DR5 agonist and IAP antagonist combination therapy

Abstract

Provided herein are methods of treating cancer with a DR5 agonist in combination with an IAP antagonist.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to 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; each of which is incorporated herein by reference in its entirety for any purpose. Sequence Listing Incorporated by Reference

[0002] This application incorporates by reference a sequence listing in electronic format, entitled 01202-0058-00PCT_Sequence_Listing, created on May 5, 2023, and 14,058 bytes in size, filed with this application. Technical Field

[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 a cell surface receptor of the TNF receptor superfamily that binds TNF-related apoptosis-inducing ligand (TRAIL). TRAIL evolved to play a key role in mammalian development and host defense by selectively eradicating unwanted, infected, and malignant cells from healthy cell populations. Upon binding to TNF receptor family members DR4 or DR5, TRAIL induces cell death via apoptosis-dependent apoptosis. DR5 appears to be the primary receptor on tumor cells that promotes the observed tumor-biased activity of the TRAIL pathway. DR5 is activated by the natural ligand TRAIL, which brings three DR5 receptors into close proximity, thereby activating intracellular apoptosis proteases-8 and initiating activation of other death-inducing apoptosis proteases, such as apoptosis proteases-9 and apoptosis proteases-3. Therefore, initiation of this cell death pathway requires aggregation of DR5 receptors to achieve effective cell death. DR5 agonists are promising therapeutic candidates for the treatment of cancer.

[0005] Inhibitor of Apoptosis Protein (IAP) is a class of proteins that negatively regulate apoptotic proteases and apoptosis. IAPs are often dysregulated in many cancers and have been shown 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 an important role in immune regulation. Examples of IAPs include cellular IAP1 (cIAP1), cellular IAP2 (cIAP2) and X-linked inhibitor of apoptosis protein (XIAP). Due to its significant biological functions in cell death and immune response, IAPs are emerging as drug targets in a wide range of malignancies. Several IAP antagonists have been developed and their clinical benefits are currently being studied. For example, the second mitochondria-derived activator of caspase (SMAC) protein inhibits IAP function, and a variety of small molecules that mimic SMAC function (called SMAC mimetics) have been developed. Summary of the invention

[0006] Provided herein are methods of treating cancer in a subject with a death receptor 5 (DR5) agonist and an inhibitor of apoptosis protein (IAP) antagonist. In some embodiments, the method comprises administering a multivalent death receptor 5 (DR5) binding polypeptide and an inhibitor of apoptosis 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. Embodiment 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject (a) a death receptor 5 (DR5) agonist, wherein the DR5 agonist is tetravalent, and (b) an inhibitor of apoptosis protein (IAP) antagonist. Embodiment 2. The method of embodiment 1, wherein the DR5 agonist is a DR5 binding polypeptide. Embodiment 3. A 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. Embodiment 4. A method as described in 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. Embodiment 5. The method of 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. Embodiment 6. The method of any one of embodiments 2 to 5, wherein the DR5 binding polypeptide comprises an Fc region. Embodiment 7. The method of embodiment 6, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:6. Embodiment 8. The method of any one of embodiments 2 to 7, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc. Embodiment 9. The method of any one of embodiments 2 to 8, wherein each VHH domain comprises: 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. Embodiment 10. The method of any one of embodiments 2 to 9, wherein the VHH-linker-VHH 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. Embodiment 11. The method of embodiment 10, wherein the VHH-linker-VHH comprises the amino acid sequence SEQ ID NO:5. Embodiment 12. The method of any one of embodiments 2 to 11, 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. Embodiment 13. The method of any one of embodiments 2 to 12, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7. Embodiment 14. The method of any one of embodiments 2 to 12, wherein the DR5 binding polypeptide consists of the amino acid sequence SEQ ID NO: 7. Embodiment 15. The method of embodiment 1 or embodiment 2, wherein the DR5 agonist is INBRX-109. Embodiment 16. The method of any one of embodiments 1 to 15, wherein the IAP antagonist is a small molecule. Embodiment 17. The method of 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.). Embodiment 18. The method of embodiment 17, wherein the IAP antagonist is APG-1387. Embodiment 19. The method of embodiment 17, wherein the IAP antagonist is birenapam. Embodiment 20. The method of embodiment 17, wherein the IAP antagonist is Debio 1143. Embodiment 21. The method of embodiment 17, wherein the IAP antagonist is LCL161. Embodiment 22. The method of any one of embodiments 1 to 21, wherein the DR5 agonist and the IAP antagonist are administered separately. Embodiment 23. The method of embodiment 22, wherein the DR5 agonist and the IAP antagonist are administered sequentially. Embodiment 24. The method of embodiment 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered prior to the IAP antagonist. Embodiment 25. The method of embodiment 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered after the IAP antagonist. Embodiment 26. The method of any one of embodiments 1 to 21, wherein the DR5 agonist and the IAP antagonist are administered simultaneously. Embodiment 27. The method of any one of embodiments 1 to 26, wherein the DR5 agonist and the IAP antagonist act synergistically. Embodiment 28. The method of embodiment 27, wherein the synergy is determined in an in vitro cell survival assay. Embodiment 29. The method of any one of embodiments 1 to 28, wherein administration of the DR5 agonist and the IAP antagonist produces a synergistic effect compared to each agent administered alone. Embodiment 30. The method of 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 and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; chondrosarcoma; Ewing's sarcoma; sarcoma); cancer of the colon and rectum (colorectal cancer); cancer of connective tissue; cancer of the digestive system; cancer of the endometrium; cancer of the esophagus; cancer of the eye; cancer of the head and neck; cancer of the stomach; cancer of the gastrointestinal tract; glioblastoma; hepatocarcinoma; liver tumor; intraepithelial neoplasia; kidney cancer or renal cancer; cancer of the larynx; leukemia; liver cancer; lung cancer; small cell lung cancer; non-small cell lung cancer; lung adenocarcinoma; lung squamous cell carcinoma; melanoma; myeloma; neuroblastoma; cancer of the oral cavity (lip cancer, tongue cancer, mouth cancer and / or pharynx cancer); cancer of the ovary; cancer of the pancreas, such as pancreatic adenocarcinoma; cancer of the pituitary gland; cancer of the prostate; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer systemic cancer; mesothelioma; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; and vulvar cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; bulky disease) NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and other carcinomas and sarcomas; and post-transplantation lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with macular degeneration, edema (such as that associated with brain tumors), and Meigs' syndrome. Embodiment 31. A DR5 agonist for use in a method of treating cancer, wherein the method comprises administering the DR5 agonist in combination with an IAP antagonist. Embodiment 32. Use of a DR5 agonist for the manufacture of a medicament for the treatment of cancer, wherein the medicament is for administration with an IAP antagonist. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1A-1EResults of experiments are shown 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 graph shows the number of cell lines (out of the six tested) that were 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. The IAP antagonist ASTX660 (900 nM) ( Figure 1A )、AZD5582(450nM)( Figure 1B )、Birinap(5μM)( Figure 1C )、Debio 1143(4.5μM)( Figure 1D ) and LCL-161 (5 μM) ( Figure 1E ) results.

[0008] Figure 2A-2B Results of LCL-161 titration experiments are shown, in which cancer cell lines were exposed to different concentrations of LCL-161 alone or in combination with 1 nM INBRX-109. The percent survival of cancer cells is shown on the y-axis of each graph. Figure 2A ) and SW620( Figure 2B The dashed line labeled "1 nM INBRX-109 only" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0009] Figure 3A-3B Results of a birenapam titration experiment are shown, in which cancer cell lines were exposed to varying concentrations of birenapam alone or in combination with 1 nM INBRX-109. The percent survival of the cancer cells is shown on the y-axis of each graph. Figure 3A ) and SW620( Figure 3B The dashed line labeled "1 nM INBRX-109 only" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0010] Figure 4A-4L Results of a Debio-1143 titration experiment are shown, in which cancer cell lines were exposed to varying concentrations of Debio-1143 alone or in combination with 1 nM INBRX-109. 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( Fig. 4I )、TE-1( Figure 4J )、KYSE-270( Figure 4K ). The survival percentage of cancer cells is shown on the y-axis of each figure. The dashed line labeled "1 nM INBRX-109 only" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone. Box and whisker plot ( Figure 4L ) shows the % cytotoxicity (% cell death) of all 18 cell lines of Table 8 treated with INBRX-109 and Debio-1143 in combination.

