that can generate a signal detectable visually or by instrumental means, for example, incorporation of a radioactively labeled amino acid or binding of a biotinyl moiety to a polypeptide that can be detected by a marked avidin (e.g., streptavidin that includes an enzyme activity detectable by a fluorescent marker or an optical or colorimetric method). Examples of polypeptide labels include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme 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. Representative examples of labels commonly used in immunoassays include moieties that emit light, such as acridinium compounds, and moieties that emit fluorescence, such as fluorescein. In this regard, although the moiety itself may not be detectably labeled, it can become detectable upon reaction with yet another moiety.

[0113] Exemplary DR5 agonists Provided herein are methods of treating cancer, including administering a DR5 agonist. Non-limiting exemplary DR5 agonists include INBRX-109, eftuzumab alfa (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 (HexaBody®-DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals), MM201-b (Merrimack Pharmaceuticals), KMTR2 (Kyowa Kirin Co., Ltd.), DS-8273a (Daiichi Sankyo Co., Ltd.), dulaglutide (Genentech / Amgen), lexatumumab (Human Genome Sciences / GSK), conatumumab (Amgen), drozitumab (Genentech / Roche), LBY135 (Novartis), and tigatuzumab (Daiichi Sankyo Co., Ltd.). In some embodiments, the DR5 agonist is a DR5-binding polypeptide. In some embodiments, the DR5-binding polypeptides provided herein are multivalent. In some embodiments, the DR5-binding polypeptides provided herein are at least tetravalent. In some embodiments, the DR5-binding polypeptides provided herein are tetravalent.

[0114] In various embodiments, the DR5-binding polypeptide comprises at least one VHH domain comprising a CDR1 comprising the sequence of SEQ ID NO: 1, a CDR2 comprising the sequence of SEQ ID NO: 2, and a CDR3 comprising the sequence of SEQ ID NO: 3. In some embodiments, at least one VHH domain is humanized. In some embodiments, the DR5-binding polypeptide comprises at least one VHH domain having an amino acid sequence that is at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the DR5-binding polypeptide comprises at least one VHH domain comprising the amino acid sequence of SEQ ID NO: 4.

[0115] In some embodiments, the DR5-binding polypeptide comprises at least one VHH domain that binds to DR5 and an Fc region. In some embodiments, the DR5-binding polypeptides provided herein comprise two VHH domains that bind to DR5 and an Fc region. In some embodiments, the Fc region mediates dimerization of the DR5-binding polypeptide under physiological conditions, and thus, by forming a dimer, the number of DR5-binding sites is doubled. For example, a DR5-binding polypeptide comprising two VHH domains that bind to DR5 and an Fc region is bivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization, and thus the DR5-binding polypeptide is a tetravalent dimer under such conditions.

[0116] In various embodiments, there are provided DR5-binding polypeptides in which each VHH domain comprises a CDR1 comprising the sequence of SEQ ID NO: 1, a CDR2 comprising the sequence of SEQ ID NO: 2, and a CDR3 comprising the sequence of SEQ ID NO: 3. In some embodiments, each VHH domain is humanized.

[0117] In some embodiments, the DR5-binding polypeptide comprises a VHH-linker-VHH-linker-Fc structure. In some embodiments, the VHH-linker-VHH portion of the DR5-binding polypeptide comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the VHH-linker-VHH portion of the DR5-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the Fc comprises a hinge. In some such embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the DR5-binding polypeptide comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, which comprises two VHH domains and an Fc region. In some embodiments, the DR5-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 7, which comprises two VHH domains and an Fc region. In some embodiments, the DR5-binding polypeptide consists of the amino acid sequence of SEQ ID NO: 7. A DR5-binding polypeptide consisting of the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence of SEQ ID NO: 7 lacking the terminal lysine, may be designated INBRX-109.

[0118] In some embodiments, the VHH domain that binds to DR5 can be humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules. This is because humanized antibodies reduce or eliminate the human immune response to non-human antibodies that can result in an immune response to the antibody therapeutic and reduce the effectiveness of the therapeutic. Generally, a humanized antibody comprises one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. A humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to, for example, restore or improve the specificity or affinity of the antibody.

[0119] Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and are further described, for example, in Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Patent No. 5,821,337, U.S. Patent No. 7,527,791, U.S. Patent No. 6,982,321, and U.S. Patent No. 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"), and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0120] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296), framework regions derived from consensus sequences of human antibodies of certain subgroups of the heavy chain variable region (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285, and Presta et al. (1993) J. Immunol, 151:2623), human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: 10678-10684, and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, a humanized VHH is prepared by replacing the FR region of the VHH with a human FR region. In some embodiments, replacing certain FR residues of the human FR improves one or more properties of the humanized VHH. A VHH domain having such replaced residues is also further referred to herein as "humanized".

