B7-h3 miniproteins, conjugates, and uses thereof

Abstract

Provided herein are B7-H3-binding miniproteins, conjugates, and uses thereof. B7-H3 binding peptides and conjugates, including radionuclide conjugates, as well as one or more additional proteins such as a decoy peptide are disclosed herein. Such peptides, conjugates, and / or decoys can be used in compositions and methods of treating, diagnosing, monitoring, and / or imaging a disease, disorder, or condition associated with expression of one or more targets (e.g., B7-H3).

Description

B7-H3 MINIPROTEINS, CONJUGATES, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Nos.63 / 607,988, filed on December 8, 2023; 63 / 636,076, filed on April 18, 2024; 63 / 650,364, filed on May 21, 2024; 63 / 650,365, filed on May 21, 2024; and 63 / 721,990, filed on November 18, 2024, the disclosures of each of which are incorporated by reference herein in their entireties 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 December 6, 2024, is named AKT-035WO_SL.xml, and is 931,498 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 including 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. SUMMARY

[0004] 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 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.

[0005] 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 direction of radiation towards 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 an antigen expressed Page 1 of 344 IPTS / 128790492.1at an increased level and / or density on the surface of tumor cells relative to non-tumor cells. Binding of the radionuclide to the antigen-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. Accordingly, a need remains for new approaches to specifically target tumors, and particularly solid tumors. The present disclosure provides technologies that meet this and other needs.

[0006] Among other things, the present disclosure provides polypeptides, compositions comprising the polypeptides and conjugates comprising the polypeptides (e.g., miniproteins) 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, the polypeptides specifically and strongly bind to B7-H3. In certain embodiments, a polypeptide or conjugate of the disclosure confers improved tumor penetration, decreased off-target toxicity and / or accumulation, improved stability (e.g., relative to a comparator polypeptide), as well as improved affinity for B7-H3 (e.g., relative to currently existing technologies (e.g., antibodies, e.g., antibody-drug conjugates, polypeptides, etc.) In some embodiments, such conjugates are used in treatment of cells expressing a target (e.g., B7-H3). In some such embodiments, the cells are cancer cells.

[0007] The disclosure also provides the insight that compositions and conjugates provided herein can be even further improved, such as by increasing stability (e.g., thermal stability), while maintaining or increasing strength of binding to B7-H3. For example, contemplated herein, in some embodiments, conjugates, such as radionuclide conjugates of the disclosure may be improved, such as by improvement on one or more measures including thermal stability, efficacy and / or reduction in toxicity grade and / or off-target effects (e.g., in a cell- based assay, e.g., when administered to a subject in need thereof), by modification of one or more amino acids in a polypeptide sequence of the conjugate (e.g., relative to a reference or starting sequence) and / or addition of one or more decoys.

[0008] These and other aspects and features of the disclosure are provided and described in the following detailed description and claims.

[0009] In one aspect, the disclosure provides a composition, comprising a polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at least two constraints; an arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3, a lysine at a position corresponding to amino acid 5, an isoleucine at a position Page 2 of 344 IPTS / 128790492.1corresponding to amino acid 6, a tryptophan at a position corresponding to amino acid 14, at least one modified lysine residue at a position corresponding to amino acid 24, and an alanine, arginine, or modified lysine at a position corresponding to 29, where each position is linear, from N-to-C-terminus, beginning at position 1, relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or to the Guanidino group nitrogen of the arginine side chain, wherein the at least one small alkyl group optionally comprises a methyl, dimethyl, or trimethyl; at least 48 amino acids in length; and has a binding affinity for B7-H3 stronger than 100 nM as measured in a cell-based assay.

[0010] In some embodiments, the at least two constraints comprises at least two disulfide bridges. In some embodiments, the composition further comprises at least one additional constraint. In certain embodiments, the at least one additional constraint is a lactam bridge. In some embodiments, the at least one additional constraint is a disulfide bridge.

[0011] In certain embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204 and 262-272. In some embodiments, the polypeptide is part of a compound selected from any one of C234-235 and C309-C332.

[0012] In one aspect, the disclosure provides a composition comprising a polypeptide of at least 48 amino acids in length and having an amino sequence according to Formula VI (SEQ ID NO: 541), comprising at least four cysteines and two disulfide bonds, wherein X24 is (Kme) or (Kme2); X29 is (Kme) or A or R; X32 is (Kme), D, or (Cit); and X45 is (Kme) or K.

[0013] In another aspect, the disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula III (SEQ ID NO: 538) as follows: CAX3EKIAALSEIIWLPCLX19YAQIX24AFIX28X29LNX32DPCX36SX38X39ILSEAX4 5ELCS, wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme) or (Kme2); X28 is A or (Kme); X29 is (Kme) or A or R; X32 is (Kme) or D or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; and X45 is K or (Kme). In certain embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204 and 262-272. In some embodiments, the polypeptide is part of a compound selected from any one of C234-235 and C309-C332.

[0014] In one aspect, the composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula IV (SEQ ID NO: 539) as Page 3 of 344 IPTS / 128790492.1follows: CAX3EKIAALSEIIWLPCLX19YAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme) or (Kme2); X29 is (Kme) or A; X32 is (Kme) or D; and X45 is (Kme) or K. In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204, 262, 265, 267, and 268.

[0015] In certain embodiments, the polypeptide is part of a compound selected from any one of C234, C235, C298, C299, C304, C305, C308, C309, C310, C311, C320, C323, C325, C326, and C332.

[0016] In another aspect, the disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising an amino sequence according to Formula V (SEQ ID NO: 540) as follows: CAX3EKIAALSEIIWLPCLTYAQIX24AFIX28X29LNX32DPCQSSEILSEAX45ELCS, wherein X3 is (Kme3) or (Rme) or R; X24 is (Kme) or (Kme2); X28 is A or (Kme); X29 is (Kme) or A or R; X32 is (Kme) or D or (Cit); and X45 is (Kme) or K. In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 265, 266, 267, 270, and 272. In certain embodiments, the polypeptide is part of a compound selected from any one of C304-C309, C314, C315, C318, C319, C323, C324, C325, C328, C330, and C332.

[0017] In one aspect, the disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula VI (SEQ ID NO: 541) as follows: CA(Kme3)EKIAALSEIIWLPCLTYAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, wherein X24 is (Kme) or (Kme2); X29 is (Kme) or A or R; X32 is (Kme) or D or (Cit); and X45 is (Kme) or K. In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204, 262, 265, 266, 267, 270, and 272. In certain embodiments, the polypeptide is part of a compound selected from any one of C234, C235, C298, C299, C304-C309, C314, C315, C318-C320, C323, C324, C325, C328, C330, and C332.

[0018] In another aspect, the disclosure provides a composition, comprising a B7-H3 binding polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acids include (i) a cysteine at each of four positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267 and wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme) or (Kme2); X28 is (Kme) or A; X29 is (Kme) or A or R; X32 is (Kme) or D or Page 4 of 344 IPTS / 128790492.1(Cit); X36 is Q or N; X38 is S or A; X39 is E or N; X45 is (Kme) or K, and X49 is S or absent.

[0019] In one aspect, the disclosure provides a composition, comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at least four cysteines, which form two disulfide bonds; at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, optionally comprising a methyl, dimethyl, or trimethyl; at least 48 amino acids in length; and has a binding affinity for B7-H3 stronger than 100 nM as measured in a cell-based assay. In some embodiments, the polypeptide is a miniprotein. In certain embodiments, the polypeptide is at least 48 amino acids in length, but no greater than 100 amino acids in length. In certain embodiments, the polypeptide binds to B7-H3 with an affinity of stronger than 100 nM as measured in a cell-based assay.

[0020] In some embodiments, the amino acid sequence of a polypeptide according to the present disclosure shares at least 90% identity to any one of SEQ ID NOs: 204 and 262-537, and includes at least one lysine or arginine with at least one modification comprising at least one small alkyl group bonded to the nitrogen of the lysine side chain or to the Guanidino group nitrogen of the arginine side chain, optionally selected from: trimethyl, dimethyl, and monomethyl. In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity to at least 44 amino acids of a reference polypeptide, which reference polypeptide is longer than 47 amino acids in length and binds to B7-H3 with a strength of at least 10 nM on a cell-based assay, and / or has an inhibition constant of no weaker than 10 nM. In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537, provided that the 44 amino acids include at least four cysteine residues that form at least two disulfide bridges. In some embodiments, the amino acid sequence of the polypeptide shares 100% identity to at least 44 amino acids of a reference polypeptide, which reference polypeptide is longer than 47 amino acids in length. In some embodiments, the amino acid sequence shares at least 90% identity to at least 44 amino acids as set forth in any one of SEQ ID NO: 199, 204, 241, or 262-272. In some embodiments, the amino acid sequence shares 100% identity to at least 44 amino acids as set forth in any one of SEQ ID NO: 199, 204, 241, or 262-272.

[0021] In some embodiments, the amino acid sequence comprises at least four cysteines with at least two disulfide bridges. In some embodiments, the polypeptide is at least 48 amino acids in length. In one aspect, the disclosure provides a composition comprising a Page 5 of 344 IPTS / 128790492.1polypeptide having an amino acid sequence comprising SEQ ID NO: 267. In certain embodiments, the composition comprises C234, C235, and C298-C333 as set forth in Table 2C.

[0022] In one aspect, the disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising any one of SEQ ID NOs: 204 and 262-272.

[0023] In certain embodiments, a composition of the disclosure (e.g., a B7-H3 polypeptide, e.g., a B7-H3 binding miniprotein) further comprises a radionuclide. Depending on context, the radionuclide is selected from Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra- 225, Tb-165, or At-211. In certain embodiments, a polypeptide of the disclosure comprises a C-terminus comprising an -OH or an -NH2. In some embodiments, the binding affinity of a composition provided herein, for B7-H3, is stronger than 100 nM. In certain embodiments, the inhibition constant is no weaker than 100 nM.

[0024] In some embodiments, a composition of the disclosure (e.g., comprising a polypeptide), further comprises one or more of a linker, chelator, and radionuclide. In some embodiments, the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG2, PEG, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 36, (Gly)1-10, or any fragment or combination via covalent bond thereof. In some embodiments, the chelator comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N- succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N-succinimidyl 3- trimethylstannylbenzoate (MeSTB). In some embodiments, the radionuclide is selected from Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

[0025] In certain embodiments, if the polypeptide comprises any one of SEQ ID NOs: 198-537 the polypeptide further comprises a linker, wherein the linker is PEG4, and an optional chelator, wherein the chelator is DOTA. In some embodiments, when present, the linker is attached to the N-terminus of the polypeptide. In some embodiments, the C-terminal amino acid of the polypeptide is not a cysteine. In some embodiments, when present, the chelator is attached to either the polypeptide or the linker. In some embodiments, when present, the radionuclide is attached to the chelator. Page 6 of 344 IPTS / 128790492.1

[0026] In one aspect, the disclosure provides a composition comprising a formula selected from one or more of (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 (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 198-537. In certain embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 198-537. In certain embodiments, the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 36, (Gly)1-10, or any fragment or combination via covalent bond thereof. In certain embodiments, the chelator comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N- succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N-succinimidyl 3- trimethylstannylbenzoate (MeSTB). In some embodiments, the radionuclide Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

[0027] In one aspect, the disclosure provides a composition comprising a formula selected from one or more of (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 (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 204 and 262-272. In certain embodiments, M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 204 and 262-272. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 204 and 262-272. In certain embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 204 and 262-272. In some embodiments, L is present, L comprises or consists of a polyethylene Page 7 of 344 IPTS / 128790492.1glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36, 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 dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 10, (Gly)1-10, or any fragment or combination via covalent bond thereof. In some embodiments, when C is present, C comprises or consists of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N-succinimidyl 3- trimethylstannylbenzoate (MeSTB). In some embodiments, when R is present, R comprises or consists of Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce- 134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211. In certain embodiments, when present, the linker is attached to the N-terminus of the polypeptide. In certain embodiments, the C-terminal amino acid of the polypeptide is not a cysteine. In some embodiments, when present, the chelator is attached to either the polypeptide or the linker. In some embodiments, when present, the radionuclide is attached to the chelator. In certain embodiments, the polypeptide comprises at least one disulfide bridge. In certain embodiments, the polypeptide comprises at least two disulfide bridges. In certain embodiments, the composition and / or polypeptide thereof selectively binds to B7-H3. In some embodiments, the polypeptide has a binding affinity for B7-H3 of 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM, in vivo, ex vivo, or in vitro and / or as measured in a cell- based assay. In some embodiments, the polypeptide has a binding inhibition constant of no weaker than 100 nM.

[0028] In one aspect, the disclosure provides a composition comprising a polypeptide-drug conjugate, comprising a polypeptide and at least one drug moiety, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to at least 48 amino acids a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 198-537. In certain embodiments, the polypeptide comprises at least four cysteines and two disulfide bridges. In some embodiments, the drug moiety is selected from a topoisomerase inhibitor, an auristatin (e.g., monomethyl auristatin E), 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, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRMl, a DPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, a Page 8 of 344 IPTS / 128790492.1protein 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, a DHFR inhibitor, and an immunotoxin.

[0029] In one aspect, the disclosure provides a composition comprising an isolated compound or pharmaceutically acceptable salt thereof, or a neutral molecule, comprising an optional linker (L), and one or more of a polypeptide (M), chelator (C) or radionuclide (R), wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids any one of SEQ ID NOs: 198-537 and 538-543 and 546- 550, including amino acid substitutions as set forth in Table 1E.

[0030] In another aspect, the disclosure provides a composition comprising, a compound designed to bind to B7-H3, which compound comprises or consists of a polypeptide having an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E, and further comprises a modified N and / or C-terminus. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E. In some embodiments, the modified N-terminus comprises one or more of an NH2-, Acetyl-, PEGn-, wherein n=0-36, DOTA-, or Biotin-. In certain embodiments, the C terminus comprises an -NH2 or an -OH. In certain embodiments, the polypeptide selectively binds to B7-H3. In some embodiments, the polypeptide has a binding affinity of stronger than about 100 nM to B7-H3, in vivo or in a cell-based assay.

[0031] In one aspect, the disclosure provides a compound comprising a miniprotein having an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of SEQ ID NO: 267, and further comprising one or more additional components according to a formula M-L-C-R, wherein M is the miniprotein, L is a linker, C Page 9 of 344 IPTS / 128790492.1is a chelator, and R is a radionuclide. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of SEQ ID NO: 267. In certain embodiments, M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to SEQ ID NO: 267. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to SEQ ID NO: 267. In some embodiments, L comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, any linker set forth in Table 2A or Table 2C, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 10, (Gly)1-10, or any fragment or combination via covalent bond thereof. In certain embodiments, C comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N- succinimidyl 3-trimethylstannylbenzoate (MeSTB). In some embodiments, R comprises or consists of Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

[0032] In one aspect, the disclosure provides a compound comprising a miniprotein with at least 90% identity to at least 48 amino acids of the amino acid sequence of SEQ ID NO: 267, wherein the N and / or C-terminus comprise between one and thirty additional amino acids, and / or wherein the C-terminus comprises one fewer amino acids or up to 30 additional amino acids, provided that the entire miniprotein is no greater than about 100 amino acids in length.

[0033] In one aspect, the disclosure provides a pharmaceutical composition comprising a polypeptide or compound as provided herein; and a pharmaceutically acceptable excipient.

[0034] In one aspect, the disclosure provides, in a method of improving binding affinity strength of a polypeptide to B7-H3, the improvement comprising modifying at least three amino acid residues of a polypeptide, which polypeptide has at least 48 amino acids in length and has cysteines at positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267, wherein a position corresponding to X24 is (Kme) or (Kme2); X29 is A or (Kme); and X32 is D or (Kme) or (Cit), and wherein X49 is S or absent.

[0035] In one aspect, the disclosure provides a method of treating cancer, the method comprising administering to a subject in need thereof, a composition comprising a conjugate comprising a polypeptide having an amino acid sequence, which comprises an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: Page 10 of 344 IPTS / 128790492.1198-537 and a radionuclide. In some embodiments, the polypeptide has at least four cysteines and two disulfide bridges. In certain embodiments, the radionuclide is associated with the polypeptide with a linker and / or chelator according to a formula M-L-C-R, wherein M is the polypeptide, L is a linker, C is a chelator, and R is the radionuclide. In some embodiments, the polypeptide has an amino acid sequence comprising or consisting of any of SEQ ID NOs: 204, or 262-272. In certain embodiments, L comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, any linker set forth in Table 2A, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 10, (Gly)1-10, or any fragment or combination via covalent bond thereof. In certain embodiments, C comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N- succinimidyl 3-trimethylstannylbenzoate (MeSTB). In certain embodiments, R is Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211. In some embodiments, R is a therapeutic agent and / or an imaging agent. In some embodiments, R is Cu-64, Ga-68, Lu- 177, In-111, Cu-67, La-132, or F-18.

[0036] In certain embodiments, the polypeptide is no longer than 100 amino acids in length.

