DR5 agonist and PLK1 inhibitor or CDK inhibitor combination therapy

Abstract

Provided herein are methods of treating cancer with a combination of a DR5 agonist and a PLK1 inhibitor. Also provided herein are methods of treating cancer with a combination of a DR5 agonist and a CDK inhibitor.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 328,951, filed on April 8, 2022, which is incorporated herein by reference in its entirety for any purpose. Technical Field

[0002] The present invention relates to treating cancer with 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 Art

[0003] Death receptor 5 (DR5; also known as TNFRSF10B or TRAILR2) is a member of the TNF receptor superfamily (TNFRSF) and is a cell surface receptor in the TNF receptor superfamily that binds TNF-related apoptosis-inducing ligand (TRAIL). TRAIL plays an important role in mammalian development and host defense by selectively eradicating unwanted, infected, and malignant cells from healthy cell populations. Upon binding to TNF receptor family members DR4 or DR5, TRAIL induces apoptosis through caspase-dependent apoptosis. DR5 appears to be the major receptor on tumor cells, contributing to the observed tumor-biased activity of the TRAIL pathway. DR5 is activated by the natural ligand TRAIL, which brings three DR5 receptors into close proximity, thereby activating intracellular caspase-8 and initiating the activation of other death-inducing caspases such as caspase-9 and caspase-3. Therefore, initiation of this cell death pathway requires aggregation of DR5 receptors to achieve efficient cell death. DR5 agonists are promising therapeutic candidates for the treatment of cancer.

[0004] Polo-like kinases belong to a family of mitotic serine / threonine kinases that are involved in cell cycle progression, centrosome cycle, mitosis, and cellular responses 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 regions with similarity called polo boxes (PB). Among the members of the polo-like kinase family, PLK1 is the most fully characterized. PLK1 plays a role in different stages of cell cycle progression and mitosis, including mitotic entry, G2 / M checkpoint, spindle assembly maturation, chromosome separation, and mitotic exit. PLK1 is overexpressed in several tumor types and appears to lead to enhanced proliferation. It is barely detectable in most normal adult tissues. PLK1 inhibition prevents cell proliferation and causes mitotic catastrophe, making it a therapeutic target for treating cancer.

[0005] Cyclin-dependent kinases (CDKs) are serine / threonine protein kinases activated by regulatory cyclins. The human genome encodes twenty CDKs, which play specific roles in many cellular processes (such as cell division and transcription) in response to intracellular and extracellular signals. CDK9 is a regulator of transcription and is used by certain cancer cells for constant production of proteins to maintain cancer cell survival. CDK9 combines with its main cyclin partner (cyclin T1) to form positive transcription elongation factor b (P-TEFb), and the main function of positive transcription elongation factor b in eukaryotic cells is to mediate transcriptional extension of nascent mRNA chains by phosphorylating the S2 residues of the YSPTSPS tandem repeat sequence at the C-terminal domain (CTD) of RNA polymerase II (RNAP II). CDK9 is essential for the expression of myeloid cell leukemia 1 (MCL-1). The dysregulation of the CDK9 pathway is involved in the prognosis of a large number of cancer types and resistance to anticancer therapeutics, making it a therapeutic target for treating cancer. Summary of the invention

[0006] Provided herein are methods for 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 for 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. Embodiment 1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject (a) a death receptor 5 (DR5) agonist and (b) a Polo-like kinase 1 (PLK1) inhibitor. Embodiment 2. The method according to embodiment 1, wherein the DR5 agonist is INBRX-109, eftozanermin alfa (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 ( -DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals) or MM201-b (Merrimack Pharmaceuticals). Embodiment 3. The method of embodiment 2, wherein the DR5 agonist is INBRX-109. Embodiment 4. The method of embodiment 1, wherein the DR5 agonist is a DR5 binding polypeptide. Embodiment 5. The method of embodiment 4, wherein the DR5 binding polypeptide is at least tetravalent. Embodiment 6. A method according to any one of Embodiments 3-5, wherein the DR5-binding polypeptide comprises at least one VHH domain, wherein the at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. Embodiment 7. A method according to 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. Embodiment 8. The method of any one of Embodiments 3-7, wherein the DR5-binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 4. Embodiment 9. The method of any one of Embodiments 3-8, wherein the DR5 binding polypeptide comprises an Fc region. Embodiment 10. A method according to embodiment 9, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:6. Embodiment 11. The method of any one of Embodiments 3-10, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc. Embodiment 12. A method according to any one of embodiments 3-11, 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. Embodiment 13. A method according to any one of embodiments 3-12, 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. Embodiment 14. A method according to embodiment 13, wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:5. Embodiment 15. The method of any one of Embodiments 3-14, 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. Embodiment 16. The method of any one of Embodiments 3-15, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7. Embodiment 17. The method of any one of Embodiments 3-15, wherein the DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:7. Embodiment 18. The method according to any one of embodiments 1-17, wherein the PLK1 inhibitor is a small molecule or interfering RNA (siRNA). Embodiment 19. The method according to any one of embodiments 1-18, wherein the PLK1 inhibitor is onvansertib, volasertib, rigosertib, BI2536 (Boehringer Ingelheim), N-[[4-[(6-chloro-3-pyridinyl)methoxy]-3-methoxyphenyl]methyl]-3,4-dimethoxyphenylethylamine hydrochloride (SBE 13HCl), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), poloxin, poloxin-2HT, RO3280 (CAS No. 1062243-51-9), HMN-214 (CAS No. 173529-46-9), HMN-176 (CAS No. 173529-10-7), 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-thiazolinone) or cyclapolin 9 (CAS No. 40533-25-3), 5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-[[2-(trifluoromethyl)phenyl]methoxy]-2-thiophenecarboxamide (GW 843682X). Embodiment 20. The method according to embodiment 19, wherein the PLK1 inhibitor is avensetib, volasetib, reglucicetib, BI2536 (Boehringer Ingelheim), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), CYC140 (Cyclacel), TKM-080301 (TKM-PLK1; Arbutus Biopharma) or TAK-960 (Takeda Pharmaceutical Company). Embodiment 21. The method according to any one of Embodiments 1-18, wherein the PLK1 inhibitor is Avensetib. Embodiment 22. The method according to any one of Embodiments 1-21, wherein the DR5 agonist and the PLK1 inhibitor are administered separately. Embodiment 23. The method according to embodiment 22, wherein the DR5 agonist and the PLK1 inhibitor are administered sequentially. 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 prior to the PLK1 inhibitor. 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. Embodiment 26. The method according to any one of embodiments 1-21, wherein the DR5 agonist and the PLK1 inhibitor are administered concurrently. Embodiment 27. The method of any one of Embodiments 1-26, wherein the DR5 agonist and the PLK1 inhibitor act synergistically. Embodiment 28. A method according to embodiment 27, wherein the synergistic effect is determined in an in vitro cell survival assay. Embodiment 29. The method of any one of Embodiments 1-28, wherein administration of the DR5 agonist and the PLK1 inhibitor results in a synergistic effect compared to each agent administered alone. Embodiment 30. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject (a) a death receptor 5 (DR5) agonist and (b) a cyclin-dependent kinase (CDK) inhibitor. Embodiment 31. The method according to embodiment 30, wherein the DR5 agonist is INBRX-109, etonamin alfa (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 ( -DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals) or MM201-b (Merrimack Pharmaceuticals). Embodiment 32. The method according to embodiment 31, wherein the DR5 agonist is INBRX-109. Embodiment 33. The method according to embodiment 30, wherein the DR5 agonist is a DR5 binding polypeptide. Embodiment 34. The method of Embodiment 33, wherein the DR5 binding polypeptide is at least tetravalent. Embodiment 35. A method according to any one of Embodiments 32-34, wherein the DR5-binding polypeptide comprises at least one VHH domain, wherein the at least one VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3. Embodiment 36. A method according to 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. Embodiment 37. The method of any one of Embodiments 32-36, wherein the DR5-binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO: 4. Embodiment 38. The method of any one of Embodiments 32-37, wherein the DR5 binding polypeptide comprises an Fc region. Embodiment 39. A method according to embodiment 38, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:6. Embodiment 40. The method of any one of Embodiments 32-39, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc. Embodiment 41. A method according to any one of Embodiments 32-40, wherein each VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:1, a CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a CDR3 comprising the amino acid sequence of SEQ ID NO:3. Embodiment 42. A method according to any one of embodiments 32-41, 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. Embodiment 43. A method according to embodiment 42, wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:5. Embodiment 44. The method of any one of Embodiments 32-43, 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. Embodiment 45. The method of any one of Embodiments 32-44, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:7. Embodiment 46. The method of any one of Embodiments 32-44, wherein the DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:7. Embodiment 47. The method of any one of Embodiments 30-46, wherein the CDK inhibitor is a CDK9 inhibitor. Embodiment 48. The method of any one of Embodiments 30-47, wherein the CDK inhibitor is a small molecule. Embodiment 49. The method of any one of Embodiments 30-48, wherein the CDK inhibitor is flavopiridol, seliciclib, dinaciclib, atuveciclib, enitociclib, AZD4573, i-CDK9, or NVP-2. Embodiment 50. The method according to any one of Embodiments 49, wherein the CDK inhibitor is dinicetib, NVP-2, flavopiridol, enicetib or AZD4573. Embodiment 51. The method according to any one of Embodiments 30-50, wherein the DR5 agonist and the CDK inhibitor are administered separately. Embodiment 52. The method according to embodiment 51, wherein the DR5 agonist and the CDK inhibitor are administered sequentially. 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 prior to the CDK inhibitor. 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. Embodiment 55. The method of any one of Embodiments 30-51, wherein the DR5 agonist and the CDK inhibitor are administered concurrently. Embodiment 56. The method of any one of Embodiments 30-55, wherein the DR5 agonist and the CDK inhibitor act synergistically. Embodiment 57. A method according to embodiment 56, wherein the synergistic effect is determined in an in vitro cell survival assay. Embodiment 58. The method of any one of Embodiments 30-57, wherein administration of the DR5 agonist and the CDK inhibitor results in a synergistic effect compared to each agent administered alone. Embodiment 59. A method according to any one of Embodiments 1-58, wherein the cancer is adrenal cancer; astrocytoma; basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; chondrosarcoma; Ewing's sarcoma; colorectal cancer (colorectal cancer); connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; gastrointestinal cancer; glioblastoma cancer of the liver; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer; small cell lung cancer; non-small cell lung cancer; lung adenocarcinoma; lung squamous cell carcinoma; melanoma; myeloma; neuroblastoma; oral cancer (lip, 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 malignant cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; and vulvar cancer; lymphoma; Hodgkin lymphoma; non-Hodgkin lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin 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-cleaved cell NHL; large mass 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), as well as abnormal vascular proliferation associated with nevus hamartomatosis, edema (such as that associated with brain tumors), and Meigs syndrome. Embodiment 60. A DR5 agonist for use in a method of treating cancer, wherein the method comprises administering the DR5 agonist in combination with a PLK1 inhibitor. Embodiment 61. Use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is for administration together with a PLK1 inhibitor. Embodiment 62. A DR5 agonist for use in a method of treating cancer, wherein the method comprises administering the DR5 agonist in combination with a CDK inhibitor, such as a CDK9 inhibitor. Embodiment 63. Use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is for administration together with a CDK inhibitor, such as a CDK9 inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1A-1FResults of a stepwise adjustment experiment are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, or 10 nM) and Avensetin (0.32, 1.6, 8, 40, 200, or 1000 nM). The percent survival of the cancer cells is shown on the y-axis of each graph. The cancer cell line HT-29 ( Figure 1A )、LS174T( Figure 1B )、SW620( Figure 1C )、SW837( Figure 1D )、SW1463( Figure 1E ) and LS411N( Figure 1F ) results.

[0008] Figure 2A-2F Results of INBRX-109 stepwise adjustment experiments are shown, in which different cancer cell lines were contacted with various concentrations of INBRX-109 (0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) alone or in combination with 200 nM Avensetin. The survival percentage of the cancer cells is shown on the y-axis of each graph. The cancer cell line HT-29 ( Figure 2A )、LS174T( Figure 2B )、SW620( Figure 2C )、SW837( Figure 2D )、SW1463( Figure 2E ) and LS411N( Figure 2F The dashed line labeled "Cpd alone" shows the survival percentage of cancer cells treated with 200 nM Avensetin alone.

