Targeted topoisomerase i inhibitor therapy for treating cancer

Abstract

Conjugates comprising one or more TOP1i payloads and an EETI-II based knottin peptide, comprising an engineered loop that binds to a cell surface molecule, which may be used for treating cancer in a patient.

Description

Attorney Docket Number: 01384-0002-00PCTTARGETED TOPOISOMERASE I INHIBITOR THERAPY FOR TREATING CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 765,208, filed February 28, 2025, the entire contents of which are incorporated by reference herein for all purposes.REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on February 25, 2026, is named “01384-0002-00PCT.xml” and is 60,791 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.DESCRIPTIONFIELD

[0003] Targeted topoisomerase I inhibitor therapy for treating cancer.BACKGROUND

[0004] Current cancer chemotherapies aim to eliminate cancer cells but often affect healthy tissues, leading to significant side effects. Among the chemotherapeutic agents, topoisomerase I inhibitors (TOP1i) have shown considerable promise due to their ability to interfere with DNA replication and subsequent cell division. TOP1 i drugs exert their action by binding to DNA-topoisomerase I complexes, and thus inhibiting DNA cutting, relaxing, and reannealing processes, ultimately leading to cell death.

[0005] These small molecule inhibitors are effective against various cancers, including colorectal, ovarian, cervical, and small cell lung cancers. Despite their efficacy, the clinical application of TOP1i drugs is often limited by severe side effects, such as hematologic and gastrointestinal toxicity. These adverse effects arise from the non-specific distribution of the drugs, which affects both cancerous and healthy cells.

[0006] To overcome these challenges, there is a growing need for targeted delivery systems that can direct TOP1i drugs specifically to tumor cells. Tumor-targeted conjugates offer a promising solution by linking the inhibitors to molecules that recognize and bind to cancerspecific markers. This targeted approach enhances the concentration of the drug in tumor tissues while minimizing exposure to healthy cells, thereby reducing side effects and improving therapeutic outcomes. Despite the advancements in tumor-targeted TOP1i conjugates, particularly antibody-drug conjugates (ADCs), the majority of patients with solidAttorney Docket Number: 01384-0002-00PCTtumors are not eligible for the currently approved ADC TOP1i treatments. This underscores a critical unmet need in the field of oncology. This limitation arises, at least in part, because currently approved TOP1i ADCs are directed to a limited set of validated tumor-associated antigens and, consequently, are deployed only in biomarker-defined patient subsets and / or specific tumor indications. Accordingly, there is a need for targeted TOP1i conjugates directed to alternative tumor-associated targets to expand patient eligibility.

[0007] Integrin-targeting agents hold great promise as they can be applied to a wide range of tumor types, offering a versatile approach to cancer treatment. The conjugates described herein have the potential to significantly advance cancer therapy by providing an effective and safe means of targeting TOP1i drugs to solid tumors, expanding treatment options for broader patient populations.SUMMARY

[0008] Provided are topoisomerase I inhibitor drug conjugates comprising tumor-targeting moieties comprising engineered knottin peptides.

[0009] Embodiment 1. A conjugate comprising:a. an EETI-II based knottin peptide, comprising an engineered loop that binds to a cell surface molecule; andb. at least one topoisomerase I inhibitor (TOP1i) payload.

[0010] Embodiment 2. The conjugate of embodiment 1, wherein the at least one TOP1i payload is attached via at least one linker to form at least one linker-payload complex.

[0011] Embodiment 3. The conjugate of embodiment 2, wherein the conjugate comprises one linker-payload complex.

[0012] Embodiment 4. The conjugate of embodiment 2, wherein the conjugate comprises two, three, four, five, six, seven, eight, nine, ten, or more linker-payload complexes.

[0013] Embodiment 5. The conjugate of any one of embodiments 2-4, wherein at least one linker-payload complex comprises a single linker conjugated to a single TOP1i payload.

[0014] Embodiment 6. The conjugate of any one of embodiments 2-5, wherein at least one linker-payload complex comprises a branched linker.

[0015] Embodiment 7. The conjugate of embodiment 6, wherein the branched linker is conjugated to two, three, four, five, six, seven, eight, nine, ten, or more TOP1i payloads.

[0016] Embodiment 8. The conjugate of any one of embodiments 6-7, wherein the branched linker is conjugated to two TOP1i payloads.Attorney Docket Number: 01384-0002-00PCT

[0017] Embodiment 9. The conjugate of any one of embodiments 6-7, wherein the branched linker is conjugated to three TOP1i payloads.

[0018] Embodiment 10. The conjugate of any one of embodiments 6-7, wherein the branched linker is conjugated to four TOP1i payloads.

[0019] Embodiment 11. The conjugate of any one of embodiments 1-10, wherein the TOP1i payloads comprise the same TOP1 i.

[0020] Embodiment 12. The conjugate of any one of embodiments 1-10, comprising two or more TOP1 i payloads comprising two or more different TOP1 i.

[0021] Embodiment 13. The conjugate of any one of embodiments 1-12, wherein the conjugate is capable of treating cancer in a patient.

[0022] Embodiment 14. The conjugate of any one of embodiments 1-13, wherein the cell surface molecule is present on cancer cells.

[0023] Embodiment 15. The conjugate of any one of embodiments 1-14, wherein the cell surface molecule is an integrin.

[0024] Embodiment 16. The conjugate of embodiment 15, wherein the integrin is av|33 integrin, av05 integrin, av 6 integrin, av01 integrin, and / or a501 integrin.

[0025] Embodiment 17. The conjugate of any one of embodiments 1-16, wherein the EETI-II based knottin peptide comprises GCXiX2X3X4X5X6X7X8X9X1oX11Xi2X13X14X15X16X17X18X19X2OCX2iQDSDCX22AGCVCX23 X24 X25 X26X27X2s X2g X30 X31 X32X33CG (SED ID NO: 2), wherein Xi - X3 are any amino acid; X4-X20 if present, are any amino acid; X2i, X22, and X23- X26, if present are any amino acid; X27-X33if present, are any amino acid; further wherein each amino acid is independently selected from standard or unnatural amino acids.

[0026] Embodiment 18. The conjugate of any one of embodiments 1-16, wherein the EETI-II based knottin peptide comprises GCXiX2X3X4X5X6X7X8X9Xi0X1iXi2X13X14X15X16X17Xi8X19X20CX2iQDSDCX22AGCVCGPNGX23CG (SEQ ID NO: 3), wherein X1-X3 are any amino acid; X4-X20, if present, are any amino acid; and wherein X2i-X23are any amino acid, further wherein each amino acid is independently selected from standard or unnatural amino acids.

[0027] Embodiment 19. The conjugate of any one of embodiments 1-18, wherein the engineered loop has a sequence that differs by no more than 2 amino acids from any one of SEQ ID NO: 4 through SEQ ID NO: 8, inclusive.Attorney Docket Number: 01384-0002-00PCT

[0028] Embodiment 20. The conjugate of any one of embodiments 1-18, wherein the engineered loop has a sequence that differs by no more than 1 amino acid from any one of SEQ ID NO: 4 through SEQ ID NO: 8, inclusive.

[0029] Embodiment 21. The conjugate of any one of embodiments 1-18, wherein the engineered loop has a sequence of any one of SEQ ID NO: 4 through SEQ ID NO: 8, inclusive.

[0030] Embodiment 22. The conjugate of any one of embodiments 1-21, wherein the EETI-II based knottin peptide has a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 9 through SEQ ID NO: 35, inclusive.

[0031] Embodiment 23. The conjugate of embodiment 22, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 12 through SED ID NO: 35, inclusive.

[0032] Embodiment 24. The conjugate of any one of embodiments 1-23, wherein the engineered loop comprises the amino acid sequence RGD.

[0033] Embodiment 25. The conjugate of any one of embodiments 1-24, wherein the at least one TOP1i payload comprises a camptothecin ora derivative thereof.

[0034] Embodiment 26. The conjugate of any one of embodiments 1-25, wherein the at least one TOP1i payload comprises: belotecan (CKD-602), camptothecin, cositecan (BNP-1350), DDDXd, diflomotecan, DXd, Ed-04, exatecan, exatecan mesylate (DX-8951f), gimatecan (ST1481), GI-147211C, irinotecan (CPT-11), karenitecin, lurtotecan, MH30010008, Rezetecan (SHR9265), rubitecan, silatecan (DB-67, AR-67), SN-38, S39625, topotecan, ZD06519 (FD1), 7-ethyl camptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, or (20S)-camptothecin.

[0035] Embodiment 27. The conjugate of any one of embodiments 1-26, wherein the at least one TOP1i payload comprises exatecan or DXd.

[0036] Embodiment 28. The conjugate of any one of embodiments 2-27, wherein at least one linker is a cleavable linker.

[0037] Embodiment 29. The conjugate of embodiment 28, wherein at least one linker is acid-cleavable or enzymatically cleavable.

[0038] Embodiment 30. The conjugate of any one of embodiments 28-29, wherein the cleavable linker is a protease cleavable linker.Attorney Docket Number: 01384-0002-00PCT

[0039] Embodiment 31. The conjugate of any one of embodiments 28-30, wherein the cleavable linker is cleaved by endosomal or lysosomal proteases.

[0040] Embodiment 32. The conjugate of any one of embodiments 28-31, wherein the cleavable linker is a cathepsin-cleavable linker.

[0041] Embodiment 33. The conjugate of any one of embodiments 28-31, wherein the cleavable linker is a beta-glucuronidase-cleavable linker.

[0042] Embodiment 34. The conjugate of any one of embodiments 2-33, wherein at least one linker comprises a cleavage motif selected from: GGFG (SEQ ID NO: 52), Valine-Citrulline-PAB (Val-Cit-PAB), Glutamic Acid-Valine-Citrulline-PAB (Glu-Val-Cit-PAB), Valine-Alanine-PAB (Val-Ala-PAB).

[0043] Embodiment 35. The conjugate of any one of embodiments 2-34, wherein at least one linker is a noncleavable linker.

[0044] Embodiment 36. The conjugate of any one of embodiments 28-35, wherein at least one linker comprises at least one polyethylene glycol (PEG) unit.

[0045] Embodiment 37. The conjugate of any one of embodiments 2-36, wherein the at least one linker payload complex is conjugated to the EETI-ll-based knottin peptide.

[0046] Embodiment 38. The conjugate of any one of embodiments 1-37, further comprising an antibody or antibody fragment comprising an Fc domain fused to the EETI-II based knottin peptide.

[0047] Embodiment 39. The conjugate of embodiment 38, wherein the at least one linkerpayload complex is conjugated to the antibody or antibody fragment comprising an Fc domain.

[0048] Embodiment 40. The conjugate of embodiment 39, wherein the at least one linkerpayload complex is conjugated to the antibody or antibody fragment comprising an Fc domain at one or more glycans attached to the antibody or antibody fragment comprising an Fc domain.

[0049] Embodiment 41. A conjugate comprising:a. an EETI-II based knottin peptide comprising the amino acid sequence of SEQ ID NO: 14; andb. two TOP1 i payloads covalently attached via a branched linker; wherein the branched linker comprises at least one Val-Ala-PAB cleavage domain.

[0050] Embodiment 42. The conjugate of embodiment 41, wherein at least one of the two TOP1i payloads is selected from exatecan or DXd.Attorney Docket Number: 01384-0002-00PCT

[0051] Embodiment 43. A method of treating cancer, comprising administering the conjugate of any one of embodiments 1-42 to a patient in need thereof.

[0052] Embodiment 44. Use of the conjugate of any one of embodiments 1 -42 in the manufacture of a medicament for treating cancer in a patient in need thereof.

[0053] Embodiment 45. The conjugate of any one of embodiments 1-42 for use in treating cancer in a patient in need thereof.

[0054] Embodiment 46. The method or use of any one of embodiments 43-45, wherein the cancer is a solid tumor.

[0055] Embodiment 47. The method or use of embodiment 45, wherein the cancer is selected from blastoma, carcinoma, lymphoma, and sarcoma.

[0056] Embodiment 48. The method or use of embodiment 47, wherein the cancer is selected from colorectal cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, uterine cancer, ovarian cancer, and gastric cancer.

[0057] Embodiment 49. The method or use of embodiment 47, wherein the cancer is selected from colorectal cancer, head and neck cancer, lung cancer, esophageal cancer, uterine cancer, ovarian cancer, gastric cancer, pancreatic cancer, breast cancer, bladder cancer, melanoma, renal cancer, liver cancer, gallbladder cancer, sarcomas, and brain cancer.

[0058] Embodiment 50. The method or use of embodiment 47, wherein the cancer is selected from adrenal cancer such as but not limited to, adrenocortical carcinoma and pheochromocytoma; bladder cancers such as but not limited to, adenocarcinoma, carcinosarcoma, squamous cell cancer, and transitional cell carcinoma; basal cancers; bone cancer and connective tissue sarcomas such as but not limited to, angiosarcoma (hemangiosarcoma), bone sarcoma, cholesteatoma-induced bone osteosarcoma, chondrosarcoma, chordoma, Ewing's sarcoma, fibrosarcoma, fibrosarcoma of bone, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, malignant giant cell tumor, multiple myeloma, myeloma bone disease, neurilemmoma, osteogenic sarcoma, osteosarcoma, Paget's disease of bone, periosteal sarcoma, rhabdomyosarcoma, soft-tissue sarcomas, and synovial sarcoma; brain tumors such as but not limited to, acoustic neurinoma, astrocytoma, brain stem glioma, craniopharyngioma, ependymoma, glioblastoma multiforme, glioma, medulloblastoma, meningioma, nonglial tumor, oligodendroglioma, pineoblastoma, pineocytoma, and primary brain lymphoma; breast cancer including but not limited to, breast carcinoma, breast sarcoma, and in some embodiments, adenocarcinoma, inflammatory breast cancer, intraductal carcinoma, lobular (small cell) carcinoma, medullaryAttorney Docket Number: 01384-0002-00PCTbreast cancer, metastatic breast cancer, mucinous breast cancer, Paget's disease (including juvenile Paget's disease), papillary breast cancer, and tubular breast cancer; cervical cancers such as but not limited to, adenocarcinoma, cervical carcinoma, and squamous cell carcinoma; cholangiocarcinomas such as but not limited to, diffuse, nodular, and papillary; colorectal cancer (colon cancer and rectal cancer), including but not limited to colon carcinoma and KRAS mutated colorectal cancer; cystadenocarcinoma; endotheliosarcoma and lymphangioendotheliosarcoma; esophageal cancers such as but not limited to, adenocarcinoma, adenoid cyctic carcinoma, adenosquamous carcinoma, melanoma, mucoepidermoid carcinoma, oat cell (small cell) carcinoma, plasmacytoma, sarcoma, squamous cancer, and verrucous carcinoma; eye cancers such as but not limited to, choroidal melanoma, ciliary body melanoma, ocular melanoma such as iris melanoma, and retinoblastoma; gallbladder cancers such as adenocarcinoma; hemangioblastoma; head and neck cancer, such as but not limited to squamous cell head and neck cancer, hematological malignancies such as but not limited to benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, heavy chain disease, leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as erythroleukemia leukemias, monocytic, myeloblastic, myelodysplastic syndrome, myelomonocytic, promyelocytic, chronic leukemias such as but not limited to hairy cell leukemia, chronic lymphocytic leukemia, and chronic myelocytic (granulocytic) leukemia, lymphomas such as but not limited to Hodgkin's disease and non-Hodgkin's disease, multiple myelomas such as but not limited to, extramedullary plasmacytoma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, smoldering multiple myeloma and solitary plasmacytoma, and polycythemia vera; kidney cancers such as but not limited to, adenocarcinoma, fibrosarcoma, hypernephroma, renal cell cancer, transitional cell cancer (renal pelvis and / or uterer), and Wilms tumor; liver cancers such as but not limited to, hepatoblastoma and hepatocellular carcinoma; lung cancers such as but not limited to adenocarcinoma, bronchogenic carcinoma, KRAS-mutated non-small cell lung cancer, largecell carcinoma, lung carcinoma, non-small cell lung cancer, papillary adenocarcinoma, smallcell lung cancer and squamous cell carcinoma (epidermoid carcinoma); mesothelioma myxosarcoma; neuroblastoma; neurofibroma; neurofibromatosis; oral cancers such as but not limited to, squamous cell carcinoma; ovarian cancers such as but not limited to, borderline tumor, germ cell tumor, ovarian epithelial carcinoma, and stromal tumor; papillary adenocarcinoma and papillary carcinoma; pancreatic cancer such as but not limited to, carcinoid or islet cell tumor, gastrinoma, glucagonoma, insulinoma, somatostatin-secreting tumor, and vipoma; pediatric tumors; penile cancers; pharynx cancers such as but not limited to, squamous cell cancer, and verrucous; pituitary cancers such as but limited to acromegaly, Cushing's disease, diabetes insipidus and prolactin-secreting tumors; prostateAttorney Docket Number: 01384-0002-00PCTcancers such as but not limited to, rhabdomyosarcoma, seminoma, spermatocytic and teratoma carcinoma; renal cancer such as but not limited to renal carcinoma; salivary gland cancers such as but not limited to, adenocarcinoma, adenoid cystic carcinoma, and mucoepidermoid carcinoma; skin cancers such as but not limited to, basal cell carcinoma, carcinomas of the epidermis, epithelial carcinoma, melanoma, including acrallentiginous melanoma, lentigo malignant melanoma, nodular melanoma, sebaceous gland carcinoma , squamous cell carcinoma, superficial spreading melanoma, and sweat gland carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; synovioma; testicular cancers such as but not limited to, adenocarcinoma, anaplastic, androgen dependent prostate cancer, androgen-independent prostate cancer, choriocarcinoma (yolk-sac tumor), classic (typical), embryonal carcinoma, germinal tumor, leiomyosarcoma, and nonseminoma; thyroid cancer such as but not limited to, anaplastic thyroid cancer, medullary thyroid cancer, and papillary or follicular thyroid cancer; uterine cancers such as but not limited to, endometrial carcinoma and uterine sarcoma; vaginal cancers such as adenocarcinoma, melanoma, and squamous cell carcinoma; vulvar cancer such as adenocarcinoma, basal cell carcinoma, melanoma, Paget's disease, sarcoma, and squamous cell carcinoma; and Waldenstrom's macroglobulinemia.BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIGs. 1A-1B: Schematics of certain knottin based-TOP1i conjugates. For conjugates containing more than one TOP1i drug, the TOP1i drugs may be identical or different compounds.

[0060] FIGs. 2A-2B: Synthesis strategy for compound 5-5. FIG. 2A shows the overall synthesis and structure of compound 5-5. FIG. 2B shows the steps for synthesis of compound 5-5.