[0011] Figure 5 Results of a birenapap titration experiment are shown in which breast cancer cell lines were exposed to varying concentrations of birenapap, either alone or in combination with 1 nM INBRX-109. Box and whisker plots show the % cytotoxicity (% cell death) of the 13 cell lines of Table 9 treated with INBRX-109 or birenapap, either alone, or in combination with INBRX-109 and birenapap.

[0012] Figure 6A-6G Shown are the results of an IAP antagonist titration experiment in which cancer cell lines were exposed to varying concentrations of birenapam or Debio 1143 alone or in combination with 1 nM INBRX-109. Figure 6A-6D The box-and-whisker plots in the Figures show the incidence of chondrosarcoma ( Fig. 6A )、pancreas( Figure 6B )、Lung cancer( Figure 6C ) and the 16 cell lines listed in Table 10A ( Fig.6D ) of cytotoxicity % (cell death %). Figure 6E-6G The box-and-whisker plots in Figure 2 show the pancreases treated with INBRX-109 or Debio 1143 alone, or in combination with INBRX-109 and Debio 1143 in these studies ( Fig. 6E )、Lung cancer( Fig. 6F ) and the 12 cell lines listed in Table 10B ( Figure 6G ) of cytotoxicity % (cell death %). DETAILED DESCRIPTION

[0013] Embodiments provided herein relate to methods of treating cancer with a combination of a death receptor 5 (DR5) agonist and an IAP (inhibitor of apoptosis protein) antagonist. Definition and various implementations

[0014] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0015] All references (including patent applications, patent publications, and Genbank Accession Numbers) cited herein are incorporated by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0016] The techniques and procedures described or referred to herein are generally well understood by those skilled in the art and are routinely employed using conventional methods, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, 1998) Academic Press; Animal Cell Culture (RI Freshney), 1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wileyand Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: APractical Approach (edited by P. Shepherd and C. Dean, 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 JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., JS Lippincott Company, 1993); and updated versions thereof.

[0017] Unless otherwise defined, scientific and technical terms used in conjunction with the present invention will have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires or otherwise clearly indicated, singular terms shall include the plural and plural terms shall include the singular. With respect to any conflicts in definitions between various sources or references, the definitions provided herein shall prevail.

[0018] In general, the residue numbering in immunoglobulin heavy chains is that of the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0019] It should be understood that the embodiments of the invention described herein include "consisting of" and / or "consisting essentially of". Unless otherwise indicated, as used herein, the singular forms "a", "an", and "the" include plural references. The use of the term "or" herein is not meant to imply that alternatives are mutually exclusive.

[0020] In this application, the use of "or" means "and / or" unless explicitly stated or understood by one skilled in the art. In the case of multiple dependent claims, the use of "or" refers back to more than one preceding independent claim or dependent claim.

[0021] The phrase "reference sample", "reference cell" or "reference tissue" means a sample with at least one known characteristic that can be used as a comparison with a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to determine, for example, the amount of protein and / or mRNA present in healthy tissue, in contrast to the amount of protein and / or mRNA present in a sample with unknown characteristics. In some embodiments, the reference sample is from the same subject, but from a different part of the tested subject. In some embodiments, the reference sample is from a tissue area around or near the cancer. In some embodiments, the reference sample is not from the tested subject, but from a sample of a subject known to have or not have the disorder (e.g., a specific cancer or DR5-related disorder). In some embodiments, the reference sample is from the same subject, but from a time point before the subject suffers from cancer. In some embodiments, the reference sample is from a benign cancer sample from the same or different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule in question in the negative reference sample will indicate the level that a person skilled in the art will understand when assuming that there is no molecule and / or a molecule with a low level in the present invention. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample will be indicative of the amount that one skilled in the art would understand if a certain amount of the molecule of the present invention were present.

[0022] The terms "benefit", "clinical benefit", "response" and "therapeutic response" as used herein in the context of benefiting from or responding to the administration of a therapeutic agent can be measured by assessing various endpoints, such as inhibiting disease progression to some extent, including slowing and complete arrest; reducing the number of disease attacks and / or symptoms; reducing the size of lesions; inhibiting (i.e., reducing, slowing or completely stopping) disease cell infiltration to adjacent peripheral organs and / or tissues; inhibiting (i.e., reducing, slowing or completely stopping) disease spread; alleviating one or more symptoms associated with the disorder to some extent; increasing the length of time that disease-free (e.g., progression-free survival) is presented after treatment; increasing overall survival; improving response rate; and / or reducing mortality at a given time point after treatment. "Non-responsive" or "unresponsive" subjects or cancers are subjects or cancers that fail to meet the above-mentioned restrictions on "response".

[0023] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to nucleotide polymers. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0024] 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 may contain natural or non-natural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers having amino acid residues. Full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation and similar modifications. In addition, for the purposes of the present invention, "polypeptide" refers to a protein, which includes modifications to the native sequence, such as deletions, additions and substitutions (usually conservative in nature), as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis; or may be accidental, such as by mutations in the host producing the protein or by errors caused by PCR amplification.

[0025] As used herein, the terms "DR5", "death receptor 5", "TNFRSF10B" and "TRAILR2" refer to any natural, mature DR5 produced by processing DR5 precursors in cells. Unless otherwise specified, the term includes DR5 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of DR5, such as splice variants or allelic variants. Non-limiting exemplary precursor human DR5 amino acid sequences are shown, for example, in NCBI Accession No. NP_003833.4. See SEQ ID NO: 8. Non-limiting exemplary precursor human DR5 amino acid sequences are shown, for example, in SEQ ID NO: 9.

[0026] The terms "IAP" and "inhibitor of apoptosis protein" refer to any natural, mature protein in the IAP protein family, of which there are at least eight known members. Unless otherwise indicated, the term includes IAPs from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of IAPs, such as splice variants or allelic variants. IAPs act as key regulators of apoptotic protease activity and are defined by the presence of at least one baculovirus IAP repeat (BIR) domain. These zinc binding domains of approximately 70 residues enable interaction with and inhibition of apoptotic proteases, and thus promote inhibition of apoptosis. Most IAPs also contain a ring finger E3 ligase domain at the C-terminus, enabling these proteins to participate in different cellular processes, including signal transduction events that promote inflammation, cell cycle progression, and migration.

[0027] The term "specific binding" 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" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it does with alternative cells or substances. A single domain antibody (sdAb) or VHH-containing polypeptide is said to "specifically bind" or "preferentially bind" to a target if it binds with greater affinity, greater 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 greater affinity, greater avidity, more readily, and / or for a longer duration than it binds to other DR5 epitopes or non-DR5 epitopes. By reading this definition, it should also be 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, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0028] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the incidence, degree or likelihood of the characteristic. "Reduction" or "inhibition" is to reduce, decrease or stagnate an activity, function and / or amount compared to a reference. In some embodiments, "reduction" or "inhibition" means the ability to cause an overall reduction of 10% or more. In some embodiments, "reduction" or "inhibition" means the ability to cause an overall reduction of 50% or more. In some embodiments, "reduction" or "inhibition" means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the amounts mentioned above are inhibited or reduced over a period of time relative to a control over the same period of time.