[0009] Figure 3A-Figure 3F Results of an Avensetib stepwise adjustment experiment are shown, in which different cancer cell lines were contacted with various concentrations of Avensetib (0.32, 1.6, 8, 40, 200 or 1000 nM) alone or in combination with 1 nM INBRX-109. The percentage of cancer cell survival is shown on the y-axis of each graph. The cancer cell line HT-29 ( Figure 3A )、LS174T( Figure 3B )、SW620( Figure 3C )、SW837( Figure 3D )、SW1463( Figure 3E ) and LS411N( Figure 3F The dashed line labeled "Ab alone" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0010] Figure 4A-Figure 4I , Figure 5A-Figure 5I and Figure 6A-6L Results of an Avensetib stepwise adjustment experiment are shown, in which different colorectal cancer cell lines were contacted with various concentrations of Avensetib (274pM, 823pM, 2.47nM, 7.41nM, 22.22nM, 66.67nM, 200nM, 600nM and 1.8μM) alone or in combination with 1nM INBRX-109. Cancer cell line SK-CO-1 ( Figure 4A )、SNU-C5( Figure 4B )、LS1034( Figure 4C )、LS123( Figure 4D )、LS180( Figure 4E )、T84( Figure 4F )、SW-48( Figure 4G )、SW01116( Figure 4H )、SW-1463( Fig. 4I )、SW-837( Figure 5A )、SW-948( Figure 5B )、LS 513( Figure 5C )、NCI-H716( Figure 5D )、HCT 116( Figure 5E )、DLD-1( Fig. 5F )、HCT-15( Figure 5G )、LS174T( Figure 5H )、Ht-55( Fig.5I )、SNU-81( Fig. 6A )、HT-29( Figure 6B )、SW-620( Figure 6C )、Hu Tu 80( Fig.6D )、SW-480( Fig. 6E )、SW-403( Fig. 6F )、LoVo( Figure 6G )、SNU-C2A( Figure 6H )、NCI-H747( Fig.6I )、COLO320DM( Figure 6J )、RKO( Figure 6K )、HCT-8( Figure 6L ). The survival percentage of cancer cells is shown on the y-axis of each graph. The dashed line labeled "Ab alone" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0011] Figure 7A-Figure 7I and Figure 8A-8HResults of an Avencetib step-up experiment are shown, in which different pancreatic cancer cell lines were contacted with various concentrations of Avencetib (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. Fig. 7A )、Panc 05.24( Figure 7B )、SW-1990( Figure 7C )、HuP-T4( Fig.7D )、BxPC-3( Fig. 7E )、Capan-2( Figure 7F )、KP4( Figure 7G )、AsPC-1( Figure 7H )、PSN-1( Fig.7I )、HPAF-II( Fig. 8A )、PANC-1( Figure 8B )、Capan-1( Figure 8C )、CFPAC-1( Fig.8D )、Panc 03.27( Fig. 8E )、SU.86.86( Fig.8F )、PL45( Figure 8G )、Panc 10.05( Figure 8H ). The survival percentage of cancer cells is shown on the y-axis of each graph. The dashed line labeled "Ab alone" shows the survival percentage of cancer cells treated with 1 nM BRRX-109 alone.

[0012] Figure 9A-9E Results of a stepwise adjustment experiment are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, or 10 nM) and dinicili (0.2, 1, 5, 25, 125, or 625 nM). The percent survival of the cancer cells is shown on the y-axis of each graph. The cancer cell line CAL-78 ( Fig. 9A ), OUMS-27( Fig. 9B )、SW1353( Fig. 9C )、H-EMC-SS( Fig.9D ) and SW620( Fig.9E ) results.

[0013] Figures 10A-10EResults of INBRX-109 stepwise adjustment experiments are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) alone or in combination with 25 nM dinicili. The survival percentage of the cancer cells is shown on the y-axis of each graph. The cancer cell line CAL-78 ( Fig. 10A ), OUMS-27( Fig. 10B )、SW1353( Fig. 10C )、H-EMC-SS( Fig. 10D ) and SW620( Fig.10E The dashed line labeled "Cpd alone" shows the survival percentage of cancer cells treated with 25 nM dinisili alone.

[0014] Figures 11A-11E Results of a dinicili stepwise adjustment experiment are shown, in which different cancer cell lines were contacted with various concentrations of dinicili (0.2, 1, 5, 25, 125 or 625 nM) alone or in combination with 1 nM INBRX-109. The survival percentage of the cancer cells is shown on the y-axis of each figure. The cancer cell line CAL-78 ( Fig.11A ), OUMS-27( Fig. 11B )、SW1353( Fig. 11C )、H-EMC-SS( Fig.11D ) and SW620( Fig.11E The dashed line labeled "Ab alone" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0015] Figure 12A-12E Results of a stepwise adjustment experiment are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, or 10 nM) and NVP-2 (0.2, 1, 5, 25, 125, or 625 nM). The percent survival of the cancer cells is shown on the y-axis of each graph. The cancer cell line CAL-78 ( Fig. 12A ), OUMS-27( Fig. 12B )、SW1353( Fig. 12C )、H-EMC-SS( Fig.12D ) and SW620( Fig.12E ) results.

[0016] Figures 13A-13EResults of INBRX-109 stepwise adjustment experiments are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) alone or in combination with 125 nM NVP-2. The percent survival of the cancer cells is shown on the y-axis of each graph. The cancer cell line CAL-78 ( Fig.13A ), OUMS-27( Fig. 13B )、SW1353( Fig. 13C )、H-EMC-SS( Fig.13D ) and SW620( Fig.13E The dashed line labeled "Cpd alone" shows the survival percentage of cancer cells treated with 125 nM NVP-2 alone.

[0017] Figure 14A-14E Results of NVP-2 stepwise adjustment experiments are shown, in which different cancer cell lines were exposed to various concentrations of NVP-2 (0.2, 1, 5, 25, 125 or 625 nM) alone or in combination with 1 nM INBRX-109. The percentage of cancer cell survival is shown on the y-axis of each graph. The cancer cell line CAL-78 ( Fig.14A ), OUMS-27( Fig. 14B )、SW1353( Fig. 14C )、H-EMC-SS( Fig.14D ) and SW620( Fig.14E The dashed line labeled "Ab alone" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0018] Figures 15A-15F Results of a stepwise adjustment experiment are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, or 10 nM) and flavopiridol (0.2, 1, 5, 25, 125, or 625 nM). The percent survival of the cancer cells is shown on the y-axis of each graph. The cancer cell line 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 ) results.

[0019] Figures 16A-16FResults of INBRX-109 stepwise adjustment experiments are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) alone or in combination with 125 nM flavopiridol. The percent survival of the cancer cells is shown on the y-axis of each graph. The cancer cell line 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 dashed line labeled "Cpd alone" shows the survival percentage of cancer cells treated with 125 nM flavopiridol alone.

[0020] Figures 17A-17F Results of a stepwise adjustment experiment of flavopiridol in which different cancer cell lines were exposed to various concentrations of flavopiridol (0.2, 1, 5, 25, 125 or 625 nM) alone or in combination with 1 nM INBRX-109 are shown. The percent survival of the cancer cells is shown on the y-axis of each graph. The survival of the cancer cell line CAL-78 ( Fig.17A ), OUMS-27( Fig. 17B )、HS-SY-II( Fig. 17C )、SW1353( Fig.17D )、H-EMC-SS( Fig.17E ) and SW620( Fig.17F The dashed line labeled "Ab alone" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0021] Figure 18A-18F Results of a stepwise adjustment experiment are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, or 10 nM) and enecilib (0.2, 1, 5, 25, 125, or 625 nM). The percent survival of the cancer cells is shown on the y-axis of each graph. The cancer cell line 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 ) results.

[0022] Figure 19A-19FResults of INBRX-109 stepwise adjustment experiments are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) alone or in combination with 125 nM enecilib. The percentage of cancer cell survival is shown on the y-axis of each graph. The cancer cell line 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 dashed line labeled "Cpd alone" shows the survival percentage of cancer cells treated with 125 nM enecil alone.

[0023] Figures 20A-20F Results of an enecil stepwise adjustment experiment are shown, in which different cancer cell lines were exposed to various concentrations of enecil (0.2, 1, 5, 25, 125 or 625 nM) alone or in combination with 1 nM INBRX-109. The percentage of cancer cell survival is shown on the y-axis of each graph. The cancer cell line 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 dashed line labeled "Ab alone" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone.

[0024] Figure 21A-21B Results of a stepwise adjustment experiment are shown, in which different cancer cell lines were exposed to various concentrations of INBRX-109 (0.00001, 0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) and AZD-5991 (0.0032, 0.016, 0.08, 0.4, 2.0, 10 nM). The percentage of cancer cell survival is shown on the y-axis of each graph. The cancer cell line 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 ) results.

[0025] Figure 22A-22F Results of INBRX-109 stepwise adjustment experiments are shown, in which different cancer cell lines were contacted with various concentrations of INBRX-109 (0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) alone or in combination with 2 nM AZD-5991. The percentage of cancer cell survival is shown on the y-axis of each graph. The cancer cell line 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 dashed line labeled "Cpd alone" shows the survival percentage of cancer cells treated with 2 nM AZD-5991 alone.

[0026] Figure 23A-23F Results of AZD-5991 stepwise adjustment experiments are shown, in which different cancer cell lines were exposed to various concentrations of AZD-5991 (0.0032, 0.016, 0.08, 0.4, 2.0, 10 nM) alone or in combination with 1 nM INBRX-109. The percentage of cancer cell survival is shown on the y-axis of each graph. The cancer cell line CAL-78 ( Fig.23A ), OUMS-27( Fig. 23B )、HS-SY-II( Fig.23C )、SW1353( Fig.23D )、H-EMC-SS( Fig.23E ) and SW620( Fig.23F The dashed line labeled "Ab alone" shows the survival percentage of cancer cells treated with 1 nM INBRX-109 alone. DETAILED DESCRIPTION

[0027] Embodiments provided 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 methods of treating cancer with a combination of a death receptor 5 (DR5) agonist and a cyclin-dependent kinase (CDK) inhibitor (such as a CDK9 inhibitor). Definition and various implementations

[0028] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0029] All references, including patent applications, patent publications, and Genbank Accession Numbers, cited herein are incorporated by reference as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety.

[0030] The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using conventional methods, such as the widely used methods described in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology (Academic Press, Inc.); Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, 1998) Academic Press; Animal Cell Culture (RI Freshney), 1987; Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and TissueCulture Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wileyand Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993); and updated versions thereof.

[0031] Unless otherwise defined, scientific and technical terms used in conjunction with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by the context or clearly indicated, singular terms shall include the plural and plural terms shall include the singular. For any conflicting definitions between various sources or references, the definitions provided herein shall prevail.

[0032] Typically, the numbering of residues in immunoglobulin heavy chains 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). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0033] It should be understood that the embodiments of the invention described herein include "consisting of" and / or "consisting essentially of". As used herein, the singular forms "a", "an", and "the" include plural referents unless otherwise stated. The use of the term "or" herein does not mean that the alternatives are mutually exclusive.

[0034] In this application, the use of "or" means "and / or" unless clearly stated or understood by one skilled in the art. In the context of multiple dependent claims, the use of "or" refers back to more than one preceding independent or dependent claim.

[0035] The phrase "reference sample", "reference cell" or "reference tissue" means a sample with at least one known characteristic, which can be used as a comparator with a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. The level of protein and / or mRNA present in a sample with unknown characteristics can be used to determine the level of protein and / or mRNA present in, for example, healthy tissue. In some embodiments, the reference sample is from the same subject, but from a different part of the subject than the part being tested. In some embodiments, the reference sample is from a tissue area surrounding or adjacent to a cancer. In some embodiments, the reference sample is not from the subject being tested, but from a sample of a subject known to have or not have the disorder in question (e.g., a specific cancer or DR5-related disorder). In some embodiments, the reference sample is from the same subject, but from a time point before the subject develops cancer. In some embodiments, the reference sample is from a benign cancer sample from the same or different subject. When a negative reference sample is used for comparison, the expression level or amount of the molecule in question in the negative reference sample will indicate the level at which a person skilled in the art would consider the molecule to be absent and / or present at a low level given the present disclosure. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample will indicate the level at which one of ordinary skill in the art would consider a certain level of the molecule to be present, given the present disclosure.