[0061] FIG. 3: Synthesis strategy for GGFG-DXd

[0062] FIG. 4: Synthesis strategy for Alkyne-PEG4-GGFG-DXd

[0063] FIG. 5: Synthesis strategy for Alkyne-PEG4-(PEG4-GGFG-DXd)2

[0064] FIG. 6: Synthesis strategy for Alkyne-PEG4-Val-Ala-PAB-Exatecan

[0065] FIG. 7: Synthesis strategy for Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)2

[0066] FIG. 8: Synthesis strategies for compound 9-2 and compound 9-3

[0067] FIG. 9: Synthesis strategy for compound 11-2Attorney Docket Number: 01384-0002-00PCT

[0068] FIG. 10: Synthesis strategy for compound 11-3

[0069] FIG. 11: Synthesis strategy for Glu-Val-Cit-PAB-Exatecan

[0070] FIG. 12: Synthesis strategy for Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2

[0071] FIG. 13: Synthesis strategies for compound 10-2 and compound 10-3

[0072] FIG. 14: Synthesis strategy for Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3

[0073] FIG. 15: Synthesis strategies for compound 11-11 and compound 11-12

[0074] FIG. 16: Synthesis strategy for PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2

[0075] FIG. 17A: Chemical structure of PIP-Az (SEQ ID NO: 14)

[0076] FIG 17B: Chemical structure of NBP-Az (SEQ ID NO: 51)

[0077] FIG. 18: Chemical structure of PDC-5 (SEQ ID NO: 14)

[0078] FIG. 19: Chemical structure of PDC-6 (SEQ ID NO: 14)

[0079] FIG. 20: Chemical structure of PDC-7 (SEQ ID NO: 14)

[0080] FIG. 21: Chemical structure of PDC-8 (SEQ ID NO: 14)

[0081] FIG. 22: Chemical structure of PDC-9 (SEQ ID NO: 14)

[0082] FIG. 23: Chemical structure of PDC-10 (SEQ ID NO: 14)

[0083] FIG. 24: Chemical structure of compound 11-14 (SEQ ID NO: 14)

[0084] FIG. 25: Chemical structure of compound 11-15 (SEQ ID NO: 14)

[0085] FIG. 26: Chemical structure of PDC-11 (SEQ ID NO: 14)

[0086] FIG. 27: Chemical structure of compound 11-16 (SEQ ID NO: 51)

[0087] FIG. 28: Chemical structure of compound 11-17 (SEQ ID NO: 51)

[0088] FIG. 29: Chemical structure of NDC-11 (SEQ ID NO: 51)

[0089] FIG. 30: Chemical structure of PDC-12 (SEQ ID NO: 14)

[0090] FIGs. 31A-31B: Synthesis strategy for PFDC-2. FIG. 31 A schematic for synthesis strategy, FIG. 31 B chemical structure of linker-payload, NHS ester-PEG4-GGFG-DXd.

[0091] FIG. 32: In vitro plasma stability of PIP-TOP1i conjugates (PDC-5, PDC-6, PDC-7 and PDC-8) in various species.

[0092] FIGs. 33A-33B: % Proliferation of U87MG glioblastoma cells treated with PDC-5, PDC-6, or their respective non-binding controls. For all conjugates, the TOP1i payload isAttorney Docket Number: 01384-0002-00PCTDXd. FIG. 33A shows the % proliferation compared to the concentration of conjugate. FIG.33B shows the % proliferation compared to the concentration of the TOP1i payload.

[0093] FIGs. 34A-34B: % Proliferation of U87MG glioblastoma cells treated with PDC-7, PDC-8, or their respective non-binding controls. For all conjugates, the TOP1i payload is exatecan. FIG. 34A shows the % proliferation compared to the concentration of conjugate. FIG. 34B shows the % proliferation compared to the concentration of the TOP1i payload.

[0094] FIGs. 35A-D: % Proliferation of HCT116 colorectal cancer cells treated with PDC-8 (FIG. 35A), PDC-9 (FIG. 35B), PDC-10 (FIG. 35C), PDC-11 (FIG. 35D), or their respective non-binding controls.

[0095] FIGs. 36A-D: % Proliferation of SKOV3 ovarian cancer cells treated with PDC-8 (FIG.36A), PDC-9 (FIG. 36B), PDC-10 (FIG. 36C), PDC-11 (FIG. 36D), or their respective nonbinding controls.

[0096] FIG. 37: % Proliferation of U87MG glioblastoma cells treated with PFDC-2 or its respective non-binding control NFDC-2.

[0097] FIG. 38: % Proliferation of SKOV3 ovarian cancer cells treated with PFDC-2 or its respective non-binding control NFDC-2.

[0098] FIG. 39A: Average tumor volume over time in U87MG-tumor bearing mice treated with vehicle or various doses of PDC-7 and PDC-8.

[0099] FIG. 39B: Average % weight change overtime in U87MG-tumor bearing mice treated with PDC-7 (200 nmol conjugate, 200 nmol Exatecan) or PDC-8 (100 nmol conjugate, 200 nmol Exatecan).

[0100] FIG. 40: Average tumor volume overtime in HEC1B-tumor bearing mice treated with vehicle or PDC-8.

[0101] FIG. 41: Average tumor volume overtime in NCI-N87-tumor bearing mice treated with vehicle or PDC-8.

[0102] FIGs. 42A-C: Average tumor volume over time in tumor-bearing mice treated weekly with vehicle or PDC-8 in the three colorectal cancer models (DLD-1 (FIG. 42A), HCT116 (FIG. 42B), and SW480 (FIG. 42C)).

[0103] FIG. 43A shows the average tumor volume overtime in HT29-tumor bearing mice treated weekly with vehicle or 100 nmol conjugate (PDC-8 or NDC-8).

[0104] FIG. 43B shows the average tumor volume overtime in HT29-tumor bearing mice treated weekly with vehicle or 50 nmol conjugate (PDC-8 or NDC-8).Attorney Docket Number: 01384-0002-00PCT

[0105] FIG. 44A shows the average tumor volume overtime in Detroit 562-tumor bearing mice treated weekly with vehicle or 25 nmol conjugate (50 nmol Exatecan) of PDC-8 or NDC-8.

[0106] FIG. 44B shows the average tumor volume overtime in Detroit 562-tumor bearing mice treated weekly with vehicle or 16.7 nmol conjugate (50 nmol Exatecan) of PDC-12 or NDC-12.

[0107] FIG. 44C shows the average tumor volume over time in Detroit 562-tumor bearing mice treated weekly with vehicle or 50 nmol conjugate (100 nmol Exatecan) of PDC-8 or NDC-8.

[0108] FIG. 44D shows the average tumor volume over time in Detroit 562-tumor bearing mice treated weekly with vehicle or 33.3 nmol conjugate (100 nmol Exatecan) of PDC-12 or NDC-12.

[0109] FIG. 44E shows the average tumor volume overtime in Detroit 562-tumor bearing mice treated weekly with vehicle or 100 nmol conjugate (200 nmol Exatecan) of PDC-8 or NDC-8.

[0110] FIG. 45 shows the average tumor volume overtime in SKOV3-bearing mice treated weekly with vehicle, Topotecan, or PDC-8.

[0111] FIGs. 46A-B show the average tumor volume over time in tumor-bearing mice treated weekly with vehicle, PFDC-2 (5 mg / kg), or PFDC-2 (10 mg / kg), in the Detroit 562 xenograft model (FIG. 46A) or the U87MG xenograft model (FIG. 46B).

[0112] FIG. 47 shows the integrin-binding knottin peptide, 2.5D-Az. (SEQ ID NO: 26)

[0113] FIG. 48 Chemical structure of TDC-8. (SEQ ID NO: 26)

[0114] FIGs. 49A-B show the % Proliferation in the U87MG glioblastoma (FIG. 49A) or HCT116 colorectal cancer (FIG. 49B) after 5 days of treatment with various concentrations of TDC-8, PDC-8, or NDC-8 (non-binding peptide-drug conjugate control).DESCRIPTION OF THE SEQUENCES

[0115] Table 1 provides a listing of certain sequences referenced herein. For SEQ ID Nos: 4-35, (1) each bold letter represents a member of the integrin binding loop and (2) Z = 5-azido-L-norvaline.Attorney Docket Number: 01384-0002-00PCTAttorney Docket Number: 01384-0002-00PCTAttorney Docket Number: 01384-0002-00PCTDESCRIPTION OF THE EMBODIMENTS

[0116] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the conjugates, compositions and methods belong. Although any conjugates, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the conjugates, compositions and methods, representative illustrative conjugates, compositions and methods are now described.

[0117] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present conjugates, compositions and methods are not entitled to antedate such publication, as the date ofAttorney Docket Number: 01384-0002-00PCTpublication provided may be different from the actual publication date which may need to be independently confirmed.

[0118] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0119] It is appreciated that certain features of the conjugates, compositions and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the conjugates, compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present conjugates, compositions and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0120] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.I. Conjugates

[0121] The present disclosure provides conjugates. For example, described herein is the development and characterization of knottin-based TOP1i drug conjugates as alternative targeted agents for cancer therapy. Traditional TOP1i cancer chemotherapy agents often have narrow ranges between effective and toxic doses. To improve efficacy and minimize side effects, the knottin-based TOP1i drug conjugates find use in selectively delivering TOP1 i drugs to cancerous cells and tissue.Attorney Docket Number: 01384-0002-00PCTA.. Knottin peptide

[0122] The conjugates of the present disclosure include a knottin peptide that includes an engineered loop that binds to a cell surface molecule. The type of knottin peptide employed in the conjugates of the present disclosure may vary. In some embodiments, the knottin peptide comprises those provided in W02008045252 and WO2014063012. The three-dimensional structure of a knottin peptide is minimally defined by a particular arrangement of three disulfide bonds. This characteristic topology forms a molecular knot in which one disulfide bond passes through a macrocycle formed by the other two intra-chain disulfide bridges. Although their secondary structure content is generally low, knottins share a small triple-stranded antiparallel b-sheet, which is stabilized by the disulfide bond framework. Folding and functional activity of knottins are often mediated by loop regions that are diverse in both length and amino acid composition. While three disulfide bonds are the minimum number that defines the fold of this family of peptides, knottins can also contain additional cysteine residues, yielding molecules with four or more disulfide bonds and additional constrained loops in their structure. The term “cystine” refers to a Cys residue in which the sulfur group is linked to another amino acid though a disulfide linkage; the term “cysteine” refers to the -SH (“half cystine”) form of the residue. Binding loop portions may be adjacent to cystines, such that there are no other intervening cystines in the primary sequence in the binding loop.

[0123] The knottin peptide may be full-length (that is, the length of the wild-type peptide / polypeptide), the knottin peptide may be truncated relative to the length of the wildtype peptide / polypeptide, or the knottin peptide may include additional amino acids such that the peptide is greater in length relative to the length of the wild-type peptide / polypeptide.

[0124] The engineered loop may include amino acid substitutions, insertions, and / or deletions in an existing loop of the knottin peptide, or the engineered loop may be a loop added to the knottin protein. That is, the knottin peptide of the conjugate may include a loop in addition to the one or more loops present in the wild-type peptide. By combining directed evolution with computational covariance analysis, guidelines for introducing modifications (both in amino acid sequence and loop length) into the loop regions of the knottin scaffold have been elucidated. See, e.g., Lahti et al. (2009) PLoS Comput. Biol. 5(9): e1000499. In some embodiments, the loop of the knottin is engineered to bind to a cancer cell surface molecule.

[0125] By “cancer cell” is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independentAttorney Docket Number: 01384-0002-00PCTgrowth potential, ability to promote tumor growth and / or development in an immunocompromised non-human animal model, and / or any appropriate indicator of cellular transformation. “Cancer cell’’ may be used interchangeably herein with “tumor cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi solid tumor, a primary tumor, a metastatic tumor, and the like. Such an engineered loop confers upon the knottin peptide a cancer cell surface molecular recognition property that is not present in the wild-type peptide. In certain aspects, the cancer is a cancer known to have one or more tumor-associated or tumor-specific cell surface molecules (e.g., cell surface receptors, membrane proteases, and the like) and the engineered loop of the knottin peptide is engineered to bind to an extracellular domain of one or more such tumor- associated or tumor-specific cell surface molecules. By “tumor-associated cell surface molecule” is meant a cell surface molecule expressed on malignant cells with limited expression on cells of normal tissues, or a cell surface molecule expressed at much higher density on malignant versus normal cells.

[0126] The knottin peptide may be a peptide described in the online KNOTTIN database (dsimb.inserm.fr / KNOTTIN / ), which includes detailed amino acid sequence, structure, classification and function information for thousands of polypeptides identified as contain cystine-knot motifs. Knottins are found in a variety of plants, animals, insects and fungi.

[0127] According to certain embodiments, a knottin of the present disclosure includes a knottin peptide based on the Ecballium elaterium trypsin inhibitor II (EETI-II) peptide, also referred to as “EETI” herein. By “EETI” is meant Protein Data Bank Entry (PDB) 2ETI. Its entry in the KNOTTIN database is EETI-II. In certain aspects, a knottin peptide of a conjugate of the present disclosure is based on an EETI-II peptide having the following amino acid sequence:GCPRILMRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 1)

[0128] The knottin peptide includes an engineered loop that binds to a cell surface molecule - that is, the loop is engineered to bind to a target molecule on the surface of a cell. Knottins contain three disulfide bonds interwoven into a molecular ‘knot’ that constrain loop regions to a core of anti-parallel b-sheets. Wild-type EETI, for example, is composed of 28 amino acids with three disulfide-constrained loops: loop 1 (the trypsin binding loop, residues 3-8), loop 2 (residues 10-14), and loop 3 (residues 22-26). Knottin family members, which include protease inhibitors, toxins, and antimicrobials, share little sequence homology apart from their core cysteine residues. As a result, their disulfide-constrained loops tolerate much sequence diversity, making knottins amenable for protein engineering applications where mutations need to be introduced into a protein without abolishing its three-dimensional fold.Attorney Docket Number: 01384-0002-00PCT

[0129] The knottin peptide includes an engineered loop that binds to a cell surface molecule present on cancer cells. According to certain embodiments, the target on the cancer cell surface is a receptor, e.g., a cell adhesion receptor, a receptor for a soluble factor (e.g., a growth factor, chemokine, or other soluble factor receptor), an immune cell receptor, or the like. In certain aspects, when the receptor is a cell adhesion receptor, the receptor is an integrin. For example, a conjugate of the present disclosure may include a knottin peptide having a loop engineered to bind to any one of avpi integrin, avp3 integrin, avp5 integrin, avp6 integrin, a5pi integrin, or any combination thereof. According to certain embodiments, the engineered loop binds to each of avpi integrin, avp3 integrin, avp5 integrin, avp6 integrin, and a5p1 integrin.

[0130] In certain embodiments, a knottin peptide of a conjugate of the present disclosure is an engineered EETI-based knottin peptide having two modified loops with the following amino acid sequence structure:G CXi X2X3X4X5X6X7X8X9X10X11 Xi 2X13X15X16Xi7X18Xi 9X20CX21 Q DS DCX22AG C VC X23X24X25X26X27X28X29X30X31X32 X33 CG (SEQ ID NO: 2)whereinXi - X3= any amino acid;X4 - X20 = if present, any amino acid;X21 = any amino acid;X22 = any amino acid;X23 - X26 = any amino acid;X27 - X33 = if present, any amino acid;wherein for each numbered X position, any amino acid may include standard or unnatural amino acids.

[0131] In certain embodiments, a knottin peptide of a conjugate of the present disclosure is an engineered EETI-based knottin peptide having the following amino acid sequence structure:GCX1X2X3X4X5X6X7X8X9XioXiiXi2Xi3Xi4Xi5Xi6X17Xi8Xi9X2oCX2iQDSDCX22AGCVCG PNGX23CG (SEQ ID NO: 3)whereinXi - X3= any amino acid;X4 - X20 - if present, any amino acid;X21 = any amino acid;X22 = any amino acid;X23 = any amino acid;Attorney Docket Number: 01384-0002-00PCTwherein for each numbered X position, any amino acid may include standard or unnatural amino acids.

[0132] In certain embodiments, a knottin peptide of a conjugate of the present disclosure is an EETI-based integrin-binding peptide having an amino acid sequence selected from the following sequences shown in Table 3 (with the integrin-binding loops in bold), where Z = 5-azido-L-norvaline:Attorney Docket Number: 01384-0002-00PCTAttorney Docket Number: 01384-0002-00PCT

[0133] In certain embodiments, an EETI-based knottin peptide, 2.5F 15Z 21L 31 Y (sometimes referred to herein as “PIP-Az”) of a conjugate of the present disclosure, which binds to each of av|31 integrin, avp3 integrin, av|35 integrin, avp6 integrin, and a5p1 integrin, has the following amino acid sequence (with the integrin-binding loop in bold), where Z = 5-azido-L-norvaline:GCPRPRGDNPPLTCZQDSDCLAGCVCGPNGYCG (SEQ ID NO: 14)

[0134] In some embodiments, the knottin peptide includes one or more unnatural amino acids. Such one or more unnatural amino acids may find use, e.g., to facilitate conjugation of the drug to the knottin peptide. Unnatural amino acids which find use, e.g., for preparing the conjugates of the present disclosure, include those having a functional group selected from an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, boronic acid, or other functional groups. Unnatural amino acids which may be incorporated into a knottin peptide of a knottin-drug conjugate of the present disclosure, which unnatural amino acid may be selected to provide a functional group of interest are known and described in, e.g., Maza et al. (2015) Bioconjug. Chem. 26(9): 1884-9; Patterson et al. (2014) ACS Chem. Biol.9:592-605; Adumeau et al. (2016) Mol. Imaging Biol. (2):153-65; and elsewhere.B. Topoisomerase I inhibitors (TOP1i)

[0135] Provided herein are conjugates comprising (a) an EETI-II based knottin peptide, comprising an engineered loop that binds to a cell surface molecule; and (b) at least one topoisomerase I inhibitor (TOP1i) payload.

[0136] The term “topoisomerase I inhibitor (TOP1 i)” refers to a group of chemical compounds that inhibit the activity of topoisomerase I. The most well-known topoisomerase I inhibitors are derivatives of camptothecin. Camptothecin itself is a cytotoxic alkaloid, which was originally extracted from Camptotheca acuminata (Nyssaceae) and shows strong antitumor activity. Derivatives of camptothecin are sometimes referred to as members of the “camptothecin family”, “camptothecin payloads”, or “camptothecins” in the literature.

[0137] The camptothecin and derivatives thereof of the present application include, but are not limited to: belotecan (CKD-602), camptothecin, cositecan (BNP-1350), DDDXd, diflomotecan, DXd, Ed-04, exatecan, exatecan mesylate (DX-8951f), gimatecan (ST1481),Attorney Docket Number: 01384-0002-00PCTGI-147211C, irinotecan (CPT-11), karenitecin, lurtotecan, MH30010008, Rezetecan (SHR9265), rubitecan, silatecan (DB-67, AR-67), SN-38, S39625, topotecan, ZD06519 (FD1), 7-ethyl camptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, and (20S)-camptothecin. Additional examplary camptothecin derivatives are described in, for example, Ogitani, Bioorg. Med. Chem. Lett. 26 (2016), 5069-5072; Kumazawa, E., Cancer Chemother Pharmacol 1998, 42: 210-220; Tahara, M, Mol Cancer Ther 2014, 13(5): 1170-1180;Nakada, T., Bioorganic & Medicinal Chemistry Letters 2016, 26: 1542-1545; and Petersen, M, Mol Cancer Ther 2024, 23(5):606-618.

[0138] In some embodiments, the TOP1i payloads are non-camptothecin compounds, such as indolocarbazole, phenanthridine, indenoisoquinoline, and derivatives thereof (e.g., LMP400). Additional example TOP1i payloads and their derivatives are described in Pommier, Y. Nature Reviews Cancer 2006, 6, 789-802.C. Linkers

[0139] Aspects of the present disclosure include conjugates wherein at least one TOP1i payload is covalently linked to a knottin-based tumor-targeting agent via at least one linker to form at least one linker-payload complex.

[0140] In some embodiments, the conjugate comprises one linker-payload complex. In some embodiments, the conjugate comprises two, three, four, five, six, seven, eight, nine, ten, or more linker-payload complexes.

[0141] In some embodiments, the conjugate contains a single TOP1i payload per knottin-based tumor-targeting agent, wherein the TOP1i payload is connected to the knottin-based tumor-targeting agent via a single linker.

[0142] In some embodiments, it is preferrable to conjugate more than one TOP1i payload per knottin-based tumor-targeting agent. For example, the conjugate may contain more than one of the same TOP1i payload per knottin based-targeting agent. Alternatively, the conjugate may contain different types of TOP1i payloads per knottin based-targeting agent.

