Targeted radioligand therapy for treating cancer

Abstract

Conjugates comprising an EETI-II based knottin peptide, comprising an engineered loop that binds to a cell surface molecule; and a chelator conjugated to the knottin peptide via a linker may be used for treating cancer in a patient. Conjugates may comprise a radionuclide-chelator complex.

Description

Attorney Docket Number: 01384-0001 -00PCTTARGETED RADIOLIGAND THERAPY FOR TREATING CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 715,420, filed November 1 , 2024, 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 October 30, 2025, is named “01384-0001 -OOPCT.xml” and is 57,967 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 radioligand therapy for treating cancer.BACKGROUND

[0004] Targeted radiopharmaceuticals represent a significant advancement in cancer diagnosis and treatment. These radiopharmaceuticals typically consist of a radionuclide linked to a targeting agent (e.g., small molecules, peptides, antibodies / antibody fragments, or other protein scaffolds). By directing therapeutic radionuclides to tumors via a targeting agent, radioligand therapies aim to maximize damage to cancer cells and minimize damage to healthy tissues, enhancing the precision and effectiveness of treatments.

[0005] Desired characteristics for precise cancer diagnostics and therapy include high target selectivity and tumor retention as well as low uptake in healthy organs and tissues. However, the required criteria for a successful radioligand therapy are different from that of a successful imaging agent. First, the radioligand therapy needs to remain in the tumor long enough to deliver a sufficient radiation dose to kill cancer cells. Second, it is crucial to ensure that the radiation dose absorbed by healthy organs remains low enough to avoid toxicity, taking into account both the necessary therapeutic doses required for tumor killing as well as the radiation sensitivity of different organs that are exposed to the radiopharmaceutical.

[0006] That being the case, radiopharmaceuticals offer the advantage of early assessment for therapeutic suitability based on time-course imaging biodistribution profiles. Importantly, dose-limiting toxicity often arises from non-specific (not target-mediated) uptake in healthyAttorney Docket Number: 01384-0001 -00PCT organs, with kidney accumulation being a common issue for targeting agents with molecular weights below 30-60 kDa, which are typically cleared by renal excretion.1

[0007] Targeting agents with molecular weights above 30-60 kDa, such as antibody-based radiopharmaceuticals (-150 kDa), exhibit slower clearance, which is often beneficial for prolonging tumor retention time, but slow clearance also inherently prolongs systemic exposure, increasing the risk of damaging radiosensitive organs and tissues like bone marrow.

[0008] Integrins are a family of cell surface receptors that play crucial roles in cell adhesion, migration, and signaling. They are often overexpressed in various cancers, making them attractive targets for radioligand therapy.2Among integrin-binding peptides, small cyclic RGD peptides (~5 amino acids), multimeric cyclic RGD peptides, and other larger integrin-binding constrained peptide scaffolds have been widely studied as radiopharmaceuticals (W02013078250)3-7. However, like other peptides and protein-based targeting agents, a limitation of many of these agents is their high non-specific accumulation in healthy organs, particularly the kidneys.3-7This non-specific uptake can lead to dose-limiting toxicities, hindering their development and translation as radioligand therapies.

[0009] In contrast, engineered integrin-binding peptides based on the EETI knottin scaffold have very low non-specific kidney accumulation and low uptake in other healthy organs and tissue.8The initial design of engineered integrin-binding EETI knottins showed promise as radio-labeled imaging agents, as documented in previous publications, showing ~1-5% ID / g in the tumor at early timepoints between 0.5 to 4h post-injection, with suitably low uptake in other organs for imaging applications.8-10However, evaluation of biodistribution over longer periods of time is an important indicator for suitability for radioligand therapy applications given that the radioligand must be retained in the tumor long enough to deliver a sufficient radiation dose to kill cancer cells, and evaluation of the absorbed dose levels in healthy organs over time is critical for predicting potential toxicity.

[0010] Despite the promising imaging data of these first-generation engineered integrin- binding EETI knottins at early timepoints, evaluation of biodistribution at later timepoints (24h post-injection and later) revealed that these first-generation agents were not particularly promising for radioligand therapy in their current form.11

[0011] Despite the advancements in radioligand therapies, a significant number of patients are not eligible for currently approved treatments due to limitations in targeting specific tumor types and managing off-target effects. This underscores a critical unmet need in the field of oncology. 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. However, there isAttorney Docket Number: 01384-0001 -00PCT still a pressing need for improved integrin-targeting agents that exhibit beneficial biodistribution profiles, minimizing non-specific uptake in healthy organs while maximizing tumor targeting. Taken together, the development of such advanced tumor-targeting binders can enhance the efficacy and safety of radioligand therapies, ultimately expanding treatment options for a broader patient population.SUMMARY

[0012] Provided are radioconjugates comprising tumor-targeting moieties comprising engineered knottin peptides.

[0013] Embodiment 1 . A conjugate comprising: (a) an EETI-II based knottin peptide, comprising an engineered loop that binds to a cell surface molecule; and (b) a chelator.

[0014] Embodiment 2. The conjugate of embodiment 1 , wherein the chelator is conjugated to the EETI-II based knottin peptide via a linker.

[0015] Embodiment 3. The conjugate of any one of embodiments 1 -2, wherein the conjugate further comprises a radionuclide.

[0016] Embodiment 4. The conjugate of embodiment 3, wherein the radionuclide is selected

[0017] Embodiment 5. The conjugate of any one of embodiments 1 -4, wherein the conjugate is capable of treating cancer in a patient.

[0018] Embodiment 6. The conjugate of any one of embodiments 1 -5, wherein the conjugate comprises a dimer of EETI-II based knottin peptides.

[0019] Embodiment 7. The conjugate of any one of embodiments 1 -5, wherein the conjugate comprises a trimer of EETI-II based knottin peptides.

[0020] Embodiment 8. The conjugate of any one of embodiments 1 -7, wherein the cell surface molecule is an integrin.

[0021] Embodiment 9. The conjugate of embodiment 8, wherein the integrin is avPi integrin, avPe integrin, avPs integrin, avp3integrin, and / or a5Pi integrin.

[0022] Embodiment 10. The conjugate of any one of embodiments 1 -9, wherein the EETI-II based knottin peptide comprisesGCXiX2X3X4X5X6X7X8XgXioXiiXi2Xi3Xi4Xi5Xi6Xi7Xi8XigX2oCX2iQDSDCX22AGCVCX23X24X25X26X27X28X29X30X3iX32X33CG (SED ID NO: 2), wherein Xi - X3are any amino acid; X4Attorney Docket Number: 01384-0001 -00PCT-X2o if present, are any amino acid; X2i, 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.

[0023] Embodiment 1 1 . The conjugate of any one of embodiments 1 -9, wherein the EETI-II based knottin peptide comprises GCXiX2X3X4X5X6X7X8XgXioXiiXi2Xi3Xi4Xi5Xi6Xi7Xi8XigX2oCX2iQDSDCX22AGCVCGPNGX23 CG (SEQ ID NO: 3), wherein X1-X3 are any amino acid; X4-X20, if present, are any amino acid; and wherein X2i-X23 are any amino acid, further wherein each amino acid is independently selected from standard or unnatural amino acids.

[0024] Embodiment 12. The conjugate of any one of embodiments 1 -1 1 , 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, or SEQ ID NO: 36.

[0025] Embodiment 13. The conjugate of any one of embodiments 1 -1 1 , 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, or SEQ ID NO: 36.

[0026] Embodiment 14. The conjugate of any one of embodiments 1 -1 1 , wherein the engineered loop has a sequence of any one of SEQ ID NO: 4 through SEQ ID NO: 8, inclusive, or SEQ ID NO: 36.

[0027] Embodiment 15. The conjugate of any one of embodiments 1 -1 1 , 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: 30, inclusive.

[0028] Embodiment 16. The conjugate of embodiment 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 9.

[0029] Embodiment 17. The conjugate of embodiment 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 10.

[0030] Embodiment 18. The conjugate of embodiment 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 1 1 .

[0031] Embodiment 19. The conjugate of embodiment 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 12.

[0032] Embodiment 20. The conjugate of embodiment 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 25.

[0033] Embodiment 21 . The conjugate of embodiment 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 26.Attorney Docket Number: 01384-0001 -00PCT

[0034] Embodiment 22. The conjugate of embodiment 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 27.

[0035] Embodiment 23. The conjugate of embodiment 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 28.

[0036] Embodiment 24. The conjugate of any one of embodiments 1 -23, wherein the chelator is selected from: 1 ,4,7,10-Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA); S-2-(4-lsothiocyanatobenzyl)-1 ,4,7,10 tetraazacyclododecanetetraacetic acid (p-SCN-Bn- DOTA); 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic amide (TCMC; also known as DOTAM); diethylene triamine pentaacetic acid (DTPA); ethylene diamine tetraacetic acid (EDTA); p-isothiocyanatobenzyl-1 ,4,7,10-tetra-azacyclododecane-1 ,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA); 1 ,4,7,10-tetra-azacyclododecane-N,N',N"-triacetic acid (DO3A);1.4.7.10-tetra-azacyclododecane-1 ,4,7,10-tetrakis(2-propionic acid) (DOTMA); 3,6,9-triaza- 12-oxa-3,6,9-tricarboxymethylene-10-carboxy-13-phenyl-tridecanoic acid (“B-19036”); 1 ,4,7- triazacyclononane-N,N',N"-triacetic acid (NOTA); 1 ,4,8,11 -tetra-azacyclotetradecane-1 ,4,8,11 -tetraacetic acid (TETA); triethylene tetraamine hexaacetic acid (TTHA); trans-1 ,2- diaminohexane tetraacetic acid (CYDTA); 1 ,4,7,10-tetra-azacyclododecane-1-(2- hydroxypropyl)-4,7,10-triacetic acid (HP-DO3A); trans-cyclohexane-diamine tetraacetic acid (CDTA); trans(1 ,2)-cyclohexane dietylene triamine pentaacetic acid (CDTPA); 1-oxa-4,7,10- triazacyclododecane-N,N',N"-triacetic acid (OTTA); 1 ,4,7,10-tetra-azacyclododecane-1 .4.7.10-tetrakis{3-(4-carboxyl)-butanoic acid}; 1 ,4,7,10-tetra-azacyclododecane-1 ,4,7,10- tetrakis(acetic acid-methyl amide); 1 ,4, 7,10-tetra-azacyclododecane- 1 ,4, 7,10- tetrakis(methylene phosphonic acid); 1 ,4,7-triazacyclononane-l,4-diacetic acid (NODA);1 ,4,7- tri azacyclononane, 1 -glutaric acid-4, 7-diacetic acid (NODAGA); 1 ,4,7,10- tetraazacyclodecane, 1 -glutaric acid-4,7, 10-triacetic acid (DOTAGA); 1 ,4,8,11- tetraazabicyclo[6.6.2]hexadecane-4, 11 -diacetic acid (CB-TE2A); 2-cyclohexyl diethylene triamine pentaacetic acid (CHX-A”-DTPA); DAD; deforoxamine (DFO); l,2-[[6-carboxypyridin- 2-yl]methylamino]ethane (H2dedpa); 2- (Carboxymethylamino)acetic acid (IDA); ethylene glycol-bis(2-aminoethylether)- N,N,N',N'-tetra acetic acid (EGTA); 1 ,2-bis(o- aminophenoxy)ethane-N,N,N’,N’- tetraacetic acid (BAPTA); ethylenediamine-N,N’- disuccinic acid (EDDS); 2,2',2",2"',2"",2 -(1 ,4,7,10,13,16- hexaazacyclooctadecane-1 ,4,7,10,13,16- hexayl) hexaacetic acid (HEHA); 2,2',2",2"',2""-(1 ,4,7,10,13-pentaazacyclopentadecane- 1 ,4,7,10,13- pentayl)pentaacetic acid (PEPA); 3,3',3",3"'-(1 ,5,9,13- tetraazacyclohexadecane- 1 ,5,9, 13-tetrayl)tetrapropionic acid (TETPA); and 3, 3', 3", 3"'- (1 ,4,7,10- tetraazacyclododecane- 1 ,4, 7, 10-tetrayl)tetrapropionic acid (DOTPA);and derivatives thereof .Attorney Docket Number: 01384-0001 -00PCT

[0037] Embodiment 25. The conjugate of any one of embodiments 1 -24, wherein the chelator comprises 1 ,4,7,10-Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA).

[0038] Embodiment 26. The conjugate of any one of embodiments 1 -24, wherein the chelator comprises 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic amide (DOTAM).

[0039] Embodiment 27. The conjugate of any one of embodiments 1 -25, wherein the linker comprises a chain of about 1 to about 12 carbon atoms.

[0040] Embodiment 28. The conjugate of embodiment 27 wherein the linker comprises a chain of 4 carbon atoms.

[0041] Embodiment 29. The conjugate of any one of embodiments 1 -25, wherein the linker comprises one or more polyethylene glycol (PEG) units.

[0042] Embodiment 30. The conjugate of embodiment 29, wherein the linker comprises a chain of 4 PEG units.

[0043] Embodiment 31 . The conjugate of any one of embodiments 1 -30, wherein the chelator is conjugated to the EETI-II based knottin peptide at the N-terminal amino acid of the peptide.

[0044] Embodiment 32. The conjugate of any one of embodiments 1 -30, wherein the chelator is conjugated to the EETI-II based peptide at the C-terminal amino acid of the peptide.

[0045] Embodiment 33. The conjugate of any one of embodiments 1 -30, wherein the chelator is conjugated to an internal, non-terminal amino acid of the EETI-II based knottin peptide.

[0046] Embodiment 34. The conjugate of embodiment 33, wherein the chelator is conjugated to an amino acid that is C-terminal to the engineered loop of the EETI-II based knottin peptide.

[0047] Embodiment 35. The conjugate of any one of embodiments 33-34, wherein the chelator is conjugated to the fifteenth amino acid of the EETI-II based knottin peptide, wherein the numbering is from N- to C-terminal order.

[0048] Embodiment 36. The conjugate of any one of embodiments 1 -35, further comprising an antibody subunit or fragment thereof fused to the EETI-II based knottin peptide.

[0049] Embodiment 37. The conjugate of any one of embodiments 1 -36, further comprising a half-life extending moiety.

[0050] Embodiment 38. The conjugate of embodiment 37, wherein the half-life extending moiety is an albumin-binding moiety.Attorney Docket Number: 01384-0001 -00PCT

[0051] Embodiment 39. The conjugate of embodiment 38, wherein the albumin-binding moiety is a small molecule albumin-binding moiety.

[0052] Embodiment 40. The conjugate of embodiment 38, wherein the albumin-binding moiety is selected from: truncated Evans Blue (EB), ibuprofen, 4-p-(tolyl)butyric acid (PT), palmitic acid (C16), and 4-(p-lodophenyl)butyric acid (IP).

[0053] Embodiment 41 . The conjugate of embodiment 38, wherein the albumin-binding moiety is truncated Evans Blue (EB).

[0054] Embodiment 42. The conjugate of embodiment 38, wherein the albumin-binding moiety is a linear or branched lipophilic chain comprising 1 -40 carbon atoms.

[0055] Embodiment 43. A method of treating cancer, comprising administering the conjugate of any one of embodiments 1-28 to a patient in need thereof.

[0056] Embodiment 44. The method of embodiment 43, wherein the cancer is a solid tumor.

[0057] Embodiment 45. The method of embodiment 44, wherein the cancer is selected from blastoma, carcinoma, lymphoma, or sarcoma.

[0058] Embodiment 46. The method of embodiment 45, wherein the cancer is selected from head and neck cancer, non-small cell lung cancer, colorectal cancer, esophageal cancer, uterine cancer, ovarian cancer, gastric cancer, pancreatic cancer, breast cancer, bladder cancer, melanoma, renal cancer, liver cancer, gallbladder cancer, sarcomas, or brain tumors.

[0059] Embodiment 47. The method of embodiment 46, 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,Attorney Docket Number: 01384-0001 -00PCT 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; 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 toAttorney Docket Number: 01384-0001 -00PCT 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, 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.

[0060] Additional objects and advantages will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0061] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0062] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The present application contains at least one drawing executed in color. Copies of this patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0064] FIG. 1 : Schematic of an engineered EETI-based knottin radiopharmaceuticalAttorney Docket Number: 01384-0001 -00PCT

[0065] FIG. 2: Schematics of engineered EETI knottin peptide-based radioconjugates

[0066] FIG. 3: Schematics of engineered EETI knottin-fusion protein radioconjugates

[0067] FIG. 4: Chemical structure of PRC-1 (DOTA-C4) (including the EETI-II peptide of SEQ ID NO: 9)

[0068] FIG. 5: Chemical structure of PRC-2 (IP-DOTA) (including the EETI-II peptide of SEQ ID NO: 9)

[0069] FIG. 6: Chemical structure of PRC-3 (NH2-DOTA) (including the EETI-II peptide of SEQ ID NO: 9)

[0070] FIG. 7: Chemical structure of PIP-2 (including the EETI-II peptide of SEQ ID NO: 9)

[0071] FIGS. 8A-C: Synthesis strategy for Intermediate Compounds 4-2 (FIG. 8A), 5-2 (FIG. 8B), 6-2 (FIG. 8C).

[0072] FIG. 9: Synthesis strategy for PRC-4 (PT-DOTA) (including the EETI-II peptide of SEQ ID NO: 9)

[0073] FIG. 10: Synthesis strategy for PRC-5 (Ibu-DOTA) (including the EETI-II peptide of SEQ ID NO: 9)

[0074] FIG. 11 : Synthesis strategy for PRC-6 (EB-DOTA) (including the EETI-II peptide of SEQ ID NO: 9)

[0075] FIG. 12: Synthesis strategy for PRC-7 (C16-DOTA) (including the EETI-II peptide of SEQ ID NO: 9)

[0076] FIGS. 13A-B: Chemical intermediates used for synthesizing the branched linkers of Di-PRC-1 (FIG. 13A) and Tri-PRC-1 (FIG. 13B)

[0077] FIG. 14: Chemical structure of Di-PRC-1 (including the EETI-II peptide of SEQ ID NO: 9)

[0078] FIG. 15: Chemical structure of Tri-PRC-1 (including the EETI-II peptide of SEQ ID NO: 9)

[0079] FIGS. 16A-B: General structure (FIG. 16A) and SEC (FIG. 16B) of PFRC-1 (p-SCN- Bn-DOTA)

[0080] FIGS. 17A-B: LCMS of PFRC-1 (p-SCN-Bn-DOTA)

[0081] FIG. 18A-B: General structure (FIG. 18A) and SEC (FIG. 18B) of PARC-1 (p-SCN- Bn-DOTA)

[0082] FIG. 19A-B: LCMS of PARC-1 (p-SCN-Bn-DOTA)Attorney Docket Number: 01384-0001 -00PCT

[0083] FIGS. 20A-B: SPECT / CT images showing biodistribution at 1 h (FIG. 20A) and 4 h (FIG. 20B) after IV administration of111ln-PRC-1

[0084] FIGS. 21 A-B: SPECT / CT images showing biodistribution at 24 h (FIG. 21 A) and 48 h (FIG. 21 B) after IV administration of111ln-PRC-1

[0085] FIGS. 22A-B: SPECT / CT images showing biodistribution at 1 h (FIG. 22A) and 4 h (FIG. 22B) after IV administration of111ln-PRC-2

[0086] FIGS. 23A-B: SPECT / CT images showing biodistribution at 24 h (FIG. 23A) and 48 h (FIG. 23B) after IV administration of111ln-PRC-2

[0087] FIG. 24: Blood concentration (% ID / g) of111ln-PRC-1 and111ln-PRC-2 over time (mean ± SD)

[0088] FIG. 25: Ex vivo gamma counting quantification of organs and tumors at 48 h postinjection of111ln-PRC-1 (mean ± SD)

[0089] FIG. 26: Ex vivo gamma counting quantification of organs and tumors at 48 h postinjection of111ln-PRC-2 (mean ± SD)

[0090] FIG. 27A-F: Ex vivo quantified tumor-to-organ ratios for111ln-PRC-1 and111ln-PRC-2 at 48 h post-injection (mean ± SD). FIG. 27A tumor / kidneys ratio; FIG. 27B tumor / blood ratio; FIG. 27C tumor / bone ratio; FIG. 27D tumor / liver ratio; FIG. 27E tumor / muscle ratio; FIG. 27F tumor / lungs ratio.

[0091] FIG. 28: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, and 48 h after administration of111ln-PRC-1 (mean + SEM).

[0092] FIG. 29: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, and 48 h after administration of111ln-PRC-2 (mean + SEM).