[0121] In various embodiments, the Fc region included in the DR5-binding polypeptide is a human Fc region or is derived from a human Fc region.

[0122] In some embodiments, the Fc region contained in the DR5-binding polypeptide is derived from the human Fc region and contains three amino acid deletions corresponding to IgG1 E233, L234, and L235 in the lower hinge, and is referred to herein as "Fc xELL". Since the Fc xELL polypeptide does not bind to FcγR, it is referred to as "effector silent" or "effector null", but in some embodiments, the xELL Fc region binds to FcRn, resulting in an extended half-life and transcytosis associated with FcRn-mediated recycling. In some embodiments, the Fc region is the human IgG1 xELL Fc region.

[0123] Exemplary IAP antagonists Provided herein are methods of treating cancer that include administering an IAP antagonist. In some embodiments, the IAP antagonist is a small molecule. In some embodiments, the IAP antagonist is APG-1387 (Ascentage Pharma Group International), birinapant (IGM Biosciences, Inc.), AZD5582 (AstraZeneca), LCL161 (Novartis), Debio 1143 (Merck, Debiopharm) or ASTX660 (Astex Pharmaceuticals, Inc.).

[0124] In some embodiments, the IAP antagonist is APG-1387. APG-1387 (also known as SM-1387) has the structure:

Chemical formula

[0125] In some embodiments, the IAP antagonist is birinapant. Birinapant (also known as TL32711) has the structure:

Chemical formula

[0126] In some embodiments, the IAP antagonist is AZD5582. AZD5582 has the structure:

Chemical formula

[0127] In some embodiments, the IAP antagonist is LCL161. LCL161 has the structure:

Chemical formula

[0128] In some embodiments, the IAP antagonist is Debio 1143. Debio 1143 (also known as xebinarapant, AT-406, SM-406, and ARRY-334543) has the structure:

Chemical formula

[0129] In some embodiments, the IAP antagonist is ASTX660. ASTX660 (also known as trilinapant) has the structure:

Chemical formula

[0130] Expression and production of polypeptides A nucleic acid molecule is provided that includes a polynucleotide encoding a DR5-binding polypeptide. In some embodiments, the nucleic acid molecule can also encode a leader sequence that directs the secretion of the DR5-binding polypeptide, which leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence can be a native heavy chain (or VHH) leader sequence or another heterologous leader sequence.

[0131] The nucleic acid molecule can be constructed using recombinant DNA techniques conventional in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0132] Vectors are provided that contain nucleic acids encoding the DR5-binding polypeptides described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, a vector optimized for expression of the polypeptide in a desired cell type, such as a CHO cell or a CHO-derived cell, or an NSO cell, is selected. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).

[0133] In some embodiments, the DR5-binding polypeptide can be expressed in prokaryotic cells such as bacterial cells, or in eukaryotic cells such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression can be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used for expression of the polypeptide include, but are not limited to, COS cells including COS7 cells, 293 cells including 293-6E cells, CHO cells including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, the DR5-binding polypeptide can be expressed in yeast. See, for example, U.S. Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform desired post-translational modifications on the polypeptide. For example, in some embodiments, CHO cells produce a polypeptide with a higher level of sialylation than the same polypeptide produced in 293 cells.

[0134] The introduction of one or more nucleic acids (such as vectors) into a desired host cell can be achieved by any method including, but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acid can be transiently or stably transfected into the desired host cell according to any suitable method.

[0135] Also provided are host cells comprising any of the nucleic acids or vectors described herein. In some embodiments, host cells that express the DR5-binding polypeptide described herein are provided. The DR5-binding polypeptide expressed in the host cell can be purified by any suitable method. Such methods include, but are not limited to, the use of an affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or an antibody affinity column can be used to purify the DR5-binding polypeptide containing the Fc region by binding to the Fc region. Hydrophobic interaction chromatography, such as a butyl column or a phenyl column, may also be suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be suitable for purifying some polypeptides such as antibodies. Mixed mode chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides such as antibodies. Many methods for purifying polypeptides are known in the art.

[0136] In some embodiments, the DR5-binding polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0137] In some embodiments, a DR5-binding polypeptide produced by the above method is provided. In some embodiments, the DR5-binding polypeptide is produced in a host cell. In some embodiments, the DR5-binding polypeptide is produced in a cell-free system. In some embodiments, the DR5-binding polypeptide is purified. In some embodiments, a cell culture medium containing the DR5-binding polypeptide is provided.