[0029] As used herein, the term "epitope" refers to the site where an antigen binding molecule (e.g., sdAb or VHH-containing polypeptide) binds to a target molecule (e.g., an antigen, such as a protein, nucleic acid, carbohydrate, or lipid). Epitopes often include a group of molecules with chemical activity on the surface, such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural characteristics and specific charge characteristics. Epitopes can be formed by continuous and / or juxtaposed discontinuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed by continuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes may 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 length of the epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they can bind to the same epitope within the antigen. In some embodiments, epitopes can be identified according to a certain minimum distance relative to the CDR residues on the antigen binding molecules. In some embodiments, epitopes can be identified according to the above distance, and are further limited to those residues involved in the bond (such as hydrogen bond) between the residues of the antigen binding molecules and the antigen residues. Epitopes can also be identified by various scans, such as alanine or arginine scanning can indicate one or more residues that can interact with antigen binding molecules. Unless explicitly stated, a group of residues as an epitope does not exclude other residues as part of the epitope of a specific antigen binding molecule. In fact, the presence of such a set represents a minimum epitope series (or species set). Therefore, in some embodiments, the residue set identified as an epitope represents the minimum relevant epitope of the antigen, rather than the exclusive residue list of the epitope on the non-antigen.

[0030] The term "antibody" is used in the broadest sense and encompasses various polypeptides comprising antibody-like antigen binding domains, including but not limited to conventional antibodies (generally comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs, comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and fragments of any of the foregoing, as long as they exhibit the desired antigen binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include but are not limited to heavy chain constant domains (comprising CH1, hinge, CH2, and CH3, wherein CH1 is generally paired with a light chain constant domain CL, and the hinge mediates dimerization) and Fc regions (comprising hinge, CH2, and CH3, wherein the hinge mediates dimerization).

[0031] 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, and the like.

[0032] As used herein, the term "antigen binding domain" refers to a part of an antibody that is sufficient to bind to an antigen. In some embodiments, the antigen binding domain of a conventional antibody comprises three heavy chain CDRs and three light chain CDRs. Therefore, in some embodiments, the antigen binding domain comprises: a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required to maintain binding to an antigen; and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required to maintain binding to an antigen. In some embodiments, an sdAb or an antigen binding domain containing a VHH polypeptide comprises three CDRs of a VHH domain. Therefore, in some embodiments, an sdAb or an antigen binding domain containing a VHH polypeptide comprises a VHH domain, which comprises CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required to maintain binding to an antigen.

[0033] 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 a 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 or C-terminus so that it comprises only a portion of FR1 and / or FR4, or lacks one or both of those framework regions, as long as the VHH substantially retains antigen binding and specificity.

[0034] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one light chain-free monomer domain such as a VHH domain and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each of which comprises at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" encompass polypeptides comprising multiple VHH domains, such as polypeptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different.

[0035] The term "VHH 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, wherein each VHH domain may be the same or different. In some embodiments, the VHH polypeptide comprises an Fc region. In some such embodiments, the VHH polypeptide may be referred to as an sdAb. In addition, in some such embodiments, the VHH polypeptide may form a dimer. VHH polypeptides, which are also non-limiting structures of sdAbs, include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, wherein VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of the structure, one VHH may be connected to another VHH via a linker, or one VHH may be connected to Fc via a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids that mainly comprise glycine and optionally comprise serine. In some embodiments, when the VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, if the structure VHH1-VHH2-Fc forms a dimer, it is considered to be tetravalent (i.e., the dimer has four VHH domains). Similarly, if the structure VHH1-VHH2-VHH3-Fc forms a dimer, it is considered to be hexavalent (i.e., the dimer has six VHH domains).

[0036] The term "monoclonal antibody" refers to an antibody (including sdAb or VHH polypeptide) of a substantially homogeneous antibody population, that is, the individual antibodies comprising the population are identical except for the possible presence of a small amount of naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Therefore, a sample of a monoclonal antibody can bind to the same epitope on the antigen. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population, and should not be understood as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be manufactured by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be manufactured by a recombinant DNA method such as described in U.S. Patent No. 4,816,567. For example, monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554.

[0037] The term "CDR" means a complementarity determining region, as defined by one skilled in the art by at least one identification method. In some embodiments, CDRs may be defined according to any one of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. VHHs comprise three CDRs, referred to as CDR1, CDR2, and CDR3.

[0038] As used herein, the term "heavy chain constant region" refers to a region comprising at least three heavy chain constant domains C H 1. Hinge, C H 2 and C H 3 region. Of course, unless otherwise specified, non-functional altered deletions and changes in the domain are included within the scope of the term "heavy chain constant region". 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 an antibody type. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, and an antibody comprising an α constant region is an IgA antibody. In addition, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Certain types can be further divided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region) and IgG4 (comprising a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0039] As used herein, "Fc region" refers to the 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 any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0040] As discussed herein, an "acceptor human framework" as used herein is an immunoglobulin construct comprising a heavy chain variable domain (V H) framework. The acceptor human framework derived from a human immunoglobulin framework or a human common framework may comprise the same amino acid sequence therewith, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes in all human frameworks of a single antigen binding domain such as a VHH 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.

[0041] "Affinity" refers to the strength of the sum 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 a molecule X for its partner Y can generally be expressed by the dissociation constant (K) and the affinity of a molecule X for its partner Y, respectively. D ) or K D-表观 Affinity can be expressed by common methods known in the art (such as ELISA K D , KinExA, flow cytometry and / or surface plasmon resonance devices), including those described herein. The methods include but are not limited to those involving or flow cytometry methods.

[0042] As used herein, the term "K D ” refers to the equilibrium dissociation constant of the antigen binding molecule / antigen interaction. When the term “K D ", it includes K D and K D-表观 .

[0043] In some embodiments, the K of the antigen binding molecule is D K is measured by flow cytometry using a cell line expressing the antigen and fitting the mean fluorescence measured at each antibody concentration to a nonlinear single-site binding equation (Prism Software graphpad). In some such embodiments, K D K D-表观 .

[0044] The term "biological activity" refers to any one or more biological properties of a molecule (whether found naturally in vivo or provided or achieved by recombinant means). Biological properties include, but are not limited to, binding to a ligand, inducing or increasing cell proliferation, and inducing or increasing cytokine expression.

[0045] An "agonist" or "activating" antibody or polypeptide is an antibody or polypeptide that increases and / or activates the biological activity of its 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.

[0046] "Antagonist", "blocking" or "neutralizing" antibodies are antibodies that inhibit, reduce and / or do not activate 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.

[0047] An "affinity matured" sdAb or VHH-containing polypeptide refers to an sdAb or VHH-containing polypeptide having one or more changes in one or more CDRs, such changes resulting in an improvement in the affinity of the sdAb or VHH-containing polypeptide for an antigen, compared to a parent sdAb or VHH-containing polypeptide that does not have such changes.

[0048] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced by human framework regions. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized VHHs may include residues that are neither present in the original VHH nor in the human framework sequence, but include the residues to further improve and optimize the sdAb-containing VHH polypeptide potency. In some embodiments, humanized sdAbs or VHH-containing polypeptides include human Fc regions. As will be appreciated, humanized sequences can be identified by their primary sequence and do not necessarily represent the process of producing antibodies.

[0049] An "effector-positive Fc region" has the "effector functions" of a native sequence Fc region. 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, among others. Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0050] A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0051] "Variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification. In some embodiments, the "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of the native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or compared to the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, and preferably about one to about five amino acid substitutions. In some embodiments, the variant Fc region herein has at least about 80% sequence identity with the native sequence Fc region and / or the Fc region of the parent polypeptide, has at least about 90% sequence identity therewith, has at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith.

[0052] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is a natural human FcR. In some embodiments, FcR is an FcR (gamma receptor) that binds to an IgG antibody and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of those receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), both of which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain, and the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed in, e.g., 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 include those to be identified in the future, which are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for 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 regulates the constant expression of immunoglobulins. 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.)).