[0143] The strategy for conjugating more than one TOP1i payload per knottin based-targeting agent can vary. In some embodiments, there is a single conjugation site on the knottin-based targeting agent and the linker may be branched in a manner that enables incorporation of more than one TOP1i payload (e.g., having 1 conjugation site, 1 branched linker, >1 TOP1i payloads). In some embodiments, the branched linker supports conjugation of two, three, four, five, six, seven, eight, nine, ten, or more TOP1i payloads. Alternatively, orAttorney Docket Number: 01384-0002-00PCTin combination with branched configurations, the conjugates may contain multiple conjugation sites on the knottin-based targeting agent resulting in conjugates with more than one TOP1i payload (e.g., having >1 conjugation site, >1 TOP1i payloads). In some instances, these conjugates with multiple conjugation sites all comprise the same linker. Alternatively, these conjugates with multiple conjugation sites may employ different types of linkers and / or linker attachment strategies. Whether the linkers are the same or different, uch linkers may independently contain a single TOP1 i payload each or may be branched to enable conjugation of two, three, four, five, six, seven, eight, nine, ten, or more TOP1i payloads per linker attachment site. Linkers and linker attachment strategies described throughout this application may be used for attaching one or more TOP1i payload.

[0144] The linkers used for knottin-based TOP1i conjugates avoid steric hindrance between the TOP1i payload and the other groups or entities of the inventive conjugate and ensure sufficient mobility and flexibility. Further, the linker may be designed so as to confer, support and / or allow sufficient high affinity target binding of the engineered knottin peptide. Linker design may typically depend on the overall conjugate and may be chosen to promote the functionality of the remaining conjugate. Accordingly, linkers may, for instance, be rigid or flexible, influencing either lipophilicity or hydrophilicity of the overall conjugate, and so on. Non-limiting examples of linkers that may be employed in the conjugates of the present disclosure include ester linkers, amide linkers, maleimide or maleimide-based linkers; valinecitrulline linkers; valine-alanine linkers; glutamic acid-valine-citrulline linkers; hydrazone linkers; N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linkers; Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers; vinylsulfone-based linkers; linkers that include polyethylene glycol (PEG), such as, but not limited to tetraethylene glycol; linkers that include propanoic acid; linkers that include caproleic acid, and linkers including any combination thereof. In some embodiments, the linker may comprise regions with any of the following components or combinations thereof: varying numbers of carbon atoms, one or more sarcosine units (polysarcosine), amino acids, or other chemical groups that are biologically compatible.

[0145] In some embodiments, the linker is sufficiently stable to minimize unintended release of payloads during blood circulation and associated toxicity therein, while enabling a sufficient amount of payload to be delivered to target cells or tissues to induce therapeutic efficacy. In some embodiments, the linker is capable of releasing the payloads around or within target cells to efficiently kill target cells or block functions of target cells. In some embodiments, the linker comprises at least one cleavable functional group. In some embodiments, the cleavable functional group is sufficiently stable outside a target cell, but upon entry into the target cell (e.g., internalization), is cleaved to release its payload.Attorney Docket Number: 01384-0002-00PCTAlternatively, the cleavable functional group may be cleaved in close proximity of the target cell (e.g., extracell ularly in the tumor microenvironment), resulting in payload release in close enough proximity to the target cells that the target cells efficiently take up the payload, resulting in cell death or inhibited function of the target cells. In some embodiments, the linker is capable of releasing the payloads both around (e.g., extracellularly in the tumor microenvironment) and within (e.g., after internalization) target cells to efficiently kill target cells or block functions of target cells.

[0146] In certain aspects, the linker is a chemically labile linker, such as an acid-cleavable linker that is stable at neutral pH (bloodstream pH 7.3-7.5) but undergoes hydrolysis upon internalization into the mildly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0) of a target cell (e.g., a cancer cell). Chemically labile linkers include, but are not limited to, hydrazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers, etc. In some embodiments, the linker is a disulfide linker containing a disulfide bond. The disulfide bond may be cleaved under an intracellular reductive environment, while remaining stable in a circulatory system.

[0147] In some embodiments, the linker is an enzyme-labile linker, such as an enzyme-labile linker that is stable in the bloodstream but undergoes enzymatic cleavage upon internalization into a target cell, e.g., by an endosomal / lysosomal protease (such as cathepsin, beta-glucuronidase, or plasmin) in the endosomal / lysosomal compartments of the target cell (e.g., a cancer cell) or that undergoes enzymatic cleavage in the extracellular space around the target cell (e.g., in the tumor microenvironment). Enzyme-labile linkers include, but are not limited to, linkers that include peptidic bonds (e.g., dipeptide-based linkers such as valine-citrulline linkers) and may also include a self-immolative moiety, such as para-aminobenzoyloxycarbonyl (PAB). In certain embodiments, linkers may contain a cleavage motif comprising Valine-Citrulline-PAB (Val-Cit-PAB), Glutamic Acid-Valine-Citrulline-PAB (Glu-Val-Cit-PAB), Valine-Alanine-PAB (Val-Ala-PAB), or other Cathepsin-cleavable linkers. In some embodiments, linkers may include cleavable motifs that are cleavable by beta-glucuronidase. In some embodiments, linkers may include cleavable motifs that are cleavable by matrix metalloproteinases (MMPs) or other enzymes that are overexpressed in the tumor microenvironment.

[0148] In some embodiments, the linker is non-cleavable. Non-cleavable linkers as used herein refer to linkers which are made of stable bonds that are intended to prevent proteolytic cleavage, meaning they do not contain an intentional cleavage motif within the linker. However, once a conjugate with a non-cleavable linker is taken up in a cell and reaches the lysosome, the overall conjugate may undergo bulk degradation in the lysosome, leading to eventual release of the payload. Chemically labile linkers, enzyme-labile, and nonAttorney Docket Number: 01384-0002-00PCTcleavable linkers are known and described in detail in literature, e.g., in Ducry & Stump (2010) Bioconjugate Chem. 21:5-13, and elsewhere.D. Knottin-based TOP1I drug conjugates

[0149] Aspects of the present disclosure include knottin-based TOP1i drug conjugates. The at least one TOP1i payload employed in the conjugates that include a knottin peptide component may be any suitable agent. Non-limiting examples of such TOP1i payloads are described in the preceding section.1. Knottin peptide-TOP1i drug conjugates

[0150] Aspects of the present disclosure include engineered EETI knottin-TOP1i drug conjugates. Such conjugates include 1) an EETI-based knottin peptide including an engineered loop that binds to cell surface molecules present on cancer cells (e.g., one or more integrins), and 2) at least one TOP1i payload. TOP1i payloads can be conjugated at different sites on the knottin peptide via at least one linker to form at least one linker-payload complex. For example, the TOP1i payload(s) or linker-payload complexes can be conjugated to the N-terminus or the C-terminus of the peptide. Alternatively, TOP1 i payloads or linkerpayload complexes can be conjugated to an amino acid that is outside of the engineered binding loop. TOP1i payloads or linker-payload complexes can be conjugated to natural amino acids or can be conjugated or incorporated using unnatural amino acids.

[0151] For conjugates containing more than one TOP1 i payloads, the conjugates may contain a single conjugation site, and the linker may be branched in a manner that enables incorporation of two, three, four, five, six, seven, eight, nine, ten, or more TOP1i payloads. Alternatively, or in combination with branched configurations, for conjugates containing more than one TOP1i payloads, the conjugates may contain multiple conjugation sites. Non-limiting examples of TOP1i payloads, Linkers, and Conjugation Strategies are further described in their own corresponding sections in the application.2. Knottin-antibody or antibody fragment TOP1 i drug conjugates

[0152] In some embodiments, the conjugates can comprise an antibody or antibody fragment comprising an Fc domain in addition to the knottin and TOP1i components.Aspects of the present disclosure, thus, further include TOP1i drug conjugates containing an engineered EETI knottin-antibody fragment comprising an Fc domain. Such conjugates include 1) an EETI-based knottin peptide including an engineered loop that binds to cell surface molecules present on cancer cells (e.g., one or more integrins), 2) an antibody or antibody fragment comprising an Fc domain, which is fused to the EETI-based knottinAttorney Docket Number: 01384-0002-00PCTpeptide, and 3) at least one TOP1i payloads (optionally wherein the TOP1i payloads are in the form of linker-payload complexes).

[0153] In some embodiments, provided are dimers of such conjugates, where the antibodies or antibody fragments comprising an Fc domain dimerize (e.g., via disulfide bridges at a hinge region (if present), or the like) to form dimerized conjugates. For full antibody-like constructs that contain 2 heavy chains and 2 light chains, the antibody-like construct would assemble in the expected manner for an antibody construct.

[0154] According to some embodiments, the antibody or antibody fragment comprising an Fc domain is an antibody heavy chain or fragment thereof. In certain embodiments, the antibody heavy chain or fragment thereof includes a G, A, D, E, or M antibody heavy chain or fragment thereof. According to some embodiments, the antibody heavy chain or fragment thereof is an IgG heavy chain or fragment thereof, e.g., a human lgG1 heavy chain or fragment thereof. In certain embodiments, the antibody heavy chain or fragment thereof comprises a heavy chain variable region (VH). In some embodiments, the antibody heavy chain or fragment thereof comprises a CH1 domain.

[0155] According to some embodiments, the antibody heavy chain or fragment thereof is a full-length antibody heavy chain - that is, an antibody heavy chain that includes a VH, a CH1 domain, a CH2 domain, and a CH3 domain. In certain embodiments, the antibody or fragment comprising an Fc domain thereof is an antibody heavy chain or fragment thereof that does not include a VH and / or CH1 domain.

[0156] When a conjugate of the present disclosure includes a knottin peptide fused to an antibody heavy chain or fragment comprising an Fc domain thereof, the knottin peptide may be fused to the N- terminus of the antibody heavy chain or fragment thereof.Alternatively, the knottin peptide may be fused to the C-terminus of the antibody heavy chain or fragment thereof.

[0157] TOP1i payloads or linker-payload complexes may be conjugated stochastically throughout the protein, resulting in TOP1i payload or linker-payload complex attachments at multiple sites of the overall protein fusion. For example, N-hydroxysuccinimide (NHS) ester conjugation or similar amine-reactive bioconjugation chemistries can result in a random distribution of TOP1i payloads attached to primary amines in the protein (e.g., free amine group at the N-terminus of the protein or lysine residues).

[0158] Alternatively, for the conjugates that include a knottin peptide fused to an antibody heavy chain or fragment comprising an Fc domain thereof, the TOP1i payload(s) or linker-payload complexes may be conjugated specifically to the antibody heavy chain orAttorney Docket Number: 01384-0002-00PCTfragment thereof portion of the fusion protein. For example, the TOP1i payload(s) or linkerpayload complexes may be conjugated to the Fc region. In these embodiments, the TOP1i payload(s) or linker-payload complexes may be conjugated to the hinge of the Fc region, the CH2 domain of the Fc region, or the CH3 domain of the Fc region, e.g., at or near the C-terminus of the Fc region. Alternatively, the TOP1i payload(s) or linker-payload complexes may be conjugated to the knottin peptide portion of the fusion protein.

[0159] According to some embodiments, the antibody or fragment comprising an Fc domain thereof comprises an antibody light chain or fragment thereof. In certain embodiments, the antibody light chain or fragment thereof includes a kappa (K) light chain or fragment thereof or a lambda (I) light chain or fragment thereof. According to some embodiments, the antibody light chain or fragment thereof includes a light chain variable region (VL). Such an antibody light chain or fragment thereof may further include an antibody light chain constant region (CL) or fragment thereof. In certain embodiments, the antibody light chain or fragment thereof is a full-length antibody light chain - that is, an antibody light chain that includes a VL and a CL. When a conjugate of the present disclosure includes a knottin peptide fused to an antibody light chain or fragment thereof, the knottin peptide may be fused to the N-terminus of the antibody light chain or fragment thereof. Alternatively, the knottin peptide may be fused to the C-terminus of the antibody light chain or fragment thereof. For clarity, if the antibody or fragment comprising an Fc domain thereof comprises both heavy and light chains, then the knottin peptide may be fused to either the N-terminus or C-terminus of the heavy or light chains.

[0160] For the conjugates where the antibody or fragment comprising an Fc domain comprises an antibody light chain or fragment thereof, TOP1i payload(s) or linker-payload complexes may be conjugated stochastically throughout the protein, resulting in TOP1i payload or linker-payload complex attachments at multiple sites of the overall protein fusion. Alternatively, the TOP1i payload(s) or linker-payload complexes may be conjugated specifically to the antibody light chain or fragment thereof portion of the fusion protein. For example, the TOP1i payload(s) or linker-payload complexes may be conjugated to a VL (if present) ora CL (if present), e.g., at or near the C-terminus of a CL. Alternatively, the TOP1i payload(s) or linker-payload complexes may be conjugated to the knottin peptide portion of the fusion protein. In some embodiments, the TOP1i payload(s) or linker-payload complexes may be conjugated specifically to the antibody heavy chain or fragment thereof portion of the fusion protein.Attorney Docket Number: 01384-0002-00PCT3. Conjugation Strategies

[0161] Aspects of the present disclosure further include methods of making conjugates. Such methods include conjugating the TOP1i payload(s) (optionally wherein the TOP1i payloads are in the form of linker-payload complexes) to the knottin peptide in the case of knottin-TOP1i conjugates, or conjugating the TOP1i payload(s) to the knottin peptide or antibody or fragment comprising an Fc domain thereof in the case of the knottin-antibody subunit conjugates. In some embodiments, the methods include site-specifically conjugating the TOP1i payload(s) or linker-payload complexes to the knottin peptide or antibody or fragment comprising an Fc domain thereof. For example, the conjugation may include site-specifically conjugating the TOP1i payload(s) or linker-payload complexes to a pre-selected amino acid of the knottin peptide or antibody or fragment comprising an Fc domain thereof. In certain aspects, the pre-selected amino acid is at the N-terminus or C-terminus of the knottin peptide or antibody or fragment comprising an Fc domain thereof. In other aspects, the pre-selected amino acid is internal to the knottin peptide or antibody or fragment comprising an Fc domain thereof - that is, between the N-terminal and C-terminal amino acid of the knottin peptide or antibody or fragment comprising an Fc domain thereof. In some embodiments, the pre-selected amino acid is an unnatural amino acid. Non-limiting examples of non-natural amino acids which may be provided to the knottin peptide or antibody or fragment comprising an Fc domain thereof to facilitate conjugation include those having a functional group selected from an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde (e.g., formylglycine, e.g., SMARTag™ technology from Catalent Pharma Solutions), nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, and boronic acid functional group. Unnatural amino acids which may be incorporated and selected to provide a functional group of interest are known and described in, e.g., Maza et al. (2015) Bioconjug. Chem. 26(9): 1884-9; Patterson et al. (2014) ACS Chem. Biol.9:592-605; Adumeau et al. (2016) Mol. Imaging Biol. (2):153-65; and elsewhere.

[0162] As another example, the conjugation may include conjugating the TOP1i payload(s) or linker-payload complexes to a non-amino acid-based element of the peptide or protein. For example, conjugation via the glycans of an antibody or fragment comprising an Fc domain thereof (e.g., GlycoConnect™ technology originally developed by Synaffix).

[0163] In some embodiments, the methods may include non-site-specific conjugation of the TOP1i payload(s) or linker-payload complexes to the knottin peptide or antibody or fragment comprising an Fc domain thereof. For example, the TOP1 i payload may be derivatized with a linker containing an amine-reactive conjugation handle, such as NHS ester, p-SCN, TFP ester, PFP ester, or others, resulting in conjugation to free primary amine groups in the protein (e.g., N-terminus and / or lysine residues). Alternatively, other methodsAttorney Docket Number: 01384-0002-00PCTfor non-site-specific chemistry, such as coupling through cysteines or tyrosine amino acids, could be employed. For example, maleimide conjugation chemistry to cysteine residues.

[0164] Numerous strategies are available for conjugating the TOP1i payload(s) or linker-payload complexes and knottin peptide or antibody or fragment comprising an Fc domain thereof. For example, the TOP1i may be derivatized by covalently attaching a linker to the TOP1 i, where the linker has a functional group capable of reacting with a “chemical handle” on the knottin peptide or antibody or fragment comprising an Fc domain thereof. Also by way of example, the knottin peptide or antibody or fragment comprising an Fc domain thereof may be derivatized by covalently attaching the linker to the knottin peptide or antibody or fragment comprising an Fc domain thereof, where the linker has a functional group capable of reacting with a “chemical handle” on the TOP1i. The functional group on the linker may vary and may be selected based on compatibility with the chemical handle on the TOP1 i or knottin peptide or antibody or fragment comprising an Fc domain thereof. As another example, the TOP1i is incorporated into the knottin peptide or antibody or fragment comprising an Fc domain thereof by incorporation of an unnatural amino acid containing that TOP1I.

[0165] For conjugates containing more than one TOP1i payload, the conjugates may contain a single conjugation site, and the linker may be branched in a manner that enables incorporation of two, three, four, five, six, seven, eight, nine, ten, or more TOP1i payloads. Alternatively, or in combination, for conjugates containing more than one TOP1i payloads, the conjugates may contain multiple conjugation sites. Non-limiting examples of linkers and linker strategies are described in the preceding section relating to Linkers. Any suitable linker may be employed in the conjugates that include a knottin peptide or a knottin peptide fused to an antibody or fragment comprising an Fc domain thereof.II. Methods of UseA. Methods of Treating Cancer

[0166] In certain embodiments, when the conjugate comprises a targeting moiety that binds to an antigen on cancer cells (e.g., a tumor antigen), the method may be a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition comprising the conjugate in an amount effective to treat the cancer.