[0138] In some embodiments, a composition comprising an antibody produced by the above method is provided. In some embodiments, the composition comprises a DR5-binding polypeptide produced in a host cell. In some embodiments, the composition comprises a DR5-binding polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified DR5-binding polypeptide.

[0139] Pharmaceutical composition In some embodiments, a composition comprising a DR5 agonist and / or an IAP antagonist is provided in a formulation comprising a variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003), Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd ed., Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers are available, including vehicles, adjuvants, and diluents. Additionally, a variety of pharmaceutically acceptable auxiliary substances are available, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, etc.

[0140] In some embodiments, INBRX-109 is provided in a formulation comprising 50 mg / mL INBRX-109, 10 mM histidine HCl, 8% (w / v) sucrose, 0.2% (w / v) poloxamer 88 (pH 6.0).

[0141] Exemplary methods of treating cancer using a DR5 agonist and an IAP antagonist In some embodiments, a method of treating cancer in an individual is provided, comprising administering a DR5 agonist and an IAP antagonist.

[0142] In some embodiments, the method comprises administering to the subject an effective amount of a DR5 agonist and an IAP antagonist. Such a treatment method can be a treatment method in humans or animals. In some embodiments, a method of treating a human is provided. Non-limiting exemplary cancers that can be treated using the combination of a DR5 agonist and an IAP antagonist provided herein include adrenal cancer, astrocytoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing's sarcoma, colorectal cancer (colon cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer (lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary system cancer and vulvar cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, large lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and angiomatosis, edema (such as edema associated with brain tumors) and abnormal vascular proliferation associated with Meigs syndrome.

[0143] A DR5 agonist and an IAP antagonist can be administered to a subject as needed. The determination of the frequency of administration of each agent can be made by a person skilled in the art, such as a attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, etc. In some embodiments, one or more therapeutic agents in an effective dose are administered to the subject one or more times. In some embodiments, an effective dose of a DR5 agonist and / or an IAP antagonist is administered to the subject daily, twice a week, weekly, every two weeks, once a month, etc. An effective dose of a DR5 agonist and / or an IAP antagonist is administered to the subject at least once. In some embodiments, an effective dose of a DR5 agonist and / or an IAP antagonist can be administered in multiple doses, including multiple doses over at least one month, at least six months, or at least one year.

[0144] In some embodiments, the DR5 agonist is administered in an amount effective to treat (including preventing) cancer. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of that subject, the extent of the condition being treated, or the age of the subject being treated. Generally, the DR5-binding polypeptide can be administered in an amount in the range of about 0.05 mg / kg (body weight) to about 100 mg / kg (body weight) per dose, or in the range of about 10 μg / kg (body weight) to about 100 mg / kg (body weight) per dose, or in the range of about 50 μg / kg (body weight) to about 5 mg / kg (body weight) per dose, or in the range of about 100 μg / kg (body weight) to about 10 mg / kg (body weight) per dose, or in the range of about 100 μg / kg (body weight) to about 20 mg / kg (body weight) per dose, or in the range of about 0.5 mg / kg (body weight) to about 20 mg / kg (body weight) per dose, or in the range of about 1 mg / kg (body weight) to about 10 mg / kg (body weight) per dose.

[0145] In some embodiments, INBRX-109 is administered at a dose of about 1 mg / kg (body weight) to about 30 mg / kg (body weight). In some embodiments, INBRX-109 is administered at a dose of about 1 mg / kg (body weight) to about 10 mg / kg (body weight). In some embodiments, INBRX-109 is administered at a dose of about 1 mg / kg (body weight) to about 2 mg / kg (body weight). In some embodiments, INBRX-109 is administered at a dose of about 2 mg / kg (body weight) to about 3 mg / kg (body weight). In some embodiments, INBRX-109 is administered at a dose of about 3 mg / kg (body weight). In some embodiments, INBRX-109 is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, INBRX-109 is administered once every three weeks.

[0146] In some embodiments, the IAP antagonist (or a pharmaceutically acceptable salt or hydrate thereof) is, for example, from about 10 mg / m 2 to about 500 mg / m 2 from about 10 mg / m 2 to about 300 mg / m 2 or from about 10 mg / m 2 to about 200 mg / m 2 and is administered at a dose of about 1 mg / m 2 to about 1000 mg / m 2 In some embodiments, the IAP antagonist (or a pharmaceutically acceptable salt or hydrate thereof) is administered at a dose of about 0.1 mg to about 10000 mg, including, for example, 10 mg to 5000 mg or 10 mg to 1000 mg or 10 mg to 500 mg.