[0036] The terms "benefit", "clinical benefit", "responsiveness" and "therapeutic responsiveness" 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 inhibition of disease progression to some extent, including slowing and complete cessation; reduction in the number of disease attacks and / or symptoms; reduction in lesion size; inhibition (i.e., reduction, slowing or complete cessation) of disease cell infiltration into adjacent peripheral organs and / or tissues; inhibition (i.e., reduction, slowing or complete cessation) of disease spread; alleviation to some extent of one or more symptoms associated with the disorder; extension of disease-free time after treatment, such as progression-free survival; increased overall survival; higher response rate; and / or reduced mortality at a given time point after treatment. "Non-responsive" or "failed to respond" subjects or cancers are subjects or cancers that fail to meet the above-mentioned "responsive" qualifications.

[0037] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to polymers of nucleotides. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0038] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. The definition encompasses both full-length proteins and fragments thereof. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of this disclosure, "polypeptide" refers to a protein including modifications (such as deletions, additions and substitutions, which are generally conservative in nature) to the native sequence, as long as the protein maintains the desired activity. These modifications may be intentional (such as by site-directed mutagenesis), or may be accidental (such as by mutations of the host producing the protein or errors caused by PCR amplification).

[0039] As used herein, the terms "DR5", "death receptor 5", "TNFRSF10B" and "TRAILR2" refer to any natural mature DR5 produced by processing of DR5 precursors in cells. Unless otherwise indicated, the terms include DR5 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys) and rodents (e.g., mice and rats). The terms also include naturally occurring DR5 variants, such as splice variants or allelic variants. Non-limiting exemplary precursor human DR5 amino acid sequences are, for example, shown in NCBI Accession No. NP_003833.4. See SEQ ID NO: 8. Non-limiting exemplary precursor human DR5 amino acid sequences are, for example, shown in SEQ ID NO: 9.

[0040] As used herein, the terms "PLK1" and "polo-like kinase 1" refer to any native mature PLK1. Unless otherwise indicated, the terms include PLK1 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys) and rodents (e.g., mice and rats). The terms also include naturally occurring PLK1 variants, 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.

[0041] As used herein, the terms "CDK9" and "cyclin-dependent kinase 9" refer to any natural mature CDK9. Unless otherwise indicated, the terms include CDK9 from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus monkeys or rhesus monkeys) and rodents (e.g., mice and rats). The terms also include naturally occurring CDK9 variants, 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.

[0042] The term "specifically binds" an antigen or epitope is a term well known in the art, and methods for determining such specific binding are also well known in the art. A molecule is considered to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration, and / or with greater affinity than it reacts or associates with alternative cells or substances. An antibody or polypeptide "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and / or for a longer duration than a single domain antibody (sdAb) or a VHH-containing polypeptide binds to other substances. For example, an sdAb or a VHH-containing polypeptide that specifically or preferentially binds to a DR5 epitope is an sdAb or a VHH-containing polypeptide that binds to this epitope with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other DR5 epitopes or non-DR5 epitopes. It should also be understood by reading this definition that, for example, a 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. Typically, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0043] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the incidence, degree or likelihood of the characteristic. "Reduce" or "inhibit" refers to a reduction, decrease or retardation of activity, function and / or amount compared to a reference. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 10% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 50% or more. In some embodiments, "reduce" or "inhibit" means the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more. In some embodiments, the above amounts are inhibited or reduced over a period of time relative to a control over the same period of time.

[0044] 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) that is bound to an antigen binding molecule (e.g., sdAb or a polypeptide containing VHH). Epitopes generally include chemically active surface groupings 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 by continuous and / or juxtaposed non-continuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) of the target molecule. Epitopes formed by continuous residues (e.g., amino acids, nucleotides, sugars, lipid moieties) are usually retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are usually lost when treated with denaturing solvents. Epitopes may include, but are not limited to, at least 3, at least 5 or 8-10 residues (e.g., amino acids or nucleotides). In some embodiments, the epitope length is less than 20 residues (e.g., amino acids or nucleotides), less than 15 residues or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they can bind to the same epitope in the antigen. In some embodiments, epitope can be identified by a certain minimum distance with the CDR residues on antigen binding molecules. In some embodiments, epitope can be identified by the above-mentioned distance, and further limited to those residues of the bond (for example, hydrogen bond) between the residues participating in the antigen binding molecules and the antigen residues. Epitope can also be identified by various scans, such as alanine or arginine scanning can indicate one or more residues that antigen binding molecules can interact with. Unless explicitly stated, other residues are not excluded as a part of the epitope of a specific antigen binding molecule as a group of residues of epitope. On the contrary, the existence of such a group represents the minimum series (or species group) of epitope. Therefore, in some embodiments, a group of residues accredited as epitope represents the minimum epitope related to antigen, rather than the exclusive list of residues of epitope on antigen.

[0045] The term "antibody" is used in the broadest sense and encompasses various polypeptides comprising an antibody-like antigen-binding domain, including but not limited to conventional antibodies (generally comprising at least one heavy chain and at least one light chain), single domain antibodies (sdAbs, comprising at least one VHH domain and an Fc region), VHH-containing polypeptides (polypeptides comprising at least one VHH domain), and fragments of any of the foregoing, as long as they exhibit the desired antigen-binding activity. In some embodiments, the antibody comprises a dimerization domain. Such dimerization domains include but are not limited to heavy chain constant domains (comprising CH1, hinge, CH2, and CH3, wherein CH1 is generally paired with a light chain constant domain CL, and the hinge mediates dimerization) and Fc regions (comprising hinge, CH2, and CH3, wherein the hinge mediates dimerization).

[0046] The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies from various species such as camelids (including llamas), sharks, mice, humans, cynomolgus monkeys, etc.

[0047] As used herein, the term "antigen binding domain" 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. Therefore, in some embodiments, the antigen binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region contains CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required for maintaining binding to an antigen, and the light chain variable region contains CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required for maintaining binding to an antigen. In some embodiments, the antigen binding domain of an sdAb or a polypeptide containing VHH comprises three CDRs of a VHH domain. Therefore, in some embodiments, the antigen binding domain of an sdAb or a polypeptide containing VHH comprises a VHH domain, and the VHH domain contains CDR1-FR2-CDR2-FR3-CDR3 and any part of FR1 and / or FR4 required for maintaining binding to an antigen.

[0048] As used herein, the term "VHH" or "VHH domain" or "VHH antigen binding domain" refers to the antigen binding portion of a single domain antibody (such as a camelid antibody or a shark antibody). In some embodiments, the VHH comprises three CDRs and four framework regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the VHH can be truncated at the N-terminus or C-terminus so that it only comprises part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as the VHH substantially maintains antigen binding and specificity.

[0049] The terms "single domain antibody" and "sdAb" are used interchangeably herein to refer to an antibody comprising at least one monomeric domain (e.g., a VHH domain) without a light chain and an Fc region. In some embodiments, an sdAb is a dimer of two polypeptides, each of which comprises at least one VHH domain and an Fc region. As used herein, the terms "single domain antibody" and "sdAb" encompass polypeptides comprising multiple VHH domains, such as a polypeptide having a structure VHH 1 -VHH 2 -Fc or VHH 1 -VHH 2 -VHH 3 -Fc polypeptide, wherein VHH 1 、VHH 2 and VHH 3Can be the same or different.

[0050] The term "VHH-containing polypeptide" refers to a polypeptide comprising at least one VHH domain. In some embodiments, the VHH polypeptide comprises two, three, or four or more VHH domains, wherein each VHH domain may be the same or different. In some embodiments, the VHH-containing polypeptide comprises an Fc region. In some such embodiments, the VHH-containing polypeptide may be referred to as an sdAb. In addition, in some such embodiments, the VHH polypeptide may form a dimer. Non-limiting structures of VHH-containing polypeptides (which are also sdAbs) include VHH. 1 -Fc, VHH 1 -VHH 2 -Fc and VHH 1 -VHH 2 -VHH 3 -Fc, where VHH 1 、VHH 2 and VHH 3 Can be the same or different. In some embodiments of such structures, one VHH can be connected to another VHH by a linker, or one VHH can be connected to Fc by a linker. In some such embodiments, the linker comprises 1-20 amino acids, preferably 1-20 amino acids mainly composed of glycine and optionally serine. In some embodiments, when the polypeptide containing VHH comprises Fc, it forms a dimer. Therefore, if the structure VHH 1 -VHH 2 -Fc forms a dimer, it is considered tetravalent (ie, the dimer has four VHH domains). Similarly, if the structure VHH 1 -VHH 2 -VHH 3 -Fc forms a dimer, it is considered hexavalent (ie, the dimer has six VHH domains).

[0051] The term "monoclonal antibody" refers to an antibody (including a polypeptide containing sdAb or VHH) in a substantially homogeneous antibody population, i.e., except for a small amount of possible naturally occurring mutations, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific for a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. Therefore, a monoclonal antibody sample can bind to the same epitope on the antigen. The modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, monoclonal antibodies can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by a recombinant DNA method as described in U.S. Patent No. 4,816,567, etc. For example, monoclonal antibodies can also be isolated from a phage library produced using the technology described in McCafferty et al., 1990, Nature 348:552-554.

[0052] The term "CDR" means a complementary determining region defined by at least one identification method of a person skilled in the art. In some embodiments, CDRs may be defined according to any one of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. A VHH comprises three CDRs, designated as CDR1, CDR2, and CDR3.

[0053] As used herein, the term "heavy chain constant region" refers to a heavy chain constant region comprising at least three heavy chain constant domains C H 1. Hinge, C H 2 and C H 3. Of course, unless otherwise specified, non-functional altering deletions and changes within the domain are all covered by the term "heavy chain constant region". Non-limiting exemplary heavy chain constant regions include γ, δ and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. In addition, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε 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 (containing γ 1 constant region), IgG2 (containing γ 2 constant region), IgG3 (containing γ 3 constant region) and IgG4 (containing γ 4constant region) antibodies; IgA antibodies include but are not limited to IgA1 (containing α 1 constant region) and IgA2 (containing α 2 constant region) antibodies; and IgM antibodies including but not limited to IgM1 and IgM2.

[0054] As used herein, "Fc region" refers to a portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0055] As used herein, an "acceptor human framework" is a heavy chain variable domain (V H ) framework, as discussed herein. An acceptor human framework derived from a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes across all human frameworks in a single antigen binding domain (such as a VHH) is less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less than 4, or less than 3.

[0056] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody such as an sdAb or a VHH-containing polypeptide) and its binding partner (e.g., an antigen). The affinity or apparent affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (KD) or K D-表观 It can be expressed by common methods known in the art (e.g. ELISA K D , KinExA, flow cytometry and / or surface plasmon resonance devices) (including those described herein). Such methods include, but are not limited to, or flow cytometry methods.

[0057] As used herein, the term "K D ” refers to the equilibrium dissociation constant of the antigen binding molecule / antigen interaction. When the term “K D ", it includes KD and K D-表观 .

[0058] In some embodiments, the K of the antigen binding molecule is DK is measured by flow cytometry using a cell line expressing the antigen and fitting the mean fluorescence measured at each antibody concentration to a nonlinear single-site binding equation (Prism Software graphpad). In some such embodiments, K D It's K D-表观 .

[0059] The term "biological activity" refers to any one or more biological properties of a molecule (whether found naturally in vivo or provided or achieved by recombinant means). Biological properties include, but are not limited to, binding to a ligand, inducing or increasing cell proliferation, and inducing or increasing the expression of cytokines.

[0060] An "agonist" or "activating" antibody or polypeptide is an antibody or polypeptide that increases and / or activates the biological activity of its target antigen. In some embodiments, an agonist antibody or polypeptide binds to an antigen and increases the biological activity of the antigen by at least about 20%, 40%, 60%, 80%, 85% or more.

[0061] "Antagonist," "blocking," or "neutralizing" antibodies are antibodies that inhibit, reduce, and / or inactivate the biological activity of a target antigen. In some embodiments, a neutralizing antibody binds to an antigen and reduces the biological activity of the antigen by at least about 20%, 40%, 60%, 80%, 85%, 90%, 95%, 99%, or more.

[0062] An "affinity matured" sdAb or VHH-containing polypeptide refers to a sdAb or VHH-containing polypeptide having one or more alterations in one or more CDRs, compared to a parent sdAb or VHH-containing polypeptide that does not have such alterations, such alterations resulting in an improvement in the affinity of the sdAb or VHH-containing polypeptide for an antigen.