[0167] Accordingly, in certain embodiments, the subject has cancer. The methods may be employed for the treatment of a large variety of cancers. “T umor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to orAttorney Docket Number: 01384-0002-00PCTdescribe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancers that may be treated using the subject methods include, but are not limited to, cancers comprising a solid tumor, e.g., such as, blastoma, carcinoma, lymphoma, or sarcoma. Solid tumors also include any hematological malignancy that forms a mass. In certain embodiments, the cancer is selected from selected from adrenal cancer such as but not limited to, adrenocortical carcinoma and pheochromocytoma; bladder cancers such as but not limited to, adenocarcinoma, carcinosarcoma, squamous cell cancer, and transitional cell carcinoma; basal cancers; bone cancer and connective tissue sarcomas such as but not limited to, angiosarcoma (hemangiosarcoma), bone sarcoma, cholesteatoma-induced bone osteosarcoma, chondrosarcoma, chordoma, Ewing's sarcoma, fibrosarcoma, fibrosarcoma of bone, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, malignant giant cell tumor, multiple myeloma, myeloma bone disease, neurilemmoma, osteogenic sarcoma, osteosarcoma, Paget's disease of bone, periosteal sarcoma, rhabdomyosarcoma, soft-tissue sarcomas, and synovial sarcoma; brain tumors such as but not limited to, acoustic neurinoma, astrocytoma, brain stem glioma, craniopharyngioma, ependymoma, glioblastoma multiforme, glioma, medulloblastoma, meningioma, nonglial tumor, oligodendroglioma, pineoblastoma, pineocytoma, and primary brain lymphoma; breast cancer including but not limited to, breast carcinoma, breast sarcoma, and in some embodiments, adenocarcinoma, inflammatory breast cancer, intraductal carcinoma, lobular (small cell) carcinoma, medullary breast cancer, metastatic breast cancer, mucinous breast cancer, Paget's disease (including juvenile Paget's disease), papillary breast cancer, and tubular breast cancer; cervical cancers such as but not limited to, adenocarcinoma, cervical carcinoma, and squamous cell carcinoma; cholangiocarcinoma, such as but not limited to, diffuse, nodular, and papillary; colorectal cancer (colon cancer and rectal cancer), including but not limited to colon carcinoma and KRAS mutated colorectal cancer; cystadenocarcinoma; endotheliosarcoma and lymphangioendotheliosarcoma; esophageal cancers such as but not limited to, adenocarcinoma, adenoid cyctic carcinoma, adenosquamous carcinoma, melanoma, mucoepidermoid carcinoma, oat cell (small cell) carcinoma, plasmacytoma, sarcoma, squamous cancer, and verrucous carcinoma; eye cancers such as but not limited to, choroidal melanoma, ciliary body melanoma, ocular melanoma such as iris melanoma, and retinoblastoma; gallbladder cancers such as adenocarcinoma; hemangioblastoma; head and neck cancer, such as but not limited to squamous cell head and neck cancer, hematological malignancies such as but not limited to benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, heavy chain disease, leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as erythroleukemia leukemias, monocytic, myeloblastic, myelodysplastic syndrome,Attorney Docket Number: 01384-0002-00PCTmyelomonocytic, promyelocytic, chronic leukemias such as but not limited to hairy cell leukemia, chronic lymphocytic leukemia, and chronic myelocytic (granulocytic) leukemia, lymphomas such as but not limited to Hodgkin's disease and non-Hodgkin's disease, multiple myelomas such as but not limited to, extramedullary plasmacytoma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, smoldering multiple myeloma and solitary plasmacytoma, and polycythemia vera; kidney cancers such as but not limited to, adenocarcinoma, fibrosarcoma, hypernephroma, renal cell cancer, transitional cell cancer (renal pelvis and / or ureter), and Wilms tumor; liver cancers such as but not limited to, hepatoblastoma and hepatocellular carcinoma; lung cancers such as but not limited to adenocarcinoma, bronchogenic carcinoma, KRAS-mutated non-small cell lung cancer, largecell carcinoma, lung carcinoma, non-small cell lung cancer, papillary adenocarcinoma, smallcell lung cancer and squamous cell carcinoma (epidermoid carcinoma); mesothelioma myxosarcoma; neuroblastoma; neurofibroma; neurofibromatosis; oral cancers such as but not limited to, squamous cell carcinoma; ovarian cancers such as but not limited to, borderline tumor, germ cell tumor, ovarian epithelial carcinoma, and stromal tumor; papillary adenocarcinoma and papillary carcinoma; pancreatic cancer such as but not limited to, carcinoid or islet cell tumor, gastrinoma, glucagonoma, insulinoma, somatostatin-secreting tumor, and vipoma; pediatric tumors; penile cancers; pharynx cancers such as but not limited to, squamous cell cancer, and verrucous; pituitary cancers such as but limited to acromegaly, Cushing's disease, diabetes insipidus and prolactin-secreting tumors; prostate cancers such as but not limited to, rhabdomyosarcoma, seminoma, spermatocytic and teratoma carcinoma; renal cancer such as but not limited to renal carcinoma; salivary gland cancers such as but not limited to, adenocarcinoma, adenoidcystic carcinoma, and mucoepidermoid carcinoma; skin cancers such as but not limited to, basal cell carcinoma, carcinomas of the epidermis, epithelial carcinoma, melanoma, including acrallentiginous melanoma, lentigo malignant melanoma, nodular melanoma, sebaceous gland carcinoma , squamous cell carcinoma, superficial spreading melanoma, and sweat gland carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; synovioma; testicular cancers such as but not limited to, adenocarcinoma, anaplastic, androgen dependent prostate cancer, androgen-independent prostate cancer, choriocarcinoma (yolk-sac tumor), classic (typical), embryonal carcinoma, germinal tumor, leiomyosarcoma, and nonseminoma; thyroid cancer such as but not limited to, anaplastic thyroid cancer, medullary thyroid cancer, and papillary or follicular thyroid cancer; uterine cancers such as but not limited to, endometrial carcinoma and uterine sarcoma; vaginal cancers such as adenocarcinoma, melanoma, and squamous cell carcinoma; vulvar cancer such as adenocarcinoma, basal cell carcinoma, melanoma,Attorney Docket Number: 01384-0002-00PCTPaget's disease, sarcoma, and squamous cell carcinoma; and Waldenstrom's macroglobulinemia.EXPERIMENTAL EXAMPLESExample 1 - Overview of certain knottin-TOP1i conjugates

[0168] FIGs. 1A-1B show schematics of knottin based-TOP1i conjugates.

[0169] A variety of knottin peptide-TOP1 i conjugates were synthesized for evaluation as shown below in Table 4 and Table 5 below.Attorney Docket Number: 01384-0002-00PCTExample 2 - Synthesis of linker-payloads containing GGFG-DXdGeneral procedure for preparation of GGFG-DXd

[0170] Step 1 : General procedure for preparation of compound 5-5

[0171] FIGs. 2A-2B show the chemical strategy for synthesizing compound 5-5.

[0172] Compound 5-5 is a peptide synthesized by solid-phase peptide synthesis (SPPS) using standard Fmoc chemistry. Compound 5-5 was synthesized according to the following steps:

[0173] Step 1.1: DCM was added to the vessel containing CTC Resin (7.50 mmol, 13.2 g, Sub=17.1 mmol / g) and Compound 5-4 (12.5 g, 32.5 mmol, 1.0 eq) with N2 bubbling.

[0174] Step 1.2: DIEA (6.0 eq) was added dropwise and mixed for 2 hours.

[0175] Step 1.3: MeOH (0.8 ml_) was added and mixed for 30 minutes.

[0176] Step 1.4: The resin was drained and washed with DMF 5 times.

[0177] Step 1.5: 20% piperidine / DMF was added and reacted for 30 minutes.

[0178] Step 1.6: The resin was drained and washed with DMF 3 times.

[0179] Step 1.7: Fmoc-amino acid solution was added and mixed 30 seconds, then activation buffer was added, followed by N2bubbling for about 1 hour.

[0180] Step 1.8: Steps 1.5 to 1.7 were repeated for next amino acid couplings. See Table 6 below.

[0181] The resin was washed with DMF for 5 times.

[0182] Peptide Cleavage: Cleavage buffer (20% HFIP / DCM) was added to the peptide Resin stirring for 3 minutes for 3 times. The DCM solution was concentrated under reduced pressure. The peptide was dried in high vacuum for 2 hours. Compound 5-5 (8.00 g, crude) was obtained as a solid.

[0183] Step 2: Final step for preparing GGFG-DXd

[0184] FIG. 3 shows the chemical strategy for synthesizing GGFG-DXd.Attorney Docket Number: 01384-0002-00PCT

[0185] To a solution of compound 5-5 (8.00 g, 7.44 mmol, 1.00 eq.) and Exatecan (5.60 g, 6.70 mmol, 0.90 eq.) in DMF (80.0 mL) was added HOBt (1.10 g, 8.18 mmol, 20.2 mL, 1.10 eq.), DIEA (2.47 mL, 14.9 mmol, 2.00 eq.) and DIC (2.31 mL, 14.9 mmol, 2.00 eq.). The mixture was stirred at 25°C for 23 hours. The reaction was monitored by LCMS. Then TEA (20.0 mL) was added to the reaction. The mixture was stirred at 25°C for 4 hours. The reaction was monitored by LCMS. The reaction mixture was dropped into ice isopropyl ether 400 mL, then filtrate and concentrated to give a crude product. The residue was purified by prep-HPLC (TFA condition) to give GGFG-Dxd (3.20 g, 3.23 mmol) as a yellow solid.General procedure for preparation of Alkyne-PEG4-GGFG-DXd

[0186] FIG. 4 shows the chemical strategy for synthesizing Alkyne-PEG4-GGFG-DXd.

[0187] To a solution of GGFG-DXd (1.00 g, 1.16 mmol, 1.00 eq.) and Alkyne-PEG4-NHSester(414 mg, 1.16 mmol, 1.00 eq.) in DMF (10.0 mL) was added DIEA (383 pL, 2.31 mmol, 2.00 eq.). The mixture was stirred at 25°C for 1 hour. LCMS showed GGFG-DXd was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition). Alkyne-PEG4-GGFG-DXd (878 mg, 698 pmol, 86.1% purity, 60.3% yield) as a white solid. This linker-payload is used for synthesis of PDC-5 and NDC-5 in Example 6.General procedure for preparation of Alkyne-PEG4-(PEG4-GGFG-DXd)2

[0188] FIG. 5 shows the chemical strategy for synthesizing Alkyne-PEG4-(PEG4-GGFG-DXd)2.

[0189] To a solution of compound 6-4 (720 mg, 0.99 mmol, 1.00 eq.) and GGFG-DXd (1.75 g, 2.08 mmol, 2.10 eq.) in DMF (20.0 mL) was added EDCI (748 mg, 3.96 mmol, 4.00 eq.), HOBt (534 mg, 3.96 mmol, 4.00 eq.) and DIEA (982 pL, 5.93 mmol, 6.00 eq.). The mixture was stirred at 25°C for 20 hours. The reaction was monitored by LCMS and showed compound 6-4 was consumed completely. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (TFA condition) directly. Alkyne-PEG4-(PEG4-GGFG-DXd)2 (999 mg, 421 pmol, 42.5% yield, 94.7% purity) as a white solid. This linker-payload is used for synthesis of PDC-6 and NDC-6 in Example 7.Attorney Docket Number: 01384-0002-00PCTExample 3 - Synthesis of linker-payloads containing Val-Ala-PAB-Exatecan General procedure for preparation of Alkyne-PEG4-Val-Ala-PAB-Exatecan

[0190] FIG. 6 shows the chemical strategy for synthesizing Alkyne-PEG4-Val-Ala-PAB-Exatecan.

[0191] To a solution of Val-Ala-PAB-Exatecan (1.00 g, 1.32 mmol, 1.00 eq.) and Alkyne-PEG4-NHS ester (521 mg, 1.45 mmol, 1.10 eq.) in DMF (10.0 mL) was added DIEA (438 pL, 2.65 mmol, 2.00 eq.). The mixture was stirred at 25°C for 1 hour. LCMS showed Val-Ala-PAB-Exatecan was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition). Alkyne-PEG4-Val-Ala-PAB-Exatecan (731 mg, 710 pmol, 96.8% purity, 53.5% yield) as a yellow solid. This linker-payload is used for synthesis of PDC-7 and NDC-7 in Example 8.General procedure for preparation of Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)2

[0192] FIG. 7 shows the chemical strategy for synthesizing Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)2.

[0193] To a solution of compound 6-4 (650 mg, 889 pmol, 1.00 eq.) and Val-Ala-PAB-Exatecan (1.44 g, 1.87 mmol, 2.10 eq.) in DMF (20.0 mL) was added EDCI (682 mg, 3.56 mmol, 4.00 eq.), HOBt (480 mg, 3.56 mmol, 4.00 eq.) and DIEA (882 pL, 5.34 mmol, 6.00 eq.). The mixture was stirred at 25°C for 18 hours. The reaction was monitored by LCMS and showed compound 6-4 was consumed completely. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (TFA condition) directly. Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)2 (913 mg, 414 pmol, 46.4% yield, 91.6% purity) as a white solid. This linker-payload is used for synthesis of PDC-8 and NDC-8 in Example 9.General procedure for preparation of Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)3

[0194] Step 1 : General procedure for preparation of Fmoc-NH-PEG4-Val-Ala-PAB-Exatecan

[0195] To a solution of Val-Ala-PAB-Exatecan (3.10 g, 4.10 mmol, 1.0 eq.) and compound Fmoc-NH-PEG4-Propionic Acid [CAS: 557756-85-1] (2.0 g, 4.10 mmol, 1.0 eq.) in DMF (30 mL) was added HATU (1.56 g, 4.10 mmol, 1.0 eq.) and DIPEA (1.06 g, 8.20 mmol, 1.36 mL, 2.0 eq.). The mixture was stirred at 20°C for 1 hr. LCMS showed one peak corresponding with the desired mass of Fmoc-NH-PEG4-Val-Ala-PAB-Exatecan. The reaction mixture was purified by prep-HPLC (TFA condition) to give Fmoc-NH-PEG4-Val-Attorney Docket Number: 01384-0002-00PCTAla-PAB-Exatecan (2.9 g, 2.37 mmol, 57.7% yield, 99.2% purity) as a yellow solid, confirmed by LCMS.

[0196] Step 2: Final step for preparing Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)3

[0197] To a solution of Fmoc-NH-PEG4-Val-Ala-PAB-Exatecan (1.0 g, 817 pmol, 3.0 eq.) in DMF (10 mL) was added TEA (1.82 g, 18.0 mmol, 2.5 mL, 66.2 eq.). The mixture was stirred at 30°C for 2 hrs. LCMS was used to confirm removal of the Fmoc protecting group, then compound 4 (293 mg, 272 pmol, 1.0 eq.) was added. The mixture was stirred at 30°C for 2 hrs. LCMS showed one peak corresponding with the desired mass of Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)3. The reaction mixture was purified by prep-HPLC (TFA condition) to give Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)3 (678 mg, 192 pmol, 70.5% yield, 94.1% purity) as a yellow solid, which was confirmed via LCMS. This linkerpayload is used for synthesis of PDC-12 and NDC-12 in Example 13.Example 4 - Synthesis of linker-payloads containing Glu-Val-Cit-PAB-Exatecan General procedure for preparation of Glu-Val-Cit-PAB-Exatecan

[0198] Step 1 : General procedure for preparation of compound 9-2 and compound 9-3

[0199] FIG. 8 shows the chemical strategy for synthesizing compound 9-2 and compound 9-3.

[0200] To a solution of compound 9-1 (30.0 g, 39.1 mmol, 1 eq.) and Exatecan (19.7 g, 37.1 mmol, 0.95 eq.) in DMF (260 mL) was added HOBt (6.34 g, 46.9 mmol,1.2 eq.) and DIEA (12.9 mL, 78.2 mmol, 2 eq.). The mixture was stirred at 25°C for 3 hours. LCMS and HPLC showed the compound 9-2 was formed. TEA was then added (65 mL) andstirred at 25°C for 16 hours. LCMS showed compound 9-2 was consumed completely. The reaction mixture was dropped into isopropyl ether (4000 mL) to give crude. The residue was purified by prep-HPLC (TFA condition) to give compound 9-3 (28.9 g, 34.3 mmol) was obtained as a yellow solid, confirmed by LCMS and HPLC.

[0201] Step 2: General procedure for preparation of compound 11-2

[0202] FIG. 9 shows the chemical strategy for synthesizing compound 11-2.

[0203] To a solution of compound 9-3 (25.2 g, 30.0 mmol, 1 eq.), compound 11-1 (12.8 g, 30.0 mmol, 1 eq.) and HOBt (8.92 g, 66.0 mmol, 2.2 eq.) in DMF (300 mL) was added DIG (15.1 g, 120 mmol, 18.6 mL, 4 eq.) and DIEA (29.8 mL,180 mmol, 6 eq.). The mixture was stirred at 25°C for 4 hours. LCMS showed compound 9-3 was consumed completely. The reaction mixture was added TFA to adjusted pH, and then the reactionAttorney Docket Number: 01384-0002-00PCTmixture was dropped into MTBE to give compound 11-2 (37.45 g, crude) was obtained as a yellow solid, confirmed by LCMS and HPLC.

[0204] Step 3: General procedure for preparation of compound 11-3

[0205] FIG. 10 shows the chemical strategy for synthesizing compound 11-3.

[0206] To a solution of compound 11-2 (37.45 g, 30.0 mmol, 1.0 eq.) in DMF (277.5 mL) was added TEA (92.5 ml_). The mixture was stirred at 25°C for 3 hours. LCMS showed compound 11-2 was consumed completely. The reaction mixture was added TFAto adjusted pH, and then dropped into MTBE to give crude. The mixture was purified by prep-HPLC (TFA condition) to give compound 11-3 (22.77 g, 22.2 mmol, 74.0% yield) as a yellow solid, confirmed by LCMS and HPLC.

[0207] Step 4: Final step for preparing Glu-Val-Cit-PAB-Exatecan

[0208] FIG. 11 shows the chemical strategy for synthesizing Glu-Val-Cit-PAB-Exatecan.

[0209] To a solution of compound 11-3 (5.66 g, 5.52 mmol, 1.0 eq.) in DMF (30 mL) and added TFA (30 mL). The mixture was stirred at 0°C for 5.5 hrs. LCMS showed compound 11-3 was consumed completely. The reaction mixture was dropped into isopropyl ether to give crude. The residue was purified by prep-HPLC (TFA condition) to give Glu-Val-Cit-PAB-Exatecan (4.34 g, 4.47 mmol, 81.1% yield) was obtained as a yellow solid, confirmed by LCMS and HPLC.General procedure for preparation of Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2

[0210] FIG. 12 shows the chemical strategy for synthesizing Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2.

[0211] To a solution of compound 9-8 (0.90 g, 976 pmol, 1.00 eq.) and Glu-Val-Cit-PAB-Exatecan (1.90 g, 1.96 mmol, 2.00 eq.) in DMF (30.0 mL) was added DIEA (645 pL, 3.90 mmol, 4.00 eq.). The mixture was stirred at 25°C for 0.5 hours. LCMS showed compound 9-8 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to obtain Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 (970 mg, 368 pmol, 93.7% purity, 37.7% yield) as a white solid. This linker-payload is used in the synthesis of PDC-9 and NDC-9 in Example 10 as well as PDC-11 and NDC-11 in Example 12.General procedure for preparation of Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3

[0212] FIG. 13 shows the chemical strategy for synthesizing compound 10-2 and compound 10-3.Attorney Docket Number: 01384-0002-00PCT

[0213] Step 1 : General procedure for preparation of compound 10-2

[0214] To a solution of compound 10-1 (687 mg, 1.18 mmol, 0.95 eq.) and Glu-Val- Cit-PAB-Exatecan (1.2 g, 1.24 mmol, 1 eq.) in DMF (12 ml_) was added DIEA (479.7 mg, 3.71 mmol, 613.3 L, 3 eq.). The mixture was stirred at 25°C for 1 hour. LCMS showed Glu-Val-Cit-PAB-Exatecan was consumed completely and then added 0.1% TFA / H2O to stirred 1 hour at 40 °C. LCMS showed compound 10-1 was consumed completely. The residue was purified by prep-HPLC (TFA condition). Compound 10-2 (1214 mg, 843.3 pmol) was obtained as a yellow solid, confirmed by LCMS and HPLC.

[0215] Step 2: General procedure for preparation of compound 10-3

[0216] To a solution of compound 10-2 (2.09 g, 1.45 mmol, 1 eq.) in DMF (14 mL) was added TEA (6 mL). The mixture was stirred at 25°C for 4 hours. LCMS showed compound 10-2 was consumed completely. The reaction mixture was added TFA to adjusted pH. The residue was purified by prep-HPLC (TFA condition). Compound 10-3 (1.36 g, 1.12 mmol) was obtained as a yellow solid, confirmed by LCMS and HPLC.

[0217] Step 3: Final step for preparing Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3

[0218] FIG. 14 shows the chemical strategy for synthesizing Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3.

[0219] To a solution of compound 10-3 (1.36 g, 1.02 mmol, 3.00 eq, TFA salt) and compound 4 (367 mg, 341 pmol, 1.00 eq) in DMF (3.6 mL) was added DIEA (441 mg, 3.41 mmol, 563 L, 10.0 eq). The mixture was stirred at 25°C for 2 hours. LCMS showed compound 10-3 was consumed completely and a peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give compound Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3 (876 mg, 210 pmol, 61.5% yield) as a yellow solid. This linker-payload is used for synthesis of PDC-10 and NDC-10 in Example 11.General procedure for preparation ofPFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2

[0220] FIG. 15 shows the chemical strategy for synthesizing compound 11-11 and compound 11-12.

[0221] Step 1: General procedure for preparation of compound 11-11

[0222] To a solution of compound 11-10 (1.0 g, 1.25 mmol, 1.0 eq.) in DMF (37.0 mL) was added compound 11-3 (2.69 g, 2.62 mmol, 2.1 eq.), HOBt (674 mg, 4.99 mmol, 4.0 eq.), DIEA (967 mg, 7.48 mmol, 1.24 mL, 6.0 eq.) and EDCI (956 mg, 4.99 mmol, 4.0 eq.). The mixture was stirred at 25°C for 4 hours. The reaction was monitored by LCMS andAttorney Docket Number: 01384-0002-00PCTshowed compound 11-10 was consumed and compound 11-11 was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to afford compound 11-11 (2.15 g, 763 pmol, 61.2% yield, 91.2% purity, TFA salt) was obtained as a yellow solid.