[0147] In some embodiments, APG-1387 is administered at a dose of about 0.3 mg to about 60 mg. In some embodiments, APG-1387 is administered once a week. In some embodiments, birinapant is from about 2.8 mg / m 2 to about 47 mg / m 2It is administered at a dose of. In some embodiments, LCL161 is administered at a dose of about 10 mg to about 3000 mg. In some embodiments, LCL161 is administered at a dose of about 900 mg to about 1800 mg. In some embodiments, LCL161 is administered weekly. In some embodiments, Debio 1143 is administered at a dose of about 5 mg to about 900 mg. In some embodiments, Debio 1143 is administered at a dose of about 100 mg to about 200 mg. In some embodiments, Debio 1143 is administered at a dose of about 200 mg. In some embodiments, Debio 1143 is administered daily. In some embodiments, ASTX660 is administered at a dose of about 15 mg to about 270 mg. In some embodiments, ASTX660 is administered daily, optionally on a 7 days on / 7 days off schedule.

[0148] In some embodiments, the therapeutic agent can be administered in vivo by various routes including, but not limited to, oral, intramuscular, intravenous, intraarterial, parenteral, intraperitoneal or subcutaneous. Appropriate formulations and administration routes can be selected according to the intended use.

[0149] In some embodiments, the DR5 agonist and the IAP antagonist are administered separately. In some embodiments, the DR5 agonist and the IAP antagonist are administered sequentially. In some embodiments, at least one dose of the DR5 agonist is administered before the IAP antagonist. In some embodiments, at least one dose of the DR5 agonist is administered after the IAP antagonist.

[0150] In some embodiments, the DR5 agonist and the IAP antagonist are administered in combination.

[0151] In some embodiments, the DR5 agonist and the IAP antagonist act synergistically. In some embodiments, the synergistic effect is determined in an in vitro cell survival assay. In some embodiments, administration of the DR5 agonist and the IAP antagonist produces a synergistic effect as compared to administration of each agent alone.

[0152] In some embodiments, there is provided a DR5 agonist for use in a method of treating cancer, the method comprising administering the DR5 agonist in combination with an IAP antagonist.

[0153] In some embodiments, there is provided use of a DR5 agonist in the manufacture of a medicament for treating cancer, the medicament being administered together with an IAP antagonist.

[0154] Kit Also provided are articles of manufacture and kits comprising any of the DR5 agonists and / or IAP antagonists provided herein and a suitable package. In some embodiments, the invention includes a kit comprising (i) a formulation comprising a DR5 agonist, (ii) a formulation comprising an IAP antagonist, and (iii) instructions for using the kit to administer the formulations to a subject. In some embodiments, the invention includes a kit comprising (i) a formulation comprising a DR5 agonist and (ii) instructions for using the kit to administer the formulation in combination with an IAP antagonist to a subject. In some embodiments, the invention includes a kit comprising (i) a formulation comprising an IAP antagonist and (ii) instructions for using the kit to administer the formulation in combination with a DR5 agonist to a subject.

[0155] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These manufactured articles can be further sterilized and / or sealed. Unit dosage forms containing the compositions described herein are also provided. These unit dosage forms can be stored in suitable packaging as single or multiple unit dosage forms and can be further sterilized and sealed. The instructions provided in the kits of the present invention are typically instructions written on a label or an accompanying document (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions held on a magnetic storage disk or an optical storage disk) are also acceptable. Instructions regarding the use of a DR5 agonist and / or an IAP antagonist generally include information regarding dosage, dosing schedule, and route of administration for the intended therapeutic or industrial use. The kit can further include an explanation for selecting an individual appropriate treatment.

[0156] The container can be a unit dose, bulk packaging (e.g., multi-dose packaging), or sub-unit dose. For example, kits can be provided that contain an amount of the molecules disclosed herein sufficient to provide effective treatment to an individual over a long period of time, such as any approximate period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. The kit can also include multiple unit doses of the molecule and instructions, and can be packaged in an amount sufficient for storage and use in a pharmacy, such as a hospital pharmacy and a dispensing pharmacy. In some embodiments, the kit includes a dry (e.g., lyophilized) composition that can generally form a stable aqueous solution of a DR5 agonist upon reconstitution, resuspension, or rehydration.

Examples

[0157] The examples discussed below are intended to purely illustrate the present invention and should in no way be construed as limiting the present invention. These examples are not intended to represent that the following experiments are all or the only experiments conducted. Efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0158] Example 1: Combinatorial Activity of INBRX-109 with Various IAP Antagonists The combination of INBRX-109 with various IAP antagonists was tested in various cancer cell lines to determine the cytotoxicity against cancer cells.