[0063] As used herein, "humanized VHH" refers to a VHH in which one or more framework regions have been substantially replaced by human framework regions. In some cases, certain framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized VHHs may include residues that are not found in the original VHH and human framework sequences, but are included to further improve and optimize the performance of sdAbs and polypeptides containing VHHs. In some embodiments, humanized sdAbs or polypeptides containing VHHs include human Fc regions. It should be understood that humanized sequences can be identified by their primary sequences and do not necessarily represent the process of producing antibodies.

[0064] An "effector-positive Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, among others. Such effector functions generally require combining an Fc region with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays.

[0065] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A allotypes 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.

[0066] A "variant Fc region" comprises an amino acid sequence that is different from the amino acid sequence of the native sequence Fc region due to at least one amino acid modification. In some embodiments, the amino acid sequence included in the "variant Fc region" is different from the amino acid sequence of the native sequence Fc region due to at least one amino acid modification, but still retains at least one effector function of the native sequence Fc region. In some embodiments, compared with the native sequence Fc region or with the Fc region of the parent polypeptide, the variant Fc region has at least one amino acid substitution in the native sequence Fc region or in the Fc region of the parent polypeptide, for example, about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions. In some embodiments, the variant Fc region herein will have at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region of the parent polypeptide, at least about 90% sequence identity therewith, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity therewith.

[0067] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcγR is a natural human FcR. In some embodiments, FcR is a receptor (gamma receptor) that binds to an IgG antibody, and includes receptors of FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, the main difference being their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). For example, FcRs are reviewed 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). The term "FcR" herein encompasses other FcRs, including those to be identified in the future. For example, the term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulating the homeostasis of immunoglobulins. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0068] As used herein, the term "substantially similar" or "substantially identical" refers to a sufficiently high similarity between two or more values ​​that a person skilled in the art would consider the difference between the two or more values ​​to have little or no biological and / or statistical significance in the context of the biological characteristics measured by the values. In some embodiments, the difference between two or more substantially similar values ​​does not exceed about any of 5%, 10%, 15%, 20%, 25%, or 50%.

[0069] Polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity with a native sequence polypeptide after aligning the sequences and introducing spaces (if necessary) to achieve the maximum sequence identity percentage and not 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, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0070] As used herein, "percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing spaces (if necessary) to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0071] Amino acid substitutions may include, but are not limited to, replacing one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. Table 1 Original residue Exemplary substitutions Ala(A) Val; Leu; Ile Arg(R) Lys; Gln; Asn Asn(N) Gln; His; Asp, Lys; Arg Asp(D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn; Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn; Gln; Lys; Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg; Gln; Asn Met(M) Leu; Phe; Ile Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Pro(P) Ala Ser(S) Thr Thr(T) Val; Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe; Thr; Ser Val(V) Ile; Leu; Met; Phe; Ala; norleucine

[0072] Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: norleucine, Met, A1a, 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.

[0073] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0074] The term "vector" is used to describe a polynucleotide that can be engineered to contain one or more cloned polynucleotides that can be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as promoters and / or enhancers) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (such as antibiotic resistance genes and genes that can be used for colorimetric assays, such as β-galactosidase). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

[0075] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. A host cell can be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, Host cells include 293 and CHO cells and their derivatives (such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells). Host cells include progeny of a single host cell, and due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical to the original parent cell (in terms of morphology or genomic DNA complementary sequence). Host cells include cells transfected in vivo with one or more polynucleotides provided herein.

[0076] As used herein, the term "isolated" refers to a molecule that has been separated from at least some of the components that are typically found or produced in nature. For example, a polypeptide is said to be "isolated" when it is separated from at least some of the components of the cell that produced it. When a polypeptide is secreted by a cell after expression, the supernatant containing the polypeptide is physically separated from the cell that produced it, and it is considered to be "isolated" the polypeptide. Similarly, when a polynucleotide is not part of a larger polynucleotide typically found in nature (such as, for example, genomic DNA or mitochondrial DNA in the case of a DNA polynucleotide), or, for example, in the case of an RNA polynucleotide, is separated from at least some of the components of the cell that produced it, the polynucleotide is referred to as "isolated". Therefore, a DNA polynucleotide contained in a vector within a host cell can be referred to as "isolated".

[0077] The terms "individual" and "subject" are used interchangeably herein to refer to animals, such as mammals. In some embodiments, methods for treating mammals are provided, including but not limited to humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sports animals, and mammalian pets. In some examples, an "individual" or "subject" refers to an individual or subject in need of treatment for a disease or disorder. In some embodiments, the subject being treated can be a patient indicating the fact that the subject has been identified as suffering from a disorder associated with the treatment, or is at sufficient risk of developing the disorder.

[0078] As used herein, "disease" or "disorder" refers to a condition for which treatment is needed and / or desired.

[0079] Unless otherwise indicated, the terms "tumor cell," "cancer cell," "cancer," "tumor," and / or "neoplasm" are used interchangeably herein and refer to a cell (or cells) that exhibits uncontrolled growth and / or abnormally increased cell survival and / or inhibition of apoptosis that interferes with the normal functioning of body organs and systems. This definition includes benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases.

[0080] The terms "cancer" and "tumor" encompass both solid cancers and blood / lymphatic cancers, and also encompass malignant, premalignant, and benign growths, such as dysplasia. Exemplary cancers include, but are not limited to: adrenal cancer; astrocytoma; basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; chondrosarcoma; Ewing's sarcoma; colorectal cancer (colorectal cancer); connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancer); glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer; cancer of the liver; cancer of the lung (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer, such as pancreatic adenocarcinoma; pituitary cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colorectal cancer; cancer of the respiratory system; mesothelioma; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer ; thyroid cancer; uterine or endometrial cancer; urological cancers; vulvar cancer; lymphomas, including Hodgkin and non-Hodgkin lymphomas, and B-cell lymphomas (including low-grade / follicular non-Hodgkin 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-cleaved cell NHL; large mass 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 disorder (PTLD), as well as the abnormal vascular proliferation associated with nevus hamartomatosis, edema (such as that associated with brain tumors), and Meigs syndrome.

[0081] In some embodiments, "increase" or "decrease" refers to a statistically significant increase or decrease, respectively. It will be clear to the technician that "modulation" can also involve changes (which may be increases or decreases) in affinity, avidity, specificity and / or selectivity of a target or antigen to one or more of its ligands, binding partners, partners for association as homopolymers or heteropolymers, or substrates compared to the same conditions but without the test agent; changes (which may be increases or decreases) in sensitivity of a target or antigen to one or more conditions (such as pH, ionic strength, presence of cofactors, etc.) in the medium or environment in which the target or antigen is present; and / or cell proliferation or cytokine production. 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.

[0082] As used herein, "treatment" is a method for obtaining a beneficial or desired clinical outcome. As used herein, "treatment" encompasses any administration or application of a therapeutic agent for a disease in a mammal, including humans. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, any one or more of the following: alleviating one or more symptoms, reducing the extent of the disease, preventing or delaying the spread of the disease (e.g., metastasis, such as metastasis to the lungs or lymph nodes), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, inhibiting the disease or disease progression, inhibiting or slowing the disease or disease progression, stagnating the development of the disease and alleviating (whether in part or in whole). "Treatment" also encompasses the pathological consequences of reducing proliferative diseases. The method provided herein takes into account any one or more of these treatment aspects. Consistent with the above, the term treatment does not require 100 percent removal of all aspects of the obstacle.

[0083] "Improvement" refers to a reduction or improvement in one or more symptoms compared to when no therapeutic agent is administered."Improvement" also includes shortening or reducing the duration of symptoms.

[0084] The term "anticancer agent" is used in its broadest sense herein to refer to an agent used to treat one or more cancers. Exemplary classes of such agents include, but are not limited to, chemotherapeutic agents, anticancer biologics (such as cytokines, receptor extracellular domain-Fc fusions, and antibodies), radiotherapy, CAR-T therapy, therapeutic oligonucleotides (such as antisense oligonucleotides and siRNA), and oncolytic viruses.

[0085] As used herein, the terms "synergistic," "synergistically," and "synergistic effect" refer to more than additive effects of two or more agents. Determination of a synergistic effect 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.

[0086] The term "biological sample" means an amount of a substance from a living thing or a once living thing. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0087] The term "control" or "reference" refers to a composition known to contain the analyte (a "negative control") or to contain the analyte (a "positive control"). A positive control may comprise a known concentration of the analyte.

[0088] As used herein, "delaying the development of a disease" means postponing, hindering, slowing, delaying, stabilizing, inhibiting and / or postponing the development of a disease (e.g., cancer). This delay can be of varying lengths of time, depending on the medical history and / or the individual being treated. It will be apparent to one skilled in the art that a sufficient or significant delay may actually encompass prevention, in that the individual will not develop the disease. For example, the development of an advanced cancer, such as metastasis, may be delayed.

[0089] As used herein, "preventing" includes providing prevention of the occurrence or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease. Unless otherwise indicated, the terms "reducing," "inhibiting," or "preventing" do not imply or require complete prevention at all times, but only during the measured time period.

[0090] A "therapeutically effective amount" of a substance / molecule, agonist or antagonist may vary according to factors such as the disease state, age, sex and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutic beneficial effects outweigh any toxic or deleterious effects of the substance / molecule, agonist or antagonist. A therapeutically effective amount may be delivered in one or more administrations. A "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic and / or preventive results at the necessary dosage and for the necessary period of time.

[0091] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation that is in a form that renders the biological activity of one or more active ingredients effective, and that contains no other components that are unacceptably toxic to a subject to which the formulation is administered. Such formulations may be sterile.

[0092] "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 together with a therapeutic agent, which together constitutes a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation used.

[0093] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0094] The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents wherein at least a portion of the administration overlaps in time or wherein the administration of one therapeutic agent occurs within a short period of time relative to the administration of another therapeutic agent, or wherein the therapeutic effects of the two agents overlap for at least a period of time.

[0095] The term "sequentially" is used herein to refer to administration of two or more therapeutic agents that do not overlap in time, or where the therapeutic effects of the agents do not overlap.

[0096] As used herein, "in combination with" means administering another therapeutic modality in addition to one therapeutic modality. Thus, "in combination with" means administering another therapeutic modality before, during, or after one therapeutic modality is administered to an individual.

[0097] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings regarding the use of such therapeutic products.

[0098] An "article of manufacture" is any article of manufacture (e.g., a package or container) or kit comprising at least one agent (e.g., a medicament for treating a disease or disorder (e.g., cancer)) or a probe for specifically detecting a biomarker described herein. In some embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for practicing the methods described herein.

[0099] The terms "label" and "detectable label" mean a moiety attached, for example, to an antibody or antigen, so that a reaction (e.g., binding) between members of a specific binding pair is detectable. The labeled member of the specific binding pair is said to be "detectably labeled." Thus, the term "labeled binding protein" refers to a protein into which a label is incorporated for identification of the binding protein. In some embodiments, the label is a detectable label that produces a signal that can be detected by visual or instrumental means, for example, incorporation of a radiolabeled amino acid or attachment of a biotinyl moiety to a polypeptide that can be detected by labeled avidin (e.g., streptavidin contains a fluorescent marker or enzymatic activity that can be detected optically or colorimetrically). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H. 14 C. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I. 177 Lu, 166 Ho or 153Sm); chromogen, fluorescent label (for example, FITC, rhodamine, lanthanide phosphor), enzyme label (for example, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent label; biotin group; predetermined polypeptide epitope recognized by second reporter molecule (for example, leucine zipper pair sequence, binding site of second antibody, metal binding domain, epitope tag); and magnetic agent such as gadolinium chelate. Representative examples of labels commonly used in immunoassays include light-generating moieties, such as acridinium compounds; and fluorescence-generating moieties, such as fluorescein. In this regard, the moiety itself may not be detectably labeled, but may become detectable when reacted with yet another moiety. Exemplary DR5 agonists

[0100] Provided herein are methods of treating cancer comprising administering a DR5 agonist. Non-limiting exemplary DR5 agonists include INBRX-109, etonamin alfa (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 ( -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 polypeptide provided herein is multivalent. In some embodiments, the DR5 binding polypeptide provided herein is at least tetravalent.

[0101] In various embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising a CDR1 comprising the sequence of SEQ ID NO: 1, a CDR2 comprising the sequence of SEQ ID NO: 2, and a CDR3 comprising the sequence of SEQ ID NO: 3. In some embodiments, at least one VHH domain is humanized. In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising an amino acid sequence that is at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain comprising the amino acid sequence of SEQ ID NO: 4.