[0223] Step 2: General procedure for preparation of compound 11-12

[0224] To a solution of compound 11-11 (2.15 g, 763 pmol, 1.0 eq.) in DMF (20.0 ml_) was added phenylsilane (82.6 mg, 763 pmol, 94.2 pL, 1.0 eq.) and palladium;triphenylphosphane (88.2 mg, 76.3 pmol, 0.1 eq.). The mixture was stirred at 25°C for 1 hour. The reaction was monitored by LCMS and showed compound 11-11 was consumed and compound 11-12 was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to afford compound 11-12 (1.74 g, 626 pmol, 82.1% yield, 96.3% purity) was obtained as a yellow solid.

[0225] Step 3: Final step for preparing PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2

[0226] FIG. 16 shows the chemical strategy for synthesizing PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2.

[0227] To a solution of compound 11-12 (2.58 g, 928 pmol, 1.0 eq.) in DMF (25.0 mL) was added 2,3,4,5,6-pentafluorophenol (341 mg, 1.86 mmol, 2.0 eq.), DMAP (113 mg, 928 pmol, 1.0 eq.) and EDCI (533 mg, 2.78 mmol, 3.0 eq.). The mixture was stirred at 25°C for 6 hours. The reaction was monitored by LCMS and showed compound 11-12 was consumed and PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to afford PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 (2.0 g, 679 pmol, 73.2% yield, 97.2% purity, TFA salt.) was obtained as a yellow solid. This linker-payload PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2, along with Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2, are used for the synthesis of PDC-11 and NDC-11 in Example 12.Example 5 - Synthesis and Characterization ofPIP-Az and NBP-Az peptides

[0228] FIGs. 17A-17B show the knottin peptides, PIP-Az (FIG. 17A) and NBP-Az (FIG. 17B), where PIP-Az is a polyspecific integrin-binding peptide and NBP-Az is a nonbinding peptide control. Both peptides contain an unnatural amino acid bearing an azide group for click chemistry conjugation.Attorney Docket Number: 01384-0002-00PCTPeptide Synthesis of PIP- Az

[0229] The peptide was synthesized via solid phase peptide synthesis (SPPS) using standard Fmoc chemistry. Resin preparation: The Rink Amide MBHA Resin (165 mmol, 550 g, 1.00 eq, Sub 0.3 mmol / g) in DMF (1.3 L) was agitated with N2for 2 hours at 20°C.

[0230] Deprotection: 20% piperidine in DMF (1.3 L) was added and agitated the resin with N2at 25 °C for 15 minutes. The resin was washed with DMF (1.3 L * 5) and filtered to get the resin.

[0231] Coupling: A solution of Oxyma or HOAt (3.00 eq, 495 mmol) and the relevant Fmoc amino acid (3.00 eq, 495 mmol) in DMF (1 L) was added to the resin, then the DIC (3.00 eq, 495 mmol) was added, the mixture was agitated with N2at 25°C for 30 minutes. The resin was washed with DMF (1.5 L * 5).

[0232] These deprotection and coupling steps were used for the amino acids 1-33, which were synthesized in the order from C-terminal amino acid to N-terminal amino acid, as shown in the table below. Note: To incorporate the unnatural amino acid, 5-azido-L-norvaline, in the peptide sequence, the appropriate Fmoc amino acid “Fmoc-5-azido-L-norvaline” (also known as Fmoc-Orn(N3)-OH) was used in one of the coupling steps as shown in the table below; for this unnatural amino acid coupling step only, 1.5 eq were used for Fmoc-5-azido-L-norvaline, DIC, and HOAt.Attorney Docket Number: 01384-0002-00PCT

[0233] Resulting in the following PIP-Az peptide sequence (from N-terminus to C-terminus), where the binding loop is shown in bold font and Z = 5-azido-L-norvaline:GCPRPRGDNPPLTCZQDSDCLAGCVCGPNGYCG (SEQ ID NO: 14)

[0234] After the 33rdamino acid coupling step, 20% piperidine in DMF (2 L) was added and agitated the resin with N2at 25 °C for 15 minutes. The resin was washed with DMF (2 L * 5) and filtered to get the resin.Peptide Cleavage and Purification of PIP-Az

[0235] After all the peptide synthesis steps were completed, the resin was washed with MeOH (2 L) * 3, then dried under reduced pressure to afford peptide resin peptide (981 g). Cleavage solution was added (9.8 L, 90.0% TFA / 7.5% DTT / 2.5% H2O) to the flask containing resin at room temperature and stirred for 2.5 hours. The peptide was precipitated with cold isopropyl ether (98 L), filtered and the filter cake was collected. The filter cake was washed with isopropyl ether (10 L * 3). The crude peptide was dried under vacuum for 4 hours to get the crude peptide (262 g).

[0236] Typical batch large scale refolding: the crude linear peptide (5 g per refolding batch) was dissolved in 70 mL of DMSO. While stirring, the crude linear peptide solution was added to the 5 L of buffer A in dropwise.

[0237] Buffer A: 0.2 M Arg HCI was dissolved in H2O, 1M NaOH was then added slowly and the pH was adjusted to 8.5-9.0, the equivalent of three reagents were added in order of the following concentration: 0.1 M NH4HCO3, 1.5 mM GSH, 0.5 mM GSSG.

[0238] The refolding reaction was allowed to stir for 12 hours. The completion of the refolding was confirmed by LCMS. Upon completion, the crude peptide from parallel refolding batches was firstly quenched by TFA until reaching pH 6 and was then filtered through 0.45pm membrane filter and was then purified by prep-HPLC (TFA condition) to giveAttorney Docket Number: 01384-0002-00PCTthe final product PIP-Az (23.93 g, 26.12 mmol, 97.11% purity, TFA) as a white solid. The PIP-Az was confirmed via LCMS and HPLC.

[0239] For purification, the peptide (in buffer A) was purified via prep HPLC on a Gilson GX-281 instrument. The method used for HPLC purification was a linear gradient from 10% solvent B to 40% solvent B over 95 minutes (solvent A: water + 0.075% TFA; solvent B: acetonitrile) at a 30°C column oven temperature. A C18 column (Luna, 10pm, 100A, 150*250mm) was used at a 20 mL / min flow rate.Peptide Synthesis of NBP-Az

[0240] The peptide was synthesized via solid phase peptide synthesis (SPPS) using standard Fmoc chemistry. Resin preparation: The Rink Amide MBHA Resin (25 mmol, 83.3 g, 1.00 eq, Sub 0.3 mmol / g) in DMF (0.7 L) was agitated with N2for 2 hours at 20°C.

[0241] Deprotection-. 20% piperidine in DMF (0.7 L) was added and agitated the resin with N2at 25°C for 15 minutes. The resin was washed with DMF (0.7 L * 5) and filtered to get the resin.

[0242] Coupling: A solution of Oxyma or HOAt (3.00 eq, 75 mmol) and the relevant Fmoc amino acid (3.00 eq, 75 mmol) in DMF (0.4 L) was added to the resin, then the DIC (3.00 eq, 75 mmol) was added, the mixture was agitated with N2at 25°C for 30 minutes. The resin was washed with DMF (0.7 L * 5).

[0243] These deprotection and coupling steps were used for the amino acids 1-33, which were synthesized in the order from C-terminal amino acid to N-terminal amino acid, as shown in the table below. Note: To incorporate the unnatural amino acid, 5-azido-L-norvaline, in the peptide sequence, the appropriate Fmoc amino acid “Fmoc-5-azido-L-norvaline” (also known as Fmoc-Orn(N3)-OH) was used in one of the coupling steps as shown in the table below; for this unnatural amino acid coupling step only, 1.5 eq were used for Fmoc-5-azido-L-norvaline, DIC, and HOAt.Attorney Docket Number: 01384-0002-00PCT

[0244] Resulting in the following NBP-Az peptide sequence (from N-terminus to C-terminus), where the binding loop is shown in bold font and Z = 5-azido-L-norvaline:GCVTGRDGSPASSCZQDSDCLAGCVCGPNGYCG (SEQ ID NO: 51) The NBP-Az peptide is similar to the PIP-Az peptide but the binding loop of NBP-Az is mutated such that it does not bind integrins, thus allowing this NBP-Az peptide to serve as a non-binding peptide control.

[0245] After the 33rdamino acid coupling step, 20% piperidine in DMF (1 L) was added and agitated the resin with N2at 25°C for 15 minutes. The resin was washed with DMF (1 L * 5) and filtered to get the resin.Peptide Cleavage and Purification of NBP-Az

[0246] After all the peptide synthesis steps were completed, the resin was washed with MeOH (1 L) * 3, then dried under reduced pressure to afford peptide resin peptide (180 g). Cleavage solution was added (1.8 L, 90.0% TFA / 7.5% DTT / 2.5% H2O) to the flask containing resin at room temperature and stirred for 2.5 hours. The peptide was precipitated with cold isopropyl ether (18 L), filtered and the filter cake was collected. The filter cake was washed with isopropyl ether (1 L * 3). The crude peptide was dried under vacuum for 4 hours to get the crude peptide (80 g).Attorney Docket Number: 01384-0002-00PCT

[0247] Typical batch large scale refolding: the crude linear peptide (5 g per refolding batch) was dissolved in 70 mL of DMSO. While stirring, the crude linear peptide solution was added to the 5 L of buffer A in dropwise.

[0248] Buffer A: 0.2 M Arg HCI was dissolved in H2O, 1M NaOH was then added slowly and pH was adjusted to 8.5-9.0, the equivalent of three reagents were added in order of the following concentration: 0.1 M NH4HCO3, 1.5 mM GSH, 0.5 mM GSSG.

[0249] The refolding reaction was allowed to stir for 12 hours. The completion of the refolding was confirmed by LCMS. Upon completion, the crude peptide from parallel refolding batches was firstly quenched by TFA until reaching pH 6 and was then filtered through 0.45pm membrane filter and was then purified by prep-HPLC (TFA condition) to give the final product NBP-Az (2.99 g, 0.94 mmol, 95.16% purity, TFA) as a white solid. The NBP-Az was confirmed via LCMS and HPLC.

[0250] For purification, the NBP-Az peptide was purified via prep HPLC on a Gilson GX-281 instrument. The method used for HPLC purification was a linear gradient from 10% solvent B to 40% solvent B over 39 minutes (solvent A: water + 0.075% TFA; solvent B: acetonitrile) at a 30°C column oven temperature. A dual C18 column system (Gemini C18, 5um, 110A, + luna C18, 10um, 100A) was used at a 20 mL / minute flow rate.Example 6- Synthesis ofknottins conjugated to 1 linker-payload (GGFG-DXd) General procedure for preparation of PDC-5

[0251] FIG. 18 Chemical structure of PDC-5.

[0252] To a solution of Alkyne-PEG4-GGFG-DXd (250 mg, 231 pmol,1.00 eq.) and PIP-Az (775.5 mg, 231 pmol, 1.00 eq.) in 0.2 M NH4HCO3-H2O / t-BuOH (1:1) (10.0 mL) were added VcNa (91.5 mg, 462 pmol 2.00 eq.), THPTA (100 mg, 231 pmol, 1.00 eq.) and CuSO4(0.4 M, 577 pL, 231 pmol, 1.00 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 1 minute and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 1 hour. LCMS showed PIP-Az was consumed completely and one main peak with desired was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly. PDC-5 (317 mg, 71.3 pmol, 95.6% purity by HPLC, 30.9% yield) was obtained as a white solid, which was confirmed via LCMS and analytical HPLC.General procedure for preparation of NDC-5

[0253] To a solution of Alkyne-PEG4-GGFG-DXd (130 mg, 120 pmol,1.00 eq.) and NBP-Az (381 mg, 120 pmol, 1.00 eq.) in DMF (5.00 mL) were added THPTAAttorney Docket Number: 01384-0002-00PCT(104 mg, 240 pmol, 2.00 eq.), Cui (45.7 mg, 240 pmol 2.00 eq.), and DIEA (39.7 pL, 240 pmol, 2.00 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 1 minute and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 2 hours. LCMS showed NBP-Az was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly. NDC-5 (121 mg, 28.3 pmol, 99.6% purity by HPLC, 23.6% yield) was obtained as a white solid, which was confirmed via LCMS and analytical HPLC.Example 7 - Synthesis ofknottins conjugated to 2 linker-payloads (GGFG-DXd)

[0254] FIG. 19 Chemical structure of PDC-6.General procedure for preparation of PDC-6

[0255] To a solution of Alkyne-PEG4-(PEG4-GGFG-DXd)2 (750 mg, 316 pmol, 1.00 eq.) and PIP-Az (1.06 g, 316 pmol, 1.00 eq.) in DMF (17.0 mL) were added THPTA (275 mg, 633 pmol, 2.00 eq.), Cui (121 mg, 635 pmol 2.01 eq.), and DIEA (104 pL, 632 pmol, 2.00 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 3 minutes and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 20 hours. LCMS showed PIP-Az was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly. PDC-6 (413 mg, 72.0 pmol, 99.3% purity by HPLC, 22.7% yield) was obtained as a white solid, which was confirmed via LCMS and analytical HPLC.General procedure for preparation of NDC-6

[0256] To a solution of Alkyne-PEG4-(PEG4-GGFG-DXd)2 (200 mg, 84.2 pmol, 1.00 eq.) and NBP-Az (268 mg, 84.4 pmol, 1.00 eq.) in DMF (5.00 mL) were added THPTA (73.3 mg, 168 pmol, 2.00 eq.), Cui (32.1 mg, 168 pmol 2.00 eq.), and DIEA (27.8 pL, 168 pmol, 2.00 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 1 minute and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 2 hours. LCMS showed NBP-Az was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly. NDC-6 (120 mg, 21.6 pmol, 99.8% purity by HPLC, 25.6% yield) was obtained as a white solid, which was confirmed via LCMS and analytical HPLC.Attorney Docket Number: 01384-0002-00PCTExample 8- Synthesis ofknottins conjugated to 1 linker-payload (Val-Ala-PAB-Exatecan)General procedure for preparation of PDC-7

[0257] FIG. 20 Chemical structure of PDC-7.

[0258] To a solution of Alkyne-PEG4-Val-Ala-PAB-Exatecan (200 mg, 200 pmol, 1.00 eq.) and PIP-Az (674 mg, 200 pmol, 1.00 eq.) in DMF (8.00 mL) were added THPTA (174 mg, 400 pmol, 2.00 eq.), Cui (76.0 mg, 400 pmol 2.00 eq.), and DIEA (66.0 pL, 400 pmol, 2.00 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 3 minutes and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 3 hours. LCMS showed PIP-Az was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly. PDC-7 (480.4 mg, 110 pmol, 98.9% purity by HPLC, 55.0% yield) was obtained as a white solid, which was confirmed via LCMS and analytical HPLC.General procedure for preparation of NDC-7

[0259] To a solution of Alkyne-PEG4-Val-Ala-PAB-Exatecan (120 mg, 120 pmol, 1.00 eq.) and NBP-Az (382 mg, 120 pmol, 1.00 eq.) in DMF (5.00 mL) were added THPTA (105 mg, 241 pmol, 2.00 eq.), Cui (45.9 mg, 241 pmol, 2.00 eq.), and DIEA (40.0 pL, 242 pmol, 2.01 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 1 minute and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 1 hour. LCMS showed NBP-Az was consumed completely and one main peak (Rt= 0.37 minutes) with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly. NDC-7 (124 mg, 29.7 pmol, 99.8% purity by HPLC, 24.8% yield) was obtained as a white solid, which was confirmed via LCMS and analytical HPLC.Example 9 - Synthesis ofknottins conjugated to 2 linker-payloads (Val-Ala-PAB-Exatecan)General procedure for preparation of PDC-8

[0260] FIG. 21 Chemical structure of PDC-8.

[0261] To a solution of Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)2 (200 mg, 90.8 pmol, 1.00 eq.) and PIP-Az (305 mg, 90.7 pmol, 1.00 eq.) in DMF (5.00 mL) were added THPTA (79.0 mg, 181 pmol, 2.00 eq.), Cui (34.0 mg, 178 pmol 2.00 eq.), and DIEA (30.0 pL, 181 pmol, 2.00 eq.), the pH of this solution maintained weakly alkaline. TheAttorney Docket Number: 01384-0002-00PCTreaction mixture was purged with N2for 3 minutes and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 20 hours. LCMS showed PIP-Az was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly. PDC-8 (303.2 mg, 54.5 pmol, 96.5% purity by HPLC, 60.0% yield) was obtained as a white solid, which was confirmed via LCMS and analytical HPLC.General procedure for preparation of NDC-8

[0262] To a solution of Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)2 (200 mg, 90.8 pmol, 1.00 eq.) and NBP-Az (289 mg, 91.0 pmol, 1.00 eq.) in DMF (5.00 mL) were added THPTA (79.0 mg, 182 pmol, 2.00 eq.), Cui (34.6 mg, 182 pmol, 2.00 eq.), and DIEA (30.0 pL, 181 pmol, 2.00 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 1 minute and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 1 hour. LCMS showed NBP-Az was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly. NDC-8 (122 mg, 22.6 pmol, 99.7% purity by HPLC, 24.9% yield) was obtained as a white solid, which was confirmed via LCMS and analytical HPLC.Example 10- Synthesis of knottins conjugated to 2 linker-payloads (Glu-Val-Cit-PAB-Exatecan)General procedure for preparation of PDC-9

[0263] FIG. 22 Chemical structure of PDC-9.

[0264] To a solution of Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 (200 mg, 76.0 pmol, 1.00 eq.) and PIP-Az (307 mg, 91.4 pmol, 1.20 eq.) in DMF (4.00 mL) were added Cui (59.0 mg, 310 pmol 4.00 eq.), THPTA (132 mg, 304 pmol, 4.08 eq.) and DIEA (50.4 pL, 305 pmol, 4.01 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 1 minute and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 3.0 hours. LCMS showed one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly to obtain PDC-9 (200 mg, 33.3 pmol, 96.7% purity by HPLC, 43.8% yield) as a yellow solid, which was confirmed via LCMS and analytical HPLC.Attorney Docket Number: 01384-0002-00PCTGeneral procedure for preparation of NDC-9

[0265] To a solution of Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 (108.3 mg, 41.1 pmol, 1.00 eq.) and NBP-Az (130.5 mg, 41.1 pmol, 1.00 eq.) in DMF (2.00 ml_) were added Cui (15.7 mg, 82.4 pmol 2.00 eq.), THPTA (35.7 mg, 82.1 pmol, 2.00 eq.) and DIEA (13.6 pL, 82.2 pmol, 2.00 eq.), the pH of this solution maintained weakly alkaline. The reaction mixture was purged with N2for 1 minute and the solution turned to light yellow, then the reaction mixture was stirred at 25°C for 2.0 hours. LCMS showed one main peak with desired mass was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (AcOH condition) directly to obtain NDC-9 (120 mg, 20.6 pmol, 99.2% purity by HPLC, 50.1% yield) as a white solid, which was confirmed via LCMS and analytical HPLC.Example 11 - Synthesis ofknottins conjugated to 3 linker-payloads (Glu-Val-Cit-PAB-Exatecan)General procedure for preparation of PDC-10

[0266] FIG. 23 Chemical structure of PDC-10.