[0093] FIG. 30: Description of reaction conditions and results from test radiolabeling studies for111ln-PFRC-1 and111ln-PARC-1

[0094] FIG. 31 : Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 72 h, and 168 h after administration of111ln-PFRC-1 (mean + SEM).

[0095] FIG. 32: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 72 h, and 168 h after administration of111ln-PARC-1 (mean + SEM).

[0096] FIG. 33: Blood concentration (% ID / g) of111ln-PRC-1 ,111ln-PRC-2,111ln-PFRC-1 , and111ln-PARC-1 at 4.5 h post-injection (mean + SD)

[0097] FIG. 34: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-1 (mean + SEM).Attorney Docket Number: 01384-0001 -00PCT

[0098] FIG. 35: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-3 (mean + SEM).

[0099] FIG. 36: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-4 (mean + SEM).

[0100] FIG. 37: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-5 (mean + SEM).

[0101] FIG. 38: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111In-Di-PRC-1 (mean + SEM).

[0102] FIG. 39: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111In-Tri-PRC-1 (mean + SEM).

[0103] FIG. 40: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 168 h after administration of111ln-PRC-7 (mean + SEM).

[0104] FIG. 41 : Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-1 (mean + SEM).

[0105] FIG. 42: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 168 h after administration of111ln-PRC-7 (mean + SEM).

[0106] FIG. 43A-B: Blood concentration (% ID / g) of111ln-PRC-1 ,111ln-PRC-3,111ln- PRC-4,111ln-PRC-5,111ln-PRC-7,111ln-Di-PRC-1 , and111ln-Tri-PRC-1 at 5 min (FIG. 43A) and 4.5 h (FIG. 43B) post-injection.

[0107] FIG. 44: Comparison of tumor uptake quantification over time derived from SPECT / CT imaging for111ln-PRC-1 ,111ln-PRC-3,111ln-PRC-4, and111ln-PRC-5 (mean + SEM).

[0108] FIG. 45: Comparison of tumor uptake quantification over time derived from SPECT / CT imaging for111ln-PRC-1 ,111ln-Di-PRC-1 , and111ln-Tri-PRC-1 (mean + SEM).

[0109] FIG. 46: Chemical structure of PRC-9 (internal DOTA-C4, including the EETI- II peptide of SEQ ID NO: 12)[001 10] FIG. 47: Chemical structure of PRC-10 (N-acetylated, internal DOTA-C4, including the EETI-II peptide of SEQ ID NO: 12)Attorney Docket Number: 01384-0001 -00PCT[001 11 ] FIG. 48: Chemical structure of PRC-1 1 (DOTA-PEG4, including the EETI-II peptide of SEQ ID NO: 9)[001 12] FIG. 49: Chemical structure of PRC-12 (DOTA-C2, including the EETI-II peptide of SEQ ID NO: 9)[001 13] FIG. 50: Chemical structure of PRC-13 (DOTA-C6, including the EETI-II peptide of SEQ ID NO: 9)[001 14] FIG. 51 : Chemical structure of PRC-14 (DOTAM-C4, including the EETI-II peptide of SEQ ID NO: 9)[001 15] FIG. 52: Chemical structure of PRC-1 .4L (DOTA-C4, including the EETI-II peptide of SEQ ID NO: 25)[001 16] FIG. 53: Chemical structure of TRC-1 (DOTA-C4, including the EETI-II peptide of SEQ ID NO: 20)[001 17] FIG. 54: Chemical structure of TRC-1 .31 F (DOTA-C4, including the EETI-II peptide of SEQ ID NO: 19)[001 18] FIG. 55: Chemical structure of NRC-1 (DOTA-C4, including the EETI-II peptide of SEQ ID NO: 34)[001 19] FIG. 56: Chemical structure of NRC-1 .4R (DOTA-C4, including the EETI-II peptide of SEQ ID NO: 35)

[0120] FIG. 57A-B: Competition binding assay in HEC1 A endometrial cancer cells showing monomeric (PRC-1 , PRC-10, PRC-11 ), dimeric (Di-PRC-1 ), and trimeric (Tri-PRC- 1 ) peptide-chelator constructs’ binding profiles relative to PIP-2 (no chelator control). FIG. 57A: PRC-1 , Di-PRC-1 and Tri-PRC-1 as compared to PIP-2. FIG. 57B: PRC-10 and PRC- 1 1 as compared to PIP-2.

[0121] FIG. 58A-B: Competition binding assays with PRC-1 , PRC-1 .4L, and TRC-1 for representative integrins, avpi (FIG. 58A) and avp3 (FIG. 58B).

[0122] FIG. 59A-B: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-1 (mean + SEM). FIG. 59A %l D / g over time in tumor and kidney. FIG. 59B: %l D / g over time in bone, brain, heart, kidney, liver, lungs, muscle, spleen, stomach, and tumor.

[0123] FIG. 60A-B: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-1 .4L (mean + SEM). FIG. 60A %l D / g over time in tumor and kidney. FIG. 60B: %l D / g over time in bone, brain, heart, kidney, liver, lungs, muscle, spleen, stomach, and tumor.Attorney Docket Number: 01384-0001 -00PCT

[0124] FIG. 61A-B: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-10 (mean + SEM). FIG. 61 A %ID / g over time in tumor and kidney. FIG. 61 B: %ID / g over time in bone, brain, heart, kidney, liver, lungs, muscle, spleen, stomach, and tumor.

[0125] FIG. 62A-B: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-11 (mean + SEM). FIG. 62A %l D / g over time in tumor and kidney. FIG. 62B: %l D / g over time in bone, brain, heart, kidney, liver, lungs, muscle, spleen, stomach, and tumor.

[0126] FIG. 63: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-12 (mean + SEM).

[0127] FIG. 64: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-13 (mean + SEM).

[0128] FIG. 65: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-TRC-1 (mean + SEM).

[0129] FIG. 66: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-NRC-1 (mean + SEM).

[0130] FIG. 67: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-NRC-1 .4R (mean + SEM).

[0131] FIG. 68A-B: Average tumor volume over time in U87MG-tumor bearing mice treated with vehicle or 2 doses (FIG. 68A) or 3 doses (FIG. 68B) of177Lu-PRC-1 (mean + SEM).

[0132] FIG. 69A-B: Average % body weight change over time in U87MG-tumor bearing mice treated with vehicle or 2 doses (FIG. 69A) or 3 doses (FIG. 69B) of177Lu-PRC- 1 (mean + SD).DESCRIPTION OF THE SEQUENCES

[0133] Table 1 provides a listing of certain sequences referenced herein. For SEQ ID Nos: 9-24, (1 ) each bold letter represents a member of the integrin binding loop; (2) Z = unnatural amino acid.Attorney Docket Number: 01384-0001 -00PCTAttorney Docket Number: 01384-0001 -00PCTAttorney Docket Number: 01384-0001 -00PCTDESCRIPTION OF THE EMBODIMENTS

[0134] 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.

[0135] 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 of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0136] 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.

[0137] 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 andAttorney Docket Number: 01384-0001 -00PCT methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0138] 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. ConjugatesA. Knottin peptide

[0139] 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.

[0140] 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.

[0141] 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 loopAttorney Docket Number: 01384-0001 -00PCT 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. 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-independent growth 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.

[0142] 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.

[0143] 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 )Attorney Docket Number: 01384-0001 -00PCT

[0144] 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.

[0145] 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:GCXiX2X3X4X5X6X7X8XgXioXiiXi2Xi3Xi4Xi5Xi6Xi7Xi8XigX2oCX2iQDSDCX22AGCVCX23X24X25X26X27X28X2gX3oX3iX32X33CG (SEQ ID NO: 2) whereinXi - X3= any amino acid;X4- X20= if present, any amino acid;X2i= 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.

[0146] 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:GCXiX2X3X4X5X6X7X8XgXioXiiXi2Xi3Xi4Xi5Xi6Xi7Xi8XigX2oCX2iQDSDCX22AGCVCG PNGX23CG (SEQ ID NO: 3) whereinXi - X3= any amino acid;X4- X20= if present, any amino acid;X2i= any amino acid;X22= any amino acid;Attorney Docket Number: 01384-0001 -00PCTX23 = any amino acid; wherein for each numbered X position, any amino acid may include standard or unnatural amino acids.

[0147] 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 = an unnatural amino acid:Attorney Docket Number: 01384-0001 -00PCT

[0148] In certain embodiments, an EETI-based knottin peptide, 2.5F F31Y (sometimes referred to herein as “PIP-2”) of a conjugate of the present disclosure, which binds to each of avpi integrin, avp3 integrin, avp5 integrin, avp6 integrin, and a5pi integrin, has the following amino acid sequence (with the integrin-binding loop in bold):GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGYCG (SEQ ID NO: 9)B. Chelators and radionuclides

[0149] The terms “chelator" or “chelating agent" refer to polydentate (multiple bonded) ligands capable of forming two or more separate coordinate bonds with (“coordinating") a central (metal) ion. The central (metal) ion is usually coordinated by two or more electron pairs to the chelating agent. Usually, the electron pairs of a chelating agent forms coordinate bonds with a single central (metal) ion; however, in certain embodiments, a chelating agent may form coordinate bonds with more than one metal ion, with a variety of binding modes being possible. The terms “coordinating" and “coordination” refer to an interaction in which one multi-electron pair donor coordinatively bonds (“is coordinated”) to, i.e. shares two or more unshared pairs of electrons with, one central (metal) ion. The chelating agent may be chosen based on its ability to coordinate the desired central (metal) ion, usually a radionuclide as specified herein. In some embodiments, the chelator is complexed with a radionuclide. In some embodiments, the conjugate comprises a chelator, and further comprises a radionuclide.

[0150] The term "radionuclide" or "radioisotope" refers to isotopes of natural or artificial origin with an unstable neutron to proton ratio that disintegrates with the emission ofAttorney Docket Number: 01384-0001 -00PCT corpuscular (i .e. protons (alpha-radiation) or electrons (beta-radiation)) or electromagnetic radiation (gamma-radiation). In other words, radionuclides undergo radioactive decay. Such radionuclides include, without limitation:94Tc,99mTc,90ln,111ln,67Ga,68Ga,86Y,88Y,90Y,177Lu,151Tb,223Ra,186Re,188Re,61Cu,62Cu,64Cu,67Cu,55Co,57Co,43Sc,44Sc,46Sc,47Sc,85Sr,89Sr,90Sr,117mSn,145Sm,235Ac,213Bi,212Bi,32P,33P,203Pb,212Pb,227Th,153Sm,166Ho,152Gd,153Gd,157Gd,225Ac,211At,166Ho,161Tb or166Dy. In some embodiments, the radionuclide is selected from99mTc,111ln,67Ga,68Ga,86Y,90Y,177Lu,151Tb,223Ra,186Re,188Re,61Cu,62Cu,64Cu,67Cu,55Co,57Co,43Sc,44Sc,46Sc,47Sc,85Sr,89Sr,90Sr,117mSn,145Sm,235Ac,213Bi,212Bi,32P,33P,203Pb,212Pb,227Th,153Sm,166Ho,225Ac,211At,166Ho, and161Tb. The choice of suitable radionuclides may depend on the chemical structure and chelating capability of the chelating agent, and the intended application of the resulting drug (e.g. diagnostic vs. therapeutic).

[0151] Aspects of the present disclosure include conjugates of knottin-peptide or knottin peptide-protein fusions that may be covalently linked to a chelator component directly or via a linker. Exemplary chelators include compounds having the dual functionality of sequestering metal ions, such as the radionuclide, plus the ability to be covalently attached to the targeting agent. In some embodiments, the chelating agent comprises 1 ,4,7,10- Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA). In some embodiments, DOTA is conjugated to the targeting agent using a linker consisting of 1 -12 carbon atoms.Alternatively, DOTA is conjugated to the targeting agent using a linker consisting of one or more polyethylene glycol (PEG) units. DOTA may be conjugated using a linker comprising amino acids or other chemical groups that are biologically compatible. Other derivatives of DOTA may also be used, including S-2-(4-lsothiocyanatobenzyl)-1 ,4,7,10 tetraazacyclododecanetetraacetic acid (p-SCN-Bn-DOTA). In some embodiments, 1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic amide (TCMC) and its derivatives may be used. TCMC is also known as DOTAM.

[0152] Additional exemplary chelators that may be used include, but are not limited to: diethylene triamine pentaacetic acid (DTPA); ethylene diamine tetraacetic acid (EDTA); p-isothiocyanatobenzyl-1 ,4,7,10-tetra-azacyclododecane-1 ,4,7,10-tetraacetic acid (p-SCN- Bz-DOTA); 1 ,4,7,10-tetra-azacyclododecane-N,N',N"-triacetic acid (DO3A); 1 ,4,7, 10-tetra- azacyclododecane-1 ,4,7,10-tetrakis(2-propionic acid) (DOTMA); 3,6,9-triaza-12-oxa-3,6,9- tricarboxymethylene-10-carboxy-13-phenyl-tridecanoic acid (“B- 19036”); 1 ,4,7- triazacyclononane-N,N',N"-triacetic acid (NOTA); 1 ,4,8,1 1 -tetra-azacyclotetradecane- 1 ,4,8,11 -tetraacetic acid (TETA); triethylene tetraamine hexaacetic acid (TTHA); trans-1 ,2-Attorney Docket Number: 01384-0001 -00PCT diaminohexane tetraacetic acid (CYDTA); 1 ,4,7,10-tetra-azacyclododecane-1-(2- hydroxypropyl)-4,7,10-triacetic acid (HP-DO3A); trans-cyclohexane-diamine tetraacetic acid (CDTA); trans(1 ,2)-cyclohexane dietylene triamine pentaacetic acid (CDTPA); 1-oxa-4,7,10- triazacyclododecane-N,N',N"-triacetic acid (OTTA); 1 ,4,7,10-tetra-azacyclododecane- 1 ,4,7,10-tetrakis{3-(4-carboxyl)-butanoic acid}; 1 ,4,7,10-tetra-azacyclododecane-1 ,4,7,10- tetrakis(acetic acid-methyl amide); 1 ,4, 7,10-tetra-azacyclododecane- 1 ,4, 7,10- tetrakis(methylene phosphonic acid); 1 ,4,7-triazacyclononane-l,4-diacetic acid (NODA);1 ,4,7- tri azacyclononane, 1 -glutaric acid-4, 7-diacetic acid (NODAGA); 1 ,4,7,10- tetraazacyclodecane, 1 -glutaric acid-4,7, 10-triacetic acid (DOTAGA); 1 ,4,8,11- tetraazabicyclo[6.6.2]hexadecane-4, 11 -diacetic acid (CB-TE2A); 2-cyclohexyl diethylene triamine pentaacetic acid (CHX-A”-DTPA); DAD; deforoxamine (DFO); l,2-[[6-carboxypyridin- 2-yl]methylamino]ethane (H2dedpa); 2- (Carboxymethylamino)acetic acid (IDA); ethylene glycol-bis(2-aminoethylether)- N,N,N',N'-tetra acetic acid (EGTA); 1 ,2-bis(o- aminophenoxy)ethane-N,N,N’,N’- tetraacetic acid (BAPTA); ethylenediamine-N,N’- disuccinic acid (EDDS); 2,2',2",2"',2"",2 -(1 ,4,7,10,13,16- hexaazacyclooctadecane-1 ,4,7,10,13,16- hexayl) hexaacetic acid (HEHA); 2,2',2",2"',2""-(1 ,4,7,10,13-pentaazacyclopentadecane- 1 ,4,7,10,13- pentayl)pentaacetic acid (PEPA); 3,3',3",3"'-(1 ,5,9,13- tetraazacyclohexadecane- 1 ,5,9, 13-tetrayl)tetrapropionic acid (TETPA); and 3, 3', 3", 3"'- (1 ,4,7,10- tetraazacyclododecane- 1 ,4, 7, 10-tetrayl)tetrapropionic acid (DOTPA); and derivatives thereof. Additional example chelators and their derivatives are listed in WO2023191839.C. Linkers

[0153] In some embodiments, the chelator is conjugated to the targeting agent using a linker consisting of 1 -12 carbon atoms. Alternatively, the chelator is conjugated to the targeting agent using a linker consisting of one or more ethylene glycol units (polyethylene glycol: PEG). Alternatively, the chelator is conjugated to the targeting agent using a linker consisting of one or more sarcosine units (polysarcosine). The chelator may be conjugated using a linker comprising amino acids or other chemical groups that are biologically compatible. In some embodiments, the linker comprises 4 carbon atoms.D. Knottin-based chelator conjugates

[0154] Aspects of the present disclosure include knottin-based chelator conjugates. The chelator employed in the conjugates that include a knottin peptide component may be any suitable agent and can vary depending on the application for which the conjugate is employed, e.g., imaging or therapeutic purposes, etc. Non-limiting examples of such chelators are described in the preceding section relating to chelators.Attorney Docket Number: 01384-0001 -00PCT1. Knottin peptide-chelator conjugates

[0155] We describe engineered integrin-binding EETI knottin peptide-chelator conjugates. Such conjugates include an EETI-based knottin peptide including an engineered loop that binds to one or more integrins, conjugated to a radio-chelator. The chelator can be conjugated at different sites on the knottin peptide. For example, the chelator can be conjugated to the N-terminus of the peptide. Alternatively, the chelator can be conjugated to an amino acid that is outside of the engineered binding loop. The chelator can be conjugated to natural amino acids or can be incorporated using unnatural amino acids.

[0156] In some embodiments, the chelator will be connected to the knottin peptide using a linker. The linker may avoid steric hindrance between the chelator 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. Suitable linkers may be stable in vivo. 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.2. Knottin-antibody subunit chelator conjugates

[0157] We describe integrin-binding EETI knottin-Fc and knottin-antibody radiochelator conjugates. Aspects of the present disclosure further include engineered integrin- binding EETI knottin-antibody subunit-chelator conjugates. Such conjugates include a fusion protein that includes an EETI-based knottin peptide including an engineered loop that binds to one or more integrins, fused to an antibody subunit or fragment thereof. Such conjugates further include a chelator conjugated to the fusion protein. In some embodiments, provided are dimers of such conjugates, where the antibody subunits or fragments thereof dimerize (e.g., via disulfide bridges at a hinge region (if present), or the like) to form dimerized knottin chelator conjugates.

[0158] According to some embodiments, the antibody subunit or fragment thereof 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 IgG 1 heavy chain or fragment thereof. In certain embodiments, the antibody heavy chain or fragment thereof comprises a heavy chain variable region (VH). Such an antibody heavy chain or fragment thereof may further include a heavy chain constant region or fragment thereof. For example, when a heavy chainAttorney Docket Number: 01384-0001 -00PCT constant region or fragment thereof is included in the fusion protein, the antibody heavy chain constant region or fragment thereof may include one or more of a CH 1 domain, CH2 domain, and / or CH3 domain. 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 subunit or fragment thereof is an antibody heavy chain or fragment thereof that does not include a VH. Such an antibody heavy chain or fragment thereof may include, consist essentially of, or consist of an Fc region.

[0159] When a conjugate of the present disclosure includes a knottin peptide fused to an antibody heavy chain or fragment 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.

[0160] Chelators may be conjugated stochastically throughout the protein, resulting in chelator 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 chelators attached to primary amines in the protein (e.g., free amine group at the N-terminus of the protein or lysine residues).

[0161] Alternatively, for the conjugates that include a knottin peptide fused to an antibody heavy chain or fragment thereof, the chelator may be conjugated specifically to the antibody heavy chain or fragment thereof portion of the fusion protein. For example, when the antibody heavy chain or fragment thereof includes, consists essentially of, or consists of Fc region, the chelator may be conjugated to the Fc region. In these embodiments, the chelator 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 chelator may be conjugated to the knottin peptide portion of the fusion protein.

[0162] According to some embodiments, the antibody subunit or fragment 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 anAttorney Docket Number: 01384-0001 -00PCT 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.