[0102] In some embodiments, the DR5 binding polypeptide comprises at least one VHH domain that binds to DR5 and an Fc region. In some embodiments, the DR5 binding polypeptide provided herein comprises two VHH domains that bind to DR5 and an Fc region. In some embodiments, the Fc region mediates dimerization of the DR5 binding polypeptide under physiological conditions, so that a dimer is formed that doubles the number of DR5 binding sites. For example, a DR5 binding polypeptide comprising two VHH domains that bind to DR5 and an Fc region is bivalent as a monomer, but under physiological conditions, the Fc region can mediate dimerization, so that the DR5 binding polypeptide is a tetravalent dimer under such conditions.

[0103] 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.

[0104] In some embodiments, the DR5 binding polypeptide comprises the structure VHH-linker-VHH-linker-Fc. In some embodiments, the VHH-linker-VHH portion of the DR5 binding polypeptide comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:5. In some embodiments, the VHH-linker-VHH portion of the DR5 binding polypeptide comprises an amino acid sequence of SEQ ID NO:5. In some embodiments, Fc comprises a hinge. In some such embodiments, Fc comprises an amino acid sequence of SEQ ID NO:6. In some embodiments, the DR5 binding polypeptide comprises an amino acid sequence that is at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, which comprises two VHH domains and an Fc region. In some embodiments, the DR5 binding polypeptide comprises an amino acid sequence of SEQ ID NO:7, which comprises two VHH domains and an Fc region. In some embodiments, the DR5 binding polypeptide consists of an amino acid sequence of SEQ ID NO:7. The 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.

[0105] In some embodiments, the VHH domain that binds DR5 can be humanized. Humanized antibodies (such as sdAb or polypeptides containing VHH) can be used as therapeutic molecules because humanized antibodies reduce or eliminate the immune response of people to non-human antibodies, which may lead to the immune response of antibody therapeutics and the effectiveness of reduced therapeutic agents. Generally, humanized antibodies include one or more variable domains, wherein CDR (or part thereof) is derived from non-human antibodies, and FR (or part thereof) is derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of human constant regions. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0106] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson (2008) Front. Biosci. 13:1619-1633, and are further described, for example, in the following references: Riechmann et al., (1988) Nature 332:323-329; Queen et al., (1989) Proc. Natl Acad. Sci. USA 86:10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., (2005) Methods 36:25-34; Padlan, (1991) Mol. Immunol. 28:489-498 (describing "resurfacing"); Dall'Acqua et al., (2005) Methods 36:43-60 (describing "FR shuffling"); and Osbourn et al., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer, 83:252-260 (describing the "guided selection" method for FR shuffling).

[0107] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296); framework regions derived from the consensus sequence of human antibodies having a particular subgroup of heavy chain variable regions (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151:26 23); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson (2008) Front.Biosci.13:1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca et al., (1997) J.Biol.Chem.272:10678-10684 and Rosok et al., (1996) J.Biol.Chem.271:22611-22618). Typically, the FR region of a VHH is replaced with a human FR region to prepare a humanized VHH. In some embodiments, certain FR residues of a human FR are replaced to improve one or more properties of a humanized VHH. VHH domains with such replacement residues are still referred to herein as "humanized".

[0108] In various embodiments, the Fc region included in the DR5 binding polypeptide is a human Fc region, or is derived from a human Fc region.

[0109] In some embodiments, the Fc region included in the DR5 binding polypeptide is derived from a human Fc region and comprises a deletion of three amino acids corresponding to IgG1 E233, L234 and L235 in the lower hinge, referred to herein as "Fc xELL". FcxELL polypeptides do not bind to FcγRs and are therefore referred to as "effector silent" or "effector null", however in some embodiments, the xELL Fc region binds to FcRn and therefore has an extended half-life associated with FcRn-mediated recycling and transcytosis. In some embodiments, the Fc region is a human IgG1 xELL Fc region. Exemplary PLK1 Inhibitors

[0110] Provided herein are methods for treating cancer comprising administering a PLK1 inhibitor. In some embodiments, the PLK1 inhibitor is a small molecule. In some embodiments, the PLK1 inhibitor is RNAi. In some embodiments, the PLK1 inhibitor is Avensetib, Volasetib, Regsetib, BI2536 (Boehringer Ingelheim), N-[[4-[(6-chloro-3-pyridinyl)methoxy]-3-methoxyphenyl]methyl]-3,4-dimethoxyphenylethylamine hydrochloride (SBE 13HCl), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), CYC140 (Cyclacel), TKM-080301 (TKM-PLK1; Arbutus Biopharma), TAK-960 (Takeda Pharmaceutical Company), poloxin, poloxin-2HT, RO3280 (CAS No. 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-thiazolinone), cycloboline 9 (CAS No. 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 avensetib, volasetib, regrosetib, BI2536 (Boehringer Ingelheim), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), CYC140 (Cyclacel), TKM-080301 (TKM-PLK1; Arbutus Biopharma), or TAK-960 (Takeda Pharmaceutical Company).

[0111] In some embodiments, the PLK1 inhibitor is Avensetib. Avensetib (also known as PCM-075 or NMS-1286937) is a selective ATP-competitive PLK1 inhibitor having the following structure: or a pharmaceutically acceptable salt or hydrate thereof. See, for example, U.S. Patent No. 8,927,530. Avenseti is specific for PLK1 and has potent anti-tumor activity both in vitro and in vivo in models of solid and hematological malignancies.

[0112] In some embodiments, the PLK1 inhibitor is volacetib. Volacetib is a selective PLK1 inhibitor having the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, WO 04 / 076454 and WO 07 / 090844.

[0113] In some embodiments, the PLK1 inhibitor is regocereti. Regocereti is an inhibitor of multiple kinases (including PI3-K and PLK1) and has the following structure: or a pharmaceutically acceptable salt or hydrate thereof. See, e.g., U.S. Pat. No. 7,598,232 (Compound 4).

[0114] In some embodiments, the PLK1 inhibitor is BI2536. BI2536 is a selective inhibitor of PLK1 and has the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Steegmaier et al., Current Biology, 17:316-322 (2007).

[0115] In some embodiments, the PLK1 inhibitor is MLN0905. MLN0905 is a selective inhibitor of PLK1 and has the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Mol Cancer Ther, 11: 2045-53 (2012).

[0116] In some embodiments, the PLK1 inhibitor is TAK-960. TAK-960 is a selective inhibitor of PLK1 and has the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Mol Cancer Ther, 11: 700-9 (2012).

[0117] In some embodiments, the PLK1 inhibitor is GSK461364. GSK461364 is an ATP-competitive PLK1 inhibitor with the following structure: or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Clin Cancer Res. 17(10):3420-30(2011).

[0118] In some embodiments, the PLK1 inhibitor is CYC140. In some embodiments, the PLK1 inhibitor has the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, WO 2009 / 040556.

[0119] In some embodiments, the PLK1 inhibitor is TKM-080301. TKM-080301 is a lipid nanoparticle (LNP) formulation comprising four lipids and a synthetic double-stranded siRNA directed against human PLK1 mRNA. Synthetic siRNA is a duplex of complementary RNA oligonucleotides designed to achieve post-transcriptional gene inhibition through an RNA interference mechanism. See, for example, Oncologist, 24(6):747-e218(2019); WO 2008 / 342535. Exemplary CDK Inhibitors

[0120] Provided herein are methods for treating cancer comprising 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), cericisili (roscovitine / CYC202), dinisili (Merck), otociclib (Bayer), anisili (Vincerx Pharma), AZD4573 (AstraZeneca), i-CDK9 or NVP-2.

[0121] In some embodiments, the CDK inhibitor is flavopiridol. Flavopiridol (also known as L86-8275, alvocidib, NSC 649890, or HMR-1275; Tolero Pharmaceuticals) is a selective ATP-competitive CDK9 inhibitor with the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. Flavin is a potent and selective inhibitor of CDK9 and has antitumor activity against various tumor cell lines (such as human lung cancer and breast cancer), and also inhibits tumor growth in xenograft models. See, for example, Anshabo et al., Frontiers in Oncology, 11 (2021).

[0122] In some embodiments, the CDK inhibitor is sericilib (also known as roscovitine or CYC202). Sericilib is a selective CDK inhibitor having the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Anshabo et al., Frontiers in Oncology, 11 (2021).

[0123] In some embodiments, the CDK inhibitor is dinisili (also known as SCH 727965; Merck). Dinisili is a potent, selective small molecule inhibitor of CDK (including CDK1, CDK2, CDK5 and CDK9) with the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Anshabo et al., Frontiers in Oncology, 11 (2021).

[0124] In some embodiments, the CDK inhibitor is otociclib (also known as BAY1143572; Bayer). Otociclib is a potent and highly selective inhibitor of positive transcription elongation factor b (PTEF-b) (composed of CDK9 and cyclin-T (CycT)) with the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Anshabo et al., Frontiers in Oncology, 11 (2021).

[0125] In some embodiments, the CDK inhibitor is Anicili (also known as BAY1251152 or VIP152; VincerxPharma). Anicili is a potent and highly selective PTEF-b / CDK9 inhibitor with the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Anshabo et al., Frontiers in Oncology, 11 (2021).

[0126] In some embodiments, the CDK inhibitor is AZD4573 (AstraZeneca). AZD4573 is a selective inhibitor of CDK9 and has the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Anshabo et al., Frontiers in Oncology, 11 (2021).

[0127] In some embodiments, the CDK inhibitor is i-CDK9. The i-CDK9 CDK inhibitor is 600 times more selective for CDK9 than other CDKs and has the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. i-CDK9 inhibits dual specificity tyrosine phosphorylation-regulated kinase (DYRK) 1A and 1B, although with lower potency than CDK9. See, e.g., Anshabo et al., Frontiers in Oncology, 11 (2021).

[0128] In some embodiments, the CDK inhibitor is NVP-2. NVP-2 is an ATP-competitive, aminopyrimidine-based inhibitor and a chemical analog of i-CDK9 and has the following structure: Or a pharmaceutically acceptable salt or hydrate thereof. See, for example, Nat Chem Biol 14, 163-170 (2018). Peptide expression and production

[0129] A nucleic acid molecule comprising a polynucleotide encoding a DR5 binding polypeptide is provided. In some embodiments, the nucleic acid molecule may also encode a leader sequence that directs secretion of the DR5 binding polypeptide, the leader sequence being typically cleaved so that it is not present in the secreted polypeptide. The leader sequence may be a native heavy chain (or VHH) leader sequence, or may be another heterologous leader sequence.

[0130] Nucleic acid molecules can be constructed using recombinant DNA techniques routine in the art. In some embodiments, the nucleic acid molecule is an expression vector suitable for expression in a selected host cell.

[0131] Provided are vectors comprising nucleic acids encoding DR5 binding polypeptides described herein. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, and the like. In some embodiments, vectors optimized for expressing polypeptides in desired cell types such as CHO or CHO-derived cells, or NSO cells are selected. Exemplary vectors of this type are described, for example, in Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004).

[0132] In some embodiments, the DR5 binding polypeptide can be expressed in prokaryotic cells (such as bacterial cells); or in eukaryotic cells (such as fungal cells (such as yeast), plant cells, insect cells and mammalian cells). Such expression can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express the polypeptide include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells and FUT8 CHO cells; cells (Crucell); and NSO cells. In some embodiments, the DR5 binding polypeptide can be expressed in yeast. See, for example, U.S. Publication No. US2006 / 0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modification on the polypeptide. For example, in some embodiments, the sialylation level of a polypeptide produced by CHO cells is higher than the sialylation level of the same polypeptide produced in 293 cells.

[0133] One or more nucleic acids (such as vectors) can be introduced into the desired host cells by any method, including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition Cold Spring Harbor Laboratory Press (2001). Nucleic acids can be transiently or stably transfected in the desired host cells according to any suitable method.

[0134] Host cells comprising any nucleic acid or vector described herein are also provided. In some embodiments, host cells expressing the DR5 binding polypeptides described herein are provided. The DR5 binding polypeptides expressed in the host cells can be purified by any suitable method. Such methods include, but are not limited to, using affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include ROR1 ECD and agents that bind to the Fc region. For example, protein A, protein G, protein A / G or antibody affinity columns can be used to bind to the Fc region and purify the DR5 binding polypeptides comprising the Fc region. Hydrophobic interaction chromatography, such as butyl or phenyl columns, may also be applicable to purifying some polypeptides, such as antibodies. Ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography) may also be applicable to purifying some polypeptides, such as antibodies. Mixed mode chromatography (e.g., reverse phase / anion exchange, reverse phase / cation exchange, hydrophilic interaction / anion exchange, hydrophilic interaction / cation exchange, etc.) may also be applicable to purifying certain polypeptides, such as antibodies. Many methods for purifying polypeptides are known in the art.

