Combination therapy of DR5 agonist and PLK1 inhibitor or CDK inhibitor

Abstract

Provided herein is a method of treating cancer using a combination of a DR5 agonist and a PLK1 inhibitor.Also provided herein is a method of treating cancer using a combination of a DR5 agonist and a CDK inhibitor.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 328,951, filed April 8, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] The present invention relates to the treatment of cancer using a combination of a DR5 agonist and a PLK1 inhibitor or a combination of a DR5 agonist and a CDK inhibitor, such as a CDK9 inhibitor. [Background technology]

[0003] 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 to TNF-related apoptosis-inducing ligand (TRAIL). TRAIL has evolved to play an important role in mammalian development and host defense by selectively eliminating unwanted, infected, and malignant cells from healthy cell populations. TRAIL induces cell death by caspase-dependent apoptosis upon binding to TNF receptor family members DR4 or DR5. DR5 appears to be the primary receptor on tumor cells that drives the tumor-biasing activity observed in the TRAIL pathway. DR5 is activated by the natural ligand TRAIL, which brings three DR5 receptors into close proximity, activating intracellular caspase-8 and initiating the activation of other cell death-inducing caspases such as caspase-9 and caspase-3. Thus, initiation of this cell death pathway requires clustering of DR5 receptors for efficient cell death. DR5 agonists are promising therapeutic candidates for the treatment of cancer.

[0004] Polo-like kinases belong to the mitotic serine / threonine kinase family that is involved in cell cycle progression, centrosome cycle, mitosis, and cellular response to DNA damage. To date, five polo-like kinases have been identified in mammalian cells: PLK1, PLK2, PLK3, PLK4, and PLK5. Polo-like kinases are highly conserved in many eukaryotic cells and are characterized by the presence of an N-terminal serine / threonine protein kinase domain (except PLK5) and one or two C-terminal similar regions called polo boxes (PBs). Among the members of the polo-like kinase family, PLK1 is the best characterized. PLK1 plays a role in various stages of cell cycle progression and mitosis, including mitotic entry, G2 / M checkpoint, spindle assembly maturation, chromosome segregation, and mitotic exit. PLK1 is overexpressed in several tumor types and appears to cause enhanced proliferation. PLK1 is nearly undetectable in most normal adult tissues. Inhibition of PLK1 arrests cell proliferation and induces mitotic catastrophe, making it a therapeutic target for the treatment of cancer.

[0005] Cyclin-dependent kinases (CDKs) are serine / threonine protein kinases that are activated by regulatory cyclin proteins. There are 20 CDKs encoded in the human genome, which play specialized roles in numerous cellular processes, such as cell division and transcription, in response to intracellular and extracellular signals. CDK9 is a transcription regulator that is utilized by certain cancer cells for the continuous production of proteins that maintain cancer cell survival. CDK9, together with its major cyclin partner, cyclin T1, forms Positive Transcription Elongation Factor b (P-TEFb). The main function of P-TEFb in eukaryotic cells is to mediate transcriptional elongation of nascent mRNA strands by phosphorylating the S2 residue of the YSPTSPS tandem repeat in the C-terminal domain (CTD) of RNA polymerase II (RNAP II). CDK9 is required for the expression of myeloid cell leukemia-1 (MCL-1). Dysregulation of the CDK9 pathway is involved in prognosis and resistance to anti-cancer therapies in many cancer types, making it a therapeutic target for the treatment of cancer. Summary of the Invention

[0006] Provided herein are methods of treating cancer in a subject with a death receptor 5 (DR5) agonist and a polo-like kinase 1 (PLK1) inhibitor. In some embodiments, the method comprises administering a multivalent death receptor 5 (DR5) binding polypeptide and a polo-like kinase 1 (PLK1) inhibitor. Also provided herein are methods of treating cancer in a subject with a death receptor 5 (DR5) agonist and a cyclin-dependent kinase 9 (CDK9) inhibitor. In some embodiments, the method comprises administering a multivalent death receptor 5 (DR5) binding polypeptide and a CDK9 inhibitor. In some embodiments, the multivalent DR5 binding polypeptide is at least tetravalent. In various embodiments, the DR5 binding polypeptide is a DR5 agonist.

[0007] Embodiment 1. A method of treating cancer in a subject in need of treatment, comprising administering to the subject (a) a death receptor 5 (DR5) agonist and (b) a polo-like kinase 1 (PLK1) inhibitor.

[0008] Embodiment 2. The method of embodiment 1, wherein the DR5 agonist is INBRX-109, eftozanermin alfa (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 (HexaBody™-DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals) or MM201-b (Merrimack Pharmaceuticals).

[0009] Embodiment 3. The method of embodiment 2, wherein the DR5 agonist is INBRX-109.

[0010] Embodiment 4. The method of embodiment 1, wherein said DR5 agonist is a DR5 binding polypeptide.

[0011] Embodiment 5. The method of embodiment 4, wherein the DR5 binding polypeptide is at least tetravalent.

[0012] Embodiment 6. The method according to any one of embodiments 3 to 5, 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.

[0013] Embodiment 7. The method described in embodiment 6, 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.

[0014] Embodiment 8. The method of any one of embodiments 3 to 7, wherein said DR5 binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:4.

[0015] Embodiment 9. The method of any one of embodiments 3 to 8, wherein said DR5 binding polypeptide comprises an Fc region.

[0016] Embodiment 10. The method of embodiment 9, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:6.

[0017] Embodiment 11. The method according to any one of embodiments 3 to 10, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc.

[0018] Embodiment 12. A method according to any one of embodiments 3 to 11, 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.

[0019] Embodiment 13. A method according to any one of embodiments 3 to 12, 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.

[0020] Embodiment 14. The method described in embodiment 13, wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:5.

[0021] Embodiment 15. The method of any one of embodiments 3 to 14, 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.

[0022] Embodiment 16. The method of any one of embodiments 3 to 15, wherein said DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7.

[0023] Embodiment 17. The method according to any one of embodiments 3 to 15, wherein said DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:7.

[0024] Embodiment 18. A method according to any one of embodiments 1 to 17, wherein the PLK1 inhibitor is a small molecule or an interfering RNA (siRNA).

[0025] Embodiment 19. The PLK1 inhibitor is onvansertib, volasertib, rigosertib, BI2536 (Boehringer Ingelheim), N-[[4-[(6-chloro-3-pyridinyl)methoxy]-3-methoxyphenyl]methyl]-3,4-dimethoxybenzeneethanamine hydrochloride (SBE 13 HCl), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), poloxin, poloxin-2HT, RO3280 (CAS number 1062243-51-9), HMN-214 (CAS number 173529-46-9), HMN-176 (CAS number 173529-10-7), 2-cyano-2-[3-ethyl-4-oxo-5-[[3-(2-pyrrolidin-1-yl 19. The method according to any one of the preceding embodiments, wherein the compound is selected from the group consisting of 5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-[[2-(trifluoromethyl)phenyl]methoxy]-2-thiophenecarboxamide (GW 843682X), ...

[0026] Embodiment 20. The method of embodiment 19, wherein the PLK1 inhibitor is onvansertib, volasertib, rigosertib, BI2536 (Boehringer Ingelheim), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), CYC140 (Cyclacel), TKM-080301 (TKM-PLK1; Arbutus Biopharma) or TAK-960 (Takeda Pharmaceutical Company Limited).

[0027] Embodiment 21. The method of any one of embodiments 1 to 18, wherein the PLK1 inhibitor is onvansertib.

[0028] Embodiment 22. The method according to any one of embodiments 1 to 21, wherein the DR5 agonist and the PLK1 inhibitor are administered separately.

[0029] Embodiment 23 The method according to embodiment 22, wherein the DR5 agonist and the PLK1 inhibitor are administered sequentially.

[0030] Embodiment 24 The method according to embodiment 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered before the PLK1 inhibitor.

[0031] Embodiment 25 The method according to embodiment 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered after the PLK1 inhibitor.

[0032] Embodiment 26. The method according to any one of embodiments 1 to 21, wherein the DR5 agonist and the PLK1 inhibitor are administered in combination.

[0033] Embodiment 27. The method according to any one of embodiments 1 to 26, wherein said DR5 agonist and said PLK1 inhibitor act synergistically.

[0034] Embodiment 28 The method of embodiment 27, wherein synergy is determined in an in vitro cell viability assay.

[0035] Embodiment 29. The method of any one of embodiments 1 to 28, wherein administration of the DR5 agonist and the PLK1 inhibitor results in a synergistic effect compared to administration of each agent alone.

[0036] Embodiment 30. A method of treating cancer in a subject in need of treatment, comprising administering to the subject (a) a death receptor 5 (DR5) agonist and (b) a cyclin-dependent kinase (CDK) inhibitor.

[0037] Embodiment 31. The method according to embodiment 30, wherein the DR5 agonist is INBRX-109, eftozanermin alfa (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 (HexaBody™-DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals) or MM201-b (Merrimack Pharmaceuticals).

[0038] Embodiment 32 The method of embodiment 31, wherein the DR5 agonist is INBRX-109.

[0039] Embodiment 33 The method of embodiment 30, wherein the DR5 agonist is a DR5 binding polypeptide.

[0040] Embodiment 34 The method of embodiment 33, wherein the DR5 binding polypeptide is at least tetravalent.

[0041] Embodiment 35. A method according to any one of embodiments 32 to 34, 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.

[0042] Embodiment 36. The method described in embodiment 35, 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.

[0043] Embodiment 37. The method of any one of embodiments 32 to 36, wherein said DR5 binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:4.

[0044] Embodiment 38 The method of any one of embodiments 32 to 37, wherein said DR5 binding polypeptide comprises an Fc region.

[0045] Embodiment 39. The method described in embodiment 38, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:6.

[0046] Embodiment 40. The method according to any one of embodiments 32 to 39, wherein said DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc.

[0047] Embodiment 41. A method according to any one of embodiments 32 to 40, wherein each VHH domain comprises a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3.

[0048] Embodiment 42. A method described in any one of embodiments 32 to 41, 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.

[0049] Embodiment 43. The method described in embodiment 42, wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:5.

[0050] Embodiment 44. The method of any one of embodiments 32 to 43, 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.

[0051] Embodiment 45. The method of any one of embodiments 32 to 44, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7.

[0052] Embodiment 46 The method according to any one of embodiments 32 to 44, wherein said DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:7.

[0053] Embodiment 47. A method according to any one of embodiments 30 to 46, wherein the CDK inhibitor is a CDK9 inhibitor.

[0054] Embodiment 48. The method of any one of embodiments 30 to 47, wherein the CDK inhibitor is a small molecule.

[0055] Embodiment 49. The method according to any one of embodiments 30 to 48, wherein the CDK inhibitor is flavopiridol, seliciclib, dinaciclib, atubeciclib, enitociclib, AZD4573, i-CDK9 or NVP-2.

[0056] Embodiment 50. The method of embodiment 49, wherein the CDK inhibitor is dinaciclib, NVP-2, flavopiridol, enitociclib or AZD4573.

[0057] Embodiment 51. A method according to any one of embodiments 30 to 50, wherein the DR5 agonist and the CDK inhibitor are administered separately.

[0058] Embodiment 52. The method according to embodiment 51, wherein the DR5 agonist and the CDK inhibitor are administered sequentially.

[0059] Embodiment 53. The method according to embodiment 51 or 52, wherein at least one dose or the first dose of the DR5 agonist is administered before the CDK inhibitor.

[0060] Embodiment 54 The method according to embodiment 51 or 52, wherein at least one dose or the first dose of the DR5 agonist is administered after the CDK inhibitor.

[0061] Embodiment 55. The method according to any one of embodiments 30 to 51, wherein the DR5 agonist and the CDK inhibitor are administered in combination.

[0062] Embodiment 56. A method according to any one of embodiments 30 to 55, wherein the DR5 agonist and the CDK inhibitor act synergistically.

[0063] Embodiment 57 The method of embodiment 56, wherein the synergy is determined in an in vitro cell viability assay.

[0064] Embodiment 58. The method according to any one of embodiments 30 to 57, wherein administration of the DR5 agonist and the CDK inhibitor results in a synergistic effect compared to administration of each agent alone.

