Decoy miniproteins, compositions, & methods of use

Abstract

Provided herein are compositions and methods of treating, preventing, diagnosing, monitoring, and / or imaging a disease, disorder, or condition using a target-binding agent in combination with a decoy. More specifically, certain compositions and methods disclosed herein (e.g., decoys) can be combined with a target-binding agent to reduce uptake and / or retention of the target-binding agent by and in non-tumor tissues without reducing efficacy of the target-binding agent against a tumor.

Description

Docket No.: AKT-033WO DECOY MINIPROTEINS, COMPOSITIONS, & METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 636,087, filed on April 18, 2024, the disclosure of which is incorporated by reference herein in its entirety for all purposes. SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on April 10, 2025, is named AKT-033WO_SL.xml, and is 165,028 bytes in size. BACKGROUND

[0003] Cancer is a leading cause of death worldwide. Classical cancer therapies such as radiotherapy, chemotherapy, and surgical procedures can be accompanied by severe side effects, due to killing of healthy non-cancerous cells. Newer therapeutics enhance the targeting of cytotoxic drugs to tumor cells relative to earlier therapies, including those that use biologics conjugates. Such therapeutics can also rapidly accumulate in the kidney, rather than reaching the biological target. SUMMARY

[0004] In one aspect, the present disclosure provides a decoy for use in cross-scaffold decoying with a target-binding agent, wherein the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 16, 17, and 34, wherein the decoy and the target- binding agent do not share a scaffold, and the target-binding agent is optionally selected from a compound selected from C170, C144, and C143.

[0005] In one aspect, the present disclosure provides a method of treating or preventing cancer in a subject comprising administering a decoy and a target-binding agent to the subject, wherein the decoy and the target-binding agent together are present in an effective amount, and wherein the decoy is a Scaffold A decoy and the target-binding agent is (i) a Scaffold A miniprotein that is already optimized for reduced kidney uptake or (ii) a non- Scaffold A target-binding agent.

[0006] In some embodiments, the scaffold of the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 16, 17, and 34.

[0007] In some embodiments, the target-binding agent is selected from any one of C1-C6, C8-C77, C109-C166 or C167-C169; and / or has an amino acid sequence comprising or Page 1 of 248 IPTS / 128939095.1Docket No.: AKT-033WO consisting of any one of SEQ ID NOs: 1-3, 5-15, 47-68, 87-111 and 112-114. In some embodiments, the target-binding agent binds to a target selected from somatostatin receptor 2 (SSTR2), prostate-specific membrane antigen (PSMA), Nectin-4, or B7-H3. In some embodiments, the scaffold of the target-binding agent is not Scaffold A.

[0008] In one aspect, the present disclosure provides a decoy for co-administration with a target-binding agent, wherein, the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 4 and 16-46, or compounds C7 and C78-C108.

[0009] In one aspect, the present disclosure provides a decoy for blocking uptake of a target-binding agent into a non-tumor tissue, wherein the decoy and the target-binding agent do not share a scaffold and the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 4 and 16-46 or compounds C7 and C78-C108.

[0010] In one aspect, the present disclosure provides a decoy for blocking uptake of a target-binding agent into a non-tumor tissue, wherein the decoy and the target-binding agent are of the same scaffold, but the decoy does not bind to the target, or binds to the target with at least about 100-1,000-fold weaker affinity than the target-binding agent binds to the target, and the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 4 or 16-46 or compounds C7 and C78-C108.

[0011] In some embodiments, the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 16, 17, and 34. In some embodiments, the target-binding agent and the decoy have the same scaffold. In some embodiments, the target-binding agent and the decoy do not share a scaffold.

[0012] In some embodiments, the target-binding agent binds to a target on a cell, and optionally, the cell is a cancer cell and / or the target is selected from TABLE 10. In some embodiments, the target-binding agent binds to a target selected from somatostatin receptor 2 (SSTR2), prostate-specific membrane antigen (PSMA), Nectin-4, or B7-H3.

[0013] In some embodiments, the combination of the decoy and the target-binding agent effectively decoys the target-binding agent as measured by reduction in kidney cell uptake and / or retention, in vitro or in vivo.

[0014] In some embodiments, the target-binding agent is a polypeptide, which polypeptide optionally comprises a miniprotein. In some embodiments, the target-binding agent further comprises one or more of a linker, chelator, and radionuclide. In some embodiments, the radionuclide is selected from Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La- 135, In-111, Ce-134, F-18, and At-211. Page 2 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0015] In some embodiments, the target-binding agent (i) comprises a compound comprising or consisting of any one of C1-C6, C8-C77, and C109-C169; and / or (ii) has an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 1-3, 5-15, 47-68, and 87-114.

[0016] In some embodiments, the target-binding agent has a first binding affinity for the target and the decoy has a second binding affinity for the target and the second binding affinity is at least about 100-1,000-fold weaker than the first binding affinity. In some embodiments, the decoy reduces the uptake and / or retention in kidney cells, in vitro or in vivo, by about 20% to about 100% by 24 hours post-contact with the decoy (in vitro) or after administration of the decoy (in vivo).

[0017] In one aspect, the present disclosure provides a method of blocking uptake of a radiolabeled target-binding agent into a non-target tissue, the method comprising administering to a subject in need thereof a target-binding agent and a decoy, wherein the decoy does not share a scaffold with the target-binding agent.

[0018] In one aspect, the present disclosure provides a method of blocking uptake of a radiolabeled target-binding agent into a non-target tissue, the method comprising administering to a subject in need thereof a target-binding agent and a decoy, wherein the decoy is of the same scaffold as the target-binding agent.

[0019] In one aspect, the present disclosure provides a method of blocking uptake of a radiolabeled target-binding agent into a non-target tissue, the method comprising administering to a subject in need thereof a target-binding agent and a decoy, wherein the decoy is of the same scaffold as the target-binding agent, but the decoy does not bind to the target or binds to the target with at least about 100-1,000-fold weaker affinity than the target- binding agent binds to the target.

[0020] In some embodiments, the target-binding agent and the decoy are administered concomitantly or sequentially. In some embodiments, the target-binding agent and the decoy are administered sequentially, and the decoy is administered before the target-binding agent. In some embodiments, the target-binding agent and the decoy are administered sequentially, and the target-binding agent is administered before the decoy. In some embodiments, the administration of the target-binding agent and the administration of the decoy are no greater than 4 hours apart.

[0021] In one aspect, the present disclosure provides a pharmaceutical composition comprising: (a) a Scaffold A target-binding agent; and (b) a Scaffold A decoy. Page 3 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0022] In one aspect, the present disclosure provides a pharmaceutical composition comprising: (a) a non-Scaffold A target-binding agent; and (b) a Scaffold A decoy.

[0023] In some embodiments, the Scaffold A target-binding agent comprises a polypeptide that binds to Nectin-4 In some embodiments, the target-binding agent comprises or consists of any one of C1-C6, C8-C77, and C109-C169; and / or (ii) has an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 1-3, 5-15, 47-68, and 87-114. In some embodiments, the decoy comprises or consists of any one of SEQ ID NOs: 4, and 16-46 or compounds C7 and C78-C108.

[0024] In one aspect, the present disclosure provides a pharmaceutical composition comprising: (a) a Scaffold B, C, J, or L target-binding agent; and (b) a Scaffold A decoy. In some embodiments, the target-binding agent comprises or consists of C12-C35, C45-C56, C58-C77, C109-C139, and C145-C169 and / or has an amino acid sequence selected from any of SEQ ID NOs: 1-3, 5-6, 8, and 90-114. In some embodiments, the decoy comprises or consists of any one of SEQ ID NOs: 4 and 16-46 or compounds C7 and C78-C108.

[0025] In some embodiments, the target-binding agent and the decoy are supplied in separate containers. In some embodiments, the target-binding agent and the decoy are supplied in the same container. In some embodiments, the target-binding agent and the decoy are co-formulated. In some embodiments, the target-binding agent and / or the decoy are administered to a subject in need thereof, optionally concomitantly or sequentially.

[0026] In one aspect, the present disclosure provides a kit comprising: (a) a decoy for co- administration with a target-binding agent, wherein the decoy blocks uptake and / or retention of the composition into a non-tumor tissue (e.g., a kidney tissue, a liver tissue, etc.); and (b) instructions for use.

[0027] In one aspect, the present disclosure provides a kit comprising: (a) a composition represented by the formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a target-binding agent (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R); and (b) a decoy, wherein the decoy blocks uptake and / or retention of the composition into a non-tumor tissue (e.g., a kidney tissue, a liver tissue, etc.).

[0028] In some embodiments, the decoy and the composition are administered to a subject in need thereof. In some embodiments, after the administration, the decoy appears in a higher concentration in the non-tumor tissue than in the tumor tissue as measured by %ID / g. In some embodiments, when R is present, it is supplied separately from any of M, C, L, or the decoy. In some embodiments, the decoy is selected from a Scaffold A decoy or a Scaffold B decoy. Page 4 of 248 IPTS / 128939095.1Docket No.: AKT-033WO In some embodiments, the decoy is supplied in a molar or mass excess as compared to the composition, which molar or mass excess may optionally be selected from a 10, 20, 100, 200, 250, 300, 1000, or more molar or mass excess. In some embodiments, the R, if present, is added just prior to use.

[0029] In one aspect, the present disclosure provides a method comprising improving a cancer treatment by reducing kidney uptake of a target-binding polypeptide by administering the target-binding agent and a decoy, wherein the target-binding agent and the decoy are both Scaffold A.

[0030] In some embodiments, the target-binding agent is optimized for reduced kidney uptake, prior to combination with the decoy. In some embodiments, the target-binding agent is or comprises a target-binding polypeptide comprising or consisting of C116, C117, C144, C146, C148, C150, C152, C154, C156, C158, C160, C162, C164, and C166 or an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 54, 55, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, and 111. In some embodiments, the target-binding agent further comprises a radionuclide selected from Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La- 132, La-135, In-111, Ce-134, F-18, and At-211.

[0031] In some embodiments, the target-binding agent has significantly higher kidney uptake in absence of combination with the decoy as compared to combination with the decoy.

[0032] In one aspect, the present disclosure provides a method comprising improving a cancer treatment by reducing kidney uptake of a target-binding agent by administering the target-binding agent and a decoy, wherein the target-binding agent is not from Scaffold A (e.g., but is from Scaffold B, C, L, J, or a target-binding agent such as disclosed in TABLE 11 or TABLE 17), and the decoy is a Scaffold A polypeptide.

[0033] In one aspect, the present disclosure provides an improvement in a method of treating an individual with cancer comprising administering (a) a composition comprising a target-binding agent (M) and a radionuclide (R); and (b) a decoy, wherein (i) the decoy and the target-binding agent do not share a scaffold; (ii) the decoy and the target-binding agent are of the same scaffold; or (iii) the target-binding agent is of the same scaffold as the decoy but already optimized for reduced kidney uptake, the improvement comprising reducing one or more off-target effects or toxicity measures after administration of the composition and the decoy as compared to administration of the composition in the absence of the decoy.

[0034] In some embodiments, the composition comprises an M that targets somatostatin receptor 2 (SSTR2), prostate-specific membrane antigen (PSMA), Nectin-4, or B7-H3. Page 5 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0035] In some embodiments, the reduction in the one or more off-target effects or toxicity measures is measured as a reduction in one or more toxicity grades of each of the one or more off-target effects or toxicity measures.

[0036] In one aspect, the present disclosure provides an improvement in a method of treating an individual with cancer by administering: (a) a composition comprising a target- binding agent (M) and a radionuclide (R); and (b) a decoy, wherein (i) the decoy and the target-binding agent do not share a scaffold; (ii) the decoy and the target-binding agent are of the same scaffold; or (iii) the target-binding agent is of the same scaffold as the decoy but already optimized for reduced kidney uptake, the improvement comprising achieving a reduction in concentration of R in a non-tumor tissue in the presence of the decoy as compared to the concentration of R in the non-tumor tissue in the absence of the decoy.

[0037] In some embodiments, the non-tumor tissue is a liver tissue or a kidney tissue. In some embodiments, the reduction in concentration of R in the kidney tissue is determined by maintenance of eGFR over at least the period that the subject is receiving treatment. In some embodiments, the administration of the composition can be repeated at least twice as many times in the presence of the decoy as in the absence of the decoy before a dose-limiting toxicity is reached.

[0038] In one aspect, the present disclosure provides an improvement in a method of reducing uptake by a population of kidney cells of a composition, the improvement comprising administering a composition comprising (a) a radionuclide therapeutic comprising at least a target-binding agent (M) and a radionuclide (R); and (b) a decoy, such that in the presence of the decoy, the radionuclide is less concentrated in the population of kidney cells than in the absence of the decoy.

[0039] In one aspect, the present disclosure provides a combination composition comprising an effective amount of each of: (i) a therapeutic comprising a composition represented by the formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a target-binding agent (M), L comprises a linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the M is of a particular scaffold; and (ii) a decoy comprising or consisting of an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NOs: 16-46, and SEQ ID NOs: 68 or 87.

[0040] In some embodiments, the decoy does not comprise the same scaffold as the M. In some embodiments, the decoy comprises a Scaffold A decoy. Page 6 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0041] In some embodiments, the decoy and the target-binding agent are supplied and / or administered separately, but the combination exists in the subject to whom the administration has occurred.

[0042] In some embodiments, the M comprises or consists of (i) any one of C1-C6, C8- C77, and C109-C170; and / or (ii) has an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 1-3, 5-15, 47-68, and 87-114.

[0043] In one aspect, the present disclosure provides a method of treating an individual with a cancer, the method comprising administering to the individual an effective amount of each of: (a) a means for blocking uptake of a radiotherapeutic to one or more non-tumor tissues, and (b) a radionuclide therapeutic comprising a composition represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a target-binding agent (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the M shares or does not share a scaffold with the means for blocking uptake of the radiotherapeutic.

[0044] In some embodiments, the non-tumor tissue is a kidney tissue or a liver tissue. In some embodiments, the radionuclide therapeutic is targeted to a tumor. In some embodiments, the radionuclide therapeutic is targeted to a cell expressing a target. In some embodiments, under otherwise identical conditions, the radionuclide therapeutic targeted to the tumor is at a greater concentration than in the absence of the means for blocking uptake of the radiotherapeutic to the one or more non-tumor tissues.

[0045] In one aspect, the present disclosure provides a method of improving a cancer treatment in an individual experiencing a decrease in efficacy and / or one or more off-target effects, the method comprising administering an effective amount of each of: (a) a decoy; and (b) a radionuclide therapeutic, wherein the decrease in efficacy is improved relative and / or the one or more off-target effects is prevented or reduced as compared to administering the radionuclide therapeutic without administering the decoy.

[0046] In one aspect, the present disclosure provides a method of improving a cancer treatment in an individual, the method comprising administering an effective amount of each of: (a) a decoy; and (b) a radionuclide therapeutic, wherein an off-target effect is prevented or reduced as compared to administering the radiotherapeutic in the absence of the decoy.

[0047] In some embodiments, the individual has not been previously treated with at least one cancer treatment (e.g., chemotherapy, radiation, immunotherapy) prior to administration of the decoy and the radionuclide therapeutic. In some embodiments, the individual has been Page 7 of 248 IPTS / 128939095.1Docket No.: AKT-033WO previously treated with at least one cancer treatment (e.g., chemotherapy, radiation, immunotherapy) prior to administration of the decoy and the radionuclide therapeutic.

[0048] In one aspect, the present disclosure provides a method of improving a cancer treatment in an individual with a refractory cancer, the method comprising administering: (a) a decoy; and (b) a radionuclide therapeutic, wherein an off-target effect is prevented or reduced as compared to administering the radiotherapeutic in the absence of the decoy.

[0049] In some embodiments, the radionuclide therapeutic comprises a target-binding agent that targets a protein, which protein is optionally selected from TABLE 10. In some embodiments, the radionuclide therapeutic comprises a target-binding agent that targets somatostatin receptor 2 (SSTR2), prostate-specific membrane antigen (PSMA), Nectin-4, or B7-H3. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing’s sarcoma, extracranial germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, hairy cell leukemia, Hodgkin lymphoma, non- Hodgkin lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous neck cell carcinoma, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and the Sezary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, non- small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancers, including osteosarcoma and malignant fibrous histiocytoma of bone, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumors, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor and other childhood kidney tumors.

[0050] The present disclosure provides technologies such as compositions and methods of use and manufacture thereof to address needs in the field of cancer. For example, in contrast to classical cancer diagnostics or therapies, targeted molecules can be designed to increase Page 8 of 248 IPTS / 128939095.1Docket No.: AKT-033WO specificity and decrease toxicity of, e.g., imaging or therapeutic modalities. For instance, delivery of a therapeutic such as a chelator and / or radionuclide (e.g., alpha emitter) using a polypeptide to specifically target the therapeutic to the tumor microenvironment provides focused treatment to tumor cells and avoids or reduces risk of toxicity to surrounding healthy tissues by the therapeutic targeted to, e.g., a tumor.

[0051] In contrast to classical cancer therapies, radionuclide therapies are more targeted and less toxic. For instance, delivery of a radionuclide specifically to a tumor microenvironment allows for selective radiation of tumor tissue, effectively killing malignant cells while preserving the surrounding healthy tissue. For example, a radionuclide can employ a targeting molecule that specifically binds to target protein expressed at an increased level and / or density on the surface of tumor cells relative to non-tumor cells. Binding of the radionuclide to the target-positive tumor cells targets radiation to those cells without targeting healthy tissue. Full-length antibodies have previously been evaluated as targeting moieties; however, due to considerations such as their large size, full length antibodies can have several challenges such as having poor tumor tissue penetration and a long circulating half-life leading to irradiation of normal tissues. A key drawback of radionuclide therapies is the potential for systemic exposure or renal toxicity in radionuclide therapy. Accordingly, a need remains for new approaches to reduce undesirable effects of radionuclide therapy, while maintaining therapeutic effectiveness by specifically targeting tumors, and particularly solid tumors. The present disclosure provides technologies that meet this and other needs.

[0052] Among other things, the present disclosure provides conjugates comprising a target-binding agent (e.g., a polypeptide (e.g., miniproteins), e.g., a small molecule, e.g., a small molecule receptor ligand) that target tumor microenvironments and / or tumor cells conjugated to one or more additional components including, for example, a linker, chelator, and / or radionuclide (e.g., an alpha emitter). In some embodiments, such conjugates are used in treatment of cells expressing a target (e.g., any one of the target proteins in TABLE 10). In some such embodiments, the cells are cancer cells.

[0053] In some embodiments, a composition comprises a linker and a chelator.

[0054] In some embodiments, a composition comprises a linker, chelator, and radionuclide.

[0055] In some embodiments, a composition comprises a target-binding agent (e.g., a polypeptide (e.g., miniproteins), e.g., a small molecule, e.g., a small molecule receptor ligand), an optional linker, a chelator and / or a radionuclide. Page 9 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0056] In some embodiments, a composition is represented by the formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a target-binding agent (e.g., a polypeptide (e.g., miniproteins), e.g., a small molecule, e.g., a small molecule receptor ligand) (M), L comprises a linker (L), C comprises a chelator (C), and R comprises a radionuclide (R).

[0057] In some embodiments, the present disclosure provides compositions that have various advantages over compositions comprising a full-length protein. For example, in some embodiments, a composition of the present disclosure comprises one or more improved properties selected from increased miniprotein expression (e.g., as compared to antibody expression such as on yeast-display based assays), increased thermostability, increased receptor binding specificity and / or affinity, increased chemical stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (e.g., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and / or reduced immunogenicity.

[0058] In some embodiments, a composition of the present disclosure specifically binds to at least one epitope of a target in, on, or near a cell. In some embodiments, the target is any one of the target proteins in TABLE 10. In some embodiments, the cell is a tumor cell (which may or may not have properties of having cancer, but be derived from a tumor sample). In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a primary cell. In some embodiments, the primary cell is taken from a sample from a subject (e.g., from a biopsy, e.g., from a tumor). In some embodiments, the cell is from a cell line.

[0059] In some embodiments, a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) or conjugate thereof selectively binds to a target. In some embodiments, the target is a protein or portion thereof expressed on the surface of a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cancer cell. In some such embodiments, the cancer cell is in (i.e., part of) or near a tumor. In some embodiments, the cancer cell is a circulating cell.

[0060] In some embodiments, a target is a cell adhesion receptor, a cytokine receptor, a chemokine receptor, a growth factor receptor, an immune cell receptor, or a tumor associated extracellular matrix polypeptide. In some such embodiments the target comprises or consists of 5T4, ADAM9, AG-7, AGS-16 / ENPP3, ALPV, ASCT2, AXL, B7-H3 / CD276, B7H4, BCMA, C4.4a, CA6, CA9, CAIX, CCR2, CCR7, CD123, CD138, CD142, CD166, CD19, CD20, CD205, CD22, CD228, CD25, CD30, CD33, CD352, CD37, CD38, CD44v6, CD45, Page 10 of 248 IPTS / 128939095.1Docket No.: AKT-033WO CD46, CD47, CD48, CD5, CD51, CD56, CD7, CD70, CD71, CD74, CD79B, CDH6, CEACAM5, Cholecystokinin 2 receptor, cKIT, CLDN18, CLDN18.2, CLDN6, CLDN6+CLDN9, CLL-1, cMET, cMET+EGFR, Cripto, CXCR4, DLL3, DPEP3, EFNA4, EGFR, EGFR+HER3, EGFR+MUC1, EGFRvIII, EPHA2, ETBR, FAP, FAPI, FCRH5, FGFR2, FGFR3, FLT3, FRα, GCC, GD2, GD3, Globo H, GPC3, GPCR5D, gpNMB, GPR20, HER2, HER2+HER3, IGF1R, IL13Ra, IL-4R, Integrin β-6, KAAG1, L1CAM, LAMP1, Lewis Y Ag, LHRH receptor, LIV1, LIV1A, LRRC15, LY6E, Ly75 / CD205, MC1R, MELTF, Mesothelin, MSLN, MT1-MMP, MUC1, MUC16 / CA-125, NaPi-2b, Nectin-4, Neurokinin 1 receptor, NKG2D, Norepinephrine transporter, NOTCH3, NTSR1, P- Cadherin, PDL1, PRLR, PSMA, PTK7, RNF43, ROR1, ROR2, SEZ6, SLAMF7, SLC44A4, SLITRK6, SS2R, STEAP1, TF, TIM1, TNFSF9, Trop-2, or a portion thereof.

[0061] In related aspects, the present disclosure provides pharmaceutical compositions comprising a conjugate. In some embodiments, a conjugate comprises one or more of a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand), linker, chelator, and radionuclide.

[0062] In some embodiments, one or more components of a conjugate are provided using a vector, e.g., a recombinant expression vector comprising a nucleic acid encoding a polypeptide.

[0063] In some embodiments the present disclosure provides a host cell comprising a vector encoding one or more components of a conjugate as provided herein.

[0064] In some embodiments, the present disclosure provides a composition that is capable of binding to, modulating, and / or inhibiting any one of the target proteins in TABLE 10. In some embodiments, the target protein selected from TABLE 10 is the human isoform. In some embodiments, a composition of the present disclosure is or comprises, peptide therapy, peptide receptor radionuclide therapy.

[0065] In some embodiments, a composition of the present disclosure is formulated for administration to a subject in need thereof (e.g., a pharmaceutical composition). In some embodiments, a composition is administered for the treatment of one or more diseases, disorders, or conditions. In some embodiments, a disease, disorder or condition is cancer. In some embodiments, the treatment comprises providing a composition (e.g., by administering, e.g., by contacting a cell or population of cells, e.g., a tumor), wherein the composition comprises a component that specifically binds to any one of the target proteins in TABLE 10. In some embodiments, the target proteins selected from TABLE 10 is expressed on a cell or Page 11 of 248 IPTS / 128939095.1Docket No.: AKT-033WO population of cells. In some embodiments, providing the composition treats the disease, disorder or condition.

[0066] In some embodiments, the present disclosure provides methods for modulating any one of the target proteins in TABLE 10. That is, in some embodiments, a method of modulating biological activity of any one of the target proteins in TABLE 10 comprises providing (e.g., by administering, e.g., by contacting a cell or population of cells) a composition comprising one or more components in an amount effective to modulate any one of the target proteins in TABLE 10 and its activity. For example, in some embodiments, a composition comprises a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand)that specifically binds to any one of the target proteins in TABLE 10 and one or more of a linker, chelator, and / or radionuclide. In some such embodiments, the composition binds to cells expressing any one of the target proteins in TABLE 10 and targets one or more therapies (e.g., a chelator, e.g., a radionuclide) to a cell expressing the target protein selected from TABLE 10, such as, for example, by internalization of the composition or a component thereof into the cell.

[0067] In some embodiments, the present disclosure provides methods and compositions for use therein that are capable of activating or inhibiting immune cell response. In some embodiments, provided compositions are administered for the treatment of cancer. In some embodiments, the cancer is associated with expression or overexpression of any one of the target proteins in TABLE 10. In some embodiments, the cancer is selected from non-small- cell lung cancer (NSCLC), cutaneous squamous cell carcinoma, pancreatic cancer, primary hepatocellular carcinoma, colorectal carcinoma, clear cell renal carcinoma, breast cancer and prostate cancer.

[0068] In some embodiments, the present disclosure provides methods and compositions for use in detecting the presence or extent of a disease, disorder, or condition in a subject. In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, the subject has been diagnosed as having cancer. In some embodiments, the subject is suspected as having or at risk of having cancer. In some embodiments, the subject diagnosed with cancer has been treated with one or more treatments, such as a composition as provided herein. In some embodiments, a method of detecting (e.g., monitoring / determining prognosis, diagnosing, etc.) comprises measuring a level of a target or a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) that specifically binds to a target in a sample comprising one or more cells from a subject (e.g., using a cell-based assay) or in a subject (e.g., using an in vivo scan or measurement). In Page 12 of 248 IPTS / 128939095.1Docket No.: AKT-033WO some such embodiments, a level of the target or of the target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) is used to determine presence and / or extent of cancer in the subject by comparing a level to a control level or to a level from the same patient at a different point in time.

[0069] In some embodiments, the present disclosure provides kits. In some such embodiments, a kit comprises one or more components such as a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand), linker, chelator, and / or radionuclide that may be combined in one or more methods for use in binding to a target (e.g., any one of the target proteins in TABLE 10). In some embodiments, the present disclosure provides compositions comprising a target- binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R), represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R.

[0070] In some embodiments, M is a polypeptide and comprises or consists of a linear polypeptide, a folded polypeptide (e.g., covalently linked polypeptide, non-covalently linked polypeptide, or polypeptide include a di-sulfide linkage), cysteine-dense peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, the binder comprises or consists of a linear polypeptide, a folded polypeptide, and / or a non-disulfide sequence. In some embodiments, M is a miniprotein and characterized in that it comprises 10-100 amino acids, and no more than 100 amino acids. In some preferred embodiments, M is characterized in that it comprises (i) no more than 100 amino acids and / or 12 kDa; (ii) at least one secondary structure elements; (iii) a sequestered hydrophobic core; and / or displays cooperative folding. In some embodiments, M comprises no more than about 100 amino acids or less, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, or 10 amino acids. In some embodiments, the miniprotein comprises at least one disulfide bridge. In some embodiments, the miniprotein comprises zero, one, two or more disulfide bonds.

[0071] In some embodiments, the present disclosure provides compositions set forth as L- C, wherein L comprises or consists of a linker, C comprises or consists of a chelator, and wherein the linker is designed to be conjugated to a polypeptide. Page 13 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0072] In some embodiments, the present disclosure provides compositions set forth as L- C-R, wherein L comprises or consists of a linker, C comprises or consists of a chelator, and R comprises or consists of a radionuclide, and wherein the composition is capable of being conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand). In some embodiments, when L is present, L comprises or consists of a polyethylene glycol (PEG) linker of PEG2, PEG4, PEG6, PEG8, PEG12, PEG24, PEG36, an ester linker, an amide linker, a maleimide linker, a succinimidyl- 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a caproleic acid linker, or (Gly)n-(^Glu)n- (SEQ ID NO: 79) or (PEG)n, wherein n is from 1 to 10, (Gly)1-10(SEQ ID NO: 80), or any fragment or combination via covalent bond thereof. In some embodiments, when C is present, C comprises or consists of: i) NOPOPage 14 of 248 IPTS / 128939095.1Docket No.: AKT-033WO iv) Macropa.