[0053] As used herein, the term "substantially similar" or "substantially identical" means that the similarity between two or more values ​​is sufficiently high that, in the case of a biological characteristic measured by the values, a person skilled in the art would consider the difference between the two or more values ​​to be minimal or non-biologically and / or statistically significant. In some embodiments, two or more substantially similar values ​​differ by no more than about any of the following: 5%, 10%, 15%, 20%, 25%, or 50%.

[0054] A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. The 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 will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0055] As used herein, "percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences 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 or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved by various means within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0056] Amino acid substitutions may 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 may be introduced into the antibody of interest, and the products screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. Table 1

[0057] Amino acids can be grouped based on common side chain properties: (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0058] Non-conservative substitutions will result in exchanging a member of one of these classes for another class.

[0059] The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences that regulate expression of a polypeptide of interest (such as, for example, a promoter and / or enhancer), and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, such as β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0060] "Host cell" refers to a cell that can be or has been a recipient of a vector or 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 cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, Host cells include 293 and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutations. Host cells include cells transfected in vivo with the polynucleotides provided herein.

[0061] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are commonly found or produced together in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the components of the cell that produced the polypeptide. If the polypeptide is secreted by a cell after expression, then physically separating the supernatant containing the polypeptide from the cell that produced the polypeptide is considered to be "isolated" the polypeptide. Similarly, a polynucleotide is said to be "isolated" when it is not part of a larger polynucleotide in which the polynucleotide is commonly found in nature (such as, for example, genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or is separated from at least some of the components of the cell that produced the polynucleotide (for example, in the case of an RNA polynucleotide). Thus, a DNA polynucleotide contained in a vector inside a host cell can be said to be "isolated".

[0062] The terms "individual" and "subject" are used interchangeably herein to refer to an animal; for example, a mammal. In some embodiments, methods of treating mammals are provided, including but not limited to humans, rodents, monkeys, felines, canines, equines, bovines, porcines, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. In some embodiments, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or condition. In some embodiments, the subject receiving treatment may be a patient, indicating the fact that the subject has been identified as having a condition associated with the treatment, or is at great risk of contracting the condition.

[0063] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0064] Unless otherwise specified, the terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein and refer to a cell (or cells) that exhibits uncontrolled growth and / or abnormally increased cell survival and / or inhibition of apoptosis, which interferes with the normal function of body organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasias, and latent tumors or micrometastases.

[0065] The terms "cancer" and "tumor" encompass solid cancers and blood / lymphatic cancers, and also encompass malignant, precancerous, and benign growths, such as dysplasia. 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 cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; chondrosarcoma, Ewing's sarcoma, colon and rectal cancer (colorectal cancer); connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; hepatocarcinoma; liver tumors; epithelial cancer. endometrial neoplasms; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cancer (lip cancer, tongue cancer, mouth cancer, and pharyngeal cancer); ovarian cancer; pancreatic cancer, such as pancreatic adenocarcinoma; pituitary cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colorectal cancer; respiratory system cancer; mesothelioma; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; Stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; vulvar cancer; lymphomas, including Hodgkin's and non-Hodgkin's lymphomas, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; bulky lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with macular degeneration, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0066] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be apparent to those skilled in the art, "modulation" may also involve a change (which may be an increase or decrease) in affinity, avidity, specificity and / or selectivity for a ligand, binding partner, one or more of the partners binding to a homopolymer or heteropolymer form or substrate of a target or antigen compared to the same conditions but without the test agent; a change (which may be an increase or decrease) in the sensitivity of a 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 cell proliferation or cytokine production. Depending on the target involved, this may be determined by any suitable means and / or using any suitable assay known per se or described herein.

[0067] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. "Treatment" as used herein encompasses any administration or administration of a therapeutic regimen for a disease in a mammal, including humans. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, any one or more of the following: alleviating one or more symptoms, weakening the extent of the disease, preventing or delaying disease spread (e.g., metastasis, such as metastasis to the lungs or lymph nodes), preventing or delaying disease recurrence, delaying or slowing disease progression, improving disease state, inhibiting disease or disease progression, inhibiting or slowing disease or its progress, blocking its development and alleviating (whether partially or completely). "Treatment" also encompasses mitigation of the pathological consequences of proliferative diseases. The method provided herein encompasses any one or more of the treatment aspects. According to the above, the term treatment does not require all aspects of the disease to be removed one hundred percent.

[0068] "Relief" means that one or more symptoms are alleviated or improved compared to when no therapeutic agent is administered. "Improvement" also includes shortening or reducing the duration of symptoms.

[0069] The term "anticancer agent" is used herein in its broadest sense to refer to an agent used to treat 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), radiotherapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0070] As used herein, the terms "synergistically," "synergistically," and "synergistic effect" refer to a more than additive effect of two or more agents. Synergistic effects between DR5 agonists and IAP antagonists can be determined using the assays described herein.

[0071] The term "biological sample" means a quantity of a substance from a living or formerly living organism. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0072] The term "control" or "reference" refers to a composition known to not contain the analyte (a "negative control") or a composition known to contain the analyte (a "positive control"). A positive control may comprise a known concentration of the analyte.

[0073] As used herein, "delaying disease progression" means delaying, hindering, slowing, stifling, stabilizing, inhibiting and / or postponing the development of a disease (such as cancer). This delay may be of varying lengths of time, depending on the disease being treated and / or the individual's medical history. As will be apparent to one skilled in the art, a sufficient or significant delay may actually encompass prevention, such that the subject does not develop the disease. For example, the development of advanced cancers, such as cancer metastases, may be delayed.

[0074] As used herein, "preventing" includes providing a preventive effect on the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms "reducing," "inhibiting," or "preventing" do not imply or require complete prevention at all times, but only over a measured period of time.

[0075] A "therapeutically effective amount" of a substance / molecule (agonist or antagonist) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule (agonist or antagonist) to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the substance / molecule (agonist or antagonist). A therapeutically effective amount may be delivered in one or more administrations. A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic and / or preventive result at the necessary dosage and for the necessary period of time.

[0076] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to preparations that are in a form that permits the biological activity of the active ingredient to be effective, and that contain no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such preparations may be sterile.

[0077] "Pharmaceutically acceptable carrier" refers to a conventional non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation adjuvant or carrier in the art, which is used together with the therapeutic agent to constitute a "pharmaceutical composition" administered to a subject. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used and are compatible with the other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for the formulation used.

[0078] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0079] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, wherein at least portions of the administration overlap in time, or wherein the administration of one therapeutic agent falls within a shorter interval relative to the administration of another therapeutic agent, or wherein the therapeutic effects of the two agents overlap for at least one period of time.

[0080] The term "sequentially" is used herein to refer to administration of two or more therapeutic agents that do not overlap in time, or where the therapeutic effects of the agents do not overlap.

[0081] As used herein, "in combination" refers to administering another treatment modality in addition to one treatment modality. Thus, "in combination" refers to administering another treatment modality before, during, or after administering one treatment modality to a subject.

[0082] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0083] An "article of manufacture" is any article (e.g., package or container) containing at least one agent, such as a drug for treating a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is marketed, distributed, or sold in unit form for performing the methods described herein.

[0084] The terms "label" and "detectable label" mean a moiety attached to, for example, an antibody or antigen so that a reaction (e.g., binding) between members of a specific binding pair is detectable. A labeled member of a specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a label is incorporated to facilitate identification of the binding protein. In some embodiments, the label is a detectable marker that produces a signal that can be detected visually or instrumentally, such as a polypeptide that incorporates a radiolabeled amino acid or is attached to a biotinyl moiety that can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of polypeptide labels include, but are not limited to, the following: 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 153Sm); chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent labels; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelators. Representative examples of labels commonly used in immunoassays include light-generating moieties, such as acridine compounds, and fluorescence-generating moieties, such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable after reaction with another moiety. Exemplary DR5 agonists

[0085] Provided herein are methods of treating cancer comprising administering a DR5 agonist. Non-limiting exemplary DR5 agonists include INBRX-109, eftozanermin alfa (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 ( -DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals), MM201-b (Merrimack Pharmaceuticals), KMTR2 (Kyowa Hakko Kirin), DS-8273a (Daiichi Sankyo), dulanermin (Genentech / Amgen), lexatumumab (Human Genome Sciences / GSK), conatumumab (Amgen), drozitumab (Genentech / Roche), LBY135 (Novartis) and tigatuzumab (Daiichi Sankyo). In some embodiments, the DR5 agonist is a DR5 binding polypeptide. In some embodiments, the DR5 binding polypeptide provided herein is multivalent. In some embodiments, the DR5 binding polypeptide provided herein is at least tetravalent. In some embodiments, the DR5 binding polypeptide provided herein is tetravalent.