[0163] For the conjugates that include a knottin peptide fused to an antibody light chain or fragment thereof, chelators may be conjugated stochastically throughout the protein, resulting in chelator attachments at multiple sites of the overall protein fusion. Alternatively, the chelator may be conjugated specifically to the antibody light chain or fragment thereof portion of the fusion protein. For example, the chelator may be conjugated to a VL (if present) or a CL (if present), e.g., at or near the C-terminus of a CL. Alternatively, the chelator may be conjugated to the knottin peptide portion of the fusion protein. In some embodiments, the chelator may be conjugated specifically to the antibody heavy chain or fragment thereof portion of the fusion protein. In certain embodiments, when a conjugate of the present disclosure includes a knottin peptide fused to an antibody subunit or fragment thereof that includes a heavy chain variable region (VH) or a light chain variable region (VL), the VH or VL does not bind to the same antigens that are bound by the engineered knottin peptide.3. Knottin-chelator conjugates with other half-life extending moieties

[0164] A strategy that can potentially lead to increased tumor retention time of a radiopharmaceutical involves conjugation or fusion to other types of half-life extending moieties besides antibodies and antibody fragments. In some embodiments, these half-life extending moieties are small molecules, which can be beneficial for small targeting agents like peptides because the overall molecular weight can be kept low, which is beneficial for achieving good penetration into the tumor tissue. However, if the molecular weight of the overall construct is still lower than the glomerular filtration cut-off (30-50 kDa), then kidneys still remain a high-risk organ for accumulation and toxicity for certain constructs.

[0165] In some embodiments, the inventive conjugates comprise an albumin binding moiety (ABM) as described herein, which may be capable of selectively binding to human serum albumin (HSA). HSA is the most abundant protein in human plasma and constitutes about half of serum protein. The term "Human Serum Albumin" or "HSA" as used herein refers to the serum albumin protein encoded by the human ALB gene. The ABM may also bind to HSA functional variants, isoforms, fragments or (post-translationally or otherwise modified) derivatives thereof.

[0166] The ABM may be any albumin binding moiety. Certain ABMs are described herein below. The ABM may bind non-covalently to serum albumin, such as HSA. WithoutAttorney Docket Number: 01384-0001 -00PCT wishing to be bound by specific theory, it is hypothesized that the ABM of the inventive conjugates extends circulation half-life of the conjugates, and effects compartmentalization of the inventive conjugates in the blood and improved delivery to the target-expressing (tumor) cells or tissues, resulting in increased tumor to healthy organ uptake ratios. The ABM is thus envisaged to confer improved pharmacokinetic properties to the inventive conjugate, without interfering with (reducing or abolishing) the desired function of the chelating agent and the target binding entity.

[0167] We describe engineered integrin-binding EETI knottin peptide-chelator conjugates containing different ABMs, including: truncated Evans Blue (EB), Ibuprofen, 4-p- (tolyl)butyric acid (PT), palmitic acid (C16), and 4-(p-lodophenyl)butyric acid (IP). In some embodiments, ABMs may comprise linear and branched lipophilic chains comprising 1-40 carbon atoms. Additional suitable ABMs are described in LIS20100172844, WO2013024035, and other publications.12In some embodiments, the ABMs may be derivatives of the previously described ABMs.

[0168] Linkers described throughout this application may be used for attaching an ABM. The linker may avoid steric hindrance between the ABM 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 HSA binding, high affinity target binding, and rapid and optionally selective penetration of target positive cells through internalization of the targeted radioligand-chelator complex. Suitable linkers may be stable in vivo. Linker design may typically depend on the overall conjugate and may be chosen to promote the functionality of the remaining conjugate (e.g. target binding, HSA binding, internalization etc.). Accordingly, linkers may, for instance, be rigid or flexible, influencing either lipophilicity or hydrophilicity of the overall conjugate, and so on.4. Multimeric knottin-chelator conjugates

[0169] Another strategy that has the potential to improve biodistribution profiles of radiopharmaceuticals is multimerization of the targeting agent. In some embodiments, this strategy involves the making of conjugates consisting of more than one identical targeting agent, usually 2-4, but higher numbers are possible. Constructs containing 2 identical targeting moieties are referred to as dimeric constructs, those containing 3 are referred to as trimeric constructs, those containing 4 are referred to as tetrameric constructs, and so on. This is an attractive strategy especially for weaker binders because multimerization increases the valency, resulting in stronger binding in many cases. Noting that the affinities reported for these types of constructs vary considerably depending on the methods used for measurement, so relative head-to-head comparisons are most accurate.Attorney Docket Number: 01384-0001 -00PCT

[0170] In some embodiments, multimeric knottin peptide constructs consist of one or more linkers to attach knottin peptides to one another. In some of embodiments, the linkers joining the knottin peptides are branched and also comprise the chelator. Alternatively, the chelator may have its own distinct linker and attachment strategy separate from the multimerization linkage strategy.E. Conjugation Strategies

[0171] Aspects of the present disclosure further include methods of making conjugates. Such methods include conjugating the chelator to the knottin peptide in the case of knottin-chelator conjugates, or conjugating the chelator to the knottin peptide or antibody subunit or fragment thereof in the case of the knottin-antibody subunit conjugates. In some embodiments, the methods include site-specifically conjugating the chelator to the knottin peptide or antibody subunit or fragment thereof. For example, the conjugating may include site-specifically conjugating the chelator to a pre-selected amino acid of the knottin peptide or antibody subunit or fragment thereof. In certain aspects, the pre-selected amino acid is at the N-terminus or C-terminus of the knottin peptide or antibody subunit or fragment thereof. In other aspects, the pre-selected amino acid is internal to the knottin peptide or antibody subunit or fragment thereof - that is, between the N-terminal and C-terminal amino acid of the knottin peptide or antibody subunit or fragment thereof. In some embodiments, the preselected amino acid is a non-natural amino acid.

[0172] Numerous strategies are available for conjugating the chelator and knottin peptide or antibody subunit or fragment thereof. For example, the chelator may be derivatized by covalently attaching a linker to the chelator, where the linker has a functional group capable of reacting with a “chemical handle” on the knottin peptide or antibody subunit or fragment thereof. Also by way of example, the knottin peptide or antibody subunit or fragment thereof may be derivatized by covalently attaching the linker to the knottin peptide or antibody subunit or fragment thereof, where the linker has a functional group capable of reacting with a “chemical handle” on the chelator. The functional group on the linker may vary and may be selected based on compatibility with the chemical handle on the chelator or knottin peptide or antibody subunit or fragment thereof. According to one embodiment, the chelator is provided by incorporation of an unnatural amino acid containing that chelator, which is then incorporated into the knottin peptide or antibody subunit or fragment thereof.

[0173] In some embodiments, the methods may include non-site-specific conjugation of the chelator to the knottin peptide or antibody subunit or fragment thereof. For example, the chelator may be derivatized with a linker containing an amine-reactive conjugation handle, such as NHS ester, p-SCN, TFP ester, or others, resulting in conjugation to freeAttorney Docket Number: 01384-0001 -00PCT primary amine groups in the protein. Alternatively, other methods for non-site-specific chemistry, such as maleimide bioconjugation chemistry, could be employed.II. Methods of UseA. Methods of Treating Cancer

[0174] 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.

[0175] Accordingly, in certain embodiments, the subject has cancer. The methods may be employed for the treatment of a large variety of cancers. “Tumor”, 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 or describe 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 cellAttorney Docket Number: 01384-0001 -00PCT 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, 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, adenoidcystic carcinoma, andAttorney Docket Number: 01384-0001 -00PCT 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.B. Combination Therapy

[0176] In certain embodiments, a composition of the present disclosure is administered to a subject receiving an immune checkpoint inhibitor therapy. In some instances, the immune checkpoint inhibitor therapy comprises administration to the subject of a therapeutically effective amount of an inhibitor of B7-H3, CTLA-4, LAG-3, PD-1 , PD-L1 , TIGIT, TIM-3, VISTA, or any combination thereof. According to some embodiments, the immune checkpoint inhibitor therapy comprises administration to the subject of a therapeutically effective amount of a PD-1 inhibitor, e.g., an anti-PD1 antibody. In certain embodiments, when the immune checkpoint inhibitor therapy comprises administration to the subject of a therapeutically effective amount of a PD-1 inhibitor, the PD-1 inhibitor is an anti- PD1 antibody, non-limiting examples of which include camrelizumab, cemiplimab, dostarlimab, nivolumab, pembrolizumab, pidilizumab, pimivalimab, retifanlimab, sintilimab, spartalizumab, tislelizumab and toripalimab. According to some embodiments, the immune checkpoint inhibitor therapy comprises administration to the subject of a therapeutically effective amount of a PD-L1 inhibitor, e.g., an anti-PD-L1 antibody. In certain embodiments, when the immune checkpoint inhibitor therapy comprises administration to the subject of a therapeutically effective amount of a PD-L1 inhibitor, the PD-L1 inhibitor is an anti-PD-L1 antibody, non-limiting examples of which include Atezolizumab, Avelumab, and Durvalumab. In embodiments where a composition of the present disclosure is administered to a subjectAttorney Docket Number: 01384-0001 -00PCT receiving an immune checkpoint inhibitor therapy, the methods may comprise administering the immune checkpoint inhibitor therapy to the subject.

[0177] A conjugate of the present disclosure may be administered to the subject alone or in combination with a second agent, e.g., any desired second agent, including but not limited to an immune checkpoint inhibitor as described elsewhere herein. Second agents of interest include, but are not limited to, agents approved by the United States Food and Drug Administration and / or the European Medicines Agency (EMA) for use in treating cancer.EXPERIMENTAL EXAMPLESExample 1 - Synthesis and Characterization of PRC-1 (DOTA-C4), PRC-2 (IP-DOTA), PRC-3 (NH2-D0TA)

[0178] Peptides and peptide-chelator conjugates were synthesized by WuXi AppTec STA.LCMS methods for PRC-1 (DOTA-C4), PRC-2 (IP-DOTA), PRC-3 (NH2-D0TA):

[0179] For QC, peptides were analyzed by LCMS using an Agilent 6230 LC / TOF & G6230B with the following method: 10-80-2MIN-QC. This method uses a linear gradient from 10% solvent B to 80% solvent B over 2 minutes (solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.075% TFA) at ambient column temperature. A C18 column (Xbridge C18, 3.5um, 2.1 *30mm) was used at a 0.4 mL / min flow rate.Peptide Synthesis of PRC-1 (DOTA-C4):

[0180] The peptide was synthesized via solid phase peptide synthesis (SPPS) using standard Fmoc chemistry. Resin preparation: The Rink Amide MBHA Resin (0.5 mmol, 1 .67 g, 1 .00 eq, Sub 0.3 mmol / g) in DMF (15 mL) was agitated with N2for 2 h at 20°C.

[0181] Deprotection: 20% piperidine in DMF (15 mL) was added and agitated the resin with N2at 25 °C for 15 min. The resin was washed with DMF (15 mL * 5) and filtered to get the resin.

[0182] Coupling: A solution of Oxyma or HOAt (3.00 eq, 1 .5 mmol) and the relevant Fmoc amino acid (3.00 eq, 1.5 mmol) in DMF (10 mL) was added to the resin, then the DIC (3.00 eq, 1 .5 mmol) was added, the mixture was agitated with N2at 25°C for 30 min. The resin was washed with DMF (15 mL * 5).

[0183] 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 Table 4.Attorney Docket Number: 01384-0001 -00PCT

[0184] Resulting in the following peptide sequence (from N-terminus to C-terminus):GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGYCG (SEQ ID NO: 9)

[0185] After the 33rdamino acid coupling step, 20% piperidine in DMF (15 mL) was added and agitated the resin with N2 at 25 °C for 15 min. The resin was washed with DMF (15 mL * 5) and filtered to get the resin.

[0186] A solution of HOAt (3.00 eq., 1 .5 mmol) and Fmoc-5-Ava-OH (3.00 eq., 1 .5 mmol) in DMF (10 mL) was added to the resin, then the DIG (3.00 eq., 1.5 mmol) wasAttorney Docket Number: 01384-0001 -00PCT added, the mixture was agitated with N2 at 25°C for 30 min. The resin was washed with DMF (15 mL * 5). 20% piperidine in DMF (15 mL) was added and agitated the resin with N2at 25 °C for 15 min. The resin was washed with DMF (15 mL * 5) and filtered to get the resin.

[0187] A solution of HATU (2.85 eq., 1.425 mmol) and DOTA-Tris(tBu) [CAS number: 137076-54-1] (3.00 eq., 1.5 mmol) in DMF (10 mL) was added to the resin, then the DIEA (6.00 eq., 3.0 mmol) was added, the mixture was agitated with N2at 25 °C for 60 min. The resin was washed with DMF (15 mL * 5).Peptide Cleavage and Purification of PRC-1 (DOTA-C4):

[0188] After all the peptide synthesis steps were completed, the resin was washed with DMF (20 mL * 5) and MeOH (20 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (3.6 g). Add cleavage solution (36 mL, 87.5% TFA / 7.5% DTT / 2.5% H2O / 2.5% TIS) to the flask containing resin at room temperature and stirred for 2.5 h. Precipitated the peptide with cold isopropyl ether (360 mL). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (40 mL * 3). Dry the crude peptide under vacuum 2 h to get the crude peptide. Then add cleavage solution (36 mL, 87.5% TFA / 7.5% DTT / 2.5% H2O / 2.5% TIS) to the flask containing crude peptide at room temperature and stirred for 0.5 h. Precipitated the peptide with cold isopropyl ether (360 mL). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (40 mL * 3). Dry the combined portions of crude linear peptide under vacuum 2 h to get the crude linear peptide (1.15 g).

[0189] The crude linear peptide (1.15 g) was dissolved in 50 mL of DMSO. While stirring, the crude linear peptide solution was added to the 1 .2L of buffer A in dropwise. Buffer A: 0.2 M Arg-HCI was dissolved in H2O, 1 M NaOH was then added slowly and adjust PH to 8.5-9.0, Add the equivalent of three reagents in order of the following concentration: 0.1 M NH4HCO3, 1 .5 mM GSH, 0.5 mM GSSG. The refolding reaction was allowed to stir for 12 hrs. The completion of the refolding was confirmed by LCMS. Upon completion, the crude peptide from refolding was firstly quenched by TFA until reaching PH 6 and was then filtered through 0.45pm membrane filter.

[0190] For purification, the peptide was diluted in 5% ACN-H2O and 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 60 minutes (solvent A: water + 0.075% TFA; solvent B: acetonitrile) at a 30°C column oven temperature. A C18 column (Kromasil 100-5-C18, 30*150mm) was used at a 20 mL / min flow rate.

[0191] The final product, PRC-1 (DOTA-C4), was a white solid (108.6 mg, 28.63 pmol, 9.71% yield, 97.43% purity) and was confirmed by LCMS using the 10-80-2MIN-QCAttorney Docket Number: 01384-0001 -00PCT method described above and by analytical HPLC. Analytical HPLC methods: linear gradient from 10% solvent B to 40% solvent B over 20 minutes (solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.075% TFA) at a 50°C column oven temperature. A C18 column (Gemini C18, 5um, 110A, 150*4.6mm) was used at a 1 mL / min flow rate.

[0192] FIG. 4: Chemical structure of PRC-1 (DOTA-C4)Peptide Synthesis of PRC-2 (IP-DOTA):

[0193] For PRC-2 (IP-DOTA), the SPPS was carried out in the same manner as it was for PRC-1 (DOTA-C4) for the first 33 amino acids, as listed in Table 4. After the 33rdamino acid coupling step, 20% piperidine in DMF (15 mL) was added and agitated the resin with N2 at 25 °C for 15 min. The resin was washed with DMF (20 mL * 5) and filtered to get the resin.

[0194] A solution of HOAt (3.00 eq., 1 .5 mmol) and Fmoc-Lys(Dde)-OH (3.00 eq., 1 .5 mmol) in DMF (15 mL) was added to the resin, then the DIC (3.00 eq., 1 .5 mmol) was added, the mixture was agitated with N2 at 25 °C for 30 min. The resin was washed with DMF (15 mL * 5). 20% piperidine in DMF (15 mL) was added and agitated the resin with N2at 25 °C for 15 min. The resin was washed with DMF (15 mL * 5) and filtered to get the resin. A solution of HATU (2.85 eq., 1.425 mmol) and 4-(p-lodophenyl)butyric acid (3.00 eq., 1.5 mmol) in DMF (15 mL) was added to the resin, then the DIEA (6.00 eq., 3.0 mmol, 533.7 pL) was added, the mixture was agitated with N2 at 25 °C for 60 min. The resin was washed with DMF (20 mL * 5).

[0195] 3% H2N-NH2 / DMF (20.0 mL) was added to the resin and allowed to react for30 min, this step was repeated for one more time. Drain and wash with DMF (15.0 mL) five times. A solution of DOTA-Tris(tBu) [CAS number: 137076-54-1] (3.00 eq., 1.5 mmol) and DIEA (6.00 eq., 3.0 mmol, 533.8 pL) in DMF (15 mL) was added to the resin, then the HATU (2.85 eq., 1 .425 mmol, 541 .5 mg) was added, the mixture was agitated with N2at 25 °C for 30 min. The resin was washed with DMF (20 mL * 5).Peptide Cleavage and Purification of PRC-2 (IP-DOTA):

[0196] After all the peptide synthesis steps were completed, the resin was washed with DMF (20 mL * 5) and MeOH (20 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (3.9 g). Add cleavage solution (40 mL, 87.5% TFA / 7.5% DTT / 2.5% H2O / 2.5% TIS) to the flask containing resin at room temperature and stirred for 2.5 h. Precipitated the peptide with cold isopropyl ether (400 mL). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (40 mL * 3). Dry the crude peptide under vacuum 2 h to get the crude peptide. Then add cleavage solution (40 mL, 87.5% TFA / 7.5% DTT / 2.5% H2O / 2.5% TIS) to the flask containing crude peptide at room temperature andAttorney Docket Number: 01384-0001 -00PCT stirred for 0.5 h. Precipitated the peptide with cold isopropyl ether (400 mL). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (40 mL * 3). Dry the crude peptide under vacuum 2 h to get the crude peptide (1 .21 g).

[0197] The crude linear peptide (1 .21 g) was dissolved in 50 mL of DMSO. While stirring, the crude linear peptide solution was added to the 1 .2L of buffer A in dropwise.Buffer A: 0.2 M Arg-HCI was dissolved in H2O, 1 M NaOH was then added slowly and adjust pH to 8.5-9.0, Add the equivalent of three reagents in order of the following concentration: 0.1 M NH4HCO3, 1 .5 mM GSH, 0.5 mM GSSG. The refolding reaction was allowed to stir for 12 hrs. The completion of the refolding was confirmed by LCMS. Upon completion, the crude peptide from refolding was firstly quenched by TFA until reaching pH 6 and was then filtered through 0.45pm membrane filter.

[0198] For purification, the peptide was diluted in 5% ACN-H2O and was purified via prep HPLC on a Gilson GX-281 instrument. The method used for HPLC purification was a linear gradient from 15% solvent B to 35% solvent B over 60 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, 110A) was used at a 20 mL / min flow rate.

[0199] The final product, PRC-2 (IP-DOTA), was a white solid (53.3 mg, 13.02 pmol, 2.47 % yield, 95.17% purity) and was confirmed by LCMS using the 10-80-2MIN-QC method described above and by analytical HPLC. Analytical HPLC methods: linear gradient from 15% solvent B to 45% solvent B over 20 minutes (solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.075% TFA) at a 50°C column oven temperature. A C18 column (Gemini C18, 5um, 110A, 150*4.6mm) was used at a 1 mL / min flow rate.

[0200] FIG. 5: Chemical structure of PRC-2 (IP-DOTA)Peptide Synthesis of PRC-3 (NH2-D0TA):

[0201] The peptide was synthesized via SPPS using standard Fmoc chemistry. Resin preparation: The Rink Amide MBHA Resin (5.0 mmol, 16.6 g, 1 .00 eq, Sub 0.3 mmol / g) in DMF (150 mL) was agitated with N2 for 2 h at 20°C.

[0202] Deprotectioir. 20% piperidine in DMF (150 mL) was added and agitated the resin with N2 at 25 °C for 15 min. The resin was washed with DMF (150 mL * 5) and filtered to get the resin.

[0203] Coupling-. A solution of Oxyma or HOAt (3.00 eq, 15 mmol) and the relevant Fmoc amino acid (3.00 eq, 15 mmol) in DMF (100 mL) was added to the resin, then the DICAttorney Docket Number: 01384-0001 -00PCT(3.00 eq, 15 mmol) was added, the mixture was agitated with N2at 25°C for 30 min. The resin was washed with DMF (150 mL * 5).

[0204] 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 Table 4.

[0205] After the 33rdamino acid coupling step, 20% piperidine in DMF (200 mL) was added and agitated the resin with N2at 25 °C for 15 min. The resin was washed with DMF (1 .0 L * 5) and filtered to get the resin. A solution of HOAt (3.00 eq., 15.0 mmol) and Boc- Lys(Fmoc)-OH (3.00 eq., 15.0 mmol) in DMF (120 mL) was added to the resin, then the DIG (3.00 eq., 15.0 mmol) was added, the mixture was agitated with N2at 25°C for 30 min. The resin was washed with DMF (1 .0 L * 5). 20% piperidine in DMF (200 mL) was added and agitated the resin with N2at 25 °C for 15 min. The resin was washed with DMF (1 .0 L * 5) and filtered to get the resin.