[0073] In some embodiments, when C is present, C comprises or consists of derivative of NOPO, Crown, Macropa, or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, when L is present and C is absent, L is covalently attached to M. In some embodiments, when R is present, R comprises or consists of Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some embodiments, a composition binds to the target with an affinity of 1 pM to 100 nM as measured by an in vitro binding assay. In some embodiments, when M is present the target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand)binding to the target modulates biological function. In some embodiments, when M is present, M selectively binds to any one of the target proteins in TABLE 10 or a portion thereof.

[0074] In some embodiments, the present disclosure provides isolated constructs or pharmaceutically acceptable salts thereof comprising a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) (M), optional linker (L), and at least one of a chelator (C) or radionuclide (R).

[0075] In some embodiments, the present disclosure provides pharmaceutical compositions comprising a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) (M), wherein the target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand)selectively binds to a target.

[0076] In some embodiments, the composition displays adherence in a tumor. In some embodiments, the composition displays passage through a kidney, a liver, a bone marrow, or a spleen. Page 15 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0077] In some embodiments, the pharmaceutical composition comprises a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) (M), an optional linker (L), and one or both of a chelator (C) and radionuclide (R). In some embodiments, when C is present, C covalently attaches to M. In some embodiments, the chelation efficiency is > 90%. In some embodiments, the pharmaceutical composition further comprises a radionuclide R. In some embodiments, when R is present, it is an alpha-emitter. In some embodiments, R, when present, is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In various embodiments, the radionuclide R is directly conjugated to M through a prosthetic group. In some embodiments, M specifically binds to a target. In some embodiments, the target is any one of the target proteins in TABLE 10. In some embodiments, the target protein selected from TABLE 10 is expressed on a cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a tumor cell. In some embodiments, the tumor cell is a solid tumor cell.

[0078] In some embodiments, the target-binding agent is a miniprotein and comprises or consists of a linear polypeptide, a folded polypeptide (e.g., covalently linked polypeptide, non-covalently linked polypeptide, or polypeptide include a di-sulfide linkage), cysteine- dense peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, the miniprotein comprises one or more disulfide bonds. In some embodiments, the miniprotein is characterized in that it has nM or sub-nM binding affinity on the target in vivo or in a cell-based assay. In some embodiments, the miniprotein has a binding affinity of 1 pM to 100 nM to any one of the target proteins in TABLE 10 on a cell line expressing human isoform of the target protein selected from TABLE 10. In some embodiments, the miniprotein has an amino acid sequence no more than about 100 amino acids or less, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, or 10 amino acids.

[0079] In some embodiments, administration of the pharmaceutical composition to a subject in need thereof does not elicit an immune response or wherein the immune response elicited is tolerable to the subject. In some embodiments, the composition comprises high tumor tissue penetration. In some embodiments, the composition is not taken up and / or Page 16 of 248 IPTS / 128939095.1Docket No.: AKT-033WO retained in the kidney or liver. In some embodiments, the composition is internalized in a cell expressing human isoform of the target protein selected from TABLE 10.

[0080] In some embodiments, a composition comprises a miniprotein-drug conjugate, comprising a miniprotein and at least one drug moiety. In some embodiments, a pharmaceutical composition comprises a miniprotein-drug conjugate, comprising a miniprotein and at least one drug moiety. In certain embodiments, the drug moiety includes but is not limited to a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRMl, a DPP-IV inhibitor, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder and a DHFR inhibitor.

[0081] In other embodiments, the drug moiety includes but is not limited to alkylating agents, such a thioTEPA and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogues) and derivatives thereof; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin, auristatins (including analogues monomethyl-auristatin E and monomethyl-auristatin F (see, e.g., U.S. Published Application No.2005-0238649, published Oct.27, 2005, incorporated herein in its entirety); duocarmycins (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine; trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calichemicin gamma1I and calicheamicin phill, see for example, Agnew, Chem. Intl. Ed. Engl.33:183-186; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; as well as neocarzinostatin Page 17 of 248 IPTS / 128939095.1Docket No.: AKT-033WO chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin™) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubucin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycine, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such a methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adranals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; democolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone, mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2(R)-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitabronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhone- Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Page 18 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston™); aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace™), exemestane, formestane, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0082] In some embodiments, the pharmaceutical composition comprises one or more of an antioxidant molecule, wherein the antioxidant molecule neutralizes a free radical. In some embodiments, the pharmaceutical composition comprises a stabilizer. In further embodiments, the stabilizer comprises gentisic acid or salts thereof, ascorbic acid or salts thereof, methionine, N-acetyl cysteine, histidine, melatonin, ethanol, Se-methionine, or combinations thereof. In some embodiments, the pharmaceutical composition is characterized to exhibit an improved resistance to a peptidase, a protease, or heat.

[0083] In some embodiments, a method of producing a composition represented by the formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M- R, wherein M comprises a miniprotein (M), L comprises a linker (L), C comprises a chelator (C), and R comprises a radionuclide (R) comprises synthesizing a miniprotein (M) and / or linker (L), and optionally reacting a chelator (C) and / or a radionuclide (R), and conjugating one or more of the miniprotein (M) to the linker (L), one or more of the miniprotein (M) to the chelator (C), one or more of the miniprotein (M) to the radionuclide (R), one or more of the miniprotein (M) to the linker (L) to the chelator (C), one or more of the miniprotein (M) to the linker (L) to the chelator (C) and the radionuclide (R), one or more of the miniprotein (M) to the chelator (C) to the radionuclide (R), or one or more of the miniprotein (M) to the linker (L) to the radionuclide (R).

[0084] In some embodiments, the method involves reacting a chelator (C) and a radionuclide (R) at a temperature of between about 25°C and 75°C during an incubation period. In some embodiments, reacting a chelator (C) and a radionuclide (R) is performed during an incubation period of about 5 minutes to about 30 minutes. In further embodiments, reacting a chelator (C) and a radionuclide (R) is performed at a pH in the range of about 5.0 to 7.4. In some embodiments, reacting a chelator (C) and a radionuclide (R) is performed in an aqueous solution that is substantially free of alcohol.

[0085] In some embodiments, a method of delivering a radionuclide to a selected location within a patient involves administering a composition represented by the formula selected Page 19 of 248 IPTS / 128939095.1Docket No.: AKT-033WO from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a miniprotein (M), L comprises a linker (L), C comprises a chelator (C), and R comprises a radionuclide (R). In some embodiments, the radionuclide is selected from Ac- 225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At- 211. In some embodiments, the method further comprises carrying out an imaging procedure to evaluate the localization of the radionuclide within the body, wherein the imaging procedure optionally comprises positron emission tomography (PET) imaging or single- photon emission computerized tomography (SPECT) imaging. In further embodiments, the imaging procedure allows for selecting patients. In certain embodiments, the imaging procedure allows for monitoring patients. In some embodiments, the imaging procedure allows for determining an appropriate dose for treating a patient in need of a pharmaceutical composition comprising one or more miniprotein.

[0086] In some embodiments, the present disclosure provides methods of treating a subject in need thereof comprising administering a composition comprising a miniprotein (M), an optional linker (L), and one or both of a chelator (C) and a radionuclide (R).

[0087] In some embodiments, L comprises or consists of a polyethylene glycol (PEG) linker, an ester linker, an amide linker, a maleimide linker, a valine-citrulline linker, a hydrazone linker, a N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, a vinylsulfone-based linker, a propanoic acid linker, a caproleic acid linker, or any fragment or combination thereof. In some embodiments, C comprises or consists of: i) NOPOPage 20 of 248 IPTS / 128939095.1Docket No.: AKT-033WO iii). some R comprises or consists of Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some embodiments, M comprises or consists of a linear polypeptide, a folded polypeptide (e.g., covalently linked polypeptide, non-covalently linked polypeptide, or polypeptide include a di-sulfide linkage), cysteine-dense peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, M is characterized in that it comprises between 10-100 amino acids, and no more than 100 amino acids. In some preferred embodiments, M is characterized in that it comprises (i) no more than 100 amino acids and / or 12 kDa; (ii) at least two secondary structure elements; (iii) a sequestered hydrophobic core; and / or displays Page 21 of 248 IPTS / 128939095.1Docket No.: AKT-033WO cooperative folding. In some embodiments, the composition comprises at least one additional component. In some embodiments, the composition can penetrate tumor tissue. In some embodiments, the miniprotein comprises no more than about 100 amino acids or less, 90 amino acids, 85 amino acids, 80 amino acids, 75 amino acids, 70 amino acids, 65 amino acids, 60 amino acids, 55 amino acids, 50 amino acids, 45 amino acids, 40 amino acids, 35 amino acids, 30 amino acids, 25 amino acids, 20 amino acids, 15 amino acids, or 10 amino acids. In some embodiments, the miniprotein comprises at least one disulfide bridge. In some embodiments, the miniprotein specifically binds to a target. In some embodiments, the composition displays ^m or nM binding affinity to the target in an in vitro assay. In some embodiments, the composition binds to the target with an affinity of 1 pM to 100 nM as measured by an in vitro binding assay. In some embodiments, the composition is characterized in that it has high tissue penetrating properties relative to a composition comprising a full-size protein that binds to the same target. In some embodiments, the miniprotein binding to the target modulates biological function. In some embodiments, administration of the composition to a subject in need thereof does not elicit an immune response or wherein the immune response elicited is tolerable to the subject. In some embodiments, the tolerable immune response includes a systemic immune response or a local immune response. In some embodiments, the subject is diagnosed as having cancer. In some embodiments, a cancer cell from the subject expresses any one of the target proteins in TABLE 10, or a portion thereof. In some embodiments, the expression of the target is higher in the cancer cell than in a non-cancer cell. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing’s sarcoma, extracranial germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous neck cell carcinoma, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and the Sezary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, Page 22 of 248 IPTS / 128939095.1Docket No.: AKT-033WO head and neck cancer, skin cancer, oropharyngeal cancer, bone cancers, including osteosarcoma and malignant fibrous histiocytoma of bone, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumors, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumors, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor and other childhood kidney tumors. In some embodiments, the composition is administered intravenously or subcutaneously. In some embodiments, the cancer is treated after administration of the composition.

[0089] Additionally disclosed herein are uses of a composition disclosed herein for treating cancer in a subject. Additionally disclosed herein is an isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from SEQ ID NO: 1-68 or 87; or a nucleic acid sequence encoding a polypeptide comprising at least 90%, 95%, 96%, 97%, 98%, 99% or greater identity to SEQ ID NO: 1-68 or 87. Additionally disclosed herein is a vector comprising an isolated polynucleotide disclosed herein. Additionally disclosed herein is a host cell transformed with an isolated polynucleotide disclosed herein or a vector disclosed herein.

[0090] Additionally disclosed herein is a method for characterizing kidney uptake of a composition, the method comprising: providing a plurality of kidney cells; contacting the plurality of kidney cells with a composition represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a miniprotein (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the composition further comprises a biotin group complexed with a fluorescently labeled streptavidin; and lysing the plurality of kidney cells and measuring kidney uptake of the composition by detecting fluorescence of the fluorescently labeled streptavidin. In various embodiments, the plurality of kidney cells are provided in a well. In various embodiments, M comprises an amino acid sequence that shares at least 90% identity to any one of SEQ ID NOs: 1-68 or 87. In various embodiments, M comprises an amino acid sequence that shares 100% identity to any one of SEQ ID NOs: 1-68 or 87.

[0091] In some embodiments, the contacting further comprises adding a decoy.

[0092] In some embodiments, the decoy is selected from C7 or C78-108 and / or has an amino acid sequence of any of SEQ ID NOs: 4 or 16-46. Page 23 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0093] In some embodiments, the decoy is added with and without the composition.

[0094] In some embodiments, the characterization is repeated and kidney uptake of the composition is measured in presence of (i) composition alone; (ii) decoy alone; and / or (iii) composition and decoy together.

[0095] Additionally disclosed herein is a method for characterizing liver uptake of a composition, the method comprising: providing a plurality of liver cells; contacting the plurality of liver cells with a composition represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a miniprotein (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the composition further comprises a biotin group complexed with a fluorescently labeled streptavidin; and lysing the plurality of liver cells and measuring liver uptake of the composition by detecting fluorescence of the fluorescently labeled streptavidin. In various embodiments, the plurality of liver cells are provided in a well. In various embodiments, M comprises an amino acid sequence that shares at least 90% identity to any one of SEQ ID NOs: 1-68 or 87. In various embodiments, M comprises an amino acid sequence that shares 100% identity to any one of SEQ ID NOs: 1-68 or 87. In some embodiments, the plurality of liver cells are provided in a well.

[0096] In some embodiments, M comprises an amino acid sequence that shares at least 90% identity to any one of SEQ ID NOs: 1-68 or 87. In some embodiments, M comprises an amino acid sequence that shares 100% identity to any one of SEQ ID NOs: 1-68 or 87.

[0097] In some embodiments, the contacting further comprises adding a decoy.

[0098] In some embodiments, the decoy is selected from C7 or C78-108 and / or has an amino acid sequence of any of SEQ ID NOs: 4 or 16-46.

[0099] In some embodiments, the decoy is added with and without the composition.

[0100] In some embodiments, the characterization is repeated and liver uptake of the composition is measured in presence of (i) composition alone; (ii) decoy alone; and / or (iii) composition and decoy together. BRIEF DESCRIPTION OF FIGURES

[0101] FIG.1 depicts analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice treated with exemplary Nectin-4 charge variant conjugates (Compound ID NOs: C14, C45, C48, and C52). Page 24 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0102] FIG.2 depicts analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Co-administration of an exemplary decoy (Compound ID NO: C15) reduces kidney uptake of an exemplary Nectin-4 targeting miniprotein conjugate (Compound ID NO: C14) scaffolds.

[0103] FIG.3 depicts analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Exemplary B7-H3 charge variant conjugates (Compound ID NOs: C1, C3, C5, C9, and C43) demonstrate reduced levels of kidney retention in mouse biodistribution.

[0104] FIG.4 depicts analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Co-administration of an exemplary decoy (Compound ID NO: C7) reduces kidney uptake of an exemplary B7-H3 targeting affibody conjugate (Compound ID NO: C9).

[0105] FIG.5 depicts analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Comparison of an exemplary conjugate with an added exemplary Version 1 cleavable linker (Compound ID NO: C64) and an exemplary Nectin-4 conjugate (Compound ID NO: C14) showed minimal alterations in kidney uptake and retention.

[0106] FIG.6 depicts a DELFIA saturation binding curve-fitting of an exemplary conjugate (Compound ID NO: C34) for estimating KD.

[0107] FIG.7 depicts a DELFIA competitive binding curve of an exemplary conjugate (Compound ID NO: C40) for estimating Ki.

[0108] FIG.8 depicts binding kinetics of an exemplary conjugate (Compound ID NO: C11) to immobilized ligand. The change in signal over time is proportional to peptide binding to the ligand, generating a sensorgram. Black lines are fitted by the 1:1 binding model to calculate a KD(M). Concentrations correspond to labels on the diagram as follows: a: 25 nM; b: 12.5 nM; c: 6.25 nM; d1: 3.13 nM; d2: 3.13 nM; e: 1.56 nM; and f: 0.78 nM.

[0109] FIG.9A depicts quantitative uptake of an exemplary AF647-labeled conjugate (Compound ID NO: C9) in the Opossum kidney proximal tubule cell (OK-PTC) uptake assay.

[0110] FIG.9B depicts quantitative uptake in the Opossum kidney proximal tubule cell (OK-PTC) uptake assay. Co-treatment with an exemplary decoy (Compound ID NO: C15) at 100X and 10X molar excess reduces the uptake of an exemplary AF647-labeled conjugate (Compound ID NO: C14). Page 25 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0111] FIG.9C depicts quantitative uptake in the Opossum kidney proximal tubule cell (OK-PTC) uptake assay. Uptake of an exemplary AF647-labeled control conjugate (Compound ID NO: C14) is reduced in a dose-dependent manner by pre-treatment with a mixture of lysine and arginine amino acids.

[0112] FIG.10 depicts quantitative uptake in the Opossum kidney proximal tubule cell (OK-PTC) uptake assay. Co-treatment with a 20-fold excess of exemplary decoys C79, C96, and C78 reduces the uptake of the test agents C132 – C139.

[0113] FIG.11 depicts quantitative uptake in the Opossum kidney proximal tubule cell (OK-PTC) uptake assay. Co-treatment with 10X molar excess of exemplary decoys (Compound ID NOs: C15, C80, and C83) reduces the uptake of an exemplary biotinylated test agent (Compound ID NO: C14).

[0114] FIG.12 depicts analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Co-administration of an exemplary decoy (Compound ID NO: C79) reduces kidney uptake and retention of an exemplary Nectin-4 targeting miniprotein conjugate (Compound ID NO: C67) scaffolds.

[0115] FIG.13 depicts analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Comparison of an exemplary conjugate with an added exemplary Version 2 cleavable linker (Compound ID NO: C68) and an exemplary Nectin-4 conjugate (Compound ID NO: C67) showed no alterations in early kidney uptake and modest reduction in kidney retention at later timepoints.

[0116] FIG.14 depicts analysis generated from SPECT / CT scans to quantify the injected dose per gram (%ID / g) of kidney tissue in mice. Comparison of an exemplary conjugate with an added albumin-binding motif (Compound ID NO: C69) and an exemplary Nectin-4 conjugate (Compound ID NO: C67) showed reduction in kidney uptake.

[0117] FIG.15 depicts tumor volume measurements after single dose treatment of test articles in mouse efficacy studies. HT-1376 cells with exogenously expressed target were treated with either vehicle or225Ac-labeled test article (Compound ID NO: C116) at X or 2X nCi and showed reduction in tumor volume.

[0118] FIG.16 depicts body weight measurements after single dose treatment of test articles in mouse efficacy studies. HT-1376 cells with exogenously expressed target were treated with either vehicle or225Ac-labeled test article (Compound ID NO: C116) at X or 2X nCi showed no change in body weight.

[0119] FIGs.17A and 17B are graphs depicting data showing reduction in cellular uptake of exemplary target-binding Scaffold B miniprotein conjugates when combined with an Page 26 of 248 IPTS / 128939095.1Docket No.: AKT-033WO exemplary Scaffold A decoy in vitro. FIG.17A is a bar graph showing percent uptake (on the y-axis) of an exemplary B7-H3-targeting Scaffold B miniprotein conjugate (C140) alone / without a decoy (20 ^M; column 1, x-axis) or in combination (column 2, x-axis) with a 20-fold molar excess of an exemplary Scaffold A decoy (C78). FIG.17B is a bar graph showing percent uptake (on the y-axis) of 20 ^M of an exemplary B7-H3-targeting Scaffold B miniprotein (C141) alone (20 ^M; column 1, x-axis) or in combination (column 2; x-axis) with a 20-fold molar excess of an exemplary Scaffold A decoy (C78). Error bars represent standard error of the mean (SEM).

[0120] FIGs.18A and 18B are graphs depicting data showing reduction in cellular uptake of exemplary target-binding miniprotein compounds when combined with an exemplary Scaffold A decoy in vitro. FIG.18A is a bar graph showing percent uptake (on the y-axis) of an exemplary Nectin-4-targeting bicyclic miniprotein (C143) alone / without a decoy (20 ^M; column 1, x-axis) or in combination (column 2, x-axis) with a 20-fold molar excess of an exemplary Scaffold A decoy (C78). FIG.18B is a bar graph showing percent uptake (on the y-axis) of an exemplary somatostatin-2-receptor-targeting miniprotein (C144) alone (20 ^M; column 1, x-axis) or in combination (right column, “2” on the x-axis) with a 20-fold molar excess of an exemplary Scaffold A decoy (C78). Error bars represent standard error of the mean (SEM).

[0121] FIG.19 is a graph depicting data showing reduction in cellular uptake of an exemplary target-binding Scaffold B miniprotein conjugate when combined with one of three exemplary Scaffold A decoys in vitro. The bar graph shows percent uptake (on the y-axis) of an exemplary B7-H3-targeting Scaffold B miniprotein conjugate (C141) alone / without a decoy (20 ^M; column 1, on the x-axis) or in combination with a 20-fold molar excess of one of three exemplary Scaffold A decoys (C79, column 2; C96, column 3; and C78, column 4 on the x-axis). Error bars represent standard error of the mean (SEM).

[0122] FIGs.20A and 20B are graphs depicting data showing reduction in cellular uptake of exemplary Scaffold B target-binding miniprotein compounds when combined with an exemplary Scaffold B decoy in vitro. FIG.20A is a bar graph showing percent uptake (on the y-axis) of an exemplary B7-H3-targeting Scaffold B miniprotein conjugate (C141) alone / without a decoy (20 ^M; column 1, x-axis) or in combination (column 2, x-axis) with a 20-fold molar excess of an exemplary Scaffold B decoy (C7). FIG.20B is a bar graph showing percent uptake (on the y-axis) of an exemplary B7-H3-targeting Scaffold B miniprotein conjugate (C142) alone / without a decoy (1 ^M; column 1, x-axis) or in Page 27 of 248 IPTS / 128939095.1Docket No.: AKT-033WO combination (column 2, x-axis) with a 100-fold molar excess of an exemplary Scaffold B decoy (C7). Error bars represent standard error of the mean (SEM).

[0123] FIGs.21A and 21B are graphs depicting results from analysis of SPECT / CT scans to quantify in vivo kidney (FIG.21A) or liver (FIG.21B) uptake and retention of a scaffold or scaffold plus decoy in mice, shown as percent injected dose per gram (%ID / g; y-axis) after injection of an111In-labeled exemplary B7-H3-targeting Scaffold B miniprotein conjugate (111In-C9) alone / without a decoy (C9; solid line with solid circles in FIG.21A and line labeled A in FIG.21B) or co-administered with 850-fold molar excess of an exemplary Scaffold B decoy (C7; dotted line with open circles in FIG.21A and line labeled B in FIG. 21B) from 4-24 hours post-injection (x-axis). Error bars represent standard deviation (SD).

[0124] FIGs.22A and 22B are graphs depicting analyses of in vivo kidney (FIG.22A) or tumor (FIG.22B) retention, in an exemplary mouse xenograft model, shown as %ID / g, from 25-32 hours post-injection of an111In-labeled exemplary Nectin-4-targeting Scaffold A miniprotein conjugate (111In-C67) alone / without a decoy or co-administered with a 1,000-fold molar excess of an exemplary Scaffold A decoy (C79). FIG.22A is a line graph showing %ID / g in kidney of an exemplary Nectin-4-targeting Scaffold A miniprotein conjugate (111In- C67) alone / without a decoy (solid circles) or in combination with an exemplary Scaffold A decoy (solid squares). FIG.22B is a line graph showing %ID / g in tumor of an exemplary Nectin-4-targeting Scaffold A miniprotein conjugate (111In-C67) alone / without a decoy (solid circles) or in combination with an exemplary Scaffold A decoy (solid squares).

[0125] FIGS.23A and 23B are graphs depicting analyses of in vivo tumor volume (mm3; FIG.23A) and bodyweight (% of initial weight; FIG.23B) measurements in an exemplary mouse xenograft model between days 0 and 30 of treatment with: (i) a non-binding, non- radiolabeled control vehicle (solid circles); (ii) an exemplary Scaffold A decoy (C78; 1 mg; 1000X, solid triangles); (iii) an225Ac-labeled exemplary Nectin-4-targeting Scaffold A radionuclide conjugate (225Ac-C116; 1 ^g; 1,000 nCi; open circles); and (iv) a combination of the exemplary Scaffold A decoy (C78; 1000x; open triangles) and the exemplary Nectin-4- targeting Scaffold A radionuclide conjugate (225Ac-C116; 1,000 nCi). In FIG.23A, the dotted line at 2,000 mm3marks the maximum tumor growth threshold for pre-defined humane endpoint of experimental mice. In FIG.23B, the dotted line at 80% of initial body weight marks the body weight decrease threshold for pre-defined humane endpoint of experimental mice. Error bars represent standard error of the mean (SEM). Page 28 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0126] FIGS.24A, 24B, and 24C are graphs depicting results from analysis of SPECT / CT scans to quantify in vivo presence in kidney of a target-binding agent of a particular scaffold with and without administration of decoy in mice, shown as percent injected dose per gram (%ID / g) present 24 hours after injection of an111In-labeled exemplary miniprotein from Scaffolds A, B, C, J, or L alone or co-administered with 100-1000X molar excess of an exemplary Scaffold A decoy at 24 hours post-injection (x-axis). Error bars represent standard deviation (SD). FIG.24A shows an exemplary Scaffold A target-binding miniprotein (C67; SEQ ID NO: 13) radioconjugate, without (left side of graph, no decoy, open circle) and with (right side of graph) one of two exemplary decoys (C79, and C96; polypeptides of SEQ ID NOs: 16, 17, and 34, respectively). FIG.24B shows an exemplary Scaffold A target-binding miniprotein (C116; SEQ ID NO: 54) radioconjugate that has already been optimized for reduced kidney uptake without (left side of graph, no decoy, open circle) and with (right side of graph) one of three exemplary decoys (C78, C79, and C96; polypeptides of SEQ ID NOs: 16, 17, and 34, respectively). FIG.24C shows exemplary target-binding miniproteins from various different scaffolds (A, B, C, J, and L; A: C67; (SEQ ID NO: 13); B: C169 (SEQ ID NO: 114) C: C168 (SEQ ID NO: 113); J: adnectin-based binding protein; L: C156 (SEQ ID NO: 101) without (left side of graph, no decoy, open circle) and with (right side of graph) one of three exemplary decoys (C78, C79, and C96; polypeptides of SEQ ID NOs: 6, 17, and 34, respectively).

[0127] FIGs.25A-25H is a bar graph showing results from analysis of SPECT / CT scans to quantify in vivo kidney uptake and retention of an exemplary target-binding protein with or without decoy, shown as percent injected dose per gram (%ID / g) present 24 hours after injection of an exemplary labeled target-binding protein alone or co-administered with 20X molar excess of an exemplary Scaffold A decoy (C78; SEQ ID NO: 16). The x-axis represents the target-binding protein with or without decoy and the y-axis shows uptake in kidney, measured as (%ID / g), normalized to 100% for each no decoy condition. Error bars represent standard deviation (SD). The exemplary target-binding protein in each condition is111In-labeled, as follows: FIG.25A: Scaffold A, C133 (SEQ ID NO: 68); FIG.25B: Scaffold B, C141 (SEQ ID NO: 87); FIG.25C: Scaffold C, C168 (SEQ ID NO: 113); FIG.25D: Scaffold L, C145 (SEQ ID NO: 90); FIG.25E: Scaffold J; FIG.25F: C143; FIG.25G: C170;; and FIG.25H: C144. Page 29 of 248 IPTS / 128939095.1Docket No.: AKT-033WO DETAILED DESCRIPTION

[0128] Among other things, the present disclosure provides compositions and methods of use thereof. In some embodiments, a composition selectively binds to a target. In some embodiments, the target is on a tumor cell. In some such embodiments, the tumor cell is part of a population of tumor cells (e.g., a solid tumor). In some embodiments, the tumor cells are circulating (e.g., a hematologic cancer, circulating tumor cells, etc.). In some embodiments the composition comprises one or more therapeutic agents (e.g., a chelator, a radionuclide), wherein the therapeutic agent is selectively targeted to a cell, e.g., expressing any one of the targets in TABLE 10, such that the target-expressing cell is treated and cells not expressing the target are not treated. In various embodiments, the target is any one of the targets in TABLE 10. In certain embodiments, the target is Nectin-4. In certain embodiments, the target is B7-H3. The present disclosure recognizes that a source of a problem in treating cells expressing a target (e.g., cancer cells) is that traditional therapies are not selective enough to specifically target cells such as tumor cells and to deliver a therapeutic in a way that minimizes damage to surrounding cells such as in non-tumor tissue. Surrounding cells (e.g., as in one or more non-tumor tissues) may also express the target at lower amounts or levels than target cells. The disclosure provides the insight that a combination of selective targeting with a specific therapeutic such as a chelator and / or radionuclide (e.g., an alpha emitter) provides an advantage over previously used therapeutics (e.g., antibodies, beta-emitters, etc.)