[0037] In one aspect, the disclosure provides a method of reducing kidney cell uptake and / or increasing tumor uptake of a composition comprising administering to a subject a B7- H3 binding protein having an amino acid sequence comprising at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO: 241, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, optionally comprising a methyl, dimethyl, or trimethyl, and the reduction is as compared to administering to the subject or a control subject an otherwise identical composition but not comprising the modified lysine residue at the position corresponding to X24.

[0038] In one aspect, the disclosure provides, in a method of treating cancer, the improvement comprising administering a composition comprising a B7-H3 binding protein having an amino acid sequence comprising at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO: 241, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, optionally comprising a methyl, dimethyl, or trimethyl, and the reduction is as compared to administering to the Page 11 of 344 IPTS / 128790492.1subject or a control subject an otherwise identical composition but not comprising the modified lysine residue at the position corresponding to X24.

[0039] In one aspect, the disclosure provides a method of treating a subject with refractory or recurrent cancer comprising administering a composition, compound, or pharmaceutical composition as provided herein, thereby treating the cancer.

[0040] In one aspect, the disclosure provides a method of improving biodistribution of a pharmaceutical composition for a B7-H3 positive population of cancer cells in a subject having a B7-H3-positive cancer, comprising contacting the population with a polypeptide that has a modified lysine at a position corresponding to X24 of SEQ ID NO: 241, wherein the lysine is modified by adding at least one small alkyl group to a lysine side chain and wherein the biodistribution is improved as compared to contacting the population without the modified lysine at a position corresponding to X24 of SEQ ID NO: 241.

[0041] In one aspect, the disclosure provides a method of diagnosing presence of a B7-H3 positive population of cancer cells comprising: contacting a population of cells with the composition, compound, or pharmaceutical composition as provided herein; detecting the presence of the composition, compound, or pharmaceutical composition of step (a) by measuring a signal; and comparing the detection in step (b) to a control signal; and diagnosing cancer if the composition, compound, or pharmaceutical composition of step (a) is detected above the control signal. In some embodiments, the contacting is performed by administering to a subject in need thereof. In certain embodiments, the administering is intravenous or subcutaneous. In some embodiments, the contacting is outside of the subject, optionally in vitro with a biopsy sample.

[0042] In one aspect, the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject a composition comprising a composition, compound, or pharmaceutical composition as provided herein.

[0043] In one aspect, the disclosure provides use of a composition, compound, or pharmaceutical composition in accordance with the disclosure.

[0044] In one aspect, the disclosure provides a method of treating a subject in need thereof comprising administering to the subject in need thereof a composition, compound, or pharmaceutical composition in accordance with the disclosure

[0045] In certain embodiments, the subject is diagnosed as having cancer. In certain embodiments, a cancer cell from the subject expresses B7-H3. In certain embodiments, the expression of B7-H3 is higher in the cancer cell than in a non-cancer cell, which expression can be measured by protein and / or nucleic acid levels. In certain embodiments, the non- Page 12 of 344 IPTS / 128790492.1cancer cell is obtained from the subject. In some embodiments, the composition, compound, or pharmaceutical composition is internalized in a cell expressing human 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, urothelial 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 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. In some embodiments, the composition, compound, or pharmaceutical composition is administered intravenously or subcutaneously.

[0046] In one aspect, the disclosure provides a method of targeting cancer cells expressing B7-H3, the method comprising: (i) determining or having determined a level of expression of B7-H3 in a population of cancer cells; and (ii) administering to a subject in need thereof a composition comprising a composition, compound, or pharmaceutical composition as provided herein, wherein the polypeptide of the composition, compound, or pharmaceutical composition is designed to specifically bind to human B7-H3, wherein the composition, compound, or pharmaceutical composition is attached to the surface and / or internalized into one or more B7-H3 expressing cancer cells.

[0047] In one aspect, the disclosure provides a method of targeting cancer cells expressing B7-H3, the method comprising administering to a subject in need thereof a composition Page 13 of 344 IPTS / 128790492.1comprising a composition, compound, or pharmaceutical composition as provided herein, wherein the polypeptide of the composition, compound, or pharmaceutical composition is designed to specifically bind to human B7-H3; wherein (i) the subject has cancer cells that express B7-H3; and (ii) the composition, compound, or pharmaceutical composition is attached to the surface and / or internalized into one or more B7-H3 expressing cancer cells. In some embodiments, the method further comprises determining or having determined that the cancer cells express B7-H3. In certain embodiments, the subject is treated after the administering as compared to prior to the administering.

[0048] In one aspect, the disclosure provides in a method of targeting a population of cancer cells expressing B7-H3, the improvement comprising contacting the population with the composition according to a composition, compound, or pharmaceutical composition as provided herein, wherein a position corresponding to X24 (relative to SEQ ID NO: 241) comprises a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein the composition is taken up less by kidney cells than in a composition comprising a polypeptide that does not have a small alkyl group attached to a nitrogen on the side chain of a lysine at X24, wherein, optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group.

[0049] In one aspect, the disclosure provides a conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Actinium-225.

[0050] In one aspect, the disclosure provides a conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Copper-64.

[0051] In one aspect, the disclosure provides a conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Gallium-68.

[0052] In one aspect, the disclosure provides a conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an Page 14 of 344 IPTS / 128790492.1optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Indium-111.

[0053] In one aspect, the disclosure provides a conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Lead-212.

[0054] In one aspect, the disclosure provides a conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Lutetium-177.

[0055] A conjugate comprising: (i) a miniprotein (M) that specifically binds to B7-H3; (ii) an N-terminal modification, conjugated to (M) through an optional linker (L), wherein (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) the N- terminal modification comprises a biotin.

[0056] In certain embodiments, M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 198-537. In some embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537. In certain embodiments, M has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 198-537. In certain embodiments, the amino acid sequence has an amino acid sequence comprising at least 90% identity to at least 44 amino acids of SEQ ID NO: 267, wherein the polypeptide has: at least four cysteines, which form two disulfide bonds; an arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3, a lysine at a position corresponding to amino acid 5, an isoleucine at a position corresponding to amino acid 6, a tryptophan at a position corresponding to amino acid 14, at least one modified lysine residue at a position corresponding to amino acid 24, and an alanine, arginine, or modified lysine at a position corresponding to 29, where each position is linear, from N-to-C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the Page 15 of 344 IPTS / 128790492.1nitrogen of the lysine side chain or to the Guanidino group of the arginine side chain, optionally comprising a methyl, dimethyl, or trimethyl; at least 48 amino acids in length; and has a binding affinity for B7-H3 stronger than 100 nM in a cell-based assay. In some embodiments, the amino acid sequence comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 204 or 262-272. In some embodiments, the amino acid sequence comprises an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 204 or 262-272. In certain embodiments, the an amino acid sequence comprises an amino acid sequence with 100% identity to at least 44 amino acids of SEQ ID NO: 267. In certain embodiments, the amino acid sequence comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 267. In certain embodiments, the amino acid sequence comprises an amino acid sequence with 100% identity to SEQ ID NO: 267. In some embodiments, the amino acid sequence comprises or consists of any one of SEQ ID NOs: 204 and 262-272. In some embodiments, the amino acid sequence comprises or consists of SEQ ID NO: 267.

[0057] In one aspect, the disclosure provides an isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from any one of SEQ ID NOs: 198-537; or a nucleic acid sequence encoding a polypeptide comprising at least 90%, 95%, 96%, 97%, 98%, 99% or greater identity to any one of SEQ ID NOs: 198-573. In some embodiments, the disclosure provides a vector comprising an isolated polynucleotide as provided herein. In some embodiments the disclosure provides a host cell transformed with an isolated polynucleotide or vector as provided herein.

[0058] In one aspect, the disclosure provides a method of evaluating locations of one or more populations of cancerous cells in a subject, the method comprising administering to the subject a composition, compound, pharmaceutical composition, or conjugate as provided herein, and detecting to determine location of the composition in the subject, wherein the composition, compound, or pharmaceutical composition comprises a detectable label. In certain embodiments, the detectable label comprises a radionuclide.

[0059] In one aspect, the disclosure provides, in a method of decreasing kidney uptake of a composition administered to detect and / or treat one or more populations of cancer cells, the improvement comprising administering to a subject in need thereof, a composition, compound, pharmaceutical composition, or conjugate as provided herein, wherein a position corresponding to X24 of SEQ ID NO: 241 comprises a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein the composition is taken up less by kidney cells than in a composition comprising a polypeptide that does not have a Page 16 of 344 IPTS / 128790492.1small alkyl group attached to a nitrogen on the side chain of a lysine at a position corresponding to X24 of SEQ ID NO: 241, wherein, optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group. In certain embodiments, the detecting comprises an imaging procedure allows for selecting subjects, monitoring subjects, and / or treating subjects with a therapeutic comprising a miniprotein designed to bind to B7-H3 expressed on one or more cancer cells in the one or more populations of cancer cells. In some embodiments, the therapeutic comprises a composition, compound, conjugate, or pharmaceutical composition as provided herein.

[0060] In one aspect, the disclosure provides a method of improving delivery of a radionuclide to a population of cancer cells in a subject, the method comprising administering a composition, compound, conjugate, or pharmaceutical composition as provided herein, wherein the amino acid sequence of the polypeptide comprises an amino acid at a position corresponding to X24 of SEQ ID NO: 241 comprising a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein uptake by kidney cells is less than with a polypeptide that does not have a small alkyl group attached to a nitrogen on the side chain of a lysine at a position corresponding to X24 of SEQ ID NO: 241. In some embodiments, the small alkyl group comprises a monomethyl, dimethyl, or trimethyl group.

[0061] In one aspect, the disclosure provides, in a method of treating an individual with cancer, the improvement comprising reducing one or more off-target effects or toxicity measures by administering a composition, compound, conjugate, or pharmaceutical composition as provided herein, wherein the amino acid sequences of the polypeptide comprises an amino acid at a position corresponding to X24 of SEQ ID NO: 241 comprising a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein uptake by kidney cells is less than with a polypeptide that does not have a small alkyl group attached to a nitrogen on the side chain of a lysine at a position corresponding to X24 of SEQ ID NO: 241.

[0062] In one aspect, the disclosure provides, in a method of treating an individual with cancer, the improvement comprising achieving a reduction in concentration of R in a kidney tissue in the presence of a composition, compound, conjugate, or pharmaceutical composition as provided herein, wherein the amino acid sequence of the polypeptide comprises an amino acid corresponding to position X24 of SEQ ID NO: 241, and wherein X24 comprises a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein uptake by kidney cells is less than with a polypeptide having an amino acid sequence that does not comprise a small alkyl group attached to a nitrogen on the side chain of a lysine Page 17 of 344 IPTS / 128790492.1at a position corresponding to X24 of SEQ ID NO: 241, as compared to the concentration of R in the kidney tissue in the absence the composition, compound, pharmaceutical composition, or conjugate. In some embodiments, the reduction in concentration of R in the kidney tissue is measured by urine output of R as measured by percent of administered radiation recovered or by detection as measured by a cell-based in vitro assay, or an in vivo detection assay. In some embodiments, the administering of the composition can be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more in the presence of the composition having 90% identity to at least 44 amino acids of SEQ ID NO: 241 including a modified lysine at positions corresponding to X24 of SEQ ID NO: 241 than in the presence of an Q, V, L, or K at positions corresponding to X24.

[0063] In one aspect, the disclosure provides, in a method of reducing uptake by a kidney tissue of a composition, the improvement comprising administering a composition comprising (a) a radionuclide therapeutic comprising at least a polypeptide and a radionuclide (R); wherein the polypeptide has at least 90% identity to at least 44 amino acids of SEQ ID NO: 241 and / or has a modified lysine at positions corresponding X24 of SEQ ID NO: 241, such that in the presence of the modified lysine, the radionuclide is less concentrated in the kidney tissue than in the absence of the polypeptide.

[0064] In another aspect, the disclosure provides a method comprising administering to a subject in need thereof a compound that binds to B7-H3 and includes at least one modified lysine at a position corresponding to X24 of SEQ ID NO: 241, wherein the administering of the compound having a miniprotein with the at least one modified lysine reduces one or more off target effects, toxicity grades, and / or uptake and / or retention in a kidney tissue as compared to a compound not having a modified lysine (e.g., an unmodified lysine, e.g., an L, V, or Q) at a position corresponding to X24. In some embodiments, the polypeptide has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 204 and 262-272. In some embodiments, the polypeptide has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 204 and 262-272. In some embodiments, the polypeptide has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 204 and 262-272.

[0065] In one aspect, the disclosure provides a method of treating an individual having or suspected of having a B7-H3-positive cancer, the method comprising administering to the individual: a means for blocking uptake and / or retention of a radiotherapeutic to kidney tissue, and a linker, a chelator, and a radionuclide. In some embodiments, the means for Page 18 of 344 IPTS / 128790492.1blocking uptake and / or retention of a radiotherapeutic to kidney tissue binds to B7-H3 and includes a modified lysine at a position corresponding to X24 of SEQ ID NO: 267 and / or have at least 90% identity to 40 amino acids of SEQ ID NO: 267 and / or have a modified lysine at a position corresponding to X24 of SEQ ID NO: 267. In some embodiments, the means further comprises a modified lysine at a position corresponding to X3 of SEQ ID NO: 267. In some embodiments, the means for blocking uptake and / or retention of a radiotherapeutic to the kidney tissue blocks uptake and / or retention to the kidney tissue greater than as compared to the blocking of uptake and / or retention to the kidney tissue by a means that does not include a modified lysine at a position corresponding to X24 of SEQ ID NO: 267 and / or have at least 90% identity to at least 44 amino acids of SEQ ID NO: 267 and / or have a modified lysine at a position corresponding to X24 of SEQ ID NO: 267. In some embodiments, the means for blocking uptake and / or retention of a radiotherapeutic to the kidney tissue blocks uptake and / or retention to the kidney tissue greater than as compared to the blocking of uptake and / or retention to the kidney tissue by a means that does not include a modified lysine at a position corresponding to X24 of SEQ ID NO: 267 and / or have at least 90% identity to at least 44 amino acids of SEQ ID NO: 267 and / or have a modified lysine at a position corresponding to X24 of SEQ ID NO: 267. In some embodiments, the means for blocking uptake and / or retention of a radiotherapeutic to the kidney tissue is a radiotherapeutic. In certain embodiments, the radiotherapeutic is targeted to a tumor or a population of cancer cells. In certain embodiments, the radiotherapeutic targeted to the tumor or the population of cancer cells is at a greater concentration than in the absence of the means for binding to kidney tissue. In some embodiments, the radiotherapeutic comprises a polypeptide that targets B7-H3. In some embodiments, the radiotherapeutic comprises or consists of a compound selected from C227-C608 and C611. In certain embodiments, the radionuclide of the radiotherapeutic is selected from Ac-225, Cu-64, Ga-68, In-111, Lu-177, or Pb-212.

[0066] In one aspect, the disclosure provides a kit comprising a polypeptide and instructions for use, wherein the polypeptide has an amino acid sequence as set forth in a polypeptide of a composition, compound, conjugate, or pharmaceutical composition as provided herein. In some embodiments, the polypeptide further comprises one or more of a linker, chelator, and radionuclide. In some embodiments, the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a Page 19 of 344 IPTS / 128790492.1propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n- (gGlu)n- or (PEG)n, wherein n is from 1 to 10, (Gly)1-10, or any fragment or combination via covalent bond thereof. In certain embodiments, the chelator comprises or consists of DOTA, NOPO, Crown, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n- butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB). In certain embodiments, prior to use, the compound is labeled with a radionuclide, wherein the radionuclide is chelated to the chelator to produce a composition with a formula M-L-C-R. In some embodiments, the radionuclide is selected from Ac-225, Cu-64, Ga-68, Lu-177, Pb- 212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211. In some embodiments, the radionuclide is Ac-225, Cu- 64, Ga-68, In-111, Lu-177, or Pb-212.

[0067] In certain embodiments, if the polypeptide has an amino acid comprising any of those set forth in any one of SEQ ID NOs: 199, 204, 241, or 262-272 and wherein the polypeptide further comprises a linker and / or a chelator, wherein the linker, when present, is PEG4, and the chelator, when present, is DOTA. In some embodiments, when present, the linker is attached to the N-terminus amino acid of the polypeptide. In some embodiments, the C-terminal amino acid of the polypeptide is not cysteine. In certain embodiments, when present, the chelator is attached to either the polypeptide or the linker. In certain embodiments, when present, the radionuclide is attached to the chelator. In certain embodiments, when present, the radionuclide is attached to the N-terminus amino acid of the polypeptide. BRIEF DESCRIPTION OF FIGURES

[0068] FIG.1A shows binding affinity of an exemplary miniprotein (SEQ ID NO: 3) for B7-H3 at 50 nM (a), 25 nM (b), 12.5 nM (c), 6.25 nM (d), 3.13 nM (e), 1.56 nM (f), 0.78 nM (g), and 0.39 nM (h).

[0069] FIG.1B shows the 1:1 binding model analysis for an exemplary miniprotein (SEQ ID NO: 3). Black lines are fitted by the model.

[0070] FIG.2A shows binding affinity of an exemplary miniprotein (SEQ ID NO: 4, Run #1) for B7-H3 at 50 nM (a), 25 nM (b), 12.5 nM (c), 6.25 nM (d), and 3.13 nM (e).