[0065] Embodiment 59. The cancer is adrenal cancer, astrocytoma, basal cell carcinoma, bile tract cancer, bladder cancer, bone cancer, brain cancer and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing's sarcoma, colorectal cancer. Cancer (colon cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasm, kidney cancer or kidney 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 (lips, tongue, mouth and / or pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer and vulvar cancer, lymphoma , Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with nematoses, edema (such as edema associated with brain tumors), and Meigs' syndrome.

[0066] Embodiment 60. A DR5 agonist for use in a method of treating cancer, the method comprising administering said DR5 agonist in combination with a PLK1 inhibitor.

[0067] Embodiment 61. The use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is administered together with a PLK1 inhibitor.

[0068] Embodiment 62. A DR5 agonist for use in a method of treating cancer, the method comprising administering the DR5 agonist in combination with a CDK inhibitor, such as a CDK9 inhibitor.

[0069] Embodiment 63. Use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is administered together with a CDK inhibitor, such as a CDK9 inhibitor. [Brief description of the drawings]

[0070] [Fig. 1A-1F] FIG. 1 shows the results of a titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.00001 nM, 0.0001 nM, 0.01 nM, 0.1 nM, 1 nM or 10 nM) and onvansertib (0.32 nM, 1.6 nM, 8 nM, 40 nM, 200 nM or 1000 nM). The percent viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines HT-29 (FIG. 1A), LS174T (FIG. 1B), SW620 (FIG. 1C), SW837 (FIG. 1D), SW1463 (FIG. 1E) and LS411N (FIG. 1F). [Fig. 2A-2F] FIG. 2 shows the results of an INBRX-109 titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM), either alone or in combination with 200 nM onvansertib. The % viability of cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines HT-29 (FIG. 2A), LS174T (FIG. 2B), SW620 (FIG. 2C), SW837 (FIG. 2D), SW1463 (FIG. 2E), and LS411N (FIG. 2F). The dotted line labeled "Cpd alone" indicates the % viability of cancer cells treated with 200 nM onvansertib alone. [Figure 3A-3F]FIG. 3 shows the results of an onvansertib titration experiment in which various cancer cell lines were contacted with various concentrations of onvansertib (0.32 nM, 1.6 nM, 8 nM, 40 nM, 200 nM or 1000 nM), either alone or in combination with 1 nM INBRX-109. The % viability of cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines HT-29 (FIG. 3A), LS174T (FIG. 3B), SW620 (FIG. 3C), SW837 (FIG. 3D), SW1463 (FIG. 3E) and LS411N (FIG. 3F). The dotted line labeled "Ab alone" indicates the % viability of cancer cells treated with 1 nM INBRX-109 alone. [Fig. 4A-6L] FIG. 1 shows the results of an onvansertib titration experiment in which various colon cancer cell lines were contacted with various concentrations of onvansertib (274 pM, 823 pM, 2.47 nM, 7.41 nM, 22.22 nM, 66.67 nM, 200 nM, 600 nM and 1.8 μM), alone or in combination with 1 nM INBRX-109. Cancer cell lines SK-CO-1 (Figure 4A), SNU-C5 (Figure 4B), LS 1034 (Figure 4C), LS 123 (Figure 4D), LS 180 (Figure 4E), T84 (Figure 4F), SW-48 (Figure 4G), SW01116 (Figure 4H), SW-1463 (Figure 4I), SW-837 (Figure 5A), SW-948 (Figure 5B), LS 513 (Figure 5C), NCI-H716 (Figure 5D), HCT 116 (Figure 5E), DLD-1 (Figure 5F), HCT-15 (Figure 5G), LS 174T (Figure 5H), Ht-55 (Figure 5I), SNU-81 (Figure 6A), HT-29 (Figure 6B), SW-620 (Figure 6C), Hu Tu Results are shown for 80 (Figure 6D), SW-480 (Figure 6E), SW-403 (Figure 6F), LoVo (Figure 6G), SNU-C2A (Figure 6H), NCI-H747 (Figure 6I), COLO320DM (Figure 6J), RKO (Figure 6K), and HCT-8 (Figure 6L). The viability (%) of cancer cells is shown on the y-axis of each graph. The dotted line labeled "Ab alone" indicates the viability (%) of cancer cells treated with 1 nM INBRX-109 alone. [Figure 7A-8H]FIG. 1 shows the results of an onvansertib titration experiment in which various pancreatic cancer cell lines were contacted with various concentrations of onvansertib (274 pM, 823 pM, 2.47 nM, 7.41 nM, 22.22 nM, 66.67 nM, 200 nM, 600 nM, and 1.8 μM), alone or in combination with 1 nM INBRX-109. Results are shown for the cancer cell lines MA PaCa-2 (Figure 7A), Panc 05.24 (Figure 7B), SW-1990 (Figure 7C), HuP-T4 (Figure 7D), BxPC-3 (Figure 7E), Capan-2 (Figure 7F), KP4 (Figure 7G), AsPC-1 (Figure 7H), PSN-1 (Figure 7I), HPAF-II (Figure 8A), PANC-1 (Figure 8B), Capan-1 (Figure 8C), CFPAC-1 (Figure 8D), Panc 03.27 (Figure 8E), SU.86.86 (Figure 8F), PL45 (Figure 8G), Panc 10.05 (Figure 8H). The viability (%) of the cancer cells is shown on the y-axis of each graph. The dotted line labeled "Ab alone" indicates the viability (%) of cancer cells treated with 1 nM INBRX-109 alone. [Figure 9A-9E] 9A-9E show the results of a titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.00001 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM) and dinaciclib (0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM, or 625 nM). The percent viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 9A), OUMS-27 (FIG. 9B), SW1353 (FIG. 9C), H-EMC-SS (FIG. 9D), and SW620 (FIG. 9E). [Figure 10A-10E]FIG. 10 shows the results of an INBRX-109 titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM), either alone or in combination with 25 nM dinaciclib. The % viability of cancer cells is shown on the y-axis of each graph. Results are shown for cancer cell lines CAL-78 (FIG. 10A), OUMS-27 (FIG. 10B), SW1353 (FIG. 10C), H-EMC-SS (FIG. 10D), and SW620 (FIG. 10E). The dotted line labeled "Cpd alone" indicates the % viability of cancer cells treated with 25 nM dinaciclib alone. [Figures 11A-11E] FIG. 11 shows the results of a dinaciclib titration experiment in which various cancer cell lines were contacted with various concentrations of dinaciclib (0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM or 625 nM), either alone or in combination with 1 nM INBRX-109. The % viability of cancer cells is shown on the y-axis of each graph. Results are shown for cancer cell lines CAL-78 (FIG. 11A), OUMS-27 (FIG. 11B), SW1353 (FIG. 11C), H-EMC-SS (FIG. 11D) and SW620 (FIG. 11E). The dotted line labeled "Ab alone" indicates the % viability of cancer cells treated with 1 nM INBRX-109 alone. [Figures 12A-12E] FIG. 12 shows the results of a titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.00001 nM, 0.0001 nM, 0.01 nM, 0.1 nM, 1 nM or 10 nM) and NVP-2 (0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM or 625 nM). The percent viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 12A), OUMS-27 (FIG. 12B), SW1353 (FIG. 12C), H-EMC-SS (FIG. 12D) and SW620 (FIG. 12E). [Figures 13A-13E]FIG. 13 shows the results of an INBRX-109 titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM), either alone or in combination with 125 nM NVP-2. The % viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 13A), OUMS-27 (FIG. 13B), SW1353 (FIG. 13C), H-EMC-SS (FIG. 13D), and SW620 (FIG. 13E). The dotted line labeled "Cpd alone" indicates the % viability of cancer cells treated with 125 nM NVP-2 alone. [Figures 14A-14E] Figure 14 shows the results of an NVP-2 titration experiment in which various cancer cell lines were contacted with various concentrations of NVP-2 (0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM or 625 nM), either alone or in combination with 1 nM INBRX-109. The % viability of cancer cells is shown on the y-axis of each graph. Results are shown for cancer cell lines CAL-78 (Figure 14A), OUMS-27 (Figure 14B), SW1353 (Figure 14C), H-EMC-SS (Figure 14D) and SW620 (Figure 14E). The dotted line labeled "Ab alone" indicates the % viability of cancer cells treated with 1 nM INBRX-109 alone. [Fig. 15A-15F] FIG. 15 shows the results of a titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.00001 nM, 0.0001 nM, 0.01 nM, 0.1 nM, 1 nM or 10 nM) and flavopiridol (0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM or 625 nM). The percent viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 15A), OUMS-27 (FIG. 15B), HS-SY-II (FIG. 15C), SW1353 (FIG. 15D), H-EMC-SS (FIG. 15E) and SW620 (FIG. 15F). [Figures 16A-16F]FIG. 16 shows the results of an INBRX-109 titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM), either alone or in combination with 125 nM flavopiridol. The % viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 16A), OUMS-27 (FIG. 16B), HS-SY-II (FIG. 16C), SW1353 (FIG. 16D), H-EMC-SS (FIG. 16E), and SW620 (FIG. 16F). The dotted line labeled "Cpd alone" indicates the % viability of cancer cells treated with 125 nM flavopiridol alone. [Figures 17A-17F] Figure 17 shows the results of a flavopiridol titration experiment in which various cancer cell lines were contacted with various concentrations of flavopiridol (0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM or 625 nM), either alone or in combination with 1 nM INBRX-109. The % viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (Figure 17A), OUMS-27 (Figure 17B), HS-SY-II (Figure 17C), SW1353 (Figure 17D), H-EMC-SS (Figure 17E) and SW620 (Figure 17F). The dotted line labeled "Ab alone" indicates the % viability of cancer cells treated with 1 nM INBRX-109 alone. [Fig. 18A-18F] FIG. 18 shows the results of a titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.00001 nM, 0.0001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM) and enitociclib (0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM, or 625 nM). The percent viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 18A), OUMS-27 (FIG. 18B), HS-SY-II (FIG. 18C), SW1353 (FIG. 18D), H-EMC-SS (FIG. 18E), and SW620 (FIG. 18F). [Figures 19A-19F]FIG. 19 shows the results of an INBRX-109 titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM), either alone or in combination with 125 nM enitociclib. The % viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 19A), OUMS-27 (FIG. 19B), HS-SY-II (FIG. 19C), SW1353 (FIG. 19D), H-EMC-SS (FIG. 19E), and SW620 (FIG. 19F). The dotted line labeled "Cpd alone" indicates the % viability of cancer cells treated with 125 nM enitociclib alone. [Fig. 20A-20F] FIG. 20 shows the results of an enitociclib titration experiment in which various cancer cell lines were contacted with various concentrations of enitociclib (0.2 nM, 1 nM, 5 nM, 25 nM, 125 nM or 625 nM), either alone or in combination with 1 nM INBRX-109. The % viability of cancer cells is shown on the y-axis of each graph. Results are shown for cancer cell lines CAL-78 (FIG. 20A), OUMS-27 (FIG. 20B), HS-SY-II (FIG. 20C), SW1353 (FIG. 20D), H-EMC-SS (FIG. 20E) and SW620 (FIG. 20F). The dotted line labeled "Ab alone" indicates the % viability of cancer cells treated with 1 nM INBRX-109 alone. [Figure 21A-21B] FIG. 21 shows the results of a titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.00001 nM, 0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM or 10 nM) and AZD-5991 (0.0032 nM, 0.016 nM, 0.08 nM, 0.4 nM, 2.0 nM, 10 nM). The percent viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 21A), OUMS-27 (FIG. 21B), HS-SY-II (FIG. 21C), SW1353 (FIG. 21D), H-EMC-SS (FIG. 21E) and SW620 (FIG. 21F). [Fig. 22A-22F]FIG. 22 shows the results of an INBRX-109 titration experiment in which various cancer cell lines were contacted with various concentrations of INBRX-109 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM), either alone or in combination with 2 nM AZD-5991. The % viability of cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (FIG. 22A), OUMS-27 (FIG. 22B), HS-SY-II (FIG. 22C), SW1353 (FIG. 22D), H-EMC-SS (FIG. 22E), and SW620 (FIG. 22F). The dotted line labeled "Cpd alone" indicates the % viability of cancer cells treated with 2 nM AZD-5991 alone. [Fig. 23A-23F] Figure 23 shows the results of an AZD-5991 titration experiment in which various cancer cell lines were contacted with various concentrations of AZD-5991 (0.0032 nM, 0.016 nM, 0.08 nM, 0.4 nM, 2.0 nM, 10 nM), either alone or in combination with 1 nM INBRX-109. The % viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines CAL-78 (Figure 23A), OUMS-27 (Figure 23B), HS-SY-II (Figure 23C), SW1353 (Figure 23D), H-EMC-SS (Figure 23E), and SW620 (Figure 23F). The dotted line labeled "Ab alone" indicates the % viability of cancer cells treated with 1 nM INBRX-109 alone. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] The embodiments presented herein relate to methods of treating cancer with a combination of a death receptor 5 (DR5) agonist and a polo-like kinase 1 (PLK1) inhibitor, and / or a combination of a death receptor 5 (DR5) agonist and a cyclin-dependent kinase (CDK) inhibitor, such as a CDK9 inhibitor.