[0129] Furthermore, the disclosure provides the insight that even a therapeutic such as those disclosed herein is designed to be more specific for a target and / or a tissue such as a tumor tissue), challenges can still arise. When a target is expressed by a non-tumor cell and / or when a therapeutic (e.g., a radiotherapeutic) is taken up by an organ system, such as involved in clearance of systemically administered agents (e.g., kidney, e.g., liver), efficacy can decrease and toxicity can increase. The disclosure contemplates that uptake to a tumor may be challenged by uptake, retention, and / or clearance by one or more non-target (e.g., non-tumor) tissues. For example, liver and kidney are two tissues that can play a role in clearance of administered therapeutics. In addition, such therapeutics can be taken up and / or retained in liver or kidney. For example, uptake of a therapeutic intended for a tumor can also be taken up, retained, and / or cleared by the kidney resulting in (1) faster clearance from a subject to whom it has been administered; (2) reduced tumor targeting (including because therapeutic is taken up and / or retained in a non-target tissue), and / or cleared; and / or (3) non- target tissue (e.g., kidney, liver, etc. damage). The disclosure recognizes that any or all of these challenges may be mitigated or prevented by combining administration of a therapeutic Page 30 of 248 IPTS / 128939095.1Docket No.: AKT-033WO with administration of a decoy. In some embodiments, the decoy reduces or prevents uptake by a non-tumor tissue (e.g., kidney, e.g., liver) of a composition comprising a polypeptide as provided herein (e.g., a radionuclide conjugate). Without wishing to be bound by theory, the disclosure contemplates that improvement in treatment efficacy is at least maintained while reducing damage to one or more non-tumor tissues (e.g., kidney, e.g., liver) and, in some embodiments, treatment efficacy is improved while simultaneously reducing risk of harm or actual harm to non-tumor tissue (e.g., kidney tissue and / or renal system tissues such as ureters, bladder, etc., liver tissue and / or other hepatic system components, etc.).

[0130] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0131] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Wittrup and VanAntwerp, Fine Affinity Discrimination by Yeast Surface Display and Flow Cytometry, Biotechnol. Prog. 2002, (16) 31-37; C. Queen et al., A humanized antibody that binds to the interleukin 2 receptor, Proc. Natl. Acad. Sci. USA 1989, 86 (24) 10029-10033; Scheinberg DA and McDevitt MR. Actinium-225 in targeted alpha-particle therapeutic applications. Curr Radiopharm.2011;4(4):306-320.

[0132] All publications, patents, and other references mentioned herein are hereby incorporated by reference in their entireties. In case of conflict, the present specification, including definitions, will control. Materials, methods, and examples as disclosed herein are illustrative only and not intended to be limiting. Page 31 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0133] Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0134] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 100 nucleotides” can mean “about 100 nucleotides” and also “100 nucleotides.” As used herein “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. Given context, the term “about” as used herein also includes an amount that would be expected to be within experimental error. For example, where the use of the term “about” is before a quantitative value, the disclosure also contemplates inclusion of the specific quantitative value itself, unless specifically stated otherwise. Ranges can be expressed in this disclosure as from “about” one particular value, and / or to “about” another particular value. When values are expressed as approximations by use of the antecedent “about,” it is understood that the disclosure also contemplates embodiments that specify the particular values and ranges of values without the approximations.

[0135] As used herein, the singular forms “a,” “an” and “the” include plural referents unless context clearly dictates otherwise. Thus, for example, in some embodiments, reference to, e.g., decoys includes a plurality of decoys, a single decoy, etc.

[0061] As used herein, the expression “and / or” in connection with two or more recited objects includes individually each of the recited objects and the various combinations of two or more of the recited objects, unless otherwise understood from the context and use.

[0136] Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:”, “nucleic acid comprising SEQ ID NO: 1” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 1. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.

[0137] As used herein, the term “administration” refers providing a composition to a subject or system. Administration to a subject may be by any appropriate route, dose and / or dose schedule.

[0138] As used herein, the term “affibody” refers to a subgenus of miniproteins. An affibody is derived from the Z-domain of staphylococcal protein A that consists of three alpha helices with 58 amino acids and has a molar mass of about 6 kDa. See, for exemplary details of affibody structures and uses, Orlova, A; Magnusson, M; Eriksson, T L; Nilsson, M; Page 32 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Larsson, B; Höidén-Guthenberg, I; Widström, C; Carlsson, J et al. (2006). “Tumor imaging using a picomolar affinity HER2 binding affibody molecule”, Cancer Res.66 (8): 4339-48. Exemplary Affibody® Molecules are commercially available from Abcam Corp. Cambridge Mass. An affibody is stable at high temperatures and under acidic or alkaline conditions. Target specificity is obtained by randomization of 13 amino acids located in two alpha- helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol.899:103-26).

[0139] As used herein, the term “affinity maturation” generally refers to a process whereby successive changes to a sequence (e.g., successive mutations) are made and selection of the polypeptide sequences are performed to choose one or more sequences with increased affinity relative to the “starting” sequence or another sequence with less affinity as compared to one with greater affinity.

[0140] As used herein, the term “anticalin” refers to a subgenus of miniproteins. An anticalin is an engineered protein derived from a lipocalin (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci U S A.96(5): 1898-903; Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Anticalins possess an eight-stranded b-barrel which forms a highly conserved core unit among the lipocalins and naturally forms binding sites for ligands by means of four structurally variable loops at the open end. Anticalins, although not homologous to the IgG superfamily, show features that so far have been considered typical for the binding sites of antibodies: (i) high structural plasticity as a consequence of sequence variation and (ii) elevated conformational flexibility, allowing induced fit to targets with differing shape.

[0141] As used herein, a “compound” refers at least a polypeptide having an amino acid sequence. Compounds may include miniproteins with one or more additional elements (e.g., conjugated to a chelator, such as by a linker, e.g., radiolabeled, etc.). Polypeptides of compounds can also have different modifications, such as N-terminal modification and / or C- terminal modifications. In various embodiments, a “compound” can include a miniprotein and one or more additional elements, examples of which include a linker, a chelator, and / or a radionuclide. For example, a compound may include a miniprotein conjugated to a chelator and / or a radionuclide e.g., via a linker. As denoted herein, compounds are identified with a specific compound number e.g., “C1,” “C2,” C3”, etc. Different compounds may have different sequences. In various embodiments, different compounds may have the same sequence (e.g., assigned the same SEQ ID NO), but may have one or more of different modifications (e.g., different N-terminal or C-terminal modifications), different linkers, Page 33 of 248 IPTS / 128939095.1Docket No.: AKT-033WO different chelators, and / or different radionuclides. A compound may also include or be a decoy.

[0142] As used herein, the term “attenuate” as used herein generally refers to a functional outcome due to a change, such as a structural change including but not limited to a deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, which reduces or inhibits production of the gene product, or renders the gene product non-functional. In some instances, a functional deletion is described as a knockout mutation. Attenuation also includes amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant (non- pathway specific feedback) is lessened such that the enzyme activity is not impacted by the presence of a compound. In other instances, an enzyme that has been altered to be less active can be referred to as attenuated.

[0143] As used herein, the term “avimer” refers to a subgenus of miniproteins. An avimer is a class of antibody mimetics which consist of two or more peptide sequences of preferably 30 to 35 amino acids each, which are derived from A-domains of various membrane receptors and which are connected by linker peptides. Binding of targets occurs via the A- domain and domains with the desired binding specificity can be selected, for example, by phage display techniques. The binding specificity of the different A-domains contained in an avimer may, but does not have to be identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68). For further details see Nature Biotechnology 23(12), 1556 — 1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

[0144] As used herein, the term “binder” refers to a subgenus of miniprotein. A binder is characterized in that is capable of binding or has known ability to engage and associate a target or a portion thereof. Binders generally comprise a cysteine-containing amino acid sequence and one or more disulfide bonds between cysteine residues, though some binders do not comprise cysteine-residues and / or disulfide bonds. Binders are preferably cleared rapidly from circulation when administered systemically to a mammalian subject. As will be understood, given context, reference to a binder may be or include its nucleic acid sequence or amino acid sequence encoding it. A binder may be provided, for instance, as protein, a polynucleotide, a vector, host cell, etc., and / or any combination of modalities. A binder may Page 34 of 248 IPTS / 128939095.1Docket No.: AKT-033WO be derived or manufactured using any method known to those of skill in the art. For instance, in some embodiments, a binder can be recombinant (i.e., produced using recombinant nucleic acids encoding a polypeptide). In some embodiments, a binder can be synthetic (e.g., synthesized such as using standard solid phase synthesis methods, such as solid phase peptide synthesis, known to those of skill in the art (see, e.g., Palomo, J. RSC Adv., 2014,4, 32658- 32672)) and described herein.

[0145] As used herein, the term “block” refers to preventing, slowing, suppressing, or otherwise reducing or decreasing uptake and / or retention of a compound into a tissue (e.g., a non-tumor tissue, e.g., a kidney or liver tissue). In some embodiments, a decoy blocks, suppresses, reduces, or otherwise decreases uptake of a conjugate or compound of the disclosure into a non-tumor tissue, such as kidney or liver tissue. In some embodiments, a decoy reduces retention of a compound (e.g., a radiotherapeutic compound, e.g., comprising a miniprotein, e.g., comprising a polypeptide) in a non-tumor tissue (e.g., kidney, e.g., liver, etc.).

[0146] As used herein, the term “chelator” refers to any moiety or entity capable of forming a complex (i.e., “chelates”) with a metal ion. Chelators generally have two or more unshared electron pairs that can be used to donate to a metal ion. Metal ions are usually coordinated to the chelator by two or more pairs of electrons.

[0147] As used herein, the term “conjugate” or “conjugated” refers to the joining by covalent or noncovalent means of two components. In some embodiments, a “conjugate” may refer to, for example, a polypeptide coupled to one or more of, e.g., a linker, chelator, and / or radionuclide.

[0148] As used herein, a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (herein incorporated by reference). The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); Page 35 of 248 IPTS / 128939095.1Docket No.: AKT-033WO 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0149] As used herein, the terms “cysteine-dense peptide” and “CDP” are used interchangeably and refer to a subgenus of miniproteins that generally comprise at least two independent folding domains and a high density of cysteines. For example, in some embodiments, the CDP comprises at least one, two, three, four, or more cysteine residues in a span of from about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues (see, e.g., Correnti et al. Nat. Struct. Mol. Biol.2018 Mar; 25(3): 270-78). In some embodiments, the CDP comprises a constrained distribution of cysteines, Cys-X[0–15]-Cys- X[0–15]-Cys-X[0–15]-Cys-X[0–15]-Cys-X[0–15]-Cys (wherein X represents any amino acid) (SEQ ID NO: 81).

[0150] As used herein, the term “deletion” generally refers to the removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, the regions on either side being joined together (e.g., by a bond).

[0151] As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term “degenerate oligonucleotide” or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.

[0152] As used herein, the term “derived from,” with reference to a nucleic acid sequence refers to a nucleic acid sequence that has at least 85% sequence identity to a reference naturally occurring nucleic acid sequence from which it is derived. The term “derived from,” with reference to an amino acid sequence refers to an amino acid sequence that has at least 85% sequence identity to a reference naturally occurring amino acid sequence from which it is derived. The term “derived from” as used herein does not denote any specific process or method for obtaining the nucleic acid or amino acid sequence. For example, the nucleic acid or amino acid sequence can be chemically synthesized.

[0153] As used herein, the term “designed ankyrin repeat domain (DARPin)” refers to a subgenus of miniproteins. A DARPin is a peptide derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is preferably a 33 residue motif consisting of two alpha-helices and a beta- turn. They can be engineered to bind different targets by randomizing residues in the first Page 36 of 248 IPTS / 128939095.1Docket No.: AKT-033WO alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see, e.g., J. Mol. Biol.332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1. DARPins typically provide a rigid interface and lack structural flexibility (Gebauer and Skerra, 2009).

[0154] As used herein, the term “domain” refers to a structure of a protein (e.g., a miniprotein, e.g., a polypeptide) that contributes to one or more structural features and / or one or more known or suspected function of the protein. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a protein. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Domains may also refer to secondary structural features, such as, in a protein, an alpha helix or beta sheet (e.g., alpha helical domain, beta sheet domain, etc.).

[0155] As used herein, the term “engineered Kunitz domain” refers to a subgenus of miniproteins. An engineered Kunitz domain is preferably a peptide derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI). Kunitz domains have a molecular weight of approximately 6 kDA and domains with the required target specificity can be selected by display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0156] As used herein, the term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Page 37 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0157] As used herein, the term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to one or more heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the present disclosure have particular utility. The heterologous polypeptide included within the fusion protein of the present disclosure is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein (“GFP”) chromophore-containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.

[0158] As used herein, when referring to a protein, “homology” to a second protein can exist if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences.) Homology between two regions of amino acid sequences (especially with respect to predicted structural similarities) can be interpreted as implying similarity in function. Homologous proteins or peptides with residue positions that are not identical are often recognized to differ by conservative amino acid substitutions.

[0159] As used herein the term “identical” refers to a nucleic acid sequence or amino acid sequence refers to at least two nucleic acid or at least two amino acid sequences or subsequences that have a specified percentage of nucleotides or amino acids, respectively, that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. A length of sequence identity comparison may be over a stretch of any number of nucleotides or amino acids. When using a sequence comparison algorithm, test and reference sequences are Page 38 of 248 IPTS / 128939095.1Docket No.: AKT-033WO input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. A number of algorithms are known in the art. Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in, e.g., Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res.25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. Additionally or alternatively sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (hereby incorporated by reference in its entirety). For instance, percent sequence identity can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

[0160] As used herein, the term “isolated” polynucleotide or polypeptide is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated. For instance, an isolated molecule is one that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polynucleotide or polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polynucleotide or polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polynucleotide or polypeptide may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, “isolated” does not necessarily require that any molecule so described has been physically removed from its Page 39 of 248 IPTS / 128939095.1Docket No.: AKT-033WO native environment. In some embodiments, as used in reference to an isolated construct, isolated means in the absence of a pharmaceutically acceptable salt.

[0161] As used herein, the term "KD" or "Kd" refers to the dissociation equilibrium constant for a particular entity and a target (e.g., antibody-antigen (or, e.g., targeting miniprotein-target protein), for example, a particular interaction between an entity and its target (e.g., a polypeptide-target interaction, e.g., a polypeptide as provided herein and B7- H3). Typically, the polypeptide (e.g., targeting protein, e.g., miniprotein) of the present disclosure binds to any one of the target proteins in TABLE 10 with a dissociation equilibrium constant (KD) of less than about 10-7M, such as less than about 10-8M, 10-9M or 10-10M or less, for example, as determined using surface plasmon resonance (SPR) techniques in a BIACORE instrument. Decoys of the disclosure may bind to a target protein (e.g., as in TABLE 6 or TABLE 10) with an affinity of about 10-6M, 10-5M, 10-4M, 10-3M, or greater (e.g., 10-2M, etc.), or may have binding that is not detectable using measures including those provided herein. KD= kd / ka.

[0162] As used herein, the term “Ki” (M) refers to the binding inhibition constant of a given entity and a target (e.g., a particular polypeptide-target interaction).

[0163] As used herein, the term “kd” (s-1) refers to the dissociation rate constant between a given entity and a target (e.g., of a particular polypeptide-target interaction). This value is also referred to as the koffvalue.

[0164] As used herein, the term “ka” (M-1×s-1) refers to the association rate constant of a given entity and a target (e.g., a particular polypeptide-target interaction). This value is also referred to as the kon value.

[0165] As used herein, the term “KA” (M-1) refers to the association equilibrium constant of a given entity and a target (e.g., a particular polypeptide-target interaction). KA = ka / kd.

[0166] The affinity of a molecule X for its target Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are as described above. For clarity, as known in the art, a smaller KDvalue indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).

[0167] As used herein, the term “knock out” generally refers to a gene whose level of expression or activity has been reduced to zero. In some examples, a gene is knocked out via deletion of some or all of its coding sequence. In other examples, a gene is knocked out via Page 40 of 248 IPTS / 128939095.1Docket No.: AKT-033WO introduction of one or more nucleotides into its open reading frame, which results in translation of a nonsense or otherwise nonfunctional protein product.

[0168] As used herein, the term “knottin” refers to a structural motif of a miniprotein containing three disulfide bridges.

[0169] As used herein, the term “knottin peptide” refers to a subgenus of miniproteins that comprises at least one knottin.

[0170] As used herein, the term “linker” refers to a moiety that is used to conjugate a polypeptide to another entity, such as a chelator.

[0171] As used herein, the term “modification,” with reference to a nucleic acid sequence, refers to a nucleic acid sequence that comprises at least one substitution, alteration, inversion, addition, or deletion of nucleotide compared to a reference nucleic acid sequence. As used herein, the term “modification,” with reference to an amino acid sequence refers to an amino acid sequence that comprises at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference amino acid sequence.

[0172] As used herein, the term “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as 125I, 32P, 35S, and 3H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002).

[0173] As used herein, the term “molecule” means any entity including, but not limited to, a polypeptide, miniprotein, compound, small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., natural or synthetic.

[0174] As used herein, the term “monobody” or “Adnectin” are used interchangeably and refer to a subgenus of miniproteins. A monobody is preferably based on the 10th extracellular Page 41 of 248 IPTS / 128939095.1Docket No.: AKT-033WO domain of human fibronectin III (10Fn3), which adopts an Ig-like b-sandwich fold of preferably 94 residues with 2 to 3 exposed loops, but lacks the central disulfide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectins with the desired target specificity can be genetically engineered by introducing modifications in solvent-exposed positions in the protein.

[0175] As used herein, the term “mutein” or “mutant protein” or “variant” means a protein comprising an amino acid sequence with at least one variation (e.g., an insertion, a deletion, or a substitution, which can be a conservative or non-conservative substitution) compared to a reference sequence. When applied to sequences (e.g., nucleic acid sequences, amino acid sequences) “mutated” means that nucleotides in a nucleic acid sequence or amino acids in an amino acid sequences may be inserted, deleted or changed compared to a reference sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides or amino acids may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid or amino acid sequence. A nucleic acid or amino acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as “error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic.2:28-33 (1992)); “oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)); directed evolution (e.g., exposing a polypeptide to differing sets of conditions resulting in production of different polypeptides with one or more amino acid changes that may or may not confer greater fitness upon the polypeptide); and site-directed mutagenesis (e.g., specifically directed changes in a sequence). For example, a “polypeptide mutant” or “polypeptide mutein” can refer to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a reference protein (e.g., a native protein, e.g., a wild-type protein, e.g., a reference protein of a different starting sequence than the “mutein”). A polypeptide mutant or polypeptide mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and / or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and / or truncations of the amino acid Page 42 of 248 IPTS / 128939095.1Docket No.: AKT-033WO sequence at either or both the amino or carboxy termini. A polypeptide mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein. A polypeptide mutein can have at least 85% overall sequence homology to its wild- type counterpart. Even more preferred are muteins having at least 90% overall sequence homology to the wild-type protein. In an even more preferred embodiment, a mutein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99% and even more preferably 99.9% overall sequence identity. Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit. Amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and / or (5) confer or modify other physicochemical or functional properties of the polypeptide mutant as compared to a reference polypeptide.

[0176] As used herein, the term “non-disulfide sequence” refers to an amino acid sequence encoding a polypeptide that does not comprise more than one cysteine residue and / or disulfide bonds in its folded and active form. For example, in some embodiments, a polypeptide may be a miniprotein comprising or consisting of a non-disulfide sequence.

[0177] As used herein, the term “non-peptide analog” refers to a compound with properties that are analogous to those of a reference polypeptide. A non-peptide compound may also be termed a “peptide mimetic” or a “peptidomimetic.” See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry--A Practical Textbook, Springer Verlag (1993); Synthetic Peptides: A Users Guide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med. Chem.30:1229 (1987); Fauchere, J. Adv. Drug Res.15:29 (1986); Veber and Freidinger, Trends Neurosci., 8:392-396 (1985); and references cited in each of the above. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the present disclosure may be used to produce an equivalent effect and are therefore envisioned to be part of the present disclosure.

[0178] As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably to refer to a polymer of nucleotides. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, Page 43 of 248 IPTS / 128939095.1Docket No.: AKT-033WO quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation. The nucleic acid sequence can contain natural, non-natural, or altered nucleotides; and contain a natural, non-natural, or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid sequence. Nucleic acid sequences include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means. Polynucleotides of the present disclosure may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in “locked” nucleic acids.

[0179] As used herein, the terms “operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

[0180] As used herein, the term “polypeptide fragment” as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and / or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least Page 44 of 248 IPTS / 128939095.1Docket No.: AKT-033WO 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

[0181] As used herein, the term “radionuclide” as used herein refers to an atom capable of undergoing radioactive decay.

[0182] As used herein, the term “radiotherapeutic” refers to a radionuclide-labeled protein (e.g., polypeptide, miniprotein, compound, etc.) as provided herein. A radiotherapeutic may be administered to a subject, such as a test subject (e.g., a mouse or rat, e.g., a non-human primate, e.g., a healthy volunteer), and / or a subject in need of radiotherapy, e.g., a subject with a cancer. In some embodiments, radiolabels are only added to proteins (e.g., polypeptides, miniproteins, compounds) intended for binding to a tumor.

[0183] As used herein, the term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, and / or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it. A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” Page 45 of 248 IPTS / 128939095.1Docket No.: AKT-033WO also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0184] As used herein, the term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.

[0185] As used herein, the term “region” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.

[0186] As used herein, “sequence homology” for polypeptides, also referred to as “percent sequence identity,” is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. A preferred algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet.3:266-272 (1993); Madden et al., Meth. Enzymol.266:131-141 (1996); Altschul et al., Nucleic Acids Res.25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res.25:3389- 3402 (1997)). Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOSUM62. The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more Page 46 of 248 IPTS / 128939095.1Docket No.: AKT-033WO usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol.183:63-98 (1990) (incorporated by reference herein). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.

[0187] As used herein, the term “specificity” generally refers to a protein (e.g., a miniprotein, e.g., a miniprotein having certain amino acids) that, when in a conformation that can bind, selectively or “specifically” binds to a given target (e.g., an antigen, such as expressed on a tumor, e.g., such as set forth in TABLE 10, e.g., certain cell types, e.g., kidney cells, e.g., kidney proximal tubule cells, etc.). As used herein, “specifically binds” means that the binding of a polynucleotide, polypeptide, or protein is selective for a specified target and can be discriminated from unwanted or non-specific interactions. For example, the ability of a protein (e.g., cysteine-dense peptides) to bind to a specific antigenic determinant can be measured techniques familiar to one of skill in the art, for example through an enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance. Between two entities (e.g., miniproteins, e.g., polypeptides, etc.), “specific binding” refers to the ability of two entities preferentially bind to each other as compared to binding to other entities in the environment. Typically, “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold, or even 1,000-fold. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10-7 M or stronger (e.g., about 10-8 M, 10-9 M or even stronger). Specific-binding requires specificity of a particular first entity (e.g., a polypeptide) for a particular second entity (e.g., an antigen binding sequence).

[0188] As used herein, “stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base Page 47 of 248 IPTS / 128939095.1Docket No.: AKT-033WO mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization. In general, “stringent hybridization” is performed at about 25°C below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5°C lower than the Tm for the specific DNA hybrid under a particular set of conditions. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference. For purposes herein, “stringent conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6xSSC (where 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65°C for 8-12 hours, followed by two washes in 0.2xSSC, 0.1% SDS at 65ºC for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65°C will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.

[0189] As used herein, the term “synthetic” is used to refer to an entity that is made is lab- created and not naturally produced or isolated, without modification, from a naturally occurring source. A recombinant polymer, such as a recombinant polynucleotide or polypeptide, may be synthetic. Synthetic polymers such as polynucleotides or polypeptides may be produced by any method known to those of skill in the art, including but not limited to solid phase synthesis, solution phase synthesis, biological synthesis by, e.g., host cells, etc.

[0190] As used herein, the term “subject” is a mammal. A subject may be a human or non-human mammal. Given context, a subject may be used interchangeably with patient, individual, donor, etc. In some embodiments, a subject is a healthy subject without a disease that is contemplated for treatment by a composition of the disclosure (e.g., a healthy volunteer being administered one or more compositions provided herein). In some embodiments, a subject is one suspected or diagnosed as having a disease, disorder, or condition, such as a cancer and / or tumor, as provided herein. In some such embodiments, such a subject is considered for treatment by a composition of the disclosure. In some embodiments, analyses of results achieved with technologies disclosed herein is evaluated in a population comprising a plurality of subjects.

[0191] As used herein, the terms “substantial homology” or “substantial similarity,” when referring to a polynucleotide or polypeptide, indicate that, when optimally aligned with appropriate nucleotide or amino acid insertions or deletions with another reference entity Page 48 of 248 IPTS / 128939095.1Docket No.: AKT-033WO (e.g., polypeptide, polynucleotide, etc. or complementary strand thereof when appropriate), there is sequence identity in at least about 70%, 75%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% or more of the nucleic acid or amino acid residues, as measured by any well-known algorithm of sequence identity, such as, e.g., FASTA, BLAST, Gap, etc.. Alternatively or additionally, substantial homology or similarity exists when, for example, a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions.

[0192] As used herein, the term “target” refers to a protein or functional portion or variant thereof to which another protein (e.g., a miniprotein) is designed to bind. A target may be or comprise a binding region, such as an epitope, to which a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) of the present disclosure binds. Further, the term “antigen” refers to a protein or functional portion or variant thereof to which a polypeptide (e.g., a miniprotein, etc.) or variant thereof binds to. A target may be or comprise an antigen. A target may be expressed on the surface of a particular cell (a “target cell”) or expressed within (e.g., on the surfaces of) cells in a population of cells (e.g., a tumor). In certain embodiments, a target will be targeted by a “target-binding” agent (e.g., a polypeptide, e.g., a miniprotein, a small molecule, etc.), wherein the target is selected from a protein in TABLE 10 such as, e.g., Nectin-4, e.g., B7-H3, and wherein the agent binds to the target, but is not necessarily a miniprotein. Rather, in certain embodiments, a target-binding agent is one that binds to a target and can include, without limitation, a polypeptide including, for example, a miniprotein or an adnectin, or binding agent and / or ligand for a target (including polypeptide and small molecule-based ligands) such as, for example, zelenectide pevedotin, vipivotide textrazetan, dotatate (e.g., including labeled, such as radiolabeled versions). In certain embodiments, a target will be targeted by a “target-binding” polypeptide (e.g., miniprotein), wherein the target is selected from a protein in TABLE 10 such as, e.g., Nectin-4, e.g., B7-H3. A target can have a certain percent identity to a reference protein and still be referred to as a target by a particular name (e.g., any one of the target proteins in TABLE 10). In certain embodiments that will be clear from context, a target may also refer to a protein in a pathway related to another protein. For example, if a target is any one of the target proteins in TABLE 10, a target may also be a protein in a pathway that is necessary for activity of the target protein selected from TABLE 10. A target can also be or include a particular cell type (or be localized to a particular cell type) characterized by expression of particular surface entities Page 49 of 248 IPTS / 128939095.1Docket No.: AKT-033WO such as receptors (e.g., a cell in a tissue, e.g., a proximal tubule cell in a kidney). Such targets may be different or the same as a target to which a target-binding miniprotein is designed to bind (e.g., Nectin-4, e.g., B7-H3). A “target-binding agent” is an agent that has at least one component (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) that binds to a target. Target-binding agent is understood to encompass target-binding proteins, including polypeptides (e.g., miniproteins) and small- molecule binding moieties such as those that bind to a receptor on a cell, e.g., on a cancer cell, such as for use in localizing a therapeutic (e.g., comprising a radionuclide) to a cancer cell.

[0193] As used herein, the term “decoy target” can refer to a non-tumor cell or entity thereon that takes up a decoy. For example, a decoy target can be a kidney tissue, a liver tissue, a kidney cell or population of kidney cells, a liver cell or population of liver cells, etc. In some embodiments, decoy target does not comprise a cell expressing a target selected from TABLE 10, e.g., Nectin-4, e.g., B7-H3.

[0194] As used herein, the term “treatment” (as well as “treat” or “treating”) refers to partial or complete alleviation, amelioration, mitigation, reduction in risk of onset, relief, inhibition, delay in onset of, reduction in severity of, reduction in frequency or incidence of one or more causes, features, and / or symptoms of or associated with a particular disease, disorder, and / or condition.

[0195] As used herein, the term “prevention” refers to prophylaxis of one or more causes, features, and / or symptoms of or associated with a particular disease, disorder, and / or condition, including, for example, in a patient who has previously been treated and is experiencing remission of one or more causes, features, and / or symptoms of or associated with a particular disease, disorder, and / or condition (e.g., no evidence of disease, reduced symptoms and / or features as compared to prior to treatment, etc.)