[0206] A solution of DOTA-Tris(tBu) [CAS number: 137076-54-1] (3.00 eq., 15.0 mmol) and DIEA (6.00 eq., 30 mmol) in DMF (120 mL) was added to the resin, then the HATU (2.85 eq., 14.0 mmol) was added, the mixture was agitated with N2at 25°C for 60 min. The resin was washed with DMF (1 L * 5).Peptide Cleavage and Purification of PRC-3 (NH2-D0TA):

[0207] After all the peptide synthesis steps were completed, the resin was washed with DMF (200 mL * 5) and MeOH (200 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (40 g). Add cleavage solution (0.4 L, 87.5% TFA / 7.5% DTT / 2.5% H2O / 2.5% TIS) to the flask containing resin at room temperature and stirred for 2.5 h. Precipitated the peptide with cold isopropyl ether (4 L). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (0.4 L * 3). Dry the crude peptide under vacuum 4 h to get the crude peptide. Then add cleavage solution (0.4 L, 87.5% TFA / 7.5% DTT / 2.5% H2O / 2.5% TIS) to the flask containing crude peptide at room temperature and stirred for 0.5 h. Precipitated the peptide with cold isopropyl ether (4 L). Filter and collect the filter cake.The filter cake was washed with isopropyl ether (0.4 L * 3). The crude peptide was combined and dried under vacuum for 4 h to get the crude linear peptide (16.3g).

[0208] Typical batch large scale refolding: the crude linear peptide (5 g) was dissolved in 70 mL of DMSO. While stirring, the crude linear peptide solution was added to the 5L of buffer A in dropwise. Buffer A: 0.2 M Arg-HCI was dissolved in H2O, 1 M NaOH was then added slowly and adjust PH to 8.5-9.0, Add the equivalent of three reagents in order of the following concentration: 0.1 M NH4HCO3, 1 .5 mM GSH, 0.5 mM GSSG. The refolding reaction was allowed to stir for 12 hrs. The completion of the refolding was confirmed byAttorney Docket Number: 01384-0001 -00PCTLCMS. 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.

[0209] For purification, the peptide was purified via prep HPLC on a Gilson GX-281 instrument. The method used for HPLC purification was a linear gradient from 0% solvent B to 90% solvent B over 60 minutes (solvent A: water + 0.075% TFA; solvent B: acetonitrile) at a 30°C column oven temperature. A C18 column (Luna C18, 10um, 100A, 100*250mm) was used at a 20 mL / min flow rate.

[0210] The final product, PRC-3 (NH2-DOTA), was a white solid (600.9 mg, 157.2 pmol, 2.98% yield, 95.03% purity) and was confirmed by LCMS using the 10-80-2MIN-QC method described above and by analytical HPLC. Analytical HPLC methods: linear gradient from 10% solvent B to 40% solvent B over 20 minutes (solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.075% TFA) at a 50°C column oven temperature. A C18 column (Gemini C18, 5um, 110A, 150*4.6mm) was used at a 1 mL / min flow rate.

[0211] FIG. 6: Chemical structure of PRC-3 (NH2-DOTA)Peptide Synthesis of PIP-2:

[0212] For PIP-2, the SPPS was carried out in the same manner as it was for PRC-3 (NH2-DOTA) for the first 33 amino acids, as listed in Table 4. After the 33rdamino acid coupling step, 20% piperidine in DMF (200 mL) was added and agitated the resin with N2at 25°C for 15 min. The resin was washed with DMF (200 mL * 5) and filtered to get the resin.Peptide Cleavage and Purification of PIP-2:

[0213] After all the peptide synthesis steps were completed, the resin was washed with MeOH (200 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (38 g). Add cleavage solution (0.4 L, 87.5% TFA / 7.5% DTT / 2.5% H2O / 2.5% TIS) to the flask containing resin at room temperature and stirred for 2.5 h. Precipitated the peptide with cold isopropyl ether (4 L). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (0.4 L * 3). Dry the crude linear peptide under vacuum 4 h to get the crude peptide (15.2 g).

[0214] Typical batch large scale refolding: the crude linear peptide (5 g) was dissolved in 70 mL of DMSO. While stirring, the crude linear peptide solution was added to the 5L of buffer A in dropwise. Buffer A: 0.2 M Arg-HCI was dissolved in H2O, 1 M NaOH was then added slowly and adjust pH to 8.5-9.0, Add the equivalent of three reagents in order of the following concentration: 0.1 M NH4HCO3, 1 .5 mM GSH, 0.5 mM GSSG. The refolding reaction was allowed to stir for 12 hrs. The completion of the refolding was confirmed byAttorney Docket Number: 01384-0001 -00PCTLCMS. 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.

[0215] For purification, the 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 90 minutes (solvent A: water + 0.075% TFA; solvent B: acetonitrile) at a 30°C column oven temperature. A C18 column (Luna C18, 10um, 100A, 100*250mm) was used at a 20 mL / min flow rate.

[0216] The final product, PIP-2, was a white solid (871.8 mg, 26.3 pmol, 5.06% yield, 96.08% purity) and was confirmed by LCMS using the 10-80-2MIN-QC method described above and by analytical HPLC. Analytical HPLC methods: linear gradient from 10% solvent B to 40% solvent B over 20 minutes (solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.075% TFA) at a 50°C column oven temperature. A C18 column (Gemini C18, 5um, 110A, 150*4.6mm) was used at a 1 mL / min flow rate.

[0217] FIG. 7: Chemical structure of PIP-2Example 2 - Synthesis and Characterization of PRC-4 (PT-DOTA), PRC-5 (Ibu-DOTA), PRC-6 (EB-DOTA), and PRC-7 (C16-DOTA)

[0218] PRC-3 (NH2-DOTA) was used as a starting material to synthesize PRC-4 (PT-DOTA), PRC-5 (Ibu-DOTA), PRC-6 (EB-DOTA), and PRC-7 (C16-DOTA) by conjugating different moieties to the free amine group of PRC-3 (NH2-DOTA).Synthesis of intermediates 4-2, 5-2, 6-2

[0219] FIG. 8A-C: Synthesis strategy for Intermediate Compounds 4-2, 5-2, 6-2

[0220] Synthesis of Intermediate Compound 4-2: To a solution of compound 4-1 (1 .00 g, 5.61 mmol, 1 .00 eq.) and N-Hydroxysuccinimide (1 .30 g, 11 .3 mmol, 2.00 eq.) in DCM (10.0 mL) was added EDCI (2.15 g, 11 .2 mmol, 2.00 eq.). The mixture was stirred at 25 °C for 1 hr. LCMS showed compound 4-1 was consumed completely and one main peak with desired mass (MS cal.: 275.3, MS observed: [M+Na]+= 297.9) was detected. The reaction mixture was diluted with 1 M HCI 10.0 mL and extracted with DCM 10.0 mL. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep- HPLC (TFA condition) to obtained compound 4-2 (500 mg, 1 .81 mmol, 98.4% purity, 32.3% yield) as a white solid.

[0221] Synthesis of Intermediate Compound 5-2: To a solution of compound 5-1 (1 .00 g, 4.85 mmol, 1 .00 eq.) and N-Hydroxysuccinimide (NHS) (1 .10 g, 9.56 mmol, 2.00 eq.) in DCM (10.0 mL) was added EDCI (1 .86 g, 9.70 mmol, 2.00 eq.). The mixture wasAttorney Docket Number: 01384-0001 -00PCT stirred at 25 °C for 1 hr. LCMS showed compound 5-1 was consumed completely and one main peak with desired mass (MS cal.: 303.3, MS observed: [M+H2O]+= 321 .0) was detected. The reaction mixture was diluted with 1 M HCI 10.0 mL and extracted with DCM 10.0 mL. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition) to obtained compound 5-2 (510 mg, 1.68 mmol, 99.7% purity, 34.6% yield) as a white solid.

[0222] Synthesis of Intermediate Compound 6-2: To a solution of compound 6-1 (200 mg, 305 pmol, 1.00 eq.) and Pentafluorophenol (123 mg, 670 pmol, 2.20 eq.) in DMF (2 mL) was added EDCI (140 mg, 731 pmol, 2.40 eq.) and DMAP (3.7 mg, 30 pmol, 0.10 eq.). The mixture was stirred at 25°C for 1 hr. LCMS showed one peak with desired mass detected. The reaction mixture was purified by prep-HPLC (pure CH3CN / H2O). LCMS and HPLC showed compound 6-2 (77.6 mg, 80.8 pmol, 26.5% yield, 85.7% purity) was obtained as a violet solid.Synthesis of PRC-4 (PT-DOTA)

[0223] To a solution of compound 4-2 (11 .6 mg, 42.1 pmol, 2.00 eq.) and PRC-3 (NH2-DOTA) (80.0 mg, 20.9 pmol, 1.00 eq.) in DMSO (1.00 mL) was added DIEA (34.6 pL, 209 pmol, 10.0 eq.). The reaction mixture was stirred at 25 °C for 18 hrs. LCMS showed compound 4-2 was consumed completely and one main peak with desired mass (MS cal.:3981 .4, MS observed: [M+H]+ / 3 = 1328.2) was detected. The reaction mixture was filtered to remove the undissolved residue. The residue was purified by prep-HPLC (TFA condition) to obtain PRC-4 (PT-DOTA) (55.5 mg, 13.9 pmol, 96.4% purity, 66.5% yield) as a white solid, which was confirmed via LCMS and analytical HPLC.

[0224] FIG. 9: Synthesis strategy for PRC-4 (PT-DOTA)Synthesis of PRC-5 (Ibu-DOTA)

[0225] To a solution of compound 5-2 (12.8 mg, 42.1 pmol, 2.00 eq.) and PRC-3 (NH2-DOTA) (80.0 mg, 20.9 pmol, 1.00 eq.) in DMSO (1.00 mL) was added DIEA (34.6 pL, 209 pmol, 10.0 eq.). The reaction mixture was stirred at 25 °C for 18 hrs. LCMS showed compound 5-2 was consumed completely and one main peak with desired mass (MS cal.:4009.5, MS observed: [M+H]+ / 3 = 1337.5) was detected. The reaction mixture was filtered to remove the undissolved residue. The residue was purified by prep-HPLC (TFA condition) to obtained PRC-5 (Ibu-DOTA) (50.3 mg, 12.5 pmol, 95.4% purity, 59.9% yield) as a white solid, which was confirmed via LCMS and analytical HPLC.

[0226] FIG. 10: Synthesis strategy for PRC-5 (Ibu-DOTA)Attorney Docket Number: 01384-0001 -00PCTSynthesis of PRC-6 (EB-DOTA)

[0227] To a solution of compound 6-2 (5.0 mg, 6.1 pmol, 2.3 eq.) and PRC-3 (NH2- DOTA) (10 mg, 2.6 pmol, 1 .00 eq.) in DMF (0.1 mL) was added DIEA (3.4 mg, 26 pmol, 4.3 pL, 10 eq.) at 0°C. The mixture was stirred at 25 °C for 1 hr. LCMS showed one peak with desired mass detected. The reaction mixture was purified by prep-HPLC (TFA condition). High resolution mass spectrometry and HPLC showed PRC-6 (EB-DOTA) (7.8 mg, 1.60 pmol, 61 .0% yield, 91 .3% purity) was obtained as a violet solid.

[0228] FIG. 11 : Synthesis strategy for PRC-6 (EB-DOTA)Synthesis of PRC-7 (C16-DOTA)

[0229] To a solution of compound 7-2 (14.8 mg, 41.8 pmol, 2.00 eq.) and PRC-3 (NH2-DOTA) (80.0 mg, 20.9 pmol, 1.00 eq.) in DMSO (1.00 mL) was added DIEA (34.6 pL, 209 pmol, 10.0 eq.). The reaction mixture was stirred at 25 °C for 18 hrs. LCMS showed compound 7-2 was consumed completely and one main peak with desired mass (MS cal.: 4059.6, MS observed: [M+H]+ / 3 = 1354.3) was detected. The reaction mixture was filtered to remove the undissolved residue. The residue was purified by prep-HPLC (TFA condition) to obtained PRC-7 (C16-DOTA) (56.7 mg, 15.4 pmol, 95.1% purity, 66.7% yield) as a white solid, which was confirmed via LCMS and analytical HPLC.

[0230] FIG. 12: Synthesis strategy for PRC-7 (C16-DOTA)Example 3 - Synthesis and Characterization of Multimeric Peptide Chelator Constructs

[0231] PIP-2 was used as the starting material to synthesize multimeric peptide chelator constructs by conjugating branched linkers to the free amine group at the N- terminus of PIP-2.Synthesis of intermediate compounds

[0232] FIG. 13A-B: Chemical intermediates used for synthesizing the branched linkers of Di-PRC-1 and Tri-PRC-1

[0233] Synthesis of “Di-PRC- 1 intermediate 2”: To a solution of “Di-PRC-1 intermediate 1” (400 mg, 480 pmol, 1.00 eq.) and Pentafluorophenol (442 mg, 2.40 mmol, 5.00 eq.) in DMF (4 mL) was added EDCI (460 mg, 2.40 mmol, 5.00 eq.) and DMAP (58.7 mg, 480 pmol, 1 .00 eq.). The mixture was stirred at 25 °C for 1 .0 hr. LCMS showed one peak with desired mass (MS cal.: 1164.45, MS observed: [M+H]+= 1165.6) detected. The reaction mixture was purified by prep-HPLC (FA condition). LCMS showed that the resulting material (205 mg), which was obtained as a yellow oil, contained “Di-PRC-1 intermediate 2” as well as a derivative containing only one PFP ester. That resulting material (205 mg, 205.20 pmol) was mixed with Pentafluorophenol (84.8 mg, 461 pmol, 2.10 eq.) in DCM (2Attorney Docket Number: 01384-0001 -00PCT mL) with added EDCI (88.4 mg, 461 pmol, 2.10 eq.). The mixture was stirred at 25 °C for 1.0 hr. LCMS showed one peak with desired mass (MS cal.: 1164.45, MS observed: [M+H]+= 1165.4) detected. The reaction mixture was concentrated under reduced pressure and the residue was purified by prep-HPLC (TFA condition). LCMS and HPLC showed “Di-PRC-1 intermediate 2” was obtained as a yellow oil with 98.9% purity.

[0234] Synthesis of “Tri-PRC- 1 intermediate 2”: To a solution of “T ri-PRC-1 intermediate 1” (490 mg, 343 pmol, 1.00 eq.) and Pentafluorophenol (316 mg, 1.72 mmol, 5.00 eq.) in DMF (4.9 mL) was added EDCI (329 mg, 1 .72 mmol, 5.00 eq.) and DMAP (42.0 mg, 343 pmol, 1 .00 eq). The mixture was stirred at 25 °C for 1 hr. LCMS showed “Tri-PRC-1 intermediate 1” was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give “Tri-PRC-1 intermediate 2” (320 mg, 166 pmol, 48.4% yield, 99.4% purity), which was obtained as a colorless oil.Synthesis of Di-PRC-1

[0235] To a mixture of “Di-PRC-1 intermediate 2” (44.5 mg, 38.2 pmol, 1 .2 eq.), PIP-2 (200 mg, 60.5 pmol, 1 .90 eq.) in DMF (2 mL) was added DIEA (41 .1 mg, 318 pmol, 52.6 pL, 10.0 eq.) at 0°C. The mixture was stirred at 25 °C for 2 hrs. LCMS showed one peak with desired mass (MS cal.: 7405.06, MS observed: [M / 4+H]+= 1853.9) detected. The reaction mixture was purified by prep-HPLC (TFA condition). LCMS and HPLC showed “Di-PRC-1 intermediate 3” [which is the dimeric PIP-2 peptide construct whose linker contains a Boc- protected amine] (130 mg, 17.543 pmol, 55.109% yield, 100% purity) was obtained as a white solid.

[0236] To a mixture of “Di-PRC-1 intermediate 3” (130 mg, 17.5 pmol, 1 .00 eq.) in DCM (0.8 mL) and TFA (0.2 mL) at 0 °C, and then the mixture was stirred at 0 °C for 1 hr. LCMS showed one peak with desired mass (MS cal.: 7305.01 , MS observed: [M / 4+H]+= 1828.7) detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was diluted with CH3CN / H2O (10 mL, 1 :4) and give the residue by lyophilization to give “Di-PRC-1 intermediate 4” [which is the dimeric PIP-2 peptide construct whose linker contains a free amine] (128 mg, 17.2 pmol, 98.1% yield, 98.1% purity) as a white solid, which was confirmed via LCMS and HPLC.

[0237] To a mixture of “Di-PRC-1 intermediate 4” (128 mg, 17.5 pmol, 1.00 eq.), DOTA-NHS (17.6 mg, 35.1 pmol, 2.00 eq.) in DMF (1.5 mL) was added DIEA (22.7 mg, 175 pmol, 29.0 pL, 10.0 eq.) at 0 °C, and then the mixture was stirred at 25 °C for 0.5 hr. LCMS showed one peak with desired mass (MS cal.: 7691.19, MS observed: [M / 5+H]+ = 1540.4) detected. The reaction mixture was purified by prep-HPLC (TFA condition). LCMS andAttorney Docket Number: 01384-0001 -00PCTHPLC showed Di-PRC-1 (83.6 mg, 10.8 pmol, 61.6% yield, 99.5% purity) was obtained as a white solid.

[0238] FIG. 14: Chemical structure of Di-PRC-1Synthesis of Tri-PRC-1

[0239] To a solution of PIP-2 (150 mg, 45.4 pmol, 4.50 eq.) and “Tri-PRC-1 intermediate 2” (19.4 mg, 10.1 pmol, 1.00 eq.) in DMF (1 mL) was added DIEA (13.0 mg, 101 pmol, 16.7 pL, 10.0 eq.) and the mixture was stirred at 25°C for 2 hrs. LCMS showed PIP-2 was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give “Tri-PRC-1 intermediate 3” [which is the trimeric PIP-2 peptide construct whose linker contains a Boc-protected amine] (74 mg, 6.55 pmol, 65.0% yield, 91 .0% purity) as a white solid.

[0240] To a solution of “Tri-PRC-1 intermediate 3” (74 mg, 6.56 pmol, 1 .00 eq.) in DCM (0.8 mL) was added TFA (307 mg, 2.69 mmol, 0.2 mL, 410 eq.). The mixture was stirred at 0 °C for 1 hr. LC-MS showed “Tri-PRC-1 intermediate 3” was consumed completely and one main peak with desired mass was detected, “Tri-PRC-1 intermediate 4” [which is the trimeric PIP- 2 peptide construct whose linker contains a free amine]. The reaction mixture was freeze- dried directly for the next step.

[0241] To a solution of “Tri-PRC-1 intermediate 4” (crude) and DOTA-NHS (5.68 mg, 11 .3 pmol, 2.00 eq.) in DMF (0.6 mL) was added DIEA (28.8 mg, 222 pmol, 36.8 pL, 30.0 eq.). The mixture was stirred at 25 °C for 1 hr. LC-MS showed “Tri-PRC-1 intermediate 4” was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition) to give Tri-PRC-1 (55.0 mg, 4.70 pmol, 63.4% yield, 99.7% purity) as a white solid.

[0242] FIG. 15: Chemical structure of Tri-PRC-1Example 4 - Production and Characterization of Knottin Fusion Protein Conjugates

[0243] Proteins were produced by WuXi Biologies, and the proteins were subsequently conjugated to chelators by WuXi XDC.Expression and purification of knottin-antibody subunit fusion proteins

[0244] PFc-1 consists of an engineered integrin-binding EETI knottin peptide genetically fused to the Fc domain of a human IgG 1 antibody, as shown schematically in FIG. 3. Specifically, the knottin peptide sequence for 2.5F (SEQ ID NO: 10) 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 for the antibody heavy chain is listed below in Table 5.Attorney Docket Number: 01384-0001 -00PCT

[0245] PAb-1 consists of an engineered integrin-binding EETI knottin peptide genetically fused to a human IgG 1 antibody, as shown schematically in FIG. 3. Specifically, the knottin peptide sequence for 2.5F (SEQ ID NO: 10) is connected to a GGGS spacer at N-terminus of the heavy chain of the human IgG 1 antibody "Motavizumab". Motavizumab is a humanized IgG 1 monoclonal antibody that binds respiratory syncytial virus (RSV).Motavizumab was chosen as the example antibody scaffold for these studies because its antigen is not present in the described preclinical mouse studies. This fusion protein construct does have an antibody light chain. The protein sequence for the antibody heavy chain and light chain are listed below in Table 5.