[0071] FIG.2B shows the 1:1 binding model analysis for an exemplary miniprotein (SEQ ID NO: 4, Run #1). Black lines are fitted by the model. Page 20 of 344 IPTS / 128790492.1

[0072] FIG.3A shows binding affinity of an exemplary miniprotein (SEQ ID NO: 4, Run #2) for B7-H3 at 50 nM (a), 25 nM (b), 12.5 nM (c), 6.25 nM (d), 3.13 nM (e), and 1.56 nM (f).

[0073] FIG.3B shows the 1:1 binding model analysis for an exemplary miniprotein (SEQ ID NO: 4, Run #2). Black lines are fitted by the model.

[0074] FIG.4 shows the 1:1 binding model analysis for an exemplary miniprotein (SEQ ID NO: 6). Black lines are fitted by the model.

[0075] FIGs.5A and 5B show the relative binding of an exemplary miniprotein to B7-H3 expressing cancer cells (Cancer Cell Line) in comparison to control (Target-null Line) as measured by flow cytometry. FIG.5A shows the median fluorescent intensity (MFI) plotted as a function of concentration of miniprotein administered to cancer cells. FIG.5B shows the overlay of representative histograms of MFI for cancer cells treated with different concentrations of miniprotein relative to a vehicle control.

[0076] FIG.6 shows internal localization of Cy5-labeled B7-H3-specific miniprotein in B7-H3-expressing cancer cells. Cell nuclei are labeled with DAPI stain and arrows indicate localization of the Cy5-labeled B7-H3 miniprotein.

[0077] FIG.7 depicts quantitative uptake in the Opossum kidney proximal tubule cell (OK-PTC) uptake assay. Co-treatment (bar 2, right side) with an exemplary decoy peptide (Compound ID NO: C10) at 20X molar excess reduces the uptake of biotinylated test agent (Compound ID NO: C8) as compared to biotinylated test agent, alone (C8, left side).

[0078] FIG.8 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: C5, C11-C14) demonstrate reduced levels of kidney retention in mouse biodistribution.

[0079] FIG.9 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 peptide (Compound ID NO: C10) reduces kidney uptake of an exemplary B7-H3 targeting affibody conjugate (Compound ID NO: C5).

[0080] FIGs.10A and 10B are graphs depicting data showing reduction in cellular uptake of exemplary target-binding scaffold B miniprotein conjugates when combined with an exemplary scaffold A decoy in vitro. FIG.10A is a bar graph showing percent uptake (on the y-axis) of an exemplary B7-H3-targeting scaffold B miniprotein conjugate (C4) alone / without a decoy (20 μM; column 1, x-axis) or in combination (column 2, x-axis) with a Page 21 of 344 IPTS / 128790492.120-fold molar excess of an exemplary scaffold A decoy (C120). FIG.10B is a bar graph showing percent uptake (on the y-axis) of 20 μM of an exemplary B7-H3-targeting scaffold B miniprotein (C8) alone (20 μM; column 1, x-axis) or in combination (column 2; x-axis) with a 20-fold molar excess of an exemplary scaffold A decoy (C120). Error bars represent standard error of the mean (SEM).

[0081] FIG.11 is a graph depicting data showing reduction in cellular uptake of an exemplary B7-H3-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 (C8) alone / without a decoy (20 μM; column 1, on the x-axis) or in combination with a 20-fold molar excess of one of three exemplary scaffold A decoys (C118, column 2; C119, column 3; and C120, column 4 on the x-axis). Error bars represent standard error of the mean (SEM).

[0082] FIGs.12A and 12B 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.12A is a bar graph showing percent uptake (on the y-axis) of an exemplary B7-H3-targeting scaffold B miniprotein conjugate (C8) alone / without a decoy (20 μM; column 1, x-axis) or in combination (column 2, x-axis) with a 20-fold molar excess of an exemplary scaffold B decoy (C10). FIG.12B is a bar graph showing percent uptake (on the y-axis) of an exemplary B7-H3-targeting scaffold B miniprotein conjugate (C117) alone / without a decoy (1 μM; column 1, x-axis) or in combination (column 2, x-axis) with a 100-fold molar excess of an exemplary scaffold B decoy (C10). Error bars represent standard error of the mean (SEM).

[0083] FIGs.13A and 13B are graphs depicting results from analysis of SPECT / CT scans to quantify in vivo kidney (FIG.13A) or liver (FIG.13B) 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-C5) alone / without a decoy (C5; solid line with solid circles in FIG.13A and in FIG. 13B) or co-administered with 850-fold molar excess of an exemplary scaffold B decoy (C10; dotted line with open circles in FIG.13A and FIG.13B) from 4-24 hours post-injection (x- axis). Error bars represent standard deviation (SD).

[0084] FIG.14 is a graph showing thermal stability of an exemplary compound comprising a conjugate with an exemplary constrained B7-H3 binding miniprotein. Percent Page 22 of 344 IPTS / 128790492.1of C309 (miniprotein of SEQ ID NO: 267) remaining (x-axis) was measured over time (y- axis, hours (h)) at 75°C (solid line) and 90 °C (dotted line).

[0085] FIG.15 is a graph showing on-cell binding as used to measure Ki for an exemplary constrained B7-H3 miniprotein conjugate (C234 with miniprotein of SEQ ID NO: 204).

[0086] FIGs.16A-16B are line graphs showing % ID / g of one of three miniprotein conjugates (C1B, C235, and C301) between 0-48 hours after administration. FIG.16A shows %ID / g in kidney at 1, 4, 24, and 48 hours after administration. FIG.16B shows %ID / g in tumor at 1, 4, 24, and 48 hours after administration.

[0087] FIGs.17A-17B are line graphs showing % ID / g of one of three miniprotein conjugates (C1B, C235, and C309) between 0-48 hours after administration. FIG.17A shows %ID / g in kidney at 1, 4, 24, and 48 hours after administration. FIG.17B shows %ID / g in tumor at 1, 4, 24, and 48 hours after administration.

[0088] FIGs.18A-18B are bar graphs showing %ID / g as measured 24 hours after administration of one of several exemplary 111-In labeled linear (C131 and C165) or constrained (C229, C235, C276, C301, and C333) B7-H3 miniprotein conjugates in tumor (FIG.18A) and kidney (FIG.18B), showing increased concentration in tumor and decreased concentration in kidney for exemplary constrained B7-H3 miniprotein conjugates as compared to exemplary linear B7-H3 miniprotein conjugates.

[0089] FIGs.19A-19D show line graphs of efficacy data, as measured by tumor volume in a low (H1915) and high (H358) B7-H3 expressing cell line after 17 days (FIG.19A, 1915 cells) and 42 days (FIG.19C, H358 cells) respectively, each evaluated following administration of a single dose of vehicle or one of two concentrations (500 or 1000 nCi) of an exemplary 225-Ac labeled constrained B7-H3 miniprotein conjugate (C332). Corresponding bodyweights for FIG.19A are shown in FIG.19C and bodyweights for FIG. 19B are shown in FIG.19D.

[0090] FIGs.20A-20B show line graphs of efficacy data as measured by tumor volume (FIG.20A) and bodyweight (FIG.20B) between about 0-6 weeks after administration of a single dose of vehicle or one of two concentrations (500 or 1000 nCi) of an exemplary 225- Ac labeled constrained B7-H3 miniprotein conjugate (C331). DETAILED DESCRIPTION

[0091] Among other things, the present disclosure provides compositions and methods of use thereof. In some embodiments, a composition selectively binds to a target (e.g., B7-H3). Page 23 of 344 IPTS / 128790492.1In 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 expressing B7-H3 such that the B7-H3-expressing cell is treated and cells not expressing B7- H3 are not treated. 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 (e.g., cancer cells) and to deliver a therapeutic in a way that minimizes damage to surrounding cells (e.g., non-cancer cells) including, for example, cells around cancer cells and cells in non-target organs (e.g., kidney).. Surrounding cells (e.g., as may be present in one or more non-tumor tissues) can also express the target at lower amounts or levels than the target cells.. In some such embodiments, such expression may be undetectable. The present disclosure provides the insight that a combination of selective targeting (e.g., of B7-H3, e.g., by a polypeptide, e.g., a miniprotein, e.g., as provided herein) with a specific therapeutic such as a chelator and / or radionuclide (e.g., an alpha emitter) provides an advantage over previously and / or currently used therapeutics (e.g., antibodies, beta-emitters, etc.)..

[0092] Furthermore, the present disclosure provides the insight that even with more specifically targeting therapeutics (e.g., such as those disclosed herein), certain challenges can still arise. Among other things, 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, the kidney resulting in (1) faster clearance; (2) reduced tumor targeting (e.g., including due to uptake and / or retention by non-target tissue); and / or (3) non-target tissue (e.g., kidney) damage. The present disclosure recognizes that any or all of these challenges may be mitigated or prevented (i) with miniproteins as provided herein (e.g., such as disclosed in Table 2A and / or 2C), including, for example, miniproteins having particular features (e.g., constraints, e.g., as in Table 2C) and / or (ii) by combining administration of a therapeutic with administration of a decoy (e.g., as set forth in Table 2D). In some embodiments, the decoy reduces or prevents uptake by the kidney of a composition comprising a radionuclide. Without wishing to be bound by theory, the disclosure contemplates that improvement in treatment efficacy is at least maintained while reducing damage to the kidney and, in some embodiments, treatment efficacy is improved while simultaneously reducing risk of harm or actual harm to kidney tissue and / or renal system tissues (e.g., ureters, bladder, etc.)..

[0093] 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. Furthermore, unless otherwise required by context, singular terms Page 24 of 344 IPTS / 128790492.1shall 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.

[0094] 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.

[0095] 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.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present disclosure pertains. 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.

[0097] 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.

[0098] As used herein, ranges and amounts can be expressed as “about” a particular value or range, e.g., “about” one particular value, and / or to “about” another particular value. About also includes the exact amount. Hence “about 100 nucleotides” means “about 100 Page 25 of 344 IPTS / 128790492.1nucleotides” and also “100 nucleotides.” Given context, the term “about” as used herein also includes an amount that would be expected to be within experimental error. If “about” appears before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. In such instances “about” can also refer to a ±10% variation from the nominal value unless otherwise indicated or inferred. 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] As used herein, the term “affibody” refers to a subgenus of miniproteins. An affibody is a molecule 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; 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). Page 26 of 344 IPTS / 128790492.1

[0104] 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.

[0105] As used herein, the terms “amino acid sequence” and “polypeptide” refer to a polymer of amino acids connected by one or more peptide bonds. A polypeptide of the present disclosure encompasses both naturally-occurring and non-naturally-occurring proteins, and any fragments, portions, peptides, mutants, derivatives, and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities. A polypeptide may be fully or partially synthetic or otherwise modified (i.e., comprising one or more synthetically- produced amino acids and / or modifications thereof). The term “peptide” may be used to refer to a short polypeptide, such as one comprising fewer than about 70 amino acids (e.g., between about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids).

[0106] As used herein, a “compound” refers at least to a miniprotein with an amino acid sequence. Compounds may include miniproteins with different modifications, such as a N- terminal modification or a C-terminal modification. 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, different chelators, and / or different radionuclides. N- and C- terminal modifications may include but not be limited to acetyl, acid, or amide (e.g., Acetyl, NH2, OH / COOH), such as provided in exemplary compounds and miniproteins of Table 2A or Table 2C. In some embodiments, a polypeptide according to the disclosure may have various modifications to its N-terminus (e.g., such as set forth in exemplary compounds in Table 2A or Table 2C), and can have an acid or amide group on its C-terminus (see, e.g., Table 2A or Table 2C). A given polypeptide having a particular amino acid sequence can have one or more N-terminal and / or C-terminal differences without materially changing the Page 27 of 344 IPTS / 128790492.1utility or function of the polypeptide, such as for binding to B7-H3 (e.g., for detection and / or treatment of cancer).

[0107] As used herein, the term “attenuate” as used herein generally refers to a functional 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.

[0108] As used herein, the term “binder” refers to a subgenus of miniprotein. A binder is characterized in that it comprises or consists of a polypeptide (e.g., peptide) that is capable of binding or has known ability to engage and associate a target. Binders generally comprise a cysteine-containing peptide comprising one or more disulfide bonds, 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 a polynucleotide, polypeptide, using a vector, host cell , etc., and / or any combination of modalities. A binder may 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.

[0109] 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 tissue, e.g., a liver tissue). In some embodiments, a decoy blocks uptake of a conjugate or compound of the disclosure into a non-tumor tissue, such as Page 28 of 344 IPTS / 128790492.1kidney tissue. In some embodiments, a decoy blocks by reducing retention of a compound (e.g., a radiotherapeutic compound, e.g., comprising a miniprotein) in a non-tumor tissue (e.g., kidney, e.g., liver).

[0110] As used herein, the term “chelator” refers to any molecule or moiety that is 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.

[0111] As used herein, the term “conjugated” refers to the joining by covalent or noncovalent means of two compounds or agents.

[0112] 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); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0113] 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. 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., pubmed.ncbi.nlm.nih.gov / 29483648 / . 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: 553).

[0114] 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. Page 29 of 344 IPTS / 128790492.1

[0115] 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.

[0116] As used herein, the term “derived from,” with reference to a nucleic acid sequence refers to a nucleic acid sequence that was developed using and has at least 85% sequence identity to a reference nucleic acid sequence. 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 derived nucleic acid or amino acid sequence. For example, the nucleic acid or amino acid sequence can be chemically synthesized.

[0117] As used herein, the term “having determined” refers to the process or the act of requesting from a third party (e.g., lab, hospital, nurse, physician) to carry out or provide results from a test, procedure, experiment, assay, analysis, etc., that defines the presence (or absence) of a given marker, e.g. a biomarker or a genetic mutation (e.g., the level of expression of B7-H3 in a biological sample, such as a sample from a tumor), from a patient.)

[0118] As used herein, the term “domain” as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non- contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

[0119] 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; Page 30 of 344 IPTS / 128790492.1and 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.

[0120] As used herein, the term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to 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.

[0121] 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.

[0122] As used herein the term “identical” refers to a nucleic acid sequence or of least two nucleic acid or refers to an amino acid sequence of at least two amino acid sequences or Page 31 of 344 IPTS / 128790492.1subsequences 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 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.

[0123] 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 Page 32 of 344 IPTS / 128790492.1or 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 native environment. In some embodiments, as used in reference to an isolated construct, isolated means in the absence of a pharmaceutically acceptable salt.

[0124] 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 antibody (e.g., targeting miniprotein, e.g., B7-H3 binding miniprotein) of the present disclosure binds to B7-H3 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. KD = kd / ka.

[0125] 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).

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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®). Page 33 of 344 IPTS / 128790492.1

[0130] 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 introduction of one or more nucleotides into its open reading frame, which results in translation of a nonsense or otherwise nonfunctional protein product.

[0131] The term “knottin” as used herein refers to a structural motif of a miniprotein containing three disulfide bridges.

[0132] The term “knottin peptide” as used herein refers to a subgenus of miniproteins that comprises at least one knottin.

[0133] The term “linker” as used herein refers to a moiety that is used to conjugate a miniprotein to a chelator.

[0134] As used herein, the term “miniprotein” refers to short proteins of less than or equal to 100 amino acids with well-defined folds comprising two or more secondary structure elements, a sequestered hydrophobic core, and / or cooperative folding. Affibodies, CDPs, knottins, and binders as disclosed herein are all examples of miniproteins.

[0135] 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.

[0136] 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., Page 34 of 344 IPTS / 128790492.1Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference).

[0137] As used herein, the term “molecule” means any compound, including, but not limited to, a miniprotein, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a molecule (e.g., miniprotein, compound, etc.) can be natural or synthetic or a combination of natural and synthetic.

[0138] As used herein, the term “monobody” or “adnectin” are used interchangeably and refer to a subgenus of miniproteins. A monobody is a molecule, preferably based on the 10th extracellular 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 specific loops of the protein.

[0139] 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). Page 35 of 344 IPTS / 128790492.1

[0140] As used herein, the terms “polypeptide mutant” or “mutein” 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 native or wild-type protein. A 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 sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein. A mutein has 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 (5) confer or modify other physicochemical or functional properties of such analogs.

[0141] 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 miniprotein may comprise or consist of a non-disulfide sequence.

[0142] 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 sited in each of the above, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the present Page 36 of 344 IPTS / 128790492.1disclosure may be used to produce an equivalent effect and are therefore envisioned to be part of the present disclosure.

[0143] 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, 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 phosphoramidate 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. Page 37 of 344 IPTS / 128790492.1

[0144] 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.

[0145] 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 or parent sequence. Fragments typically are at least 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.

[0146] As used herein, the term “radionuclide” refers to an atom capable of undergoing radioactive decay.

[0147] As used herein, the term “radiotherapeutic” refers to a radionuclide-labeled miniprotein or compound as provided herein, comprising a radionuclide. 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.

[0148] 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 Page 38 of 344 IPTS / 128790492.1cell 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” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0149] 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.

[0150] 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.