[0196] As used herein, the term “prevention” (as well as “prevent” or “preventing”) refers to the partial or complete inhibiting or delaying the onset of one or more causes, features, and / or symptoms of or associated with a particular disease, disorder, and / or condition.

[0197] As used herein, the term “vector” as used herein is intended to refer to a polynucleotide capable of transporting another polynucleotide to which it has been linked. One type of vector is a “plasmid,” which generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double- stranded polynucleotides such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other Page 50 of 248 IPTS / 128939095.1Docket No.: AKT-033WO vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).

[0198] As used herein, the term “decharged” refers to an entity (e.g., a molecule such as a polypeptide, e.g., a miniprotein ) that has been modified to contain fewer positively charged or polar features, greater negatively charged features, and / or both.

[0199] As used herein, the term “surface charge” refers to the electrostatic charge present at the surface of a protein (e.g., such as a polypeptide or miniprotein). For example, in certain embodiments, the surface charge of a miniprotein can influence kidney uptake of the miniprotein (e.g., increase kidney uptake of the miniprotein or reduce kidney uptake of the miniprotein).

[0200] As used herein, the term “surface patch” refers to regions on a surface of a protein (e.g., a miniprotein) with surface characterizations. For example, a surface patch can be defined according to surface charges and / or surface hydrophobicities, that may influence kidney uptake of the protein (e.g., miniprotein).

[0201] As used herein, the term “cleavable linker” refers to a linker that can be cleaved. A cleavable linker contains a cleavable bond that is cleaved in vivo, for example: by an acidic pH (pH less than 7, typically about 4 to 6), by glutathione, or where there is up-regulation of enzymes such as matrix proteases or peptidases from the proximal tubule. Non-limiting examples of cleavable linkers are linkers that contain hydrazine, or disulfide bonds, or enzymatically cleavable peptide sequences.

[0202] As used herein, the term “polypeptide” refers to a polymer of amino acids, wherein amino acids within the polymer are connected to one another by amide bonds. A polypeptide of the present disclosure encompasses both naturally-occurring and non- naturally-occurring amino acids, and any fragments, portions, peptides, mutants, derivatives, and analogs thereof. In some embodiments, a polypeptide can be monomeric. In some embodiments, a polypeptide can be multimeric (e.g., exhibit varying quaternary structure, Page 51 of 248 IPTS / 128939095.1Docket No.: AKT-033WO comprise two or more miniproteins connected through covalent bonds or non-covalent interactions). Further, a polypeptide may comprise a number of structural characteristics and / or domains (e.g., alpha helix, e.g., beta sheet, etc., e.g., bonds or constraints such as, for example, disulfide bridges). Domains can each, dependently or independently, have one or more distinct characteristics associated with its presence (e.g., target binding, structural support, etc.). A polypeptide of the disclosure can be designed to bind to a certain target (e.g., a target-binding polypeptide that can be part of a composition comprising a miniprotein provided herein, e.g., a tumor-associated antigen such as can be expressed on a surface of a tumor cell). A polypeptide may be fully or partially synthetic or otherwise modified (e.g., comprising one or more synthetically-produced amino acids and / or modifications thereof).

[0203] As used herein, the term “miniprotein” refers to short proteins comprising a polypeptide of about 20 to about 100 amino acids and certain characteristics comprising one or more secondary structure elements (e.g., alpha helix, turn, beta sheet, other loop structures, etc.), a hydrophobic core, cooperative folding, and / or cross-linking bonds such as disulfides, lactams, etc. CDPs, knottins, affibodies, engineered Kunitz domains, monobodies (Adnectins), anticalins, designed ankyrin repeat domains (DARPins), avimers, and binders as disclosed herein are all examples of miniproteins. The term “target-binding polypeptide” (e.g., a target binding miniprotein, target-binding compound, etc.) can be used to refer to a polypeptide designed to bind to a particular target (e.g., such as a target provided in TABLE 10, e.g., Nectin-4, e.g., B7-H3), though one of ordinary skill in the art will understand that if a given polypeptide (e.g., miniprotein) is said to bind to a particular target, it does not have to be explicitly referred to as a target-binding miniprotein in order to be considered one.

[0204] As used herein, the term “scaffold” is used to describe miniproteins that share a general set of structural characteristics (e.g., certain constraints, secondary structures, tertiary structures, etc.). Any individual scaffold may include varying amounts of alpha helix, turn, and / or beta sheet, e.g., all alpha helix proteins, all beta sheet proteins, blended alpha helix / beta sheet proteins (“a / b”), blended alpha and beta proteins (“a+b”), and small proteins. Examples and features of certain scaffolds are provided herein. While scaffold is used to describe miniproteins, one of ordinary skill in the art, when comparing two agents (e.g., two target-binding agents, e.g., a target-binding agent and a decoy, e.g., two decoys, etc.) will understand that when any one such agent is not a miniprotein but is, for example, a protein that is larger or smaller than a miniprotein, a small molecule, a combination of protein and small molecule, etc., the two agents will be considered not to share a scaffold (also described as being of “different scaffolds” even if one of the agents is not a miniprotein with a Page 52 of 248 IPTS / 128939095.1Docket No.: AKT-033WO “scaffold” as defined herein). The phrases “sharing a scaffold” or “not sharing a scaffold” are unrelated to physical co-localization (e.g., of target-binding agents); rather, they each refer to presence or absence of a common scaffold as between two structures, when those two structures are compared. For example, a Scaffold B target-binding miniprotein does not share a scaffold with a Scaffold A decoy. A small molecule does not share a scaffold with a Scaffold B miniprotein, etc.

[0205] As used herein, the term “decoy” refers to a subgenus of miniproteins specifically designed to (i) decrease accumulation of a compound (e.g., a miniprotein, e.g., a radiolabeled miniprotein, e.g., a radiotherapeutic as provided herein) in a non-tumor tissue (e.g., kidney tissue or liver tissue when the tumor is elsewhere); and / or (ii) have substantially no impact to minimal impact on compound uptake by a tumor (e.g., one or more tumor cells, e.g., a population of tumor cells, e.g., a tumor with cells expressing a target, e.g., a target such as set forth in TABLE 10); and / or (iii) decrease adverse (e.g., toxic) accumulation in a non-tumor containing organ (e.g., liver, e.g., kidney, etc.) of a subject. To give but one example, an exemplary decoy may be combined with a composition of the disclosure (e.g., comprising a miniprotein and a radionuclide such as a radiotherapeutic) to block the composition from (non-tumor) kidney tissue, as compared to uptake and / or retention in (non-tumor) kidney tissue in the absence of the decoy. Without wishing to be bound by theory, the disclosure describes, in some embodiments, a decoy (or decoy miniprotein) that decoys a composition such as a radiotherapeutic (e.g., comprising a target-binding agent, e.g., a target-binding polypeptide (e.g., a miniprotein), a small molecule, etc.), which means that presence of the decoy in a non-tumor tissue blocks uptake and / or retention of the radiotherapeutic into the non-tumor tissue (e.g., kidney, e.g., liver). For clarity, when a decoy is referred to as “decoying” e.g., a composition, e.g., a compound, e.g., a target-binding agent (e.g., a polypeptide such as a miniprotein or a small molecule ligand, that binds to a target (e.g., such as in TABLE 10, e.g., Nectin-4, e.g., B7-H3)), the decoy, is not acting on the composition (or compound or miniprotein), rather, it is acting on its own and, for example, even in the absence of a composition that it is decoying, if administered alone, would still be present in the non-tumor tissue (e.g., kidney, e.g., liver). A decoy can also be referred to as a “decoy- targeting” miniprotein to distinguish between decoys and their targets, “decoy targets” (e.g., non-tumor tissue, e.g., kidney, e.g., liver) as compared to “target-binding” miniproteins, such as those that bind to targets (e.g., as in TABLE 10, e.g., Nectin-4, e.g., B7-H3) on a cancer cell, a population of cancer cells, a tumor, etc. Page 53 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Compositions

[0206] Provided herein are compositions comprising at least one of a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) and / or a polypeptide (e.g., decoy) or fragment thereof as provided herein. In certain embodiments, a composition comprises one or more of a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand), linker, chelator, and / or radionuclide. In certain embodiments, a composition comprises a decoy, which decoy does not comprise a chelator and / or radionuclide. In some embodiments, a composition comprises a linker and a chelator. In some such embodiments, the composition is metalated (e.g., with a cold-metal form of an elemental label, such as provided herein). In some embodiments, the composition is radiolabeled (e.g., with a radionuclide such as provided herein). In some embodiments, a composition comprises a linker, chelator, and radionuclide. In some embodiments, a composition comprises or consists of a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand), an optional linker, and a chelator and / or radionuclide. In some embodiments, a chelator and / or radionuclide are conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) via a linker. In various embodiments, compositions disclosed herein comprise a compound. In various embodiments, a compound as provided herein can include a miniprotein. In certain embodiments, the compound can further include one or more additional components, examples of which include a linker, a chelator, and / or a cold-metal label or radionuclide. In some embodiments, a composition comprises a polypeptide with one or more of an N-and / or C-terminal modification and / or one or more positions on the polypeptide backbone.

[0207] In some aspects, a composition of the disclosure is or comprises a compound. A compound of the disclosure can comprise a target-binding agent. In certain embodiments, the compound comprises a decoy. , such as disclosed in TABLE 3, TABLE 4C, and TABLE 4D. TABLE 3 contains compounds comprising miniproteins that are target-binding agents and / or decoys from various scaffolds (e.g., A, B, C, L). Miniproteins can be more specifically identified by comparisons to, for example, TABLE 4A (e.g., target-binding agents that bind to B7-H3), TABLE 4B, TABLE 4C, and TABLE 4D. which can be more specifically identified by comparison of target-binding agents as in TABLE 4A and TABLE 4B, TABLE 11, and TABLE 17, as well as FIGs.25A-25D and FIGs.25F-25H (miniprotein compositions of which are found in TABLE 3) and decoys as in TABLE 4C and TABLE Page 54 of 248 IPTS / 128939095.1Docket No.: AKT-033WO 4D (also found in TABLE 3). In certain embodiments, a compound of the disclosure can include a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) having an amino acid sequence comprising any of those disclosed in TABLE 3 (see column entitled “Sequence”) or as disclosed in TABLE 11 or TABLE 17.

[0208] In some embodiments, a polypeptide of the disclosure includes one or more additional components such as a linker, chelator, cold-metal form of an elemental label, radionuclide, etc. In certain embodiments, a polypeptide, miniprotein, or compound comprising such polypeptide (e.g., miniprotein) of TABLE 3 can be used in a decoy- (e.g., miniprotein, e.g., as set forth in TABLE 4C and / or TABLE 4D) or a target-binding polypeptide (e.g., a target-binding miniprotein, e.g., that binds to a target of TABLE 10, e.g., Nectin-4, e.g., B7-H3, e.g., as set forth in TABLE 4A and / or 4B).

[0209] In various embodiments, a compound includes a polypeptide (e.g., a miniprotein) comprising an amino acid sequence disclosed in TABLE 3. In some embodiments, the compound comprising the polypeptide further includes a linker such as disclosed in TABLE 3. In various embodiments, a compound includes a miniprotein comprising an amino acid sequence disclosed in TABLE 3 and further includes a radionuclide disclosed herein. In certain embodiments, a compound of the disclosure includes a miniprotein comprising an amino acid sequence disclosed in TABLE 3 and further includes a chelator as provided herein. In various embodiments, a compound includes a miniprotein comprising an amino acid sequence disclosed in TABLE 3 and further includes a linker as disclosed in TABLE 3, and a chelator provided herein. In various embodiments, a compound includes a miniprotein comprising a sequence disclosed in TABLE 3 and further includes a linker disclosed in TABLE 3, and a radionuclide disclosed herein. In various embodiments, a compound includes a miniprotein comprising a sequence disclosed in TABLE 3 and further includes a linker disclosed in TABLE 3, a chelator disclosed herein, and a radionuclide disclosed herein.

[0210] In certain embodiments, a target-binding agent comprises a compound or binding portion thereof as set forth in TABLE 11 or TABLE 17. It will be understood to those of skill in the art that such compounds, as disclosed, comprise certain additional features (e.g., linkers, chelators, etc.), but a target-binding agent comprising a portion that binds to a target can be modified with any suitable additional component (e.g., radionuclide). Accordingly, in certain embodiments, a target-binding agent consists essentially of any one of the target- Page 55 of 248 IPTS / 128939095.1Docket No.: AKT-033WO binding portions (e.g., the Nectin-4, PSMA, or SSTR2-binding portions) of zelenectide pevedotin, vipivotide textrazetan, and dotatate. TABLE 3. Exemplary Miniprotein Sequences and Structures Compound N- Cleavable Sequence SEQ ID C- # terminal Linker NO: termin al C1 DOTA-AEAK(Ac)YAK(Ac)EK(Ac)IAALSEIIWLPNL1 NH2ac co pou s e e a a co pou e.g., , , , ec. a ees o e co a o o e -e a, Linker (if present), Sequence, and C-terminal components.2This column refers to linkers that are optionally cleavable, distinct from, for example, the N-terminal modifications which can include entities such as a PEG moiety between the miniprotein and, e.g., a DOTA, etc. “me” refers methyl (e.g., mono-, di-, tri-methyllysine, etc.); “K(Ac)” and “Lys(Ac)” refer to acetylated lysine; “Cit” refers to citrulline; Refers to sequences in the “Sequence” column Page 56 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Compound N- Cleavable Sequence SEQ ID C- # terminal Linker NO: termin al C19 DOTA- D-Tyr-GF-β-Ala- CEYDEEFFTALKRLRGGDICYYIKKKFKKVPKLC 7 OHSEQ ID NO: 83 refers to “yGF-β-Ala” and SEQ ID NO: 86 refers to “Gly10” Page 57 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Compound N- Cleavable Sequence SEQ ID C- # terminal Linker NO: termin al C39 DOTA- MVK-PEG4 AEAKYAKEKIAALSEIIWLPNLTHGQI(norleu 6 NH2Page 58 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Compound N- Cleavable Sequence SEQ ID C- # terminal Linker NO: termin al C67 DOTA- CEYDEEFFTALKRLRGGDICYYIQASFQYLPGLC 13 OHPage 59 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Compound N- Cleavable Sequence SEQ ID C- # terminal Linker NO: termin al C95 Acetyl CEYKEEFFTELKRLRGGKICYYIKKKFKKVPKLC 33 NH2Page 60 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Compound N- Cleavable Sequence SEQ ID C- # terminal Linker NO: termin al C129 Biotin- CEYDEEFFTALLys(Ac)(Citrulline)L(Ci 66 NH2Page 61 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Compound N- Cleavable Sequence SEQ ID C- # terminal Linker NO: termin al C162 DOTA- CA(Kme3)EKIAALSEIIWLPCLTYAQI(Kme2) 107 NH2, es that compositions provided herein are more effective than previously described compositions (e.g., such as those comprising antibodies and / or beta-emitter radionuclides). In addition, the disclosure provides additional ways to improve such compositions, including by administration with a decoy. Surprisingly, target-binding agents of the disclosure can be improved even further when used with a decoy that decreases uptake and / or retention in non- target tissues (e.g., kidney or liver as compared to tumor tissue). The target-binding agents and the decoy may, but do not have to, be of the same scaffold. Also surprisingly, target- binding agents that have already been optimized to reduce uptake and / or retention (e.g., in kidney cells) can be decoyed using decoys of the disclosure. While miniproteins (e.g., to be used in compositions as provided herein, including, for example, target-binding miniproteins and decoys) have several key features of antibody-based therapeutics (e.g., affinity, potency, specificity, and ability to disrupt protein:protein interactions), they can avoid undesirable limitations such as, e.g., large size, expensive manufacturing, and the necessity of chimerization or humanization. For instance, in some embodiments, a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) of the present disclosure is no more than about 100 amino acids in length. In some embodiments, such a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) may be or comprise a cysteine dense peptide. In some embodiments, a miniprotein of the disclosure (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) comprises one or more disulfide bridges. In some embodiments, a miniprotein of the disclosure (e.g., Page 62 of 248 IPTS / 128939095.1Docket No.: AKT-033WO CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) comprises multiple cysteine residues that crosslink to maintain a very stable, folded state for a peptide of its length (e.g., relative to a peptide of the same length without as many cysteine residues). Without wishing to be bound by theory, the disclosure contemplates that in some embodiments, a miniprotein does not comprise multiple cysteine residues such as, for example, a miniprotein comprising a single cysteine residue. The present disclosure contemplates that stability conferred by crosslinked cysteines contributes to reduced immunogenicity of miniproteins or compositions comprising such miniproteins. In some embodiments, such stability may also confer resistance to harsher conditions provided for efficient chelation (e.g., high temperature, low pH incubations, etc.), while continuing to retain biological activity (e.g., capability of binding a target).

[0212] In some embodiments, a composition comprising a linker, chelator, and / or radionuclide can efficiently penetrate a tumor. In certain embodiments, a composition comprising a target-binding agent (e.g., a polypeptide (e.g., miniproteins), e.g., a small molecule, e.g., a small molecule receptor ligand) and a radionuclide can efficiently and effectively penetrate a tumor (e.g., a solid tumor), and be further improved by combination with a decoy (e.g., administered before, during, or after administration of a target-binding agent). Targets

[0213] Targets of the disclosure can be targeted (e.g., bound) by target-binding agents (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) and compositions thereof as provided herein. Targets are generally expressed on a surface of a cell (e.g., a particular cell, e.g., a “target cell”) or in a population of cells (e.g., a tumor). A target may have a certain percent identity to a reference protein and still be referred to as a target by a particular name (e.g., any one of the target proteins in TABLE 10, e.g., Nectin-4, e.g., B7-H3). In certain embodiments that will be clear from context, a target may also refer to a protein in a pathway related to another protein. For example, if a target is any one of the target proteins in TABLE 10, a target may also be a protein in a pathway that is necessary for activity of the target protein selected from TABLE 10.

[0214] In certain embodiments that will be clear given context, a target may also be a particular cell type (or be localized to a particular cell type) characterized by expression of particular surface entities such as receptors (e.g., a surface receptor such as in TABLE 10, e.g., a cell in a tissue, e.g., a proximal tubule cell in a kidney). Page 63 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0215] In some embodiments, a target of one polypeptide (e.g., a first miniprotein, e.g., a target-binding miniprotein) may be different or the same as a target to which different miniprotein (e.g., a second miniprotein, e.g., a decoy-targeting miniprotein) is designed to bind. In some embodiments a first miniprotein targets a first target (e.g., as in TABLE 10, e.g., Nectin-4, e.g., B7-H3) on a cell surface (e.g., a cancer cell, a population of cancer cells, a tumor, etc.) and a second miniprotein (e.g., a decoy) targets a second target (e.g., on a non- tumor cell, e.g., on a kidney cell, on a liver cell, etc.).

[0216] In certain embodiments, a target-binding agent binds to a target on a cell. In certain embodiments, the cell is a cancer cell.

[0217] Any cell expressing a target may be targeted by a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein.

[0218] In some embodiments, a cell is a mammalian cell. In some embodiments, a cell is a human cell. In some embodiments, a cell is from a cell line. In some embodiments, a cell is a primary cell. In some embodiments, a primary cell is from a sample from a subject such as from a tumor or from corresponding tissue without a tumor (e.g., from another area of an organ or from a healthy donor). In some embodiments, a cell is in vitro (e.g., a primary cell, a cell line, etc.). In some embodiments, a cell is in vivo (e.g., in a subject, e.g., in a human subject, e.g., in a tumor of a human subject.) In some embodiments, a cell expresses or has been induced to express (e.g., via recombinant technology) a target. In some embodiments, the target is expressed on the surface of a cell. In some embodiments, a cell is contacted by a composition binding to a target expressed on its surface. In some embodiments, upon binding (e.g., upon binding of a target-binding agent as disclosed herein), a target and any bound proteins and / or payloads is / are internalized into the cell. In some embodiments, a cell is killed by a payload (e.g., a radionuclide and / or chelator, etc.) after internalization.

[0219] In some embodiments, a target is a protein or portion thereof that is upregulated or overexpressed on cancer cells as compared to non-cancer cells. That is, in some embodiments, a target is expressed or overexpressed in a tumor or in a tumor microenvironment relative to a level of the target in non-diseased tissue (e.g., tissue without a tumor or tumor microenvironment). In some such embodiments, the target is absent or non- detectable in non-diseased (e.g., healthy) tissue. In some embodiments, a target is a biomarker for cancer (e.g., for cancer cells, for a tumor). In certain embodiments, a target is any one of the targets identified in TABLE 10. Page 64 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0220] In some embodiments, a target is Nectin-4. In some embodiments, a polypeptide (e.g., miniprotein) that binds to Nectin-4 is or comprises a Scaffold A miniprotein. In some such embodiments, the Scaffold A miniprotein is not a decoy. In some embodiments, a Scaffold A miniprotein that binds to Nectin-4 has an amino acid sequence comprising that of any of SEQ ID NOs: 7, 9-15, or 47-68. In some embodiments, a compound of any of C12- C35, C45-C56, C58-C77, or C109-C139 binds to Nectin-4. In certain embodiments, the affinity of such compounds for Nectin-4 is much weaker as compared to the affinity of compounds such as C116 or C117 for Nectin-4.

[0221] In some embodiments, a target is B7-H3. In some embodiments, a polypeptide (e.g., miniprotein) that binds to B7-H3 is or comprises a Scaffold B miniprotein. In some such embodiments, the Scaffold B miniprotein is not a decoy. In some embodiments, a Scaffold B miniprotein that binds to B7-H3 has an amino acid sequence comprising that of any of SEQ ID NOs: 2-3, 5-6, 8, and 87. In some embodiments, a compound of any of C1- C6, C8-C11, C36-C44, C57, and C140-C142 binds to B7-H3.

[0222] In some embodiments, a target is a non-tumor cell, such as a kidney cell or a liver cell. In some embodiments, a target is a tissue, such as a kidney a liver. In some embodiments, a miniprotein that binds to a non-tumor cell is or comprises a decoy. In some embodiments, a decoy is a Scaffold A decoy. In some embodiments, a Scaffold A decoy has an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 16-46. In some embodiments, a Scaffold A decoy comprises or consists of a compound selected from any of C78-C108. In some embodiments, a decoy is a Scaffold B decoy. In some embodiments, a Scaffold B decoy has an amino acid sequence comprising, consisting essentially of, or consisting of SEQ ID NO: 4. In some embodiments, a Scaffold B decoy comprises or consists of compound C7. TABLE 10. Candidate Proteins Targeted By Each Candidate Target Target Target Target rPage 65 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Target Target Target Target BCMA CD79B Globo H Norepinephrineprotein in a pathway activated or acted upon by another protein. For instance, in some embodiments, a protein may be expressed on the surface of a cancer cell and a target may be a pathway that the surface-cell protein acts upon. In some embodiments, a protein may be expressed on a cancer cell and a target may be a protein on a different cell that causing a cancer cell to proliferate or otherwise be refractory to a treatment. In some embodiments, a tumor-associated cell surface molecule or tumor-specific cell surface molecule may be targeted by a miniprotein or composition comprising a miniprotein as provided herein.

[0224] In some embodiments, the miniprotein or composition comprising a miniprotein specifically binds a target expressed on the surface of a cell. In some embodiments, a target is Page 66 of 248 IPTS / 128939095.1Docket No.: AKT-033WO cleaved from a cell surface. In some such embodiments, if the target is in an organism, cleavage of the target results in circulation of the target throughout the system of the organism. In some such embodiments, a target is found at a particular level in, e.g., blood, serum, plasma, etc. In some embodiments, however, a substantial portion of expressed target is localized to cell surfaces; thus, in some embodiments, measurements of a level of a target may not accurately reflect the amount of target in a population of cells (e.g., a tumor).In some embodiments, a target is a secreted protein. In some such embodiments, a target is found at a particular level in, e.g., blood, serum, plasma, etc. In some such embodiments, the miniprotein binds to a region of a target such as, for example, an epitope. In some embodiments, a miniprotein or composition comprising a miniprotein specifically binds a target expressed on the surface of a cancer cell. In some embodiments, the cancer cell is in, on, or near a solid tumor. In some embodiments, the cancer cell is a circulating cancer cell. In some embodiments, a miniprotein or composition comprising a miniprotein specifically binds a target or expressed at a higher level on a cancer cell than a reference cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0225] In some embodiments, the miniprotein or composition comprising a miniprotein specifically binds to any one of the target proteins in TABLE 10. In some embodiments, the target comprises or consists of any one of the target proteins in TABLE 10. In some embodiments, the miniprotein specifically binds to a target comprising an amino acid sequence or portion thereof as set forth in TABLE 6.

[0226] In some embodiments, a target is B7-H3. In some such embodiments, a compound targeting B7-H3 is disclosed in TABLE 4A. In some embodiments, a miniprotein that targets B7-H3-4 has an amino acid sequence comprising that of any of SEQ ID NOs: 2-3, 5-6, 8, and 87. In some embodiments, a compound of any of C1-C6, C8-C11, C36-C44, C57, and C140- C142 binds to B7-H3.

[0227] In some embodiments, a target is Nectin-4. In some such embodiments, a compound targeting Nectin-4 is disclosed in TABLE 4B. In some embodiments, a miniprotein that targets Nectin-4 has an amino acid sequence comprising that of any of SEQ ID NOs: 7, 9-15, or 47-68. In some embodiments, a compound of any of C12-C35, C45-C56, C58-C77, or C109-C139 targets Nectin-4.

[0228] Given context, in some embodiments, a target may be related to a protein such as, for example, a protein in a pathway activated or acted upon by another protein. For example, in some embodiments, a protein may be expressed on the surface of a cancer cell and a target may be a pathway that the surface-cell protein acts upon. In some embodiments, a protein Page 67 of 248 IPTS / 128939095.1Docket No.: AKT-033WO may be expressed on a cancer cell and a target may be a protein on a different cell that causing a cancer cell to proliferate or otherwise be refractory to a treatment. In some embodiments, a tumor-associated cell surface molecule or tumor-specific cell surface molecule may be targeted by a miniprotein or composition comprising a miniprotein as provided herein.