[0159] Assay Protocol Day 1 - Cell Seeding. Monolayer cultures of each cell line were harvested for the compound screening detailed below. The culture medium was aspirated, and the cells were washed once with PBS. Accutase was added, and the flask was incubated at 37 °C until the cells detached. After adding an equal volume of complete medium to quench Accutase, the cells were pipetted up and down several times to generate a uniform single-cell suspension. The cell density and viability were determined by trypan blue using a TC20 Automated Cell Counter. The experimental cells were resuspended in EMEM / 10% FBS / Anti-Anti medium (complete EMEM) at a concentration of 0.17×10 6 cells / mL and seeded at 15 μL / well (final 2500 cells / well) into the inner wells of a 384-well luminescence plate. Each cell line was plated in duplicate on separate plates. After filling the outer wells with 50 μL of PBS, the plates were incubated for 16 hours overnight in a humidified temperature-controlled 37 °C tissue culture incubator with 5% CO 2 2.

[0160] Preparation of test substances for IAP antagonists: All small molecules were purchased from MedChemExpress as 10 mM solutions in DMSO. These test substances were then aliquoted and stored at -80 °C. The aliquots were thawed immediately prior to dilution for use in the assay. A 500-fold master plate of serial dilutions (6-point 2-fold dilutions in 100% DMSO + DMSO-only control) was prepared and gently mixed by pipetting. To make a 5-fold standard (working) dilution plate of the small molecules, 100-fold dilutions were made from the 500-fold plate with complete medium (EMEM).

[0161] Preparation of test substances for INBRX-109. The INBRX-109 assay concentration range was selected to include the minimum and maximum activities seen in previous cytotoxicity assays using several cancer cell lines, and 1 nM was defined as the maximum effective concentration. A 50-fold master plate of INBRX-109 serial dilutions (6-point 10-fold dilutions in complete EMEM starting from 500 nM + complete EMEM-only control) was prepared and gently mixed by pipetting. To make a 5-fold standard dilution plate of INBRX-109, each well of the 50-fold master plate was diluted 10-fold with complete EMEM.

[0162] Day 2 - Addition of test substances and controls. The IAP antagonist small molecule standard dilutions (5-fold, 5 μL), INBRX-109 standard dilutions (5-fold, 5 μL), or staurosporine positive control (5-fold, 5 μL) were added to their respective experimental wells. The IAP antagonist titration was performed horizontally across the plate and the INBRX-109 titration was performed vertically down the plate to obtain a matrix of all possible combinations of the two test substances. These test substances were added in duplicate to each cell line. The plate was then centrifuged at 400 × g for 1 minute and then incubated in a humidified tissue culture incubator at 37 °C (5% CO 2 ) for 48 hours.

[0163] Day 4 - Survival rate measurement. After equilibrating the plates at room temperature for 10 minutes, 25 μL of CellTiter - Glo 2.0™ was added to each well. The plates were rotated at 400 × g for 1 minute, then covered and incubated at room temperature in the dark for 10 minutes. After removing all visible bubbles with 100% ethanol vapor, luminescence (RLU) was read on a Spectra Max M5e plate reader with a 384 - well opaque plate setting and SoftMaxPro v5.4 software using an integration time of 50 milliseconds. To determine the effect of the test substance on cell viability, the raw RLU values were exported to Excel and the survival rate (%) was calculated as a percentage relative to the vehicle control (0.2% DMSO in EMEM) (with the vehicle control set as 100%). The data was graphed using GraphPad Prism 7.

[0164] Results Figures 1A - 1E show the results of experiments in which six different cancer cell lines were contacted with INBRX - 109 (1 nM), an IAP antagonist, or a combination of INBRX - 109 (1 nM) and an IAP antagonist. The bar graphs show the number of cell lines (out of six tested) that are resistant (0% - 25% cytotoxicity), intermediate (25% - 75% cytotoxicity), or sensitive (75% - 100% cytotoxicity). The six cell lines tested were LOVO, LS174T, SW620, SNU - C2B, HT - 29, and LS411N. The number of cancer cell lines is shown on the y - axis of each bar graph. Results for the IAP antagonists ASTX660 (900 nM) (Figure 1A), AZD5582 (450 nM) (Figure 1B), birinapant (5 μM) (Figure 1C), Debio 1143 (4.5 μM) (Figure 1D), and LCL - 161 (5 μM) (Figure 1E) are shown.

[0165] These results are also shown in Tables 2 - 6. Cytotoxicity (%) was calculated by subtracting the survival rate (%) from 100.