[0151] As used herein the phrase “secondary structure elements” refers to local folded structures that form within a polypeptide due to interactions between atoms of its backbone. Examples of secondary structure elements can include an alpha helix, a beta sheet, a 310 helix, a pi helix, and a random coil. A miniprotein of the present disclosure may comprise one or more of any of such secondary structures (e.g., one or more alpha helix, one or more alpha helices and one or more beta sheets). It will be understood by those of skill in the art that secondary structure elements may be joined by loop regions, which may or may not be modified to change the interactions of secondary structure elements of the polypeptide. As will be understood to those of skill in the art, in some embodiments, loops may be secondary structural elements. In some embodiments, loops may be interstructural elements that are not necessarily considered secondary structural elements.

[0152] As used herein, “sequence homology” for polypeptides, also referred to as “percent sequence identity,” is typically measured using sequence analysis software. See, Page 39 of 344 IPTS / 128790492.1e.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 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.

[0153] As used herein, the term “specific activity” generally refers to the activity per unit (e.g., mass, e.g., moles) of a radionuclide. Units of specific activity may include units of Page 40 of 344 IPTS / 128790492.1megabecquerel per microgram (MBq / μg), microcurie per microgram (μCi / μg), microcurie per nanomole (μCi / nmole), etc.

[0154] As used herein, the term “specificity” generally refers to a sequence (e.g., of a protein, e.g., of a miniprotein, .e.g., a miniprotein having certain amino acids) that, when in a conformation that can bind, selectively or “specifically” binds to a specific target (e.g., an antigen, such as expressed on a tumor, e.g., B7-H3, e.g., certain cell types, e.g., kidney cells, e.g., kidney proximal tubule cells, etc.).

[0155] As used herein, “specifically binds” means that the binding of a polynucleotide, polypeptide, or protein is selective for a specified antigen (e.g., target) and can be discriminated from unwanted or non-specific interactions. Hallmarks of “specific binding” include saturability of binding to the target as well as a demonstrable ability of that binding to be competed by the introduction of additional specifically binding molecules against that same target. 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 molecules (e.g., entities such as miniproteins), “specific binding” refers to the ability of two molecules to bind to each other in preference to binding to other molecules 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).

[0156] 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 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 Page 41 of 344 IPTS / 128790492.15°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.

[0157] 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.

[0158] 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 are evaluated in a population comprising a plurality of subjects.

[0159] 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 molecule (or its complementary strand 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 Page 42 of 344 IPTS / 128790492.1nucleic 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.

[0160] As used herein, the term “target” refers to a protein or functional portion or variant thereof. A target is a protein 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., affibody, CDP, knottin, binder, 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. 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., 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 B7-H3, a target may also be a protein in a pathway that is necessary for B7-H3 activity. A target may be or comprise a binding region, such as an epitope, to which a miniprotein (e.g., affibody, CDP, knottin, binder, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) of the present disclosure binds. 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 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 miniprotein (M) is designed to bind; in some embodiments, a target (e.g., a non-tumor cell, e.g., a kidney cell, a liver cell, etc.) is bound by a decoy rather than a polypeptide (e.g., a miniprotein) of a composition (e.g., a radiotherapeutic composition) provided herein.

[0161] As used herein, the term “treatment” (as well as “treat” or “treating”) refers to partial or complete alleviation, amelioration, mitigation, prevention, 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.

[0162] As used herein, the term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which generally refers to a circular double stranded DNA loop Page 43 of 344 IPTS / 128790492.1into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other 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”).

[0163] As used herein, the term “decharged molecules” refers to molecules that have been modified to contain fewer positively charged or polar features, greater negatively charged features, and / or both as compared to a parent molecule.

[0164] As used herein, the term “surface charge” refers to the electrostatic charge present at the surface of a protein (e.g., such as a miniprotein). In various embodiments, the surface charge of a miniprotein can influence the kidney uptake of the miniprotein (e.g., increase the kidney uptake of the miniprotein or reduce the kidney uptake of the miniprotein).

[0165] As used herein, the term “surface patch” refers to regions on the 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 the kidney uptake of the protein (e.g., miniprotein).

[0166] 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. Examples of cleavable linkers are linkers that contain hydrazine, or disulfide bonds, or enzymatically cleavable peptide sequences.

[0167] As used herein, the term “scaffold” refers to a particular set of structural characteristics of a given protein (e.g., a miniprotein, e.g., a binder, e.g., a decoy peptide). Scaffold is also 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., Page 44 of 344 IPTS / 128790492.1all alpha helix proteins (“a”), all beta sheet proteins (“b”), 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. To give but one example, a scaffold may be an “affibody” scaffold as provided herein, and the affibody may be a target-binding affibody (e.g., B7-H3 binding) or an affibody decoy, where each entity has an affibody scaffold comprising, e.g., certain secondary structural features (e.g., alpha helices and / or beta sheets, disulfide bridges at particular, consistent cysteine residues, etc.). Scaffolds of the disclosure may be characterized as “Scaffold A” and “Scaffold B” types. Exemplary Scaffold A decoys include compounds C118-C120. Exemplary Scaffold B decoys have affibody scaffolds and include compounds C10; B7-H3-targeting polypeptides of the disclosure are also Scaffold B affibodies, such as C1-C9, C11-C117.

[0168] As used herein, the term “decoy peptides” or “decoys” refers to molecules specifically designed to mimic the role of a certain receptor protein and interact with certain target entities. One such type of decoy peptides or decoys is 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., a tumor expressing a target, e.g., B7-H3); 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 kidney tissue, as compared to uptake and / or retention in kidney tissue in the absence of the decoy peptide. Without wishing to be bound by theory, the disclosure describes, in some embodiments, a decoy peptide (or decoy) that decoys a composition such as a radiotherapeutic, 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 peptide is referred to as “decoying,” e.g., a composition, e.g., a compound, e.g., a miniprotein that binds to a target (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). Page 45 of 344 IPTS / 128790492.1Compositions

[0169] Provided herein are novel compositions comprising one or more of a polypeptide, linker, 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 polypeptide (i.e., a miniprotein), an optional linker, and a chelator and / or radionuclide. In some embodiments, a chelator and / or radionuclide are conjugated to a miniprotein via a linker. In some embodiments, a miniprotein of the present disclosure comprises or consists of an affibody, a CDP, a knottin and / or a binder. In some embodiments, the miniprotein comprises or consists of an affibody. 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 embodiments the miniprotein (e.g., affibody, CDP, knottin, binder, 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., affibody, CDP, knottin, binder, 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.

[0170] Without wishing to be bound by any particular theory, the present disclosure contemplates that compositions of the present disclosure are more effective than previously described compositions (e.g., such as those comprising antibodies and / or beta-emitter radionuclides). For example, while miniproteins (e.g., to be used in compositions as provided herein) 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., affibody, CDP, knottin, binder, 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., affibody, CDP, Page 46 of 344 IPTS / 128790492.1knottin, binder, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) may be or comprise a cysteine dense peptide. In some embodiments, a miniprotein (e.g., affibody, CDP, knottin, binder, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) comprises one or more disulfide bridges. In some embodiments, a miniprotein (e.g., affibody, CDP, knottin, binder, 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).

[0171] Among other things, the present 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. In some such embodiments, the miniprotein may form a dimer, such as with another miniprotein (e.g., self-dimerization). In certain embodiments, a miniprotein (e.g., that binds to B7-H3) comprises at least two cysteine residues. In some embodiments, a miniprotein comprises at least four cysteine residues.. In certain embodiments, where a miniprotein comprises at least two or at least four cysteine residues, the miniprotein also comprises a disulfide bridge between pairs of the cysteine residues. In certain embodiments, a miniprotein must have at least one or two disulfide bridges. In some embodiments, the miniprotein comprises three disulfide bridges (and at least six cysteine residues). In certain embodiments, constraints include more than one type of constraint (e.g., a disulfide bridge, a lactam bridge, an alkyl stapled bridge, and any combinations thereof).. For example, in some embodiments, the disclosure provides a polypeptide comprising at least one or two disulfide bridges and a lactam bridge. Without wishing to be bound by theory, the present disclosure contemplates that stability conferred by crosslinked cysteines contributes to reduced immunogenicity of miniproteins or 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) and drug- like physicochemical properties (e.g., stability, solubility, monomeric).

[0172] 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, miniproteins of the present disclosure may be formulated such that they are combined with other components such as a therapeutic Page 47 of 344 IPTS / 128790492.1molecule (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.

[0173] In some embodiments a miniprotein (e.g., affibody, CDP, knottin, binder, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), 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).

[0174] In some embodiments, a composition comprising a linker, chelator, and / or radionuclide can efficiently penetrate a tumor.

[0175] In some embodiments, miniproteins (e.g., affibody, CDP, knottin, binder, 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..

[0176] As described herein, miniproteins (e.g., affibody, CDP, knottin, binder), 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., affibody, CDP, knotting, binder) with a radionuclide. That is, without being bound by any particular theory, the present disclosure provides a conjugate wherein, in some embodiments, a miniprotein (e.g., affibody, CDP, knottin, binder, engineered Kunitz domain, monobody, Page 48 of 344 IPTS / 128790492.1anticalin, designed ankyrin repeat domain (DARPin), avimer) targets a radioisotope to which it’s 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 B7-H3. In some embodiments, the cell is a tumor cell. In some embodiments, the conjugate binds to the B7-H3 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.. Targets

[0177] Any cell expressing a target may be targeted by a miniprotein (e.g., affibody, CDP, knottin, binder, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) as provided herein.

[0178] 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 miniprotein provided by the present disclosure), 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.

[0179] 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).

[0180] 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 instance, in some Page 49 of 344 IPTS / 128790492.1embodiments, 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.

[0181] In some embodiments, the miniprotein or composition comprising a miniprotein specifically binds a target (e.g., B7-H3) 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.

[0182] In some embodiments, the miniprotein or composition comprising a miniprotein specifically binds to B7-H3. In some embodiments, the target comprises or consists of B7- H3. In some embodiments, the miniprotein specifically binds to a target comprising an amino acid sequence or portion thereof as set forth in Table 1A. B7-H3

[0183] B7-H3 is also known as CD276. B7-H3 is considered an immune checkpoint protein and is expressed by immune cells (e.g., APCs, macrophages) and tumor cells. B7-H3 has also been shown to play an inhibitory role on T cells and has been found to contribute to tumor cell immune evasion. (Dong et al., Front Oncol.2018 Jul 6;8:264). Page 50 of 344 IPTS / 128790492.1

[0184] B7-H3 overexpression has been documented in many cancers including, for example, bladder, breast, cervical, colorectal, esophageal, glioma, kidney, liver, lung, ovarian, pancreatic, prostate, intrahepatic cholangiocarcinoma, liver, oral squamous cell carcinoma, endometrial cancer, and squamous cell carcinoma and gastric cancer, glioma, and melanoma. (Dong et al., Front Oncol.2018 Jul 6;8:264). Studies of B7-H3 overexpression have also shown association of B7-H3 levels with advanced tumor stage, high tumor grade, and poor clinical prognosis. Without being bound by any particular theory, such overexpression of B7-H3 is thought to contribute to metastasis in a number of different cancer types and by a number of possible mechanisms. For example, in some embodiments, B7-H3 may play a role in cancer cell proliferation and invasiveness. (Id.). In studies documenting B7-H3 knockdown, significant suppression of cell migration and invasion was observed in cells from prostate, breast, gastric, liver, pancreatic, colorectal and melanoma cancers. (Id.).

[0185] In some embodiments, overexpressed B7-H3 on tumor cells has been shown to be successfully targeted including with T-cell-mediated immunotherapy. For example, one study showed that specific cytotoxic activity of activated T cell (ATC) also having a novel anti- CD3 x anti-B7-H3 bispecific antibody showed increase in cytotoxicity relative to ATC alone, resulting in inhibition of tumor growth and increased survival in xenograft models. (Ma J., et al, Oncotarget (2016) 7(20):29480–91). In some embodiments, a binder of the present disclosure binds to B7-H3 expressed on one or more cells (e.g., cancer cells). In some such embodiments, the present disclosure contemplates targeting of B7-H3 with one or more binders of the present disclosure, wherein the B7-H3 is expressed at higher levels on cancer cells relative to non-cancer cells, and wherein the binding of the miniprotein is specific for cancer cells.

[0186] In some embodiments, compositions provided by the present disclosure more specifically and effectively target a cell overexpressing B7-H3 while minimizing or eliminating damage to surrounding cells not expressing or lowly expressing B7-H3 by providing a targeted composition including, in some embodiments, a chelator and / or alpha- emitter, which when combined with a miniprotein as provided herein provide specific, efficient and effective approaches to target cells overexpressing B7-H3. As used herein, cells that “lowly express B7-H3” refer to cells that do not express B7-H3 and / or cells that express B7-H3 at a level less than 50% of the level of expression of B7-H3 of cells that overexpress B7-H3. In particular embodiments, cells that “lowly express B7-H3” refer to cells that Page 51 of 344 IPTS / 128790492.1express B7-H3 at a level less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the level of expression of B7-H3 of cells that overexpress B7-H3.

[0187] In some embodiments, there is a relationship between B7-H3 levels and metastasis. For example, some studies have documented a relationship between B7-H3 and metastasis such as increased expression levels of metastasis-associated proteins such as MMP2, STAT3, and IL-8 (Tekle et al., Int J Cancer (2012) 130(10):2282–90) and increased levels of CXCR4 and activation of AKT, ERK, and JAK2 / STAT3 pathways. (Wang et al., Tumour Biol (2016) 37(3):2961–71). In some embodiments, changes in B7-H3 can impact cancer treatment. For example, studies have shown that changes in B7-H3 can impact efficacy of some cancer treatments. In breast cancer, specifically, B7-H3 has been shown to reduce efficacy of paclitaxel through activation of JAK2 / STAT3 and silencing of B7-H3 both abrogates JAK2 / STAT3 phosphorylation and improves paclitaxel sensitivity. (Liu et al., Mol Cancer Ther (2011) 10(6):960–71). Similarly, in colorectal cancer, overexpression of B7-H3 has been shown to reduce efficacy of 5-fluorouracil through JAK2 / STAT3 activation. (Zhang et al., World J Gastroenterol (2015) 21(6):1804–13). In some embodiments, immunoglobulin- like-transcript 4 (ILT4), an inhibitory receptor of certain immune cells, may upregulate B7- H3 expression through the PI3K / AKT / mTOR pathway in lung cancer cells (see, e.g., Zhang P et al., FEBS Lett (2015) 589(17):2248–56). In some embodiments, co-expression of ILT4 and B7-H3 may be positively associated with lymph node metastasis, advanced tumor stage and poor clinical prognosis in NSCLC (see, e.g., Zhang P et al., FEBS Lett (2015) 589(17):2248–56).

[0188] Without wishing to be bound by theory, B7-H3 may play a role in cell transitions and potential. For instance, in some embodiments, B7-H3 may increase cell transitions and stemness. For example, B7-H3 has been shown to increase epithelial-mesenchymal transition and cancer “stemness” through increased expression of several proteins including vimentin, CD133, CD44 and OCT4 and decreased expression of E-cadherin. (Jiang et al., Oncotarget (2016) 7(22):31755–71). In some embodiments, B7-H3 overexpression may occur due to genomic DNA amplification. In some embodiments, B7-H3 overexpression may occur due to increased transcription. In some embodiments, B7-H3 overexpression may occur due to decreased degradation.

[0189] By way of non-limiting example, though it is not presently known whether B7-H3 overexpression is due to, e.g., genomic DNA amplification or increased transcription, chromatin immunoprecipitation has demonstrated an androgen receptor-binding site upstream of B7-H3, as well as B7-H3 expression reduction in presence of androgens (Benzon B. et al., Page 52 of 344 IPTS / 128790492.1Prostate Cancer Prostatic Dis (2017) 20(1):28–35). Along similar lines, in some embodiments, B7-H3 may increase expression of genes (e.g., thymidylate synthase) via PI3K / AKT pathway. (see, e.g., Jiang et al., Tumour Biol (2016) 37(7):9465–72). Furthermore, data have shown that oxaliplatin treatment in colorectal cancer can be made more efficacious through inhibition. In some embodiments, B7-H3 is contemplated as contributing to chemoresistance by increasing expression of XRCC1 via PI3K / AKT pathway (see, e.g., Zhang et al., Biochem Biophys Res Commun (2017) 490(3):1132–8), which may, in some embodiments, increase expression of BRCC3, which also opposes DNA damaging effects of 5-FU (see, e.g., Sun et al., Oncol Rep (2016) 36(1):231–8).

[0190] Without limitation, in some embodiments, B7-H3 may play a role in cancer metabolism. Specifically, it has been contemplated that B7-H3 promotes the “Warburg Effect” of preferential metabolism of glucose to lactate when abundance oxygen is present. This Warburg Effect phenomenon has been observed through HIF1α in breast cancer cells and in a mouse model of breast cancer. (Lim et al., Cancer Res (2016) 76(8):2231–42). Similarly, in some embodiments, B7-H3-mediated decrease may impact proliferation and Warburg effect. Specifically, data have shown that in metastatic melanoma, B7-H3-mediated decrease during treatment of metastatic melanoma has been shown to be overcome through use of a monoclonal B7-H3 antibody, which decreased proliferation and the Warburg effect in melanoma. (Flem-Karlsen K, et al., Pigment Cell Melanoma Res (2017) 30(5):467–76). Treatment with monoclonal anti-B7-H3 has also demonstrated efficacy in the rare stage IV pediatric neuroblastoma, DIPG. (Zhou Z, J Neurooncol (2013) 111(3):257–64).