[0229] In some embodiments, the miniprotein or composition comprising a miniprotein specifically binds a target expressed on the surface of a cell. In some embodiments, a target is cleaved from a cell surface. In some such embodiments, if the target is in an organism, cleavage of the target results in circulation of the target throughout the system of the organism. In some such embodiments, a target is found at a particular level in, e.g., blood, serum, plasma, etc. In some embodiments, however, a substantial portion of expressed target is localized to cell surfaces; thus, in some embodiments, measurements of a level of a target may not accurately reflect the amount of target in a population of cells (e.g., a tumor).In some embodiments, a target is a secreted protein. In some such embodiments, a target is found at a particular level in, e.g., blood, serum, plasma, etc. In some such embodiments, the miniprotein binds to a region of a target such as, for example, an epitope. In some embodiments, a miniprotein or composition comprising a miniprotein specifically binds a target expressed on the surface of a cancer cell. In some embodiments, the cancer cell is in, on, or near a solid tumor. In some embodiments, the cancer cell is a circulating cancer cell. In some embodiments, a miniprotein or composition comprising a miniprotein specifically binds a target or expressed at a higher level on a cancer cell than a reference cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0230] In some embodiments, the miniprotein or composition comprising a miniprotein specifically binds to any one of the target proteins in TABLE 10. In some embodiments, the target comprises or consists of any one of the target proteins in TABLE 10. In some embodiments, the miniprotein specifically binds to a target comprising an amino acid sequence or portion thereof as set forth in TABLE 6. TABLE 6: Exemplary Target Protein Amino Acid Sequences Target Protein Amino Acid Sequence SEQ ID NO: (Uniprot Acc. No.)age o IPTS / 128939095.1Docket No.: AKT-033WO Target Protein Amino Acid Sequence SEQ ID NO: (Uniprot Acc. No.) NRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVPage 69 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Target Protein Amino Acid Sequence SEQ ID NO: (Uniprot Acc. No.) Murine FAP MKTWLKTVFGVTTLAALALVVICIVLRPSRVYKPEGNTKRALTLPage 70 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Target Protein Amino Acid Sequence SEQ ID NO: (Uniprot Acc. No.) RQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGP

[0231] In some embodiments, a polypeptide (e.g., a miniprotein, e.g., a decoy) in accordance with the present disclosure is manufactured using solid phase peptide synthesis methods. In some embodiments, the polypeptide is recombinant. In some embodiments, the polypeptide is a folded polypeptide held together by disulfide bonds, covalent, or non- covalent interactions. In some embodiments, the polypeptide comprises or consists of a miniprotein. In certain embodiments, a miniprotein comprises or consists of a binder. In certain embodiments, a miniprotein comprises or consists of an affibody. In some embodiments, a miniprotein comprises or consists of a CDP, monobody, binder, affibody, engineered Kunitz domain, anticalin, avimer, or combinations thereof. In some embodiments, polypeptides of the present disclosure (including muteins, allelic variants, fragments, derivatives, and analogs) are encoded by polynucleotides as described and provided herein. In certain embodiments, a polypeptide is of a particular scaffold as disclosed herein (e.g., Scaffold A, Scaffold B, another scaffold, etc.). In some embodiments, the miniprotein comprises or consists of a linear polypeptide, a folded polypeptide (e.g., covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide including a disulfide linkage), cysteine-dense peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer. In some embodiments, the miniprotein comprises or consists of approximately 100 amino acids or less. In some embodiments, the miniprotein is a cysteine-dense protein. In some embodiments, the miniprotein comprises at least one cysteine-dense region. In some Page 71 of 248 IPTS / 128939095.1Docket No.: AKT-033WO embodiments, the miniprotein comprises one or more disulfide bridges. In some embodiments, the miniprotein comprises or consists of a non-disulfide-containing amino acid sequences. In some embodiments, the miniprotein comprises three alpha helices with 58 amino acids and has a molar mass of about 6 kDa. In some embodiments, the miniprotein is stable at high temperatures and under acidic or alkaline conditions. In some embodiments, the miniprotein comprises an engineered protein derived from a lipocalin. In some embodiments, the miniprotein comprises an eight-stranded ^-barrel. In some embodiments, the miniprotein displays (i) high structural plasticity as a consequence of sequence variation and (ii) elevated conformational flexibility, allowing induced fit to targets with differing shape. In some embodiments, the miniprotein comprises a class of antibody mimetics which consist of two or more peptide sequences of 30 to 70 amino acids each, which are connected by linker peptides. In some embodiments, the miniprotein comprises a peptide derived from Ankyrin. In some embodiments, the miniprotein comprises an ankyrin repeat, a 33 residue motif consisting of two alpha-helices and a beta-turn. In some embodiments, the miniprotein comprises a peptide derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI).

[0232] In some embodiments, a target-binding agent (e.g., a polypeptide (e.g., miniproteins), e.g., a small molecule, e.g., a small molecule receptor ligand) of the present disclosure comprises or consists of an amino acid sequence as shown in TABLE 3, TABLE 4A, TABLE 4B, TABLE 11, and TABLE 17 and is capable of binding to a target (e.g., a target of a protein or portion thereof as provided in TABLE 6, e.g., a target of TABLE 10).

[0233] In some embodiments, the disclosure provides target-binding proteins (e.g., miniproteins, e.g., binders, etc.) comprising or consisting of a fragment of an amino acid sequence as provided herein. In some such embodiments, fragments include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more contiguous amino acids.

[0234] In some embodiments, the disclosure provides affibodies comprising or consisting of a fragment of an amino acid sequence as provided herein. In some such embodiments, fragments include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more contiguous amino acids.

[0235] In some embodiments, target-binding agents (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand)of the present Page 72 of 248 IPTS / 128939095.1Docket No.: AKT-033WO disclosure can also include fusions or conjugates with one or more other components, such as heterologous polypeptides. For example, in some embodiments, heterologous sequences can comprise or consist of sequences designed to facilitate purification, e.g., histidine tags, and / or visualization of recombinantly-expressed proteins. Other non-limiting examples of such fusions or conjugates include those that permit display of the encoded protein on the surface of a phage or a cell, including any detectable or visualizable component such as, e.g., green fluorescent protein (GFP), and fusions to the IgG Fc region.

[0236] In some embodiments, a polypeptide of the disclosure can be a linear polypeptide, a folded polypeptide (e.g., covalently linked polypeptide, non-covalently linked polypeptide, or polypeptide include a di-sulfide linkage), cysteine-dense peptide, a knottin peptide, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), or an avimer.

[0237] In some embodiments, the polypeptide comprises at least one disulfide bridge. In some embodiments, the polypeptide comprises at least two disulfide bridges. In some embodiments, the polypeptide comprises one or two disulfide bridges. In some embodiments, the polypeptide comprises at least one non-natural amino acid (e.g., methylated lysine, citrulline, etc.). In some embodiments, the polypeptide comprises at least one modified amino acid. In some such embodiments, a modified amino acid is an amino acid that comprises a small alkyl group on the side chain of the amino acid (e.g., methylated lysine, e.g., mono, di, tri-methyllysine, etc.). For example, in some embodiments, a lysine can have a small alkyl group (e.g., a methyl group, e.g., mono, di, tri-methyl, etc.) on the nitrogen on its side chain. In some embodiments, an arginine can have a small alkyl group on the Guanidino group of its side chain.

[0238] Polypeptides of the disclosure can be monomers or multimers. For example, in certain embodiments, compositions of the disclosure can comprise a single miniprotein or a multimeric protein, such as according to one or more formulas as follows (M)x-L-C-R, (M)x- L-C, (M)x-C-R, (M)x-L-(R), (M)x-C, (M)x-L, and (M)x-R, wherein M comprises a polypeptide (e.g., a miniprotein) (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4. In some embodiments, x is the same or a different miniprotein. Miniproteins of a multimer can be linked such as disclosed herein (e.g., one or more linkers, e.g., covalent bonds, e.g., non-covalent interactions, etc.)

[0239] In some embodiments, a miniprotein of the disclosure comprises or consists of a specific amino acid sequence. In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, Page 73 of 248 IPTS / 128939095.1Docket No.: AKT-033WO 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 1-68 and 87.

[0240] As used herein and known to those of skill in the art, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed.1991), which is incorporated herein by reference. In some embodiments, an amino acid of the present disclosure may be a stereoisomer (e.g., D-amino acids) of the twenty conventional amino acids. In some embodiments, an amino acid in a polypeptide of the present disclosure may be a non-natural amino acid. For example, amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine (Lys(Ac)), O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, norleucine (including, e.g., 6-hydroxynorleucine), citrulline, L-citrulline, methylated arginine (Rme, Rme2), symmetrically dimethylated arginine (sRme2, Rme2s, or SDMA), nitroarginine (Arg(NO2)), Leu-13C6,15N (an enriched stable isotope version of Leucine), and other similar amino acids and imino acids (e.g., 4-hydroxyproline). Arrangements of polypeptide sequence notations used herein have a left-side end corresponding to the amino terminal and a right-side end corresponding to the carboxy- terminal end, in accordance with standard usage and convention.

[0241] In some embodiments, miniproteins of the present disclosure comprising two or more cysteine residues, such as those set forth in SEQ ID NOs: 1-84 or 90-111, have cysteine residues connected via disulfide bridges (e.g., via natural folding). Miniproteins

[0242] In some embodiments, a target-binding agent of the disclosure comprises a miniprotein. In certain embodiments, miniproteins (e.g., as a monomer, as a multimer, etc.) having amino acid sequences comprising or consisting of those provided herein and methods of use of such miniproteins. In some embodiments, a multimeric miniprotein can comprise one or more monomers that may be the same monomer, in duplicate, triplicate, or more, or may be two or more distinct monomers that are connected such as through covalent bonds or non-covalent interactions. Page 74 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0243] In some embodiments, a polypeptide of the disclosure comprises or consists of a miniprotein (e.g., having an amino acid sequence as provided herein). In some embodiments, the miniprotein has a particular set of characteristics. For example, in certain embodiments, the miniprotein comprises or consists of a CDP, knottin, and / or binder. In some embodiments the miniprotein is designed to be linked to one or more other components. For example, in some embodiments, a miniprotein may be linked (conjugated) to another component such as a chelator and / or a radionuclide. In some embodiments, conjugation is via a lysine or cysteine residue. To give but one example, in some embodiments, a miniprotein is engineered to remove all lysine residues except for one, which is, in some embodiments, used for conjugation. In some embodiments, conjugation occurs via an optional linker. In some embodiments, conjugation between a miniprotein and a chelator and / or radionuclide is direct.

[0244] Without wishing to be bound by theory, the disclosure contemplates that therapeutics comprising compositions provided herein are characterized by several features relative to other (e.g., antibody-based) therapeutics. For example, in some embodiments, miniproteins display several key features of antibody-based therapeutics (e.g., affinity, potency, specificity, and ability to disrupt protein:protein interactions) but also have several advantages as compared to antibody-based therapeutics such as smaller size, cheaper manufacturing, and elimination of need to chimerize or humanize the proteins. In addition, the size and specificity of binding increases tumor penetrance and uptake into cells expressing the target of the miniprotein or composition (e.g., conjugate) comprising a miniprotein.

[0245] In some embodiments, a miniprotein of the present disclosure is no more than about 100 amino acids in length. In some embodiments, a miniprotein is about 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more amino acids in lengths. In some such embodiments, however, a miniprotein of the present disclosure does not exceed about 100 amino acids in length. In some embodiments, a miniprotein is between about 20 to about 40, about 30 to about 50, about 40 to about 60, about 45 to about 65, about 50 to about 70, about 55 to about 75, about 65 to about 85 or more amino acids in length, but not exceeding about 100 amino acids in length. In some preferred embodiments, a miniprotein is about 65 amino acids or less. In some preferred embodiments, a miniprotein is about 50 amino acids or less.

[0246] In some embodiments, a miniprotein of the present disclosure is not larger than about 12 kDa. In some embodiments, a miniprotein of the present disclosure is about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or more kDa. In some such Page 75 of 248 IPTS / 128939095.1Docket No.: AKT-033WO embodiments, however, a miniprotein of the present disclosure does not exceed about 12 kDa.

[0247] In some embodiments, a miniprotein of the present disclosure comprises or consists of a CDP, a knottin, a binder, an affibody, an engineered Kunitz domain, a monobody, an anticalin, a designed ankyrin repeat domain (DARPin), and / or an avimer. In some embodiments, the miniprotein comprises or consists of a CDP. In some such embodiments, the miniprotein comprises or consists of a knottin. In some such embodiments, the miniprotein comprises or consists of a binder. In some such embodiments, the miniprotein comprises or consists of an affibody. In some such embodiments, the miniprotein comprises or consists of an engineered Kunitz domain. In some such embodiments, the miniprotein comprises or consists of a monobody. In some such embodiments, the miniprotein comprises or consists of an anticalin. In some such embodiments, the miniprotein comprises or consists of a designed ankyrin repeat domain (DARPin). In some such embodiments, the miniprotein comprises or consists of an avimer. In some embodiments the miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) is designed to be linked to one or more other components. For example, in some embodiments, a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) may be linked (conjugated) to another component such as a chelator and / or a radionuclide. In some embodiments, a radionuclide of the present disclosure is an alpha emitter. In some such embodiments, a chelator and / or radionuclide are conjugated to a miniprotein via a linker. In some embodiments, miniproteins as provided herein function as targeting moieties, e.g., specifically binding to a target expressed on the surface of a tumor cell. In some such embodiments, a miniprotein is designed such that it may be joined to one or more additional components. For example, without being bound by any particular theory, miniproteins of the present disclosure may be formulated such that they are combined with other components such as a therapeutic molecule (e.g., chelator compositions and / or radionuclide) and / or a detectable agent (e.g., a visualizable agent, e.g., a metabolizable and visualizable agent, etc.). In some such embodiments, such miniproteins conjugated to one or more additional components may be used, for example, in diagnosis, prognosis, monitoring and / or treatment of one or more diseases, disorders or conditions such as those with expression of particular targets on particular populations of cells.

[0248] In some embodiments, a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), Page 76 of 248 IPTS / 128939095.1Docket No.: AKT-033WO avimer) has low immunogenicity relative to a larger protein. In some such embodiments, the lower immunogenicity increases amenability to harsher environmental conditions (e.g., high temperature and low pH incubations) while retaining biological activity. Thus, in some embodiments, a conjugate comprising a miniprotein has lower immunogenicity than a composition comprising a larger protein or different targeting moiety (i.e., other than a miniprotein).

[0249] In some embodiments, miniproteins (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) have superior penetration efficiency relative to larger proteins. That is, in some embodiments, a miniprotein or composition comprising a miniprotein can penetrate a solid tumor better than a larger protein or composition comprising a protein larger than a miniprotein. For example, in some embodiments, a binder has superior tumor penetration efficiency with a hydrodynamic radius on the order of about 1 nm – 25 nm. In some embodiments, the hydrodynamic radius is between about 1 nm -5 nm. In some embodiments, the hydrodynamic radius is between about 1 nm – 3 nm. In some embodiments, the hydrodynamic radius is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nm.

[0250] As described herein, in some embodiments, miniproteins (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer), are conjugated to a chelator. In some embodiments, the chelator binds a radionuclide (e.g., an alpha-emitter radionuclide, e.g., actinium). In some such embodiments, such radionuclide conjugates combine specific-binding capabilities and properties of a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) with a radionuclide. In some embodiments, a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) that targets a radioisotope to which its conjugated to a cell expressing a target. In some embodiments, the target is expressed on the surface of a cell. In some embodiments, the target is any one of the target proteins in TABLE 10. In some embodiments, the cell is a tumor cell. In some embodiments, the conjugate binds to any one of the target proteins in TABLE 10 on the surface of the tumor cell. In some such embodiments, the radionuclide is targeted to the tumor cell. In some embodiments, the radionuclide is an alpha-emitter radionuclide and when internalized, serves to specifically target (e.g., without damaging surrounding tissue / cells) the tumor cell. Page 77 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0251] In some embodiments, a miniprotein comprises one or more disulfide bridges. In some embodiments, a miniprotein comprises multiple cysteine residues. In some such embodiments, cysteine residues crosslink to maintain a very stable, folded state for a peptide of its length (e.g., relative to a peptide of the same length without as many cysteine residues). The present disclosure contemplates that such crosslinking confers improved stability with reduced (i.e., very low to no) immunogenicity and / or sustains or improves ability to maintain biological activity in harsh but efficient chelation conditions (e.g., high temperature and low pH).

[0252] In some embodiments, a miniprotein or composition comprising a miniprotein (e.g., a radionuclide conjugate) has low immunogenicity relative to a larger protein or composition comprising or consisting of a larger protein (e.g., an antibody).

[0253] In some embodiments, miniproteins (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) have superior penetration efficiency relative to larger proteins. That is, in some embodiments, a miniprotein or composition comprising a miniprotein can penetrate a solid tumor better than a larger protein or composition comprising a protein larger than a miniprotein. For example, in some such embodiments, a miniprotein or composition comprising a miniprotein has a hydrodynamic radius of about 1 to about 25 nm. In some embodiments, a hydrodynamic radius is in a range of about 1-25 nm, 10-20 nm, 5-15 nm, 1-5 nm, 2-4 nm, or 1-3 nm. In some embodiments, hydrodynamic radius is measured using light scatter methods known to those of skill in the art.

[0254] In some embodiments, a miniprotein of the present disclosure is characterized in that it has one or more properties relative to a protein larger than 100 amino acids like an antibody, antibody fragment, VHH domain, single chain antibody or other protein or binder greater than 12 kDA. In some embodiments, a property is selected from increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased activity, increased receptor binding specificity and / or affinity, increased specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (e.g., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, and altered temperature profile, increased resistance to liver uptake, kidney uptake or healthy tissue binding, increased tumor penetration, and / or increased volume of distribution. Page 78 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0255] In some embodiments, a miniprotein or composition comprising a miniprotein (e.g., conjugate, e.g., radionuclide conjugate) provided by the present disclosure exhibits binding affinity to any one of the target proteins in TABLE 10. In some embodiments, the target proteins selected from TABLE 10 is the human isoform. In some embodiments, the human isoform of any one of the target proteins in TABLE 10 is on a cell. In some embodiments, the cell is a cell line, a primary cell, or a cell in a human (e.g., in a tumor).

[0256] In some embodiments, a miniprotein or composition comprising a miniprotein (e.g., conjugate, e.g., radionuclide conjugate) displays nM or sub-nM binding affinity to any one of the target proteins in TABLE 10. In some embodiments, the affinity is measured in an in vitro assay. In some embodiments, the in vitro assay is a cell-based assay. In some embodiments, affinity is measured in an in vivo assay (e.g., a PET scan) or using a sample from a subject (e.g., an in vitro assay using a biological specimen such as blood or a cell biopsy from a subject).

[0257] In some embodiments, a miniprotein or conjugate thereof displays a binding affinity to any one of the target proteins in TABLE 10. In some embodiments, the binding affinity of a miniprotein or conjugate thereof to the human isoform of any one of the target proteins in TABLE 10 is about 500 nM In some embodiments, the miniprotein comprises picomolar binding affinity. In some embodiments, the miniprotein or conjugate thereof comprises a binding affinity characterized by a dissociation constant ranging from about 900 nM to about 1 nM, e.g., 900, 800, 700, 600, 500, 400, 300.200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, .9, .8, .7, .6, .5, .4 nM or less binding affinity to the human isoform of any one of the target proteins in TABLE 10. In some embodiments, the binding is selective to the human isoform of any one of the target proteins in TABLE 10 and, not, e.g., non-human target proteins selected from TABLE 10.

[0258] In some embodiments, a miniprotein or conjugate thereof provided by the present disclosure has high affinity for any one of the target proteins in TABLE 10. In some such embodiments, the target protein selected from TABLE 10 is the human isoform of the selected protein from TABLE 10. In some embodiments, a miniprotein of the present disclosure is stable, including in the presence of one or more additional molecules (e.g., a cytotoxic molecule, e.g., radiation).

[0259] In some embodiments, binding ability of a miniprotein or conjugate thereof to a target is improved by one or more modifications. For example, in some embodiments, the binding ability of a miniprotein or conjugate thereof as provided herein to any one of the target proteins in TABLE 10, is improved using chemical crosslinking. In some Page 79 of 248 IPTS / 128939095.1Docket No.: AKT-033WO embodiments, binding may be enhanced by using one or more of lysine residues, fusion proteins, non-natural amino acids, or other chemical moieties to enhance binding and / or functional activity.

[0260] In some embodiments, to ensure proper folding and connectivity, selected cysteine pairs can be replaced with selenocysteines. It is contemplated that, in some embodiments, diselenide crosslinks form more readily than disulfide crosslinks due to their lower redox potential and such a replacement may cross-couple remaining cysteine residues.

[0261] In some embodiments, a miniprotein of the present disclosure comprises or consists of an antigen for use in generating an antibody that specifically binds to at least one epitope on any one of the target proteins in TABLE 10. In some embodiments, such an antibody may be used for, e.g., diagnostic purposes, blocking (e.g., antagonism), etc.

[0262] In some embodiments, the miniprotein comprises one or more disulfide bridges.

[0263] In some embodiments, a miniprotein or conjugate thereof as provided herein does not comprises one or more cysteine residues. In some embodiments, the miniprotein does not comprise one or more disulfide bridges.

[0264] In some embodiments, a miniprotein or conjugate thereof as provided herein is specific for a target. In some embodiments, a miniprotein is specific for any one of the target proteins in TABLE 10 or a fragment thereof.

[0265] In some embodiments, a target is represented by an amino acid sequence or a portion thereof as set forth in any one of SEQ ID NOS: 69-72 as set forth in TABLE 6.

[0266] In some embodiments, a miniprotein or conjugate thereof as provided herein comprises or consists of a specific amino acid sequence.

[0267] In some embodiments, miniproteins or compositions comprising miniproteins (e.g., radionuclide conjugates) are conjugated to a chelator that optionally binds a radionuclide (e.g., actinium). In some embodiments, the conjugation is via a linker. In some embodiments, conjugation is direct conjugation. In some embodiments, such radionuclide conjugates combine and synergize to provide target specificity (e.g., via the miniprotein) and superior treatment (e.g., via directed radioisotope delivery to the cell expressing the target).

[0268] As used herein and known to those of skill in the art, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed.1991), which is incorporated herein by reference. In some embodiments, an amino acid of the present disclosure may be a stereoisomer (e.g., D-amino acids) of the twenty conventional amino acids. In some embodiments, an amino acid in a polypeptide of the present disclosure may be Page 80 of 248 IPTS / 128939095.1Docket No.: AKT-033WO a non-natural amino acid. For example, amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N- methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). Arrangements of polypeptide amino acid sequence notations used herein have a left-side end corresponding to the amino terminal and a right-side end corresponding to the carboxy- terminal end, in accordance with standard usage and convention.

[0269] In some embodiments, a miniprotein as provided herein has an amino acid sequence comprising or consisting of an amino acid sequence as set forth in any of TABLE 3, TABLE 4A or TABLE 4B.

[0270] In some embodiments, a miniprotein comprises or consists of a specific amino acid sequence. In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 1-68 and 87.

[0271] As used herein and known to those of skill in the art, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed.1991), which is incorporated herein by reference. In some embodiments, an amino acid of the present disclosure may be a stereoisomer (e.g., D-amino acids) of the twenty conventional amino acids. In some embodiments, an amino acid in a polypeptide of the present disclosure may be a non-natural amino acid. For example, amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N- methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). Arrangements of polypeptide sequence notations used herein have a left-side end corresponding to the amino terminal and a right-side end corresponding to the carboxy- terminal end, in accordance with standard usage and convention. DOTA-PEG4: alpha-(1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetate)-4(ethylene glycol) Page 81 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Biotin-PEG4: FITCI_PEG4 :Norleucine: (S)-(+)-2-Aminohexanoic acid, (S)-2-AminocaproicHomoleucine: (S)-3-Amino-5-methylhexanoic acid

[0272] In some embodiments, a miniprotein of the presenttryptophan, leucine, phenylalanine, alanine, 3-Pyridyl Alanine, substituted tryptophan, or substituted phenylalanine. In some embodiments, a miniprotein of the present disclosure has asparagine, aspartic acid, serine, lysine, glutamine, glutamic acid, leucine, alanine, norleucine, homo-leucine, homo serine, or substituted phenylalanine. In some embodiments, a miniprotein of the present disclosure has glycine, alanine, lysine, glutamic acid, leucine, serine, proline, phenylalanine, norleucine, homo-leucine, homo serine, or substituted phenylalanine. In some embodiments, a miniprotein of the present disclosure has glutamic acid, leucine, aspartic acid, methionine, glutamine, tyrosine, phenylalanine, norleucine, homo-leucine, homo serine, or substituted phenylalanine.

[0273] In some embodiments, a miniprotein of the present disclosure exhibits binding specificity to the human isoform of any one of the target proteins in TABLE 10. For example, in some embodiments a miniprotein provided by the present disclosure, such as, for example, those represented by any one of SEQ ID NOs: 1-68 and 87, demonstrates binding when expressed on the surface of yeast and binding to any one of the target proteins in TABLE 10 tested by flow cytometry. In some embodiments, a miniprotein provided by the present disclosure, such as, for example, those represented by any one of SEQ ID NOs: 1-68 and 87, demonstrates binding specificity via flow cytometry when, for example, such as miniprotein of any one of the target proteins in TABLE 10 (e.g., as represented by any of SEQ ID NOs: 1-68 and 87) only binds to the selected target protein from TABLE 10 and not to other target proteins.

[0274] In some embodiments, a miniprotein of the present disclosure such as, for example, any of those represented by SEQ ID NOs 1-68 and 87, shows greater than 10 nM potency. In some embodiments, a miniprotein shows potency greater than 1, 2, 3, 4, 5, 6, 7, 8, 9 nM or more. Page 82 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Scaffolds

[0275] Scaffolds of the disclosure may be characterized as “Scaffold A” “Scaffold B”, “Scaffold C”, “Scaffold L”, “Scaffold J”, etc. Any target-binding agent (e.g., a polypeptide e.g., miniprotein) of the disclosure can be based on the same scaffold or different scaffold as another target-binding agent (e.g., a polypeptide (e.g., miniproteins), e.g., a small molecule, e.g., a small molecule receptor ligand). Two entities that have different scaffolds from one another are said to “not share a scaffold”. One of ordinary skill in the art will also understand that even if a target-binding agent is not a protein (such as, e.g., a small molecule), it will be considered to not share a scaffold with a decoy. For example, as provided herein, a Scaffold A decoy (e.g., C78, C79, C96, etc.) is a different scaffold than the small molecule of compound C170, as set forth herein, or, in other words, a Scaffold A decoy and C170 do not share a scaffold. Similarly, a Scaffold B target-binding agent and a Scaffold A decoy do not share a scaffold, and a Scaffold A target-binding protein and Scaffold B decoy do not share a scaffold. For clarity, not sharing a scaffold does not refer to lack of physical colocalization, rather, that the arrangement and structure (e.g., atomic structure) of a given entity is different than that of a comparator entity. Furthermore, even if two agents share a scaffold, such as a Scaffold A target-binding protein and a Scaffold A decoy, it does not mean that their function (e.g., binding to a target, e.g., decoying) or specificity (e.g., for a target) is the same. That is, for example, certain Scaffold A target-binding proteins of the disclosure bind with strong affinity and specificity to Nectin-4, whereas certain Scaffold A decoys (e.g., C78, C79, C96) do not bind to Nectin-4 at any detectable levels.

[0276] One of ordinary skill in the art, given context (e.g., of a particular scaffold), and using in silico tools such as, e.g., AlphaFold2, or in vitro tools such as NMR, x-ray crystallography, cryo-electron microscopy, will be able to determine whether a particular entity (e.g., a target-binding agent, a decoy, etc.) is of the same or of a different scaffold as another entity. That is, it will be understood or readily decipherable without any undue burden and when considering a given decoy, and target-binding agent, (e.g., or two target- binding agents, etc.) whether they are of the same or different scaffolds.

[0277] If the scaffold is the same, functions (e.g., of a decoy versus a target-binding miniprotein) can differ depending upon the amino acid sequence contained in each therein (e.g., in a decoy, e.g., in a target-binding miniprotein). To give an example, a decoy and a target-binding polypeptide (e.g., miniprotein) can have the same scaffold (e.g., Scaffold A, e.g., Scaffold B, Scaffold C, Scaffold J, Scaffold L, etc. where each scaffold has a similar general structure, e.g., secondary structure, e.g., bonds such as disulfide bonds, etc.) but bind Page 83 of 248 IPTS / 128939095.1Docket No.: AKT-033WO (e.g., preferentially, e.g., with a particular affinity, etc.) to different locations such as a target on a tumor (e.g., a target-binding miniprotein) or a non-tumor cell, such as on a kidney (e.g., a decoy).

[0278] Scaffolds are meant to describe certain structural characteristics of a given polypeptide or miniprotein, but not necessarily describe any particular function, such as binding characteristics (e.g., to a particular target, e.g., as a decoy, etc.). Scaffolds are to be understood to refer to a core binding component of a given agent (e.g., miniprotein), for example, a miniprotein that binds to a target such as Nectin-4 and independent of decorations, such as, e.g., N- and / or C-terminal extension and / or labels, for example, detectable labels and / or therapeutic labels, including, without limitation, linkers, chelators, and / or radionuclides, such as shown in various exemplary compounds of the disclosure as set forth in TABLE 3.

[0279] A target-binding agent can be of any scaffold. In certain embodiments, a target- binding polypeptide can also be a Scaffold A, B, C, L, or J scaffold. In certain embodiments, a target-binding agent is not a protein (see, e.g., C170 in TABLE 17), and is understood to be of a “different scaffold” than a Scaffold A or B decoy. In some embodiments, an exemplary comparator molecule with a particular scaffold is a protein, but not a miniprotein (e.g., C143 and C144 of TABLE 11 and TABLE 17). In some embodiments, a target-binding agent is a polypeptide, such as a miniprotein, and can be of any scaffold. In certain embodiments, an exemplary comparator molecule is a small molecule (e.g., C170).

[0280] A decoy of the disclosure can be a Scaffold A decoy or a Scaffold B decoy.