[0072] Definitions and Various Embodiments The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0073] All references cited in this specification, including patent applications, patent publications, and Genbank accession numbers, are incorporated herein by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0074] The techniques and procedures described or referenced herein are generally well understood and generally described in, for example, 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)), the series METHODS IN ENZYMOLOGY (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 (JE Cellis, ed., 1998) Academic Press, Animal Cell Culture (RI Freshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell eds., 1993-8) J. Wiley and Sons, Handbook of Experimental Immunology (DM Weir and C.C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JE Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using The antibodies are used using routine methodologies by those skilled in the art, such as the widely used methodologies described in Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993) and their latest editions.

[0075] Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context or expressly indicated otherwise, singular nouns shall include the plural and plural nouns shall include the singular. In the event of discrepancies in definitions among various sources or references, the definitions set forth herein shall prevail.

[0076] Generally, the numbering of residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0077] Embodiments of the invention described herein are understood to include "consisting of" and / or "consisting essentially of." As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. Use of the term "or" herein is not to be construed as implying that options are mutually exclusive.

[0078] In this application, the use of "or" means "and / or" unless expressly stated otherwise or understood by a person skilled in the art. In the context of a multiple dependent claim, the use of "or" refers to more than one preceding independent or dependent claim.

[0079] The phrases "reference sample", "reference cell" or "reference tissue" refer to 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 establish, for example, the level of protein and / or mRNA present in a healthy tissue relative to the level of protein and / or mRNA present in a sample with an unknown characteristic. In some embodiments, the reference sample is a sample from the same subject, but from a part of the subject different from the part being tested. In some embodiments, the reference sample is a sample from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but from a subject known to have or not have a disorder of interest (e.g., a particular cancer or DR5-associated disorder). In some embodiments, the reference sample is from the same subject, but at a time before the subject developed cancer. In some embodiments, the reference sample is a sample from a benign cancer sample from the same subject or a different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule of interest in the negative reference sample indicates a level at which one skilled in the art would recognize, in view of the present disclosure, that the molecule is absent and / or present at a low level.When a positive reference sample is used for comparison, the expression level or amount of the molecule of interest in the positive reference sample indicates a level at which one skilled in the art would recognize, in view of the present disclosure, that the molecule is present at a certain level.

[0080] The terms "benefit", "clinical benefit", "responsiveness" 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 a degree of inhibition of disease progression, including slowing and complete halt, a reduction in the number of disease episodes and / or symptoms, a reduction in lesion size, an inhibition (i.e., reduction, slowing, or complete halt) of disease cell invasion into adjacent peripheral organs and / or tissues, an inhibition (i.e., reduction, slowing, or complete halt) of disease spread, a degree of relief of one or more symptoms associated with the disorder, a disease-free presentation after treatment, such as an increase in the length of progression-free survival, an increase in overall survival, a higher response rate, and / or a reduction in mortality at a given time point after treatment. A "non-responsive" or "non-responsive" subject or cancer is one that does not meet the above criteria of "responding".

[0081] The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably and may refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, including, but not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0082] 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 may include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. This definition includes both full-length proteins and fragments thereof. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to proteins that contain modifications (generally conservative in nature), such as deletions, additions, and substitutions to the native sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host that produces the protein or by errors due to PCR amplification.

[0083] As used herein, the terms "DR5", "death receptor 5", "TNFRSF10B" and "TRAILR2" refer to any naturally occurring mature DR5 resulting from processing of a DR5 precursor in a cell. The term includes DR5 from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes naturally occurring variants of DR5, such as splice variants or allelic variants. A non-limiting exemplary precursor human DR5 amino acid sequence is shown, for example, in NCBI Accession No. NP_003833.4. See SEQ ID NO: 8. A non-limiting exemplary precursor human DR5 amino acid sequence is shown, for example, in SEQ ID NO: 9.

[0084] As used herein, the terms "PLK1" and "Polo-like kinase 1" refer to any naturally occurring mature PLK1. The terms include PLK1 from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The terms also include naturally occurring variants of PLK1, such as splice variants or allelic variants. Non-limiting PLK1 amino acid sequences are shown, for example, in UniProtKB / Swiss-Prot Accession No. P53350.1. See SEQ ID NO: 10.

[0085] As used herein, the terms "CDK9" and "cyclin-dependent kinase 9" refer to any naturally occurring mature CDK9. The terms include CDK9 from any vertebrate origin, including mammals such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The terms also include naturally occurring variants of CDK9, such as splice variants or allelic variants. Non-limiting amino acid sequences are shown, for example, in UniProtKB / Swiss-Prot Accession No. P50750-1. See SEQ ID NO: 11.

[0086] The term "specifically binds" to an antigen or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, with a longer duration and / or with a higher affinity with a particular cell or substance than it reacts or associates with another cell or substance. A single domain antibody (sdAb) or VHH-containing polypeptide "specifically binds" or "preferentially binds" to a target if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. For example, an sdAb or VHH-containing polypeptide that specifically or preferentially binds to the DR5 epitope is an sdAb or VHH-containing polypeptide that binds to this epitope with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other DR5 epitopes or non-DR5 epitopes. It is also understood by interpreting this definition 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.

[0087] The term "inhibition" or "inhibiting" refers to the reduction or cessation of any phenotypic characteristic, or the reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "inhibit" refers to the decrease, reduction, or cessation of an activity, function, and / or amount compared to a reference. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 10% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the amount is inhibited or reduced over a period of time relative to a control over the same period of time.

[0088] As used herein, the term "epitope" refers to a site on a target molecule (e.g., an antigen such as a protein, nucleic acid, carbohydrate, or lipid) to which an antigen-binding molecule (e.g., an sdAb or VHH-containing polypeptide) binds. Epitopes often comprise chemically active surface arrangements of molecules such as amino acids, polypeptides, or sugar side chains, and have specific three-dimensional structural features and specific charge characteristics. Epitopes can be formed from both contiguous and / or juxtaposed non-contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 residues (e.g., amino acids or nucleotides). In some embodiments, an epitope is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues, or less than 12 residues in length. Two antibodies may bind to the same epitope in an antigen if they exhibit competitive binding to the antigen. In some embodiments, an epitope may be specified by a certain minimum distance to the CDR residues on the antigen-binding molecule. In some embodiments, an epitope may be specified by the above distance and further limited to those residues involved in binding (e.g., hydrogen bonding) between the antigen-binding molecule residues and the antigen residues. Epitopes may also be specified by various scans. For example, an alanine scan or an arginine scan may indicate one or more residues with which an antigen-binding molecule may interact. Unless explicitly indicated, a set of residues as an epitope does not exclude other residues from being part of the epitope for a particular antigen-binding molecule. Rather, the existence of such a set indicates a minimum epitope sequence (or set of types). Thus, in some embodiments, the set of residues identified as an epitope is not an exclusive list of residues for the epitope on the antigen, but rather represents a minimal epitope associated with the antigen.

[0089] The term "antibody" is used in the broadest sense and includes various polypeptides, including, but not limited to, conventional antibodies (typically 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 antibody-like antigen-binding domains, including fragments of any of the above, so 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, a heavy chain constant domain (comprising CH1, hinge, CH2, and CH3, where CH1 is typically paired with a light chain constant domain CL, while the hinge mediates dimerization) and an Fc region (comprising hinge, CH2, and CH3, where the hinge mediates dimerization).

[0090] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species such as camel (including llama), shark, mouse, human, cynomolgus monkey, etc.

[0091] The term "antigen-binding domain" as used herein refers to a portion of an antibody 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. Thus, in some embodiments, the antigen-binding domain comprises a heavy chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen, and a light chain variable region comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen. In some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises the three CDRs of a VHH domain. Thus, in some embodiments, the antigen-binding domain of an sdAb or VHH-containing polypeptide comprises a VHH domain comprising CDR1-FR2-CDR2-FR3-CDR3 and any portion of FR1 and / or FR4 required to maintain binding to the antigen.

[0092] The term "VHH" or "VHH domain" or "VHH antigen-binding domain" as used herein 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 to include only a partial FR1 and / or FR4, or to lack one or both of these framework regions, so long as the VHH substantially retains antigen binding and specificity.

[0093] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one monomer domain, such as a VHH domain, without a light chain and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each polypeptide comprising at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" encompass polypeptides comprising multiple VHH domains, for example, polypeptides having the structure VHH1-VHH2-Fc or VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different.

[0094] The term "VHH-containing polypeptide" refers to a polypeptide that comprises at least one VHH domain. In some embodiments, a VHH polypeptide comprises two, three, or four or more VHH domains, where each VHH domain may be the same or different. In some embodiments, a VHH-containing polypeptide comprises an Fc region. In some such embodiments, a VHH-containing polypeptide may be referred to as an sdAb. Furthermore, in some such embodiments, a VHH polypeptide may form a dimer. Non-limiting structures of a VHH-containing polypeptide, also referred to as an sdAb, include VHH1-Fc, VHH1-VHH2-Fc, and VHH1-VHH2-VHH3-Fc, where VHH1, VHH2, and VHH3 may be the same or different. In some embodiments of such structures, a VHH may be linked to another VHH by a linker, or a VHH may be linked to an Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids consisting mainly of glycine and optionally serine. In some embodiments, when a VHH-containing polypeptide comprises an Fc, it forms a dimer. Thus, the structure VHH1-VHH2-Fc is considered to be tetravalent when it forms a dimer (i.e., the dimer has four VHH domains). Similarly, the structure VHH1-VHH2-VHH3-Fc is considered to be hexavalent when it forms a dimer (i.e., the dimer has six VHH domains).

[0095] The term "monoclonal antibody" refers to an antibody (including sdAb or VHH-containing polypeptide) of a substantially homogeneous antibody population. That is, the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, 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. Thus, a sample of monoclonal antibodies is capable of binding to the same epitope on the antigen. The modifier "monoclonal" indicates the nature of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. The monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0096] The term "CDR" refers to a complementarity determining region defined by at least one specific manner to one skilled in the art. In some embodiments, the CDRs may be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. A VHH comprises three CDRs designated CDR1, CDR2, and CDR3.

[0097] As used herein, the term "heavy chain constant region" refers to a region that comprises at least three heavy chain constant domains, i.e., H 1, hinge, C H 2, and C H3. Of course, deletions and modifications that do not change the function within the domain are included within the scope of the term "heavy chain constant region" unless otherwise specified. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to one antibody isotype. For example, an antibody containing a gamma constant region is an IgG antibody, an antibody containing a delta constant region is an IgD antibody, and an antibody containing an alpha constant region is an IgA antibody. Furthermore, an antibody containing a mu constant region is an IgM antibody, and an antibody containing an epsilon constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibodies (containing a gamma 1 constant region), IgG2 antibodies (containing a gamma 2 constant region), IgG3 antibodies (containing a gamma 3 constant region), and IgG4 antibodies (containing a gamma 4 constant region); IgA antibodies include, but are not limited to, IgA1 antibodies (containing an alpha 1 constant region) and IgA2 antibodies (containing an alpha 2 constant region); and IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0098] As used herein, "Fc region" refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In various embodiments, when the Fc region comprises a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0099] As used herein, an "acceptor human framework" refers to a heavy chain variable domain (V) derived from a human immunoglobulin framework or a human consensus framework, as discussed herein. H) framework amino acid sequence. The acceptor human framework derived from a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence or can comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3 across all human frameworks within a single antigen-binding domain such as a VHH.