[0166]

Table 2

[0167]

Table 3

[0168]

Table 4

[0169]

Table 5

[0170]

Table 6

[0171] The combination of INBRX-109 and an IAP antagonist showed an increase in cell death in almost all cancer cell lines tested in this screening and for all IAP antagonists, compared to either INBRX-109 or the IAP antagonist alone.

[0172] This data suggests that combinations of DR5 agonists such as INBRX-109 and IAP antagonists such as ASTX660, AZD5582, birinapant, Debio 1143 or LCL-161 result in improved or synergistic cancer cell death compared to the individual drugs alone.

[0173] Example 2: Combinatorial activity of INBRX-109 with LCL-161 or INBRX-109 with birinapant Using an assay similar to that described in Example 1, the combination of INBRX-109 with the IAP antagonists LCL-161 or birinapant was tested in LS174T and SW620 cancer cell lines to determine cytotoxicity against cancer cells.

[0174] Results Figures 2A and 2B show the results of an LCL-161 titration experiment in which cancer cell lines were contacted with different concentrations of LCL-161, either alone or in combination with 1 nM INBRX-109. The survival rate (%) of cancer cells is shown on the y-axis of each graph. Results for the cancer cell lines LS174T (Figure 2A) and SW620 (Figure 2B) are shown. The dotted line labeled "1 nM INBRX-109 only" shows the survival rate (%) of cancer cells treated with 1 nM INBRX-109 alone.

[0175] Figures 3A and 3B show the results of a birinapant titration experiment in which cancer cell lines were contacted with different concentrations of birinapant, either alone or in combination with 1 nM INBRX-109. The survival rate (%) of cancer cells is shown on the y-axis of each graph. Results for the cancer cell lines LS174T (Figure 3A) and SW620 (Figure 3B) are shown. The dotted line labeled "1 nM INBRX-109 only" shows the survival rate (%) of cancer cells treated with 1 nM INBRX-109 alone.

[0176] The combination of INBRX-109 with either LCL-161 or birinapant showed an increase in cell death in the cancer cell lines tested in this screening compared to either agent alone.

[0177] This data suggests that the combination of a DR5 agonist such as INBRX-109 with an IAP antagonist such as LCL-161 or birinapant results in improved or synergistic cancer cell death compared to the individual drugs alone.

[0178] Example 3: Combinatorial Activity of INBRX-109 and Debio-1143 Against Additional Cancer Cell Lines The combination of INBRX-109 and Debio-1143 was tested in various head and neck cancer cell lines to determine cytotoxicity against cancer cells.

[0179] Assay Protocol Day 1 - Cell seeding. Monolayer cultures of each cancer cell line (see Table 7) were harvested during the logarithmic growth phase. The cell density and viability were determined by trypan blue using a Vi-cell counter, and the cells were diluted in the appropriate growth medium (see Table 7). For compound screening described in detail below, 90 μL of the cell suspension was added to a 96-well plate. Two replicate plates were set up for reading at day 0 (T0) and at the endpoint (T48).

[0180]

Table 7

[0181] Test substance preparation: A 1000-fold master plate of serial dilutions (9-point 2-fold dilutions of Debio-1143 in 100% DMSO starting from 9000 μM + DMSO-only control) was prepared and gently mixed with a pipette. To make a 10-fold standard dilution plate of the small molecule, 100-fold dilutions were made from the 1000-fold plate with the appropriate medium. A 10-fold stock of INBRX-109 (10 nM) was prepared.

[0182] Day 2 - Compound treatment. 10 μL (10-fold) and / or 10 μL (10-fold) of INBRX-109 (final concentration 1 nM) from the 10-fold standard plate were added in triplicate to each experimental well. The plate was then incubated in a humidified tissue culture incubator at 37 °C (5% CO 2 )).

[0183] 20 μL of culture medium containing 1% DMSO and 50 μL of CellTiter-Glo™ reagent was added to each well of the T0 plate, the contents were mixed, the plate was incubated at room temperature for 30 minutes, and read on an EnVision Multi Label Reader.

[0184] Day 4 - Survival rate measurement. 50 μL of CellTiter-Glo (trademark) reagent was added to each well of a T48 plate, the contents were mixed, the plate was incubated at room temperature for 30 minutes, and read with an EnVision Multi Label Reader. The formula for calculating the survival rate (%) is survival rate (%) = (Lum 被験物質 -Lum 培地対照 ) / (Lum 非処理 -Lum 培地対照 )×100%. The cytotoxicity (%) was calculated by subtracting the survival rate (%) from 100.