[0267] A mixture of Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3 (250 mg, 59.8 pmol, 1.00 eq.), PIP-Az (200.9 mg, 59.8 pmol, 1.00 eq.), Cui (18.2 mg, 95.8 pmol, 2.00 eq.), (12.4 mg, 95.8 pmol, 15.8 pL, 2.00 eq.) and THPTA (52.0 mg, 120 pmol, 2.00 eq.) in DMF (4 mL) was degassed and purged with N2for 3 times, and then the mixture was stirred at 25 °C for 2 hours under N2atmosphere. LCMS showed Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC to give PDC-10 (173 mg, 22.2 pmol, 98.1% purity by HPLC, 37.2% yield, AcOH salt) as a white solid, which was confirmed via LCMS and analytical HPLC.General procedure for preparation of NDC-10

[0268] A mixture of Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3 (100 mg, 23.9 pmol, 1.00 eq.), NBP-Az (76.0 mg, 23.9 pmol, 1.00 eq.), Cui (9.12 mg, 47.9 pmol, 2.00 eq.), DIEA (6.19 mg, 47.9 pmol, 7.91 pL, 2.00 eq.) and THPTA (20.8 mg, 47.9 pmol, 2.00 eq.) in DMF (1 mL) was degassed and purged with N2for 3 times, and then the mixture was stirred at 25°C for 2 hours under N2atmosphere. LCMS showed Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)3 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC to give NDC-10 (87 mg, 11.4 pmol, 99.8% purity by HPLC, 47.9% yield, AcOH salt) as a white solid, which was confirmed via LCMS and analytical HPLC.Attorney Docket Number: 01384-0002-00PCTExample 12- Synthesis of knottins conjugated to 4 linker-payloads (Glu-Val-Cit-PAB-Exatecan)General procedure for preparation of PDC-11

[0269] Step 1 : To a solution of PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 (200 mg, 68.0 pmol, 1.0 eq.) in DMF (4.50 mL) was added PIP-Az (251 mg, 74.7 pmol, 1.1 eq.) and DIEA (70.2 mg, 544 pmol, 89.8 pL, 8.0 eq.). The mixture was stirred at 25 °C for 2 hours. The reaction was monitored by LCMS and showed PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 was consumed and compound 11-14 was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to afford compound 11-14 (284 mg, 46.4 pmol, 94.3% purity by HPLC, 68.3% yield, TFA salt.) as a yellow solid, which was confirmed via LCMS and analytical HPLC.

[0270] FIG. 24 Chemical structure of compound 11-14.

[0271] Step 2: To a solution of compound 11-14 (284 mg, 46.4 pmol, 1.0 eq.) in DCM (1.5 mL) was added TFA (2.19 g, 19.2 mmol, 1425 pL, 415 eq.) and triisopropylsilane (57.8 mg, 365 pmol, 75.0 pL, 7.87 eq.). The mixture was stirred at 0°C for 4.0 hours. The reaction was monitored by LCMS and showed compound 11-14 was consumed and compound 11-15 was detected. The resulting reaction mixture was triturated with isopropyl ether (30.0 mL *2), the precipitated solid was centrifuged and filtered, then the solid was dried to get the crude product. The residue was purified by prep-HPLC (TFA condition) to afford compound 11-15 (214 mg, 35.6 pmol, 97.2% purity by HPLC, 76.8% yield, TFA salt.) as a white solid, which was confirmed via LCMS and analytical HPLC.

[0272] FIG. 25 Chemical structure of compound 11-15.

[0273] Step 3: To a solution of compound 11-15 (227 mg, 37.8 pmol, 1.0 eq.) in DMF (3.0 mL) was added Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 (89.5 mg, 34.0 pmol, 0.9 eq.), Cui (14.4 mg, 75.6 pmol, 2.0 eq.), THPTA (32.8 mg, 75.6 pmol, 2.0 eq.) and DIEA (9.77 mg, 75.6 pmol, 12.5 pL, 2.0 eq.). The mixture was stirred at 25°C for 1.0 hour. The reaction was monitored by LCMS and showed PDC-11 was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (TFA condition) directly to afford the pure fraction. The pure fraction was injected to the prep-HPLC and change to the Acetate salt (follow the standard procedure). The material was then lyophilized, affording PDC-11 (162 mg, 18.8 pmol, 98.4% purity by HPLC, 55.3% yield) as a white solid, which was confirmed via LCMS and analytical HPLC.

[0274] FIG. 26 Chemical structure of PDC-11.Attorney Docket Number: 01384-0002-00PCTGeneral procedure for preparation of NDC-11

[0275] Step 1 : To a solution of PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 (200 mg, 68.0 pmol, 1.0 eq.) in DMF (3.30 mL) was added NBP-Az (237 mg, 74.7 pmol, 1.1 eq.) and DIEA (70.2 mg, 543 pmol, 89.8 pL, 8.0 eq.). The mixture was stirred at 25°C for 2 hours. The reaction was monitored by LCMS and showed PFP-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 was consumed and compound 11-16 was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to afford compound 11-16 (275 mg, 46.3 pmol, 68.1% yield, 99.5% purity by HPLC, TFA salt.) as a white solid, which was confirmed via LCMS and analytical HPLC.

[0276] FIG. 27 Chemical structure of compound 11-16.

[0277] Step 2: To a solution of compound 11-16 (275 mg, 46.3 pmol, 1.0 eq.) in DCM (2.50 mL) was added TFA (3.65 g, 32.0 mmol, 2.38 mL, 690 eq.) and triisopropylsilane (96.4 mg, 609 pmol, 125 pL, 13.1 eq.). The mixture was stirred at 0°C for 4.0 hours. The reaction was monitored by LCMS and showed compound 11-16 was consumed and compound 11-17 was detected. The resulting reaction mixture was triturated with isopropyl ether (50.0 mL *2), the precipitated solid was centrifuged and filtered, then the solid was dried to get the crude product. The residue was purified by prep-HPLC (TFA condition) to afford compound 11-17 (140 mg, 24.1 pmol, 52.0% yield, 95.7% purity by HPLC, TFA salt.) as a yellow solid, which was confirmed via LCMS and analytical HPLC.

[0278] FIG. 28 Chemical structure of compound 11-17.

[0279] Step 3: To a solution of compound 11-17 (140 mg, 24.1 pmol, 1.0 eq.) in DMF (2.0 mL) was added Alkyne-PEG4-(PEG4-Glu-Val-Cit-PAB-Exatecan)2 (63.295 mg, 24.050 pmol, 1 eq), Cui (9.16 mg, 48.1 pmol, 2.0 eq.), THPTA (20.9 mg, 48.1 pmol, 2.0 eq.) and DIEA (6.22mg, 48.1 pmol, 7.95 pL, 2.0 eq.). The mixture was stirred at 25°C for 4 hours. The reaction was monitored by LCMS and showed NDC-11 was detected. The reaction mixture was filtered to remove the undissolved residue and purified by prep-HPLC (TFA condition) directly to afford the pure fraction. The pure fraction was injected to the prep-HPLC and change to the Acetate salt (follow the standard procedure). The material was then lyophilized, affording NDC-11 (98.0 mg, 11.6 pmol, 48.2% yield) as a white solid, which was confirmed via LCMS and analytical HPLC.

[0280] FIG. 29 Chemical structure of NDC-11.Attorney Docket Number: 01384-0002-00PCTExample 13- Synthesis ofknottins conjugated to 3 linker-payloads (Val-Ala-PAB-Exatecan)General procedure for preparation of PDC-12

[0281] To a solution of Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)3 (550 mg, 156 pmol, 1.0 eq.) and PIP-Az (523 mg, 156 pmol, 1.0 eq.) in DMF (5.5 mL) was added Cui (59.3 mg, 311 pmol, 2.0 eq.), THPTA (135 mg, 311 pmol, 2.0 eq.) and DIPEA (40.2 mg, 311 pmol, 51.6 pL, 2.0 eq.). The mixture was stirred at 25°C for 2 hrs. LCMS showed one peak with the desired mass detected. The reaction mixture was purified by prep-HPLC (TFA condition, the pure fractions were collected and directly converted to the AcOH salt before lyophilization.) to give PDC-12 (700 mg, 102 pmol, 32.6% yield, 95.8% purity) as a white solid, which was confirmed via LCMS and analytical HPLC.

[0282] FIG. 30 Chemical structure of PDC-12.General procedure for preparation of NDC-12

[0283] To a solution of Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)3 (150 mg, 42.3 pmol, 1.0 eq.) and NBP-Az (135 mg, 42.5 pmol, 1.0 eq.) in DMF (1.5 mL) was added Cui (16.2 mg, 84.9 pmol, 2.0 eq.), THPTA (36.9 mg, 84.9 pmol, 2.0 eq.) and DIPEA (11.0 mg, 84.9 pmol, 14.08 pL, 2.0 eq.). The mixture was stirred at 25°C for 1 hr. LCMS showed one peak with the desired mass detected. The reaction mixture was purified by prep-HPLC (TFA condition, the pure fractions were collected and directly converted to the AcOH salt before lyophilization.) to give NDC-12 (107 mg, 15.8 pmol, 37.2% yield, 98.7% purity) as a white solid which was confirmed via LCMS and analytical HPLC.Example 14- Expression and purification of Knottin-Fc Fusions

[0284] PIP-hFc is an engineered integrin-binding EETI knottin peptide genetically fused to the Fc domain of a human IgG 1 antibody, as shown schematically in FIG. 31 A. Specifically, the knottin peptide sequence for 2.5F 15S 21 L 31 F (SEQ ID NO: 13) is connected to the hinge region of the Fc domain of a human IgG 1 antibody. This fusion protein construct does not have an antibody light chain. The protein sequence is listed below in Table 9.

[0285] NBP-hFc is an non-binding EETI knottin peptide genetically fused to the Fc domain of a human lgG1 antibody, serving as a non-binding control for PIP-hFc. Specifically, a non-binding knottin peptide sequence is connected to the hinge region of the Fc domain of a human IgG 1 antibody. This fusion protein construct does not have an antibody light chain. The protein sequence is listed below in Table 9.Attorney Docket Number: 01384-0002-00PCT

[0286] In Table 9, the knottin peptide portions of the knottin-Fc fusions are shown in bold.

[0287] Both PIP-hFc and NBP-hFc were transiently expressed in CHO-K1 cells. The desired protein sequences were codon optimized, synthesized and cloned in protein production vectors. CHO-K1 cells were transiently transfected and cultured for 7 days with regular feeding as recommended by the manufacturer. After expression, cell culture supernatant was collected for purification and proteins were purified with a protein A column and SEC column to ensure at least 98% purity. The final protein samples were formulated in PBS, pH 7.4 at >10 mg / mL. The final protein samples were analyzed using A280, SDS-PAGE, SEC-HPLC, LC-MS and endotoxin testing.Example 15 - Production of Knottin-Fc Fusions conjugated to an average of 4 linker¬ payloads (GGFG-DXd)Attorney Docket Number: 01384-0002-00PCTConjugation Procedure for PFDC-2

[0288] Protein PIP-hFc (10.23 mg / mL in original PBS, pH 7.4 buffer) was pipetted into an Eppendorf tube. Conjugation buffer with 50 mM PBS, 2 mM EDTA, pH 7.0 was added to the tube to make the final protein reaction concentration at 6 mg / mL. The linkerpayload (NHS ester-PEG4-GGFG-DXd) prepared in DMSO (10 mM stock) was added into the solution to make the drug to protein ratio at 9.9 equivalents. The DMSO solvent was added into the tube to make the final organic solvent percentage in this solution was 10% (v / v).

[0289] The reaction solution vial was placed in an incubator-shaker at 22°C with rotation speed 60 rpm for 3 hours. After 3 hours, the DAR results were determined by LC-MS. After reaction, the sample was purified via zeba spin desalting column (7K). Then the buffer was exchanged and concentrated into formulation buffer (PBS, pH 7.4) via Amicon (10K MWCO). The final conjugate PFDC-2 sample was filtered through a 0.22 pm pore size, PES membrane filter. The conjugate was characterized by SEC, MS, and quantified by BCA. FIGs. 31A-31B show the chemical strategy for synthesizing PFDC-2.Conjugation Procedure for NFDC-2

[0290] Protein NBP-hFc (10.84 mg / mL in original PBS, pH 7.4 buffer) was pipetted into an Eppendorf tube. Conjugation buffer with 50 mM PBS, 2 mM EDTA, pH 7.0 was added to the tube to make the final protein reaction concentration at 6 mg / mL. The linkerpayload (NHS ester-PEG4-GGFG-DXd) prepared in DMSO (10 mM stock) was added into the solution to make the drug to protein ratio at 9.5 equivalents. The DMSO solvent was added into the tube to make the final organic solvent percentage in this solution was 10% (v / v).

[0291] The reaction solution vial was placed in an incubator-shaker at 22°C with rotation speed 60 rpm for 3 hours. After 3 hours, the DAR results were determined by LC-MS. After reaction, the sample was purified via zeba spin desalting column (7K). Then the buffer was exchanged and concentrated into formulation buffer (PBS, pH 7.4) via Amicon (10K MWCO). The final conjugate NFDC-2 sample was filtered through a 0.22 m pore size, PES membrane filter. The conjugate was characterized by SEC, MS, and quantified by BCA.Example 16 - Plasma stability testing with PIP-TOPH conjugates

[0292] This example describes a study performed to analyze the stability of PIP-TOPI i conjugates (PDC-5, PDC-6, PDC-7 and PDC-8) in plasma (Heparin) derived from mice (CD-1), rats (Sprague-Dawley “SD”), monkeys (cynomolgus macaque) and humans.Attorney Docket Number: 01384-0002-00PCTMouse (CD-1) and rat (Sprague-Dawley “SD”) plasma were purchased from BiolVT (catalog numbers: MSE00PLNHY2N and RAT00PLNHY2N, respectively).

[0293] PDC-5 and PDC-7 were resuspended in 100 uM PBS, pH 7.4. PDC-6 and PDC-8 were resuspended in 100 uM DMSO. All PIP-TOP1i conjugates were subsequently diluted into plasma 50xto result in a final concentration of 2 uM in plasma. These samples were incubated at 37°C in a water bath for several time points up to and including 48 hours. At the end of incubation, 800uL of stop solution (methanol containing tolbutamide and labetalol as internal standards) was added to all samples and the mixtures were centrifuged at 3220 x g for 20 minutes. Intact conjugates were analyzed in the samples by LC-MS / MS and the in vitro plasma half-lives (ti / 2) were calculated.

[0294] FIG. 32 shows the in vitro plasma half-lives (t1 / 2) of PIP-TOP1i conjugates (PDC-5, PDC-6, PDC-7 and PDC-8) in plasma derived from mice (CD-1), rats (Sprague-Dawley “SD”), monkeys (cynomolgus macaque) and humans.

[0295] All conjugates were very stable in monkey and human plasma for days. While PDC-7 and PDC-8 had higher stability in rat plasma than PDC-5 and PDC-6, PDC-5 and PDC-6 had higher stability in mouse plasma than PDC-7 and PDC-8. Importantly, due to their small size, these conjugates are expected to have much shorter in vivo plasma halflives due to renal clearance, meaning that they are expected to remain mostly intact while circulating in the blood based on these in vitro plasma stability results.Example 17 - In vitro proliferation assays with PIP-TOPH conjugates

[0296] This example describes a series of studies performed to analyze the in vitro efficacy of PIP-TOP1i conjugates, tested against various cancer cell lines.

[0297] Cancer cells were plated in a 384-well plate at a seeding density of 500 cells per well, then allowed to adhere overnight. Following overnight incubation, cells were treated with serial dilutions (ranging from 0.01-750 nM) of PIP-TOP1i conjugates (also referred to as “PDCs” here) and their respective non-binding peptide-drug conjugate controls, NBP-TOP1i conjugates (also referred to as “NDCs” here). Proliferation of treated and untreated cells were tracked using live cell imaging (Sartorius Incucyte S3) over a 5 to 6-day period following drug treatment, with phase images taken every 4 hours. To determine in vitro efficacy, Incucyte live imaging software was used to calculate confluence (measured as percentage of image area covered by cells) across the 5 to 6-day time course, at which time the confluence of untreated control wells generally plateaued. % Proliferation was calculated for each well by normalizing the confluence data as follows: For a given well (example “well X”) in a 5-day study, % Proliferation of well X = 100*[(% confluence of a well X at 5 days) -Attorney Docket Number: 01384-0002-00PCT(% confluence of well X at 0 days)] / [(average % confluence of untreated wells at 5 days) -(% confluence of well X at 0 days)]. For a given well (example “well X”) in a 6-day study, % Proliferation of well X = 100*[(% confluence of a well X at 6 days) - (% confluence of well X at 0 days)] I [(average % confluence of untreated wells at 6 days) - (% confluence of well X at 0 days)].

[0298] FIGs. 33A-33B show the % Proliferation in the U87MG glioblastoma model after 5 days of treatment with various concentrations of PDC-5 or PDC-6, which includes one DXd or two DXd molecules per PIP peptide, respectively. These results are displayed on two plots, one where the x-axis is concentration with respect to the conjugate (FIG. 33A) and the otherwhere the x-axis is the concentration of the payload (DXd, FIG. 33B). The U87MG response to treatment with corresponding non-binding peptide-DXd controls, NDC-5 and NDC-6, are also shown. Both PDC-5 and PDC-6 conjugates display strong target-mediated potency in this model.

[0299] FIGs. 34A-34B show the % Proliferation in the U87MG glioblastoma model after 5 days of treatment with various concentrations of PDC-7 or PDC-8, which includes one Exatecan or two Exatecan molecules per PIP peptide, respectively. These results are displayed on two plots, one where the x-axis is concentration with respect to the conjugate (FIG. 34A) and the otherwhere the x-axis is the concentration of the payload (Exatecan, FIG. 34B). The U87MG response to treatment with corresponding non-binding peptide-Exatecan controls, NDC-7 and NDC-8, are also shown. Consistent with the responses seen with the DXd payload, both PDC-7 and PDC-8 both display strong target-mediated potency in this model.

[0300] FIGs. 35A-D show the % Proliferation in the HCT 116 colorectal cancer model after 5 days of treatment with various concentrations of PDC-8 (FIG. 35A), PDC-9 (FIG. 35B), PDC-10 (FIG. 35C), or PDC-11 (FIG. 35D). PDC-8 contains two Exatecan payloads using Val-Ala-PAB as the cleavable linker moiety, whereas PDC-9 contains two Exatecan payloads using Glu-Val-Cit-PAB as the cleavable linker moiety. PDC-10 contains three Exatecan payloads using Glu-Val-Cit-PAB as the cleavable linker moiety. PDC-11 contains four Exatecan payloads using Glu-Val-Cit-PAB as the cleavable linker moiety. PDC-8, PDC-9, and PDC-10 each use a branched linker conjugated at a single residue of the knottin peptide. PDC-11 uses branched linkers conjugated to two sites on the knottin peptide. Each PDC is shown on a plot with its respective non-binding peptide-drug conjugate control (NDC-8, NDC-9, NDC-10, and NDC-11). PDC-8, PDC-9, PDC-10, and PDC-11 all displayed strong target-mediated efficacy in this colorectal cancer model.Attorney Docket Number: 01384-0002-00PCT

[0301] FIGs. 36A-D show the % Proliferation in the SKOV3 ovarian cancer model after 6 days of treatment with various concentrations of PDC-8 (FIG. 36A), PDC-9 (FIG. 36B), PDC-10 (FIG. 36C), or PDC-11 (FIG. 36D). Each PDC is shown on a plot with its respective non-binding peptide-drug conjugate control (NDC-8, NDC-9, NDC-10, and NDC-11). PDC-8, PDC-9, PDC-10, and PDC-11 all displayed strong target-mediated efficacy in this ovarian cancer model.Example 18 - In vitro proliferation assays with a PIP-Fc-TOP1i conjugate

[0302] This example describes a series of studies performed to analyze the in vitro efficacy of a PIP-Fc fusion-TOP1i conjugate, tested against various cancer cell lines.

[0303] PDFC-2 (PIP-hFc-DXd) and its corresponding non-binding control “NFDC-2” (NBP-hFc-DXd) were evaluated in an in vitro proliferation assay using identical methods to those described in Example 17.