[0100] "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 is generally expressed as the dissociation constant (K D ) or K D(見かけ) Affinity can be expressed by any of the conventional methods known in the art, including those described herein (e.g., ELISA K D , KinExA, flow cytometry, and / or surface plasmon resonance devices, etc. Such methods include, but are not limited to, BIAcore™, Octet™, or methods involving flow cytometry.

[0101] As used herein, "K D As used herein, the term "K" refers to the equilibrium dissociation constant of an antigen-binding molecule / antigen interaction. D When the term "K" is used, it includes D and K D(見かけ) Includes:

[0102] In some embodiments, the K of the antigen binding molecule D is measured by flow cytometry using antigen-expressing cell lines and fitting the mean fluorescence measured at each antibody concentration to a nonlinear one-site binding equation (Graphpad's Prism Software).D is K D(見かけ) It is.

[0103] The term "biological activity" refers to any one or more biological properties of a molecule (whether naturally occurring as found in vivo or provided or made possible by recombinant means), including, but not limited to, binding of a ligand, induction or increase in cell proliferation, and induction or increase in expression of a cytokine.

[0104] An "agonist" or "activating" antibody or polypeptide is an antibody or polypeptide that increases and / or activates the biological activity of a target antigen. In some embodiments, an agonist antibody or polypeptide binds to an antigen and increases its biological activity by at least about 20%, 40%, 60%, 80%, 85% or more.

[0105] An "antagonist," "blocking," or "neutralizing" antibody is an antibody that inhibits, reduces and / or inactivates the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces its biological activity by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99% or more.

[0106] An "affinity matured" sdAb or VHH containing polypeptide refers to an sdAb or VHH containing polypeptide which has one or more modifications in one or more CDRs which result in an improvement in the affinity of the sdAb or VHH containing polypeptide for its antigen compared to a parent sdAb or VHH containing polypeptide which does not possess such modifications.

[0107] "Humanized VHH" as used herein refers to a VHH in which one or more framework regions have been substantially replaced with human framework regions. In some cases, certain framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized VHH may contain residues that are not found in the original VHH or in the human framework sequence, but are included to further improve and optimize the performance of the sdAb or VHH-containing polypeptide. In some embodiments, the humanized sdAb or VHH-containing polypeptide comprises a human Fc region. As will be appreciated, a humanized sequence may be identified by its primary sequence and does not necessarily indicate the process by which the antibody was made.

[0108] An "effector positive Fc region" has an "effector function" 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, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Such effector functions generally require combining the Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0109] A "native sequence Fc region" comprises an amino acid sequence identical to that 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.

[0110] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification. In some embodiments, a "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification, but that retains at least one effector function of the native sequence Fc region. In some embodiments, a variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of the parent polypeptide compared to the native sequence Fc region or the Fc region of the parent polypeptide. In some embodiments, a variant Fc region herein has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the native sequence Fc region and / or the Fc region of the parent polypeptide.

[0111] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, the FcγR is a native human FcR. In some embodiments, the FcR binds an IgG antibody (gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, e.g., in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. For example, the term "Fc receptor" or "FcR" also includes the fetal receptor FcRn, which plays a role in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulating immunoglobulin homeostasis.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.)).

[0112] As used herein, the terms "substantially similar" or "substantially the same" refer to a sufficiently high degree of similarity between two or more numerical values ​​such that one of skill in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance within the context of the biological characteristic measured by the values. In some embodiments, two or more substantially similar values ​​differ by no more than the approximate value of any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0113] A polypeptide "variant" refers to 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 percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. In some embodiments, a variant has at least about 80% amino acid sequence identity. In some embodiments, a variant has at least about 90% amino acid sequence identity. In some embodiments, a variant has at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0114] As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of one of ordinary skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0115] Amino acid substitutions can include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions can be introduced into a subject antibody and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0116] TIFF2025512950000001.tif108170

[0117] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: 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.

[0118] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0119] The term "vector" is used to describe a polynucleotide that can be manipulated to contain a cloned polynucleotide or polynucleotides that can be propagated in a host cell. A vector can contain one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters and / or enhancers, etc.) that control the expression of a polypeptide of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactosidase, etc.). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0120] "Host cell" refers to a cell that can be or has been the recipient of a vector or isolated polynucleotide. Host cells can be prokaryotic or eukaryotic. 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, PER.C6™ cells (Crucell), and 293 and CHO cells, and their derivatives, such as 293-6E cells, CHO-DG44 cells, CHO-K1 cells, CHO-S cells, and CHO-DS cells. Host cells include the progeny of a single host cell, although 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 also include cells transfected in vivo with a polynucleotide(s) provided herein.

[0121] The term "isolated" as used herein refers to a molecule that is separated from at least some of the components that it is typically found or produced with in nature. For example, a polypeptide is referred to as "isolated" when it is separated from at least some of the components of the cell that produced it. If the polypeptide is secreted by the cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to "isolate" the polypeptide. Similarly, a polynucleotide is referred to as "isolated" when it is not part of a larger polynucleotide that it is typically found in nature (e.g., in the case of a DNA polynucleotide, genomic DNA or mitochondrial DNA, etc.) or when it is separated from at least some of the components of the cell that produced it, for example, in the case of an RNA polynucleotide. Thus, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0122] The terms "individual" and "subject" are used interchangeably herein to refer to animals, e.g., mammals. In some embodiments, methods are provided for treating mammals, including, but not limited to, humans, rodents, monkeys, cats, dogs, horses, cows, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets. In some instances, "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject to be treated may be a patient, which means the subject has been identified as having or being at sufficient risk of suffering from a disorder relevant to the treatment.

[0123] As used herein, "disease" or "disorder" refers to a condition for which treatment is necessary and / or desirable.

[0124] The terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein, unless otherwise specified, to refer to a cell (or cells) that exhibit uncontrolled proliferation and / or abnormally increased cell survival and / or inhibited apoptosis that interferes with the normal functioning of bodily organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasias, and occult tumors or micrometastases.

[0125] The terms "cancer" and "tumor" include solid cancers and hematological / lymphatic cancers, as well as malignant tumors such as dysplasia, premalignant tumors, and benign tumors. Exemplary cancers include, but are not limited to, adrenal gland 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, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, renal 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 (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, secretory cancer, These include urinary system cancer, vulvar cancer, lymphomas including Hodgkin's lymphoma and non-Hodgkin's lymphoma, 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-dividing cell NHL, bulky disease NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), and abnormal blood vessel growth associated with nematoses, edema (such as edema associated with brain tumors), and Meigs syndrome.

[0126] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. As will be clear to the skilled artisan, "modulation" can also include causing a change (which can be either an increase or a decrease) in the affinity, avidity, specificity and / or selectivity of a target or antigen to one or more of its ligands, binding partners, partners that associate into homo- or heteromultimeric forms or substrates, compared to the same conditions except for the absence of the test agent, causing a change (which can be either an increase or a decrease) in the sensitivity of the target or antigen to one or more conditions (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. This can be determined in any suitable manner and / or using any suitable assay known per se or described herein, depending on the target involved.

[0127] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. As used herein, "treatment" covers any administration or application of a therapeutic agent for a disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, reduction in the extent of the disease, prevention or delay of disease progression (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slowing of disease progression, amelioration of disease state, inhibition of disease or disease progression, inhibition or slowing of disease or its progression, arrest of its development, and remission (whether partial or total). "Treatment" also includes reduction of pathological consequences of proliferative diseases. The methods provided herein contemplate any one or more of these aspects of treatment. In accordance with the above, the term treatment does not require 100 percent elimination of all aspects of the disorder.

[0128] "Amelioration" means that one or more symptoms are lessened or improved compared to when the therapeutic agent is not administered. "Amelioration" also includes a shortening or reduction in the duration of the symptoms.

[0129] The term "anti-cancer 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, anti-cancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiotherapy agents, CAR-T therapy agents, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNAs), and oncolytic viruses.

[0130] As used herein, the terms "synergistic," "synergistically," and "synergy" refer to a greater than additive effect of two or more agents. Determination of synergistic effects between a DR5 agonist and a PLK1 inhibitor, or between a DR5 agonist and a CDK inhibitor, such as a CDK9 inhibitor, can be performed using the assays described herein.

[0131] The term "biological sample" refers to a quantity of material from a living or once living thing, including, but 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.

[0132] The term "control" or "reference" refers to a composition known to be free of the analyte (a "negative control") or to a composition known to contain the analyte (a "positive control"). A positive control may contain a known concentration of the analyte.

[0133] As used herein, "delaying the onset of disease" means delaying, preventing, slowing, retarding, stabilizing, inhibiting, and / or prolonging the onset of a disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. As will be apparent to one of skill in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, late-stage cancer, such as the onset of metastases, can be delayed.

[0134] "Prevention" as used herein includes providing prevention against the occurrence or recurrence of a disease in a subject who may have a predisposition to the disease, but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reduce," "inhibit," or "prevent" do not indicate or require complete prevention over the entire period of time, but only prevention over the period being measured.

[0135] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary depending on 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 any toxic or detrimental effects of the substance / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective to achieve a desired therapeutic and / or prophylactic result at the required dosages for the required time.

[0136] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in a form that allows the biological activity of the active ingredient(s) to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0137] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or carrier conventional in the art used with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation in which it is used.

[0138] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0139] The term "in combination" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administration overlaps in time, or where the administration of one therapeutic agent is brief relative to the administration of the other therapeutic agent, or where the therapeutic effects of both therapeutic agents overlap for at least some period of time.

[0140] The term "sequential" is used herein to refer to the administration of two or more therapeutic agents that do not overlap in time or where the therapeutic effects of the therapeutic agents do not overlap.

[0141] As used herein, "in combination with" refers to the administration of one therapy in addition to another. Thus, "in combination with" refers to the administration of one therapy before, during, or after the administration of another therapy to an individual.

[0142] The term "package insert" is used to refer to instructions typically included in commercial packaging for a therapeutic product, which contain information regarding directions, usage, dosage, administration, concomitant therapy, contraindications and / or warnings regarding the use of such therapeutic product.

[0143] An "article of manufacture" is any manufacture (e.g., package or container) or kit that contains at least one reagent, e.g., an agent for treating a disease or disorder (e.g., cancer), or a probe that specifically detects a biomarker described herein. In some embodiments, the article of manufacture or kit is advertised, delivered, or sold as a unit for performing a method described herein.

[0144] The terms "label" and "detectable label" refer to a moiety that, for example, is attached to an antibody or an antigen to render the reaction (e.g., binding) between members of a specific binding pair 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 that incorporates a label that provides for the identification of the binding protein. In some embodiments, the label is a detectable marker that can generate a signal that is detectable visually or by instrumental means, such as the incorporation of a radiolabeled amino acid or the attachment of a biotinyl moiety to the polypeptide that can be detected by marked avidin (e.g., streptavidin that contains a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I, 177 Lu, 166 Ho, or 153 Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates. Representative examples of labels commonly used in immunoassays include moieties that emit light, e.g., acridinium compounds, and moieties that emit fluorescence, e.g., fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable upon reaction with yet another moiety.

[0145] Exemplary DR5 Agonists 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 (HexaBody™-DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals) and MM201-b (Merrimack Pharmaceuticals). In some embodiments, the DR5 agonist is a DR5 binding polypeptide. In some embodiments, the DR5 binding polypeptides provided herein are multivalent. In some embodiments, the DR5 binding polypeptides provided herein are at least tetravalent.

[0146] 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 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.

[0147] In some embodiments, a DR5 binding polypeptide comprises at least one VHH domain that binds to DR5 and an Fc region. In some embodiments, a 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, thus forming a dimer and doubling 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 divalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization, such that the DR5 binding polypeptide is a tetravalent dimer under such conditions.