[0135] In some embodiments, the DR5 binding polypeptide is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0136] In some embodiments, a DR5 binding polypeptide prepared by the above method is provided. In some embodiments, the DR5 binding polypeptide is prepared in a host cell. In some embodiments, the DR5 binding polypeptide is prepared in a cell-free system. In some embodiments, the DR5 binding polypeptide is purified. In some embodiments, a cell culture medium comprising the DR5 binding polypeptide is provided.

[0137] In some embodiments, a composition comprising an antibody prepared by the above method is provided. In some embodiments, the composition comprises a DR5 binding polypeptide prepared in a host cell. In some embodiments, the composition comprises a DR5 binding polypeptide prepared in a cell-free system. In some embodiments, the composition comprises a purified DR5 binding polypeptide. Pharmaceutical composition

[0138] In some embodiments, compositions comprising a DR5 agonist, a PLK1 inhibitor, and / or a CDK inhibitor are provided as formulations with various pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th Edition (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd Edition, Pharmaceutical Press (2000)). Various pharmaceutically acceptable carriers, including vehicles, adjuvants, and diluents, may be used. In addition, various pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity adjusters, stabilizers, wetting agents, and the like may also be used.

[0139] 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. Exemplary methods for treating cancer using DR5 agonists and PLK1 inhibitors

[0140] In some embodiments, a method of treating cancer in an individual is provided, the method comprising administering a DR5 agonist and a PLK1 inhibitor.

[0141] In some embodiments, the method comprises administering to an individual an effective amount of a DR5 agonist and a PLK1 inhibitor. Such treatment methods can be used in humans or animals. In some embodiments, methods of treating humans are provided. Non-limiting exemplary cancers that can be treated with a combination of a DR5 agonist and a PLK1 inhibitor provided herein include adrenal cancer; astrocytoma; basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; chondrosarcoma; Ewing's sarcoma; colorectal cancer (colorectal cancer); connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; gastrointestinal cancer; colloid cancer Glandular blastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer; small cell lung cancer; non-small cell lung cancer; lung adenocarcinoma; lung squamous cell carcinoma; melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth, and 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 or endometrial cancer; urological cancer; and vulvar cancer; lymphoma; Hodgkin lymphoma; non-Hodgkin lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin 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-cleaved cell NHL L; bulky NHL; mantle cell lymphoma; AIDS-associated 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-transplantation lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with nevus hamartomatosis, edema (such as that associated with brain tumors), and Meigs syndrome.

[0142] The DR5 agonist and PLK1 inhibitor can be administered to the subject as needed. The determination of the frequency of administration of each agent can be carried out by a person skilled in the art (such as an attending physician) based on consideration of the following factors: the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health status of the subject being treated, etc. In some embodiments, an effective dose of one or more therapeutic agents is administered to the subject once or more. In some embodiments, an effective dose of a DR5 agonist and / or a PLK1 inhibitor is administered to the subject every day, every half week, every week, every two weeks, once a month, etc. An effective dose of a DR5 agonist and / or a PLK1 inhibitor is administered to the subject at least once. In some embodiments, an effective dose of a DR5 agonist and / or a PLK1 inhibitor can be administered multiple times, including multiple administrations over a period of at least one month, at least six months, or at least one year.

[0143] In some embodiments, the DR5 agonist is administered in an amount effective for treating (including preventing) cancer. The therapeutically effective amount generally depends on the weight of the subject being treated, the physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated. Generally, the DR5 binding polypeptide can be administered in the following amounts: 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.

[0144] In some embodiments, the PLK1 inhibitor (or a pharmaceutically acceptable salt or hydrate thereof) is administered at 1 mg / m 2 Up to 1000 mg / m 2 (including, for example, 10 mg / m 2 Up to 500 mg / m 2 , 10mg / m 2 Up to 300 mg / m 2 or 10 mg / m 2 Up to 200 mg / m 2 In some embodiments, the PLK1 inhibitor (or a pharmaceutically 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).

[0145] In some embodiments, Avensetin is administered at 2 mg / m 2 With 100mg / m 2 In some embodiments, volacetib is administered at a dose of between 10 and 500 mg. In some embodiments, regaxetib is administered at a dose of between 10 and 1,000 mg.

[0146] In some embodiments, the therapeutic agent can be administered in vivo by various routes, including but not limited to oral, intramuscular, intravenous, intraarterial, parenteral, intraperitoneal or subcutaneous. Appropriate formulations and routes of administration can be selected according to the intended application.

[0147] 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.

[0148] In some embodiments, the DR5 agonist and the PLK1 inhibitor are administered concurrently.

[0149] In some embodiments, the DR5 agonist and the PLK1 inhibitor act synergistically. In some embodiments, the synergistic effect is determined in an in vitro cell survival assay. In some embodiments, administration of the DR5 agonist and the PLK1 inhibitor results in a synergistic effect compared to each agent administered alone.

[0150] In some embodiments, a DR5 agonist for use in a method of treating cancer is provided, wherein the method comprises administering the DR5 agonist in combination with a PLK1 inhibitor.

[0151] In some embodiments, there is provided a use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is for administration with a PLK1 inhibitor. Exemplary methods for treating cancer using DR5 agonists and CDK inhibitors

[0152] In some embodiments, a method of treating cancer in an individual is provided, the method comprising administering a DR5 agonist and a CDK inhibitor (such as a CDK9 inhibitor).

[0153] In some embodiments, the method comprises administering to an individual an effective amount of a DR5 agonist and a CDK inhibitor. Such treatment methods can be used for humans or animals. In some embodiments, methods for treating humans are provided. Non-limiting exemplary cancers that can be treated with a combination of a DR5 agonist and a CDK inhibitor provided herein include adrenal cancer; astrocytoma; basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; chondrosarcoma; Ewing's sarcoma; colorectal cancer (colorectal cancer); connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; gastrointestinal cancer; glioma blastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer; small cell lung cancer; non-small cell lung cancer; lung adenocarcinoma; lung squamous cell carcinoma; melanoma; myeloma; neuroblastoma; oral cancer (lip, 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; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; and vulvar cancer; lymphoma; Hodgkin lymphoma; non-Hodgkin lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin 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-cleaved cell NHL ; bulky NHL; mantle cell lymphoma; AIDS-associated 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-transplantation lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with nevus hamartomatosis, edema (such as that associated with brain tumors), and Meigs syndrome.

[0154] DR5 agonists and CDK inhibitors can be administered to subjects as needed. The determination of the frequency of administration of each agent can be carried out by a person skilled in the art (such as an attending physician) based on considerations of the following factors: the condition being treated, the age of the subject being treated, the severity of the condition being treated, the general health status of the subject being treated, etc. In some embodiments, an effective dose of one or more therapeutic agents is administered to a subject once or more. In some embodiments, an effective dose of DR5 agonists and / or CDK inhibitors are administered to a subject every day, every half week, every week, every two weeks, once a month, etc. An effective dose of DR5 agonists and / or CDK inhibitors are administered to a subject at least once. In some embodiments, an effective dose of DR5 agonists and / or CDK inhibitors can be administered multiple times, including multiple administrations over a period of at least one month, at least six months, or at least one year.

[0155] In some embodiments, the DR5 agonist is administered in an amount effective for treating (including preventing) cancer. The therapeutically effective amount generally depends on the weight of the subject being treated, the physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated. Generally, the DR5 binding polypeptide can be administered in the following amounts: 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.

[0156] In some embodiments, the CDK inhibitor (or a pharmaceutically acceptable salt or hydrate thereof) is administered at 1 mg / m 2 Up to 1000 mg / m 2 (including, for example, 10 mg / m 2 Up to 500 mg / m 2 , 10mg / m 2 Up to 300 mg / m 2 or 10 mg / m 2 Up to 200 mg / m 2 In some embodiments, the CDK inhibitor (or a pharmaceutically 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).

[0157] In some embodiments, flavopiridol is administered at 2 mg / m 2 With 100mg / m 2 In some embodiments, sericilib is administered at a dose of between 10 and 500 mg. In some embodiments, dinicili is administered at a dose of between 10 and 2,000 mg. In some embodiments, otociclib is administered at a dose of between 10 and 1,000 mg. In some embodiments, enecil is administered at a dose of between 10 and 500 mg. In some embodiments, AZD4573 is administered at a dose of between 1 and 100 mg.

[0158] In some embodiments, the therapeutic agent can be administered in vivo by various routes, including but not limited to oral, intramuscular, intravenous, intraarterial, parenteral, intraperitoneal or subcutaneous. Appropriate formulations and routes of administration can be selected according to the intended application.

[0159] 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.

[0160] In some embodiments, the DR5 agonist and the CDK inhibitor are administered concurrently.

[0161] In some embodiments, the DR5 agonist and the CDK inhibitor act synergistically. In some embodiments, the synergistic effect is determined in an in vitro cell survival assay. In some embodiments, the administration of the DR5 agonist and the CDK inhibitor results in a synergistic effect compared to each agent administered alone.

[0162] In some embodiments, a DR5 agonist for use in a method of treating cancer is provided, wherein the method comprises administering the DR5 agonist in combination with a CDK inhibitor.

[0163] In some embodiments, there is provided a use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is for administration with a CDK inhibitor. Reagent test kit

[0164] Also provided are articles and kits comprising any DR5 agonist and / or PLK1 inhibitor provided herein and suitable packaging. In some embodiments, the present invention includes a kit having (i) a formulation comprising a DR5 agonist, (ii) a formulation comprising a PLK1 inhibitor, and (iii) instructions for administering the formulation to an individual using the kit. In some embodiments, the present invention includes a kit having (i) a formulation comprising a DR5 agonist, and (ii) instructions for administering a combination of a formulation and a PLK1 inhibitor to an individual using the kit. In some embodiments, the present invention includes a kit having (i) a formulation comprising a PLK1 inhibitor, and (ii) instructions for administering a combination of a formulation and a DR5 agonist to an individual using the kit.

[0165] Also provided are preparations and kits comprising any DR5 agonist and / or CDK inhibitor provided herein and suitable packaging. In some embodiments, the present invention includes a kit having (i) a preparation comprising a DR5 agonist, (ii) a preparation comprising a CDK inhibitor, and (iii) instructions for using the kit to administer the preparation to an individual. In some embodiments, the present invention includes a kit having (i) a preparation comprising a DR5 agonist, and (ii) instructions for using the kit to administer a combination of a preparation and a CDK inhibitor to an individual. In some embodiments, the present invention includes a kit having (i) a preparation comprising a CDK inhibitor, and (ii) instructions for using the kit to administer a combination of a preparation and a DR5 agonist to an individual.

[0166] Suitable packaging for the compositions described herein is known in the art and includes, for example, vials (e.g., sealed vials), vessels, ampoules, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. These products may be further sterilized and / or sealed. Unit dosage forms comprising the compositions described herein are also provided. These unit dosage forms may be stored in suitable packaging in single or multiple unit doses and may also be further sterilized and sealed. The instructions provided in the kit of the present invention are typically written instructions on a label or drug instructions (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Instructions related to the use of DR5 agonists, PLK1 inhibitors and / or CDK inhibitors typically include information about dosages, dosing schedules, and routes of administration for intended treatment or industrial use. The kit may also include a description of selecting a suitable individual or treatment.