[0304] FIG. 37 shows the % Proliferation in the U87MG glioblastoma model after 5 days of treatment with various concentrations of PFDC-2 or NFDC-2. These results demonstrate strong target-mediated efficacy of PFDC-2 in this model.

[0305] FIG. 38 shows the % Proliferation in the SKOV3 ovarian cancer model after 6 days of treatment with various concentrations of PFDC-2 or NFDC-2. Consistent with the data obtained in U87MG, PFDC-2 also shows potent target-mediated efficacy in the SKOV3 model.Example 19 - In vivo efficacy study of PDC-7 and PDC-8 in the U87MG (glioblastoma) xenograft model in mice

[0306] This example describes a study performed to analyze the in vivo efficacy of PDC-7 and PDC-8 after IV administration in mice bearing U87MG tumors.

[0307] PDC-7 has one Exatecan payload per peptide, while PDC-8 has two Exatecan payloads per peptide, conjugated at the same amino acid residue as PDC-7 using a branched linker. PDC-7 was resuspended in PBS, pH 7.4 and diluted to the appropriate concentrations for dosing. PDC-8 was resuspended in 20mM Histidine, 8% Sucrose, pH 6 and diluted to the appropriate concentrations for dosing.

[0308] U87MG cancer cells (5x106, in 0.2 ml_ PBS) were implanted subcutaneously into the right flank of 6-8 week-old female BALB / c nude mice. When tumors reached volumes of approximately 135 mm3, mice were randomized into different treatment groups (nAttorney Docket Number: 01384-0002-00PCT= 3 per group) and IV injected (200 uL) with vehicle or peptide-drug conjugates at indicated doses weekly for 4 total doses.

[0309] FIG. 39A shows the average tumor volume over time in U87MG-tumor bearing mice treated with vehicle or various doses of PDC-7 and PDC-8. While PDC-7 showed inhibited tumor growth relative to vehicle treatment, PDC-8 was much more effective than either vehicle or PDC-7. Furthermore, 100 nmol of PDC-8 (delivering 200 nmol Exatecan) dramatically outperformed 200 nmol of PDC-7 (also delivering 200 nmol of Exatecan) in terms of therapeutic efficacy.

[0310] FIG. 39B shows that % Weight Change was similar in U87MG tumor-bearing mice treated with 100 nmol of PDC-8 (delivering 200 nmol Exatecan) relative to U87MG tumor-bearing mice treated with 200 nmol of PDC-7 (also delivering 200 nmol of Exatecan). Both treatments were found to be well tolerated with a small amount of transient weight loss after each dose, which recovered overtime.Example 20 - In vivo efficacy studies of PDC-8 in the NCI-N87 (gastric cancer) and HEC1B (endometrial cancer) xenograft models in mice

[0311] This example describes studies performed to analyze the in vivo efficacy of IV-administered PDC-8 in mice bearing NCI-N87 or HEC1B tumors.

[0312] PDC-8 was resuspended in 20mM Histidine, 8% Sucrose, pH 6 and diluted to the appropriate concentration for dosing.

[0313] Forthe HECIB model, HEC1B cancer cells (10x10®, in 0.2mL PBS) were implanted subcutaneously into the right flank of 6-8 week-old female BALB / c nude mice. When tumors reached volumes of approximately 140 mm3, mice were randomized into different treatment groups (n = 4 per group) and IV injected (200 uL) with either vehicle or 200 nmol of PDC-8 weekly for 5 total doses.

[0314] For the NCI-N87 model, NCI-N87 cancer cells (10x10®, in 0.2 ml_ PBS mixed 50:50 with Matrigel) were implanted subcutaneously into the right flank of 6-8 week-old female BALB / c nude mice. When tumors reached volumes of approximately 150 mm3, mice were randomized into different treatment groups (n = 4 per group) and IV injected (200 uL) with either vehicle or 200 nmol of PDC-8 weekly for 4 total doses.

[0315] FIG. 40 shows the average tumor volume over time in HEC1 B-tumor bearing mice treated weekly with vehicle or 200 nmol of PDC-8.

[0316] FIG. 41 shows the average tumor volume overtime in NCI-N87-tumor bearing mice treated weekly with vehicle or 200 nmol of PDC-8.Attorney Docket Number: 01384-0002-00PCT

[0317] In both the HEC1B endometrial cancer and the NCI-N87 gastric cancer models, PDC-8 leads to dramatic tumor regression with weekly IV dosing.Example 21 - In vivo efficacy studies of PDC-8 in the DLD-1, HCT116, and SW480 (colorectal cancers) xenograft models in mice

[0318] This example describes studies performed to analyze the in vivo efficacy of IV-administered PDC-8 in mice bearing colorectal tumors (DLD-1, HCT116, or SW480). PDC-8 was resuspended in 20mM Histidine, 8% Sucrose, pH 6 and diluted to the appropriate concentration for dosing.

[0319] For the DLD-1 model, DLD-1 cancer cells (5x106, in 0.1 mL PBS with 50% Matrigel) were implanted subcutaneously into the right flank of 6-8 week-old female BALB / c nude mice.

[0320] For the HCT116 model, HCT 116 cancer cells (5x106, in 0.1 mL) were implanted subcutaneously into the right flank of 6-8 week-old female BALB / c nude mice.

[0321] Forthe SW480 model, SW480 cancer cells (10x106, in 0.1 mL PBS with 50% Matrigel) were implanted subcutaneously into the right flank of 6-8 week-old female BALB / c nude mice.

[0322] For all three models, when tumors reached volumes of approximately 130-150 mm3, mice were randomized into different treatment groups (n = 4 per group) and IV injected (200 uL) with 100 nmol of PDC-8 weekly for 4 total doses.

[0323] FIGs. 42A-C show the average tumor volume over time in tumor-bearing mice treated weekly with vehicle or 100 nmol of PDC-8 in the three colorectal cancer models (DLD-1 (FIG. 42A), HCT116 (FIG. 42B), and SW480 (FIG. 42C)). In all three colorectal models, PDC-8 shows remarkable therapeutic efficacy.Example 22 - In vivo efficacy study comparing PDC-8 vs. NDC-8 in the HT29 (colorectal cancer) xenograft model in mice

[0324] Target-mediated potency of PIP-TOP1i conjugates was previously confirmed through in vitro proliferation assays in Examples 18-19. This example describes a study performed to analyze the target-mediated potency of PDC-8 in vivo in mice bearing HT29 colorectal tumors.

[0325] For the HT29 model, HT29 cancer cells (5x106, in 0.1 mL PBS) were implanted subcutaneously into the right flank of 6-8 week-old female BALB / c nude mice.Attorney Docket Number: 01384-0002-00PCTWhen tumors reached volumes of approximately 150 mm3, mice were randomized into different treatment groups (n = 3 per group) and IV injected (200 uL) with either vehicle, PDC-8 (100 nmol conjugate), NDC-8 (100 nmol conjugate), PDC-8 (50 nmol conjugate), or NDC-8 (50 nmol conjugate) weekly for 4 total doses.

[0326] Across both dose levels (50 nmol and 100 nmol conjugate), PDC-8 showed spectacular therapeutic efficacy, whereas the non-binding control (NDC-8) was ineffective. These results show that PDC-8 exhibits target-mediated potency in vivo.

[0327] FIG. 43A shows the average tumor volume over time in HT29-tumor bearing mice treated weekly with vehicle or 100 nmol conjugate (PDC-8 or NDC-8).

[0328] FIG. 43B shows the average tumor volume over time in HT29-tumor bearing mice treated weekly with vehicle or 50 nmol conjugate (PDC-8 or NDC-8).Example 23 - In vivo efficacy study evaluating the effects of TOP1i payload / peptide ratio in the Detroit 562 (head and neck cancer) xenograft model in mice

[0329] Previously in Example 19, it was found that there was an incredible improvement in therapeutic efficacy in vivo for the PIP-TOP1i conjugate containing a 2:1 TOP1i payload / peptide ratio (PDC-8) relative to the version with a 1:1 TOP1i payload / peptide ratio (PDC-7). Specifically, even when the same molar amount of payload was administered, PDC-8 and PDC-7 showed a similar safety profile in terms of % Body Weight Change, and yet, PDC-8 was substantially more effective than PDC-7 in terms of inhibiting tumor growth.

[0330] This example describes a study performed to analyze the differences in therapeutic window between a PIP-TOP1i conjugate containing a 2:1 TOP1i payload / peptide ratio (PDC-8) relative to a version with a 3:1 TOP1i payload / peptide ratio (PDC-12); both conjugates utilize the same Val-Ala-PAB cleavable linker moiety, but they utilize different branched linker structures to attach 2 Exatecan payloads vs. 3 Exatecan payloads.Corresponding non-binding peptide controls, NDC-8 and NDC-12, were also included in this study.

[0331] Detroit 562 cancer cells (5x106cells, in 0.2 mL PBS mixed 50:50 with Matrigel) were implanted subcutaneously in the right flank of 6-8 week-old female BALB / c nude mice. When tumors reached volumes of approximately 150 mm3, mice were randomized into different treatment groups (n = 5 per group) and IV injected (200 uL) with vehicle or drug conjugates at indicated doses weekly for 4 total doses.Attorney Docket Number: 01384-0002-00PCT

[0332] Across dose levels, PDC-8 and PDC-12 were substantially more effective than their respective non-binding controls (NDC-8 and NDC-12), confirming target-mediated efficacy was observed for both PIP-TOP1i conjugates in vivo.

[0333] FIG. 44A shows the average tumor volume over time in Detroit 562-tumor bearing mice treated weekly with vehicle or 25 nmol conjugate (50 nmol Exatecan) of PDC-8 or NDC-8.

[0334] FIG. 44B shows the average tumor volume over time in Detroit 562-tumor bearing mice treated weekly with vehicle or 16.7 nmol conjugate (50 nmol Exatecan) of PDC-12 or NDC-12.

[0335] FIG. 44C shows the average tumor volume over time in Detroit 562-tumor bearing mice treated weekly with vehicle or 50 nmol conjugate (100 nmol Exatecan) of PDC-8 or NDC-8.

[0336] FIG. 44D shows the average tumor volume over time in Detroit 562-tumor bearing mice treated weekly with vehicle or 33.3 nmol conjugate (100 nmol Exatecan) of PDC-12 or NDC-12.

[0337] FIG. 44E shows the average tumor volume over time in Detroit 562-tumor bearing mice treated weekly with vehicle or 100 nmol conjugate (200 nmol Exatecan) of PDC-8 or NDC-8.

[0338] The lowest dose level tested was equivalent to 50 nmol Exatecan for both 2:1 and 3:1 ratio conjugates, which corresponded to 25 nmol of conjugate for 2:1 ratio conjugates (PDC-8 and NDC-8) and 16.7 nmol of conjugate for 3:1 ratio conjugates (PDC-12 and NDC-12). At this dose level, PDC-8 showed durable tumor growth inhibition up until day 21 with slight outgrowth noted by day 28; in contrast, PDC-12 showed durable tumor growth inhibition until the end of the study (day 28).

[0339] The next dose level tested was equivalent to 100 nmol Exatecan for both 2:1 and 3:1 ratio conjugates, which corresponded to 50 nmol of conjugate for 2:1 ratio conjugates (PDC-8 and NDC-8) and 33.3 nmol of conjugate for 3:1 ratio conjugates (PDC-12 and NDC-12). At this dose level, both PDC-8 and PDC-12 showed strong and durable tumor regression.

[0340] For the 50 nmol and 100 nmol Exatecan payload equivalent doses (equivalent for both 2:1 and 3:1 ratio conjugates), both PDC-8 and PDC-12 were well tolerated showing minimal and transient body weight loss (generally < 10% change with swift recovery between dosing days).Attorney Docket Number: 01384-0002-00PCT

[0341] The highest dose level tested in this experiment was equivalent to 200 nmol Exatecan, which corresponded to 100 nmol of conjugate for 2:1 ratio conjugates (PDC-8 and NDC-8) and 66.7 nmol of conjugate for 3:1 ratio conjugates (PDC-12 and NDC-12). At this dose level, PDC-8 was again highly effective, leading to durable tumor regression, and it was also well tolerated (generally < 10% body weight change with swift recovery between dosing days). Interestingly, despite having the same molar amount of Exatecan administered as PDC-8 (200 nmol Exatecan equivalent), the mice treated with PDC-12 did not tolerate this dose level well and were euthanized early due to more substantial body weight loss that was not showing signs of recovery.

[0342] Collectively, these data suggest that the 3: 1 TOP1 i / peptide ratio version “PDC-12” shifts the therapeutic window to be slightly more potent at lower doses, but also more toxic at higher doses compared to equimolar payload amounts (200 nmol Exatecan equivalent) of the 2:1 TOP1 payload / peptide ratio version “PDC-8”. Overall, this shift in efficacy is much less dramatic than the improvement in efficacy previously observed for the 2:1 ratio (PDC-8) vs. 1:1 ratio (PDC-7). This suggests the 2:1 TOP1i payload / peptide ratio offers optimal benefits (balancing potency and toxicity) and strong therapeutic activity for PIP-TOP1i conjugates; TOP1i payload / peptide ratios > 2:1 are also efficacious, but the therapeutic window shifts due to increased potency.Example 24 - In vivo benchmarking studies of PDC-8 vs. standard of care chemotherapy in the SKOV3 (ovarian cancer) xenograft model in mice

[0343] This example describes a study performed to benchmark the in vivo efficacy of IV-administered PDC-8 against topotecan, a standard of care chemotherapy for ovarian cancerthat is in the same class of topoisomerase I inhibitors as Exatecan, in mice bearing SKOV3 tumors.

[0344] SKOV3 cancer cells (10x106, in 0.2 mL PBS mixed 50:50 with Matrigel) were implanted subcutaneously in the right flank of 6-8 week-old female BALB / c nude mice. When tumors reached volumes of approximately 150 mm3, mice were randomized into different treatment groups (n = 5 per group) and IV injected (200 uL) with either vehicle, 100 nmol PDC-8, or 15 mg / kg topotecan (a high but non-toxic dose) weekly for 5 total doses.

[0345] FIG. 45 shows the average tumor volume over time in SKOV3-bearing mice treated weekly with vehicle, Topotecan, or PDC-8. Topotecan treatment only led to a delay in tumor growth, whereas PDC-8 treatment led to durable tumor regression.Attorney Docket Number: 01384-0002-00PCTExample 25 - In vivo efficacy studies ofPFDC-2 in the Detroit 562 (head and neck) and U87MG (glioblastoma) xenograft models in mice

[0346] This example describes studies performed to analyze the in vivo efficacy of IV administered PFDC-2, which possess an average of 4 DXd TOP1i payloads per molecule, in mice bearing head and neck cancer tumors (Detroit 562) or glioblastoma (U87MG).

[0347] For the Detroit 562 model, Detroit 562 cancer cells (5x106cells, in 0.2 mL PBS mixed 50:50 with Matrigel) were implanted subcutaneously in the right flank of 6-8 week-old female BALB / c nude mice.

[0348] For the U87MG model, U87MG cancer cells (5x106, in 0.2 mL PBS) were implanted subcutaneously into the right flank of 6-8 week-old female BALB / c nude mice.

[0349] For both models, when tumors reached volumes of approximately 140-150 mm3, mice were randomized into different treatment groups (n = 3-5 per group) and IV injected (200 uL) with vehicle or PFDC-2 at indicated doses weekly for 4 total doses.

[0350] FIGs. 46A-B show the average tumor volume over time in tumor-bearing mice treated weekly with vehicle, PFDC-2 (5 mg / kg), or PFDC-2 (10 mg / kg), in the Detroit 562 xenograft model (FIG. 46A) or the U87MG xenograft model (FIG. 46B). PDFC-2 shows therapeutic efficacy in both models.Example 26 - Synthesis and Characterization of integrin-binding knottin, 2.5D-Az

[0351] FIG. 47 shows the integrin-binding knottin peptide, 2.5D-Az. 2.5D-Az is the same as PIP-Az, except 2.5D-Az contains a different engineered integrin-binding loop (between sites 3-13), which imparts different selectivity toward particular RGD-binding integrins.Peptide Synthesis of 2.5D-Az

[0352] The peptide was synthesized via solid phase peptide synthesis (SPPS) using standard Fmoc chemistry. Resin preparation: The Sieber Resin (3.0 mmol, 11.6 g, 1.00 eq, Sub 0.26 mmol / g) in DMF (240 mL) was agitated with N2for 2 hrs at 20 °C.

[0353] Deprotection: 20% piperidine in DMF (240 mL) was added and agitated the resin with N2at 25°C for 15 min. The resin was washed with DMF (240 mL * 5) and filtered to get the resin.

[0354] Coupling: A solution of HOAt (2.00 eq, 6.0 mmol, 0.82 g) and the relevant Fmoc amino acid (2.00 eq, 6.0 mmol, 1.79 g) in DMF (120 mL) was added to the resin, thenAttorney Docket Number: 01384-0002-00PCTthe DIC (2.00 eq, 6.0 mmol) was added, the mixture was agitated with N2at 25°C for 30 min. The resin was washed with DMF (120 mL * 5).

[0355] These deprotection and coupling steps were used for the following amino acids, which were synthesized in the order from C-terminal amino acid to N-terminal amino acid, as shown in the table below. Note: To incorporate the unnatural amino acid, 5-azido-L-norvaline, in the peptide sequence, the appropriate Fmoc amino acid “Fmoc-5-azido-L-norvaline” (also known as Fmoc-Orn(N3)-OH) was used in step 18 as shown in the table below. Note for step 16, “Fmoc-Asp(OtBu)-SerPsi(Me,Me)Pro-OH” was used to incorporate two adjacent amino acids, Asp-Ser [N to C-term], Note for step 20, “Fmoc-Thr(tBu)-SerPsi(Me,Me)Pro-OH” was used to incorporate two adjacent amino acids, Thr-Ser [N to C-term].Attorney Docket Number: 01384-0002-00PCT

[0356] Resulting in the following 2.5D-Az peptide sequence (from N-terminus to C-terminus), also known as “2.5D 15Z 21L 31 Y” (SEQ ID NO: 26), where the binding loop is shown in bold font and Z = 5-azido-L-norvaline:GCPQGRGDWAPTSCZQDSDCLAGCVCGPNGYCG (SEQ ID NO: 26) Peptide Cleavage and Purification of2.5D-Az

[0357] After all the peptide synthesis steps were completed including the final deprotection step, the resin was washed with MeOH (240 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (20.0 g).

[0358] Cleavage solution was added (200 mL, 5% DTT / 2.5% H2O / 92.5% TFA) to the flask containing resin at room temperature and stirred for 2 hours. The peptide was precipitated with cold isopropyl ether (1 L), filtered and the filter cake was collected. The filter cake was washed with isopropyl ether (1 L * 3). The crude peptide was dried under vacuum for 3 hours to get the crude peptide (10 g).

[0359] Refolding: the crude linear peptide (10 g) was dissolved in 500 mL of DMSO. While stirring, the crude linear peptide solution was added to the 9.5 L of buffer A in dropwise. Buffer A: Arg HCI was dissolved in miliQ H2O to reach the final concentration of 0.2 M, 1 M NaOH was then added slowly and adjust pH to 8.5-9.0. Three reagents were added in order to reach the following concentration: 0.05 M NH4HCO3, 1.5 mM GSH, 0.5 mM GSSG. The refolding reaction was allowed to stir for 8 hours.

[0360] Upon completion, the crude reaction mixture was filtered through 0.45 pm membrane filter and was purified by prep-HPLC (TFA condition: A: 0.075 % TFA in H2O, B: ACN) to give the final product 2.5D-Az (506.3 mg, 140.8 pmol, 95.29% purity, TFA) as a white solid, which was confirmed via LCMS and HPLC.Example 27 - Synthesis of2.5D knottin variant conjugated to 2 linker-payloads (Val-Ala-PAB-Exatecan)General procedure for preparation of TDC-8

[0361] FIG. 48 Chemical structure of TDC-8.