[0281] In certain embodiments, Scaffold A can generally be characterized as comprising at least 40 amino acids in length, at least one structural domain comprising three alpha helices, wherein at least two of the alpha helices are anti-parallel, and at least one disulfide bridge. For example, a given amino acid sequence, when entered into a program such as AlphaFold2, can be characterized as a Scaffold A protein if it has a Root Mean Square Deviation (RMSD), as determined by comparing polypeptide backbone atoms across two structures, e.g., such as to a Scaffold A protein as disclosed herein (e.g., TABLE 4B, TABLE 4C) of <3.5 angstroms as compared to a reference Scaffold A miniprotein. Polypeptide backbone refers to the repeating sequence of atoms along the core of a polypeptide chain in a protein. A polypeptide backbone is considered a “main chain” of a protein, formed by linking amino acid residues through peptide bonds. A polypeptide backbone of a protein is composed of a repeating sequence of -N-alpha-C -C -, where the N represents the amine group of an amino acid and the C represents the carbonyl group of the amino acid. In some embodiments, a Scaffold A Page 84 of 248 IPTS / 128939095.1Docket No.: AKT-033WO protein is a binder. In certain embodiments, the Scaffold A target-binding miniprotein binds to Nectin-4. As provided herein, a Scaffold A Nectin-4 binding protein has a particular secondary structure, and binds to Nectin-4 with a particular affinity (e.g., such as compared to any decoys as described herein).

[0282] Exemplary Scaffold A decoys include miniproteins as in, e.g., compounds C78, C79, C96, e.g., polypeptides having amino acid sequences comprising any one of SEQ ID NOs: 16, 17, and 34, and / or as set forth in TABLE 4C.

[0283] As provided herein, Scaffold A decoys have the same scaffold as exemplary target- binding miniproteins of the disclosure that are designed to and / or bind to Nectin-4 (with a certain affinity) such as C67 (polypeptide of SEQ ID NO: 13), C116 (polypeptide of SEQ ID NO: 54), C117 (polypeptide of SEQ ID NO: 55), etc. (such as in TABLE 4B), but, for example, Scaffold A decoys do not bind to Nectin-4 with the same affinity as a target-binding miniprotein (as described herein), or, in some embodiments, may not bind to Nectin-4 at all (as measured by methods provided herein and / or known to those of skill in the art). Exemplary Scaffold A miniproteins of the disclosure that bind to Nectin-4 include, e.g., C67, C116, and C117, as well as those set forth in TABLE 4B. Exemplary Scaffold A decoys of the disclosure can be found, for example, in TABLE 4C.

[0284] As provided herein, Scaffold B comprises affibody miniproteins. An affibody has a particular secondary structure comprising at least two anti-parallel alpha helices. For example, a given amino acid sequence, when entered into a program such as AlphaFold2, it can be characterized as a Scaffold B protein if it has an RMSD, as determined by comparing backbone atoms across two structures, e.g., such as to a Scaffold B protein as disclosed herein (e.g., TABLE 4A, TABLE 4D) of <3.5 angstroms as compared to a reference Scaffold B miniprotein. Affibodies can derive target specificity by randomization of 13 amino acids located in two alpha-helices involved in binding activity (see, e.g., Feldwisch J, Tolmachev V.; (2012) supra). In certain embodiments, an affibody is no fewer than 50 amino acids, though generally in the 58-59 amino acid range. In some embodiments, a Scaffold B miniprotein binds to B7-H3. Certain exemplary B7-H3-targeting binding agents (e.g., miniproteins) of the disclosure are set forth as in TABLE 4A, including C9, C140, C141, and C142 (e.g., polypeptides of SEQ ID NOs: 6 or 87). As used herein Scaffold B decoys have affibody-based scaffolds and include miniproteins as in TABLE 4D, e.g., C7 (SEQ ID NO: 4).

[0285] In some embodiments, a Scaffold C miniprotein is a GP2-based protein. In certain embodiments, a Scaffold C protein binds to a target. GP2 scaffolds can be used in target- Page 85 of 248 IPTS / 128939095.1Docket No.: AKT-033WO binding agents (e.g., miniproteins) of the disclosure, such as to demonstrate the cross- scaffolding capabilities decoys of a different scaffold. An exemplary miniprotein of a GP2 scaffold is set forth in C168 (SEQ ID NO: 113). The GP2 scaffold was identified in a search for small, evolvable protein domains that could be used as a binding scaffold (Kruziki et al. A 45-Amino-Acid Scaffold Mined from the PDB for High-Affinity Ligand Engineering. Chem Biol.2015 Jul 23;22(7):946-56). In certain embodiments, a Scaffold C miniprotein is no fewer than 40 amino acids. As discovered, GP2-based miniproteins are generally 45 amino acids in length with a topology of an α helix opposite a three-strand β sheet underpinning two diversified loops. Scaffold C (e.g., GP2 miniproteins) has been found to have thermal stability and has been used to evolve high-affinity binding proteins. A given amino acid sequence, when entered into a program such as AlphaFold2, can be characterized as a Scaffold C protein if it has a RMSD, as determined by comparing backbone atoms across two structures, e.g., such as to a Scaffold C protein as disclosed herein (e.g., TABLE 3, e.g., C168 (SEQ ID NO: 113).

[0286] As used herein, the term “Scaffold L” refers to a subgenus of miniproteins comprising at least two to three alpha helices and at least one or two constraints. A Scaffold L miniprotein is shorter and constrained, as compared to an affibody-based miniprotein, and, while affibodies can derive target specificity by randomization of 13 amino acids located in two alpha-helices involved in binding activity (see, e.g., Feldwisch J, Tolmachev V.; (2012) supra), Scaffold L miniproteins comprise one or more mutations in the helix 1 and / or helix 2 binding site, conferring binding specificity; furthermore, Scaffold L miniproteins can have one or more residues in a third alpha helix which can be changed such as for physiochemical reasons and / or to mitigate off-target binding. In certain embodiments, a Scaffold L miniprotein comprises at least 40 amino acids in length, three alpha helices, with at least one or two constraints (e.g., such as one or more disulfide bonds and / or lactam bridges), and displays increased melting temperature, stability, and specificity for a target such as compared to the same protein without the constraints or a protein that binds the same target as a Scaffold L miniprotein (e.g., such as an affibody). A given amino acid sequence, when entered into a program such as AlphaFold2, can be characterized as a Scaffold L protein if it has a RMSD, as determined by comparing backbone atoms across two structures, e.g., such as to a Scaffold B protein as disclosed herein (e.g., TABLE 3, e.g., C145-C166 (SEQ ID NOs: 90-111), in combination with features of at least 40 amino acids in length, and at least one or two constraints. That is, the disclosure recognizes that RMSD between Scaffold B and Scaffold L miniproteins may also be < 3.5 angstroms; however, to be considered Scaffold L Page 86 of 248 IPTS / 128939095.1Docket No.: AKT-033WO miniprotein, a given miniprotein must also characteristics of Scaffold L miniproteins as disclosed herein. In some embodiments, a Scaffold L miniprotein is a triple-helical, constrained miniprotein that is shorter than, e.g., a Scaffold B miniprotein. In some embodiments, the Scaffold L miniprotein binds to B7-H3. Exemplary Scaffold L target- binding proteins are set forth in TABLE 3, e.g., C145-C166 (SEQ ID NOs: 90-111).

[0287] In certain embodiments, the Scaffold J protein is an adnectin. In some embodiments, the adnectin is designed to bind to a particular cell-surface receptor (e.g., on a cancer cell).

[0288] In certain embodiments, a target-binding agent is not considered to be of Scaffold A, B, C, J, or L, as provided herein (i.e., it does not share a scaffold with any molecule identified as belonging to one of those scaffolds). For example, in some embodiments, a scaffold can be a bicycle-based binding protein having binding affinity for Nectin-4 such as zelenectide-pevedotin. In some embodiments, a scaffold can be a short protein that is smaller than a miniprotein (e.g., 8-20 amino acids), for example, dotatate. In some embodiments, a scaffold can comprise protein that is a miniprotein (e.g., up to 100 amino acids), that is not of Scaffold A, B, C, J, or L. In certain embodiments, a scaffold can be or comprise a protein that is greater than 100 amino acids.

[0289] In certain embodiments a scaffold is a ligand for a particular target, but is not a miniprotein or peptide (e.g., vipivotide textrazetan).

[0290] When a target-binding protein is said to be of the same scaffold as a decoy, each scaffold may be, for example, a Scaffold A protein (e.g., a miniprotein). If a target-binding agent (e.g., a target-binding ligand, e.g., a target-binding protein, e.g., a miniprotein) is of a different scaffold than a decoy, the decoy may be a Scaffold A decoy (e.g., such as exemplified herein, e.g., compounds C78-C108 (polypeptides having amino acid sequences comprising SEQ ID NOs: 16-46) compounds C78, C79, C96 or polypeptides having amino acid sequences of SEQ ID NOs: 16, 17, or 34) and a target-binding agent may be, for example, a Scaffold B, C, J, or L miniprotein, or, in some embodiments, target-binding agent that binds to Nectin-4 (e.g., zelenectide-pevedotin, e.g., C143, e.g., SEQ ID NO: 88), a binding agent that binds to prostate-specific membrane antigen (e.g., vipivotide textrazetan, e.g., C170), or a binding agent that binds to somatostatin receptor 2 (e.g., dotatate, e.g., C144, e.g., SEQ ID NO: 89).

[0291] In certain embodiments, a scaffold comprises a comparator compound, comprising or according to those in TABLE 17. Page 87 of 248 IPTS / 128939095.1Docket No.: AKT-033WO TABLE 17. Exemplary Comparator Compounds Compound N-Term. Sequence or Structure7SEQ ID C-Term. ID NO6NO:8

[0292] In some embodiments, miniproteins of the present disclosure comprise or consist of a cysteine-dense peptides (CDPs). In some embodiments, conjugates provided herein comprise a CDP. In some embodiments, a CDP functions as a targeting moiety, e.g., specifically binding to a protein target expressed on the surface of a target tumor cell. In some embodiments, a CDP comprises or consists of at least two independent folding domains and a high density of cysteines. In some embodiments, the CDP comprises at least one, two, three, four, five, six, or more than six cysteine residues in a span of from about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues. (See, e.g., Correnti et al., Nat Struct Mol Biol.2018 Mar;25(3):270-278, for exemplary CDPs and characteristics thereof). Knottins

[0293] In some a embodiments, miniproteins of the present disclosure comprise or consist of knottin peptides. In some embodiments, conjugates provided herein comprise a knottin peptide. In some embodiments, a knottin peptide functions as a targeting moiety, e.g., specifically binding to a target protein expressed on the surface of a target tumor cell. In some embodiments, a knottin comprises at least three disulfide bonds connected in an arrangement that generates the so-called “cysteine-knot” for which knottins are named. (See, e.g., Kintzing & Cochran et al., Curr Opin Chem Biol.2016 Oct;34:143-150.). In some embodiments, knottins have high stability (e.g., thermal, proteolytic, chemical, etc.). In some6Each compound is identified via a compound # (e.g., “C1”, “C2”, C3”, etc.) and refers to the combination of the N-terminal, Linker (if present), Sequence, andIPTS / 128939095.1Docket No.: AKT-033WO embodiments, a knottin can be further engineered to modify binding, folding, and / or related properties.

[0294] In some embodiments, a given knottin is highly specific for a given target. In some embodiments, a knottin specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the knottin specifically binds to any one of the target proteins in TABLE 10 or a fragment thereof. In some embodiments, a knottin is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is via a linker. It will be understood by those of skill in the art, that in some embodiments, the particular knottin employed in a conjugate of the present disclosure may vary depending on the target protein of interest.

[0295] In some embodiments, folded structures of miniproteins (e.g., CDPs, knottins, binders, affibodies, engineered Kunitz domains, monobodies, anticalins, designed ankyrin repeat domains (DARPins), avimers) make them rigid, providing for very tight and potent binding to the target protein or antigen (relative to less structured peptides). In some such embodiments, a miniprotein (e.g., CDP, knottin, binder, affibody, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) exhibits extraordinary stability with resistance to heat, peptidase cleavage, and pH. Binders

[0296] In some embodiments, a miniprotein of the present disclosure comprises or consists of a binder. In some embodiments, the binder functions as a targeting moiety, e.g., specifically binding to a target expressed on the surface of a tumor cell.

[0297] In some embodiments, a binder has certain structural features; for example, in some embodiments, a binder may be rich in alpha-helices, such as a helix-helix-helix structure (see, e.g., Crook et al., Nat Commun. (2017) 8, 2244; Berger et al, Elife (2016) 5, e20352; and Procko et al., Cell (2014), 157, 1644-1656). In some embodiments, a binder comprises sufficient surface to functionalize the molecule on a disparate surface to a binding surface. In some embodiments, a binder comprises a sequestered hydrophobic core. In some embodiments, a binder displays cooperative folding. In some embodiments, a binder has two or more of the following features: (i) represented by an amino acid sequence of 100 amino acids or fewer; (ii) at least two secondary structure elements; (iii) a sequestered hydrophobic core; and / or (iv) cooperative folding.

[0298] In some embodiments, a given binder is highly specific for a given target. In some embodiments, a binder specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the binder specifically binds to any one Page 89 of 248 IPTS / 128939095.1Docket No.: AKT-033WO of the target proteins in TABLE 10 or a fragment thereof. In some embodiments, a binder is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is via a linker. It will be understood by those of skill in the art, that in some embodiments, the particular binder employed in a conjugate of the present disclosure may vary depending on the target protein or antigen of interest. Affibodies

[0299] In some embodiments, miniproteins of the present disclosure comprise or consist of affibodies. In some embodiments, conjugates provided herein comprise an affibody. In some embodiments, an affibody functions as a targeting moiety, e.g., specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, an affibody comprises or consists of no more than 100 amino acids, 90 amino acids, 80 amino acids, 70 amino acids, 60 amino acids, 50 amino acids, 40 amino acids, 30 amino acids, 20 amino acids, or 10 amino acids. In some embodiments, an affibody comprises or consists of at least three alpha helices with 58 amino acids. In some embodiments, the affibody comprises target specificity that is obtained by randomization of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol.899:103-26). In some embodiments, an affibody can be further engineered to modify binding, folding, and / or related properties.

[0300] In some embodiments, an affibody specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the affibody specifically binds to any one of the target proteins in TABLE 10 or a fragment thereof. In some embodiments, an affibody is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is via a linker. It will be understood by those of skill in the art, that in some embodiments, the particular affibody employed in a conjugate of the present disclosure may vary depending on the target protein or antigen of interest. Engineered Kunitz Domains

[0301] In some embodiments, miniproteins of the present disclosure comprise or consist of engineered Kunitz domains. In some embodiments, conjugates provided herein comprise an engineered Kunitz domain. In some embodiments, an engineered Kunitz domain functions as a targeting moiety, e.g., specifically binding to a protein target expressed on the surface of a target tumor cell. In some embodiments, an engineered Kunitz domain comprises or consists of at least one peptide derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) Page 90 of 248 IPTS / 128939095.1Docket No.: AKT-033WO or tissue factor pathway inhibitor (TFPI). In some embodiments, an engineered Kunitz domain can be further engineered to modify binding, folding, and / or related properties.

[0302] In some embodiments, an engineered Kunitz domain specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the engineered Kunitz domain specifically binds to any one of the target proteins in TABLE 10 or a fragment thereof. In some embodiments, an engineered Kunitz domain is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is via a linker. It will be understood by those of skill in the art, that in some embodiments, the particular engineered Kunitz domain employed in a conjugate of the present disclosure may vary depending on the target protein of interest. Monobodies / Adnectins

[0303] In some embodiments, miniproteins of the present disclosure comprise or consist of monobodies, which is also used interchangeably with the term “Adnectins”. In some embodiments, conjugates provided herein comprise an monobody. In some embodiments, an monobody functions as a targeting moiety, e.g., specifically binding to a protein target expressed on the surface of a target tumor cell. In some embodiments, an monobody comprises or consists of a molecule based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts an Ig-like b-sandwich fold of about 94 residues with 2 to 3 exposed loops, but lacks the central disulfide bridge. In some embodiments, an monobody can be further engineered to modify binding, folding, and / or related properties. For example, in certain embodiments, modifications can be introduced on solvent-exposed residues such as to impact binding affinity and / or specificity.

[0304] In some embodiments, a monobody specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the monobody specifically binds to any one of the target proteins in TABLE 10 or a fragment thereof. In some embodiments, a monobody is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is via a linker. It will be understood by those of skill in the art, that in some embodiments, the particular monobody employed in a conjugate of the present disclosure may vary depending on the target protein of interest. Anticalins

[0305] In some embodiments, miniproteins of the present disclosure comprise or consist of anticalins. In some embodiments, conjugates provided herein comprise an anticalin. In some embodiments, an anticalin functions as a targeting moiety, e.g., specifically binding to a protein target expressed on the surface of a target tumor cell. In some embodiments, an Page 91 of 248 IPTS / 128939095.1Docket No.: AKT-033WO anticalin comprises or consists of an eight-stranded ^-barrel which forms a highly conserved core unit among the lipocalins and naturally forms binding sites for ligands by means of four structurally variable loops at the open end. In some embodiments, an anticalin can be further engineered to modify binding, folding, and / or related properties.

[0306] In some embodiments, an anticalin specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the anticalin specifically binds to any one of the target proteins in TABLE 10 or a fragment thereof. In some embodiments, an anticalin is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is via a linker. It will be understood by those of skill in the art, that in some embodiments, the particular anticalin employed in a conjugate of the present disclosure may vary depending on the target protein of interest. Designed Ankyrin Repeat Domains

[0307] In some embodiments, miniproteins of the present disclosure comprise or consist of designed Ankyrin repeat domains. In some embodiments, conjugates provided herein comprise a designed Ankyrin repeat domain. In some embodiments, a designed Ankyrin repeat domain functions as a targeting moiety, e.g., specifically binding to a protein target expressed on the surface of a target tumor cell. In some embodiments, a designed Ankyrin repeat domain comprises a peptide derived from Ankyrin. In some embodiments, a designed Ankyrin repeat domain comprises a single ankyrin repeat, preferably comprising a 33 residue motif comprising two alpha-helices and a beta-turn. In some embodiments, a designed Ankyrin repeat domain provides a rigid interface and lacks structural flexibility. In some embodiments, a designed Ankyrin repeat domain can be further engineered to modify binding, folding, and / or related properties.

[0308] In some embodiments, a designed Ankyrin repeat domain specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the designed Ankyrin repeat domain specifically binds to any one of the target proteins in TABLE 10 or a fragment thereof. In some embodiments, a designed Ankyrin repeat domain is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is via a linker. It will be understood by those of skill in the art, that in some embodiments, the particular designed Ankyrin repeat domain employed in a conjugate of the present disclosure may vary depending on the target protein of interest. Page 92 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Avimers

[0309] In some embodiments, miniproteins of the present disclosure comprise or consist of avimers. In some embodiments, conjugates provided herein comprise an avimer. In some embodiments, an avimer functions as a targeting moiety, e.g., specifically binding to a protein target expressed on the surface of a target tumor cell. In some embodiments, an avimer comprises a peptide of about 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, 80 amino acids, 90 amino acids, or 100 amino acids. In some embodiments, an avimer comprises at least one peptide sequence of about 30 to 35 amino acids. In some embodiments, an avimer comprises two or more of two peptide sequences of about 30 to 35 amino acids. In some embodiments, an avimer comprises one or more peptide sequences derived from A-domains of various membrane receptors. (Weidle UH,–et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68). For further details see Nature Biotechnology 23(12), 1556 - 1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). In some embodiments, an avimer can be further engineered to modify binding, folding, and / or related properties.

[0310] In some embodiments, an avimer specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the avimer specifically binds to any one of the target proteins in TABLE 10 or a fragment thereof. In some embodiments, an avimer is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is via a linker. It will be understood by those of skill in the art that in some embodiments, the particular avimer employed in a conjugate of the present disclosure may vary depending on the target protein of interest. Albumin binding domains

[0311] In exemplary aspects, such as to reduce or prevent kidney localization (e.g., retention, e.g., uptake, etc.), in some embodiments, a composition provided herein comprises amino acid sequences comprising albumin binding domains. Thus, such compositions can bind to albumin, which, in circulation, can extend the circulating half-life of the amino acid sequence and reduce kidney retention. In some embodiments, the composition comprising amino acid sequences comprising albumin binding domains comprises an amino acid sequence of any one of SEQ ID NOs: 7 or 13. In some embodiments, the composition comprising amino acid sequences comprising albumin binding domains comprises a compound selected from any of C12, C23-C25, C29, C30, C32, C33, C66, C69, or C71. Page 93 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Decharged Molecules

[0312] In exemplary aspects, including such as to reduce or prevent kidney localization (e.g., retention, e.g., uptake, etc.) a composition of the disclosure comprises a decharged molecule or molecules that have fewer charges that enable rapid clearance through the kidney. In some embodiments, a reduction in positively charged or polar molecules, an increase in negatively charged molecules, and / or both results in reduced kidney retention.

[0313] In some embodiments, the composition comprising a decharged molecule or molecules comprises an amino acid sequence comprising any one of SEQ ID NOs: 1-3, 8-11, or 65-67. In some embodiments, the composition comprising a decharged molecule or molecules comprises a compound selected from any of C1-C6, C43-54, or C155-C157.

[0314] In some embodiments, the composition wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 1-5% of the total amino acid sequence, wherein the charged amino acids are selected from Lys, Arg, or His, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 5-10% of the total amino acid sequence, wherein the charged amino acids are selected from Lys, Arg, or His, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 10-15% of the total amino acid sequence, wherein the charged amino acids are selected from Lys, Arg, or His, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 15-20% of the total amino acid sequence, wherein the charged amino acids are selected from Lys, Arg, or His, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 20-25% of the total amino acid sequence, wherein the charged amino acids are selected from Lys, Arg, or His, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises an amino acid sequence comprising a percentage of charged amino acids between 25-30% of the total amino acid sequence, wherein the charged amino acids are selected from Lys, Arg, or His, is characterized to exhibit reduced kidney uptake. In some embodiments, the reduced percentage of positively charged amino acids in M results in decreased reabsorption of M at the negatively charged membrane of the renal proximal tubule cells. Page 94 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0315] In some embodiments, the composition wherein M comprises wherein an amino acid sequence comprising a percentage of charged amino acids between 1-5% of the total amino acid sequence, wherein the charged amino acids are selected from Asp or Glu, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises wherein an amino acid sequence comprising a percentage of charged amino acids between 5-10% of the total amino acid sequence, wherein the charged amino acids are selected from Asp or Glu, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises wherein an amino acid sequence comprising a percentage of charged amino acids between 10-15% of the total amino acid sequence, wherein the charged amino acids are selected from Asp or Glu, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises wherein an amino acid sequence comprising a percentage of charged amino acids between 15-20% of the total amino acid sequence, wherein the charged amino acids are selected from Asp or Glu, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises wherein an amino acid sequence comprising a percentage of charged amino acids between 20-25% of the total amino acid sequence, wherein the charged amino acids are selected from Asp or Glu, is characterized to exhibit reduced kidney uptake. In some embodiments, the composition wherein M comprises wherein an amino acid sequence comprising a percentage of charged amino acids between 25-30% of the total amino acid sequence, wherein the charged amino acids are selected from Asp or Glu, is characterized to exhibit reduced kidney uptake. In some embodiments, the reduced percentage of polar amino acids in M results in decreased reabsorption of M at the negatively charged membrane of the renal proximal tubule cells.

[0316] Example 10 and Example 11 and FIG.1 and FIG.3 provide exemplary data on the level of kidney retention measured in mice treated with radioactively-labeled peptides and imaged via SPECT / CT. In some embodiments, the percentage of ID / g in the kidney at 4 hours is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% when the composition comprises a percentage of charged amino acids compared to a composition in which the percentage of charged amino acids has not been decreased. In some embodiments, the percentage of ID / g in the kidney at 24 hours is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% when the composition comprises a percentage of charged amino acids compared to a composition in which the percentage of charged amino acids has not been increased. Page 95 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Linkers

[0317] In some embodiments, the present disclosure provides linkers for use in one or more conjugates. For example, in some embodiments, a linker is linked to a chelator. In some embodiments, a linker is linked to a chelator, which itself is coupled to a radionuclide. In some embodiments, a target-binding agent (e.g., a target-binding polypeptide, e.g., a miniprotein) is conjugated to a chelator and / or radionuclide. In some embodiments, the target-binding agent is conjugated to a chelator, optionally, through a linker. In some embodiments, a composition as provided herein comprises one or more linkers.

[0318] As described herein, in some embodiments, a conjugate of the disclosure comprises a linker. In some embodiments, the linker functions to connect the chelator to target-binding agent. In some embodiments, a linker is non-cleavable. In some embodiments, a linker is cleavable. For example, in certain embodiments, a linker can be a cleavable linker as set forth in compounds of TABLE 3.

[0319] In some embodiments, selection and placement of one or more linkers and chelators on a target-binding agent (e.g., polypeptide, e.g., miniprotein) aids to maintain desired potency and receptor engagement profile, enhance binder affinity and optimize physicochemical and pharmacokinetic properties of a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) or conjugate thereof. Any suitable linker known in the art can be utilized. Exemplary linkers include, but are not limited to polyethylene glycol (PEG) linkers, an ester linker, an amide linker, a maleimide linker, a valine-citrulline linker, a hydrazone linker, a N-succinimidyl-4- (2-pyridyldithio)butyrate (SPDB) linker, a succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, a vinylsulfone-based linker, a propanoic acid linker, a caproleic acid linker, or a linker including any combination thereof. One or more additional linkers may be contemplated as will be known to those of skill in the art and chosen given the context and components of a given composition.

[0320] In some embodiments, a linker (e.g., of a compound, e.g., of a conjugate provided herein) is a PEG linker. In some embodiments, the linker is a non-cleavable PEG linker. In some embodiments, the PEG linker is PEG (4-24).

[0321] In some embodiments, linkers are used to assess lead polypeptide sequences binding to a target, a target expressed on cells, and target selectivity and / or affinity. For instance, in some embodiments, confirmation of in vitro on-target binding and affinity for lead polypeptide sequences and lead polypeptide sequences-linker-fluorophore reagent can be Page 96 of 248 IPTS / 128939095.1Docket No.: AKT-033WO assessed using Biacore. In some embodiments, other linkers such as a fast clear linker or a halogen linker are also contemplated. Cleavable Linker

[0322] In certain aspects to reduce or prevent kidney localization (e.g., retention, e.g., uptake, etc.), in some embodiments, a composition of the disclosure comprises M-L-C-R, wherein L is a cleavable linker and M is a miniprotein. In some embodiments, the cleavable linker is distal to the miniprotein to not interfere with tumor binding. In some embodiments, the cleavable linker is engineered to optimize physicochemical characteristics. In some embodiments, a cleavable linker has a short length. In some embodiments, the cleavable linker is selectively cleaved in the proximal tubule of the kidney. In some embodiments, selective cleavage of the cleavable linker in the proximal tubule of the kidney is due to the presence of peptidases in the proximal tubule. In some embodiments, cleavage of the cleavable linker in the proximal tubule releases chelator-radionuclide-miniprotein that filters into the bladder and avoids kidney reuptake. See Arano, Y. Nuclear Medicine and Biology 2021, 92, 149-55; Zhang, M. et. Al. Bioconjugate Chemistry 2019, 30, 1745-53.

[0323] In some embodiments, the composition comprising a cleavable linker comprises an amino acid sequence comprising any one of SEQ ID NOs: 7-11, 17-23, 26, or 27. In some embodiments, the composition comprising a cleavable linker comprises a compound selected from any of C15, C17-C22, C26-C28, C34-C42, C55-66, C68, C70, or C72-C77.

[0324] In some embodiments, the cleavable linker is connected to M at the Na-carboxyl of lysine. In some embodiments, the cleavable linker comprises hydrogen, (PEG4)1-4, 4- aminomethyl-phenylacetic acid (AmPA), aminomethylbenzoyl (AmBz), (succinic acid- (PEG4)1-4, norleucine, ileucine, glutamine, methoxinine, phenylalanine, tyrosine, beta alanine, MWK or MVK, glycine, citrulline (Cit), or sarcosine (Sar). In some embodiments, the linker is cleaved by cathepsin B in a lysosome or a neutral endopeptidase, metalloprotease, or dipeptidyl peptidase in a kidney brush border membrane.

[0325] Described in Example 10 and Example 11 are the level of kidney retention measured in mice treated with radioactively-labeled peptides and imaged via SPECT / CT. In some embodiments, the cleavable linker reduces the uptake of M in the kidney, as shown in FIG.5 and FIG.13.