[0191] In some embodiments, B7-H3 interacts with other cellular components or units, such as miRNA. For example, B7-H3 has also been found to interact with many miRNAs that are thought to be involved in cancer. (Dong et al., Front Oncol.2018 Jul 6;8:264). For example, the 3’ UTR of B7-H3 transcripts has been observed to interact with miR-214, miR- 363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593 in breast cancer, resulting in reduced B7-H3 expression (Nygren MK, et al., Br J Cancer (2014) 110(8):2072–80). In osteosarcoma, the 3’ UTRs of B7-H3 transcripts have been shown to interact with miR-124 and a study in colorectal cancer cells showed that iASPP-mediated p53 repression led to downregulation of miR-124, which was accompanied by increased expression of B7-H3. (Dong et al., Front Oncol.2018 Jul 6;8:264). Furthermore, in a colorectal cancer study, oncogenesis was promoted via miR-155 / miR-143 axis (Zhou et al., Oncotarget (2016) 7(41):67196–211). Page 53 of 344 IPTS / 128790492.1Specifically, in colorectal cancer cells, TGFβ elevated miR-155 expression (via SMAD3 and SMAD4) resulted in reduced CEPBP expression, which led to reduced miR-143 expression in colorectal cancer cells. B7-H3 is a target of miR-143, thus reduced miR-143 expression resulted in increased B7-H3 expression supporting the possibility that in some contexts, TGFB may contribute to cancer immune escape by upregulating B7-H3. (Zhou et al., Oncotarget (2016) 7(41):67196–211; Dong et al., Front Oncol.2018 Jul 6;8:264).

[0192] Importantly, novel compositions provided by the present disclosure are capable of specifically, efficiently, and effectively targeting B7-H3 overexpressing cells with reduced toxicity as compared to presently available treatments. That is, in some embodiments, a composition targeting B7-H3 as provided by the present disclosure provides improved treatment as compared to presently available treatments.

[0193] In some embodiments, a target of compositions of the present disclosure comprises or consists of B7-H3 (e.g., human B7-H3).

[0194] In certain embodiments, a miniprotein is selected from any of Tables 1B-1E , 2A and 2C, or portions or fragments thereof.. In certain embodiments, a miniprotein binds to B7-H3 and is selected from any of Tables 1B-1E, 2A, and 2C. In some embodiments, the miniprotein that binds to B7-H3 is not selected from any of Tables 1B, 1C, or 2A.

[0195] In some embodiments, a B7-H3 binding miniprotein has an amino acid sequence comprising or consisting of an amino acid sequence according to any one of SEQ ID NOs: 4- 6, 8-94 and 100-537 and can have different N- and / or C-terminal ends, such as, for example, an Acetyl, NH2, Biotin-PEG4, DOTA-PEG4, radiolabel, etc. on its N-terminus and an - OH / COOH or -NH2 on its C-terminus. N- and / or C-termini of B7-H3 binding miniproteins of the disclosure can include but are not limited to acetyl, acid, or amide (e.g., Acetyl, NH2, OH / COOH), such as provided in exemplary compounds and miniproteins of Table 2A or Table 2C.

[0196] In some embodiments, a B7-H3 binding miniprotein does not have an amino acid sequence comprising or consisting of an amino acid sequence according to any one of SEQ ID NOs: 4-6, 8-94 and 100-197.

[0197] In some embodiments, a polypeptide according to the disclosure may have various modifications to its N-terminus (e.g., a linker, chelator, and / or radionuclide, e.g., such as set forth in exemplary compounds in Table 2A or Table 2C) or its C-terminus (e.g., a linker, chelator, and / or radionuclide). In some embodiments, the C-terminus of a given polypeptide can have an acid or amide group on its C-terminus (see, e.g., Table 2A, e.g., Table 2C). A Page 54 of 344 IPTS / 128790492.1given polypeptide having a particular amino acid sequence can have one or more N-terminal and / or C-terminal differences without materially changing the utility or function of the polypeptide, such as for binding to B7-H3 (e.g., for detection and / or treatment of cancer).

[0198] In some embodiments, a target of compositions of the present disclosure comprises or consists of B7-H3. In some embodiments, a material change in a basic and novel characteristic of a polypeptide (e.g., miniprotein) provided herein is the ability to strongly and specifically bind to its intended target (e.g., B7-H3, e.g., B7-H3 on a cancer cell) with minimal or no off-target effects and minimal kidney uptake (e.g., as compared to kidney uptake of previously developed B7-H3 binding molecules). In some embodiments, B7-H3 is expressed on the surface of a cell. Alternatively, in some embodiments, B7-H3 is released from tumor associated cells and present in the tumor microenvironment. (see, e.g., Zhang et al., Immunology.2008 Apr; 123(4): 538–546). In some embodiments, the cell is a cancer cell. In some such embodiments, the cancer cell is a tumor cell and the tumor is a solid tumor. In some embodiments, a level of B7-H3 expressed in a tumor cell or population of tumor cells is higher than that expressed in non-tumor cells. In some embodiments, targeting of B7- H3 by a miniprotein or composition comprising a miniprotein as provided by the present disclosure specifically targets a composition or one or more components thereof (e.g., a chelator and / or radionuclide) to a cancer cell or a tumor microenvironment (e.g., a location comprising a population of cancer cells or cells at risk of becoming cancer cells).

[0199] In some embodiments, compositions in accordance with the present disclosure specifically bind to B7-H3 (e.g., through a polypeptide that binds to B7-H3, e.g., through a miniprotein that specifically binds to B7-H3). In some embodiments, a compound targeting B7-H3 is disclosed in Table 2A or Table 2C.

[0200] In some embodiments, a composition comprising a miniprotein comprises or consists of a protein comprising a specific an amino acid sequence that binds to B7-H3 or a portion thereof. In some embodiments, certain exemplary B7-H3 binding miniproteins are engineered to have an amino acid sequence comprising one or more non-natural amino acids (e.g., such as set forth herein, e.g., such as set forth in Tables 2A and / or 2C, e.g., an amino acid with a small alkyl group on its side chain, etc.). In some such embodiments, such a B7- H3 miniprotein comprises or consists of an amino acid sequence selected from any of SEQ ID NOs: 4-6, 8-94 and 100-537 or a functional variant or portion thereof (e.g., a functional fragment, e.g., a miniprotein that folds and binds to B7-H3 or a portion thereof). In some embodiments, such a B7-H3 miniprotein is a binding protein or part of a conjugate comprising such a binding protein as set forth in Table 2A or Table 2C. In certain Page 55 of 344 IPTS / 128790492.1embodiments, a B7-H3 miniprotein has an amino acid sequence comprising that set forth according to any of Tables 1B-1E.

[0201] In some embodiments, a composition comprising a miniprotein comprises or consists of a protein comprising a specific an amino acid sequence that binds to B7-H3 or a portion thereof.

[0202] In some embodiments, the present disclosure provides a polynucleotide encoding a polypeptide that comprises or consists of one or more portions of a composition as provided herein. In some embodiments, the present disclosure provides a vector and / or host cell comprising a sequence encoding one or more components of a composition as provided herein.

[0203] In some embodiments, the present disclosure provides methods of detecting a target. In some embodiments, a method as provided herein comprises detecting presence of a target for, e.g., imaging, e.g., diagnostic, prognostic, and / or monitoring purposes, e.g., treatment. In some embodiments, the present disclosure provides methods of treatment and / or methods of manufacturing using composition as provided herein (e.g., a miniprotein, e.g., a linker-chelator, e.g., a miniprotein comprising one or more of a linker, chelator, and radionuclide, etc.). In some embodiments, a method of treatment comprises administering a composition as provided herein to a subject in need thereof.

[0204] In some embodiments, when present, the linker is attached to the N-terminus of the polypeptide (e.g., the B7-H3 binding miniprotein). In some embodiments, when present, the linker is attached to the C-terminus of the polypeptide (e.g., the B7-H3 binding miniprotein).

[0205] In some embodiments, the C-terminal amino acid of the polypeptide is not a cysteine.

[0206] In some embodiments, when present, the chelator is attached to either the polypeptide or the linker. In some embodiments, when the chelator is present without a linker, the chelator is attached to the N-terminus of the polypeptide. In some embodiments, when the chelator is present without a linker, the chelator is attached to the C-terminus of the polypeptide.

[0207] In some embodiments, when present, the radionuclide is attached to the chelator. In some embodiments, when the radionuclide is present without a linker or chelator, the radionuclide is attached to the N-terminus of the polypeptide. In some embodiments, when the radionuclide is present without a linker or chelator, the radionuclide is attached to the C- terminus of the polypeptide. Page 56 of 344 IPTS / 128790492.1

[0208] In some embodiments, the disclosure provides a composition comprising a formula selected from one or more of (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 (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M comprises an amino acid sequence of any one of SEQ ID NO: 4-6, 8-94 and 100-537.

[0209] In some embodiments, the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is from 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment or combination via covalent bond thereof.

[0210] In some embodiments, the chelator comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

[0211] In some embodiments, the radionuclide Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm- 153, Ra-225, Tb-165, or At-211.

[0212] In some embodiments, the disclosure provides a composition comprising a formula selected from one or more of (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 (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising any one of those set forth in SEQ ID NOs: 198-537.

[0213] In some embodiments, when L is present, L comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36, 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 dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n (SEQ ID NO: 551) - or (PEG)n, wherein n is from 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment or combination via covalent bond thereof.

[0214] In some embodiments, when C is present, C comprises or consists of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n- butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB). Page 57 of 344 IPTS / 128790492.1

[0215]

[0238] In some embodiments, when R is present, R comprises or consists of Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

[0216] In some embodiments, when present, the linker is attached to the N-terminus of the polypeptide (e.g., the B7-H3 binding miniprotein). In some embodiments, when present, the linker is attached to the C-terminus of the polypeptide (e.g., the B7-H3 binding miniprotein).

[0217] In some embodiments, the C-terminal amino acid of the polypeptide is not a cysteine.

[0218] In some embodiments, when present, the chelator is attached to either the polypeptide or the linker. In some embodiments, when the chelator is present without a linker, the chelator is attached to the N-terminus of the polypeptide. In some embodiments, when the chelator is present without a linker, the chelator is attached to the C-terminus of the polypeptide.

[0219] In some embodiments, when present, the radionuclide is attached to the chelator. In some embodiments, when the radionuclide is present without a linker or chelator, the radionuclide is attached to the N-terminus of the polypeptide. In some embodiments, when the radionuclide is present without a linker or chelator, the radionuclide is attached to the C- terminus of the polypeptide.

[0220] In some embodiments, a polypeptide provided herein (e.g., a miniprotein that binds to B7-H3) comprises at least one disulfide bridge.

[0221] In some embodiments, a polypeptide provided herein (e.g., a miniprotein that binds to B7-H3) comprises at least two disulfide bridges.

[0222] In some embodiments, the composition and / or polypeptide thereof selectively binds to B7-H3 or a portion thereof.

[0223] In some embodiments, the polypeptide has a binding affinity for B7-H3 or a portion thereof of 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM, in vivo, ex vivo, or in vitro and / or as measured in a cell-based assay.

[0224] In some embodiments, the polypeptide has a binding inhibition constant of no greater than 100 nM.

[0225] A composition comprising a polypeptide-drug conjugate, comprising a polypeptide and at least one drug moiety, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to at least 44 amino acids a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 198-537. In some embodiments, the polypeptide comprises at least one disulfide bridge. In some embodiments, the polypeptide Page 58 of 344 IPTS / 128790492.1comprises 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.

[0226] In some embodiments, a B7-H3 binding miniprotein comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 4-6, 8-94 and 100-537 and can have different N- and / or C-terminal ends, such as, for example, an Acetyl, NH2, Biotin- PEG4, DOTA-PEG4, radiolabel, etc. on its N-terminus and an -OH or -NH2 on its C- terminus. N- and / or C-termini of B7-H3 binding miniproteins of the disclosure can include but are not limited to acetyl, acid, or amide (e.g., Acetyl, NH2, OH), such as provided in exemplary compounds and miniproteins of Table 2A or Table 2C. In some embodiments, a polypeptide according to the disclosure may have various modifications to its N-terminus (e.g., a linker, chelator, and / or radionuclide, e.g., such as set forth in exemplary compounds in Table 2A and / or Table 2C) or its C-terminus (e.g., a linker, chelator, and / or radionuclide). In some embodiments, the C-terminus of a given polypeptide can have an acid or amide group on its C-terminus (see, e.g., Table 2A and / or Table 2C). A given polypeptide having a particular amino acid sequence can have one or more N-terminal and / or C-terminal differences without materially changing the utility or function of the polypeptide, such as for binding to B7-H3 (e.g., for detection and / or treatment of cancer).

[0227] In some embodiments, a B7-H3 binding miniprotein comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 198-537 and can have different N- and / or C-terminal ends, such as, for example, an Acetyl, NH2, Biotin-PEG4, DOTA- PEG4, radiolabel, etc. on its N-terminus and an -OH or -NH2 on its C-terminus. N- and / or C- termini of B7-H3 binding miniproteins of the disclosure can include but are not limited to acetyl, acid, or amide (e.g., Acetyl, NH2, OH), such as provided in exemplary compounds and miniproteins of Table 2A or Table 2C. In some embodiments, a polypeptide according to the disclosure may have various modifications to its N-terminus (e.g., a linker, chelator, and / or radionuclide, e.g., such as set forth in exemplary compounds in Table 2C) or its C- terminus (e.g., a linker, chelator, and / or radionuclide). In some embodiments, the C-terminus Page 59 of 344 IPTS / 128790492.1of a given polypeptide can have an acid or amide group on its C-terminus (see, e.g., Table 2C). A given polypeptide having a particular amino acid sequence can have one or more N- terminal and / or C-terminal differences without materially changing the utility or function of the polypeptide, such as for binding to B7-H3 (e.g., for detection and / or treatment of cancer).

[0228] In certain embodiments, the disclosure provides a composition comprising a polypeptide that binds to B7-H3, has at least 48 amino acids, two disulfide bonds, and a modified amino acid at positions corresponding to 3, 24, and 29 relative to SEQ ID NO: 267, wherein the modification is a small alkyl group on the side chain of the amino acid (e.g., on the nitrogen of a lysine side chain, e.g., on the Guanidino group of an arginine side chain, etc.).

[0229] In one aspect, the disclosure provides a composition with one or more modified amino acids. In some such embodiments, the one or more modifications comprises a small alkyl group. In one aspect, the disclosure provides a composition comprising a B7-H3 binding polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acids include (i) a cysteine at each of four positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267 and wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; X45 is K or (Kme), and X49 is S or absent.

[0230] In one aspect, the disclosure provides a composition, comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at least four cysteines, which form two disulfide bonds; an arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3, a lysine at a position corresponding to amino acid 5, an isoleucine at a position corresponding to amino acid 6, a tryptophan at a position corresponding to amino acid 14, at least one modified lysine residue at a position corresponding to amino acid 24, and an alanine, arginine, or modified lysine at a position corresponding to 29, where each position is linear, from N-to-C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or to the Guanidino group of the arginine side chain, optionally comprising a methyl, dimethyl, or trimethyl group; at least 48 amino acids in length; and has a binding affinity for B7-H3 stronger than 100 nM as measured in a cell- based assay.

[0231] In one aspect, the disclosure provides a composition, comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at Page 60 of 344 IPTS / 128790492.1least four cysteines, which form two disulfide bonds; at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, optionally comprising a methyl, dimethyl, or trimethyl group; at least 48 amino acids in length; and has a binding affinity for B7-H3 stronger than 100 nM as measured in a cell-based assay.

[0232] In some embodiments, the polypeptide is at least 48 amino acids in length, but no greater than 100 amino acids in length. In some embodiments, the polypeptide binds to B7- H3 with an affinity of stronger than 10 nM in a cell-based assay.

[0233] In some embodiments, overexpressed B7-H3 on tumor cells has been shown to be successfully targeted including with T-cell-mediated immunotherapy. For example, one study showed that specific cytotoxic activity of activated T cell (ATC) also having a novel anti- CD3 x anti-B7-H3 bispecific antibody showed increase in cytotoxicity relative to ATC alone, resulting in inhibition of tumor growth and increased survival in xenograft models. (Ma J., et al., Oncotarget (2016) 7(20):29480–91). In some embodiments, a binder of the present disclosure binds to B7-H3 expressed on one or more cells (e.g., cancer cells). In some such embodiments, the present disclosure contemplates targeting of B7-H3 with one or more binders of the present disclosure, wherein the B7-H3 is expressed at higher levels on cancer cells relative to non-cancer cells, and wherein the binding of the miniprotein is specific for cancer cells. the amino acid sequence of the polypeptide shares at least 90% identity to any one of SEQ ID NOs: 198-537, but includes at least one lysine and / or arginine with at least one modification comprising at least one small alkyl group bonded to the nitrogen of the lysine side chain or the Guanidino group of the arginine side chain, optionally selected from: trimethyl, dimethyl, and monomethyl. In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity to at least 44 amino acids of a reference polypeptide, which reference polypeptide is longer than 48 amino acids in length and binds to B7-H3 with a strength of at least 10 nM on a cell-based assay, and / or has an inhibition constant of no greater than 10 nM.