[0148] 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.

[0149] 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 at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the VHH-linker-VHH portion of the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the Fc comprises a hinge. In some such embodiments, the Fc comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the DR5 binding polypeptide comprises an amino acid sequence at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:7 comprising two VHH domains and an Fc region. In some embodiments, the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7 comprising two VHH domains and an Fc region. In some embodiments, the DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:7. A DR5 binding polypeptide consisting of the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:7 lacking the terminal lysine, may be referred to as INBRX-109.

[0150] In some embodiments, the VHH domain that binds DR5 may be humanized. Humanized antibodies (such as sdAbs or VHH-containing polypeptides) are useful as therapeutic molecules because they reduce or eliminate human immune responses to non-human antibodies that may result in immune responses to antibody therapeutics and reduce the efficacy of the therapeutics. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0151] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and described, e.g., in Riechmann et al., (1988) Nature 332:323-329, Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033, U.S. Pat. No. 5,821,337, U.S. Pat. No. 7,527,791, U.S. Pat. No. 6,982,321, and U.S. Pat. No. 7,087,409, Kashmiri et al., (2005) Methods 36:25-34, Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"), Dall'Acqua, et al. et al., (2005) Methods 36:43-60 (describing "FR shuffling"), as well as Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing a "guided selection" approach to FR shuffling).

[0152] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296), framework regions derived from consensus sequences of human antibodies of particular subgroups 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, 151:2623), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633), and framework regions obtained from screening of FR libraries (see, e.g., Baca et al., (1997) J. Biol. Chem. 272: 10678-10684, and Rosok et al., (1996) J. Biol. Chem. 271:22611-22618). Typically, the FR regions of a VHH are replaced with human FR regions to generate a humanized VHH. In some embodiments, certain FR residues of the human FR are replaced to improve one or more properties of the humanized VHH. A VHH domain having such replaced residues is also referred to herein as "humanized".

[0153] In various embodiments, the Fc region comprised in the DR5 binding polypeptide is a human Fc region or is derived from a human Fc region.

[0154] In some embodiments, the Fc region included in the DR5 binding polypeptide is derived from a human Fc region and includes a three amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235, referred to herein as "Fc xELL". Fc xELL polypeptides do not bind FcγR and are therefore referred to as "effector silent" or "effector null", however, in some embodiments, the xELL Fc region binds to FcRn, with associated transcytosis associated with extended half-life and FcRn mediated recycling. In some embodiments, the Fc region is a human IgG1 xELL Fc region.

[0155] Exemplary PLK1 Inhibitors Provided herein are methods of treating cancer, comprising administering a PLK1 inhibitor. In some embodiments, the PLK1 inhibitor is a small molecule. In some embodiments, the PLK1 inhibitor is an RNAi. In some embodiments, the PLK1 inhibitor is onvansertib, volasertib, rigosertib, BI2536 (Boehringer Ingelheim), N-[[4-[(6-chloro-3-pyridinyl)methoxy]-3-methoxyphenyl]methyl]-3,4-dimethoxybenzeneethanamine hydrochloride (SBE 13 HCl), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), CYC140 (Cyclacel), TKM-080301 (TKM-PLK1; Arbutus Biopharma), TAK-960 (Takeda Pharmaceutical Co., Ltd.), poloxin, poloxin-2HT, RO3280 (CAS number 1062243-51-9), 2-cyano-2-[3-ethyl-4-oxo-5-[[3-(2-pyrrolidin-1-ylethyl)anilino]methyl]-1,3-thiazolidin-2-yl]-N-(2,2,2-trifluoroethyl)acetamide (ZK-thiazolidinone), cyclaporin 9 (CAS number 40533-25-3), 5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-[[2-(trifluoromethyl)phenyl]methoxy]-2-thiophenecarboxamide (GW 843682X), HMN-214 (CAS No. 173529-46-9), or HMN-176 (CAS No. 173529-10-7). In some embodiments, the PLK1 inhibitor is onvansertib, volasertib, rigosertib, BI2536 (Boehringer Ingelheim), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), CYC140 (Cyclacel), TKM-080301 (TKM-PLK1; Arbutus Biopharma), or TAK-960 (Takeda Pharmaceutical Co., Ltd.).

[0156] In some embodiments, the PLK1 inhibitor is onvansertib. Onvansertib (also known as PCM-075 or NMS-1286937) has the structure: [ka] or a pharmaceutically acceptable salt or hydrate thereof. See, for example, U.S. Patent No. 8,927,530. Onvansertib is specific for PLK1 and has potent in vitro and in vivo antitumor activity in models of both solid and hematological malignancies.

[0157] In some embodiments, the PLK1 inhibitor is volasertib. Volasertib has the structure: [ka] or a pharma- ceutically acceptable salt or hydrate thereof. See, e.g., WO 04 / 076454 and WO 07 / 090844.

[0158] In some embodiments, the PLK1 inhibitor is rigosertib. Rigosertib has the structure: [ka] or a pharma- ceutically acceptable salt or hydrate thereof. See, for example, U.S. Patent No. 7,598,232 (compound 4).

[0159] In some embodiments, the PLK1 inhibitor is BI2536. BI2536 has the structure: [ka] or a pharma- ceutically acceptable salt or hydrate thereof. See, e.g., Steegmaier et al., Current Biology, 17: 316-322 (2007).

[0160] In some embodiments, the PLK1 inhibitor is MLN0905. MLN0905 has the structure: [ka] or a pharma- ceutically acceptable salt or hydrate thereof. See, e.g., Mol Cancer Ther, 11: 2045-53 (2012).

[0161] In some embodiments, the PLK1 inhibitor is TAK-960. TAK-960 has the structure: [ka] or a pharma- ceutically acceptable salt or hydrate thereof. See, e.g., Mol Cancer Ther, 11: 700-9 (2012).

[0162] In some embodiments, the PLK1 inhibitor is GSK461364. GSK461364 has the structure: [ka] or a pharma- ceutical acceptable salt or hydrate thereof. See, for example, Clin Cancer Res. 17(10):3420-30 (2011).

[0163] In some embodiments, the PLK1 inhibitor is CYC140. In some embodiments, the PLK1 inhibitor, or a pharma- ceutically acceptable salt or hydrate thereof, has the structure: [ka] See, for example, WO 2009 / 040556.

[0164] In some embodiments, the PLK1 inhibitor is TKM-080301. TKM-080301 is a lipid nanoparticle (LNP) formulation that contains four lipids and synthetic double-stranded siRNA against human PLK1 mRNA. Synthetic siRNA is a duplex of complementary RNA oligonucleotides designed to achieve post-transcriptional gene silencing by RNA interference mechanism. See, for example, Oncologist, 24(6):747-e218 (2019), WO 2008 / 342535.

[0165] Exemplary CDK Inhibitors Provided herein are methods of treating cancer, including administering a CDK inhibitor, such as a CDK9 inhibitor. In some embodiments, the CDK inhibitor is a small molecule. In some embodiments, the CDK inhibitor is flavopiridol (Tolero Pharmaceuticals), seliciclib (roscovitine / CYC202), dinaciclib (Merck), atubeciclib (Bayer), enitociclib (Vincerx Pharma), AZD4573 (AstraZeneca), i-CDK9, or NVP-2.

[0166] In some embodiments, the CDK inhibitor is flavopiridol. Flavopiridol (also known as L86-8275, alvocidib, NSC 649890 or HMR-1275; Tolero Pharmaceuticals) has the structure: [ka] or a pharma- ceutical acceptable salt or hydrate thereof. Flavopiridol is a potent selective inhibitor of CDK9, and has antitumor activity against various tumor cell lines, such as human lung and breast cancer, and also inhibits tumor growth in xenograft models. See, for example, Anshabo, et al., Frontiers in Oncology, 11 (2021).

[0167] In some embodiments, the CDK inhibitor is seliciclib (also known as roscovitine or CYC202). Seliciclib has the structure: [ka] or a pharma- ceutical acceptable salt or hydrate thereof. See, for example, Anshabo, et al., Frontiers in Oncology, 11 (2021).

[0168] In some embodiments, the CDK inhibitor is dinaciclib (also known as SCH 727965; Merck). Dinaciclib has the structure: [ka] or a pharma- ceutical acceptable salt or hydrate thereof. See, e.g., Anshabo, et al., Frontiers in Oncology, 11 (2021).

[0169] In some embodiments, the CDK inhibitor is atubeciclib (also known as BAY1143572; Bayer). Atubeciclib has the structure: [ka] The present invention relates to a potent and highly selective inhibitor of transcription elongation factor b (PTEF-b), which is composed of CDK9 and cyclin-T (CycT), or a pharma- ceutically acceptable salt or hydrate thereof. See, for example, Anshabo, et al., Frontiers in Oncology, 11 (2021).

[0170] In some embodiments, the CDK inhibitor is enitociclib (also known as BAY1251152 or VIP152; Vincerc Pharma). Enitociclib has the structure: [ka] or a pharma- ceutical acceptable salt or hydrate thereof. See, for example, Anshabo, et al., Frontiers in Oncology, 11 (2021).

[0171] In some embodiments, the CDK inhibitor is AZD4573 (AstraZeneca). AZD4573 has the structure: [ka] or a pharma- ceutical acceptable salt or hydrate thereof. See, for example, Anshabo, et al., Frontiers in Oncology, 11 (2021).

[0172] In some embodiments, the CDK inhibitor is i-CDK9. i-CDK9 has the structure: [ka] i-CDK9 is a CDK inhibitor having 600-fold selectivity for CDK9 over other CDKs, or a pharma- ceutically acceptable salt or hydrate thereof. i-CDK9 inhibits dual specificity tyrosine phosphorylation-regulated kinases (DYRK) 1A and 1B, but with lower potency compared to CDK9. See, for example, Anshabo, et al., Frontiers in Oncology, 11 (2021).

[0173] In some embodiments, the CDK inhibitor is NVP-2. NVP-2 is an ATP-competitive aminopyrimidine inhibitor, or a pharma- ceutically acceptable salt or hydrate thereof, which is a chemical analog of i-CDK9 and has the structure: [ka] See, for example, Nat Chem Biol 14, 163-170 (2018).

[0174] Polypeptide Expression and Production Nucleic acid molecules are provided that comprise a polynucleotide encoding a DR5 binding polypeptide. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of the DR5 binding polypeptide, and the leader sequence is typically cleaved so that it is not present in the secreted polypeptide. The leader sequence may be the native heavy chain (or VHH) leader sequence, or may be another heterologous leader sequence.

[0175] The nucleic acid molecule 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.

[0176] Vectors are provided that contain a nucleic acid encoding a DR5 binding polypeptide as described herein. 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 for expression of the polypeptide in a desired cell type, such as CHO cells 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).

[0177] In some embodiments, DR5 binding polypeptides may 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 may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express the polypeptide include, but are not limited to, COS cells, including COS7 cells, 293 cells, including 293-6E cells, CHO cells, including CHO-S, DG44, Lec13 CHO cells, and FUT8 CHO cells, PER.C6™ cells (Crucell), and NSO cells. In some embodiments, DR5 binding polypeptides may be expressed in yeast. See, for example, US Patent Application Publication No. 2006 / 0270045. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to a polypeptide. For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0178] Introduction of one or more nucleic acids (e.g., vectors) into a desired host cell 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, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3 rd ed. Cold Spring Harbor Laboratory Press (2001). The nucleic acid may be transiently or stably transfected into the desired host cell according to any suitable method.

[0179] Also provided is a host cell comprising any of the nucleic acids or vectors described herein. In some embodiments, a host cell is provided that expresses a DR5 binding polypeptide described herein. The DR5 binding polypeptide expressed in the host cell can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrix or hydrophobic interaction chromatography. Suitable affinity ligands include agents that bind to the ROR1 ECD and Fc region. For example, Protein A, Protein G, Protein A / G, or antibody affinity columns can be used to purify DR5 binding polypeptides comprising an Fc region by binding to the Fc region. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be suitable for purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange and / or cation exchange chromatography) may also be suitable for purifying some polypeptides, such as antibodies. Mixed mode chromatography (e.g., reversed phase / anion exchange, reversed phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be suitable for purifying some polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0180] In some embodiments, the DR5 binding polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009), Spirin, Trends Biotechnol. 22: 538-45 (2004), and Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0181] In some embodiments, a DR5 binding polypeptide produced by the above method is provided. In some embodiments, the DR5 binding polypeptide is produced in a host cell. In some embodiments, the DR5 binding polypeptide is produced in a cell-free system. In some embodiments, the DR5 binding polypeptide is purified. In some embodiments, a cell culture medium comprising the DR5 binding polypeptide is provided.