[0185] Results Table 8 shows the cytotoxicity (%) for each cell line tested. Figures 4A - 4L show the results of Debio-1143 titration experiments in which cancer cell lines were contacted with different concentrations of Debio-1143, alone or in combination with 1 nM INBRX-109. Figures 4A - 4K show the results for cancer cell lines that showed less than 90% killing with INBRX-109 alone and at least 25% maximum cytotoxicity after treatment in combination: A253 (Figure 4A), CAL-27 (Figure 4B), COLO-680N (Figure 4C), Detroit 562 (Figure 4D), FADU (Figure 4E), KYSE-70 (Figure 4F), SCC-9 (Figure 4G), SCC-25 (Figure 4H), T.Tn (Figure 4I), TE-1 (Figure 4J), KYSE-270 (Figure 4K). The survival rate (%) of the cancer cells is shown on the y-axis of each graph, and the dotted line labeled "1 nM INBRX-109 only" shows the survival rate (%) of the cancer cells treated with 1 nM INBRX-109 alone. The results shown in Table 8 are summarized in Figure 4L, and the cytotoxicity (%) (cell death (%)) is plotted for all 18 cell lines treated with INBRX-109 or Debio-1143 alone, or in combination with INBRX-109 and Debio-1143.

[0186]

Table 8

[0187] The combination of INBRX-109 and Debio-1143 showed an increase in cell death in almost all cancer cell lines tested in this screening, compared to either INBRX-109 or the IAP antagonist alone. From this data, it is further suggested that the combination of a DR5 agonist such as INBRX-109 and an IAP antagonist such as Debio 1143 results in improved or synergistic cancer cell death compared to the individual drugs alone.

[0188] Example 4: Combinatorial Activity of INBRX-109 and Birinapant Against Additional Cancer Cell Lines Using an assay similar to that described in Example 3, the combination of INBRX-109 and birinapant was tested in various breast cancer cell lines (BT-20, BT-549, CAL-120, CAL-148, CAL-51, DU4475, HCC1143, HCC1187, HCC1395, HCC1806, HCC1937, HCC38, HCC70, HDQ-P1, Hs 578T, MX-1 and SUM159PT) to determine cytotoxicity against cancer cells. In additional studies, using an assay similar to that described in Example 1, the combination of INBRX-109 and birinapant was tested in various chondrosarcoma (CAL-78, OUMS-27, SW1353 and H-EMC-SS), gastric cancer (NCI-N87, SNU-5 and SNU-1), pancreatic cancer (AsPC-1, Panc-1, MIA-PaCa-2 and SW1990) and lung cancer (NCI-H460, HCC2935, NCI-H23, NCI-H2452 and A549) cell lines, and the combination of INBRX-109 and Debio 1143 was tested in gastric cancer (NCI-N87, SNU-5 and SNU-1), pancreatic cancer (AsPC-1, Panc-1, MIA-PaCa-2 and SW1990) and lung cancer (NCI-H460, HCC2935, NCI-H23, NCI-H2452 and A549) cell lines to determine cytotoxicity against cancer cells.

[0189] Results Table 9 shows the cytotoxicity (%) for each breast cancer cell line tested. The results in Table 9 are summarized in Figure 5, and the cytotoxicity (%) (cell death (%)) is plotted for all 13 breast cancer cell lines treated with INBRX-109 or birinapant alone or in combination.

[0190]

Table 9

[0191] Table 10A shows the cytotoxicity (%) for chondrosarcoma, gastric cancer, pancreatic cancer, and lung cancer cell lines treated with INBRX-109 or birinapant alone or in combination. Table 10B shows the cytotoxicity (%) for gastric cancer, pancreatic cancer, and lung cancer cell lines treated with INBRX-109 or Debio 1143 alone or in combination. The results in Table 10A are summarized in Figures 6A - 6D, and the cytotoxicity (%) (cell death (%)) is plotted for the cell lines treated with INBRX-109 or birinapant alone or in combination. The results for chondrosarcoma, pancreatic cancer, and lung cancer cell lines are plotted in Figures 6A - 6C respectively, and the results for all cell lines shown in Table 10A are plotted in Figure 6D. The results in Table 10B are summarized in Figures 6E - 6G, and the cytotoxicity (%) (cell death (%)) is plotted for the cell lines treated with INBRX-109 or Debio 1143 alone or in combination. The results for pancreatic cancer and lung cancer cell lines are plotted in Figures 6E and 6F respectively, and the results for all cell lines shown in Table 10B are plotted in Figure 6G.

[0192]

Table 10A

[0193]

Table 10B

[0194] The combination of INBRX-109 and an IAP antagonist (birinapant or Debio 1143) showed an increase in cell death in almost all cancer cell lines tested in these screenings compared to either INBRX-109 or the IAP antagonist alone. From this data, it is further suggested that the combination of a DR5 agonist such as INBRX-109 and an IAP antagonist such as birinapant or Debio 1143 results in improved or synergistic cancer cell death compared to the individual drugs alone.