[0234] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537, provided that the 44 amino acids include at least two cysteine residues that form one disulfide bridge.

[0235] In some embodiments, the amino acid sequence shares 90% identity to at least 44 amino acids as set forth in any one of SEQ ID NO: 199, 204, 241, or 262-272.

[0236] In some embodiments, the amino acid sequence shares 100% identity to at least 44 amino acids as set forth in any one of SEQ ID NO: 199, 204, 241, or 262-272. Page 61 of 344 IPTS / 128790492.1

[0237] In some embodiments, the disclosure provides composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO: 267.

[0238] In some embodiments, the disclosure provides a composition comprising a compound as set forth in C234, C235, C309, C325, or C332 of Table 2C and / or a compound having an amino acid sequence comprising SEQ ID NO: 204 or 267.

[0239] In some embodiments, the composition further comprises a radionuclide. In some embodiments, the radionuclide is Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La- 132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

[0240] In some embodiments, the disclosure provides a composition comprising a polypeptide having an amino acid sequence of at least 48 amino acids in length, but with four cysteines positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267, wherein the following amino acids are present; an arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3, a lysine at a position corresponding to amino acid 5, an isoleucine at a position corresponding to amino acid 6, a tryptophan at a position corresponding to amino acid 14, at least one modified lysine residue at a position corresponding to amino acid 24, and an alanine, arginine, or modified lysine at a position corresponding to 29, where each position is linear, from N-to-C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or to the Guanidino group of the arginine side chain, optionally comprising a methyl, dimethyl, or trimethyl.

[0241] In some embodiments, the C-terminus has an -OH or an -NH2.

[0242] In some embodiments, the binding affinity of the composition (e.g., the polypeptide) for B7-H3 is stronger than 100 nM.

[0243] In some embodiments, the inhibition constant is no greater than 100 nM.

[0244] In some embodiments, the composition further comprises one or more of a linker, chelator, and radionuclide.

[0245] In some embodiments, the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is from 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment or combination via covalent bond thereof. Page 62 of 344 IPTS / 128790492.1

[0246] In some embodiments, the chelator comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

[0247] In some embodiments, the radionuclide is selected from Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

[0248] In some embodiments, if the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 198-537. In some embodiments, the polypeptide sequence comprises any one of SEQ ID NOs: 199, 204, 241, or 262-272 and further comprises a linker, wherein the linker is PEG4, and an optional chelator, wherein the chelator is DOTA.

[0249] In some embodiments, when present, the linker is attached to the N-terminus of the polypeptide. In some embodiments, when present, the linker is attached to the C-terminus of the polypeptide.

[0250] In some embodiments, the C-terminal amino acid of the polypeptide is not a cysteine.

[0251] In some embodiments, when present, the chelator is attached to either the polypeptide or the linker.

[0252] In some embodiments, when present, the radionuclide is attached to the chelator.

[0253] In some embodiments, the disclosure provides a composition comprising a formula selected from one or more of (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 (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M comprises an amino acid sequence of any one of SEQ ID NO: 4-6, 8-94 and 100-537 or according to any of SEQ ID NOs: 538-543 and 546-550 and Table 1E. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 204 and 262-272.

[0254] In some embodiments, the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is from 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment or combination via covalent bond thereof. Page 63 of 344 IPTS / 128790492.1

[0255] In some embodiments, the chelator comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

[0256] In some embodiments, the radionuclide Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm- 153, Ra-225, Tb-165, or At-211.

[0257] In some embodiments, the disclosure provides a composition comprising a formula selected from one or more of (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 (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising any one of those set forth in SEQ ID NOs: 4-6 and 8- 94 and 100-537 or according to any of SEQ ID NOs: 95 and 96 and Tables 1B and 1C, and SEQ ID NOs: 538-543 and 546-550 (Table 1D) and Table 1E. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 204 and 262-272.

[0258] In some embodiments, when L is present, L comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36, 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 dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is from 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment or combination via covalent bond thereof.

[0259] In some embodiments, when C is present, C comprises or consists of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n- butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

[0260] In some embodiments, when R is present, R comprises or consists of Ac-225, Cu- 64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb- 203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

[0261] In some embodiments, when present, the linker is attached to the N-terminus of the polypeptide. In some embodiments, when present, the linker is attached to the C-terminus of the polypeptide.

[0262] In some embodiments, the C-terminal amino acid of the polypeptide is not a cysteine. Page 64 of 344 IPTS / 128790492.1

[0263] In some embodiments, when present, the chelator is attached to either the polypeptide or the linker.

[0264] In some embodiments, when present, the radionuclide is attached to the chelator.

[0265] In some embodiments, the polypeptide comprises at least one disulfide bridge.

[0266] In some embodiments, the polypeptide comprises at least two disulfide bridges.

[0267] In some embodiments, the composition and / or polypeptide thereof selectively binds to B7-H3 or a portion thereof.

[0268] In some embodiments, the polypeptide has a binding affinity for B7-H3 or a portion thereof of 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM, in vivo, ex vivo, or in vitro and / or as measured in a cell-based assay.

[0269] In some embodiments, the polypeptide has a binding inhibition constant of no greater than 100 nM.

[0270] In one aspect, the disclosure provides a composition comprising a polypeptide-drug conjugate, comprising a polypeptide and at least one drug moiety, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to at least 44 amino acids a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 4-6, 8-94 and 100-197 or according to SEQ ID NOs: 95 or 96 and Tables 1B and 1C.

[0271] A composition comprising a polypeptide-drug conjugate, comprising a polypeptide and at least one drug moiety, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to at least 44 amino acids a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 198-537, Table 2C, or according to any of SEQ ID NOs: 538-543 and 546-550 (Table 1D) and Table 1E.

[0272] In some embodiments, the drug moiety is selected from a topoisomerase inhibitor, an auristatin (e.g., monomethyl auristatin E), 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, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRMl, a DPPIV 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, a DHFR inhibitor, and an immunotoxin.

[0273] In some embodiments, the disclosure provides a composition comprising an isolated compound, or a pharmaceutically acceptable salt thereof or a neutral molecule comprising an optional linker (L), and one or more of a polypeptide (M), chelator (C) or Page 65 of 344 IPTS / 128790492.1radionuclide (R), wherein M has an amino acid sequence comprising any one of SEQ ID NOs: 4-6, 8-94 and 100-197 or SEQ ID NOs: 95 and 96 and Tables 1B and 1C, and / or wherein M has an amino acid sequence comprising any one of SEQ ID NOs: 198-537, Table 2C, or according to any of SEQ ID NOs: 538-543 and 546-550 (Table 1D) and Table 1E.

[0274] In some embodiments, the disclosure provides a composition comprising, a compound designed to bind to B7-H3, which compound comprises or consists of a polypeptide having an amino acid sequence comprising any one of SEQ ID NOs: 4-6, 8-94 and 100-197 or SEQ ID NOs: 95 and 96 and Tables 1B and 1C, or an amino acid sequence comprising any one of SEQ ID NOs: 198-537, Table 2C, or according to any of SEQ ID NOs: 538-543 and 546-550 (Table 1D) and Table 1E.

[0275] In some embodiments, the modified N-terminus comprises one or more of an NH2, Acetyl, PEGn, wherein n=1-36, DOTA, or Biotin.

[0276] In some embodiments, the C terminus comprises an -NH2 or an -OH.

[0277] In some embodiments, the polypeptide selectively binds to B7-H3 or a portion thereof.

[0278] In some embodiments, the polypeptide has a binding affinity of stronger than about 100 nM to B7-H3, or a portion thereof, in vivo or in a cell-based assay. Polypeptides

[0279] Among other things, the present disclosure provides polypeptides. In some embodiments a polypeptide is assembled using solid phase synthesis methods. In some embodiments, a polypeptide is recombinant. In some embodiments, a polypeptide comprises or consists of a miniprotein. In some such embodiments, a miniprotein comprises or consists of a binder. 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.

[0280] In some embodiments, a miniprotein of the present disclosure comprises or consists of a polypeptide capable of binding to target as shown in Table 1A.

[0281] In some embodiments, the present disclosure provides binders comprising or consisting of a fragment of a polypeptide 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.

[0282] In some embodiments, miniproteins of the present disclosure can also include fusions or conjugates with one or more other components, such as heterologous polypeptides. Page 66 of 344 IPTS / 128790492.1For 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.

[0283] 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: 4-6, SEQ ID NOs: 12-94, or SEQ ID NOs: 100-537, and / or according to any of 1B, 1C, 1D, 1E, 2A, 2C, or 2D.

[0284] 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 amino acid sequences set forth in Table 2A.

[0285] In some embodiments, a miniprotein does not have an amino acid sequence selected from any one of those according to Tables 1B, 1C, and / or 2A.

[0286] In some embodiments, a miniprotein has an amino acid sequence that is 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 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 contiguous amino acids of a given polypeptide such as those set forth in Table 2A or Table 2C. In some embodiments, the miniprotein does not have an amino acid sequence as set forth in Table 2A.

[0287] 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 amino acid sequences set forth in Table 2A or Table 2C. Page 67 of 344 IPTS / 128790492.1

[0288] In some embodiments, a miniprotein has an amino acid sequence that is 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 a portion or all of an amino acid sequence set forth in Table 2A. For example, in some embodiments, a miniprotein has an amino acid sequence that is 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 20, 21, 22, 23, 24, 25, 26, 27, 28.29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids of a given polypeptide such as those set forth in Table 2A.

[0289] In some embodiments, a miniprotein has an amino acid sequence that is 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 a portion or all of an amino acid sequence set forth in Table 2C. For example, in some embodiments, a miniprotein has an amino acid sequence that is 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 20, 21, 22, 23, 24, 25, 26, 27, 28.29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids of a given polypeptide such as those set forth in Table 2C.

[0290] In some embodiments, a miniprotein has an amino acid sequence that is 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 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 contiguous amino acids of a given polypeptide such as those set forth in Table 2C.

[0291] In some embodiments, a polypeptide (e.g., a miniprotein) has an amino acid sequence with a certain percent identity over a certain percent coverage (e.g., of a reference sequence). Unless otherwise stated, percent identity refers to a maximum percent identity measured according to any one of the approaches set forth herein. That is, a B7-H3 binding polypeptide (e.g., a reference molecule) as provided herein can have 70, 75, 80, 85, 90, 95, 99, or 100 percent identity to a given query but over a percent coverage of that molecule (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%), where if the query molecule is shorter it may have a percent identity and a percent coverage that are different (e.g., 100% identity and 90% coverage). If the query molecule is longer, than the percent identity and coverage could each be 100% with respect to the reference molecule, and the reference molecule would have a Page 68 of 344 IPTS / 128790492.1percent identity over a length (e.g., at least 20, 25, 30, amino acids) of the query molecule. For example, in some embodiments, if a reference sequence (e.g., a miniprotein as provided herein) is shorter than a query sequence, such a query sequence is within the scope of the present disclosure if it has a length of reference sequence that aligns with the query sequence, wherein the percent identity is determined over at least a minimum length of the alignment between the two sequences (query and reference). That is, if a polypeptide disclosed herein is longer than a query sequence, a percent identity is determined by aligning the reference and query and determining the percent identity as between the query and the portion of the reference sequence over which it aligns. Conversely, where a query sequence is longer than a reference sequence, percent identity equals an identity over an aligned portion with the reference sequence. That is, if the reference sequence is shorter, the query sequence can fall within the scope of a reference sequence if it aligns at a claimed percent identity over the aligned portion between the two polypeptides (reference and query).

[0292] 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.

[0293] In some embodiments, miniproteins of the present disclosure comprising two or more cysteine residues, such as those set forth in Table 2C, have cysteine residues connected via disulfide bridges (e.g., via natural folding). In some embodiments a miniprotein (e.g., that binds to B7-H3) comprises one disulfide bridge. In some embodiments, a miniprotein comprises two disulfide bridges. Page 69 of 344 IPTS / 128790492.1

[0294] In some embodiments, the one disulfide bridge can be between cysteine residues corresponding to, for example, Cys4 and Cys37; Cys5 and Cys34; Cys5 and Cys37; Cys12 and Cys26; Cys12 and Cys44; Cys17 and Cys48, with positions relative to linear position from N-to-C-terminus with reference to SEQ ID NO: 267.

[0295] In some embodiments, cysteine connections can be between two different pairs of cysteine residues. For example, in some embodiments, two disulfide bridges can be between positions corresponding to pairs of cysteines such as Cys1 and Cys35 and Cys17 and Cys52, with reference to the miniprotein of SEQ ID NO: 213. For example, in some embodiments, disulfide bridges can be between positions corresponding to Cys1 and Cys35; and Cys17 and Cys48 of a reference sequence such as set forth in Table 2C (e.g., SEQ ID NO: 267). In some embodiments, cysteine connections are between Cys1 and Cys17; and Cys35 and Cys48. In some embodiments, cysteine connections are between Cys1 and Cys48; and Cys17 and Cys35.

[0296] In some embodiments, In some embodiments, the disulfide bridge or bridges comprise two or four cysteines at positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267, wherein the cysteine corresponding to position 1 can form a disulfide bridge with the cysteine corresponding to position 17, 35, or 48. In some embodiments, the cysteine corresponding to position 17 can form a disulfide bridge with the cysteine corresponding to position 1, 35, or 48. In some embodiments, the cysteine corresponding to position 35 can form a disulfide bridge with the cysteine corresponding to position 1, 17, or 48. In some embodiments, the cysteine corresponding to position 48 can form a disulfide bridge with the cysteine corresponding to position 1, 17, or 35. In some embodiments, where four cysteines are present and correspond to positions 1, 17, 35, and 48 of SEQ ID NO: 267, pairings can comprise 1 paired with 35 and 17 paired with 48, 1 paired with 17 and 35 paired with 48, or 1 paired with 48 and 17 paired with 35 (e.g., disulfide bridges between the two cysteines of the pair).

[0297] In some embodiments, the present disclosure provides a miniprotein as set forth in Tables 1B, 1C, 1D, 1E, 2A, 2C and / or 2D or a portion or functional variant thereof and / or comprising or consisting of an amino acid sequence set forth in any of SEQ ID NOs: 4-6, 8- 94 and 100-537.

[0298] In some embodiments, a miniprotein comprises or consists of an amino acid sequence having at least 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 greater sequence identity to as set forth in any of Page 70 of 344 IPTS / 128790492.1SEQ ID NOs: 4-6, 8-94 and 100-537 and / or Tables 1B, 1C, 1D, 1E, 2A, 2C or 2D or a portion or functional variant thereof.

[0299] In some embodiments, the miniprotein comprises or consists of an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more amino acid residue differences from SEQ ID NOs: 4-6, 8-94 and 100-537 and / or as set forth in Tables 1B, 1C, 1D, 1E, 2A, 2C, 2D, or a portion or functional variant thereof.

[0300] In some embodiments, the miniprotein comprises or consists of an amino acid sequence having no more than 1, no more than 2, no more than 3, no more than 4, no more than 5, no more than 6, no more than 7, no more than 8, no more than 9, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, or no more than 35 amino acid residue differences from SEQ ID NOs: 4-6, 8-94 and 100-537 and / or as set forth in Tables 1B, 1C, 1D, 1E, 2A, 2C, 2D, or a portion or functional variant thereof.

[0301] In some embodiments, the miniprotein has an amino acid sequence comprising or consisting of any of SEQ ID NOs: 4-6, SEQ ID NOs: 12-94, or SEQ ID NOs: 100-537, and / or as set forth in Tables 1B, 1C, 1D, 1E, 2A, 2C, 2D, or a portion or functional variant thereof selectively binds to the target B7-H3.

[0302] Polypeptides of the disclosure may have one or more modifications. A modification can refer 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. An alteration can include but is not limited to a change to or of one or more atoms of a side chain, such as, for example addition of a methyl-group (e.g., methylated versions of lysine). In some embodiments, a natural amino acid is modified such as set forth herein. In some embodiments, a modification includes addition of at least one small alkyl group attached to the nitrogen of an amino acid side chain, such as, for example, a lysine side chain. As used herein, a “small alkyl group” refers to an alkyl group with a short carbon chain, typically having one to four carbon atoms, such as methyl, ethyl, propyl, or butyl, and also including, for example, dimethyl, trimethyl, isopropyl, etc. In some embodiments, for example, one or more small alkyl groups can be added to the nitrogen of a lysine side chain to produce monomethyl, dimethyl, or trimethyllysine. In some embodiments, one, two, three, four or more small alkyl groups may be added to a given amino acid (e.g., through attachment to the nitrogen of the side chain). In some embodiments no more than five, four, three, two, or one small alkyl groups are added. Miniproteins Page 71 of 344 IPTS / 128790492.1

[0303] The present disclosure provides a polypeptide, comprising: an amino acid sequence, wherein the amino acid sequences comprises Formula I: X1X2X3X4YX6X7EX9IX11ALX14EIIWLPNX22X23X24X25QIX28AFIAALNX36DPSQ SSELLSEAX49X50LX52DSX55X56X57X58 (SEQ ID NO: 95), wherein X1 is A, N, or absent; X2 is A, E, or absent; X3 is A, Q, or absent; X4 is K, (KAc), L, or absent; X6 is A, D, E, I, L, N, Q, S, T, or Y; X7 is A, E, K, (KAc), ( Kme3), L, Q, or S; X9 is K, (KAc), or (Kme3); X11 is A, Q, S, T, or Y; X14 is E, Q, S, or Y; X22 is A, D, F, (homo-leucine), I, L, N, (Nle), T, or Y; X23 is T or V; X24 is H or Y; X25 is A or G; X28 is A, (homo-leucine), M, M(O2), (Nle), S, T, or V; X36 is A, (Cit), D, E, L, N, Q, S, or T; X49 is A, E, G, K, (KAc), L, Q, S, or Y; X50 is A, (Cit), D, E, G, (hSer), K, (KAc), L, Q, S, or Y; X52 is A, D, G, N, Q, T, or Y; X55 is D, E, L, Q, S, Y, or absent; X56 is A or absent; X57 is P or absent; and X58 is G, K, (KAc), or absent; wherein if X28 is A, (homo-leucine), M(O2), S, T, or V, then X24 is Y; or wherein if X28 is M, then X7 is A, E, (Kme3), L, Q, or S.