[0182] In some embodiments, a composition is provided that comprises the antibody produced by the above method. In some embodiments, the composition comprises a DR5 binding polypeptide produced in a host cell. In some embodiments, the composition comprises a DR5 binding polypeptide produced in a cell-free system. In some embodiments, the composition comprises a purified DR5 binding polypeptide.

[0183] Pharmaceutical Compositions In some embodiments, compositions comprising a DR5 agonist, a PLK1 inhibitor and / or a CDK inhibitor are provided in a formulation comprising a wide variety of pharma- ceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7 th ed., Lippencott Williams and Wilkins (2004), Kibbe et al., Handbook of Pharmaceutical Excipients, 3 rd (See, e.g., J.D., Pharmaceutical Press (2000)). A variety of pharma- ceutically acceptable carriers, including vehicles, adjuvants, and diluents, are available. In addition, a variety of pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are also available.

[0184] In some embodiments, INBRX-109 is provided in a formulation comprising 50 mg / mL INBRX-109, 10 mM histidine HCl, 8% (w / v) sucrose, 0.2% (w / v) poloxamer 88, pH 6.0.

[0185] Exemplary Methods of Treating Cancer with DR5 Agonists and PLK1 Inhibitors In some embodiments, a method of treating cancer in an individual is provided comprising administering a DR5 agonist and a PLK1 inhibitor.

[0186] In some embodiments, the method comprises administering an effective amount of a DR5 agonist and a PLK1 inhibitor to an individual. Such a method of treatment may be in humans or animals. In some embodiments, a method of treating a human is provided. Non-limiting exemplary cancers that may be treated with the combination of a DR5 agonist and a PLK1 inhibitor provided herein include adrenal gland 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, colorectal cancer (colon cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, Glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal cell carcinoma, laryngeal cancer, leukemia, liver cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, adenocarcinoma, uterine or endometrial cancer, urinary system 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-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, These include cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorders (PTLDs), and abnormal blood vessel growth associated with phacomas, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0187] The DR5 agonist and the PLK1 inhibitor 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 the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, 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 the DR5 agonist and / or the PLK1 inhibitor is administered to the subject daily, twice weekly, weekly, biweekly, monthly, and the like. An effective dose of the DR5 agonist and / or the PLK1 inhibitor is administered to the subject at least once. In some embodiments, an effective dose of the DR5 agonist and / or the PLK1 inhibitor may be administered multiple times, including multiple times over at least one month, at least six months, or at least one year.

[0188] In some embodiments, the DR5 agonist is administered in an amount effective to treat (including prevent) cancer. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated. In general, the DR5 binding polypeptide may 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.

[0189] In some embodiments, the PLK1 inhibitor (or a pharma- ceutically acceptable salt or hydrate thereof) is administered at a dose of, for example, 10 mg / m 2 ~500mg / m 2 , 10 mg / m 2 ~300mg / m 2 or 10 mg / m2 ~200mg / m 2 Contains 1 mg / m 2 ~1000mg / m 2 In some embodiments, the PLK1 inhibitor (or a pharma- ceutically acceptable salt or hydrate thereof) is administered at a dose of 1 mg to 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.

[0190] In some embodiments, onvansertib is administered at a dose of 2 mg / m 2 ~100mg / m 2 In some embodiments, volasertib is administered at a dose of 10 mg to 500 mg. In some embodiments, rigosertib is administered at a dose of 10 mg to 1000 mg.

[0191] In some embodiments, the therapeutic agent may be administered in vivo by various routes, including, but not limited to, oral, intramuscular, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Depending on the intended use, the appropriate formulation and route of administration may be selected.

[0192] In some embodiments, the DR5 agonist and the PLK1 inhibitor are administered separately. In some embodiments, the DR5 agonist and the PLK1 inhibitor are administered sequentially. In some embodiments, at least one dose of the DR5 agonist is administered before the PLK1 inhibitor. In some embodiments, at least one dose of the DR5 agonist is administered after the PLK1 inhibitor.

[0193] In some embodiments, the DR5 agonist and the PLK1 inhibitor are administered in combination.

[0194] In some embodiments, the DR5 agonist and the PLK1 inhibitor act synergistically. In some embodiments, the synergy is determined in an in vitro cell viability assay. In some embodiments, administration of the DR5 agonist and the PLK1 inhibitor results in a synergistic effect compared to administration of each agent alone.

[0195] In some embodiments, there is provided a DR5 agonist for use in a method of treating cancer, the method comprising administering the DR5 agonist in combination with a PLK1 inhibitor.

[0196] In some embodiments, there is provided the use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is administered in combination with a PLK1 inhibitor.

[0197] Exemplary methods of treating cancer with DR5 agonists and CDK inhibitors In some embodiments, a method of treating cancer in an individual is provided comprising administering a DR5 agonist and a CDK inhibitor, such as a CDK9 inhibitor.

[0198] In some embodiments, the method comprises administering an effective amount of a DR5 agonist and a CDK inhibitor to an individual. Such a method of treatment may be in a human or an animal. In some embodiments, a method of treating a human is provided. Non-limiting exemplary cancers that may be treated with a combination of a DR5 agonist and a CDK inhibitor provided herein include adrenal gland 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, colorectal cancer (colon cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glial cancer, and glioma. blastoma, liver cancer, hepatocellular carcinoma, 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 (lips, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, adenocarcinoma, uterine or endometrial cancer, urinary system 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-dividing cell NHL, bulky disease NHL, mantle cell lymphoma, These include cell lymphoma, AIDS-related lymphoma, Waldenstrom's macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorders (PTLDs), and abnormal blood vessel growth associated with phacomas, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0199] The DR5 agonist and CDK inhibitor 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 the attending physician, based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health of the subject being treated, 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 the DR5 agonist and / or CDK inhibitor is administered to the subject daily, twice weekly, weekly, biweekly, monthly, and the like. An effective dose of the DR5 agonist and / or CDK inhibitor is administered to the subject at least once. In some embodiments, an effective dose of the DR5 agonist and / or CDK inhibitor may be administered multiple times, including multiple times over at least one month, at least six months, or at least one year.

[0200] In some embodiments, the DR5 agonist is administered in an amount effective to treat (including prevent) cancer. The therapeutically effective amount typically depends on the weight of the subject being treated, the physical or health condition of the subject, the extent of the condition being treated, or the age of the subject being treated. In general, the DR5 binding polypeptide may 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.

[0201] In some embodiments, the CDK inhibitor (or a pharma- ceutically acceptable salt or hydrate thereof) is administered at a dose of, for example, 10 mg / m 2 ~500mg / m 2 , 10 mg / m 2 ~300mg / m 2 or 10 mg / m2 ~200mg / m 2 Contains 1 mg / m 2 ~1000mg / m 2 In some embodiments, the CDK inhibitor (or a pharma- ceutically acceptable salt or hydrate thereof) is administered in a dose of 1 mg to 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.

[0202] In some embodiments, flavopiridol is administered at a dose of 2 mg / m 2 ~100mg / m 2 In some embodiments, seliciclib is administered at a dose of 10 mg to 500 mg. In some embodiments, dinaciclib is administered at a dose of 10 mg to 2000 mg. In some embodiments, atubeciclib is administered at a dose of 10 mg to 1000 mg. In some embodiments, enitociclib is administered at a dose of 10 mg to 500 mg. In some embodiments, AZD4573 is administered at a dose of 1 mg to 100 mg.

[0203] In some embodiments, the therapeutic agent may be administered in vivo by various routes, including, but not limited to, oral, intramuscular, intravenous, intraarterial, parenteral, intraperitoneal, or subcutaneous. Depending on the intended use, the appropriate formulation and route of administration may be selected.

[0204] In some embodiments, the DR5 agonist and the CDK inhibitor are administered separately. In some embodiments, the DR5 agonist and the CDK inhibitor are administered sequentially. In some embodiments, at least one dose of the DR5 agonist is administered before the CDK inhibitor. In some embodiments, at least one dose of the DR5 agonist is administered after the CDK inhibitor.

[0205] In some embodiments, the DR5 agonist and the CDK inhibitor are administered in combination.

[0206] In some embodiments, the DR5 agonist and the CDK inhibitor act synergistically. In some embodiments, the synergy is determined in an in vitro cell viability assay. In some embodiments, administration of the DR5 agonist and the CDK inhibitor results in a synergistic effect compared to administration of each agent alone.

[0207] In some embodiments there is provided a DR5 agonist for use in a method of treating cancer, the method comprising administering the DR5 agonist in combination with a CDK inhibitor.

[0208] In some embodiments there is provided the use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is administered in combination with a CDK inhibitor.

[0209] kit Also provided are articles of manufacture and kits comprising any of the DR5 agonists and / or PLK1 inhibitors provided herein and suitable packaging. In some embodiments, the invention comprises a kit comprising (i) a formulation comprising a DR5 agonist, (ii) a formulation comprising a PLK1 inhibitor, and (iii) instructions for administering the formulation to an individual using the kit. In some embodiments, the invention comprises a kit comprising (i) a formulation comprising a DR5 agonist, and (ii) instructions for administering the formulation in combination with a PLK1 inhibitor to an individual using the kit. In some embodiments, the invention comprises a kit comprising (i) a formulation comprising a PLK1 inhibitor, and (ii) instructions for administering the formulation in combination with a DR5 agonist to an individual using the kit.

[0210] Also provided are articles of manufacture and kits comprising any of the DR5 agonists and / or CDK inhibitors provided herein and suitable packaging. In some embodiments, the invention comprises a kit comprising (i) a formulation comprising a DR5 agonist, (ii) a formulation comprising a CDK inhibitor, and (iii) instructions for administering the formulation to an individual using the kit. In some embodiments, the invention comprises a kit comprising (i) a formulation comprising a DR5 agonist, and (ii) instructions for administering the formulation in combination with a CDK inhibitor to an individual using the kit. In some embodiments, the invention comprises a kit comprising (i) a formulation comprising a CDK inhibitor, and (ii) instructions for administering the formulation in combination with a DR5 agonist to an individual using the kit.

[0211] Suitable packaging for the compositions described herein are known in the art and include, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture can be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms can be stored in suitable packaging in single or multiple unit dosage forms and can also be further sterilized and sealed. The instructions provided in the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions for use of the DR5 agonist, PLK1 inhibitor and / or CDK inhibitor generally include information regarding dosage, administration schedule, and route of administration for the intended therapeutic or industrial use. The kits can further include instructions for selecting an appropriate individual treatment.

[0212] The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Kits may also be provided that contain a sufficient dosage of the molecules disclosed herein to provide effective treatment to an individual for an extended period of time, such as any approximate period of 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more. Kits may also contain multiple unit doses of the molecules and instructions for use, and may be packaged in sufficient quantities for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. In some embodiments, the kits include a dried (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to generally form a stable aqueous solution of the DR5 agonist. EXAMPLES

[0213] The examples discussed below are intended to be purely illustrative of the present invention and should not be considered as limiting the present invention in any way. These examples are not intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0214] Example 1: Combination Activity of INBRX-109 and Onvansertib The combination of INBRX-109 and onvansertib was tested in various cancer cell lines to determine cytotoxicity against cancer cells.