[0086] 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 comprising 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.

[0087] 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 polypeptide provided herein comprises 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, so that a dimer is formed that doubles the number of DR5 binding sites. 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, so that the DR5 binding polypeptide is a tetravalent dimer under such conditions.

[0088] In various embodiments, a DR5 binding polypeptide is provided, wherein 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.

[0089] In some embodiments, the DR5 binding polypeptide comprises the structure VHH-linker-VHH-linker-Fc. 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 SEQ ID NO:5. In some embodiments, the VHH-linker-VHH portion of the DR5 binding polypeptide comprises the amino acid sequence SEQ ID NO:5. In some embodiments, the Fc comprises a hinge. In some such embodiments, the Fc comprises the amino acid sequence 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 SEQ ID NO:7, which includes two VHH domains and an Fc region. In some embodiments, the DR5 binding polypeptide comprises the amino acid sequence SEQ ID NO:7, which includes two VHH domains and an Fc region. In some embodiments, the DR5 binding polypeptide consists of the amino acid sequence SEQ ID NO:7. The DR5-binding polypeptide consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 without the terminal lysine may be referred to as INBRX-109.

[0090] In some embodiments, the VHH domain that binds to DR5 can be humanized. Humanized antibodies (such as sdAbs or VHH polypeptides) are suitable for use as therapeutic molecules because humanized antibodies reduce or eliminate human immune responses to non-human antibodies, which can cause immune responses to antibody therapeutics and reduce the effectiveness of therapeutics. In general, humanized antibodies include one or more variable domains, wherein CDR (or a portion thereof) is derived from non-human antibodies, and FR (or a portion thereof) is derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies from which CDR residues are derived), thereby, for example, restoring or improving antibody specificity or affinity.

[0091] Humanized antibodies and methods for making them are reviewed in, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633, and further described in, e.g., Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 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 the "guided selection" method of FR shuffling).

[0092] 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 the consensus sequence of human antibodies having a particular subgroup of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4285; and Presta et al. (1993) J. Immunol, 152: 2376). 1:2623); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front.Biosci.13:1619-1633); and framework regions derived from screening 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, the FR regions of a VHH are replaced with human FR regions to generate a humanized VHH. In some embodiments, certain FR residues of a human FR are replaced in order to improve one or more properties of a humanized VHH. VHH domains with such replaced residues are still referred to herein as "humanized".

[0093] 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.

[0094] In some embodiments, the Fc region included in the DR5 binding polypeptide is derived from a human Fc region and comprises three amino acid deletions corresponding to IgG1 E233, L234 and L235 in the lower hinge, referred to herein as "Fc xELL". Fc xELL polypeptides do not bind FcγRs and are therefore referred to as "effector silent" or "effector void", however in some embodiments, the xELL Fc region binds FcRn and therefore has an extended half-life and endocytic trafficking associated with FcRn-mediated recycling. In some embodiments, the Fc region is a human IgG1 xELL Fc region. Exemplary IAP Antagonists

[0095] Provided herein are methods of treating cancer comprising 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), birenapam (IGM Biosciences, Inc.), AZD5582 (AstraZeneca), LCL161 (Novartis), Debio 1143 (Merck, Debiopharm), or ASTX660 (Astex Pharmaceuticals, Inc.).

[0096] In some embodiments, the IAP antagonist is APG-1387. APG-1387 (also known as SM-1387) is a bivalent IAP antagonist with the following structure: or a pharmaceutically acceptable salt or hydrate thereof. See, for example, US8,883,771 and WO2014 / 031487. APG-1387 is a SMAC mimetic and has potent in vitro and in vivo antitumor activity in solid malignancies.

[0097] In some embodiments, the IAP antagonist is birenapam. Birenapam (also known as TL32711) is a bivalent IAP antagonist having the following structure: or a pharmaceutically acceptable salt or hydrate thereof. See, e.g., U.S. Pat. No. 8,283,372. Birenapam is a SMAC mimetic that binds to XIAP, CIAP1 and CIAP2 and has in vitro and in vivo antitumor activity in solid malignancies.

[0098] In some embodiments, the IAP antagonist is AZD5582. AZD5582 is a bivalent IAP antagonist having the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, WO2010 / 142994A1. AZD5582 is a SMAC mimetic.

[0099] In some embodiments, the IAP antagonist is LCL161. LCL161 is a monovalent IAP antagonist having the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, WO 2011 / 019782. LCL161 is a SMAC mimetic that binds to XIAP, CIAP1 and CIAP2.

[0100] In some embodiments, the IAP antagonist is Debio 1143. Debio 1143 (also known as xevinapant, AT-406, SM-406, and ARRY-334543) is a monovalent IAP antagonist with the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, PLoS ONE 14(2):e0211746(2019). Debio 1143 is a SMAC mimetic that binds to XIAP, CIAP1, and CIAP2.

[0101] In some embodiments, the IAP antagonist is ASTX660. ASTX660 (also known as tolinapant) is a monovalent IAP antagonist having the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, J. Med. Chem., 61(16):7314-7329 (2018); U.S. Pat. No. 9,783,538; WO 2021 / 225955A1. ASTX660 is a non-peptide mimetic that antagonizes XIAP, CIAP1, and CIAP2. Peptide expression and production

[0102] A nucleic acid molecule comprising a polynucleotide encoding a DR5 binding polypeptide is provided. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs 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 may be a native heavy chain (or VHH) leader sequence, or may be another heterologous leader sequence.

[0103] Nucleic acid molecules can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0104] Vectors comprising nucleic acids encoding DR5 binding polypeptides described herein are provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, vectors are selected that are optimized to express the polypeptide in a desired cell type such as CHO or CHO-derived cells or NSO cells. Exemplary such vectors are described, for example, in Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004).

[0105] 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 (such as yeast), plant cells, insect cells and mammalian cells. Such expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express the polypeptide include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells and FUT8 CHO cells; cells (Crucell); and NSO cells. In some embodiments, the DR5 binding polypeptide can be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to produce the desired post-translational modification of the polypeptide. For example, in some embodiments, CHO cells produce a polypeptide that has a higher degree of sialylation than the same polypeptide produced in 293 cells.

[0106] The introduction of one or more nucleic acids (such as vectors) into the desired host cells can be accomplished 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 in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected into the desired host cells according to any suitable method.

[0107] Host cells comprising any nucleic acid or vector described herein are also provided. In some embodiments, host cells expressing the DR5 binding polypeptides described herein are provided. The DR5 binding polypeptides expressed in the host cells can be purified by any suitable method. Such methods include, but are not limited to, using affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind to the Fc region. For example, protein A, protein G, protein A / G or antibody affinity columns can be used to bind to the Fc region and purify the DR5 binding polypeptides comprising the Fc region. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be applicable to purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be applicable to 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 applicable to purifying some polypeptides, such as antibodies. A variety of methods for purifying polypeptides are known in the art.

[0108] In some embodiments, the DR5-binding polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, e.g., 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).