[0304] In some embodiments, if X4 is K or (KAc), then X24 is Y.

[0305] In some embodiments, the amino acid sequence comprises Formula II: X1X2X3X4YAX7EKIAALSEIIWLPNX22TX24X25QIX28AFIAALNX36DPSQSSELLSE AX49X50LNDSQAP(SEQ ID NO: 96), wherein X1 is A or absent; X2 is E or absent; X3 is A or absent; X4 is L or absent; X7 is K or Q; X22 is D or L; X24 is H or Y; X25 is A or G; X28 is ((homo-leucine)) or M; X36 is D or N; X49 is E or K; and X50 is E.

[0306] In some embodiments, the amino acid sequence comprises Formula III (SEQ ID NO: 538) as set forth in Tables 1D and 1E.

[0307] In some embodiments, the amino acid sequence comprises Formula IV (SEQ ID NO: 539) as set forth in Tables 1D and 1E.

[0308] In some embodiments, the amino acid sequence comprises Formula V (SEQ ID NO: 540) as set forth in Tables 1D and 1E:

[0309] In some embodiments, the amino acid sequence comprises Formula VI (SEQ ID NO: 541) as set forth in Tables 1D and 1E.

[0310] In some embodiments, the amino acid sequence comprises Formula VII (SEQ ID NO: 542) as set forth in Tables 1D and 1E.

[0311] In some embodiments, the amino acid sequence comprises Formula VIII (SEQ ID NO: 543) as set forth in Tables 1D and 1E.

[0312] In some embodiments, the amino acid sequence comprises Formula IX (SEQ ID NO: 546) as set forth in Tables 1D and 1E. Page 72 of 344 IPTS / 128790492.1

[0313] In some embodiments, the amino acid sequence comprises Formula X (SEQ ID NO: 547) as set forth in Tables 1D and 1E.

[0314] In some embodiments, the amino acid sequence comprises Formula XI (SEQ ID NO: 548) as set forth in Tables 1D and 1E.

[0315] In some embodiments, the amino acid sequence comprises Formula XII (SEQ ID NO: 549) as set forth in Tables 1D and 1E.

[0316] In some embodiments, the amino acid sequence comprises Formula XIII (SEQ ID NO: 550) as set forth in Tables 1D and 1E.

[0317] In some embodiments, the amino acid sequence shares at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or more percent) identity to any one of the SEQ ID NOs: 4-6 or SEQ ID NOs: 12-94.

[0318] In some embodiments, the amino acid sequence shares 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or more percent) identity to any one of SEQ ID NOs: 6, 21, or 30.

[0319] In some embodiments, the amino acid sequence shares 100% identity to any one of the SEQ ID NOs: 4-6 or SEQ ID NOs: 12-94.

[0320] In some embodiments, the amino acid sequence shares 100% identity to any one of SEQ ID NOs: 6, 21, or 30.

[0321] In some embodiments, the amino acid sequence shares 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or more percent) identity to SEQ ID NO: 6.

[0322] In some embodiments, the amino acid sequence shares 100% identity to SEQ ID NO: 6.

[0323] The present disclosure also provides a polypeptide that has an amino acid sequence comprising or consisting of any of SEQ ID NOs: 4-6, 8-94, or 100-537.

[0324] The present disclosure also provides a polypeptide that has an amino acid sequence comprising or consisting of any of SEQ ID NOs: 4-6, 8-94, or 100-197.

[0325] The present disclosure also provides a polypeptide that has an amino acid sequence comprising or consisting of any of SEQ ID NOs: 198-537. In some embodiments, the miniprotein comprises at least one constraint, wherein the constraint is a disulfide bridge.

[0326] In some embodiments, the polypeptide further comprises one or more of a linker, chelator, and radionuclide.

[0327] In some embodiments, when present, the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a Page 73 of 344 IPTS / 128790492.1propanoic acid linker, a caproleic acid linker, an aminocaproic acid (Ahx) linker, or (Gly)n- (γGlu)n- or (PEG)n, wherein n is from 1 to 36, (Gly)1-10,or any fragment or combination via covalent bond thereof.

[0328] In some embodiments, when present, the chelator comprises or consists of: i) NOPOPage 74 of 344 IPTS / 128790492.1.

[0329] In some embodiments, the chelator comprises or consists of derivative of NOPO, Crown, Macropa, or tetrazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0330] In some embodiments, the radionuclide comprises or consists of Ac-225, Ga-68, In-111, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, Ce-134, F-18, or At-211.

[0331] In some embodiments, the linker, when present, is attached to the N-terminal or C- terminal end of the polypeptide.

[0332] In some embodiments, the chelator, when present is attached to either the polypeptide or the linker.

[0333] In some embodiments, the radionuclide, when present, is attached to the chelator.

[0334] In some embodiments, the polypeptide 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.

[0335] In some embodiments, the polypeptide comprises at least one disulfide bridge.

[0336] In some embodiments, the polypeptide selectively binds to B7-H3 or a portion thereof.

[0337] In some embodiments, the polypeptide exhibits a binding affinity of 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM to B7-H3, or a portion thereof, in vivo or in a cell-based assay.

[0338] In some embodiments, the polypeptide exhibits a binding affinity of 10 pM to 50 nM (e.g., 10 pM to 10 nM, e.g., 20 pM to 40 pM, e.g., 25 pM to 50 to B7-H3 in a cell-based assay. Page 75 of 344 IPTS / 128790492.1Miniproteins

[0339] Provided herein are novel polypeptides (e.g., miniproteins) and methods of use thereof. In some embodiments, a polypeptide comprises or consists of a miniprotein. The present disclosure recognizes that a source of a problem in therapeutics (e.g., miniprotein therapeutics) is binding to B7-H3 with sufficient specificity and affinity. Thus, provided herein are miniproteins that bind strongly, efficiently, and specifically to B7-H3 (e.g., on a cell, e.g., on a cancer cell).

[0340] Another source of a problem includes toxicity (e.g., renal toxicity). In some embodiments, specificity and strength of binding for B7-H3 in cancer cells reduces uptake into kidney.

[0341] In some such embodiments, the miniprotein comprises or consists of an affibody, a CDP, a knottin, a binder, a monobody, an anticalin, a designed ankyrin repeat, an engineered Kunitz domain, and / or an avimer. 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. For 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.

[0342] Without wishing to be bound by any particular theory, the present disclosure contemplates that therapeutics comprising compositions provided by the present disclosure 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.

[0343] 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, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more amino acids in length, up to Page 76 of 344 IPTS / 128790492.1about 100 amino acids in length. 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 10 to about 30, 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.

[0344] 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 embodiments, however, a miniprotein of the present disclosure does not exceed about 12 kDa.

[0345] In some embodiments, a miniprotein of the present disclosure comprises or consists of a cysteine-dense peptide, knottin, and / or binder.

[0346] 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).

[0347] 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).

[0348] In some embodiments, miniproteins (e.g., affibody, CDP, knottin, binder, 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 Page 77 of 344 IPTS / 128790492.1nm, 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.

[0349] 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 (i.e., 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.

[0350] 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 B7-H3. In some embodiments, the B7-H3 is human B7-H3. In some embodiments, the human B7-H3 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).

[0351] In some embodiments, the binding affinity of a miniprotein or conjugate thereof to human B7-H3 is about 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, or stronger (e.g., 90 nM, 75 nM, 50 nM, 25 nM, 10 nM, 5 nM, etc.). 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, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or stronger (e.g., 0.3, 0.2, 0.1, 0.09 nM, etc.) binding affinity to human B7-H3. In some embodiments, the binding is selective to human B7-H3 and, not, e.g., non-human B7-H3.

[0352] In some embodiments, a miniprotein or conjugate thereof displays a binding inhibition constant. In some embodiments, the binding inhibition constant (Ki) to human B7- H3 is about 300 nM, 200 nM, 100 nM, 50 nM, 25 nM, 10 nM, 5 nM, or less (e.g., 1 nM, etc.). In some embodiments, the miniprotein comprises picomolar binding affinity. In some embodiments, the miniprotein or conjugate thereof comprises a binding affinity characterized Page 78 of 344 IPTS / 128790492.1by 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, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or stronger (e.g., 0.3, 0.2, 0.1, 0.09 nM, etc.) binding affinity to human B7-H3. In certain embodiments, the binding inhibition constant of a B7-H3 binding polypeptide of the disclosure is between about 100 pM to about 50 nM (e.g., in a cell-based assay, e.g., DELFIA). In some embodiments, the binding is selective to human B7-H3 and, not, e.g., non-human B7-H3.

[0353]

[0173] In some embodiments, a miniprotein or conjugate thereof provided by the present disclosure has high affinity for B7-H3 (e.g., as measured by binding affinity and / or inhibition constant, etc.). In some such embodiments, the B7-H3 is human B7-H3. 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., radionuclide).

[0354] 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 B7- H3. 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).

[0355] In some embodiments, a miniprotein or conjugate thereof displays a binding affinity to B7-H3. In some embodiments, the binding affinity of a miniprotein or conjugate thereof to human B7-H3 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, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or less binding affinity to human B7-H3. In some embodiments, the binding is selective to human B7-H3 and, not, e.g., non-human B7-H3.

[0356] In some embodiments, a miniprotein or conjugate thereof provided by the present disclosure has high affinity for B7-H3. In some such embodiments, the B7-H3 is human B7- H3. 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).

[0357] 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, binding ability of a miniprotein or conjugate thereof as provided herein to B7-H3, is Page 79 of 344 IPTS / 128790492.1improved using chemical crosslinking. In some 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.

[0358] 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.

[0359] In some embodiments, a miniproteins or conjugates thereof provided by the present disclosure comprises or consists of monomers that make up a dimer, polymer or a multimer. In some such embodiments, the monomers all bind to the same target. For example, in some embodiments, where more than one miniprotein is present, each miniprotein is no greater than about 30-40 amino acids in length or a total of about 8 kDa in size (with both miniproteins). In some embodiments, the monomers each bind to a different target. In some embodiments, some monomers bind to one target and others bind to one or more additional targets.

[0360] 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 B7-H3. In some embodiments, such an antibody may be used for, e.g., diagnostic purposes, blocking (e.g., antagonism), etc.

[0361] In some embodiments, the miniprotein comprises one or more disulfide bridges.

[0362] 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.

[0363] In some embodiments, a miniprotein or conjugate thereof as provided herein is specific for a target. In some embodiments, a binder is specific for B7-H3 or a fragment thereof.

[0364] In some embodiments, a miniprotein or conjugate thereof as provided herein comprises or consists of a specific amino acid sequence.

[0365] 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). Page 80 of 344 IPTS / 128790492.1

[0366] In some embodiments, miniproteins or compositions comprising miniproteins are conjugated to a chelator that optionally binds a cold-metal surrogate. In some embodiments, a cold-metal surrogate is a natural isotope of an element that is not radioactive. In some embodiments, an element may have more than one natural isotope that is not radioactive. In some embodiments, “cold” is used to refer to an isotype of an element that is not radioactive. In some embodiments, “hot” refers to an isotope of an element that is radioactive.

[0367] 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, trimethyllysine, ε-N,N,N-trimethyllysine, ε-N- acetyllysine (Lys(Ac)), O-phosphoserine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine, N-methylarginine, norleucine, 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.

[0368] In some embodiments, a miniprotein disclosed herein has one or more of the following unconventional amino acids: trimethyllysine, dimethyllysine, monomethyllysine, isopropyl-lysine, Lys(Ac), norleucine, citrulline, L-citrulline, methylated arginine (Rme, Rme2), symmetrically dimethylated arginine (sRme2, Rme2s, or SDMA), nitroarginine (Arg(NO2)), or Leu-13C6,15N. 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. Page 81 of 344 IPTS / 128790492.1

[0369] 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: 4-6, 8- 94 and 100-537. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in any of SEQ ID NOs: 4-6, 8-94 and 100- 537. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 4-6, 8-94 and 100-537.

[0370] 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%, 91%, 92%, 93%, 94%, 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: 4-6, 8-94 or 100-537. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in any of SEQ ID NOs: 4-6, 8-94 or 100-537. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 4-6, 8-94 or 100-537.

[0371] A polypeptide (e.g., a miniprotein) in accordance with the present disclosure can have an amino acid sequence with a certain percent identity over a certain percent coverage (e.g., of a reference sequence). That is, for example, a B7-H3 binding polypeptide (e.g., a reference molecule) as provided herein can have 70, 75, 80, 85, 90, 95, 99, or 100 percent identity to a given query but over a percent coverage of that molecule (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%), where if the query molecule is shorter it may have a percent identity and a percent coverage that are different (e.g., 100% identity and 90% coverage). If the query molecule is longer, than the percent identity and coverage could each be 100% with respect to the reference molecule, and the reference molecule would have a percent identity over a length (e.g., at least 20, 25, 30, amino acids) of the query molecule. For example, in some embodiments, if a reference sequence (e.g., a miniprotein as provided herein) is shorter than a query sequence, such a query sequence is within the scope of the present disclosure if it has a length of reference sequence that aligns with the query sequence, wherein the percent identity is determined over at least a minimum length of the alignment between the two sequences (query and reference). That is, if a polypeptide disclosed herein is longer than a query Page 82 of 344 IPTS / 128790492.1sequence, a percent identity is determined by aligning the reference and query and determining the percent identity as between the query and the portion of the reference sequence over which it aligns. Conversely, where a query sequence is longer than a reference sequence, percent identity equals an identity over an aligned portion with the reference sequence. That is, if the reference sequence is shorter, the query sequence can fall within the scope of a reference sequence if it aligns at a claimed percent identity over the aligned portion between the two polypeptides (reference and query).

[0372] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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: 4-6, SEQ ID NOs: 12-94, or SEQ ID NOs: 100-537. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in any of SEQ ID NOs: 4-6, SEQ ID NOs: 12-94, or SEQ ID NOs: 100-537. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 4-6, SEQ ID NOs: 12- 94, or SEQ ID NOs: 100-537.

[0373] In some embodiments, a miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99 % or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in any one of SEQ ID NOs: 4-6, SEQ ID NOs: 12- 94, or SEQ ID NOs: 100-537. In some embodiments, a miniprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in any one of SEQ ID NOs: 4-6, SEQ ID NOs: 12-94, or SEQ ID NOs: 100-537.

[0374] In some embodiments, a miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99 % or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in any one of SEQ ID NOs: 198-537. In some embodiments, a miniprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in any one of SEQ ID NOs: 198-537.

[0375] In some embodiments, a miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99 % or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in any one of SEQ ID NOs: 204 and 262-272. In some embodiments, a miniprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in any one of SEQ ID NOs: 204 and 262-272. Page 83 of 344 IPTS / 128790492.1

[0376] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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: 6, 21, 30, 130-131, 183, 199, 204, 241 or 267. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in any of SEQ ID NOs: 6, 21, or 30. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 6, 21, 30, 130-131, 183, 199, 204, 241, or 267.

[0377] 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 SEQ ID NO: 6. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 6. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 6.

[0378] 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 SEQ ID NO: 21. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 21. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 21.

[0379] 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 SEQ ID NO: 30. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 30. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 30. Page 84 of 344 IPTS / 128790492.1

[0380] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 130. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 130. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 130.

[0381] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 131. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 131. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 131.

[0382] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 183. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 183. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 183.

[0383] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 199. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 199. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 199. In some embodiments, a miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99 % or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in SEQ ID NO: 199. In some Page 85 of 344 IPTS / 128790492.1embodiments, a miniprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in SEQ ID NO: 199.

[0384] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 204. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 204. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 204. In some embodiments, a miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99 % or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in SEQ ID NO: 204. In some embodiments, a miniprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in SEQ ID NO: 204.

[0385] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 241. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 241. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 241. In some embodiments, a miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99 % or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in SEQ ID NO: 241. In some embodiments, a miniprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in SEQ ID NO: 241.