[0246] In Table 5, the 2.5F knottin peptide sequence is shown in bold.

[0247] Both PFc-1 and PAb-1 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 manufacturer. After expression, cell culture supernatant was collected for purification and proteins were purified with a protein A column and SECAttorney Docket Number: 01384-0001 -00PCT 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.Chelator Conjugation for PFRC-1 (p-SCN-Bn-DOTA)

[0248] PFc-1 (10.23 mg / mL in PBS pH 7.4 buffer) was pipetted into an Eppendorf tube. Conjugation buffer with PBS, pH 7.4 was added to the tube to make the final protein reaction concentration at 7 mg / mL. EDTA (200 mM in stock) was added to a final of 2 mM concentration. 0.5% of total volume of 1 M NaHCO3 pH 9.5 buffer was added into the solution to adjust the reaction pH to 8.0.

[0249] The linker-payload (p-SCN-Bn-DOTA) [Macrocyclics #B-205] prepared in DMSO (10 mM stock) was added into the solution to make the drug to protein ratio at 6 equivalents. And the final organic solvent percentage in this solution was 7.2% (v / v). The reaction solution vial was placed in an incubator-shaker at 22°C with rotate speed 60 rpm for 18 hours. After 18 hours, a small aliquot was analyzed by LC-MS.

[0250] After reaction, the sample was purified and buffer exchanged into formulation buffer (PBS, pH 7.4) via Amicon (10K MWCO). The final conjugate sample was filtered through a 0.22 pm pore size, PES membrane filter. The conjugate was characterized by SEC, MS, and quantified by BCA. The target chelator-to-protein ratio for this reaction was 2 ± 0.2. The actual chelator-to-protein ratio for this lot was 1 .85. The purity of the final product, referred to as PFRC-1 (p-SCN-Bn-DOTA), was 99.66% by SEC.

[0251] FIG. 16A-B: General structure and SEC of PFRC-1 (p-SCN-Bn-DOTA)

[0252] FIG. 17A-B: LCMS of PFRC-1 (p-SCN-Bn-DOTA)Chelator Conjugation for PARC-1 (p-SCN-Bn-DOTA)

[0253] PAb-1 (10.99 mg / mL in PBS pH 7.4 buffer) was pipetted into an Eppendorf tube. Conjugation buffer with PBS, pH 7.4 was added to the tube to make the final protein reaction concentration at 7 mg / mL. EDTA (200 mM in stock) was added to a final of 2 mM concentration. 0.5% of total volume of 1 M NaHCO3 pH 9.5 buffer was added into the solution to adjust the reaction pH to 8.0.

[0254] The linker-payload (p-SCN-Bn-DOTA) [Macrocyclics #B-205] prepared in DMSO (10 mM stock) was added into the solution to make the drug to protein ratio at 9.5 equivalents. And the final organic solvent percentage in this solution was 4.4% (v / v). The reaction solution vial was placed in an incubator-shaker at 22°C with rotate speed 60 rpm for 18 hours. After 18 hours, a small aliquot was analyzed by LC-MS. After reaction, the sample was purified and buffer exchanged into formulation buffer (PBS, pH 7.4) via Amicon (10KAttorney Docket Number: 01384-0001 -00PCTMWCO). The final conjugate sample was filtered through a 0.22 pm pore size, PES membrane filter. The conjugate was characterized by SEC, MS, and quantified by BCA. The target chelator-to-protein ratio for this reaction was 3 ± 0.2. The actual chelator-to-protein ratio for this lot was 2.92. The purity of the final product, referred to as PARC-1 (p-SCN-Bn- DOTA) was 97.97% by SEC.

[0255] FIG. 18A-B: General structure and SEC of PARC-1 (p-SCN-Bn-DOTA)

[0256] FIG. 19A-B: LCMS of PARC-1 (p-SCN-Bn-DOTA)Example 5:111ln-PRC-1 and111ln-PRC-2 Radiolabeling and Stability Testing

[0257] Studies involving radiolabeling and stability assessment of111In radiolabeled constructs were performed at Invicro.Radiolabeling PRC-1 and PRC-2 with111In

[0258] Test articles, PRC-1 (DOTA-C4) and PRC-2 (IP-DOTA), were dissolved in reaction buffer (0.2 M sodium acetate buffer with pH 5.5) at a 1 mg / mL concentration.[111ln]lnCI3 in 0.1 N HCI was mixed with over 10x volume of reaction buffer and mixed with test articles. The final concentration of the test articles in the reaction mixture was 0.11 mg / mL. Each crude test article mixture was incubated at 95°C for 10 minutes. After incubation,111ln-PRC-1 and111ln-PRC-2 were analyzed via radio-TLC and HPLC. Radio- TLC and HPLC sample preparation:111ln-PRC-1 and111ln-PRC-2 were diluted with formulation buffer (PBS) and 10% 5 mM of EDTA.Results

[0259] Specific activity at 120.0 pCi / pg for111ln-PRC-1 and 111.5 pCi / pg for111ln- PRC-2 was achieved. The labeling efficiencies for the reactions are shown in Table 6.Methods for assessing stability of111ln-PRC-1 and111ln-PRC-2

[0260] Formulation Stability:111ln-PRC-1 and111ln-PRC-2, were stored at 4°C in formulation buffer (PBS). Formulation stability was confirmed via HPLC at 2 and 24 hour time points post sample preparation.Attorney Docket Number: 01384-0001 -00PCT

[0261] Serum Stability: 100 |_iL of mouse serum was added to 100 |_iL of each test article,111ln-PRC-1 and111ln-PRC-2 in formulation buffer (PBS). The resulting serum stability test solutions were mixed by pipette.111ln-PRC-1 and111ln-PRC-2 test solutions were incubated at 37°C for the designated time points. A 50 pL sample of each test solution was mixed with 50 pL of cold acetonitrile and centrifuged at 10,000 g for 10 minutes. Serum stability was confirmed via HPLC at 2 and 24 hour time points post sample preparation.Results

[0262] The radiochemical purity (RCP) of111ln-PRC-1 and111ln-PRC-2 showed >99% by HPLC, and it was stable up to 24 hours in formulation buffer and with mouse serum, as shown in Table 7.Example 6: Time Course Blodlstributlon Study of111ln-PRC-1 and111In PRC-2 in Tumor Bearing Mice

[0263] This example describes experiments that were performed to investigate the tissue accumulation of111ln-PRC-1 and111ln-PRC-2 after IV administration into mice bearing U87MG tumors. All biodistribution imaging experiments were performed at Invicro. The retention kinetics of111ln-PRC-1 and111ln-PRC-2 upon administration in tumor-bearing mice were assessed in a time course biodistribution experiment. PRC-1 (DOTA-C4) and PRC-2 (IP-DOTA) were radiolabeled with111In using similar methods as described in Example 5.

[0264] U87MG cancer cells (5x106, in PBS with 50% Matrigel) were implanted subcutaneously into the right axilla of athymic nude female mice (Charles River: 490). When tumors reached volumes of approximately 100-250 mm3, mice were randomized into different treatment groups and IV injected (single dose) with radiolabeled constructs.

[0265] Shown are SPECT / CT maximal intensity projection (MIP) images at 1 h, 4 h, 24 h, and 48 h after dosing with111In labeled test articles (approximately 300 pCi injected activity per mouse; 3 mice per group). Uptake is presented as percent injected dose per gram tissue (% I D / g) . Both test articles show rapid accumulation and prolonged retention in tumors and have profiles indicative of renal clearance (FIG. 20A-B - FIG. 23A-B), with111ln- PRC-1 showing lower accumulation in the kidneys relative to111ln-PRC-2 (FIG.27A, FIG. 28, FIG. 29). FIG. 20A-B: SPECT / CT images showing biodistribution at 1 h and 4 h after IVAttorney Docket Number: 01384-0001 -00PCT administration of111ln-PRC-1 . At 1 h (FIG. 20A) and 4 h post-injection (FIG. 20B), signal is highest in the tumor (near the right shoulder) and in tissues associated with renal excretion, the kidneys and bladder. The signal in the bladder is from the urine and is expected to be high and quite variable at early timepoints with a substantial decrease in signal at later timepoints (>12 h) due to urination.

[0266] FIG. 21 A-B: SPECT / CT images showing biodistribution at 24 h and 48 h after IV administration of111ln-PRC-1 . At 24 h (FIG. 21 A) and 48 h post-injection (FIG. 21 B), the signal in the tumor is still strong, whereas the signals in both the kidneys and bladder have decreased substantially.

[0267] FIG. 22A-B: SPECT / CT images showing biodistribution at 1 h and 4 h after IV administration of111ln-PRC-2. At 1 h (FIG.22A) and 4 h (FIG.22B) post-injection, the signal is highest in the tumor (near the right shoulder) and in tissues associated with renal excretion, the kidneys and bladder. The kidney levels observed for111ln-PRC-2 appear higher than the kidney levels observed for111ln-PRC-1.

[0268] FIG. 23A-B: SPECT / CT images showing biodistribution at 24 h and 48 h after IV administration of111ln-PRC-2. At 24 h (FIG. 23A) and 48 h post-injection (FIG. 23B), the signal in the tumor and kidneys is still strong, whereas the signal in the bladder has decreased substantially. Overall, the kidney uptake and retention were higher for111ln-PRC- 2 compared to111ln-PRC-1 .Whole blood was collected for gamma counting at 4.5 h, 24.5 h, and 48 h post-injection. As expected for a peptide construct bearing an ABM,111ln-PRC-2 exhibits longer blood circulation time relative to111ln-PRC-1. By 48h post-injection, blood concentrations for both test articles are very low (< 0.05 % I D / g).

[0269] FIG. 24: Blood concentration (% ID / g) of111ln-PRC-1 and111ln-PRC-2 over time (mean ± SD)

[0270] At 48h post-injection, mice were euthanized. Organs and tumors were collected, and the radioactivity was determined with a gamma counter.

[0271] FIG. 25: Ex vivo gamma counting quantification of organs and tumors at 48 h post-injection of111ln-PRC-1 (mean ± SD)

[0272] FIG. 26: Ex vivo gamma counting quantification of organs and tumors at 48 h post-injection of111ln-PRC-2 (mean ± SD)

[0273] FIG. 27A-F: Ex vivo quantified tumor-to-organ ratios for111ln-PRC-1 and111ln- PRC-2 at 48 h post-injection (mean ± SD)

[0274] Consistent with111ln-PRC-1 's high tumor uptake and short blood circulation time,111ln-PRC-1 showed a higher tumor-to-blood ratio than111ln-PRC-2 at 48h postAttorney Docket Number: 01384-0001 -00PCT injection (FIG. 27B). In terms of tumor uptake relative to other healthy tissues,111ln-PRC-1 generally showed a more favorable or similar profile as111ln-PRC-2 as seen with tumor / healthy tissue ratios for bone (FIG. 27C), liver (FIG. 27D), muscle (FIG. 27E), and lungs (FIG. 27F).

[0275] Overall, these data indicate that the PRC-1 (DOTA-C4) construct had a more desirable biodistribution profile relative to PRC-2 (IP-DOTA). While the albumin binding capability of the (p-lodophenyl)butyric acid (IP) group in PRC-2 did successfully increase blood circulation time, that property did not translate to higher tumor retention with the PRC- 2 construct; in addition, the PRC-2 construct showed much higher undesirable kidney accumulation than PRC-1 .

[0276] FIG. 28: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, and 48 h after administration of111ln-PRC-1 (mean + SEM).

[0277] FIG. 29: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, and 48 h after administration of111ln-PRC-2 (mean + SEM).Example 7:111ln-PFRC-1 and111ln-PARC-1 Radiolabeling and Stability Testing

[0278] Studies involving radiolabeling and stability assessment of111In radiolabeled constructs were performed at Invicro.Radiolabeling PFRC-1 and PARC-1 with111In

[0279] For test articles, PFRC-1 and PARC-1 (in PBS): [111ln]lnCh in 0.1 N HCI was mixed with over 10x volume of reaction buffer (0.2 M sodium acetate buffer pH 5.5) and mixed with test articles. For Study 240801 , reaction mixtures of111ln-PFRC-1 and111ln- PARC-1 were incubated at 37°C for 30, 60, and 120 minutes. For Study 240808, reaction mixtures of111ln-PFRC-1 and111ln-PARC-1 were incubated at 37°C for 30 minutes.

[0280] After each incubation time point, the Radiochemical Yield of111ln-PFRC-1 and111ln-PARC-1 were analyzed via radio-TLC and HPLC. Radio-TLC and HPLC sample preparation:111ln-PFRC-1 and111ln-PARC-1 were diluted with formulation buffer (PBS) and 10% 5 mM of EDTA.

[0281] Purification: Zeba spin columns (7kDa MWCO) were placed into 1 .5 mL collection tubes and centrifuged to remove storage solution. The Zeba spin columns were buffer exchanged into PBS with pH 7.4 per the manufacturer’s protocol. Columns were placed into new collection tubes, and111ln-PFRC-1 and111ln-PARC-1 were applied to the top of each column. Following the application of each test article, columns were centrifuged at 1 ,000 x g for 2 minutes.111ln-PFRC-1 and111ln-PARC-1 samples were collected from eachAttorney Docket Number: 01384-0001 -00PCT of the collection tubes and columns were discarded. The final products were analyzed via radio-TLC and HPLC.Results

[0282] PFRC-1 and PARC-1 were labeled with [111ln]lnCh and the radiochemical yield and purity was assessed via radio-TLC and HPLC over multiple test labeling trials. Crude products of radiolabeled test articles,111ln-PFRC-1 and111ln-PARC-1 , exhibited 54%- 72% radiochemical yield at 37°C when assessed at 30, 60, and 120 minutes. Both radiolabeled crude products were then purified using Zeba Spin Desalting columns. The final purified products of111ln-PFRC-1 and111ln-PARC-1 were analyzed and showed over 90% radiochemical purity (RCP), meeting in vivo production criteria.

[0283] FIG. 30: Description of reaction conditions and results from test radiolabeling studies for111ln-PFRC-1 and111ln-PARC-1Methods for assessing stability of111ln-PFRC-1 and111ln-PARC-1

[0284] Formulation Stability:111ln-PFRC-1 and111ln-PARC-1 , were stored at 4°C in formulation buffer (PBS). Formulation stability was confirmed via HPLC at 2 and 24 hour time points post sample preparation.

[0285] Serum Stability: A 60 pL of mouse serum aliquot was added to 60 pL of each test article,111ln-PFRC-1 and111ln-PARC-1 in formulation buffer (PBS). The resulting serum stability test solutions were mixed by pipette. Both test solutions were incubated at 37°C for the designated time points. Serum stability was confirmed via HPLC at 2 and 24 hour time points post sample preparation.

[0286] Results: Stability test in formulation buffer at 4°C and with mouse serum at 37°C were assessed at 2 and 24 hours via HPLC.111ln-PFRC-1 and111ln-PARC-1 showed RCP over 90% in formulation buffer at 4°C, as shown in Table 8. For serum stability, the majority of main product peak remained stable.Attorney Docket Number: 01384-0001 -00PCTExample 8: Time Course Biodistribution Study of111ln-PFRC-1 and111ln PARC-1 in Tumor Bearing Mice

[0287] This example describes experiments that were performed to investigate the tissue accumulation of111ln-PFRC-1 and111ln-PARC-1 after IV administration into mice bearing LI87MG tumors. The retention kinetics of111ln-PFRC-1 and111ln-PARC-1 upon administration in tumor-bearing mice were assessed in a time course biodistribution experiment. PFRC-1 and PARC-1 were radiolabeled with111In using similar methods as described in Example 7. U87MG-tumor bearing mice inoculation and preparation was the same as described in Example 6.

[0288] Tumor-bearing mice were imaged by SPECT / CT at 4 h, 24 h, 72 h, and 168 h after dosing with111In labeled test articles (approximately 300 pCi injected activity per mouse; 3 mice per group). Uptake is presented as percent injected dose per gram tissue (% ID / g).

[0289] In the imaging biodistribution study described in Example 6, the smaller PRC constructs (111ln-PRC-1 ,111ln-PRC-2) showed rapid tumor accumulation with maximum levels observed at the first timepoint (1 h post-injection). In contrast, PFRC-1 and PARC-1 showed maximum tumor uptake levels at 24 h. The observed delay for reaching maximum tumor uptake levels is expected for a Fc fusion / Antibody fusion-based construct given the longer circulation time and larger size, which reduces rapid tumor penetration.

[0290] FIG. 31 : Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 72 h, and 168 h after administration of111ln-PFRC-1 (mean + SEM).

[0291] FIG. 32: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 72 h, and 168 h after administration of111ln-PARC-1 (mean + SEM).

[0292] 111ln-PRC-1 ,111ln-PRC-2,111ln-PFRC-1 , and111ln-PARC-1 were the first set of test articles selected for biodistribution studies in order to evaluate constructs with different expected blood circulation times, where blood circulation time was expected to be the shortest for111ln-PRC-1 , then111ln-PRC-2, then111ln-PFRC-1 , and finally111ln-PARC-1 . Blood concentration was measured by gamma counting at 4.5 h for these four test articles, and the results appeared consistent with the expected trends as shown in FIG. 33.

[0293] FIG. 33: Blood concentration (% ID / g) of111ln-PRC-1 ,111ln-PRC-2,111ln- PFRC-1 , and111ln-PARC-1 at 4.5 h post-injection (mean + SD)Attorney Docket Number: 01384-0001 -00PCTExample 9:111ln-PRC-1,111ln-PRC-3,111ln-PRC-4,111ln-PRC-5,111ln-PRC-7,111ln-Di-PRC- 1, and111In-Tri-PRC-1 Radiolabeling and Stability TestingRadiolabeling PRC-1, PRC-3, PRC-4, PRC-5, PRC-7, Di-PRC-1, and Tri-PRC-1 with111ln

[0294] Test articles (PRC-1 (DOTA-C4), PRC-4 (PT-DOTA), PRC-5 (Ibu-DOTA), and Di-PRC-1 ), were dissolved in reaction buffer (0.2 M sodium acetate buffer with pH 5.5) at a 1 mg / mL concentration. [111ln]lnCI3 in 0.1 N HCI was mixed with over 10x volume of reaction buffer and mixed with test articles. The final concentration of the test articles in the reaction mixture was as follows: 0.173 mg / mL for PRC-1 , 0.140 mg / mL for PRC-4, 0.155 mg / ml for PRC-5, and 0.268 mg / ml for Di-PRC-1 . Each crude test article mixture was incubated at 95°C for 10 minutes.

[0295] PRC-3 (NH2-DOTA) was provided to Invicro in PBS at 1 mM (-3.82 mg / mL). [111ln]lnCI3 in 0.1 N HCI was mixed with over 10x volume of reaction buffer (0.2 M sodium acetate buffer with pH 5.5) and mixed with test article. The final concentration of PRC-3 in the reaction mixture was 0.26 mg / mL. The crude test article mixture was incubated at 80°C for 10 minutes.

[0296] For Tri-PRC-1 , various reaction conditions (different reaction temperatures, reaction times, and target specific activities) were evaluated, but labeling efficacy remained suboptimal, and thus a purification step was added after synthesis to increase Radiochemical Purity. Ultimately, Tri-PRC-1 was dissolved in reaction buffer (0.2 M sodium acetate buffer with pH 5.5) at a 1 mg / mL concentration. [111ln]lnCI3 in 0.1 N HCI was mixed with over 10x volume of reaction buffer and mixed with test article. The final concentration of Tri-PRC-1 in the reaction mixture was 0.411 mg / ml. The crude test article mixture was incubated at 95°C for 10 minutes. The crude test article mixture was then purified using two sequential elutions from 7 kDa Zeba spin columns in PBS.

[0297] For all test articles,111In-labeled test articles were analyzed via radio-TLC and HPLC. Radio-TLC and HPLC sample preparation:111ln-labeled test articles were diluted with formulation buffer (PBS) and 10% 5 mM of EDTA.Results

[0298] The specific activities and labeling efficiencies for the reactions are shown in Table 9.Attorney Docket Number: 01384-0001 -00PCT

[0299] As described in the labeling methods for111In-Tri-PRC-1 , a Zeba spin column purification step was added, resulting in a Radiochemical Purity of 95.85% and a Radiochemical Yield of 68.98%.Methods for assessing stability of111In-labeled test articles

[0300] Formulation Stability:1111n-labeled test articles were stored at 4°C in formulation buffer (PBS). Formulation stability was evaluated via HPLC at 2 and 24 hour time points post sample preparation.