[0109] In some embodiments, a DR5-binding polypeptide prepared by the methods described above is provided. In some embodiments, the DR5-binding polypeptide is prepared in a host cell. In some embodiments, the DR5-binding polypeptide is prepared in a cell-free system. In some embodiments, the DR5-binding polypeptide is purified. In some embodiments, a cell culture medium comprising the DR5-binding polypeptide is provided.

[0110] In some embodiments, a composition comprising an antibody prepared by the methods described above is provided. In some embodiments, the composition comprises a DR5 binding polypeptide prepared in a host cell. In some embodiments, the composition comprises a DR5 binding polypeptide prepared in a cell-free system. In some embodiments, the composition comprises a purified DR5 binding polypeptide. Pharmaceutical composition

[0111] In some embodiments, compositions comprising a DR5 agonist and / or an IAP antagonist are provided in the form of a formulation containing a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). A variety of pharmaceutically acceptable carriers, including vehicles, adjuvants, and diluents, are available. In addition, a variety of pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, and the like, are also available.

[0112] In some embodiments, INBRX-109 is provided as 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. Exemplary methods for treating cancer using DR5 agonists and IAP antagonists

[0113] In some embodiments, methods of treating cancer in a subject are provided, comprising administering a DR5 agonist and an IAP antagonist.

[0114] In some embodiments, the method comprises administering to an individual an effective amount of a DR5 agonist and an IAP antagonist. Such treatment methods may be for humans or animals. In some embodiments, methods of treating humans are provided. Non-limiting exemplary cancers that can be treated with a 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 and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; chondrosarcoma; Ewing's sarcoma; colon and rectal cancer (colorectal cancer); connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; gastrointestinal cancer cancer; glioblastoma; hepatocarcinoma; liver tumor; intraepithelial neoplasia; kidney cancer or renal cancer; 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 cancer, tongue cancer, mouth cancer and pharynx cancer); 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; urological cancer; and vulvar cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL L; bulky lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and other carcinomas and sarcomas; and post-transplantation lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with macular degeneration, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0115] The DR5 agonist and IAP antagonist may be administered to the subject as needed. The frequency of administration of each agent may be determined by a person skilled in the art, such as an attending physician, based on consideration of 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, and the like. In some embodiments, an effective dose of one or more therapeutic agents is 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, semi-weekly, weekly, biweekly, monthly, 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 may be administered multiple times, including multiple administrations over a period of at least one month, at least six months, or at least one year.

[0116] In some embodiments, the DR5 agonist is administered in an amount that is effective for treating (including preventing) cancer. The therapeutically effective amount generally depends on the weight of the subject being treated, its physiological or health condition, the scalability of the condition being treated, or the age of the subject being treated. In general, the DR5 binding polypeptide can be administered in an amount ranging from about 0.05 mg / kg body weight to about 100 mg / kg body weight per dose, or from about 10 μg / kg body weight to about 100 mg / kg body weight per dose, or from about 50 μg / kg body weight to about 5 mg / kg body weight per dose, or from about 100 μg / kg body weight to about 10 mg / kg body weight per dose, or from about 100 μg / kg body weight to about 20 mg / kg body weight per dose, or from about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose, or from about 1 mg / kg body weight to about 10 mg / kg body weight per dose.

[0117] In some embodiments, INBRX-109 is administered at a dose between about 1 mg / kg body weight and about 30 mg / kg body weight. In some embodiments, INBRX-109 is administered at a dose between about 1 mg / kg body weight and about 10 mg / kg body weight. In some embodiments, INBRX-109 is administered at a dose between about 1 mg / kg body weight and about 2 mg / kg body weight. In some embodiments, INBRX-109 is administered at a dose between about 2 mg / kg body weight and 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 every 1 week, once every 2 weeks, once every 3 weeks, or once every 4 weeks. In some embodiments, INBRX-109 is administered once every 3 weeks.

[0118] In some embodiments, the IAP antagonist (or a pharmaceutically acceptable salt or hydrate thereof) is administered at about 1 mg / m2 About 1000 mg / m 2 , including, for example, about 10 mg / m 2 About 500 mg / m 2 , about 10mg / m 2 About 300 mg / m 2 or about 10 mg / m 2 About 200 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 10,000 mg, including, for example, 10 mg to 5,000 mg, or 10 mg to 1,000 mg, or 10 mg to 500 mg.

[0119] In some embodiments, APG-1387 is administered at a dose between about 0.3 mg and about 60 mg. In some embodiments, APG-1387 is administered once every week. In some embodiments, birinapa is administered at a dose between about 2.8 and about 47 mg / m 2 In some embodiments, LCL161 is administered at a dose of about 10 to about 3,000 mg. In some embodiments, LCL161 is administered at a dose of about 900 mg to about 1,800 mg. In some embodiments, LCL161 is administered weekly. In some embodiments, Debio 1143 is administered at a dose of about 5 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 optionally administered daily on a 7-day activation / 7-day deactivation schedule.

[0120] 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 administration. Appropriate formulations and administration routes can be selected according to the intended application.

[0121] 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.

[0122] In some embodiments, the DR5 agonist and the IAP antagonist are administered simultaneously.

[0123] 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 a DR5 agonist and an IAP antagonist produces a synergistic effect compared to each agent administered alone.

[0124] In some embodiments, a DR5 agonist is provided for use in a method of treating cancer, wherein the method comprises administering the DR5 agonist in combination with an IAP antagonist.

[0125] In some embodiments, there is provided a use of a DR5 agonist for the manufacture of a medicament for treating cancer, wherein the medicament is administered with an IAP antagonist. Reagent test kit

[0126] Also provided are articles of manufacture and kits comprising any of the DR5 agonists and / or IAP antagonists provided herein and suitable packaging. In some embodiments, the present invention includes a kit having (i) a formulation comprising a DR5 agonist, (ii) a formulation comprising an IAP antagonist, and (iii) instructions for administering the formulation to an individual using the kit. In some embodiments, the present invention includes a kit having (i) a formulation comprising a DR5 agonist and (ii) instructions for administering the formulation in combination with an IAP antagonist to an individual using the kit. In some embodiments, the present invention includes a kit having (i) a formulation comprising an IAP antagonist and (ii) instructions for administering the formulation in combination with a DR5 agonist to an individual using the kit.

[0127] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), containers, ampoules, bottles, cans, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles may be further sterilized and / or sealed. Also provided are unit dosage forms comprising the compositions described herein. These unit dosage forms may be stored in suitable packaging as single or multiple unit doses and may be further sterilized and sealed. The instructions supplied in the kits of the present invention are written instructions, usually on a label or drug insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions on a magnetized or optical storage disk) are also acceptable. Instructions associated with the use of DR5 agonists and / or IAP antagonists generally include information on dosage, dosing schedule, and route of administration for intended treatment or industrial use. The kit may further include a description of selecting a suitable subject or treatment.

[0128] The container may be a unit dose, bulk (e.g., multi-dose packaging), or sub-unit dose. For example, kits containing sufficient doses of the molecules disclosed herein to provide effective treatment to a subject for an extended period of time, such as any of about 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 longer, may also be provided. The kit may also include multiple unit doses of the molecule and instructions for use and be packaged in sufficient quantities for storage and use in pharmacies (e.g., hospital pharmacies and dispensing pharmacies). In some embodiments, the kit includes a dried (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form a stable aqueous solution of a general DR5 agonist. Example

[0129] The examples discussed below are intended only to illustrate the present invention and should not be considered to limit the present invention in any way. The examples are not intended to represent that the following experiments are all experiments or the only experiments performed. Every effort has been made to ensure the accuracy of the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Example 1: Combination activity of INBRX-109 with various IAP antagonists

[0130] INBRX-109 was tested in combination with various IAP antagonists on various cancer cell lines to determine cytotoxicity towards cancer cells. Assay protocol

[0131] Day 1 - cell inoculation. Harvest the monolayer culture of each cell line for compound screening as described in detail below. Aspirate the culture medium, and wash the cells once with PBS. Add cell digestion solution (Accutase) and cultivate the bottle at 37°C until the cells become detached. Add an equal volume of complete culture medium to quench the cell digestion solution, and then pipette the cells up and down several times to produce a homogenous single cell suspension. The density and viability of the cells are determined using a TC20 automated cell counter using Trypan Blue. The experimental cells are resuspended in EMEM / 10% FBS / Anti-Anti culture medium (complete EMEM) to a concentration of 0.17x10^6 / mL, and are inoculated in the inner wells of a 384-well luminescent plate with 15 μL / well (final 2,500 / well). Each cell line is spread on a single plate in duplicate. The outer wells were filled with 50 μL PBS and the plates were then incubated overnight in a humidified temperature controlled 37° C. tissue culture incubator at 5% CO 2 for 16 hours.