[0215] Assay protocol On day 1, cells were seeded as follows: Monolayer cultures of each cell line were harvested for compound screening as detailed below. Culture medium was aspirated and cells were washed once with PBS. Accutase was added and flasks were incubated at 37°C until cells detached. After adding an equal volume of complete medium to quench the Accutase, cells were pipetted up and down several times to generate a uniform single cell suspension. Cell density and viability were determined by trypan blue using a TC20 Automated Cell Counter. Experimental cells were seeded at 0.17 × 10 in Eagle's Minimum Essential Medium (EMEM) / 10% FBS / Anti-Anti medium (complete EMEM). 6 The cells were resuspended to a concentration of 1000 cells / mL and seeded into the inner wells of a 384-well luminescence plate at 15 μL / well (final 2500 cells / well). Each cell line was plated in duplicate on separate plates. After filling the outer wells with 50 μL of PBS, the plates were incubated overnight in a humidified, temperature-controlled 37°C tissue culture incubator with 5% CO2 for 16 hours.

[0216] On day 2, the following test and control articles were prepared: onvansertib, INBRX-109, and staurosporine.

[0217] Onvansertib: A 10 mg stock of onvansertib was purchased from Selleck Chemicals and resuspended to 10 mM in DMSO. It was then aliquoted and stored at -80°C. Aliquots were thawed immediately prior to dilution and use in the assay. A 500x master plate of serial dilutions (6-point 5-fold dilutions in 100% DMSO starting at 500 μM + DMSO only control) was prepared and mixed gently with a pipette. A 100x dilution was made from the 500x plate in complete medium (EMEM) to generate a 5x working dilution plate of onvansertib.

[0218] INBRX-109: The INBRX-109 assay concentration range was chosen to include the minimum and maximum activity seen in previous cytotoxicity assays with several cancer cell lines, with 1 nM defined as the maximum effective concentration. A 50x master plate of INBRX-109 serial dilutions (6-point 10-fold dilutions in complete EMEM starting at 500 nM + complete EMEM only control) was prepared and mixed gently with a pipette. To generate a 5x standard dilution plate of INBRX-109, each well of the 50x master plate was diluted 10-fold in complete EMEM.

[0219] Staurosporine: Staurosporine was included in the assay as a positive control for cytotoxicity. 10 mM DMSO stocks purchased from the manufacturer were thawed, aliquoted, and stored at -80°C. Aliquots were thawed immediately prior to dilution and use in the assay. 5x staurosporine standard dilutions (100 μM) were made by adding 5 μL of 10 mM staurosporine stock solution to 495 μL of complete EMEM followed by thorough mixing.

[0220] Test and control articles were also added on day 2. Onvansertib small molecule standard dilutions (5x at 5μL), INBRX-109 standard dilutions (5x at 5μL) or staurosporine positive control (5x at 5μL) were added to each experimental well. Onvansertib titrations were performed across the plate and INBRX-109 titrations were performed down the plate to obtain 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×g for 1 minute before being incubated at 37° C. in a humidity-controlled tissue culture incubator (5% CO2) for 48 hours.

[0221] Viability measurements were performed on day 4. After equilibrating the plates to room temperature for 10 minutes, 25 μL of CellTiter-Glo 2.0™ was added to each well. The plates were spun at 400×g for 1 minute, then covered and incubated in the dark at room temperature for 10 minutes. After removing any visible bubbles with 100% ethanol vapor, luminescence (RLU) was read on a Spectra Max M5e plate reader using a 384-well opaque plate setting and SoftMaxPro v5.4 software with an integration time of 50 ms. To determine the effect of test articles on cell viability, raw RLU values ​​were exported to Excel and viability (%) was calculated as a percentage of vehicle control (0.5% DMSO in EMEM), where vehicle control is set to 100%. Data was graphed in GraphPad Prism 9.

[0222] result Figures 1A-1F show the results of a titration experiment in which various cancer cell lines were contacted with different concentrations of INBRX-109 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM or 10 nM) and onvansertib (0 nM, 0.32 nM, 1.6 nM, 8 nM, 40 nM, 200 nM or 1000 nM). The percent viability of the cancer cells is shown on the y-axis of each graph. Results are shown for the cancer cell lines HT-29, LS174T, SW620, SW837, SW1463 and LS411N.

[0223] Figures 2A-F show the results of an INBRX-109 titration experiment in which various cancer cell lines were contacted with different concentrations of INBRX-109 (0.0001 nM, 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, or 10 nM), either alone or in combination with 200 nM onvansertib. The percent viability of cancer cells is shown on the y-axis of each graph. Results are shown for cancer cell lines HT-29, LS174T, SW620, SW837, SW1463, and LS411N. The dotted line labeled "Cpd alone" indicates the percent viability of cancer cells treated with 200 nM onvansertib alone.

[0224] Figures 3A-F show the results of an onvansertib titration experiment in which various cancer cell lines were contacted with different concentrations of onvansertib (0.32 nM, 1.6 nM, 8 nM, 40 nM, 200 nM or 1000 nM), either alone or in combination with 1 nM INBRX-109. The percent viability of the cancer cells is shown on the y-axis of each graph. Results are shown for cancer cell lines HT-29, LS174T, SW620, SW837, SW1463 and LS411N. The dotted line labeled "Ab alone" indicates the percent viability of cancer cells treated with INBRX-109 alone.

[0225] Table 1 shows the EC of INBRX-109 titration curves with and without 200 nM ombansertib. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0226] TIFF2025512950000018.tif50170

[0227] Table 2 shows the EC values ​​of the onvansertib titration curves with and without 1 nM INBRX-109. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0228] TIFF2025512950000019.tif55170

[0229] The combination of INBRX-109 and onvansertib demonstrated increased cell killing in all cancer cell lines tested in this screen. In general, greater benefit was observed at onvansertib concentrations higher than 40 nM. At these concentrations, the EC 50 EC of INBRX-109 compared to 50 The combination resulted in a synergistic effect, as evidenced by a shift in the β-terminal region of the β-terminal region of the cytoplasmic endothelial cell (C-Tc) ...

[0230] Additional cancer cell lines were assayed for cell killing in the presence of 1 nM INBRX-109 alone, onvansertib alone, or 1 nM INBRX-109 in combination with different concentrations of onvansertib (274 pM, 823 pM, 2.47 nM, 7.41 nM, 22.22 nM, 66.67 nM, 200 nM, 600 nM, and 1.8 μM) substantially as described above. Tables 3 and 4 and Figures 4-8 show the maximum cytotoxicity of 1 nM INBRX-109 alone, and the EC50 values ​​of the onvansertib titration curves with and without 1 nM INBRX-109. 50 The results for the panel of colon cancer cell lines are shown in Table 3 and Figures 4 to 6, and the results for the panel of pancreatic cell lines are shown in Table 4 and Figures 7 and 8.

[0231] TIFF2025512950000020.tif178170

[0232] TIFF2025512950000021.tif121170

[0233] The combination of INBRX-109 and onvansertib demonstrated increased cell killing in the cancer cell lines tested in this screen compared to either agent alone, and in some cases, the addition of 1 nM INBRX-109 increased EC 50 has dropped significantly.

[0234] This data suggests that the combination of a DR5 agonist, such as INBRX-109, with a PLK1 inhibitor, such as onvansertib, results in improved or synergistic cancer cell killing compared to either drug alone.

[0235] Example 2: Combination activity of INBRX-109 with CDK9 inhibitors Additional assays were performed to determine cytotoxicity against cancer cells by testing combinations of INBRX-109 with cyclin-dependent kinase 9 (CDK9) inhibitors (dinaciclib, NVP-2, flavopiridol, and enitociclib) or downstream inhibitors of MCL-1 (AZD5991) in various cell lines.

[0236] Assay protocol On day 1, cells were seeded as follows: Monolayer cultures of each cell line (chondrosarcoma: CAL-78, OUMS-27, SW1353, and H-EMC-SS, colon cancer: SW620, and synovial sarcoma: HS-SY-II) were harvested for compound screening. Culture medium was aspirated and cells were washed once with PBS. Accutase was added and flasks were incubated at 37°C until cells detached. After adding an equal volume of complete medium to quench the Accutase, cells were pipetted up and down several times to generate a uniform single cell suspension. Cell density and viability were determined by trypan blue using a TC20 Automated Cell Counter. Experimental cells were seeded at 0.17 × 10 in Eagle's Minimum Essential Medium (EMEM) / 10% FBS / Anti-Anti medium (complete EMEM). 6 The cells were resuspended to a concentration of 1000 cells / mL and seeded into the inner wells of a 384-well luminescence plate at 15 mL / well (final 2500 cells / well). Each cell line was plated in duplicate on separate plates. After filling the outer wells with 50 mL of PBS, the plates were incubated overnight in a humidified, temperature-controlled 37°C tissue culture incubator with 5% CO2 for 16 hours.

[0237] On day 2, the following test articles and INBRX-109 were prepared as follows: Dinaciclib (purchased from MedChemExpress): Prepare a 5-fold standard serial dilution plate in complete medium (EMEM) (6-point 5-fold dilutions starting from 312.5 µM with a final concentration range of 625 nM to 0.2 nM). NVP-2 (purchased from MedChemExpress): Prepare a 5-fold standard serial dilution plate in complete medium (EMEM) (6-point 5-fold dilutions starting from 312.5 µM with a final concentration range of 625 nM to 0.2 nM). Flavopiridol (purchased from MedChemExpress): Prepare a 5-fold standard serial dilution plate in complete medium (EMEM) (6-point 5-fold dilutions starting from 312.5 µM with a final concentration range of 625 nM - 0.2 nM). Enitociclib (purchased from MedChemExpress): Prepare a 5-fold standard serial dilution plate in complete medium (EMEM) (6-point 5-fold dilutions starting from 312.5 µM with a final concentration range of 625 nM to 0.2 nM). AZD-5991 (purchased from MedChemExpress): Prepare a 5-fold standard serial dilution plate in complete medium (EMEM) (6-point 5-fold dilutions starting from 5 mM with a final concentration range of 10 µM to 3.2 nM). INBRX-109: 5-fold standard serial dilution plate in complete medium (EMEM) (6-point 10-fold dilutions starting from 50 nM with final concentration range 10 nM - 0.0001 nM).

[0238] Test articles were added to the cells as follows: small molecule compound standard dilutions (5x at 5μL), INBRX-109 standard dilutions (5x at 5μL) or medium alone were added to each experimental well. Small molecule compound titrations were performed across the plate and INBRX-109 titrations were performed down the plate to obtain 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×g for 1 minute and then incubated at 37° C. in a humidity-controlled tissue culture incubator (5% CO2) for 48 hours.

[0239] Viability measurements were performed briefly after incubation with the test article. After equilibrating the plate to room temperature for 10 minutes, 25 μL of CellTiter-Glo 2.0™ was added to each well. The plate was spun at 400×g for 1 minute, then covered and incubated in the dark at room temperature for 10 minutes. After removing any visible bubbles with 100% ethanol vapor, luminescence (RLU) was read on a Spectra Max M5e plate reader using a 384-well opaque plate setting and SoftMax™ Pro v5.4 software with an integration time of 50 ms. To determine the effect of the test article on cell viability, raw RLU values ​​were exported to Excel and viability (%) was calculated as a percentage of vehicle control (0.5% DMSO in EMEM), where vehicle control is set to 100%. Data was graphed in GraphPad Prism 9.

[0240] result The results of these titration experiments are shown in Figures 9A-9E (INBRX-109 and dinaciclib titrated), 10A-10E (INBRX-109 titrated alone or with 25 nM dinaciclib), 11A-11E (dinaciclib titrated alone or with 1 nM INBRX-109), 12A-12E (INBRX-109 and NVP-2 titrated), 13A-13E (INBRX-109 titrated alone or with 125 nM NVP-2), 14A-14E (NVP-2 titrated alone or with 1 nM 15A-15F (INBRX-109 and flavopiridol titrated), 16A-16F (INBRX-109 titrated alone or with 125 nM flavopiridol), 17A-17F (flavopiridol titrated alone or with 1 nM INBRX-109), 18A-18F (INBRX-109 and enitociclib titrated), 19A-19F (INBRX-109 titrated alone or with 125 nM enitociclib), 20A-20F (enitociclib titrated alone or with 1 nM 21A-21F (titration of INBRX-109 and AZD-5991), 22A-22F (titration of INBRX-109 alone or with 2 nM AZD-5991) and 23A-23F (titration of AZD-5991 alone or with 1 nM INBRX-109). The dotted line labeled "Ab alone" indicates the % viability of cancer cells treated with 1 nM INBRX-109 alone and the dotted line labeled "Cpd alone" indicates the % viability of cancer cells treated with the indicated small molecule inhibitor.