[0301] Serum Stability: 100 pL of mouse or human serum was added to 100 pL of each111In-labeled test article in formulation buffer (PBS). The resulting serum stability test solutions were mixed by pipette. Samples were incubated at 37°C for the designated time points. A 50 pL sample of each test solution was mixed with 50 pL of cold acetonitrile and centrifuged at 10,000 x g for 10 minutes. Serum stability was evaluated via HPLC at 2 and 24 hour time points post sample preparation.Results

[0302] In general,111In-labeled test articles appeared highly stable up to 24 hours in formulation buffer, mouse serum, and human serum as shown in the table below. For111ln- PRC-7, the RCP was over 90% in formulation buffer and human serum for up to 24 hours; however, the RCP dropped from 95% (2 hours) to 88% (24 hours) in mouse serum.Attorney Docket Number: 01384-0001 -00PCTExample 10: Time Course Biodistribution Study of111ln-PRC-1,111ln-PRC-3,111ln-PRC-4,111ln-PRC-5,111ln-PRC-7,111In-Di-PRC-1, and111In-Tri-PRC-1 in Tumor Bearing Mice

[0303] This example describes experiments that were performed to investigate the tissue accumulation of111ln-PRC-1 (DOTA-C4),111ln-PRC-3 (NH2-DOTA),111ln-PRC-4 (PT- DOTA),111ln-PRC-5 (Ibu-DOTA),111ln-PRC-7 (C16-DOTA),111In-Di-PRC-1 , and111In-Tri- PRC-1 after IV administration into mice bearing LI87MG tumors. The retention kinetics of111In-labeled test articles upon administration in tumor-bearing mice were assessed in a time course biodistribution experiment. Test articles were radiolabeled with111In using similar methods as described in Example 9.

[0304] LI87MG cancer cells (5x106, in PBS with 50% Matrigel) were implanted subcutaneously into the right axilla of athymic nude female mice (Charles River: 490). When tumors reached volumes of approximately 150-350 mm3, mice were randomized into different treatment groups and IV injected (single dose) with radiolabeled constructs.

[0305] Tumor-bearing mice (3 mice per group) were imaged by SPECT / CT at multiple time points after dosing with1111n labeled test articles. For all groups, mice were IV injected with 1 .462 nmol of test article, resulting in the following approximate injected activities: 660 pCi (for111ln-PRC-1 ,111ln-PRC-4), 600 pCi (for111ln-PRC-5,11 1ln-PRC-7,111In-Di-PRC-1 ), and 450 pCi (for111ln-PRC-3,111In-Tri-PRC-1 ). Uptake is presented as percent injected dose per gram tissue (% ID / g). Mice were imaged at 1 h, 4 h, 24 h, 72 h, and 192 h in this study; the only exception was the last imaging time point for111ln-PRC-7 was 168 h instead of 192 h due to scheduling constraints.

[0306] Tumor uptake was observed for all test articles, and maximum uptake levels were generally observed around 1 h or 4 h post-injection.111ln-PRC-7 was the exception where maximum tumor uptake was observed around 24 h post-injection. In accordance withAttorney Docket Number: 01384-0001 -00PCT this observation,111ln-PRC-7 appeared to have substantially higher blood levels and circulation time than the other test articles evaluated in this study.

[0307] For the other test articles except111ln-PRC-7, tumors and kidneys were the main tissues showing notable uptake. High non-specific kidney accumulation was observed for111ln-PRC-3 and111In-Tri-PRC-1 , whereas other test articles showed substantially lower kidney accumulation. While111ln-PRC-7 showed higher tumor uptake than the other test articles, there was also much higher non-specific accumulation in all other healthy organs as well.

[0308] FIG. 34: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-1 (mean + SEM)

[0309] FIG. 35: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-3 (mean + SEM)

[0310] FIG. 36: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-4 (mean + SEM)[0031 1] FIG. 37: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-5 (mean + SEM)

[0312] FIG. 38: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111In-Di-PRC-1 (mean + SEM).

[0313] FIG. 39: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111In-Tri-PRC-1 (mean + SEM).

[0314] FIG. 40: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 168 h after administration of111ln-PRC-7 (mean + SEM)

[0315] FIG. 41 : Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 192 h after administration of111ln-PRC-1 (mean + SEM).

[0316] FIG. 42: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 1 h, 4 h, 24 h, 72 h, and 168 h after administration of111ln-PRC-7 (mean + SEM).

[0317] FIG. 43A-B: Blood concentration (% ID / g) of111ln-PRC-1 ,111ln-PRC-3,111ln- PRC-4,111ln-PRC-5,111ln-PRC-7,111ln-Di-PRC-1 , and111ln-Tri-PRC-1 at 5 min and 4.5 h post-injection.Attorney Docket Number: 01384-0001 -00PCT

[0318] Blood concentration was measured by gamma counting.111ln-PRC-7,111ln- PRC-5, and111ln-PRC-4 all have half-life extension moieties, and both111ln-PRC-7 and111ln- PRC-5 do appear to have longer blood circulation half-life than the other test articles. In general,111ln-PRC-4 blood levels at 4.5 h were slightly higher than the test articles without half-life extension, but substantially lower than111ln-PRC-7 and111ln-PRC-5.

[0319] As previously noted,111ln-PRC-7 showed the highest tumor uptake and retention, although this was coupled with higher levels of non-specific accumulation in other healthy tissues. In terms of the other test articles,111ln-PRC-1 had the most favorable tumor uptake and retention profile, although111ln-PRC-3,111ln-PRC-4, and111In-Di-PRC-1 also had good tumor uptake and retention as well.111ln-PRC-5 initially showed fairly high tumor uptake, but the levels dropped faster than other test articles over time.111In-Tri-PRC-1 showed the opposite trend where the initial tumor uptake was lower than the other test articles, but the tumor retention was favorable. Overall,111ln-PRC-1 demonstrated the most favorable tumor-to-kidney ratio of the test articles evaluated in this study.

[0320] FIG. 44: Comparison of tumor uptake quantification over time derived from SPECT / CT imaging for111ln-PRC-1 ,111ln-PRC-3,111ln-PRC-4, and111ln-PRC-5 (mean + SEM).

[0321] FIG. 45: Comparison of tumor uptake quantification over time derived from SPECT / CT imaging for111ln-PRC-1 ,111ln-Di-PRC-1 , and111ln-Tri-PRC-1 (mean + SEM).Example 11 - Synthesis and Characterization of PRC-9 and PRC-10Peptide Synthesis of PRC-9 (internal DOTA-C4) and PRC-10 (N-acetylated, internal DOTA-C4):

[0322] This example describes the synthesis and characterization of PRC-9 (internal DOTA-C4) and PRC-10 (N-acetylated, internal DOTA-C4), which were developed to investigate the impact of the chelator conjugation site. The previously described peptide constructs have the chelator conjugated to the N-terminal amino acid of the knottin peptide, whereas PRC-9 and PRC-10’s chelators are attached at amino acid site 15 of the peptide sequence. PRC-10 differs from PRC-9 by the addition of an acetyl group at the N-terminal amino acid of the knottin peptide.

[0323] The peptide was synthesized via solid phase peptide synthesis (SPPS) using standard Fmoc chemistry. Resin preparation: The Sieber Resin (1 .0 mmol, 3.8g, 1 .00 eq, Sub 0.26 mmol / g) in DMF (80.0 mL) was agitated with N2 for 2 h at 20°C.Attorney Docket Number: 01384-0001 -00PCT

[0324] Deprotection-. 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0325] Coupling-. A solution of HOAt (6.00 eq, 6.0 mmol, 0.82 g) and the relevant Fmoc amino acid (6.00 eq, 6.0 mmol) in DMF (40.0 mL) was added to the resin, then the DIG (6.00 eq, 6.0 mmol) was added. The mixture was agitated with N2 at 25°C for 30 min. The resin was washed with DMF (80.0 mL * 5).

[0326] 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 for step 23, “Fmoc-Pro-Pro-OH” was used to incorporate two adjacent Prolines.Attorney Docket Number: 01384-0001 -00PCT

[0327] Deprotection after step 32: 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0328] The subsequent synthesis steps now diverge for PRC-9 versus PRC-10.Final peptide synthesis steps, peptide cleavage, and purification for PRC-9:

[0329] Boo Protection: A solution of DIEA (3.00 eq) and Boc2O (3.00 eq) in DMF (40 mL) was added to the resin and agitated with N2 for 1 hr at 25°C. Then the resin was washed with DMF (80 mL*5).

[0330] Dde Deprotection: 3% N2H4-H2O / DMF (80.0 mL) was added to the resin, and the de-Dde reaction was undergone for 20 min and then the process was repeated 3 times. The mixture was drained and washed with DMF (80.00 mL*5).

[0331] DOTA Coupling: DOTA-3tBu (6.00 eq), DIG (6.00 eq) and HOAt (6.00 eq) in DMF (40 mL) was added to the resin and agitated with N2 for 1 hr at 25°C. The resin was then washed with DMF (80 mL*5).

[0332] Peptide Cleavage: After all the steps were completed, the resin was washed with MeOH (80.0 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (6.0 g). Cleavage solution (80.0 mL, 10% DTT / 2.5% TIS / 2.5% H2O / 85% TFA) was added to the flask containing resin at room temperature and stirred for 3 h. The peptide intermediate was precipitated with cold isopropyl ether (800.0 mL). It was then filtered, and the filter cake was collected. The filter cake was washed with isopropyl ether (800.0 mL * 3). The crude peptide was dried under vacuum for 3 h to get the crude peptide intermediate (1 .0 g)-

[0333] Peptide Folding: The crude linear peptide (1 .0 g) was dissolved in 50 mL DMSO solution. While stirring, the crude linear peptide solution was added to 1 .0 L of Buffer A dropwise. The refolding reaction was allowed to stir for 8 hrs. Methods for making Buffer A: Arg-HCI was dissolved in miliQ H2O to reach the final concentration of 0.2 M, NaOH was then added slowly and adjust pH to 8.5-9.0. Add three reagents in order to reach the following concentration: 0.1 M NH4HCO3, 1.5 mM GSH, 0.5 mM GSSG.

[0334] Purification: 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) on a C18 column. The final product, PRC-9, was a white solid (77.3 mg, 20.15 pmol, 97.32% purity) and was confirmed via LCMS and analytical HPLC.Attorney Docket Number: 01384-0001 -00PCT

[0335] FIG. 46: Chemical structure of PRC-9Final peptide synthesis steps, peptide cleavage, and purification for PRC-10:

[0336] Acetylation: A solution of DIEA (3.00 eq) and Ac2O (3.00 eq) in DMF (40 mL) was added to the resin and agitated with N2 for 1 hr at 25°C. Then the resin was washed with DMF (80 mL*5).

[0337] Dde Deprotection: 3% N2H4-H2O / DMF (80.0 mL) was added to the resin, and the de-Dde reaction was undergone for 20 min and then the process was repeated 3 times. The mixture was drained and washed with DMF (80.00 mL*5).

[0338] DOTA Coupling: DOTA-3tBu (6.00 eq), DIG (6.00 eq) and HOAt (6.00 eq) in DMF (40 mL) was added to the resin and agitated with N2 for 1 hr at 25°C. The resin was then washed with DMF (80 mL*5).

[0339] Peptide Cleavage: After all the steps were completed, the resin was washed with MeOH (80.0 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (6.0 g). Cleavage solution (80.0 mL, 10% DTT / 2.5% TIS / 2.5% H2O / 85% TFA) was added to the flask containing resin at room temperature and stirred for 3 h. The peptide intermediate was precipitated with cold isopropyl ether (800.0 mL). It was then filtered, and the filter cake was collected. The filter cake was washed with isopropyl ether (800.0 mL * 3). The crude peptide was dried under vacuum for 3 h to get the crude peptide intermediate (1 .3 g)-

[0340] Peptide Folding: The crude linear peptide (1 .3 g) was dissolved in 60 mL DMSO solution. While stirring, the crude linear peptide solution was added to 1 .5 L of Buffer A dropwise. The refolding reaction was allowed to stir for 8 hrs. Methods for making Buffer A: Arg-HCI was dissolved in miliQ H2O to reach the final concentration of 0.2 M, NaOH was then added slowly and adjust pH to 8.5-9.0. Add three reagents in order to reach the following concentration: 0.1 M NH4HCO3, 1.5 mM GSH, 0.5 mM GSSG.

[0341] Purification: 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) on a C18 column. The final product, PRC-10, was a white solid (89.6 mg, 22.76 pmol, 95.86% purity) and was confirmed via LCMS and analytical HPLC.

[0342] FIG. 47: Chemical structure of PRC-10Attorney Docket Number: 01384-0001 -00PCTExample 12 - Synthesis and Characterization of PRC-11, PRC-12, PRC-13, and PRC-14Peptide Synthesis of PRC-11 (D0TA-PEG4), PRC-12 (D0TA-C2), PRC-13 (D0TA-C6), and PRC-14 (D0TAM-C4):

[0343] The peptide was synthesized via solid phase peptide synthesis (SPPS) using standard Fmoc chemistry. Resin preparation: The Sieber Resin (1 .0 mmol, 3.8g, 1 .00 eq, Sub 0.26 mmol / g) in DMF (80.0 mL) was agitated with N2 for 2 h at 20°C.

[0344] Deprotection: 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0345] Coupling: A solution of HOAt (6.00 eq, 6.0 mmol, 0.82 g) and the relevant Fmoc amino acid (6.00 eq, 6.0 mmol) in DMF (40.0 mL) was added to the resin, then the DIG (6.00 eq, 6.0 mmol) was added. The mixture was agitated with N2 at 25°C for 30 min. The resin was washed with DMF (80.0 mL * 5).

[0346] 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 for step 23, “Fmoc-Pro-Pro-OH” was used to incorporate two adjacent Prolines.Attorney Docket Number: 01384-0001 -00PCT

[0347] Deprotection after coupling step 32: 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0348] The subsequent synthesis steps now diverge for PRC-11 , PRC-12, PRC-13, and PRC-14.Final Synthesis Steps for PRC-11:

[0349] Coupling step 33: A solution of HOAt (6.00 eq, 6.0 mmol, 0.82 g) and Fmoc- NH-PEG4-CH2CH2COOH (6.00 eq, 6.0 mmol) in DMF (40.0 mL) was added to the resin, then the DIG (6.00 eq, 6.0 mmol) was added. The mixture was agitated with N2 at 25°C for 30 min. The resin was washed with DMF (80.0 mL * 5).

[0350] Deprotection after coupling step 33: 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0351] DOTA Coupling: DOTA-3tBu (6.00 eq), DIG (6.00 eq) and HOAt (6.00 eq) in DMF (40 mL) was added to the resin and agitated with N2 for 1 hr at 25°C. The resin was then washed with DMF (80 mL*5).

[0352] Peptide Cleavage: After all the steps were completed, the resin was washed with MeOH (80.0 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (6.0 g). Cleavage solution (80.0 mL, 10% DTT / 2.5% TIS / 2.5% H2O / 85% TFA) was added to the flask containing resin at room temperature and stirred for 3 h. The peptide intermediate was precipitated with cold isopropyl ether (800.0 mL). It was then filtered, and the filter cake was collected. The filter cake was washed with isopropyl ether (800.0 mL * 3). The crude peptide was dried under vacuum for 3 h to get the crude peptide intermediate (1 .2 g)-

[0353] Peptide Folding: The crude linear peptide (1 .2 g) was dissolved in 60 mL DMSO solution. While stirring, the crude linear peptide solution was added to 1 .2 L of BufferAttorney Docket Number: 01384-0001 -00PCTA dropwise. The refolding reaction was allowed to stir for 8 hrs. Methods for making Buffer A: Arg-HCI was dissolved in miliQ H2O to reach the final concentration of 0.2 M, NaOH was then added slowly and adjust pH to 8.5-9.0. Add three reagents in order to reach the following concentration: 0.1 M NH4HCO3, 1.5 mM GSH, 0.5 mM GSSG.

[0354] Purification: 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) on a C18 column.

[0355] The final product, PRC-11 , was a white solid (158.1 mg, 38.439 pmol, 98.55% purity) and was confirmed via LCMS and analytical HPLC.

[0356] FIG. 48: Chemical structure of PRC-11Final Synthesis Steps for PRC-12:

[0357] Coupling step 33: A solution of HOAt (6.00 eq, 6.0 mmol, 0.82 g) and Fmoc- pAla-OH (6.00 eq, 6.0 mmol) in DMF (40.0 mL) was added to the resin, then the DIC (6.00 eq, 6.0 mmol) was added. The mixture was agitated with N2 at 25°C for 30 min. The resin was washed with DMF (80.0 mL * 5).

[0358] Deprotection after coupling step 33: 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0359] DOTA Coupling: DOTA-3tBu (6.00 eq), DIC (6.00 eq) and HOAt (6.00 eq) in DMF (40 mL) was added to the resin and agitated with N2 for 1 hr at 25°C. The resin was then washed with DMF (80 mL*5).

[0360] Peptide cleavage, peptide folding, and purification followed the same protocol as described above for PRC-11 .

[0361] The final product, PRC-12, was a white solid (194.8 mg, 51.54 pmol, 95.28% purity) and was confirmed via LCMS and analytical HPLC.

[0362] FIG. 49: Chemical structure of PRC-12Final Synthesis Steps for PRC-13:

[0363] Coupling step 33: A solution of HOAt (6.00 eq, 6.0 mmol, 0.82 g) and Fmoc- 7-Ahp-OH (6.00 eq, 6.0 mmol) in DMF (40.0 mL) was added to the resin, then the DIC (6.00 eq, 6.0 mmol) was added. The mixture was agitated with N2 at 25°C for 30 min. The resin was washed with DMF (80.0 mL * 5).Attorney Docket Number: 01384-0001 -00PCT

[0364] Deprotection after coupling step 33: 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0365] DOTA Coupling: DOTA-3tBu (6.00 eq), DIG (6.00 eq) and HOAt (6.00 eq) in DMF (40 mL) was added to the resin and agitated with N2 for 1 hr at 25°C. The resin was then washed with DMF (80 mL*5).

[0366] Peptide cleavage, peptide folding, and purification followed the same protocol as described above for PRC-11 .

[0367] The final product, PRC-13, was a white solid (177.5 mg, 46.46 pmol, 97.00% purity) and was confirmed via LCMS and analytical HPLC.

[0368] FIG. 50: Chemical structure of PRC-13Final Synthesis Steps for PRC-14:

[0369] PRC-14 is similar to PRC-1 except PRC-14 has a different chelator (DOTAM), whereas PRC-1 has a DOTA chelator. DOTAM, also known as TCMC, is appropriate for chelating different types of metals, such as Lead isotopes.

[0370] Coupling step 33: A solution of HOAt (6.00 eq, 6.0 mmol, 0.82 g) and Fmoc- 5-Ava-OH (6.00 eq, 6.0 mmol) in DMF (40.0 mL) was added to the resin, then the DIG (6.00 eq, 6.0 mmol) was added. The mixture was agitated with N2 at 25°C for 30 min. The resin was washed with DMF (80.0 mL * 5).

[0371] Deprotection after coupling step 33: 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0372] DOTAM Coupling: DOTAM(3Trt)-OH (6.00 eq), DIG (6.00 eq) and HOAt (6.00 eq) in DMF (40 mL) was added to the resin and agitated with N2 for 1 hr at 25°C. The resin was then washed with DMF (80 mL*5).

[0373] Peptide cleavage, peptide folding, and purification followed the same protocol as described above for PRC-11 .

[0374] The final product, PRC-14, was a white solid (85 mg, 22.43 pmol, 95.21% purity) and was confirmed via LCMS and analytical HPLC.

[0375] FIG. 51 : Chemical structure of PRC-14Attorney Docket Number: 01384-0001 -00PCTExample 13 - Synthesis and Characterization of PRC-1.4L (D0TA-C4), TRC-1 (DOTA- C4), TRC-1.31 F (D0TA-C4), NRC-1 (D0TA-C4), and NRC-1.4R (D0TA-C4):General Peptide Synthesis, Cleavage, Folding, and Purification Protocol for PRC-1.4L, TRC-1, TRC-1.31 F, NRC-1, and NRC-1.4R:

[0376] The peptide was synthesized via solid phase peptide synthesis (SPPS) using standard Fmoc chemistry. Resin preparation: The Sieber Resin (1 .0 mmol, 3.8g, 1 .00 eq, Sub 0.26 mmol / g) in DMF (80.0 mL) was agitated with N2 for 2 h at 20°C.