[0132] Test Article Preparation for IAP Antagonists: All small molecules were purchased from MedChemExpress as 10 mM solutions in DMSO. These test articles were then aliquoted and stored at -80°C. Aliquots were thawed just prior to dilution and use in the assay. A master plate of 500x serial dilutions (6 point 2-fold dilutions in 100% DMSO, plus a DMSO only control) was prepared and mixed gently with a pipette. To generate a 5x working dilution plate of the small molecules, dilute 1:100 from the 500x plate into complete medium (EMEM).

[0133] Test Article Preparation for INBRX-109. The INBRX-109 assay concentration range was chosen to encompass the minimum and maximum activity seen in previous cytotoxicity assays with several cancer cell lines, with 1 nM defined as the maximal effective concentration. A 50x plate of INBRX-109 serial dilutions was prepared (starting at 500 nM, 6 point 10-fold dilutions in complete EMEM, plus a complete EMEM only control) and mixed gently with a pipette. To generate a 5x working dilution plate of INBRX-109, each well from the 50x plate was diluted 1:10 into complete EMEM.

[0134] Day 2 - Test and control addition. IAP antagonist small molecule working dilution (5 μL of 5x), INBRX-109 working dilution (5 μL of 5x), or Staurosporine positive control (5 μL of 5x) were added to the respective experimental wells. IAP antagonist titration was performed across the plate, and INBRX-109 titration was performed down the plate to generate a matrix of all possible combinations of the two test articles. These test articles were added in duplicate to each cell line. The plates were then centrifuged at 400 x g for 1 minute and then incubated for 48 hours at 37°C in a humidity controlled tissue culture incubator (5% CO2).

[0135] Day 4 - Viability Measurement. The plates were equilibrated to room temperature for 10 minutes, followed by addition of 25 μL The plate was added to each well. The plate was rotated at 400 x g for 1 minute, then covered and incubated at room temperature for 10 minutes in the dark. Any visible bubbles were removed with 100% ethanol vapor, and then 384-well opaque plates were set and SoftMaxPro v5.4 software was used to read luminescence (RLU) on a Spectra Max M5e plate reader with a 50 millisecond integration time. To determine the effect of the test substance on cell viability, the original RLU value was exported to Excel and the survival percentage was calculated as a percentage relative to the vehicle control (0.2% DMSO in EMEM), wherein the vehicle control was set to 100%. Data are shown in Figure 7 in GraphPad Prism. result

[0136] Figure 1A-1E Results of experiments are shown 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 graph shows the number of cell lines (out of the six tested) that were 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. The IAP antagonist ASTX660 (900 nM) ( Figure 1A )、AZD5582(450nM)( Figure 1B )、Birinap(5μM)( Figure 1C )、Debio 1143(4.5μM)( Figure 1D ) and LCL-161 (5 μM) ( Figure 1E ) results.

[0137] These results are also shown in Tables 2-6. Percent cytotoxicity was calculated by subtracting % survival from 100. Table 2 Table 3 Table 4 Table 5 Table 6

[0138] The combination of INBRX-109 and an IAP antagonist demonstrated increased cell killing for nearly all cancer cell lines tested in this screen, and this was also true for each IAP antagonist individually, compared to INBRX-109 or the IAP antagonist alone.

[0139] This data suggests that the combination of a DR5 agonist (such as INBRX-109) and an IAP antagonist (such as ASTX660, AZD5582, birenapam, Debio 1143, or LCL-161) results in improved or synergistic cancer cell killing compared to either drug by itself. Example 2: Combination Activity of INBRX-109 and LCL-161, or INBRX-109 and Birinapag

[0140] Using a similar assay as described in Example 1, INBRX-109 was tested in combination with the IAP antagonists LCL-161 or birenapam on LS174T and SW620 cancer cell lines to determine cytotoxicity against cancer cells. result

[0141] Figure 2A-2B Results of LCL-161 titration experiments are shown, in which cancer cell lines were exposed to different concentrations of LCL-161 alone or in combination with 1 nM INBRX-109. The percent survival of cancer cells is shown on the y-axis of each graph. Figure 2A ) and SW620( Figure 2B The dashed line labeled "1 nM INBRX-109 only" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0142] Figure 3A-3B Results of a birenapam titration experiment are shown, in which cancer cell lines were exposed to varying concentrations of birenapam alone or in combination with 1 nM INBRX-109. The percent survival of the cancer cells is shown on the y-axis of each graph. Figure 3A ) and SW620( Figure 3B The dashed line labeled "1 nM INBRX-109 only" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0143] The combination of INBRX-109 and LCL-161 or birenapam demonstrated increased cell killing of the cancer cell lines tested in this screen compared to either agent alone.

[0144] This data suggests that the combination of a DR5 agonist (such as INBRX-109) and an IAP antagonist (such as LCL-161 or birenapam) produces improved or synergistic cancer cell killing compared to either drug by itself. Example 3: Combination activity of INBRX-109 and Debio-1143 on other cancer cell lines

[0145] The combination of INBRX-109 and Debio-1143 was tested on various head and neck cancer cell lines to determine the cytotoxicity against cancer cells. Assay protocol

[0146] Day 1 - Cell inoculation. Monolayer cultures of each cancer cell line (see Table 7) were harvested during the logarithmic growth phase. The density and viability of the cells were determined by trypan blue using a Vi cell counter, the cells were diluted in appropriate growth medium (see Table 7) and 90 μL of the cell suspension was added to a 96-well plate for compound screening as detailed below. Two replicate plates were set up for reading on day 0 (T0) and for reading at the end point (T48). Table 7

[0147] Test Article Preparation: Prepare a 1000x serial dilution plate (starting at 9000 μM, 9 point 2-fold dilutions of Debio-1143 in 100% DMSO, plus a DMSO only control) and mix gently with a pipette. To generate a 10x working dilution plate of the small molecule, dilute 1:100 from the 1000x plate into appropriate media. Prepare a 10x stock solution of INBRX-109 (10 nM).

[0148] Day 2 - Compound Treatment. 10 μL (10x) from the 10x working plate and / or 10 μL (10x) INBRX-109 (final concentration 1 nM) were added to respective experimental wells in triplicate. The plates were then incubated at 37°C in a humidity controlled tissue culture incubator (5% CO2).

[0149] Containing 1% DMSO and 50 μL 20 μL of the reagent medium was added to each well of the TO plate, and the contents were mixed, the plate was incubated at room temperature for 30 minutes, and read on an EnVision Multilabel Reader.