[0241] Table 5 shows the EC of INBRX-109 titration curves with and without 25 nM dinaciclib. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0242] TIFF2025512950000022.tif50170

[0243] Table 6 shows the EC of the dinaciclib titration curve with and without 1 nM INBRX-109. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0244] TIFF2025512950000023.tif51170

[0245] Table 7 shows the EC of INBRX-109 titration curves with and without 25 nM NVP-2. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0246] TIFF2025512950000024.tif50170

[0247] Table 8 shows the EC values ​​of NVP-2 titration curves with and without 1 nM INBRX-109. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0248] TIFF2025512950000025.tif50170

[0249] Table 9 shows the EC values ​​of INBRX-109 titration curves with and without 25 nM flavopiridol. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0250] TIFF2025512950000026.tif55170

[0251] Table 10 shows the EC of the flavopiridol titration curve with and without 1 nM INBRX-109. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0252] TIFF2025512950000027.tif55170

[0253] Table 11 shows the EC of INBRX-109 titration curves with and without 125 nM enitociclib. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0254] TIFF2025512950000028.tif55170

[0255] Table 12 shows the EC of the enitociclib titration curve with and without 1 nM INBRX-109. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0256] TIFF2025512950000029.tif55170

[0257] Table 13 shows the EC of INBRX-109 titration curve with and without 2 μM AZD-5991. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0258] TIFF2025512950000030.tif54170

[0259] Table 14 shows the EC of AZD-5991 titration curve with and without 1 nM INBRX-109. 50 Values ​​are shown. Maximum cytotoxicity was calculated by subtracting the % viability from 100.

[0260] TIFF2025512950000031.tif55170

[0261] Combinations of INBRX-109 with each of the CDK9 inhibitors tested, dinaciclib, NVP-2, flavopiridol, and enitociclib, showed increased cell killing in almost all cancer cell lines tested in these screens. Combinations of INBRX-109 with AZD-5991, an inhibitor of MCL-1 (a downstream target of CDK9), also showed increased cell killing in a number of cell lines. Greater benefits were generally observed at concentrations greater than 25 nM for dinaciclib and NVP-2, 125 nM or greater for flavopiridol and enitociclib, and greater than 0.4 μM for AZD-5991. At these concentrations, the combinations produced synergistic effects, as evidenced by the shift in the killing curves of the combination of INBRX-109 with CDK9 and downstream MCL-1 inhibitors compared to the killing curves of INBRX-109 treatment alone. See, for example, Figures 9-23 and Tables 5-14. Furthermore, the combination resulted in a decrease in total cell viability compared to treatment with either drug alone.

[0262] The present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. The above-described embodiments are therefore to be considered in all respects as illustrative and not limiting of the present disclosure. The scope of the present disclosure is therefore defined by the appended claims, rather than the above detailed description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

[0263] TIFF2025512950000032.tif181170TIFF2025512950000033.tif136170

Claims

1. A pharmaceutical composition for treating cancer in a subject, comprising a cell death receptor 5 (DR5) agonist, wherein the pharmaceutical composition is administered to the subject in combination with a polo-like kinase 1 (PLK1) inhibitor.

2. A pharmaceutical composition for treating cancer in a subject, comprising a polo-like kinase 1 (PLK1) inhibitor, wherein the pharmaceutical composition is administered to the subject in combination with a cell death receptor 5 (DR5) agonist.

3. The pharmaceutical composition according to claim 1 or 2, wherein the DR5 agonist is INBRX-109, eftozanermin alpha (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 (HexaBody™-DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals), or MM201-b (Merrimack Pharmaceuticals).

4. The pharmaceutical composition according to claim 3, wherein the DR5 agonist is INBRX-109.

5. The pharmaceutical composition according to claim 1 or 2, wherein the DR5 agonist is a DR5-binding polypeptide.

6. The pharmaceutical composition according to claim 5, wherein the DR5-binding polypeptide comprises at least one VHH domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO:

3.

7. The aforementioned at least one VHH domain, (a) 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; or, (b) Amino acid sequence of Sequence ID No. 4 The pharmaceutical composition according to claim 6, comprising:

8. The pharmaceutical composition according to claim 5, wherein the DR5-binding polypeptide comprises a VHH domain consisting solely of the amino acid sequence of SEQ ID NO:

4.

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

10. The pharmaceutical composition according to claim 5, wherein the DR5-binding polypeptide has the structure VHH-linker-VHH-linker-Fc.

11. The pharmaceutical composition according to claim 10, wherein each VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO:

3.

12. The VHH-linker-VHH is (a) an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of Sequence ID No. 5; or, (b) Amino acid sequence of Sequence ID No. 5 A pharmaceutical composition according to claim 10, comprising:

13. The pharmaceutical composition according to claim 12, wherein the VHH-linker-VHH consists only of the amino acid sequence of SEQ ID NO:

5.

14. The DR5-binding polypeptide is (a) an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of Sequence ID No. 7; or, (b) Amino acid sequence of Sequence ID No. 7 The pharmaceutical composition according to claim 5, comprising:

15. The pharmaceutical composition according to claim 5, wherein the DR5-binding polypeptide consists solely of the amino acid sequence of SEQ ID NO:

7.

16. The pharmaceutical composition according to claim 1 or 2, wherein the PLK1 inhibitor is a small molecule or interfering RNA (siRNA).

17. The PLK1 inhibitors mentioned above include onvancertib, volasertib, rigosertib, BI2536 (Boehringer Ingelheim), N-[[4-[(6-chloro-3-pyridinyl)methoxy]-3-methoxyphenyl]methyl]-3,4-dimethoxybenzeneethanamine hydrochloride (SBE 13 HCl), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), poloxin, poloxin-2HT, RO3280 (CAS number 1062243-51-9), HMN-214 (CAS number 173529-46-9), HMN-176 (CAS number 173529-10-7), 2-cyano-2-[3-ethyl-4-oxo-5-[[3-(2-pyrrolidine-1-yl) The pharmaceutical composition according to claim 1 or 2, wherein the composition is [(Tyl)anilino]methyl]-1,3-thiazolidined-2-yl]-N-(2,2,2-trifluoroethyl)acetamide (ZK-thiazolidinone), cyclaporine 9 (CAS number 40533-25-3), or 5-(5,6-dimethoxy-1H-benzimidazole-1-yl)-3-[[2-(trifluoromethyl)phenyl]methoxy]-2-thiophenecarboxamide (GW 843682X).

18. The pharmaceutical composition according to claim 17, wherein the PLK1 inhibitor is onvancertib, volasertib, rigosertib, BI2536 (Boehringer Ingelheim), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), CYC140 (Cyclacel), TKM-080301 (TKM-PLK1; Arbutus Biopharma), or TAK-960 (Takeda Pharmaceutical Company Limited).

19. The pharmaceutical composition according to claim 1 or 2, wherein the DR5 agonist and the PLK1 inhibitor are administered separately.

20. (a) At least one dose or the first dose of the DR5 agonist is administered before the PLK1 inhibitor, (b) At least one dose or the first dose of the DR5 agonist is administered after the PLK1 inhibitor, The pharmaceutical composition according to claim 19.

21. The pharmaceutical composition according to claim 1 or 2, wherein the DR5 agonist and the PLK1 inhibitor are administered simultaneously.

22. The pharmaceutical composition according to claim 1 or 2, wherein the administration of the DR5 agonist and the PLK1 inhibitor produces a synergistic effect compared to the administration of each active substance alone.

23. A pharmaceutical composition for treating cancer in a subject, comprising a cell death receptor 5 (DR5) agonist, wherein the pharmaceutical composition is administered to the subject in combination with a cyclin-dependent kinase (CDK) inhibitor.

24. A pharmaceutical composition for treating cancer in a subject, comprising a cyclin-dependent kinase (CDK) inhibitor, wherein the pharmaceutical composition is administered to the subject in combination with a cell death receptor 5 (DR5) agonist.

25. The pharmaceutical composition according to claim 23 or 24, wherein the DR5 agonist is INBRX-109, eftozanermin alpha (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 (HexaBody™-DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals), or MM201-b (Merrimack Pharmaceuticals).

26. The pharmaceutical composition according to claim 25, wherein the DR5 agonist is INBRX-109.

27. The pharmaceutical composition according to claim 23 or 24, wherein the DR5 agonist is a DR5-binding polypeptide.

28. The pharmaceutical composition according to claim 27, wherein the DR5-binding polypeptide comprises at least one VHH domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the amino acid sequence of SEQ ID NO:

3.

29. The aforementioned at least one VHH domain, (a) 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; or, (b) The pharmaceutical composition according to claim 28, comprising the amino acid sequence of Sequence ID No.

4.

30. The pharmaceutical composition according to claim 27, wherein the DR5-binding polypeptide comprises a VHH domain consisting of the amino acid sequence of SEQ ID NO:

4.

31. The pharmaceutical composition according to claim 27, wherein the DR5-binding polypeptide includes an Fc region.

32. The pharmaceutical composition according to claim 27, wherein the DR5-binding polypeptide has the structure VHH-linker-VHH-linker-Fc.

33. The pharmaceutical composition according to claim 32, wherein each VHH domain comprises CDR1 containing the amino acid sequence of SEQ ID NO: 1, CDR2 containing the amino acid sequence of SEQ ID NO: 2, and CDR3 containing the amino acid sequence of SEQ ID NO:

3.

34. The VHH-linker-VHH is (a) an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of Sequence ID No. 5; or, (b) The pharmaceutical composition according to claim 32, comprising the amino acid sequence of Sequence ID No.

5.

35. The DR5-binding polypeptide is (a) an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of Sequence ID No. 7; or, (b) The pharmaceutical composition according to claim 27, comprising the amino acid sequence of Sequence ID No.

7.

36. The pharmaceutical composition according to claim 27, wherein the DR5-binding polypeptide consists solely of the amino acid sequence of SEQ ID NO:

7.

37. (a) The CDK inhibitor is a CDK9 inhibitor; (b) The CDK inhibitor is a small molecule; (c) The CDK inhibitor is flavopyridol, sericiclib, dinacyclib, atubeciclib, enitocyclib, AZD4573, i-CDK9, or NVP-2; and / or (d) The CDK inhibitor is dinaciclib, NVP-2, flavopyridol, enitociclib, or AZD4573. The pharmaceutical composition according to claim 23 or 24.

38. The pharmaceutical composition according to claim 23 or 24, wherein the DR5 agonist and the CDK inhibitor are administered separately.

39. (a) at least one dose or first dose of the DR5 agonist is administered before the CDK inhibitor; or (b) At least one dose or the first dose of the DR5 agonist is administered after the CDK inhibitor. The pharmaceutical composition according to claim 38.

40. The pharmaceutical composition according to claim 23 or 24, wherein the DR5 agonist and the CDK inhibitor are administered simultaneously.

41. The pharmaceutical composition according to claim 23 or 24, wherein the administration of the DR5 agonist and the CDK inhibitor produces a synergistic effect compared to the administration of each active substance alone.

42. The cancer is adrenal cancer, astrocytoma, basal cell carcinoma, bile tract cancer, bladder cancer, bone cancer, brain cancer and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, chondrosarcoma, Ewing's sarcoma, colorectal cancer (colon cancer). , connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasm, kidney cancer or kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, small Cellular lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth and / or pharynx), ovarian cancer, pancreatic cancer such as pancreatic adenocarcinoma, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, mesothelioma, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine cancer or endometrial cancer, urinary tract cancer and vulvar cancer, lymphoma, The pharmaceutical composition according to claim 41, selected from Dikin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocyte (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small undivided cell NHL, giant lesion NHL, mantle cell lymphoma, AIDS-associated lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, as well as post-transplant lymphoproliferative disorders (PTLD), and nevus disorders, edema (such as edema associated with brain tumors), and abnormal angiogenesis associated with Meigs syndrome.

43. The pharmaceutical composition according to any one of claims 1, 2, 23, or 24, wherein the DR5-binding polypeptide is a tetravalent dimer.

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