[0362] To a solution of 2.5D-Az (93.504 mg, 26.906 pmol, 1.106 eq.) and Alkyne-PEG4-(PEG4-Val-Ala-PAB-Exatecan)2 (55 mg, 24.334 pmol, 1 eq.) in DMF (1 mL), Cui (9.269 mg, 48.669 pmol, 2 eq.), THPTA (21.146 mg, 48.669 pmol, 2 eq.) and DIPEA (6.290 mg, 48.669 pmol, 8.477 pL, 2 eq.) were added. The solution was degassed and purged with N2for 1 min, and then the mixture was stirred at 25°C for 100 min. LC-MS showed Alkyne-Attorney Docket Number: 01384-0002-00PCTPEG4-(PEG4-Val-Ala-PAB-Exatecan)2 was consumed completely, and one main peak with the desired mass of TDC-8 was detected. The reaction mixture was filtered.

[0363] The crude product from this reaction was combined with the crude product from a smaller pilot conjugation reaction (10 mg) that was completed first. The combined crude product mixture was then purified by prep-HPLC (TFA condition) directly to afford the pure fraction. The pure fraction was then injected into the prep-HPLC in order to exchange to the Acetate salt. TDC-8 (116 mg, 20.515 pmol, 97.5% purity) was obtained as a white solid, which was confirmed by LCMS and analytical HPLC.Example 28 - Therapeutic activity of knottin-TOPH conjugates with different target selectivity

[0364] The focus of this present disclosure is tumor-targeting TOP1 i conjugates that comprise engineered EETI-based knottin peptides to enable tumor targeting. In particular, the experimental examples focus on EETI-based knottin peptides that bind to tumor-associated RGD-binding integrins. RGD-binding integrins, which include avp1, avp3, av|35, avp6, avp8, a5pi, a8p1, and alip3, are attractive targets for therapeutic development; In particular, many of the RGD-binding integrins are tumor-associated integrins (avpi, avp3, avp5, avp6, avp8, and a5p1). The RGD-binding integrin family is named as such because these integrins can recognize the “RGD” peptide motif. See e.g., Pierschbacher et al., Nature. 1984 May;309(5963):30-3; Takada et al., Genome Biol. 2007;8(5):215; Nieberler et al. Cancers (Basel). 2017 Sep 4;9(9): 116; and Ruoslahti et al., Annu Rev Cell Dev Biol. 1996:12:697-715 which are hereby incorporated by reference in their entirety.

[0365] This propensity to bind to RGD-binding integrins can be enabled by incorporating the RGD sequence into the sequence of a protein or peptide, with the exact selectivity and affinity for each RGD-integrin influenced by the amino acid sequences flanking the “RGD” motif and the overall spatial arrangement. Moreover, incorporating the RGD sequence into the binding loop of EETI-based knottin peptides was previously found to be an effective strategy for engineering novel RGD-binding integrin targeted EETI-based knottin peptides (WG2008045252).

[0366] In the previous experimental examples, the integrin-targeted peptide-TOP1i conjugates all utilized PIP-Az as the base peptide, featuring the integrin-binding loop “PRPRGDNPPLT” (SEQ ID NO: 4), which allows it to bind to five tumor-associated RGD-binding integrins (avp3, av|31 , avp6, avp5, and a5|31). Utilizing the PIP-Az peptide was particularly useful for the prior examples as it allowed for facile demonstration of activityAttorney Docket Number: 01384-0002-00PCTacross multiple different cancer models since most cancers will overexpress at least 1 of those 5 tumor-associated integrins.

[0367] In this current example, target-mediated activity was demonstrated using a different integrin-binding knottin-drug conjugate called TDC-8. TDC-8 is the same as PDC-8, except the knottin peptide portion of TDC-8 contains a different engineered integrin-binding loop “PQGRGDWAPTS” (SEQ ID NO: 5), which imparts different selectivity toward particular RGD-binding integrins (only binds avp3, avpi, and av|36). TDC-8, PDC-8, and NDC-8 all contain the same linker-payload structure (2X Val-Ala-PAB Exatecan), enabling useful comparison.

[0368] TDC-8, PDC-8, and NDC-8 (relevant non-binding control), were evaluated via in vitro proliferation assays using similar methods to those described in Example 17.

[0369] The first cell line tested was U87MG glioblastoma, which expresses multiple tumor-associated integrins: very high levels of avp3 and ct5p 1 ; lower levels of avp5; no expression of avpi or avpe. TDC-8 and PDC-8 both exhibit strong target-mediated potency in the U87MG glioblastoma model, which has sufficiently high expression of PDC-8 and TDC-8-targetable integrins.

[0370] The second cell line tested was HCT 116 colorectal cancer, which expresses multiple tumor-associated integrins: moderate levels of avp5 and a5pi; lower levels of avp1; no expression of avp3 or avp6. The HCT116 model possess sufficient levels of PDC-8-targetable integrins (moderate levels of avp5 and a5pi; lower levels of avpi), corresponding with strong target-mediated activity with PDC-8, as expected. However, the HCT116 model has much lower levels of TDC-8-targetable integrins (only low levels avpi); consequently, the potency of TDC-8 was substantially weaker than that of PDC-8, but TDC-8 was still more potent than NDC-8.

[0371] Overall, these results show that knottin-TOP1i conjugates possessing different target selectively remain effective, and their potency is influenced by the level of target expression in a given cancer, as expected.

[0372] FIGs. 49A-B show the % Proliferation in the U87MG glioblastoma (FIG. 49A) or HCT116 colorectal cancer (FIG. 49B) after 5 days of treatment with various concentrations of TDC-8, PDC-8, or NDC-8 (non-binding peptide-drug conjugate control).

[0373] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody theAttorney Docket Number: 01384-0002-00PCTprinciples of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.EQUIVALENTS

[0374] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0375] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., + / -5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

Claims

Attorney Docket Number: 01384-0002-00PCTWHAT IS CLAIMED IS:

1. A conjugate comprising:a. an EETI-II based knottin peptide, comprising an engineered loop that binds to a cell surface molecule; andb. at least one topoisomerase I inhibitor (TOP1i) payload.

2. The conjugate of claim 1 , wherein the at least one TOP1 i payload is attached via at least one linker to form at least one linker-payload complex.

3. The conjugate of claim 2, wherein the conjugate comprises one linker-payload complex.

4. The conjugate of claim 2, wherein the conjugate comprises two, three, four, five, six, seven, eight, nine, ten, or more linker-payload complexes.

5. The conjugate of any one of claims 2-4, wherein at least one linker-payload complex comprises a single linker conjugated to a single TOP1 i payload.

6. The conjugate of any one of claims 2-5, wherein at least one linker-payload complex comprises a branched linker.

7. The conjugate of claim 6, wherein the branched linker is conjugated to two, three, four, five, six, seven, eight, nine, ten, or more TOP1i payloads.

8. The conjugate of any one of claims 6-7, wherein the branched linker is conjugated to two TOP1i payloads.

9. The conjugate of any one of claims 6-7, wherein the branched linker is conjugated to three TOP1 i payloads.

10. The conjugate of any one of claims 6-7, wherein the branched linker is conjugated to fourTOPIi payloads.

11. The conjugate of any one of claims 1-10, wherein the TOP1i payloads comprise the same TOP1L12. The conjugate of any one of claims 1-10, comprising two or more TOP1i payloads comprising two or more different TOP1L13. The conjugate of any one of claims 1-12, wherein the conjugate is capable of treating cancer in a patient.

14. The conjugate of any one of claims 1-13, wherein the cell surface molecule is present on cancer cells.

15. The conjugate of any one of claims 1-14, wherein the cell surface molecule is an integrin.

16. The conjugate of claim 15, wherein the integrin is avp3 integrin, avPs integrin, avPe integrin, av|3i integrin, and / or a5pi integrin.Attorney Docket Number: 01384-0002-00PCT17. The conjugate of any one of claims 1-16, wherein the EETI-II based knottin peptide comprises GCX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20CX21QDSDCX22AGCVCX23 X24 X25 X26X27 X28 X29 X30 X31 X32 X33 CG (SED ID NO: 2), wherein Xi - X3 are any amino acid; X4 -X20 if present, are any amino acid; X21, X22, and X23- X26, if present are any amino acid; X27 -X33 if present, are any amino acid; further wherein each amino acid is independently selected from standard or unnatural amino acids.

18. The conjugate of any one of claims 1-16, wherein the EETI-II based knottin peptide comprises GCXlX2X3X4X5X6X7X8X9X1oXl1Xl2X13Xl4Xl5Xl6Xl7X18Xl9X2oCX2lQDSDCX22AGCVCGPNGX23 CG (SEQ ID NO: 3), wherein X1-X3 are any amino acid; X4-X20, if present, are any amino acid; and wherein X21-X23 are any amino acid, further wherein each amino acid is independently selected from standard or unnatural amino acids.

19. The conjugate of any one of claims 1-18, wherein the engineered loop has a sequence that differs by no more than 2 amino acids from any one of SEQ ID NO: 4 through SEQ ID NO: 8, inclusive.

20. The conjugate of any one of claims 1-18, wherein the engineered loop has a sequence that differs by no more than 1 amino acid from any one of SEQ ID NO: 4 through SEQ ID NO: 8, inclusive.

21. The conjugate of any one of claims 1-18, wherein the engineered loop has a sequence of any one of SEQ ID NO: 4 through SEQ ID NO: 8, inclusive.

22. The conjugate of any one of claims 1-21, wherein the EETI-II based knottin peptide has a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 9 through SEQ ID NO: 35, inclusive.

23. The conjugate of claim 22, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 12 through SED ID NO: 35, inclusive.

24. The conjugate of any one of claims 1 -23, wherein the engineered loop comprises the amino acid sequence RGD.

25. The conjugate of any one of claims 1-24, wherein the at least one TOP1i payload comprises a camptothecin or a derivative thereof.

26. The conjugate of any one of claims 1-25, wherein the at least one TOP1i payload comprises: belotecan (CKD-602), camptothecin, cositecan (BNP-1350), DDDXd, diflomotecan, DXd, Ed-04, exatecan, exatecan mesylate (DX-8951f), gimatecan (ST1481), GI-147211C, irinotecan (CPT-11), karenitecin, lurtotecan, MH30010008, Rezetecan (SHR9265), rubitecan, silatecan (DB-67, AR-67), SN-38, S39625, topotecan, ZD06519 (FD1), 7-ethyl camptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin,Attorney Docket Number: 01384-0002-00PCT9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, or(20S)-camptothecin.

27. The conjugate of any one of claims 1-26, wherein the at least one TOP1i payload comprises exatecan or DXd.

28. The conjugate of any one of claims 2-27, wherein at least one linker is a cleavable linker.

29. The conjugate of claim 28, wherein at least one linker is acid-cleavable or enzymatically cleavable.

30. The conjugate of any one of claims 28-29, wherein the cleavable linker is a protease cleavable linker.

31. The conjugate of any one of claims 28-30, wherein the cleavable linker is cleaved by endosomal or lysosomal proteases.

32. The conjugate of any one of claims 28-31 , wherein the cleavable linker is a cathepsin-cleavable linker.

33. The conjugate of any one of claims 28-31 , wherein the cleavable linker is a beta-glucuronidase-cleavable linker.

34. The conjugate of any one of claims 2-33, wherein at least one linker comprises a cleavage motif selected from: GGFG (SEQ ID NO: 52), Valine-Citrulline-PAB (Val-Cit-PAB), Glutamic Acid-Valine-Citrulline-PAB (Glu-Val-Cit-PAB), Valine-Alanine-PAB (Val-Ala-PAB).

35. The conjugate of any one of claims 2-34, wherein at least one linker is a noncleavable linker.

36. The conjugate of any one of claims 28-35, wherein at least one linker comprises at least one polyethylene glycol (PEG) unit.

37. The conjugate of any one of claims 2-36, wherein the at least one linker payload complex is conjugated to the EETI-ll-based knottin peptide.

38. The conjugate of any one of claims 1 -37, further comprising an antibody or antibody fragment comprising an Fc domain fused to the EETI-II based knottin peptide.

39. The conjugate of claim 38, wherein the at least one linker-payload complex is conjugated to the antibody or antibody fragment comprising an Fc domain.

40. The conjugate of claim 39, wherein the at least one linker-payload complex is conjugated to the antibody or antibody fragment comprising an Fc domain at one or more glycans attached to the antibody or antibody fragment comprising an Fc domain.

41. A conjugate comprising:a. an EETI-II based knottin peptide comprising the amino acid sequence of SEQ ID NO: 14; andb. two TOP1i payloads covalently attached via a branched linker; wherein the branched linker comprises at least one Val-Ala-PAB cleavage domain.Attorney Docket Number: 01384-0002-00PCT42. The conjugate of claim 41 , wherein at least one of the two TOP1 i payloads is selected from exatecan or DXd.

43. A method of treating cancer, comprising administering the conjugate of any one of claims 1-42 to a patient in need thereof.

44. Use of the conjugate of any one of claims 1 -42 in the manufacture of a medicament for treating cancer in a patient in need thereof.

45. The conjugate of any one of claims 1-42 for use in treating cancer in a patient in need thereof.

46. The method or use of any one of claims 43-45, wherein the cancer is a solid tumor.

47. The method or use of claim 45, wherein the cancer is selected from blastoma, carcinoma, lymphoma, and sarcoma.

48. The method or use of claim 47, wherein the cancer is selected from colorectal cancer, head and neck cancer, non-small cell lung cancer, esophageal cancer, uterine cancer, ovarian cancer, and gastric cancer.

49. The method or use of claim 47, wherein the cancer is selected from colorectal cancer, head and neck cancer, lung cancer, esophageal cancer, uterine cancer, ovarian cancer, gastric cancer, pancreatic cancer, breast cancer, bladder cancer, melanoma, renal cancer, liver cancer, gallbladder cancer, sarcomas, and brain cancer.

50. The method or use of claim 47, wherein the cancer is selected from adrenal cancer such as but not limited to, adrenocortical carcinoma and pheochromocytoma; bladder cancers such as but not limited to, adenocarcinoma, carcinosarcoma, squamous cell cancer, and transitional cell carcinoma; basal cancers; bone cancer and connective tissue sarcomas such as but not limited to, angiosarcoma (hemangiosarcoma), bone sarcoma, cholesteatoma-induced bone osteosarcoma, chondrosarcoma, chordoma, Ewing's sarcoma, fibrosarcoma, fibrosarcoma of bone, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, malignant giant cell tumor, multiple myeloma, myeloma bone disease, neurilemmoma, osteogenic sarcoma, osteosarcoma, Paget's disease of bone, periosteal sarcoma, rhabdomyosarcoma, soft-tissue sarcomas, and synovial sarcoma; brain tumors such as but not limited to, acoustic neurinoma, astrocytoma, brain stem glioma, craniopharyngioma, ependymoma, glioblastoma multiforme, glioma, medulloblastoma, meningioma, nonglial tumor, oligodendroglioma, pineoblastoma, pineocytoma, and primary brain lymphoma; breast cancer including but not limited to, breast carcinoma, breast sarcoma, and in some embodiments, adenocarcinoma, inflammatory breast cancer, intraductal carcinoma, lobular (small cell) carcinoma, medullary breast cancer, metastatic breast cancer, mucinous breast cancer, Paget's disease (including juvenile Paget's disease), papillary breast cancer, and tubular breast cancer; cervical cancers such as but not limited to, adenocarcinoma, cervical carcinoma, and squamous cell carcinoma;Attorney Docket Number: 01384-0002-00PCTcholangiocarcinomas such as but not limited to, diffuse, nodular, and papillary; colorectal cancer (colon cancer and rectal cancer), including but not limited to colon carcinoma and KRAS mutated colorectal cancer; cystadenocarcinoma; endotheliosarcoma and lymphangioendotheliosarcoma; esophageal cancers such as but not limited to, adenocarcinoma, adenoid cyctic carcinoma, adenosquamous carcinoma, melanoma, mucoepidermoid carcinoma, oat cell (small cell) carcinoma, plasmacytoma, sarcoma, squamous cancer, and verrucous carcinoma; eye cancers such as but not limited to, choroidal melanoma, ciliary body melanoma, ocular melanoma such as iris melanoma, and retinoblastoma; gallbladder cancers such as adenocarcinoma; hemangioblastoma; head and neck cancer, such as but not limited to squamous cell head and neck cancer, hematological malignancies such as but not limited to benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, heavy chain disease, leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as erythroleukemia leukemias, monocytic, myeloblastic, myelodysplastic syndrome, myelomonocytic, promyelocytic, chronic leukemias such as but not limited to hairy cell leukemia, chronic lymphocytic leukemia, and chronic myelocytic (granulocytic) leukemia, lymphomas such as but not limited to Hodgkin's disease and non-Hodgkin's disease, multiple myelomas such as but not limited to, extramedullary plasmacytoma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, smoldering multiple myeloma and solitary plasmacytoma, and polycythemia vera; kidney cancers such as but not limited to, adenocarcinoma, fibrosarcoma, hypernephroma, renal cell cancer, transitional cell cancer (renal pelvis and / or uterer), and Wilms tumor; liver cancers such as but not limited to, hepatoblastoma and hepatocellular carcinoma; lung cancers such as but not limited to adenocarcinoma, bronchogenic carcinoma, KRAS-mutated non-small cell lung cancer, largecell carcinoma, lung carcinoma, non-small cell lung cancer, papillary adenocarcinoma, smallcell lung cancer and squamous cell carcinoma (epidermoid carcinoma); mesothelioma myxosarcoma; neuroblastoma; neurofibroma; neurofibromatosis; oral cancers such as but not limited to, squamous cell carcinoma; ovarian cancers such as but not limited to, borderline tumor, germ cell tumor, ovarian epithelial carcinoma, and stromal tumor; papillary adenocarcinoma and papillary carcinoma; pancreatic cancer such as but not limited to, carcinoid or islet cell tumor, gastrinoma, glucagonoma, insulinoma, somatostatin-secreting tumor, and vipoma; pediatric tumors; penile cancers; pharynx cancers such as but not limited to, squamous cell cancer, and verrucous; pituitary cancers such as but limited to acromegaly, Cushing's disease, diabetes insipidus and prolactin-secreting tumors; prostate cancers such as but not limited to, rhabdomyosarcoma, seminoma, spermatocytic and teratoma carcinoma; renal cancer such as but not limited to renal carcinoma; salivary gland cancers such as but not limited to, adenocarcinoma, adenoid cystic carcinoma, andAttorney Docket Number: 01384-0002-00PCTmucoepidermoid carcinoma; skin cancers such as but not limited to, basal cell carcinoma, carcinomas of the epidermis, epithelial carcinoma, melanoma, including acrallentiginous melanoma, lentigo malignant melanoma, nodular melanoma, sebaceous gland carcinoma , squamous cell carcinoma, superficial spreading melanoma, and sweat gland carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; synovioma; testicular cancers such as but not limited to, adenocarcinoma, anaplastic, androgen dependent prostate cancer, androgen-independent prostate cancer, choriocarcinoma (yolk-sac tumor), classic (typical), embryonal carcinoma, germinal tumor, leiomyosarcoma, and nonseminoma; thyroid cancer such as but not limited to, anaplastic thyroid cancer, medullary thyroid cancer, and papillary or follicular thyroid cancer; uterine cancers such as but not limited to, endometrial carcinoma and uterine sarcoma; vaginal cancers such as adenocarcinoma, melanoma, and squamous cell carcinoma; vulvar cancer such as adenocarcinoma, basal cell carcinoma, melanoma, Paget's disease, sarcoma, and squamous cell carcinoma; and Waldenstrom'smacroglobulinemia.

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