[0167] These containers can be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. For example, kits containing sufficient doses of the molecules disclosed herein can also be provided to provide effective treatment for individuals over an extended period of time, such as about one 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 any of longer periods of time. The kit can also include multiple unit doses of the molecule and instructions for use, and the packaging quantity is sufficient for storage and use in a pharmacy (e.g., a hospital pharmacy and a compounding pharmacy). In some embodiments, the kit comprises a dry (e.g., lyophilized) composition that can be reconstituted, resuspended or rehydrated to form a generally stable DR5 agonist aqueous solution. Example

[0168] The embodiments discussed below are intended only to illustrate the present invention, and therefore should not be considered to limit the present invention in any way. The embodiments are not intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure the accuracy of the numerals (e.g., amount, temperature, etc.) used, but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Example 1: Combination Activity of INBRX-109 and Avensetib

[0169] The combination of INBRX-109 and Avensetin was tested on various cancer cell lines to determine the cytotoxicity against cancer cells. Assay protocol

[0170] On the first day, cells were inoculated as follows. The monolayer culture of each cell line was harvested for compound screening as detailed below. The culture medium was aspirated, and the cells were washed once with PBS. Accutase was added and the flask was incubated at 37°C until the cells separated. An equal volume of complete culture medium was added to quench Accutase, and the cells were then pipetted up and down several times to produce a uniform single cell suspension. The density and viability of the cells were determined by trypan blue using a TC20 automated cell counter. The experimental cells were resuspended in Eagle's Extreme Essential Medium (EMEM) / 10% FBS / Anti-Anti culture medium (complete EMEM) to a concentration of 0.17x 10^6 / mL, and inoculated in the inner wells of a 384-well luminescent plate with 15 μL / well (finally 2,500 / well). Each cell line was laid on a separate plate in duplicate. The outer wells were filled with 50 μL PBS, and the plate was then placed in a humidified temperature-controlled 37°C tissue culture incubator at 5% CO 2 Incubate overnight at 4 °C for 16 h.

[0171] On day 2, the following test and control articles were prepared: Aventhiophene, INBRX-109, and Staurosporine.

[0172] Avensetib: A 10 mg stock solution of Avensetib was purchased from Selleck Chemicals and resuspended in DMSO to 10 mM. It was then divided into aliquots and stored at -80°C. Aliquots were thawed immediately prior to dilution and use in the assay. A 500x master plate of serial dilutions was prepared (6 5-fold dilutions starting from 500 μM in 100% DMSO, plus a DMSO-only control) and mixed gently with a pipette. To generate a 5x working dilution plate of Avensetib, a 1:100 dilution was made from the 500x plate into complete medium (EMEM).

[0173] INBRX-109: The INBRX-109 assay concentration range was chosen to include the minimum and maximum activity observed in previous cytotoxicity assays with several cancer cell lines, with 1 nM defined as the maximal effective concentration. A 50x master plate of serial dilutions of INBRX-109 was prepared (6 point 10-fold dilutions starting at 500 nM in complete EMEM, plus a complete EMEM only control) and mixed gently with a pipette. To generate a 5x working dilution plate of INBRX-109, dilute each well from the 50x master plate 1:10 into complete EMEM.

[0174] Staurosporine: Staurosporine was included in the assay as a positive control for cytotoxicity. A 10 mM DMSO stock solution purchased from the manufacturer was thawed, aliquoted, and stored at -80°C. Aliquots were thawed immediately prior to dilution and use in the assay. A 5x staurosporine working dilution (100 μM) was prepared by adding 5 μL of 10 mM staurosporine stock solution to 495 μL complete EMEM and then mixing thoroughly.

[0175] Also on day 2, add the test and control articles. Add the Avenset small molecule working dilution (5 μL of 5x), INBRX-109 working dilution (5 μL of 5x), or the staurosporine positive control (5 μL of 5x) to the corresponding experimental wells. Avenset stepwise adjustment is performed on the plate, and INBRX-109 stepwise adjustment is performed under the plate to obtain a matrix of all possible combinations of the two test articles. These test articles are added to each cell line in duplicate. The plate is then centrifuged at 400 x g for 1 minute and then placed in a 37°C humidity-controlled tissue culture incubator (5% CO 2 ) for 48 h.

[0176] On day 4, viability measurements were performed. The plates were equilibrated to room temperature for 10 minutes, and then 25 μL of CellTiter-Glo 4% DMSO in EMEM).Add in each well.Plate was rotated 1 minute with 400x g, then covered and incubated in the dark for 10 minutes at room temperature.Any visible bubbles were removed with 100% ethanol vapor, then 384 hole opaque plates were set and SoftMaxProv5.4 software was used, 50 milliseconds of integration time was adopted, luminescence (RLU) was read on Spectra Max M5e plate reader.In order to measure the impact of test article on cell viability, original RLU value was exported in Excel, and survival percentage was calculated as the percentage of vehicle control (0.5% DMSO in EMEM), wherein vehicle control was set to 100%.Data were mapped in GraphPad Prism9. result

[0177] Figure 1A-1F The results of a stepwise adjustment experiment are shown, in which a variety of cancer cell lines were contacted with different concentrations of INBRX-109 (0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) and Avensetin (0, 0.32, 1.6, 8, 40, 200 or 1000 nM). The survival percentage of the cancer cells is shown on the y-axis of each figure. Results for cancer cell lines HT-29, LS174T, SW620, SW837, SW1463 and LS411N are shown.

[0178] Figure 2A-2F The results of INBRX-109 stepwise adjustment experiments are shown, in which a variety of cancer cell lines are contacted with different concentrations of INBRX-109 (0.0001, 0.001, 0.01, 0.1, 1 or 10 nM) alone or in combination with 200 nM Avenset. The survival percentage of cancer cells is shown on the y-axis of each figure. Results for cancer cell lines HT-29, LS174T, SW620, SW837, SW1463 and LS411N are shown. The dotted line marked "Cpd alone" shows the survival percentage of cancer cells treated with 200 nM Avenset alone.

[0179] Figure 3A-Figure 3F The results of the stepwise adjustment experiment of Avensetib are shown, in which various cancer cell lines are contacted with different concentrations of Avensetib (0.32, 1.6, 8, 40, 200 or 1000 nM) alone or in combination with 1 nM INBRX-109. The survival percentage of the cancer cells is shown on the y-axis of each figure. The results of the cancer cell lines HT-29, LS174T, SW620, SW837, SW1463 and LS411N are shown. The dotted line marked "Ab alone" shows the survival percentage of cancer cells treated with INBRX-109 alone.

[0180] Table 1 shows the EC of the stepwise adjustment curve of INBRX-109 with and without 200 nM Avensetin 50 The maximum cytotoxicity was calculated as 100 minus the % survival. Table 1

[0181] Table 2 shows the EC of the stepwise adjustment curve of Avensetin with and without 1 nM INBRX-109 50 The maximum cytotoxicity was calculated as 100 minus the % survival. Table 2

[0182] The combination of INBRX-109 and Avensetib showed enhanced cell killing against every cancer cell line tested in this screen. Greater benefit was generally observed at Avensetib concentrations above 40 nM. At these concentrations, the combination produced a synergistic effect, as demonstrated by the increased activity of ECs treated with INBRX-109 alone. 50 In comparison, the EC of INBRX-109 50 See, e.g., Figure 2 and Table 1. In addition, this combination resulted in a decrease in overall cell survival compared to treatment with either drug alone.

[0183] Cell killing was determined essentially as described above for additional cancer cell lines in the presence of 1 nM INBRX-109 alone, Avensetib alone, or a combination of 1 nM INBRX-109 and varying concentrations of Avensetib (274 pM, 823 pM, 2.47 nM, 7.41 nM, 22.22 nM, 66.67 nM, 200 nM, 600 nM, and 1.8 μM). Tables 3 and 4 and Figures 4-8 show the maximum cytotoxicity of 1 nM INBRX-109 alone, and the EC of the Avensetib stepwise adjustment curve with and without 1 nM INBRX-109. 50 The results for a panel of colorectal cancer cell lines are shown in Table 3 and Figures 4-6 , and the results for a panel of pancreatic cell lines are shown in Table 4 and Figures 7 and 8 . Table 3 Table 4

[0184] The combination of INBRX-109 and Avensetib showed enhanced cell killing against the cancer cell lines tested in this screen compared to either agent alone, and in some cases, the addition of 1 nM INBRX-109 significantly reduced the EC50 compared to Avensetib alone.

[0185] The data suggest that the combination of a DR5 agonist (such as INBRX-109) and a PLK1 inhibitor (such as Avensetin) results in improved or synergistic cancer cell killing compared to either drug by itself. Example 2: Combination Activity of INBRX-109 and CDK9 Inhibitors

[0186] Additional assays were performed to test the combination of INBRX-109 and cyclin-dependent kinase 9 (CDK9) inhibitors (dinisili, NVP-2, flavopiridol, and enisili) or a downstream MCL-1 inhibitor (AZD5991) on various cell lines to determine cytotoxicity against cancer cells. Assay protocol

[0187] On the first day, cells were inoculated as follows. Monolayer cultures of each cell line (chondrosarcoma: CAL-78, OUMS-27, SW1353 and H-EMC-SS; colorectal cancer: SW620; and synovial sarcoma: HS-SY-II) were harvested for compound screening. The culture medium was aspirated, and the cells were washed once with PBS. Accutase was added and the flask was incubated at 37°C until the cells separated. An equal volume of complete culture medium was added to quench Accutase, and the cells were then pipetted up and down several times to produce a uniform single cell suspension. The density and viability of the cells were determined by trypan blue using a TC20 automated cell counter. The experimental cells were resuspended in Eagle's Extreme Essential Medium (EMEM) / 10% FBS / Anti-Anti Medium (complete EMEM) to a concentration of 0.17x 10^6 / mL, and inoculated in the inner wells of a 384-well luminescent plate with 15mL / well (finally 2,500 / well). Each cell line was plated in duplicate on a separate plate. The outer wells were filled with 50 mL of PBS and the plates were incubated overnight in a humidified temperature controlled 37°C tissue culture incubator at 5% CO2 for 16 hours.

[0188] On Day 2, the following test articles and INBRX-109 were prepared as follows: Dinicetine (purchased from MedChemExpress): Prepare 5X in complete medium (EMEM) Working serial dilution plate (6-point 5-fold dilutions, starting at 312.5uM, final concentration range 625nM to 0.2 nM). NVP-2 (purchased from MedChemExpress): Prepare a 5X working serial dilution plate (6-point 5-fold dilutions starting from 312.5uM, with a final concentration range of 625nM to 0.2nM) in complete medium (EMEM). Flavin (purchased from MedChemExpress): Prepare 5X in complete medium (EMEM) Working serial dilution plate (6-point 5-fold dilutions, starting at 312.5uM, final concentration range 625nM to 0.2 nM). Enisili (purchased from MedChemExpress): Prepare 5X in complete medium (EMEM) Working serial dilution plate (6-point 5-fold dilutions, starting at 312.5uM, final concentration range 625nM to 0.2 nM). AZD-5991 (purchased from MedChemExpress): Prepare 5X in complete medium (EMEM) Working serial dilution plate (6-point 5-fold dilutions, starting from 5 mM, with final concentrations ranging from 10 uM to 3.2nM). INBRX-109: 5x working serial dilution plate in complete medium (EMEM) (6 point 10-fold dilutions starting at 50 nM, with final concentrations ranging from 10 nM to 0.0001 nM).

[0189] Test articles were added to the cells as follows: small molecule compound working dilution (5 μL of 5x), INBRX-109 working dilution (5 μL of 5x), or culture medium alone was added to the corresponding experimental wells. Small molecule compounds were stepped horizontally on the plate, and INBRX-109 was stepped vertically on the plate to obtain a matrix of all possible combinations of the two test articles. These test articles were added to each cell line in duplicate. The plate was then centrifuged at 400 x g for 1 minute and then placed in a 37°C humidity controlled tissue culture incubator (5% CO 2 ) for 48 h.