[0377] Deprotection: 20% piperidine in DMF (80.0 mL) was added, and the resin was agitated with N2 at 25 °C for 15 min. The resin was washed with DMF (80.0 mL * 5) and filtered to get the resin.

[0378] Coupling: A solution of HOAt (6.00 eq, 6.0 mmol, 0.82 g) and the relevant Fmoc amino acid (6.00 eq, 6.0 mmol) in DMF (40.0 mL) was added to the resin, then the DIG (6.00 eq, 6.0 mmol) was added. The mixture was agitated with N2 at 25°C for 30 min. The resin was washed with DMF (80.0 mL * 5).

[0379] 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 tables below.

[0380] Peptide Cleavage: After all the steps were completed, the resin was washed with MeOH (80.0 mL) * 3, then dried under reduced pressure to afford peptide resin peptide (6.0 g). Cleavage solution (80.0 mL, 10% DTT / 2.5% TIS / 2.5% H2O / 85% TFA) was added to the flask containing resin at room temperature and stirred for 3 h. The peptide intermediate was precipitated with cold isopropyl ether (800.0 mL). It was then filtered, and the filter cake was collected. The filter cake was washed with isopropyl ether (800.0 mL * 3). The crude peptide was dried under vacuum for 3 h to get the crude peptide intermediate (1.2-1.4 g).

[0381] Peptide Folding: The crude linear peptide (1 .2-1 .4 g) was dissolved in 60 mL DMSO solution. While stirring, the crude linear peptide solution was added to 1 .2-1 .4 L of Buffer A dropwise. The refolding reaction was allowed to stir for 8 hrs. Methods for making Buffer A: Arg-HCI was dissolved in miliQ H2O to reach the final concentration of 0.2 M, NaOH was then added slowly and adjust pH to 8.5-9.0. Add three reagents in order to reach the following concentration: 0.1 M NH4HCO3, 1 .5 mM GSH, 0.5 mM GSSG.

[0382] Purification: 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) on a C18 column.Attorney Docket Number: 01384-0001 -00PCTSynthesis Details for PRC-1.4L:

[0383] PRC-1 .4L is the same as PRC-1 except it has a Leucine at site 4 of the sequence (see coupling step 29) instead of an Arginine like PRC-1 . Note for step 23, “Fmoc- Pro-Pro-OH” was used to incorporate two adjacent Prolines.

[0384] The final purified product, PRC-1 .4L, was a white solid (155.0 mg, 41 .342 pmol, 97.34% purity) and was confirmed via LCMS and analytical HPLC.

[0385] FIG. 52: Chemical structure of PRC-1 .4LAttorney Docket Number: 01384-0001 -00PCTSynthesis Details for TRC-1:

[0386] TRC-1 is the same as PRC-1 except it contains a different engineered integrin-binding loop (between sites 3-13) (see coupling steps 21-31 ), which imparts different selectivity toward particular RGD-binding integrins (see e.g., Example 14 for characterization of integrin binding).

[0387] The final purified product, TRC-1 , was a white solid (194.2 mg, 51 .86 pmol, 96.18% purity) and was confirmed via LCMS and analytical HPLC.

[0388] FIG. 53: Chemical structure of TRC-1Attorney Docket Number: 01384-0001 -00PCTSynthesis Details for TRC-1.31 F:

[0389] TRC-1 .31 F is the same as TRC-1 except it has a Phenylalanine at site 31 of the sequence (see coupling step 3) instead of a Tyrosine like TRC-1 .

[0390] The final purified product, TRC-1 .31 F, was a white solid (214 mg, 57.40 pmol, 97.49% purity) and was confirmed via LCMS and analytical HPLC.

[0391] FIG. 54: Chemical structure of TRC-1 .31 FAttorney Docket Number: 01384-0001 -00PCTSynthesis Details for NRC-1:

[0392] NRC-1 is the same as PRC-1 and TRC-1 , except it contains a different binding loop (between sites 3-13) (see coupling steps 21 -30) that does not contain the “RGD” motif and eliminates binding to RGD-binding integrins. NRC-1 is used as a nonbinding control, where the number of positively charged residues (i.e. , # of Arginines) matches PRC-1 .4L, TRC-1 , and TRC-1 .31 F. Note for step 21 , “Fmoc-Ser(tBu)- SerPsi(Me,Me)Pro-OH” was used to incorporate two adjacent Serines.Attorney Docket Number: 01384-0001 -00PCT

[0393] The final purified product, NRC-1 , was a white solid (166.7 mg, 46.230 pmol, 95.49% purity) and was confirmed via LCMS and analytical HPLC.

[0394] FIG. 55: Chemical structure of NRC-1Synthesis Details for NRC-1.4R:

[0395] NRC-1 .4R is the same as NRC-1 except it has an Arginine at site 4 of the sequence (see coupling step 29) instead of a Threonine like NRC-1 . This is used as a nonbinding control, where the number of positively charged residues (i.e. , # of Arginines) matches all other PRCs (not including PRC-1 .4L, TRC-1 , and TRC-1 .31 F). Note for step 21 , “Fmoc-Ser(tBu)-SerPsi(Me,Me)Pro-OH” was used to incorporate two adjacent Serines.Attorney Docket Number: 01384-0001 -00PCT

[0396] The final purified product, NRC-1 .4R, was a white solid (132.8 mg, 36.274 pmol, 95.36% purity) and was confirmed via LCMS and analytical HPLC.

[0397] FIG. 56: Chemical structure of NRC-1 .4RExample 14: Competition Binding Assays Evaluating Apparent Affinity of Peptide- Chelator Constructs

[0398] This example describes a series of studies performed to estimate the relative apparent affinity of various peptide-chelator constructs to integrins expressed on the surface of cancer cell lines. These experiments were performed at TwoStep Therapeutics.

[0399] Construct binding was evaluated in two different contexts using two different model systems. First, construct binding to endogenous integrin targets on cancer cells was evaluated via a competition binding assay on HEC1 A endometrial cancer cells. Second, construct binding to individual integrins was evaluated via competition binding assays on Ramos cells (B cell lymphoma cell line with no endogenous expression of RGD-binding integrins) that were engineered to overexpress each integrin of interest via lentiviral transduction.

[0400] Competition binding assay methods (used for both model systems): Alexa Fluor 647-labeled labeled PIP-Fc fusion (PIP-Fc-AF647) [same protein sequence as PFc-1] was used as a fluorescent binder to compare the apparent binding affinity of unlabeled integrin-binding test article competitors. Briefly, 5x104cells were incubated with 0.5 nM of PIP-Fc-AF647 and varying concentrations of unlabeled integrin-binding competitors in cold buffer (25 mM Tris pH 7.5, 150 mM NaCI, 2 mM CaCI2, 1 mM MgCI2, 1 mM MnCI2, and 0.5% BSA). Following a 2-hour incubation at 4°C, cells were then washed, and the MFI from PIP-Fc-AF647 binding was measured by flow cytometry. Note: the methods were the same for the competition binding assay with a5pi -expressing Ramos cells except 5 nM of PIP-Fc- AF647 was used instead of 0.5 nM for that particular integrin assessment.

[0401] The datasets were normalized such that the MFI from the positive control (cells treated with PIP-Fc-AF647 only) was equal to 100% Bound, and the MFI value for the negative control (cells incubated with only buffer) was equal to 0% Bound. A 5-parameter asymmetric sigmoidal curve was fit to the % Bound datasets, allowing calculation of an IC50 value for each unlabeled integrin-binding competitor. The Cheng-Prusoff equation was then used to convert IC50 values into apparent affinity (KD) values for each unlabeled integrin- binding construct.Attorney Docket Number: 01384-0001 -00PCT

[0402] Results from HEC1A cell-based competition binding assay. In order to evaluate relative apparent affinity differences between different monomeric and multimeric peptide-chelator constructs, a competition binding assay was performed in HEC1 A endometrial cancer cells, which endogenously express a variety of the target integrins (high: avp3, moderate: avp6, a5pi , low: avp5). The following test articles were evaluated in this assay: monomeric (PRC-1 , PRC-10, PRC-11 ), dimeric (Di-PRC-1 ), and trimeric (Tri-PRC-1) peptide-chelator constructs as well as a positive control peptide without a chelator (PIP-2).

[0403] These results indicate that each of the monomeric constructs (PRC-1 , PRC- 10, PRC-11) have similar apparent affinities to that of unconjugated PIP control (PIP-2). In contrast, the dimeric (Di-PRC-1 ) and trimeric (Tri-PRC-1) constructs showed similar apparent affinities relative to one another, but their apparent affinities were both stronger than PIP-2 and the monomeric constructs. The enhanced apparent affinity with multimeric constructs relative to monomeric constructs was expected to some extent, although it was surprising that Tri-PRC-1 did not appear to have stronger apparent affinity relative to Di- PRC-1 . In addition, despite the enhanced apparent affinity observed with multimeric constructs relative to monomeric constructs, this trend did not appear to translate to a more favorable tumor uptake, tumor retention, or overall biodistribution profile, as evaluated in Example 10. This would suggest that the apparent affinities of monomeric constructs are already strong enough to result in robust tumor uptake and retention in vivo.

[0404] FIG. 57A-B: Competition binding assay in HEC1 A endometrial cancer cells showing monomeric (PRC-1 , PRC-10, PRC-11), dimeric (Di-PRC-1 ), and trimeric (Tri-PRC- 1) peptide-chelator constructs’ binding profiles relative to PIP-2.

[0405] Results from cell-based competition binding assays evaluating binding to individual integrins'. While many of the integrin-binding constructs evaluated in this workAttorney Docket Number: 01384-0001 -00PCT utilize the integrin binding loop (PRPRGDNPPLT, SEQ ID NO: 4) (e.g., PRC-1), additional constructs were synthesized containing different engineered-binding loops that may impart different selectivity toward particular RGD-binding integrins. For example, TRC-1 and TRC- 1 .31 F have the following integrin binding loop (PQGRGDWAPTS, SEQ ID NO: 5) and PRC- 1 .4L has the integrin binding loop (PLPRGDNPPLT, SEQ ID NO: 7). To evaluate how these binding loop differences impact binding to different RGD integrins, PRC-1 , TRC-1 , and PRC- 1 ,4L were evaluated in competition binding assays on Ramos cell lines overexpressing individual integrin targets.

[0406] These results show that PRC-1 binds to all five individual integrins in this context with high affinity, as expected with the integrin binding loop, PRPRGDNPPLT. Interestingly, the single amino acid substitution present in the PRC-1.4L binding loop (PLPRGDNPPLT) appears to drive a slight change in affinities to different RGD-binding integrins relative to PRC-1 , but overall, PRC-1 .4L still maintained high affinity binding to multiple RGD-binding integrins. In contrast, TRC-1 showed high affinity binding for avp3, much weaker binding to avpi and avp6, and very little or no binding to avp5 and a5pi .

[0407] FIG. 58A-B: Competition binding assays with PRC-1 , PRC-1 .4L, and TRC-1 for representative integrins, avpi (FIG. 58A) and avp3 (FIG. 58B).

[0408] The table below lists the apparent KD(nM) values calculated from these assays. Note: values with an asterisk (*) may not be accurate due to weak binding (inability to fit full binding curve at tested concentrations).Attorney Docket Number: 01384-0001 -00PCTExample 15:111ln-PRC-10,111ln-PRC-11,111ln-PRC-12,111ln-PRC-13,111ln-PRC-1.4L,111In- TRC-1,111ln-NRC-1, and111ln-NRC-1.4R Radiolabeling and Stability TestingRadiolabeling test articles with111In

[0409] Test articles (PRC-10, PRC-1 1 , PRC-12, PRC-13, PRC-1 ,4L, TRC-1 , NRC-1 , and NRC-1 .4R), were dissolved in reaction buffer (0.2 M sodium acetate buffer with pH 5.5) at a 1 mg / mL concentration. [111ln]lnCI3 in 0.1 N HCI was mixed with over 10x volume of reaction buffer and mixed with test articles. The final concentration of the test articles in the reaction mixture was between 0.19 - 0.39 mg / ml. Each crude test article mixture was incubated at 95°C for 10 minutes with 300 rpm agitation.

[0410] For all test articles,1111n-labeled test articles were analyzed via radio-TLC and HPLC. Radio-TLC and HPLC sample preparation:111ln-labeled test articles were diluted with formulation buffer (PBS) and 10% 5 mM of EDTA.Results[0041 1] The specific activities and labeling efficiencies for the reactions are shown in the table below.Attorney Docket Number: 01384-0001 -00PCTMethods for assessing stability of111In-labeled test articles

[0412] Formulation and serum stability assay methods were the same as described in Example 9.Results

[0413] In general,111In-labeled test articles appeared highly stable up to 24 hours in formulation buffer, mouse serum, and human serum as shown in the table below. For111In- TRC-1 , the RCP was over 93% in mouse serum and human serum for up to 24 hours; however, the RCP dropped from 98% (2 hours) to 88.6% (24 hours) in formulation. *N / A:111ln-PRC-10 in human serum at 24h could not be evaluated due to serum sample processing issue.Example 16: Time Course Biodistribution Study of111ln-PRC-1,111ln-PRC-10,111ln-PRC- 11,111ln-PRC-12,111ln-PRC-13,111ln-PRC-1.4L,111ln-TRC-1,111ln-NRC-1, and111ln-NRC- 1.4R in Tumor Bearing Mice

[0414] This example describes experiments that were performed to investigate the tissue accumulation of111ln-PRC-1 ,111ln-PRC-10,111ln-PRC-1 1 ,111ln-PRC-12,111ln-PRC-13,111ln-PRC-1 .4L,111ln-TRC-1 ,111ln-NRC-1 and111ln-NRC-1 .4R after IV administration into mice bearing LI87MG tumors. The retention kinetics of111In-labeled test articles uponAttorney Docket Number: 01384-0001 -00PCT administration in tumor-bearing mice were assessed in a time course biodistribution experiment. Test articles were radiolabeled with111In using similar methods as described in Example 15.

[0415] LI87MG cancer cells (5x106, in PBS with 50% Matrigel) were implanted subcutaneously into the right axilla of athymic nude female mice (Charles River: 490). When tumors reached volumes of approximately 150-350 mm3, mice were randomized into different treatment groups and IV injected (single dose) with radiolabeled constructs.

[0416] Tumor-bearing mice (3 mice per group) were imaged by SPECT / CT at multiple time points after dosing with111In labeled test articles. Mice were IV injected with approximately 1 .5 nmol of111In-labeled test article, resulting in injected activities between 550-770 pCi. Note: molar dose was slightly higher for111ln-PRC-1 and111ln-TRC-1 at approximately 1 .8 nmol. Uptake is presented as percent injected dose per gram tissue (% ID / g). Mice were imaged at 4 h, 24 h, 48 h, and 96 h in this study.

[0417] All of the new test articles in this study were peptide-based constructs (monomeric) without half-life extension moieties. In this study,111ln-PRC-1 again showed a desirable biodistribution profile with prolonged tumor retention and low kidney accumulation. Some of the new test articles in this current study were designed to be close variants to PRC-1 to evaluate how different changes may impact biodistribution.

[0418] As shown in Example 10, increasing the positive charge by adding an additional amine group outside of the peptide binding loop (e.g., in PRC-3) led to increased non-specific uptake in the kidneys. A strategy that can potentially reduce kidney accumulation of peptides is to reduce the number of positively charged residues within the peptide sequence. However, this approach can also have detrimental effects since changing the amino acid sequence can also potentially impact on-target binding and / or stability, which can result in a worse profile overall.

[0419] For PRC-1 , there are only two positively charged amino acids (both are Arginine) at site 4 and site 6; the Arginine at site 6 is part of the critical “RGD motif” needed for RGD-integrin binding, whereas the Arginine at site 4 is still located within the integrin- binding loop but is not part of the “RGD” motif. Relative to PRC-1 , PRC-1 .4L features a point mutation at site 4 of the peptide sequence from Arginine to Leucine in order to evaluate the impact of mutating this positively charged amino acid to a neutral amino acid. As shown in Example 14, PRC-1 .4L showed a slight change in affinities to different RGD-binding integrins relative to PRC-1 , but overall, PRC-1.4L still maintained high affinity binding to multiple RGD-binding integrins. Furthermore,111ln-PRC-1 .4L maintained high stability in murine and human serum as shown in Example 15. In terms of biodistribution,111ln-PRC-Attorney Docket Number: 01384-0001 -00PCT1 ,4L showed an interesting profile where the tumor-to-kidney ratio was quite favorable and similar to that of111ln-PRC-1 , yet the overall uptake in both the kidneys and tumor was a bit lower than111ln-PRC-1 .

[0420] FIG. 59A-B: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-1 (mean + SEM).

[0421] FIG. 60A-B: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-1 .4L (mean + SEM).

[0422] The next construct, PRC-10, was synthesized and evaluated in order to investigate the impact of the chelator conjugation site. Most of the peptide constructs feature N-terminal chelator conjugation, whereas PRC-10’s chelator is attached at amino acid site 15 of the sequence. In this imaging study,111ln-PRC-10 showed an excellent biodistribution profile, indicating chelator conjugation at different sites is permissible.

[0423] FIG. 61 A-B: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-10 (mean + SEM).

[0424] The next set of constructs, PRC-11 (PEG4 linker), PRC-12 (C2 linker), and PRC-13 (C6 linker), were synthesized and evaluated in order to investigate how different chelator linkers may impact biodistribution relative to PRC-1 (C4 linker). Overall, these different constructs all showed decent biodistribution profiles; however,111ln-PRC-1 1 and111ln-PRC-1 showed slightly better results than111ln-PRC-12 and111ln-PRC-13. Intriguingly, the biodistribution does not seem to be based solely on the size of the linker, as the shorter C2 linker in PRC-12 was not quite as favorable compared to the longer C4 linker in PRC-1. Increasing the size to a C6 linker in PRC-13 was also less favorable than the C4 linker in PRC-1 . Moreover, the PEG4 linker, which is larger still than the C6 linker displayed a favorable biodistribution, underscoring the importance of the balancing hydrophilicity / hydrophobicity of the linker as well as its size.

[0425] FIG. 62A-B: Quantification of tumor and healthy organ uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-11 (mean + SEM).

[0426] FIG. 63: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-12 (mean + SEM).Attorney Docket Number: 01384-0001 -00PCT

[0427] FIG. 64: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-PRC-13 (mean + SEM).

[0428] The next construct, TRC-1 , is similar in design to PRC-1 except it contains a different engineered integrin-binding loop (spanning sites 3-13), which confers altered selectivity toward specific RGD-binding integrins, as seen in Example 14. In this imaging study,111ln-TRC-1 demonstrated measurable tumor uptake and retention; however, the levels were substantially lower than those observed with constructs featuring the same engineered binding loop as111ln-PRC-1 . This discrepancy is likely attributable to the LI87MG tumor model’s expression of multiple RGD-binding integrins, favoring PRC-1 (and other constructs with the same binding loop), which engages a broader spectrum of these integrins compared to TRC-1 . However, comparable tumor uptake between TRC-1 and PRC-1 may be observed in tumor models that predominantly express the narrower subset of integrins targeted by TRC-1 .

[0429] FIG. 65: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-TRC-1 (mean + SEM).

[0430] The next two constructs, NRC-1 and NRC-1 .4R, are non-binding peptide control chelator constructs. NRC-1 is similar in design to PRC-1 and TRC-1 , except it contains a different binding loop (between sites 3-13) that eliminates binding to RGD-binding integrins. This is used as a non-binding control, where the number of positively charged residues (i.e. , # of Arginines) matches PRC-1 .4L, TRC-1 , and TRC-1 .31 F.

[0431] NRC-1 .4R is the same as NRC-1 except it has an Arginine at site 4 of the sequence instead of a Threonine like NRC-1 . This is used as a non-binding control, where the number of positively charged residues (i.e., # of Arginines) matches all other integrin- binding peptide-chelator constructs tested (not including PRC-1 .4L, TRC-1 , and TRC-1 .31 F).

[0432] In this imaging study, neither111ln-NRC-1 nor111ln-NRC-1 .4R showed any appreciable tumor uptake, indicating that the tumor uptake observed for other engineered integrin-binding constructs was dependent on integrin-binding rather than non-specific uptake. In contrast, kidney accumulation was still observed for both111ln-NRC-1 and111ln- NRC-1 .4R, with111ln-NRC-1 .4R showing higher kidney accumulation than111ln-NRC-1 . The overall levels of kidney accumulation observed for these non-binding control constructs suggest that the kidney levels seen for engineered integrin-binding constructs is likely due to non-specific uptake.