[0195] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the above-described embodiments should be considered illustrative in all respects and not restrictive of the present disclosure. Accordingly, the scope of the present disclosure is indicated by the appended claims rather than the above detailed description, and all changes within the meaning and equivalent scope of the claims are intended to be included herein.

[0196] [Table 11]

Claims

1. A pharmaceutical composition comprising a cell death receptor 5 (DR5) agonist for treating cancer in a subject, wherein the DR5 agonist is a tetravalent DR5-binding polypeptide, and the pharmaceutical composition is administered to the subject in combination with an apoptosis inhibitor protein (IAP) antagonist.

2. A pharmaceutical composition comprising an apoptosis inhibitor protein (IAP) antagonist for treating cancer in a subject, wherein the pharmaceutical composition is administered to the subject in combination with a cell death receptor 5 (DR5) agonist, the DR5 agonist being a tetravalent DR5-binding polypeptide.

3. The pharmaceutical composition according to claim 1 or 2, wherein the DR5-binding polypeptide comprises at least one VHH domain including CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO:

3.

4. The aforementioned at least one VHH domain, (a) an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of Sequence ID No. 4; or (b) Amino acid sequence of Sequence ID No. 4, The pharmaceutical composition according to claim 3, comprising:

5. The pharmaceutical composition according to claim 1 or 2, wherein the DR5-binding polypeptide includes an Fc region.

6. The pharmaceutical composition according to claim 5, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:

6.

7. The pharmaceutical composition according to claim 1 or 2, wherein the DR5-binding polypeptide has the structure VHH-linker-VHH-linker-Fc.

8. The pharmaceutical composition according to claim 7, wherein each VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO:

3.

9. The pharmaceutical composition according to claim 7, wherein VHH-linker-VHH contains an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

5.

10. The pharmaceutical composition according to claim 9, wherein VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:

5.

11. The pharmaceutical composition according to claim 1 or 2, wherein the DR5-binding polypeptide comprises an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:

7.

12. The pharmaceutical composition according to claim 1 or 2, wherein the DR5-binding polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

13. The pharmaceutical composition according to claim 1 or 2, wherein the DR5-binding polypeptide consists solely of the amino acid sequence of SEQ ID NO:

7.

14. The pharmaceutical composition according to claim 1 or 2, wherein the IAP antagonist is a small molecule.

15. The pharmaceutical composition according to claim 1 or 2, wherein the IAP antagonist is APG-1387 (Ascentage Pharma Group International), virinapanth (IGM Biosciences, Inc.), AZD5582 (AstraZeneca), LCL161 (Novartis), Debio 1143 (Merck, Debiopharm), or ASTX660 (Astex Pharmaceuticals, Inc.).

16. The pharmaceutical composition according to claim 15, wherein the IAP antagonist is APG-1387.

17. The pharmaceutical composition according to claim 15, wherein the IAP antagonist is virinapanth.

18. The pharmaceutical composition according to claim 15, wherein the IAP antagonist is Debio 1143.

19. The pharmaceutical composition according to claim 15, wherein the IAP antagonist is LCL161.

20. The pharmaceutical composition according to claim 1 or 2, wherein the DR5 agonist and the IAP antagonist are administered separately.

21. (a) at least one dose or first dose of the DR5 agonist is administered prior to the IAP antagonist; or, (b) At least one dose or the first dose of the DR5 agonist is administered after the IAP antagonist. The pharmaceutical composition according to claim 20.

22. The pharmaceutical composition according to claim 1 or 2, wherein the DR5 agonist and the IAP antagonist are administered simultaneously.

23. The pharmaceutical composition according to claim 1 or 2, wherein the administration of the DR5 agonist and the IAP antagonist produces a synergistic effect compared to the administration of each active substance alone.

24. The cancer is adrenal cancer, astrocytoma, basal cell carcinoma, bile tract cancer, bladder cancer, bone cancer, brain cancer and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing's sarcoma, colorectal cancer (colon cancer). , connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasm, kidney cancer or renal cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, small cancer. Alveolar lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth and / or pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary tract cancer and vulvar cancer, lymphoma, The pharmaceutical composition according to claim 1 or 2, wherein the patient is Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small undivided cell NHL, giant lesion NHL, mantle cell lymphoma, AIDS-associated lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorders (PTLD), and nevus, edema (such as edema associated with brain tumors) and abnormal angiogenesis associated with Meigs syndrome.

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