[0190] After incubation with the test article, viability measurements were performed briefly. The plates were equilibrated to room temperature for 10 minutes, and then 25 μL of CellTiter-Glo Add to each well. Spin the plate at 400 x g for 1 minute, then cover and incubate at room temperature in the dark for 10 minutes. Remove any visible bubbles with 100% ethanol vapor, then use a 384-well opaque plate setup and Luminescence (RLU) was read on a Spectra Max M5e plate reader using Spectra Pro v5.4 software with an integration time of 50 milliseconds. To determine the effect of the test article on cell viability, the raw RLU values ​​were exported to Excel and the survival percentage was calculated as the percentage of the vehicle control (0.5% DMSO in EMEM), where the vehicle control was set to 100%. Data were plotted in GraphPad Prism 9. result

[0191] The results of these step-wise adjustment experiments are shown in the following figures: Figure 9A-9E (gradually adjusted INBRX-109 and Dinisili), Figures 10A-10E (stepwise adjusted INBRX-109, alone or with 25 nM dinisili), Figures 11A-11E (stepwise adjustment of dinisili, alone or with 1nM INBRX-109), Figure 12A-12E (gradually adjusted INBRX-109 and NVP-2), Figures 13A-13E (stepwise adjustment of INBRX-109, alone or with 125 nM NVP-2), Figure 14A-14E (stepwise adjustment of NVP-2, alone or with 1 nM INBRX-109), Figures 15A-15F (gradually adjusted INBRX-109 and Flavin), Figures 16A-16F (stepwise adjusted INBRX-109, alone or with 125 nM flavopiridol), Figures 17A-17F (stepwise adjustment of flavopiridol concentration, alone or with 1 nM INBRX-109), Figure 18A-18F (step-wise adjustment of INBRX-109 and Enisili), Figure 19A-19F (stepwise adjusted INBRX-109, alone or with 125 nM enecillib), Figures 20A-20F (stepwise titration of enecilib, alone or with 1 nM INBRX-109), Figure 21A-Figure 21F (step-wise adjustment of INBRX-109 and AZD-5991), Figure 22A-22F (stepwise adjusted INBRX-109, alone or with 2 nM AZD-5991) and Figure 23A-23F (AZD-5991 was titrated, alone or with 1 nM INBRX-109). The dashed line labeled "Ab alone" shows the percent survival of cancer cells treated with 1 nM INBRX-109 alone, and the dashed line labeled "Cpd alone" shows the percent survival of cancer cells treated with the indicated small molecule inhibitors.

[0192] EC50 values ​​of stepwise adjustment curves of INBRX-109 with and without 25 nM dinisib are shown in Table 5. Maximum cytotoxicity was calculated as 100 minus % survival. Table 5

[0193] EC50 values ​​of the stepwise adjustment curves of dinisib with and without 1 nM INBRX-109 are shown in Table 6. Maximum cytotoxicity was calculated as 100 minus % survival. Table 6

[0194] EC50 values ​​of stepwise adjustment curves of INBRX-109 with and without 25 nM NVP-2 are shown in Table 7. Maximum cytotoxicity was calculated as 100 minus % survival. Table 7

[0195] EC50 values ​​for the stepwise adjustment curves of NVP-2 with and without 1 nM INBRX-109 are shown in Table 8. Maximum cytotoxicity was calculated as 100 minus % survival. Table 8

[0196] EC50 values ​​of stepwise adjustment curves of INBRX-109 with and without 25 nM flavopiridol are shown in Table 9. Maximum cytotoxicity was calculated as 100 minus % survival. Table 9

[0197] EC50 values ​​of the stepwise adjustment curves of flavopiridol with and without 1 nM INBRX-109 are shown in Table 10. Maximum cytotoxicity was calculated as 100 minus % survival. Table 10

[0198] EC50 values ​​of stepwise adjustment curves of INBRX-109 with and without 125 nM enecillib are shown in Table 11. Maximum cytotoxicity was calculated as 100 minus % survival. Table 11

[0199] Table 12 shows the EC50 values ​​of the stepwise adjustment curves of enecil with and without 1 nM INBRX-109.Maximum cytotoxicity was calculated as 100 minus % survival. Table 12

[0200] EC50 values ​​for stepwise adjustment curves of INBRX-109 with and without 2 μM AZD-5991 are shown in Table 13. Maximum cytotoxicity was calculated as 100 minus % survival. Table 13

[0201] EC50 values ​​for the stepwise adjustment curves of AZD-5991 with and without 1 nM INBRX-109 are shown in Table 14. Maximum cytotoxicity was calculated as 100 minus % survival. Table 14

[0202] The combination of INBRX-109 with each CDK9 inhibitor tested (dinisili, NVP-2, flavopiridol and enecil) showed enhanced cell killing of almost every cancer cell line tested in these screens. The combination of INBRX-109 with AZD-5991 (an inhibitor of MCL-1 (a CDK9 downstream target)) also showed enhanced cell killing of many cell lines. Generally, when the concentrations of dinisili and NVP-2 were above 25nM, greater benefits were observed when the concentrations of flavopiridol and enecil were 125nM or higher and when the concentration of AZD-5991 was above 0.4μM. At these concentrations, the combination produced a synergistic effect, as shown by the changes in the killing curves of INBRX-109 combined with CDK9 and downstream MCL-1 inhibitors compared to the killing curves of INBRX-109 alone. See, for example, Figures 9-23 and Tables 5-14. Furthermore, this combination resulted in a decrease in overall cell survival compared to treatment with either drug alone.

[0203] The present invention may be implemented in other specific forms without departing from the spirit or essential features of the present disclosure. Therefore, the foregoing embodiments are considered to be illustrative in all respects, rather than limiting the present disclosure. The scope of the present disclosure is therefore indicated by the appended claims rather than by the foregoing description, and therefore all changes within the equivalent meaning and scope of the claims are intended to be included herein. List of some sequences

Claims

1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject (a) a death receptor 5 (DR5) agonist and (b) a Polo-like kinase 1 (PLK1) inhibitor.

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

3. The method of claim 2, wherein the DR5 agonist is INBRX-109.

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

5. The method of claim 4, wherein the DR5 binding polypeptide is at least tetravalent.

6. The method of any one of claims 3-5, 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.

7. The method of claim 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.

8. The method of any one of claims 3-7, wherein the DR5 binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:

4.

9. The method of any one of claims 3-8, wherein the DR5 binding polypeptide comprises an Fc region.

10. The method of claim 9, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:

6.

11. The method of any one of claims 3-10, wherein the DR5 binding polypeptide has the structure VHH-linker-VHH-linker-Fc.

12. The method of any one of claims 3-11, 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.

13. The method of any one of claims 3-12, 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. The method of claim 13 , wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:

5.

15. The method of any one of claims 3-14, 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.

16. The method of any one of claims 3-15, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

17. The method according to any one of claims 3 to 15, wherein the DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:

7.

18. The method of any one of claims 1-17, wherein the PLK1 inhibitor is a small molecule or interfering RNA (siRNA).

19. The method according to any one of claims 1 to 18, wherein the PLK1 inhibitor is Avensetib, Volasetib, Regosatib, BI2536 (Boehringer Ingelheim), N-[[4-[(6-chloro-3-pyridinyl)methoxy]-3-methoxyphenyl]methyl]-3,4-dimethoxyphenethylamine hydrochloride (SBE 13HCl), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), poloxin, poloxin-2HT, RO3280 (CAS No. 1062243-51-9), HMN-214 (CAS No. 173529-46-9), HMN-176 (CAS No. 173529-10-7), 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-thiazolinone) or cycloboline 9 (CAS No. 40533-25-3), 5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-[[2-(trifluoromethyl)phenyl]methoxy]-2-thiophenecarboxamide (GW 843682X).

20. The method of claim 19, wherein the PLK1 inhibitor is avensetib, volasetib, reglucicetib, BI2536 (Boehringer Ingelheim), MLN0905 (Takeda Oncology), GSK461364 (GlaxosSmithKline), CYC140 (Cyclacel), TKM-080301 (TKM-PLK1; Arbutus Biopharma), or TAK-960 (Takeda Pharmaceutical Company).

21. The method of any one of claims 1-18, wherein the PLK1 inhibitor is Avensetib.

22. The method of any one of claims 1-21, wherein the DR5 agonist and the PLK1 inhibitor are administered separately.

23. The method of claim 22, wherein the DR5 agonist and the PLK1 inhibitor are administered sequentially.

24. The method of claim 22 or 23, wherein at least one dose or a first dose of the DR5 agonist is administered prior to the PLK1 inhibitor.

25. The method of claim 22 or 23, wherein at least one dose or the first dose of the DR5 agonist is administered after the PLK1 inhibitor.

26. The method of any one of claims 1-21, wherein the DR5 agonist and the PLK1 inhibitor are administered concurrently.

27. The method of any one of claims 1-26, wherein the DR5 agonist and the PLK1 inhibitor act synergistically.

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

29. The method of any one of claims 1-28, wherein administration of the DR5 agonist and the PLK1 inhibitor results in a synergistic effect compared to each agent administered alone.

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

31. The method of claim 30, wherein the DR5 agonist is INBRX-109, etonamin alfa (ABBV-621), IGM-8444 (IGM Biosciences), BI 905711 (Boehringer Ingelheim), GEN1029 ( -DR5 / DR5; Genmab), TAS266 (Novartis), MM-201a (Merrimack Pharmaceuticals) or MM201-b (Merrimack Pharmaceuticals).

32. The method of claim 31 , wherein the DR5 agonist is INBRX-109.

33. The method of claim 30, wherein the DR5 agonist is a DR5 binding polypeptide.

34. The method of claim 33, wherein the DR5 binding polypeptide is at least tetravalent.

35. The method of any one of claims 32-34, 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.

36. The method of claim 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.

37. The method of any one of claims 32-36, wherein the DR5 binding polypeptide comprises a VHH domain comprising the amino acid sequence of SEQ ID NO:

4.

38. The method of any one of claims 32-37, wherein the DR5 binding polypeptide comprises an Fc region.

39. The method of claim 38, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:

6.

40. The method of any one of claims 32-39, wherein the DR5 binding polypeptide has the structure VHH-Linker-VHH-Linker-Fc.

41. The method of any one of claims 32-40, 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.

42. The method of any one of claims 32-41, 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.

43. The method of claim 42, wherein the VHH-linker-VHH comprises the amino acid sequence of SEQ ID NO:

5.

44. The method of any one of claims 32-43, 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.

45. The method of any one of claims 32-44, wherein the DR5 binding polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

46. ​​The method of any one of claims 32-44, wherein the DR5 binding polypeptide consists of the amino acid sequence of SEQ ID NO:

7.

47. The method of any one of claims 30-46, wherein the CDK inhibitor is a CDK9 inhibitor.

48. The method of any one of claims 30-47, wherein the CDK inhibitor is a small molecule.

49. The method of any one of claims 30-48, wherein the CDK inhibitor is flavopiridol, cericislib, dinicislib, otociclib, enecillib, AZD4573, i-CDK9 or NVP-2.

50. The method of any one of claims 49, wherein the CDK inhibitor is dinisili, NVP-2, flavopiridol, enisili or AZD4573.

51. The method of any one of claims 30-50, wherein the DR5 agonist and the CDK inhibitor are administered separately.

52. The method of claim 51, wherein the DR5 agonist and the CDK inhibitor are administered sequentially.

53. The method of claim 51 or 52, wherein at least one dose or a first dose of the DR5 agonist is administered prior to the CDK inhibitor.

54. The method of claim 51 or 52, wherein at least one dose or the first dose of the DR5 agonist is administered after the CDK inhibitor.

55. The method of any one of claims 30-51, wherein the DR5 agonist and the CDK inhibitor are administered concurrently.

56. The method of any one of claims 30-55, wherein the DR5 agonist and the CDK inhibitor act synergistically.

57. The method of claim 56, wherein synergy is determined in an in vitro cell survival assay.

58. The method of any one of claims 30-57, wherein administration of the DR5 agonist and the CDK inhibitor results in a synergistic effect compared to each agent administered alone.

59. The method of any one of claims 1-58, wherein the cancer is adrenal cancer; astrocytoma; basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; chondrosarcoma; Ewing's sarcoma; colorectal cancer (colorectal cancer); connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer; gastrointestinal cancer; glioblastoma; liver cancer; hepatoma; intraepithelial neoplasia; kidney cancer or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer; small cell lung cancer; non-small cell lung cancer; lung adenocarcinoma; lung squamous cell carcinoma; melanoma; myeloma; neuroblastoma; oral cancer (lip, tongue, mouth and / or pharynx); ovarian cancer; pancreatic cancer, such as pancreatic adenocarcinoma; pituitary cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colorectal cancer; respiratory cancer; mesothelioma; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urological cancer; and vulvar cancer; lymphoma; Hodgkin lymphoma; non-Hodgkin lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin 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-cleaved cell NHL; large mass 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-transplantation lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with nevus hamartomatosis, edema (such as that associated with brain tumors), and Meigs syndrome.

60. A DR5 agonist for use in a method of treating cancer, wherein the method comprises administering the DR5 agonist in combination with a PLK1 inhibitor.

61. Use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is for administration together with a PLK1 inhibitor.

62. A DR5 agonist for use in a method of treating cancer, wherein the method comprises administering the DR5 agonist in combination with a CDK inhibitor, such as a CDK9 inhibitor.

63. Use of a DR5 agonist in the manufacture of a medicament for treating cancer, wherein the medicament is for administration together with a CDK inhibitor, such as a CDK9 inhibitor.

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