[0433] FIG. 66: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-NRC-1 (mean + SEM).Attorney Docket Number: 01384-0001 -00PCT

[0434] FIG. 67: Quantification of tumor and kidney uptake derived from SPECT / CT imaging at 4 h, 24 h, 48 h, and 96 h after administration of111ln-NRC-1 .4R (mean + SEM).Example 17:177Lu-PRC-1 Radiolabeling and Stability Testing

[0435] Studies involving radiolabeling and stability assessment of177Lu radiolabeled constructs were performed at WuXi AppTec.Radiolabeling PRC-1 with177Lu

[0436] PRC-1 was dissolved in reaction buffer (0.2 M sodium acetate buffer with pH 5.5) at a 1 mg / mL concentration. [177Lu]LuCh was mixed with over 10x volume of reaction buffer and mixed with test article. The final concentration of PRC-1 in the reaction mixture was between 0.18 - 0.33 mg / ml. The crude test article mixture was incubated at 95°C for 10 minutes.

[0437] 177Lu-PRC-1 was analyzed via radio-TLC and HPLC. Radio-TLC and HPLC sample preparation:177Lu-PRC-1 was diluted with formulation buffer (PBS with 10 mg / mL sodium ascorbate) and 10% 5 mM of EDTA.

[0438] The specific activity was 200 pCi / pg, and the labeling efficiency was >99% by TLC and >99% by HPLC.Assessing stability of177Lu-PRC-1

[0439] Formulation and serum stability assay methods were similar to those described in Example 9.

[0440] Overall,177Lu-PRC-1 was highly stable in formulation buffer, mouse plasma, and mouse serum as shown in the table below.Example 18: Efficacy Study with177Lu-PRC-1 in Tumor Bearing Mice

[0441] This example describes a study performed to analyze the in vivo efficacy of177Lu-PRC-1 after IV administration in mice bearing U87MG tumors. Efficacy studies in mice were performed at WuXi AppTec.Attorney Docket Number: 01384-0001 -00PCT

[0442] LI87MG cancer cells (5x106cells in PBS) were implanted subcutaneously in the right flank of BALB / c nude mice. When tumors reached volumes of approximately 115 mm3, mice were randomized into different treatment groups (n = 5 per group) and IV injected with Vehicle on Days 0, 7, 14 (Group 1), 3 mCi177Lu-PRC-1 on Days 0, 14 (Group 2 - 2 doses total), or 3 mCi177Lu-PRC-1 on Days 0, 7, 14 (Group 3 - 3 doses total). PRC-1 was radiolabeled with177Lu using similar methods as described in Example 17.

[0443] In this study, the dose used for177Lu-PRC-1 was relatively high (3 mCi) so that both efficacy (as measured by tumor volume from caliper measurements) and safety (as measured by % body weight change) could be evaluated in parallel. All tumor-bearing mice treated with177Lu-PRC-1 showed strong tumor regression, regardless of dosing schedule.

[0444] Given how strong the efficacy was at this dose level and with the evaluated dosing schedules, we would expect to see strong efficacy at lower dose levels and less frequent dosing schedules as well. However, a relatively high dose level and frequent dosing schedules were selected for this initial study so that safety could also be evaluated. For tumor-bearing mice dosed on Days 0 and 14 (Group 2 - 2 doses total), a transient slight dip in body weight (average <10%) was observed after each dose, with body weight recovery observed about 7-10 days after each dose; due to the dosing frequency being every two weeks, this provided sufficient time for body weight recovery between doses. For tumorbearing mice dosed on Days 0, 7, and 14 (Group 3 - 3 doses total), a slight dip in body weight was observed after the first dose (average <5%), then decreased a bit more after the second dose (average <10%). Supplementary food was provided to these mice on Day 10. Then, after the third dose on Day 14, instead of continuing to decrease, body weight actually increased and quickly recovered after that third dose. Overall, these results suggest that177Lu-PRC-1 was very well tolerated even at a relatively high dose given that the overall magnitude of body weight loss was low (average <10%) and swift recovery was observed.

[0445] FIG. 68A-B: Average tumor volume over time in U87MG-tumor bearing mice treated with vehicle or177Lu-PRC-1 (mean + SEM).

[0446] FIG. 69A-B: Average % body weight change over time in U87MG-tumor bearing mice treated with vehicle or177Lu-PRC-1 (mean + SD).

[0447] Efficacy and tolerability of177Lu-PRC-1 was also confirmed in the SKOV3 ovarian cancer xenograft model.

[0448] 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: 01384-0001 -00PCT principles 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

[0449] 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.

[0450] 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.References

[0451] 1. Roode, K.E. de, Joosten, L., and Behe, M. (2024). Towards the MagicRadioactive Bullet: Improving Targeted Radionuclide Therapy by Reducing the Renal Retention of Radioligands. Pharmaceuticals 17, 256. https: / / doi.org / 10.3390 / ph17020256.

[0452] 2. Desgrosellier, J.S., and Cheresh, D.A. (2010). Integrins in cancer: biological implications and therapeutic opportunities. Nature Reviews Cancer 10, 9-22. https: / / doi.org / 10.1038 / nrc2748.Attorney Docket Number: 01384-0001 -00PCT

[0453] 3. Jiang, L., Kimura, R.H., Miao, Z., Silverman, A.P., Ren, G., Liu, H., Li,P., Gambhir, S.S., Cochran, J.R., and Cheng, Z. (2010). Evaluation of a 64Cu-Labeled Cystine-Knot Peptide Based on Agouti-Related Protein for PET of Tumors Expressing avp3 Integrin. Journal of Nuclear Medicine 51, 251-258. https: / / doi.Org / 10.2967 / jnumed.109.069831 .

[0454] 4. Sachindra, S., Hellberg, T., Exner, S., Prasad, S., Beindorff, N.,Rogalla, S., Kimura, R., Gambhir, S.S., Wiedenmann, B., and Grbtzinger, C. (2021). SPECT / CT Imaging, Biodistribution and Radiation Dosimetry of a 177Lu-DOTA-lntegrin avp6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model. Frontiers in Oncology 11.

[0455] 5. Kimura, R.H., Teed, R., Hackel, B.J., Pysz, M.A., Chuang, C.Z.,Sathirachinda, A., Willmann, J.K., and Gambhir, S.S. (2012). Pharmacokinetically Stabilized Cystine Knot Peptides That Bind Alpha-v-Beta-6 Integrin with Single-Digit Nanomolar Affinities for Detection of Pancreatic Cancer. Clinical Cancer Research 18, 839-849. https: / / doi.Org / 10.1158 / 1078-0432.CCR-11 -1116.

[0456] 6. Notni, J. (2023). RGD Forever! — Past, Present, and Future of a 3-Letter-Code in Radiopharmacy and Life Sciences. Pharmaceuticals 16, 56. https: / / doi.Org / 10.3390 / ph16010056.

[0457] 7. Quigley, N.G., Steiger, K., Hoberuck, S., Czech, N., Zierke, M.A.,Kossatz, S., Pretze, M., Richter, F., Weichert, W., Pox, C., et al. (2022). PET / CT imaging of head-and-neck and pancreatic cancer in humans by targeting the “Cancer Integrin” avp6 with Ga-68-Trivehexin. Eur J Nucl Med Mol Imaging 49, 1136-1147. https: / / doi.org / 10.1007 / s00259-021 -05559-x.

[0458] 8. Kimura, R.H., Cheng, Z., Gambhir, S.S., and Cochran, J.R. (2009).Engineered Knottin Peptides: A New Class of Agents for Imaging Integrin Expression in Living Subjects. Cancer Research 69, 2435-2442. https: / / doi.org / 10-1158 / 0008-5472. CAN- 08-2495.

[0459] 9. Nielsen, C.H., Kimura, R.H., Withofs, N., Tran, P.T., Miao, Z.,Cochran, J.R., Cheng, Z., Felsher, D., Kjaer, A., Willmann, J.K., et al. (2010). PET Imaging of Tumor Neovascularization in a Transgenic Mouse Model Using a Novel 64Cu-DOTA-Knottin Peptide. Cancer Res 70, 9022-9030. https: / / doi.org / 10.1158 / 0008-5472.CAN-10-1338.

[0460] 10. Liu, S., Liu, H., Ren, G., Kimura, R.H., Cochran, J.R., and Cheng, Z.(2011 ). PET Imaging of Integrin Positive Tumors Using18F Labeled Knottin Peptides. Theranostics 1, 403-412. https: / / doi.org / 10.7150 / thno / v01 p0403.Attorney Docket Number: 01384-0001 -00PCT

[0461] 11. Jiang, L., Miao, Z., Kimura, R.H., Liu, H., Cochran, J.R., Culter, C.S.,Bao, A., Li, P., and Cheng, Z. (2011 ). Preliminary evaluation of 177Lu-labeled knottin peptides for integrin receptor-targeted radionuclide therapy. Eur J Nucl Med Mol Imaging 38, 613-622. https: / / doi.Org / 10.1007 / S00259-010-1684-x.

[0462] 12. Zorzi, A., Linciano, S., and Angelini, A. (2019). Non-covalent albuminbinding ligands for extending the circulating half-life of small biotherapeutics.MedChemComm 10, 1068. https: / / doi.org / 10.1039 / c9md00018f.

[0463] 13. Kim, J.W., Cochran, F.V., and Cochran, J.R. (2015). A ChemicallyCross-Linked Knottin Dimer Binds Integrins with Picomolar Affinity and Inhibits Tumor Cell Migration and Proliferation. Journal of the American Chemical Society 137, 6-9. https: / / doi.Org / 10.1021 / ja508416e.

[0464] 14. Moore, S.J., Hayden Gephart, M.G., Bergen, J.M., Su, Y.S., Rayburn,H., Scott, M.P., and Cochran, J.R. (2013). Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma. Proceedings of the National Academy of Sciences 110, 14598-14603. https: / / doi.org / 10.1073 / pnas.1311333110.

Claims

Attorney Docket Number: 01384-0001 -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; and(b) a chelator.

2. The conjugate of claim 1 , wherein the chelator is conjugated to the EETI-II based knottin peptide via a linker.

3. The conjugate of any one of claims 1 -2, wherein the conjugate further comprises a radionuclide.

4. The conjugate of claim 3, wherein the radionuclide is selected from99mTc,111ln,67Ga,68Ga,86Y,90Y,177Lu,151Tb,223Ra,186Re,188Re,61Cu,62Cu,64Cu,67Cu,55Co,57Co,43Sc,44Sc,46Sc,47Sc,85Sr,89Sr,90Sr,117mSn,145Sm,235Ac,213Bi,212Bi,32P,33P,203Pb,212Pb,227Th,153Sm,166Ho,225Ac,211At,166Ho, and161Tb.

5. The conjugate of any one of claims 1 -4, wherein the conjugate is capable of treating cancer in a patient.

6. The conjugate of any one of claims 1 -5, wherein the conjugate comprises a dimer of EETI-II based knottin peptides.

7. The conjugate of any one of claims 1 -5, wherein the conjugate comprises a trimer of EETI-II based knottin peptides.

8. The conjugate of any one of claims 1 -7, wherein the cell surface molecule is an integrin.

9. The conjugate of claim 8, wherein the integrin is avPi integrin, avPe integrin, avPs integrin, avp3integrin, and / or a5Pi integrin.

10. The conjugate of any one of claims 1 -9, wherein the EETI-II based knottin peptide comprises GCXiX2X3X4X5X6X7X8XgXioXiiXi2Xi3Xi4Xi5Xi6Xi7Xi8XigX2oCX2iQDSDCX22AGCVCX23X24X25X26X27X28X29X30X3iX32X33CG (SED ID NO: 2), wherein Xi - X3are any amino acid; X4-X20if 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.11 . The conjugate of any one of claims 1 -9, wherein the EETI-II based knottin peptide comprises GCXiX2X3X4X5X6X7X8XgXioXiiXi2Xi3Xi4Xi5Xi6Xi7Xi8XigX2oCX2iQDSDCX22AGCVCGPNGX23CG (SEQ ID NO: 3), wherein Xi-X3are any amino acid; X4-X20, if present, are any aminoAttorney Docket Number: 01384-0001 -00PCT acid; and wherein X21-X23 are any amino acid, further wherein each amino acid is independently selected from standard or unnatural amino acids.

12. The conjugate of any one of claims 1 -11 , 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, or SEQ ID NO: 36.

13. The conjugate of any one of claims 1 -11 , 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, or SEQ ID NO: 36.

14. The conjugate of any one of claims 1 -11 , wherein the engineered loop has a sequence of any one of SEQ ID NO: 4 through SEQ ID NO: 8, inclusive, or SEQ ID NO: 36.

15. The conjugate of any one of claims 1 -11 , 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: 30, inclusive.

16. The conjugate of claim 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 9.

17. The conjugate of claim 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 10.

18. The conjugate of claim 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 1 1 .

19. The conjugate of claim 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 12.

20. The conjugate of claim 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 25.21 . The conjugate of claim 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 26.

22. The conjugate of claim 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 27.

23. The conjugate of claim 15, wherein the EETI-II based knottin peptide has an amino acid sequence comprising SEQ ID NO: 28.

24. The conjugate of any one of claims 1 -23, wherein the chelator is selected from:1.4.7.10-Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA); S-2-(4- lsothiocyanatobenzyl)-1 ,4,7,10 tetraazacyclododecanetetraacetic acid (p-SCN-Bn-DOTA);1.4.7.10-tetraazacyclododecane-1 ,4,7,10-tetraacetic amide (TCMC; also known as DOTAM); diethylene triamine pentaacetic acid (DTPA); ethylene diamine tetraacetic acid (EDTA); p- isothiocyanatobenzyl-1 ,4,7,10-tetra-azacyclododecane-1 ,4,7,10-tetraacetic acid (p-SCN-Bz- DOTA); 1 ,4,7,10-tetra-azacyclododecane-N,N',N"-triacetic acid (DO3A); 1 ,4,7, 10-tetra- azacyclododecane-1 ,4,7,10-tetrakis(2-propionic acid) (DOTMA); 3,6,9-triaza-12-oxa-3,6,9-Attorney Docket Number: 01384-0001 -00PCT tricarboxymethylene-10-carboxy-13-phenyl-tridecanoic acid (“B- 19036”); 1 ,4,7- triazacyclononane-N,N',N"-triacetic acid (NOTA); 1 ,4,8,11 -tetra-azacyclotetradecane- 1 ,4,8,11 -tetraacetic acid (TETA); triethylene tetraamine hexaacetic acid (TTHA); trans-1 ,2- diaminohexane tetraacetic acid (CYDTA); 1 ,4,7,10-tetra-azacyclododecane-1-(2- hydroxypropyl)-4,7,10-triacetic acid (HP-DO3A); trans-cyclohexane-diamine tetraacetic acid (CDTA); trans(1 ,2)-cyclohexane dietylene triamine pentaacetic acid (CDTPA); 1-oxa-4,7,10- triazacyclododecane-N,N',N"-triacetic acid (OTTA); 1 ,4,7,10-tetra-azacyclododecane- 1 ,4,7,10-tetrakis{3-(4-carboxyl)-butanoic acid}; 1 ,4,7,10-tetra-azacyclododecane-1 ,4,7,10- tetrakis(acetic acid-methyl amide); 1 ,4, 7,10-tetra-azacyclododecane- 1 ,4, 7,10- tetrakis(methylene phosphonic acid); 1 ,4,7-triazacyclononane-l,4-diacetic acid (NODA);1 ,4,7- tri azacyclononane, 1 -glutaric acid-4, 7-diacetic acid (NODAGA); 1 ,4,7,10- tetraazacyclodecane, 1 -glutaric acid-4,7, 10-triacetic acid (DOTAGA); 1 ,4,8,11- tetraazabicyclo[6.6.2]hexadecane-4, 11 -diacetic acid (CB-TE2A); 2-cyclohexyl diethylene triamine pentaacetic acid (CHX-A”-DTPA); DAD; deforoxamine (DFO); l,2-[[6-carboxypyridin- 2-yl]methylamino]ethane (H2dedpa); 2- (Carboxymethylamino)acetic acid (IDA); ethylene glycol-bis(2-aminoethylether)- N,N,N',N'-tetra acetic acid (EGTA); 1 ,2-bis(o- aminophenoxy)ethane-N,N,N’,N’- tetraacetic acid (BAPTA); ethylenediamine-N,N’- disuccinic acid (EDDS); 2,2',2",2"',2"",2 -(1 ,4,7,10,13,16- hexaazacyclooctadecane-1 ,4,7,10,13,16- hexayl) hexaacetic acid (HEHA); 2,2',2",2"',2""-(1 ,4,7,10,13-pentaazacyclopentadecane- 1 ,4,7,10,13- pentayl)pentaacetic acid (PEPA); 3,3',3",3"'-(1 ,5,9,13- tetraazacyclohexadecane- 1 ,5,9, 13-tetrayl)tetrapropionic acid (TETPA); and 3, 3', 3", 3"'- (1 ,4,7,10- tetraazacyclododecane- 1 ,4, 7, 10-tetrayl)tetrapropionic acid (DOTPA);and derivatives thereof .

25. The conjugate of any one of claims 1 -24, wherein the chelator comprises 1 ,4,7,10- Tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA).

26. The conjugate of any one of claims 1 -24, wherein the chelator comprises 1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic amide (DOTAM).

27. The conjugate of any one of claims 1 -25, wherein the linker comprises a chain of about 1 to about 12 carbon atoms.

28. The conjugate of claim 27 wherein the linker comprises a chain of 4 carbon atoms.

29. The conjugate of any one of claims 1 -25, wherein the linker comprises one or more polyethylene glycol (PEG) units.

30. The conjugate of claim 29, wherein the linker comprises a chain of 4 PEG units.31 . The conjugate of any one of claims 1 -30, wherein the chelator is conjugated to theEETI-II based knottin peptide at the N-terminal amino acid of the peptide.

32. The conjugate of any one of claims 1 -30, wherein the chelator is conjugated to the EETI-II based peptide at the C-terminal amino acid of the peptide.Attorney Docket Number: 01384-0001 -00PCT33. The conjugate of any one of claims 1 -30, wherein the chelator is conjugated to an internal, non-terminal amino acid of the EETI-II based knottin peptide.

34. The conjugate of claim 33, wherein the chelator is conjugated to an amino acid that is C-terminal to the engineered loop of the EETI-II based knottin peptide.

35. The conjugate of any one of claims 33-34, wherein the chelator is conjugated to the fifteenth amino acid of the EETI-II based knottin peptide, wherein the numbering is from N- to C-terminal order.

36. The conjugate of any one of claims 1 -35, further comprising an antibody subunit or fragment thereof fused to the EETI-II based knottin peptide.

37. The conjugate of any one of claims 1 -36, further comprising a half-life extending moiety.

38. The conjugate of claim 37, wherein the half-life extending moiety is an albuminbinding moiety.

39. The conjugate of claim 38, wherein the albumin-binding moiety is a small molecule albumin-binding moiety.

40. The conjugate of claim 38, wherein the albumin-binding moiety is selected from: truncated Evans Blue (EB), ibuprofen, 4-p-(tolyl)butyric acid (PT), palmitic acid (C16), and 4- (p-lodophenyl)butyric acid (IP).41 . The conjugate of claim 38, wherein the albumin-binding moiety is truncated Evans Blue (EB).

42. The conjugate of claim 38, wherein the albumin-binding moiety is a linear or branched lipophilic chain comprising 1 -40 carbon atoms.

43. A method of treating cancer, comprising administering the conjugate of any one of claims 1 -28 to a patient in need thereof.

44. The method of claim 43, wherein the cancer is a solid tumor.

45. The method of claim 44, wherein the cancer is selected from blastoma, carcinoma, lymphoma, or sarcoma.

46. The method of claim 45, wherein the cancer is selected from head and neck cancer, non-small cell lung cancer, colorectal cancer, esophageal cancer, uterine cancer, ovarian cancer, gastric cancer, pancreatic cancer, breast cancer, bladder cancer, melanoma, renal cancer, liver cancer, gallbladder cancer, sarcomas, or brain tumors.

47. The method of claim 46, 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,Attorney Docket Number: 01384-0001 -00PCT 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; 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, large-Attorney Docket Number: 01384-0001 -00PCT cell 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, 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.

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