[0326] In some embodiments, the cleavable linker is a disulfide bond or protease sensitive. In a further embodiment, the groups adjacent to the disulfide bond are modified to control the hindrance of the disulfide bond, and by this the rate of cleavage. Published work established Page 97 of 248 IPTS / 128939095.1Docket No.: AKT-033WO the potential for modifying the susceptibility of the disulfide bond to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction by intracellular glutathione and also extracellular (systemic) reducing agents, consequentially reducing the ease by which the rest of the conjugate is released, both inside and outside the cell. Thus, selection of the optimum in disulfide stability in the circulation (which minimizes undesirable side effects of the radionuclide) versus efficient release in the intracellular milieu (which maximizes the therapeutic effect) can be achieved by careful selection of the degree of hindrance on either side of the disulfide bond. The hindrance on either side of the disulfide bond is modulated through introducing one or more methyl groups on either the miniprotein or radionuclide side of the molecular construct. Chelators

[0327] In some embodiments, a composition (e.g., conjugate) as provided herein comprises a linker. In some embodiments, a composition comprises a linker and a chelator. In some embodiments, a composition comprises a linker, a chelator, and a radionuclide. In some embodiments, a composition comprises a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) , optional linker, chelator, and / or radionuclide. In some embodiments, a chelator is covalently attached to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) . In some embodiments, a chelator binds to a radionuclide. In some embodiments, a chelator refers to any molecule or moiety that “binds” to a metal ion, in solution (effectively collecting / binding up metal ions so that they may, e.g., no longer participate in one or more cellular activities or processes). In some embodiments, a chelator chelates one or more components of a metabolic pathway in a cell (e.g., metal ions, e.g., copper, iron, zinc, etc.). In some such embodiments, a chelator disrupts a life-cycle of a cancer cell and may, in some embodiments, reduce its viability, function, and / or ability to grow or proliferate. In some embodiments, a chelator chelates one or more toxins that are produced as a result of targeted radiotherapy (e.g., to reduce toxicity of the therapy).

[0328] In some embodiments, a chelator comprises or consists of, but is not limited to diethylenetriamine pentaacetic acid (DTPA), tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), l,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7- triazacyclononane-Ν,Ν',Ν"-triacetic acid (NOTA), ({4-[2- (bis- carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7] triazonan-l-yl}acetic acid (ΝΕΤΑ), Page 98 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Macropa, and p-bromoacetamidobenzyl-tetraethylaminetetraacetic acid (TETA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes. In some embodiments, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelator is Macropa. In some embodiments, a chelator comprises or consists of: (i) NOPO:Page 99 of 248 IPTS / 128939095.1Docket No.: AKT-033WO (iv) Macropa.

[0329] In additional embodiments, the chelation conditions are optimized using methods known to those of skill in the art (see, e.g., J Nucl Med.1998 Dec;39(12):2105-10). In some embodiments, chelation efficiency is about > 99%, > 98%, > 97%, > 96%, > 95%, > 94%, > 93%, > 92%, > 91%, > 90%, > 89%, > 88%, > 87%, > 86%, > 85%, > 84%, > 83%, > 82%, >81%, or > 80%.

[0330] In some embodiments, a chelator for use in a composition as described herein is chosen based on if and which radionuclide is present. As provided herein, in some embodiments, a chelator is DOTA, NOPO, Crown, or Macropa. In some embodiments, DOTA is the chelator and the radionuclide is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211. In some embodiments, Crown is the chelator and the radionuclide is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La- 135, In-111, Ce-134, F-18, or At-211. In some embodiments, NOPO is the chelator, and the radionuclide is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce- 134, F-18, or At-211. In some embodiments, Macropa is the chelator, and the radionuclide is Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At- 211.

[0331] In some embodiments, a particular chelator or type of chelator may be chosen for certain applications. For instance, in some embodiments, NOPO is used in diagnostic or theranostic applications. In some embodiments, Crown is used for therapeutic applications. In some embodiments, DOTA is used for diagnostic, theranostic, and / or therapeutic applications. In some embodiments, Macropa is used for diagnostic, theranostic, and / or therapeutic applications.

[0332] It is recognized that screening chelators for certain characteristics is within the scope of this disclosure and methods for such screening are known to those of skill in the art. For example, in some embodiments, chelators are screened for their ability to bind Page 100 of 248 IPTS / 128939095.1Docket No.: AKT-033WO radionuclides (e.g., Ga68, Ac225 and daughter(s) of Ac225 (Bi213)) and display serum stability.

[0333] In some embodiments, a target-binding conjugate described herein comprises a chelator. Any suitable chelator known in the art can be utilized. In some embodiments the chelator is directly conjugated to the miniprotein. In some embodiments, the chelator is indirectly connected to the target-binding agent through a linker. In some embodiments, the chelator is indirectly connected to the target-binding agent through a linker (e.g., a linker described herein). Radionuclides

[0334] In some embodiments, the present disclosure provides one or more radionuclides for use in a composition (e.g., conjugate).

[0335] In certain embodiments, a composition comprises a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) and a radionuclide.

[0336] In some embodiments, conjugates (e.g., comprising a target-binding agent) comprise a radionuclide bound to a chelator. As will be understood to those of skill in the art, any suitable radionuclide known in the art may be used. In some embodiments, a radionuclide is selected for imaging of a tumor with in a human having cancer. In some embodiments, a radionuclide is selected for its inability to kill cells in vivo. In some embodiments, the radionuclide is selected for its ability to kill cells in vivo.

[0337] In some embodiments, a composition of the present disclosure comprises one or more cytotoxic payloads including particle-emitting isotopes such as alpha-, beta-particles, and Auger electrons in radiotherapeutic applications. In some embodiments, a radionuclide of the present disclosure is an alpha emitter. As will be known to those of skill in the art, in some embodiments, an alpha emitter has a more localized area of impact such that when internalized into a cell it will act to, e.g., kill a cancer cell, but will spare surrounding tissue from extensive damage such as could occur with use of a beta or gamma emitter.

[0338] Studies have evaluated alpha nuclide therapy versus beta nuclide therapy with the stronger clinical results pointing to alpha nuclides. In some embodiments, a benefit of alpha therapy is that the short path length means patients do not have to physically distance themselves from family and health care providers making treatment more tolerable. Further, in some embodiments, alpha therapy exhibits better cell killing potency due to its ability to induce double stranded DNA breaks. Page 101 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0339] In some embodiments, a composition comprises a linker, chelator, and radionuclide. In some embodiments, a composition comprises a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) , optional linker, chelator, and a radionuclide. Without being bound by any particular theory, the present disclosure contemplates that a wide variety of radionuclides can be used in the pharmaceutical composition or as a diagnostic. Exemplary radionuclides, include but are not limited to, Actinium-225, Astatine-211, Bismuth-212, Bismuth-213, Cesium-137, Chromium-51, Cobalt-60, Copper-64 Dysprosium-165, Erbium-169, Fermium-255, Fluor-18, Gallium-67, Gallium-68, Gold-198, Holmium-166, Indium-111, Iodine-123, Iodine-124, Iodine-125, Iodine-131, Iridium-192, Iron-59, Lead-212, Lutetium-177, Molybdenum-99, Palladium-103, Phosphorus-32, Potassium-42, Rhenium-186, Rhenium-188, Samarium-153, Technetium-99m, Radium-223, Ruthenium-106, Sodium-24, Strontium-89, Terbium-149, Thorium-227, Xenon-133, Ytterbium-169, Ytterbium-177, Yttrium-90, and Zirconium-89. Accordingly, in some embodiments, a radionuclide is selected from: iodine (131I or 125I), yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (211At), rhenium (186Re), bismuth (212Bi or 213Bi), indium (111In), technetium (99Tc), phosphorus (32P), rhodium (188Rh), sulfur (35S), carbon (14C), tritium (3H), chromium (51Cr), chlorine (36Cl), cobalt (57Co or 58Co), iron (59Fe), selenium (75Se), or gallium (67Ga) or (68Ga). In some embodiments, the present disclosure contemplates that certain radioisotopes may be useful in or as therapeutic agents including but not limited to yttrium (90Y), lutetium (177Lu), actinium (225Ac), praseodymium, astatine (211At), rhenium (186Re), bismuth (212 Bi or 213Bi), and rhodium (188Rh). In some embodiments, radioisotopes are useful as labels, e.g., for use in diagnostics. In some such embodiments, such radioisotopes may include but are not limited to iodine (131I or 125I), indium (111In), technetium (99Tc), phosphorus (32P), carbon (14C), lead (212Pb) or tritium (3H). See, e.g., US Patent No. 7514078.

[0340] In some embodiments, radionuclides are conjugated to different complexing agents and chelators. In some embodiments, chelators are identified and attached / bound to a target- binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) through a linker or by acyclic, cyclic and macrocyclic chelates such as, for example, 1,4,7,10,13,16-hexaazacyclohexadecane-N,N′,N″,N‴,N′‴,N″‴-hexaacetic acid (HEHA), 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA), NOPO, Crown, etc. In some embodiments, certain chelators may be preferred for certain radionuclides such as, for example, Ac-225 with DOTA or Crown, Ga-68 with NOPO, etc. In Page 102 of 248 IPTS / 128939095.1Docket No.: AKT-033WO some embodiments, preferred combinations of chelators and radionuclides comprise one or more of the following: DOTA and Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211; Crown and Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211; NOPO and Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211; and / or Macropa and Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211.

[0341] Preferably, in some embodiments, a preferred radionuclide complex comprises Ac- 225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At- 211. In some such embodiments, such a complex with desired stability is selected. That is, in some embodiments, a complex comprising Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, or At-211 is characterized as having better stability in vivo in comparison to other complexes. Without wishing to be bound by theory, the present disclosure contemplates that, in some embodiments, a radionuclide complex comprising a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) forms with the target (e.g., any one of the target proteins in TABLE 10 or a fragment thereof). In some such embodiments, such a complex is internalized in the target cell.

[0342] In some embodiments, a radionuclide complex forms with a chelator (e.g., DOTA, NOPO, Crown, Macropa, etc.) and is considerably more stable in vivo. In some embodiments, a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) forms internalizing complexes with targets (e.g., any one of the target proteins in TABLE 10).

[0343] In some embodiments, a composition provided by the present disclosure comprises Actinium-225. In some embodiments, a composition provided by the present disclosure comprises gallium (Ga-68). In some embodiments, a composition provided by the present disclosure comprises copper (Cu-64). In some embodiments, a composition provided by the present disclosure comprises indium (In-111). In some embodiments, a composition provided by the present disclosure comprises lutetium (Lu-177). In some embodiments, a composition provided by the present disclosure comprises lead (Pb-212) In some embodiments, a composition provided by the present disclosure comprises copper (Cu-67). In some embodiments, a composition provided by the present disclosure comprises lutetium (Lu-177). In some embodiments, a composition provided by the present disclosure comprises lanthanum (La-132). In some embodiments, a composition provided by the present disclosure Page 103 of 248 IPTS / 128939095.1Docket No.: AKT-033WO comprises lanthanum (La-135). In some embodiments, a composition provided by the present disclosure comprises indium (In-111). In some embodiments, a composition provided by the present disclosure comprises cerium (Ce-134). For example, in some embodiments, radioimmunotherapy comprising Ac-225 may provide i) limited range in tissue of a few cell diameters; ii) high linear energy transfer leading to dense radiation damage along each alpha track; iii) a 10 day half-life; and / or iv) four net alpha particles emitted per decay (see, e.g., as described in Scheinberg, David A, and Michael R McDevitt. “Actinium-225 in targeted alpha-particle therapeutic applications.” Current radiopharmaceuticals vol.4,4 (2011): 306- 20).

[0344] In some embodiments, targeting constructs (e.g., 225-Ac-drug constructs, e.g., 68- Ga-constructs) have potential for use in cancer. For example, in some such embodiments, such constructs may be used in the treatment of cancer, such as, for example 225-Ac-drug constructs. In some embodiments, such constructs may be used in imaging, such as for prognostics, diagnostics, and / or monitoring, such as Ga-68 or Cu-64-based constructs.

[0345] In some embodiments, Ac-225 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the actinium is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0346] In some embodiments, Ga-68 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the gallium is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0347] In some embodiments, Cu-64 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the copper is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0348] In some embodiments, In-111 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) Page 104 of 248 IPTS / 128939095.1Docket No.: AKT-033WO as provided herein. In some embodiments, the indium is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0349] In some embodiments, Lu-177 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the lutetium is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0350] In some embodiments, Pb-212 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the lead is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0351] In some embodiments, Cu-67 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the copper is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0352] In some embodiments, La-132 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the lanthanum is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0353] In some embodiments, La-135 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the lanthanum is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10). Page 105 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0354] In some embodiments, In-111 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the indium is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0355] In some embodiments, Ce-134 is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) as provided herein. In some embodiments, the cerium is conjugated onto a chelator and may include an optional linker to link it to a target-binding agent, which target-binding agent targets the conjugate to a cell expressing the target (e.g., any one of the target proteins in TABLE 10).

[0356] For avoidance of doubt, the disclosure contemplates target-binding agents comprising radionuclides and use in combination with a decoy. In some such embodiments, the decoy reduces and / or suppresses uptake of the radionuclide-comprising agent into a non- target tissue, such that one or more aspects of the radionuclide-containing composition and / or its use are improved (e.g., reduced toxicity to a population of cells and / or an individual, e.g., more effective therapy due to improved targeting of a population of cancer cells and / or ability to give increased doses and / or cycles of the radionuclide-containing therapeutic, etc.).

[0357] In some embodiments, alpha particles (e.g., of Actinium-225, etc.) are positively charged. In some such embodiments, the range of penetration in tissue varies between 5 and 10 cell diameters (40 to 100 μm) depending on their energy (Radiobiologic principles in radionuclide therapy. Kassis AI, Adelstein SJ J Nucl Med.2005 Jan; 46 Suppl 1():4S-12S). In some such embodiments, such penetration allows for localized irradiation of target cells with minimal toxicity on surrounding normal cells, and internalization by cancer cells with as few as 1–3 tracks across the cell nucleus resulting in cell death (Humm 1987; Macklis et al 1988; Humm and Chin 1993; Couturier et al 2005) causing single- and double-stranded DNA breaks. See, e.g., Sofou S. Radionuclide carriers for targeting of cancer. Int J Nanomedicine. 2008;3(2):181-199. doi:10.2147 / ijn.s2736. Dose Calculation

[0358] In some embodiments, a dose of a radiotherapeutic is calculated. In some such embodiments, calculation of an absorbed dose (D) is necessary to quantitatively correlate tumor response to a particular radiotherapeutic modality and to project on the potential effect of other radiotherapeutic modalities or administration strategies. That is, in some Page 106 of 248 IPTS / 128939095.1Docket No.: AKT-033WO embodiments, the absorbed dose from a target site is defined as the energy (E) absorbed by a particular mass of tissue, normalized by the tissue mass (M): D = E / M (Sgouros 2005). The absorbed energy is defined as a function of three parameters: the number of disintegrations within the particular volume of interest (δ), the energy emitted per disintegration (ε), and the fraction of emitted energy that is absorbed by the particular volume of interest (the target mass) (f): E = δ × ε × f. For the relatively long range beta emitters, the dose evaluation at a target site includes not only the energy emitted by radionuclides localized within the target volume, but also the energy emitted by radionuclides accumulated in neighboring organs or areas whose emissions cross along their path the target volume of interest (Kolbert et al 2003). In other words, in some embodiments, the calculated total absorbed dose is the sum of the dose contributions from all regions containing radionuclides that act as secondary sources. In some embodiments, the adsorbed dose due to photon emissions is usually calculated separately and added to the dose due to alpha or beta particles. In some embodiments, where a composition comprises an alpha particle emitter, such cross organ absorbed doses may be of no significance due to their short recoil distances. In some embodiments, given appropriate context, at the micron-scale and at distances comparable to a few cells, microdosimetric evaluations are used to evaluate dose or ‘hits’ acquired by cancer cells within micrometastatic clusters (Palm et al 2002).

[0359] In some embodiments, a miniprotein conjugate comprising a radionuclide displays binding specificity to the human isoform of any one of the target proteins in TABLE 10. In some embodiments, the a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) comprises a binding affinity characterized by a dissociation constant ranging from about 500 nM to about 1 pM, e.g., 500, 400, 300.200, 100, 90, 80, 70, 60, 50, 40, 3020, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nM, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM binding affinity to the human isoform of any one of the target proteins in TABLE 10. Without being bound by any particular theory, the present disclosure contemplates that, in some embodiments, a preferred dissociation constant of a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) is about 10 nM or less, about 7.5 nM, about 5 nM or less, about 2.5 nM or less, about 1 nM or less (e.g., in the picomolar range).

[0360] In some embodiments, compositions as provided herein are characterized for one or more of absorbed dose, dose rate, tumor penetration profile of radionuclides, intracellular localization profiles of radionuclides of shorter range, and tumor radiosensitivity (see, e.g., Page 107 of 248 IPTS / 128939095.1Docket No.: AKT-033WO Sofou S. Radionuclide carriers for targeting of cancer. Int J Nanomedicine.2008;3(2):181- 199).

[0361] As is known to those of skill in the art, due to toxicity of radionuclides, dose needs to be carefully controlled and considered. Accordingly, in some embodiments, compositions comprising radionuclides of the present disclosure address dose-limiting toxicity of compositions such that radionuclides do not accumulate significantly (e.g., in a toxicity- limiting manner) in vital organs.

[0362] In some embodiments, alpha particle-emitting isotopes engage in on-target cell killing while minimizing toxic effects (e.g., to surrounding tissue, e.g., as compared to, e.g., beta emitters, etc.).

[0363] In some embodiments, compositions provided herein (comprising a radionuclide) are administered in a single step such as, e.g., using a ligand, e.g., a miniprotein resulting in improved biodistributions (e.g., specific targeting), pK with partial and acceptable damage or no damage to normal tissues, enhanced penetration of the pharmaceutical composition into the tumor heterogeneous interstitial space.

[0364] In some embodiments, one or more radionuclides is conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand). Relatedly, in some embodiments, radiolabeling efficiency of a target- binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) is optimized to radiolabel a desired number of radionuclides. In some embodiments, a ratio of radionuclides conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. In some embodiments, radionuclides conjugated to a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) does not present toxicity. In some embodiments, a composition comprising a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) and radionuclide does not accumulate in the liver, spleen, and the pancreas and is cleared rapidly when administered to a subject. For instance, in some embodiments, after administration to a subject, biodistribution and t 1 / 2 in the kidney is >10% of the injected dose (ID) in tumors at 24 hrs and tumors is >3% ID at 24 hrs.

[0365] Dose calculations may depend on various factors such as therapeutic indices (e.g., comparing median lethal dose and median effective dose to develop the ratio representing a therapeutic index). Dose calculations may also involve calculations based on molar or mass Page 108 of 248 IPTS / 128939095.1Docket No.: AKT-033WO excess such as of a decoy relative to a molar or mass amount of a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) of a conjugate such as a compound (e.g., a radionuclide conjugate of the disclosure), etc.)

[0366] In some embodiments, a dose calculation comprises or consists of a dose calculation for a compound comprising a radionuclide and / or a compound comprising a decoy. In some embodiments, a decoy dose calculation depends on a dose or concentration of a target-binding agent (e.g., a polypeptide (e.g., miniprotein), e.g., a small molecule, e.g., a small molecule receptor ligand) (e.g., micromolar, nanomolar, etc.). In some embodiments, a decoy dose calculation depends on a dose of a radionuclide (e.g., nCi). In some embodiments, a decoy, when administered (e.g., co-administered, e.g., before, concomitant with, after administration of a radionuclide) is dosed at an excess as compared to the radionuclide dose. For example, in some embodiments, a dose excess may be 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 750, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000, 7,500, 10,000-fold excess, or greater. In some embodiments, the excess is a fold-excess based on molarity of the concentration of a compound and / or radionuclide. In some embodiments, the excess is a fold-excess based on mass of the concentration of a compound and / or radionuclide.

[0367] In some embodiments, a dose calculation may include more than one administration of a given compound (e.g., a radioactive compound, e.g., a radionuclide- labeled compound of the disclosure, etc.). For example, in some embodiments, dose is measured as absorbed dose to a location (e.g., a tissue, e.g., an organ, e.g., kidney). In some such embodiments, dose can be measured using methods known to those of skill in the art, such as in RBE5Gy / MBq, and dose limits, such as for a particular organ, are determined by guidelines, such as accepted clinical guidelines known to those of ordinary skill in the art. Without wishing to be bound by theory, the disclosure contemplates that co-administration (e.g., of a radionuclide compound of the disclosure and a decoy of the disclosure) can allow a greater number of administrations of the radionuclide-labeled compound as compared to the radionuclide compound alone (in the absence of a decoy). In some embodiments, a number of administrations increases while absorbed dose to non-tumor tissues (e.g., kidney, e.g., liver, etc.) decreases. In some embodiments, co-administration of a decoy with a target-binding radionuclide of the disclosure reduces an absorbed dose to the kidney by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more. In some embodiments, absorbed dose to kidney is reduced 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65% or more in presence of a decoy as compared to radiotherapeutic in absence of a decoy. In some embodiments, number of administrations Page 109 of 248 IPTS / 128939095.1Docket No.: AKT-033WO achieved with co-administration increases by 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 % or more in presence of a decoy as compared to in absence of a decoy. In some embodiments, absorbed dose to the kidney decreases while number of administrations increases . In some embodiments, toxicity or risk of toxicity to the kidney in presence of a radiotherapeutic is reduced in presence of a decoy as compared to in absence of a decoy. In some embodiments, administration of the decoy will enable higher dosing with one or more radiotherapeutic treatments.as compared to dosing in the absence of a decoy with no change in toxicity grade of the therapeutic. Radionuclides and Chelation

[0368] A radionuclide can be bound to a chelator through any method known in the art. In some embodiments, chelation methods may differ based on the radionuclide and chelator selected. For example, in some embodiments, chelation can be carried out in one step by incubating the target-binding-agent-chelator conjugate with the radionuclide for a predetermined period at a predetermined temperature to achieve a sufficient amount of chelation. In some embodiments, a target-binding-agent--chelator conjugate comprises a chelator or variant thereof as provided herein (e.g., DOTA, e.g., NOPO, e.g., Crown, e.g., Macropa, etc.). In some embodiments, target-binding-agent--chelator conjugates can be chelated to a radionuclide (e.g., Actinium-225, Gallium-68, Copper-64, Lutetium-177, Indium-111, Lead-212, etc.) by incubation with the radionuclide for about 1 hour at 70°C. In some embodiments, target-binding-agent--chelator conjugates can be chelated to a radionuclide (e.g., Actinium-225, Gallium-68, Copper-64, Lutetium-177, Indium-111, Lead- 212, etc.) by incubation with the radionuclide for about 1 hour at 70°C.

[0369] In some embodiments, the chelation process yields a preparation in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the target-binding-agent-- chelator is bound to a radionuclide. In some embodiments, the chelation process yields a preparation in which more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the target-binding-agent--chelator is bound to a radionuclide. Excess radionuclide can be removed from the preparation by purification methods known in the art. Decoys

[0370] Decoys of the disclosure may be based on a particular scaffold (e.g., type A, type B, etc.). The scaffold of a given decoy may be the same or different from the scaffold of a polypeptide of the disclosure (e.g., a target-binding polypeptide, e.g., a target-binding miniprotein, e.g., a B7-H3-targeting miniprotein, e.g., a Nectin-4 targeting miniprotein) with which it can be combined (co-administered, such as in the same vial, or serially administered Page 110 of 248 IPTS / 128939095.1Docket No.: AKT-033WO vials, etc.), in order to act as a decoy (e.g., block uptake into kidney tissue). For example, a Scaffold B polypeptide that binds to a target (e.g., B7-H3) can be used for co-administration with either a Scaffold A or Scaffold B decoy(e.g., concomitantly, sequentially, etc.). In some embodiments, a Scaffold A polypeptide that binds to a target (e.g., Nectin-4) can be used for co-administration with either a Scaffold A decoy or a decoy of another scaffold (e.g., a non- Scaffold A decoy, e.g., non-Scaffold B decoy) (e.g., concomitantly, sequentially, etc.). In some embodiments, a Scaffold A polypeptide that binds to a target (e.g., Nectin-4) cannot be used for co-administration with a Scaffold B decoy. The present disclosure contemplates that compositions (e.g., comprising a miniprotein, e.g., target-binding miniprotein) do not need to be combined with a decoy of the same scaffold in order for the decoy to block uptake and / or retention of the miniprotein (e.g., a cold-labelled miniprotein, e.g., a radionuclide-labeled miniprotein) into non-tumor tissue (e.g., kidney, e.g., liver). That is, a decoy can be a cross- scaffolding decoy. Furthermore, the disclosure contemplates that decoys provided herein can decoy compositions (e.g., comprising polypeptides as provided herein) that have one or more modifications to an N-terminus and / or C-terminus, and / or, one or more positions on a polypeptide backbone (e.g., the polypeptide of the composition, e.g., the target-binding polypeptide). That is, a polypeptide that binds to a target (e.g., Nectin-4, e.g., B7-H3) can be modified by one or more N- and / or C-terminal modifications and / or one or more polypeptide backbone modifications and, in addition, decoys such as disclosed herein, can still block uptake and / or retention into a non-tumor tissue when co-administered with one or more such polypeptides. Compositions that can be combined with decoys of the disclosure include, for example, polypeptides (e.g., target-binding polypeptides) modified on N- and / or C-termini and / or their polypeptide backbones such as to include one or more modifications and / or payloads (e.g., radionuclide payloads, e.g., antimitotic payloads, etc.).

[0371] Decoys of the disclosure can be monomeric or multimeric. For example, in some embodiments, a decoy is a monomer. In some embodiments, a decoy is a multimer of one or more monomers of the same or different miniprotein. A multimer may have one or more monomers attached to one another by one or more linkers, such as peptide linkers, covalent bonds, non-covalent linkages, etc.

[0372] In one aspect, the disclosure provides a decoy having an amino acid sequence of any of SEQ ID NOs: 16-46 or 112-125.

[0373] In another aspect, the disclosure provides a decoy comprising or consisting of a compound selected from any of C78-C108. Page 111 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0374] In another aspect, the disclosure provides a decoy comprising or consisting of SEQ ID NO: 4.

[0375] In another aspect, the disclosure provides a decoy comprising or consisting of compound C7.

[0376] In some embodiments, a decoy is present at a concentration of about 2X, 5X, 10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X,100X, 250X, 300X, 500X, 750X, 1000x, 2500X, 5000X, 7500X, 10000X, or greater as compared to the concentration of a target-binding agent (M). Example 10, Example 11, Example 18, and Examples 20-26 each provide data on levels of kidney retention measured in mice treated with radioactively-labeled peptides and imaged via SPECT / CT, as provided herein. In some embodiments, the decoy blocks uptake and / or retention of M (e.g., of a composition of the disclosure) in the kidney, such as shown in, e.g., FIG.2, FIG.4, FIG.12, FIG.21A, and FIG.22 and liver, such as shown in, e.g., Example 18 and FIG.21B.

[0377] In certain embodiments, the target-binding-agent(e.g., target-binding miniprotein and / or compound) comprises or consists of any compound or amino acid sequence selected from TABLE 3, TABLE 4A, TABLE 4B, TABLE 11 and / or TABLE 17.

[0378] In some embodiments, a decoy is selected from TABLE 4C and / or 4D.

[0379] In certain embodiments, the decoy comprises or consists of a Scaffold A decoy. In some embodiments, the Scaffold A decoy comprises or consists of a compound selected from C78-C108 and / or has an amino acid sequence selected from any of SEQ ID NO: 16-46.

[0380] In some embodiments, the Scaffold B decoy comprises or consists of compound C7 and / or has an amino acid comprising or consisting of SEQ ID NO: 4.

[0381] In some embodiments, a Scaffold A decoy does not have an amino acid sequence comprising or consisting of any of SEQ ID NOs: 16-46 and / or does not comprise or consist of a compound selected from C78-C108.

[0382] In some embodiments, a Scaffold A decoy does not have an amino acid sequence comprising or consisting of SEQ ID NO: 4 and / or does not comprise or consist of compound C7.

[0383] In certain embodiments, the decoy comprises or consists of a Scaffold A decoy and the miniprotein comprises or consists of a Scaffold A target-binding miniprotein. In some embodiments, the Scaffold A decoy is selected from any of C78-C108 and the Scaffold A target-binding miniprotein comprises or consists of a Nectin-4 targeting protein selected from TABLE 3 or TABLE 4B. In some embodiments, the target-binding agent is a miniprotein binds to Nectin-4 and is selected from an amino acid sequence comprising, consisting Page 112 of 248 IPTS / 128939095.1Docket No.: AKT-033WO essentially of, or consisting of any of SEQ ID NOs: 7, 9-15, and 47-68 and / or compounds C12-C35, C45-C56, C58-C77, C109-C139 and C143.