[0150] Day 4 - Survival measurement. 50 μL The reagents were added to each well of the T48 plate and the contents were mixed, the plate was incubated at room temperature for 30 minutes, and read on the EnVision multi-label reader. The formula used to calculate the survival percentage was: Survival % = (Lum 测试物 -Lum 培养基对照 ) / (Lum 未处理者 -Lum 培养基对照 ) × 100%. The percentage of cytotoxicity was calculated by subtracting the % survival from 100. result

[0151] Table 8 presents the percent cytotoxicity for each cell line tested. Figure 4A-4L Shown are the results of a Debio-1143 titration experiment in which cancer cell lines were contacted with varying concentrations of Debio-1143 alone or in combination with 1 nM INBRX-109. Figure 4A-Figure 4K Results are presented for the following cancer cell lines that exhibited less than 90% killing with INBRX-109 alone and at least 25% maximal toxicity following treatment with the 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( Fig. 4I )、TE-1( Figure 4J )、KYSE-270( Figure 4K ). The survival percentage of cancer cells is shown on the y-axis of each graph, and the dashed line labeled "1 nM INBRX-109 only" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone. The results shown in Table 8 are summarized in Figure 4L , which plots the % cytotoxicity (% cell death) of all 18 cell lines treated with INBRX-109 or Debio-1143 alone or in combination with INBRX-109 and Debio-1143. Table 8

[0152] The combination of INBRX-109 and Debio-1143 demonstrated increased cell killing of almost all cancer cell lines tested in this screen compared to either INBRX-109 or an IAP antagonist alone. This data further suggests 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 killing compared to either drug alone. Example 4: Combination Activity of INBRX-109 and Birinapa on Other Cancer Cell Lines

[0153] Using a similar assay as described in Example 3, the combination of INBRX-109 and birenapam was tested on 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 the cytotoxicity to cancer cells. In additional studies, using a similar assay as described in Example 1, the expression of α-aspartate amino acid sequences was detected in various chondrosarcomas (CAL-78, OUMS-27, SW1353, and H-EMC-SS), stomach (NCI-N87, SNU-5, and SNU-1), pancreas (AsPC-1, Panc-1, MIA-PaCa-2, and SW1990), and lung (NCI-H460, HCC2935, NCI-H23, NCI-H24 The combination of INBRX-109 and birenapam was tested on gastric (NCI-N87, SNU-5, and SNU-1), pancreatic (AsPC-1, Panc-1, MIA-PaCa-2, and SW1990), and lung (NCI-H460, HCC2935, NCI-H23, NCI-H2452, and A549) cancer cell lines to determine cytotoxicity against cancer cells. result

[0154] Table 9 presents the percentage of cytotoxicity for each breast cancer cell line tested. The results of Table 9 are summarized in Figure 5 , which plots the % cytotoxicity (% cell death) of all 13 breast cancer cell lines treated with INBRX-109 or birenapam alone or in combination. Table 9

[0155] Table 10A presents the percent cytotoxicity of chondrosarcoma, gastric, pancreatic, and lung cancer cell lines treated with INBRX-109 or birenapam alone or in combination. Table 10B presents the percent cytotoxicity of gastric, pancreatic, and lung cancer cell lines treated with INBRX-109 or Debio 1143 alone or in combination. The results of Table 10A are summarized in Figure 6A-6D The graphs plot the % cytotoxicity (% cell death) of cell lines treated with INBRX-109 or birenapam alone or in combination. The results for chondrosarcoma, pancreatic and lung cancer cell lines are plotted in Figure 6A-6C , and all cell lines presented in Table 10A are plotted in Fig.6D The results of Table 10B are summarized in Figure 6E-6G The graphs plot the % cytotoxicity (% cell death) of cell lines treated with INBRX-109 or Debio 1143 alone or in combination. The results for pancreatic and lung cancer cell lines are plotted in Fig. 6E and Fig. 6F , and all cell lines presented in Table 10B are plotted in Figure 6G middle. Table 10A Table 10B

[0156] The combination of INBRX-109 and an IAP antagonist (Birinap or Debio 1143) demonstrated increased cell killing of almost all cancer cell lines tested in these screens compared to either INBRX-109 or the IAP antagonist alone. This data further suggests that the combination of a DR5 agonist (such as INBRX-109) and an IAP antagonist (such as Birinap or Debio 1143) results in improved or synergistic cancer cell killing compared to either drug alone.

[0157] Without departing from the spirit or essential features of the present invention, the present invention may be embodied in other specific forms. The foregoing embodiments are therefore intended to be illustrative rather than limiting in all respects. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and scope of the equivalents of the claims are therefore intended to be included herein. Tables of some sequences

Claims

1. A method of treating cancer in a subject in need thereof, comprising administering to the subject (a) a death receptor 5 (DR5) agonist, wherein the DR5 agonist is tetravalent, and (b) an inhibitor of apoptosis protein (IAP) antagonist.

2. The method of claim 1, wherein the DR5 agonist is a DR5 binding polypeptide.

3. The method of claim 1 or claim 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.

4. The method of claim 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.

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

4.

6. The method of any one of claims 2 to 5, wherein the DR5 binding polypeptide comprises an Fc region.

7. The method of claim 6, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:

6.

8. The method of any one of claims 2 to 7, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc.

9. The method of any one of claims 2 to 8, wherein each VHH domain comprises: 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.

10. The method of any one of claims 2 to 9, wherein the VHH-linker-VHH 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. The method of claim 10 , wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:

5.

12. The method of any one of claims 2 to 11, 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.

13. The method of any one of claims 2 to 12, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

14. The method of any one of claims 2 to 12, wherein the DR5 binding polypeptide consists of the amino acid sequence SEQ ID NO:

7.

15. The method of claim 1 or 2, wherein the DR5 agonist is INBRX-109.

16. The method of any one of claims 1 to 15, wherein the IAP antagonist is a small molecule.

17. The method of any one of claims 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.).

18. The method of claim 17, wherein the IAP antagonist is APG-1387.

19. The method of claim 17, wherein the IAP antagonist is birenapam.

20. The method of claim 17, wherein the IAP antagonist is Debio 1143.

21. The method of claim 17, wherein the IAP antagonist is LCL161.

22. The method of any one of claims 1 to 21, wherein the DR5 agonist and the IAP antagonist are administered separately.

23. The method of claim 22, wherein the DR5 agonist and the IAP antagonist are administered sequentially.

24. The method of claim 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered prior to the IAP antagonist.

25. The method of claim 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered after the IAP antagonist.

26. The method of any one of claims 1 to 21, wherein the DR5 agonist and the IAP antagonist are administered simultaneously.

27. The method of any one of claims 1 to 26, wherein the DR5 agonist and the IAP antagonist act synergistically.

28. The method of claim 27, wherein synergy is determined in an in vitro cell survival assay.

29. The method of any one of claims 1 to 28, wherein administration of the DR5 agonist and the IAP antagonist produces a synergistic effect compared to each agent administered alone.

30. The method of any one of claims 1 to 29, wherein the cancer is 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's sarcoma; sarcoma); cancer of the colon and rectum (colorectal cancer); cancer of connective tissue; cancer of the digestive system; cancer of the endometrium; cancer of the esophagus; cancer of the eye; cancer of the head and neck; cancer of the stomach; cancer of the gastrointestinal tract; glioblastoma; hepatocarcinoma; liver tumors; intraepithelial neoplasia; cancer of the kidney or renal cancer; cancer of the larynx; leukemia; liver cancer; lung cancer; small cell lung cancer; non-small cell lung cancer; adenocarcinoma of the lung; squamous cell carcinoma of the lung; melanoma; myeloma; neuroblastoma; cancer of the oral cavity (lip cancer, tongue cancer, mouth cancer, and / or pharynx cancer); cancer of the ovary; cancer of the pancreas, such as adenocarcinoma of the pancreas; cancer of the pituitary gland; cancer of the prostate; retinoblastoma; rhabdomyosarcoma; cancer of the rectum; cancer of the respiratory system; mesothelioma; cancer of the salivary gland; sarcoma; skin cancer; squamous cell carcinoma of the lung; malignant cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary tract cancer; and vulvar cancer; lymphoma; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; bulky lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia (Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplantation lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with macular degeneration, edema (such as that associated with brain tumors), and Meigs' syndrome.

31. A DR5 agonist for use in a method of treating cancer, wherein the method comprises administering the DR5 agonist in combination with an IAP antagonist.

32. Use of a DR5 agonist for the manufacture of a medicament for the treatment of cancer, wherein the medicament is for administration with an IAP antagonist.

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