[0384] In certain embodiments, the decoy comprises or consists of a Scaffold A decoy and the miniprotein comprises or consists of a Scaffold B target-binding miniprotein. In some embodiments, the Scaffold A decoy is selected from any of C78-C108 and the Scaffold B target-binding miniprotein comprises or consists of a B7-H3-targeting protein selected from TABLE 3 or TABLE 4A. In some embodiments, the target-binding miniprotein binds to B7- H3 and is selected from an amino acid sequence comprising, consisting essentially of, or consisting of any of SEQ ID NOs: 1-3, 5-6, 8, 87, 90-111 and 113-114 and / or compounds C1-C6, C8-C11, C36-C44, C57, C140-C142, C146-C166, and C168-C169.

[0385] In certain embodiments, the decoy comprises or consists of a Scaffold B decoy and the miniprotein comprises or consists of a Scaffold B target-binding miniprotein. In some embodiments, the decoy comprises or consists of C7 and the Scaffold B target-binding miniprotein comprises or consists of a B7-H3-targeting protein selected from TABLE 3 or TABLE 4A.

[0386] In certain embodiments, the decoy comprises or consists of a Scaffold A decoy and the miniprotein comprises or consists of a miniprotein that can bind to prostate-specific membrane antigen, somatostatin receptor 2, Nectin-4, or B7-H3. In some embodiments, the Scaffold A decoy comprises or consists of an amino acid sequence selected from any of compounds C78-C108 and the target-binding agent comprises or consists of a compound or the target-binding component of C143, C144, or C170.

[0387] In certain embodiments, the decoy comprises or consists of a Scaffold A decoy and the target-binding agent comprises or consists of a miniprotein that can bind to prostate- specific membrane antigen, somatostatin receptor 2, Nectin-4, or B7-H3. In some embodiments, the Scaffold A decoy comprises or consists of an amino acid sequence selected from any of compounds C78-C108 and the target-binding agent comprises or consists of a compound selected from any of C143, C144, and C170.

[0388] In certain embodiments, the disclosure provides decoys that are able to effectively decoy across scaffolds. That is, the disclosure provides decoys that can be used in combination with various types of target-binding agents. Surprisingly, these decoys can effectively and efficiently decoy various types of target-binding agents, including miniprotein conjugates of the same scaffold (A) as decoys disclosed herein, as well as other polypeptide and non-polypeptide based scaffolds, such as disclosed in TABLE 3, TABLE 4A, TABLE 4B, TABLE 11, and TABLE 17. In certain embodiments, decoys can even further provide Page 113 of 248 IPTS / 128939095.1Docket No.: AKT-033WO decoying against target-binding agents, such as disclosed in C116 and C117, and / or C145- C166, that have already been optimized (e.g., such as by introduction of small alkyl groups on amino acid side chains).

[0389] As provided herein, decoys can decoy within or across scaffolds and provide reduction in uptake by a population of kidney cells by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or even 100%. In certain embodiments, a decoy can reduce percent uptake of a target-binding agent in a non-target tissue (e.g., kidney cells, e.g., in vitro and / or in vivo) Percent reduction in uptake can be measured in vitro, such as described herein, or in vitro, including such as described herein (see, e.g., FIGS 25A-25H). In some aspects, the amino acid sequence of the decoys described herein can include an amino acid sequence that has substantial identity to any of the sequences of the decoys disclosed herein. As used herein, the term “substantial identity” means that two amino acid sequences, when optimally aligned and then analyzed by an algorithm normally used in the art, such as BLAST, GAP, or BESTFIT, or by visual inspection, share at least about 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity. Methods of alignment for sequence comparison are known in the art. In some embodiments, the polypeptide sequence is identical, but the decoy differs in one or more modifications on its N- and / or C-terminus. In some embodiments, an N-terminus of a decoy is acetylated. In some embodiments, an N-terminus is modified with an N-terminal blocking group that does not interfere with decoy function, including, for example, impacting the ability of the decoy to successfully block, interfering with decoy solubility, etc. By way of non-limiting example, in some embodiments, an N-terminal modification can comprise one or more of a free amin, alkyl blocking group, PEG, PEGylation with DOTA, etc.

[0390] In some aspects, the amino acid sequence of the decoys described herein can include a sequence (e.g., a nucleic acid sequence, an amino acid sequence) that has some degree of identity or homology to a sequence of any decoy disclosed herein. The degree of identity can vary and be determined by methods known to one of ordinary skill in the art. The terms “homology” and “identity” each refer to sequence similarity between two polypeptide or polynucleotide sequences. Homology and identity can each be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same amino acid residue or nucleic acid, then the polypeptides or polynucleotides can be referred to as identical at that position; when the equivalent site is occupied by the same amino acid or nucleic acid (e.g., identical) or a similar amino acid or nucleic acid (e.g., similar in steric and / or electronic nature), then the molecules Page 114 of 248 IPTS / 128939095.1Docket No.: AKT-033WO can be referred to as homologous at that position. A percentage of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences. The decoys described herein can have at least or about 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity or homology to a reference decoy.

[0391] In some embodiments, a decoy can bind with a binding affinity to a target as provided in TABLE 10, e.g., Nectin-4, e.g., B7-H3, but the binding affinity is at least about 10-10,000-fold (e.g., 10-100-fold, 50-500-fold, 100-1,000-fold, 250-2500-fold, 500-5,000- fold, 750-7500-fold, 1,000-5,000-fold, or 5,000-10,000-fold) weaker than a target-binding agent as characterized by KDe.g., as measured by SPR or DELFIA. In some embodiments, the binding affinity of the decoy for a target that a target-binding agent targets is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, or 10000-fold weaker than the binding affinity of the target-binding agent for the target. In some embodiments, a decoy does not bind (e.g., at all, or weaker than 10000, 5000, or 1000-fold weaker) to a target on a cancer cell, such as, e.g., provided in TABLE 10, e.g., Nectin-4, e.g., B7-H3.

[0392] In some embodiments, a target-binding agent and a decoy each have amino acid sequences that differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, and the decoy is characterized in that it has a binding affinity for the target of the target-binding agent that is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, or 10000-fold weaker than the binding affinity of the target- binding agent for the target. In some embodiments, the amino acid sequences between a decoy and a target-binding agent are no more than 10, 9, 8, 7, 6, or 5 amino acids. In some embodiments, a decoy that has a weaker affinity for a target is about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 or more percent identical to a target-binding agent that binds the target.

[0393] In some embodiments, a target-binding agent (e.g., miniprotein) has at least 10- 10000-fold (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, or 10000) greater binding affinity for a target than Page 115 of 248 IPTS / 128939095.1Docket No.: AKT-033WO the binding affinity of a decoy for that target. In some embodiments, the decoy with weaker binding affinity has an amino acid sequence that is about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 or more percent identical to a target-binding miniprotein that binds the target. In some such embodiments, the amino acid sequences between the decoy and the target binding miniprotein are no more than 10, 9, 8, 7, 6, or 5 amino acids.

[0394] Among other things, the disclosure provides decoys that, surprisingly, can decoy (e.g., by reducing kidney uptake and / or retention) across various different scaffolds (cross- scaffolding decoying) and target-binding agents such as those used to treat a subject with cancer. Even more surprisingly, such decoys can further improve binding agents that have been optimized to have already low (e.g., below 20%) kidney uptake and / or retention. As provided herein, a decoy can reduce uptake into a kidney cell in vitro and / or in vivo. For example, a Scaffold A decoy (e.g., as set forth in TABLE 3, TABLE 4C), when administered (e.g., before, during, or after) in combination with a target-binding agent (e.g., a protein, e.g., a miniprotein, etc., as set forth in TABLE 3 or TABLE 11, e.g., including, for example, C143 (e.g., zelenectide pevedotin), C144 (e.g., dotatate), or C170 (e.g., vipivotide textrazetan) can decoy a target-binding agent by at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, as compared to the target-binding agent in the absence of the decoy.

[0395] In certain embodiments, a Scaffold A decoy can decoy a non-Scaffold A target- binding agent (e.g., a miniprotein, e.g., an adnectin, e.g., a bicycle-based binding agent, e.g., a PSMA-binding agent, an SSTR2 binding agent, etc.) by at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or even 100%, as compared to the target-binding agent in the absence of the decoy.

[0396] Scaffold A decoys can also be used to further decoy Scaffold A miniproteins that have already been optimized to reduce kidney uptake. For example, in certain embodiments, an exemplary Scaffold A compound, C116 (SEQ ID NO: 54) has small alkyl groups on the nitrogen atom of certain lysine side chains and, in certain embodiments, has reduced kidney uptake as compared to a similar sequence without the small alkyl groups (see, e.g., PCT / US2024 / 049013; WO 2025 / 072791 A2) can decoy a non-Scaffold A target binding agent (e.g., miniprotein, etc., as set forth in TABLE 3 or TABLE 11, e.g., including, for example, C143 (e.g., zelenectide pevedotin), C144, or C170 (e.g., vipivotide textrazetan) by at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, as compared to the target binding agent in the absence of the decoy. Page 116 of 248 IPTS / 128939095.1Docket No.: AKT-033WO

[0397] As provided herein, a decoy “not binding” to a target refers to binding that is either not detectable, such as by using binding affinity measurements as provided herein, and / or has a binding affinity greater than 1000-fold weaker than a comparator reference target-binding miniprotein. Thus, as will be understood, given context, in some embodiments, a decoy does not bind to a tumor, cancer cell, or population of cancer cells. In some embodiments, a decoy does not bind to a target set forth in TABLE 10. In some embodiments, a decoy does not bind to Nectin-4. In some embodiments, a decoy does not bind to B7-H3. In certain embodiments, a decoy does not bind to PSMA. In certain embodiments, a decoy does not bind to SSTR2. In some embodiments, a decoy does not bind to a target on a cancer cell. In some such embodiments, a target can be selected from those set forth in TABLE 10.

[0398] In some embodiments, a decoy has a comparable PK profile as compared to a reference target-binding miniprotein. A decoy and target binding miniprotein pair can be considered to be any decoy that can be paired with a target-binding miniprotein. For example, in some embodiments a Scaffold A miniprotein (e.g., that binds Nectin-4) can be compared to Scaffold A decoy, and have comparable PK characteristics. In some embodiments a Scaffold B miniprotein (e.g., that binds B7-H3) can be compared to Scaffold A decoy, and have comparable PK characteristics. In some embodiments, a target-binding miniprotein of a different scaffold (e.g., a bicyclic protein, e.g., a miniprotein that binds to PSMA, SSTR2, etc.) can be compared to a Scaffold A decoy and have comparable PK characteristics.

[0399] Decoys of the disclosure do not bind to targets other than non-tumor targets such as kidney and / or liver tissue. That is, decoys do not accumulate and / or bind throughout the body at a level that could result in toxicity, rather, decoys are taken up by non-tumor tissues or cells such as in the kidney and / or liver and then metabolized and excreted. Decoys of the disclosure can be characterized, analyzed, / or assessed on one or more in vivo, ex vivo, or in vitro assays including, e.g., an OK cell binding assay for evaluation of kidney uptake, e.g., BioD analysis, a cell microarray screening for selectivity (e.g., Retrogenix), etc., and tested alone or in combination with a target-specific miniprotein as well as, in some embodiments, compared to uptake data of a target-specific miniprotein alone (in absence of a decoy).

[0400] In some embodiments, decoys have solubility of about 1-100 mg / mL in PBS and thermal stability of greater than about 80% at 75oC for at least one hour.

[0401] The disclosure also contemplates that, given context, in comparing two target- binding miniproteins and their relative binding affinities, if, for example, a first target- binding miniprotein that targets Ne...

Claims

Docket No.: AKT-033WO CLAIMS What is claimed is:

1. A decoy for use in cross-scaffold decoying with a target-binding agent, wherein the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 16, 17, and 34, wherein the decoy and the target-binding agent do not share a scaffold, and the target-binding agent is optionally selected from a compound selected from C170, C144, and C143.

2. A method of treating or preventing cancer in a subject comprising administering a decoy and a target-binding agent to the subject, wherein the decoy and the target-binding agent together are present in an effective amount, and wherein the decoy is a Scaffold A decoy and the target-binding agent is (i) a Scaffold A miniprotein that is already optimized for reduced kidney uptake or (ii) a non-Scaffold A target-binding agent.

3. The method of claim 1, wherein the scaffold of the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 16, 17, and 34.

4. The method of claim 1 or 2, wherein the target-binding agent is selected from any one of C1-C6, C8-C77, C109-C166 or C167-C169; and / or has an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 1-3, 5-15, 47-68, 87-111 and 112- 114.

5. The method of any one of claims 2-4, wherein the target-binding agent binds to a target selected from somatostatin receptor 2 (SSTR2), prostate-specific membrane antigen (PSMA), Nectin-4, or B7-H3.

6. The method of claim 5, wherein the scaffold of the target-binding agent is not Scaffold A.

7. A decoy for co-administration with a target-binding agent, wherein, the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 4 and 16-46, or compounds C7 and C78-C108.

8. A decoy for blocking uptake of a target-binding agent into a non-tumor tissue, wherein the decoy and the target-binding agent do not share a scaffold and the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 4 and 16-46 or compounds C7 and C78-C108.

9. A decoy for blocking uptake of a target-binding agent into a non-tumor tissue, wherein the decoy and the target-binding agent are of the same scaffold, but the decoy does not bind to the target, or binds to the target with at least about 100-1,000-fold weaker affinity than Page 238 of 248 IPTS / 128939095.1Docket No.: AKT-033WO the target-binding agent binds to the target, and the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 4 or 16-46 or compounds C7 and C78-C108.

10. The decoy of any one of claims 8-10, wherein the decoy has an amino acid sequence comprising any one of SEQ ID NOs: 16, 17, and 34.

11. The decoy of any one of claims 8-11, wherein the target-binding agent and the decoy have the same scaffold.

12. The decoy of any one of claims 8-11, wherein the target-binding agent and the decoy do not share a scaffold.

13. The decoy of any one of claims 8-13, wherein the target-binding agent binds to a target on a cell, optionally, wherein the cell is a cancer cell and / or the target is selected from TABLE 10.

14. The decoy of claim 13, wherein the target-binding agent binds to a target selected from somatostatin receptor 2 (SSTR2), prostate-specific membrane antigen (PSMA), Nectin-4, or B7-H3.

15. The decoy of claim 13 or 14, wherein the combination of the decoy and the target-binding agent effectively decoys the target-binding agent as measured by reduction in kidney cell uptake and / or retention, in vitro or in vivo.

16. The decoy of any one of claims 13-15, wherein the target-binding agent is a polypeptide, which polypeptide optionally comprises a miniprotein.

17. The decoy of claim 16, wherein the target-binding agent further comprises one or more of a linker, chelator, and radionuclide.

18. The decoy of claim 17, wherein the radionuclide is selected from Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, In-111, Ce-134, F-18, and At-211.

19. The decoy of any one of claims 7-18, wherein the target-binding agent (i) comprises a compound comprising or consisting of any one of C1-C6, C8-C77, and C109-C169; and / or (ii) has an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 1-3, 5-15, 47-68, and 87-114.

20. The decoy of any of one of claims 7-19, wherein the target-binding agent has a first binding affinity for the target and the decoy has a second binding affinity for the target Page 239 of 248 IPTS / 128939095.1Docket No.: AKT-033WO and the second binding affinity is at least about 100-1,000-fold weaker than the first binding affinity.

21. The decoy of any of one of claims 7-20, wherein the decoy reduces the uptake and / or retention in kidney cells, in vitro or in vivo, by about 20% to about 100% by 24 hours post-contact with the decoy (in vitro) or after administration of the decoy (in vivo).

22. A method of blocking uptake of a radiolabeled target-binding agent into a non-target tissue, the method comprising administering to a subject in need thereof a target-binding agent and a decoy, wherein the decoy does not share a scaffold with the target-binding agent.

23. A method of blocking uptake of a radiolabeled target-binding agent into a non-target tissue, the method comprising administering to a subject in need thereof a target-binding agent and a decoy, wherein the decoy is of the same scaffold as the target-binding agent.

24. A method of blocking uptake of a radiolabeled target-binding agent into a non-target tissue, the method comprising administering to a subject in need thereof a target-binding agent and a decoy, wherein the decoy is of the same scaffold as the target-binding agent, but the decoy does not bind to the target or binds to the target with at least about 100- 1,000-fold weaker affinity than the target-binding agent binds to the target.

25. The method of any one of claims 22-24, wherein the target-binding agent and the decoy are administered concomitantly or sequentially.

26. The method of claim 25, wherein the target-binding agent and the decoy are administered sequentially, and wherein the decoy is administered before the target-binding agent.

27. The method of claim 25, wherein the target-binding agent and the decoy are administered sequentially, and wherein the target-binding agent is administered before the decoy.

28. The method of claim 26 or 27, wherein the administration of the target-binding agent and the administration of the decoy are no greater than 4 hours apart.

29. A pharmaceutical composition comprising: a. a Scaffold A target-binding agent; and b. a Scaffold A decoy.

30. A pharmaceutical composition comprising: a. a non-Scaffold A target-binding agent; and Page 240 of 248 IPTS / 128939095.1Docket No.: AKT-033WO b. a Scaffold A decoy.

31. The pharmaceutical composition of claim 29, wherein the Scaffold A target-binding agent comprises a polypeptide that binds to Nectin-4.

32. The pharmaceutical composition of claim 31, wherein the target-binding agent comprises or consists of any one of C1-C6, C8-C77, and C109-C169; and / or (ii) has an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 1-3, 5-15, 47-68, and 87- 114.

33. The pharmaceutical composition of any one of claims 29-32, wherein the decoy comprises or consists of any one of SEQ ID NOs: 4, and 16-46 or compounds C7 and C78-C108.

34. A pharmaceutical composition comprising: a. a Scaffold B, C, J, or L target-binding agent; and b. a Scaffold A decoy.

35. The pharmaceutical composition of claim 34, wherein the target-binding agent comprises or consists of C12-C35, C45-C56, C58-C77, C109-C139, and C143-C169 and / or has an amino acid sequence selected from any of SEQ ID NOs: 1-3, 5-6, 8, and 87-114.

36. The pharmaceutical composition of claim 34 or 35, wherein the decoy comprises or consists of any one of SEQ ID NOs: 4 and 16-46 or compounds C7 and C78-C108.

37. The pharmaceutical composition of any one of claims 29-36, wherein the target-binding agent and the decoy are supplied in separate containers.

38. The pharmaceutical composition of any one of claims 29-37, wherein the target-binding agent and the decoy are supplied in the same container.

39. The pharmaceutical composition of any one of claims 29-37, wherein the target-binding agent and the decoy are co-formulated.

40. The pharmaceutical composition of any one of claims 29-37, wherein the target-binding agent and / or the decoy are administered to a subject in need thereof, optionally concomitantly or sequentially.

41. A kit comprising: Page 241 of 248 IPTS / 128939095.1Docket No.: AKT-033WO a. a decoy for co-administration with a target-binding agent, wherein the decoy blocks uptake and / or retention of the composition into a non-tumor tissue (e.g., a kidney tissue, a liver tissue, etc.); and b. instructions for use.

42. A kit comprising: a. a composition represented by the formula selected from one or more of M-L- C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a target-binding agent (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R); and b. a decoy, wherein the decoy blocks uptake and / or retention of the composition into a non-tumor tissue (e.g., a kidney tissue, a liver tissue, etc.).

43. The kit of claim 41 or 42, wherein the decoy and the composition are administered to a subject in need thereof.

44. The kit of claim 43, wherein after the administration, the decoy appears in a higher concentration in the non-tumor tissue than in the tumor tissue as measured by %ID / g.

45. The kit of any one of claims 41-44, when R is present, it is supplied separately from any of M, C, L, or the decoy.

46. The kit of any one of claims 41-45, wherein the decoy is selected from a Scaffold A decoy or a Scaffold B decoy.

47. The kit of any one of claims 41-46, wherein the decoy is supplied in a molar or mass excess as compared to the composition, which molar or mass excess may optionally be selected from a 10, 20, 100, 200, 250, 300, 1000, or more molar or mass excess.

48. The kit of any one of claims 41-47, wherein the R, if present, is added just prior to use.

49. A method comprising improving a cancer treatment by reducing kidney uptake of a target-binding polypeptide by administering the target-binding agent and a decoy, wherein the target-binding agent and the decoy are both Scaffold A.

50. The method of claim 49, wherein the target-binding agent is optimized for reduced kidney uptake, prior to combination with the decoy.

51. The method of claim 50, wherein the target-binding agent is or comprises a target-binding polypeptide comprising or consisting of C116, C117, C144, C146, C148, C150, C152, Page 242 of 248 IPTS / 128939095.1Docket No.: AKT-033WO C154, C156, C158, C160, C162, C164, and C166 or an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 54, 55, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, and 111.

52. The method of claim 51, wherein the target-binding agent further comprises a radionuclide selected from Ac-225, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La- 135, In-111, Ce-134, F-18, and At-211.

53. The method of any one of claims 49-52, wherein the target-binding agent has significantly higher kidney uptake in absence of combination with the decoy as compared to combination with the decoy.

54. A method comprising improving a cancer treatment by reducing kidney uptake of a target-binding agent by administering the target-binding agent and a decoy, wherein the target-binding agent is not from Scaffold A (e.g., but is from Scaffold B, C, L, J, or a target-binding agent such as disclosed in Tables 11 or 17), and the decoy is a Scaffold A polypeptide.

55. In a method of treating an individual with cancer comprising administering (a) a composition comprising a target-binding agent (M) and a radionuclide (R); and (b) a decoy, wherein (i) the decoy and the target-binding agent do not share a scaffold; (ii) the decoy and the target-binding agent are of the same scaffold; or (iii) the target-binding agent is of the same scaffold as the decoy but already optimized for reduced kidney uptake, the improvement comprising reducing one or more off-target effects or toxicity measures after administration of the composition and the decoy as compared to administration of the composition in the absence of the decoy.

56. The method of claim 55, wherein the composition comprises an M that targets somatostatin receptor 2 (SSTR2), prostate-specific membrane antigen (PSMA), Nectin-4, or B7-H3.

57. The method of claim 55 or 56, wherein the reduction in the one or more off-target effects or toxicity measures is measured as a reduction in one or more toxicity grades of each of the one or more off-target effects or toxicity measures.

58. In a method of treating an individual with cancer by administering: (a) a composition comprising a target-binding agent (M) and a radionuclide (R); and (b) a decoy, wherein (i) the decoy and the target-binding agent do not share a scaffold; (ii) the decoy and the target-binding agent are of the same scaffold; or (iii) the target-binding agent is of the Page 243 of 248 IPTS / 128939095.1Docket No.: AKT-033WO same scaffold as the decoy but already optimized for reduced kidney uptake, the improvement comprising achieving a reduction in concentration of R in a non-tumor tissue in the presence of the decoy as compared to the concentration of R in the non- tumor tissue in the absence of the decoy.

59. The method of claim 58, wherein the non-tumor tissue is a liver tissue or a kidney tissue.

60. The method of claim 59, wherein the reduction in concentration of R in the kidney tissue is determined by maintenance of eGFR over at least the period that the subject is receiving treatment.

61. The method of any one of claims 58-60, wherein the administration of the composition can be repeated at least twice as many times in the presence of the decoy as in the absence of the decoy before a dose-limiting toxicity is reached.

62. In a method of reducing uptake by a population of kidney cells of a composition, the improvement comprising administering a composition comprising (a) a radionuclide therapeutic comprising at least a target-binding agent (M) and a radionuclide (R); and (b) a decoy, such that in the presence of the decoy, the radionuclide is less concentrated in the population of kidney cells than in the absence of the decoy.

63. A combination composition comprising an effective amount of each of: (i) a therapeutic comprising a composition represented by the formula selected from one or more of M-L- C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a target- binding agent (M), L comprises a linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the M is of a particular scaffold; and (ii) a decoy comprising or consisting of an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NOs: 16-46, and SEQ ID NOs: 68 or 87.

64. The combination composition of claim 63, wherein the decoy does not comprise the same scaffold as the M.

65. The combination composition of claim 63 or 64, wherein the decoy comprises a Scaffold A decoy.

66. The combination composition of any one of claims 63-65, wherein the decoy and the target-binding agent are supplied and / or administered separately, but wherein the combination exists in the subject to whom the administration has occurred. Page 244 of 248 IPTS / 128939095.1Docket No.: AKT-033WO 67. The combination composition of any one of claims 63-66, wherein the M comprises or consists of (i) any one of C1-C6, C8-C77, and C109-C170; and / or (ii) has an amino acid sequence comprising or consisting of any one of SEQ ID NOs: 1-3, 5-15, 47-68, and 87- 114.

68. A method of treating an individual with a cancer, the method comprising administering to the individual an effective amount of each of: a. a means for blocking uptake of a radiotherapeutic to one or more non-tumor tissues, and b. a radionuclide therapeutic comprising a composition represented by a formula selected from one or more of M-L-C-R, M-L-C, M-C-R, M-L-R, M-C, M-L, and M-R, wherein M comprises a target-binding agent (M), L comprises a linear, branched, or enzymatically cleavable linker (L), C comprises a chelator (C), and R comprises a radionuclide (R), wherein the M shares or does not share a scaffold with the means for blocking uptake of the radiotherapeutic.

69. The method of claim 68, wherein the non-tumor tissue is a kidney tissue or a liver tissue.

70. The method of claim 68 or 69, wherein the radionuclide therapeutic is targeted to a tumor.

71. The method of any one of claims 68-70, wherein the radionuclide therapeutic is targeted to a cell expressing a target.

72. The method of claim 71, wherein under otherwise identical conditions, the radionuclide therapeutic targeted to the tumor is at a greater concentration than in the absence of the means for blocking uptake of the radiotherapeutic to the one or more non-tumor tissues.

73. A method of improving a cancer treatment in an individual experiencing a decrease in efficacy and / or one or more off-target effects, the method comprising administering an effective amount of each of: (a) a decoy; and (b) a radionuclide therapeutic, wherein the decrease in efficacy is improved relative and / or the one or more off-target effects is prevented or reduced as compared to administering the radionuclide therapeutic without administering the decoy.

74. A method of improving a cancer treatment in an individual, the method comprising administering an effective amount of each of: (a) a decoy; and (b) a radionuclide therapeutic, wherein an off-target effect is prevented or reduced as compared to administering the radiotherapeutic in the absence of the decoy. Page 245 of 248 IPTS / 128939095.1Docket No.: AKT-033WO75. The method of claim 74, wherein the individual has not been previously treated with atleast one cancer treatment (e.g., chemotherapy, radiation, immunotherapy) prior to administration of the decoy and the radionuclide therapeutic.

76. The method of claim 74 or 75, wherein the individual has been previously treated with atleast one cancer treatment (e.g., chemotherapy, radiation, immunotherapy) prior to administration of the decoy and the radionuclide therapeutic.

77. A method of improving a cancer treatment in an individual with a refractory cancer, themethod comprising administering: (a) a decoy; and (b) a radionuclide therapeutic, wherein an off-target effect is prevented or reduced as compared to administering the radiotherapeutic in the absence of the decoy.

78. The method of any one of claims 73-77, wherein the radionuclide therapeutic comprises atarget-binding agent that targets a protein, which protein is optionally selected from TABLE 10.

79. The method of any one of claims 73-78, wherein the radionuclide therapeutic comprises atarget-binding agent that targets somatostatin receptor 2 (SSTR2), prostate-specific membrane antigen (PSMA), Nectin-4, or B7-H3.

80. The method of any one of claims 1-7, 47-60, and 67-79, wherein the cancer is selectedfrom breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorders, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing’s sarcoma, extracranial germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gestational trophoblastic tumors, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, lip cancer, oral cavity cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic squamous neck cell carcinoma, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and the Sezary syndrome, myelodysplastic syndromes, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancers, including osteosarcoma and malignant fibrous histiocytoma of bone, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary Page 246 of 248 IPTS / 128939095.1Docket No.: AKT-033WO tumors, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumors, pineoblastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor and other childhood kidney tumors. Page 247 of 248 IPTS / 128939095.1

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