[0386] In some embodiments, a miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 267. In various embodiments, a miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence set forth in SEQ ID NO: 267. In various embodiments, a miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence set forth in SEQ ID NO: 267. In some embodiments, a miniprotein has an amino acid sequence Page 86 of 344 IPTS / 128790492.1that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99 % or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in SEQ ID NO: 267. In some embodiments, a miniprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids as set forth in SEQ ID NO: 267.

[0387] In some embodiments, the miniprotein can further comprise additions on its N- and / or C-terminus, for example, one or more additional amino acids.

[0388] In some embodiments, a B7-H3 binding miniprotein in accordance with the disclosure can have different N- and / or C-terminal ends, such as, for example, an Acetyl, NH2, Biotin-PEG4, DOTA-PEG4, radiolabel, etc. on its N-terminus and an -OH or -NH2 on its C-terminus. N- and / or C-termini of B7-H3 binding miniproteins of the disclosure can include but are not limited to acetyl, acid, or amide (e.g., Acetyl, NH2, OH), such as provided in exemplary compounds and miniproteins of Table 2A or Table 2C.

[0389] In some embodiments, a polypeptide according to the disclosure may have various modifications to its N-terminus (e.g., a linker, chelator, and / or radionuclide, e.g., such as set forth in exemplary compounds in Table 2A or Table 2C) or its C-terminus (e.g., a linker, chelator, and / or radionuclide). In some embodiments, the C-terminus of a given polypeptide can have an acid or amide group on its C-terminus (see, e.g., Table 2A, e.g., Table 2C). A given polypeptide having a particular amino acid sequence can have one or more N-terminal and / or C-terminal differences without materially changing the utility or function of the polypeptide, such as for binding to B7-H3 (e.g., for detection and / or treatment of cancer).

[0390] In some embodiments, an addition on the N and / or C-terminus can include one or more of a linker and / or chelator, etc., such as, for example, an N-terminal DOTA-PEG4. In some embodiments, the C-terminus of a miniprotein comprises and NH2, OH, and / or COOH.

[0391] In some embodiments, the miniprotein further comprising additions on its N- and / or C-terminus has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NOs: 4-6, 8-94 and 100-537. In some such embodiments, the miniprotein is included in a compound. In some embodiments, the compound is selected from any of Compound Nos: C1-C608 and C611.

[0392] In some embodiments, the miniprotein further comprising additions on its N- and / or C-terminus has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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%, Page 87 of 344 IPTS / 128790492.199.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 4-6, 8-94, or 100-537. In some such embodiments, the miniprotein is included in a compound. In some embodiments, the compound is selected from any of Compound Nos: C1-C9, C11-C117, or C121-C608 and C611.

[0393] In some embodiments, the miniprotein does not comprise one or more constraints (e.g., a disulfide bridge). In some embodiments, the miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NOs: 4-6, 8-94 or 100-197. In some embodiments, the miniprotein is part of a compound. In some embodiments, the compound is selected from any of Compound Nos: C1-C226.

[0394] In some embodiments, the miniprotein does not comprise one or more constraints (e.g., a disulfide bridge). In some embodiments, the miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NOs: 198-537. In some embodiments, the miniprotein is part of a compound. In some embodiments, the compound is selected from any of Compound Nos: C227-C608 and C611.

[0395] In some embodiments, the miniprotein comprises further changes such as, for example, one or more constraints (e.g., as disclosed herein). In some embodiments, a constraint is a disulfide bridge. In some embodiments, the miniprotein has at least one, two, or three disulfide bridges. In some embodiments, the miniprotein has one disulfide bridge. In some embodiments, the miniprotein has two disulfide bridges. In some such embodiments, such a miniprotein can be referred to as a constrained miniprotein. In some embodiments, the constrained miniprotein is more stable than, for example, a miniprotein without one or more constraints as characterized by having a higher melting temperature, e.g., such as one having the same or a similar amino acid sequence without one or more constraints (e.g., such as a disulfide bridge). In some embodiments, the constrained miniprotein has an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NOs: 198-537. In some embodiments, the miniprotein is part of Page 88 of 344 IPTS / 128790492.1a compound. In some embodiments, the compound is selected from any of Compound Nos: C227-C608 and C611.

[0396] In some embodiments, a miniprotein as provided herein is specific for or binds to a polypeptide or portion thereof having an amino acid sequence or portion thereof as set forth in Table 1A. Table 1A. Exemplary Target Protein Amino Acid Sequences Target Protein Amino Acid Sequence SEQ ID (Uniprot Acc. No.) NO. Human B7-H3 MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDA

[0397] In some embodiments, a miniprotein provided by the present disclosure is set forth in one or more consensus sequences provided in Table 1B. In some embodiments, a miniprotein with a sequence set forth in Table 1B has amino acid substitutions as provided in Table 1C. Table 1B. Consensus Sequences of Exemplary B7-H3 Miniproteins SEQ ID NO: Formula Consensus Sequence11In consensus sequences throughout the application, each X is followed by a number indicating its linear position along a miniprotein, where amino acid position 1 is the N-terminal amino acid of a particular sequence and 58 is the C-terminal amino acid of a particular amino acid sequence. For example, X1 is a substitutable Page 89 of 344 IPTS / 128790492.1SEQ ID NO: Formula Consensus Sequence196II X1X2X3X4YAX7EKIAALSEIIWLPNX22TX24X25QIX28AFIAALNX36DPSQSSELLSEAX49X50LNDSQAP TSEQ ID NO Position Amino Acids 95, 96X1 A or N when present

[0398] In some embodiments, a miniprotein provided by the present disclosure has an amino acid sequence comprising that set forth according to Formula I: X1X2X3X4YX6X7EX9IX11ALX14EIIWLPNX22X23X24X25QIX28AFIAALNX3 6DPSQSSELLSEAX49X50LX52DSX55X56X57X58 (SEQ ID NO: 95), wherein X1 is A, N, or absent; X2 is A, E, or absent; X3 is A, Q, or absent; X4 is K, (KAc), L, or absent; X6 is A, D, E, I, L, N, Q, S, T, or Y; X7 is A, E, K, (KAc), (Kme3), L, Q, or S; position at the first amino acid, X28 is a substitutable amino acid at the 28thamino acid, and so on. Amino acid positions can be determined relative to, for example, SEQ ID NO: 3. Page 90 of 344 IPTS / 128790492.1X9 is K, (KAc), or (Kme3); X11 is A, Q, S, T, or Y; X14 is E, Q, S, or Y; X22 is A, D, F, (homo-leucine), I, L, N, (Nle), T, or Y; X23 is T or V; X24 is H or Y; X25 is A or G; X28 is A, (homo-leucine), M, M(O2), (Nle), S, T, or V; X36 is A, (Cit), D, E, L, N, Q, S, or T; X49 is A, E, G, K, (KAc), L, Q, S, or Y; X50 is A, (Cit), D, E, G, (hSer), K, (KAc), L, Q, S, or Y; X52 is A, D, G, N, Q, T, or Y; X55 is D, E, L, Q, S, Y, or absent; X56 is A or absent; X57 is P or absent; and X58 is G, K, (KAc), or absent; wherein if X28 is A, (homo-leucine), (MO2), S, T, or V, then X24 is Y; or wherein if X28 is M, then X7 is A, E, (Kme3), L, Q, or S.

[0399] In various embodiments, if X4 is K or (KAc), then X24 is Y.

[0400] In various embodiments, a miniprotein provided by the present disclosure has an amino acid sequence comprising that set forth according to Formula II: X1X2X3X4YAX7EKIAALSEIIWLPNX22TX24X25QIX28AFIAALNX36DPSQSS ELLSEAX49X50LNDSQAP (SEQ ID NO: 96), wherein X1 is A or absent; X2 is E or absent; X3 is A or absent; X4 is L or absent; X7 is K or Q; X22 is D or L; X24 is H or Y; X25 is A or G; X28 is (homo-leucine) or M; X36 is D or N; X49 is E or K; and X50 is E.

[0401] In some embodiments, a miniprotein provided by the present disclosure is set forth in one or more consensus sequences provided in Table 1D. In some embodiments, a miniprotein with a sequence set forth in Table 1D has amino acid substitutions as provided in Table 1E. Table 1D. Consensus Sequences of Exemplary Constrained B7-H3 Miniproteins SEQ ID NO: Formula Consensus Sequence25386IISDC2In consensus sequences throughout the application, each X is followed by a number indicating its linear position along a miniprotein, where amino acid position 1 is the N-terminal amino acid of a particular sequence and 49 is the C-terminal amino acid of a particular amino acid sequence. For example, X3 is a substitutable position at the third amino acid (counting length from N-to-C terminus), X28 is a substitutable amino acid at the 28thamino acid, and so on. Amino acid positions can be determined relative to, for example, SEQ ID NO: 267. Page 91 of 344 IPTS / 128790492.1SEQ ID NO: Formula Consensus Sequence2546IX CAX3EKIX7ALX10EIIWLPX17LTYX21QIX24X25FIX28X29LNX32DPCQSX38X39X40LX42EAX45X46LX48S DSS3TSEQ ID Position Amino Acids NO: / FormulaPage 92 of 344 IPTS / 128790492.1SEQ ID Position Amino Acids NO: / FormulaPage 93 of 344 IPTS / 128790492.1SEQ ID Position Amino Acids NO: / FormulaPage 94 of 344 IPTS / 128790492.1SEQ ID Position Amino Acids NO: / Formula “(Kers to trimethyllysine; (Cit) refers to citrulline; and (Rme) refers to methylated arginine

[0402] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula III (SEQ ID NO: 538) as follows: CAX3EKIAALSEIIWLPCLX19YAQIX24AFIX28X29LNX32DPCX36SX38X39ILSEAX4 5ELCS, wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; and X45 is K or (Kme). In some embodiments, X49 is absent.

[0403] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 238, 239, 241, 247, 262- 273, 292, 295, 297, 298, 307, 309, 310, 314-317, 335, 336, 338, 352, 374, 389, 392, 396, 401, 460-462, 466-468, 470, 471, 497, and 528.

[0404] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula IV (SEQ ID NO: 539) as follows: Page 95 of 344 IPTS / 128790492.1CAX3EKIAALSEIIWLPCLX19YAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme2) or (Kme); X29 is A or (Kme); X32 is D or (Kme); and X45 is K or (Kme). In some embodiments, X49 is absent.

[0405] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 241, 262, 265, 267, 268, 292, 307, 314-317, 338, 352, 392, 396, and 401.

[0406] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula V (SEQ ID NO: 540) as follows: CAX3EKIAALSEIIWLPCLTYAQIX24AFIX28X29LNX32DPCQSSEILSEAX45ELCS, wherein X3 is (Kme3) or R or (Rme); X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit); and X45 is K or (Kme). In some embodiments, X49 is absent.

[0407] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 241, 262, 265-267, 270, 272, 292, 307, 314-317, 338, 352, 374, 389, 392, 396, 401, 460-462, 466, 467, 468, 470, 471, and 497.

[0408] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula VI (SEQ ID NO: 541) as follows: CA(Kme3)EKIAALSEIIWLPCLTYAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, wherein X24 is (Kme2) or (Kme); X29 is (Kme) or A or R; X32 is D or (Kme) or (Cit); and X45 is K or (Kme). In some embodiments, X49 is absent.

[0409] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 262, 265, 267, 270, 272, 314-317, 338, 352, 389, 392, 396, 401, 462, and 471.

[0410] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula VII (SEQ ID NO: 542) as follows: CAX3EKIX7ALX10EIIWLPX17LTYX21QIX24AFIX28X29LNX32DPCQSX38X39X40L X42EAX45ELCS, wherein X3 is (Kme3) or R or K; X7 is A or N; X10 is S or G; X17 is C or N; X21 is A or D; X24 is (Kme3) or (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme); X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; and X45 is K or (Kme) or (Kme3) or Q. In some embodiments, X49 is absent. Page 96 of 344 IPTS / 128790492.1

[0411] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 206, 217, 219, 221, 238, 239, 241, 247, 250, 262-267, 273, 278-280, 287, 288, 292, 295, 297- 302, 304, 305, 307, 309-319, 321-326, 328-331, 335-338, 352, 356, 366, 367, 374-377, 383- 385, 389, 392, 394-396, 401, 402, 404-409, 414-418, 426, 437-439, 449-451, 460-462, 465- 468, 470, 471, 489, 497, 502-508, 518-521, 523, and 527.

[0412] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula VIII (SEQ ID NO: 543) as follows: CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIX28X29LNX32DPCQSX38X39X40LX4 2EAX45ELCS, wherein X3 is (Kme3) or R; X7 is A or N; X10 is S or G; X21 is A or D; X24 is (Kme3) or (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme); X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; and X45 is K or (Kme) or (Kme3) or Q. In some embodiments, X49 is absent.

[0413] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 206, 238, 239, 241, 247, 250, 262-267, 273, 278-280, 287, 288, 292, 295, 297-302, 304, 305, 307, 309-319, 321-326, 328, 329, 330, 331, 335-338, 352, 356, 366, 367, 374, 375-377, 383- 385, 389, 392, 394-396, 401, 402, 404-409, 414- 418, 426, 437-439, 449-451, 460-462, 465- 468, 470, 471, 489, 497, 502-508, 518-521, 523, and 527.

[0414] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula IX (SEQ ID NO: 546) as follows: CAX3EKIX7ALX10EIIWLPX17LTYX21QIX24X25FIX28X29LNX32DPCQSX38X39X40 LX42EAX45X46LX48S, wherein X3 is (Kme3) or K or Q or R; X7 is A or N; X10 is S or G; X17 is C or N; X21 is A or D; X24 is (Kme2) or (Kme) or (homo-leucine); X25 is A or E; X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit) or A; X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; X45 is K or (Kme) or (Kme3) or Q or R; X46 is E or A; and X48 is C or N. In some embodiments, X49 is absent.

[0415] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 203- 205, 209-213, 216-220, 225, 226, 230-233, 235, 238, 239, 241, 242, 247, 248, 250, 262-267, 270, 272, 273, 278-280, 287, 288, 292, 294-302, 304, 305, 307, 309-319, 321-326, 328, 329, 330, 331, 335-338, 352, 356, 358, 366, 367, 374- 377, 380, 383-385, 389, 392, 394-396, 399, Page 97 of 344 IPTS / 128790492.1401, 402, 404-409, 414-418, 426, 434, 437-439, 447, 449, 450, 451, 460-462, 465-468, 470, 471, 474, 475, 489, 497, 499, 502-508, 515, 518-521, 523, 524, 527, 532, 533, 535, and 537.

[0416] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula X (SEQ ID NO: 547) as follows: CAX3EKIX7ALX10EIIWLPX17LTYX21QIX24AFIX28X29LNX32DPCQSX38X39X40L X42EAX45ELX48S, wherein X3 is (Kme3) or K or R; X7 is A or N; X10 is S or G; X17 is C or N; X21 is A or D; X24 is (Kme2) or (Kme) or (homo-leucine); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme); X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; X45 is K or (Kme) or (Kme3) or Q; and X48 is C or N. In some embodiments, X49 is absent.

[0417] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 209-213, 217-220, 225, 226, 238, 239, 241, 242, 247, 250, 262-267, 273, 278-280, 287, 288, 292, 295, 297-302, 304, 305, 307, 309-319, 321-326, 328, 329, 330, 331, 335- 338, 352, 356, 366, 367, 374-377, 383-385, 389, 392, 394-396, 401, 402, 404-409, 414-418, 426, 437-439, 449-451, 460-462, 465-468, 470, 471, 489, 497, 502-508, 518-521, 523, and 527.

[0418] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula XI (SEQ ID NO: 548) as follows: CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIX28X29LNDDPCQSX38X39X40LX42E AX45ELX48S, wherein X3 is (Kme3) or K or R; X7 is A or N; X10 is S or G; X21 is A or D; X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; X45 is K or (Kme) or Q; and X48 is C or N. In some embodiments, X49 is absent.

[0419] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 217-220, 238, 239, 241, 242, 247, 250, 262-266, 273, 278, 279, 280, 287, 288, 292, 295, 297- 302, 304, 305, 307, 309-319, 321-326, 328, 329, 330, 331, 335-338, 352, 356, 366, 367, 374- 377, 383-385, 389, 392, 394-396, 402, 404-409, 414-418, 426, 437-439, 449, 460, 466-468, 471, 489, 497, and 502-508.

[0420] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula XII (SEQ ID NO: 549) as follows: Page 98 of 344 IPTS / 128790492.1CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIX28X29LNDDPCQSX38X39X40LX42E AX45ELX48S, wherein X3 is (Kme3) or K or R; X7 is A or N; X10 is S or G; X21 is A or D; X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme); X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; X45 is K or (Kme); and X48 is C or N. In some embodiments, X49 is absent.

[0421] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 217-220, 238, 239, 241, 242, 247, 262-266, 273, 278, 279, 280, 287, 292, 295, 297-302, 304, 305, 307, 309-319, 321-326, 328-330, 335-338, 352, 356, 366, 367, 374, 383, 384, 392, 395, 396, 402, 404-406, 414, 415, 417, 426, 437, 439, 460, 466, 468, 502, 503, 505, and 506.

[0422] In one aspect, the disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) with an amino acid sequence comprising an amino acid sequence according to Formula XIII (SEQ ID NO: 550) as follows: CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIAX29LNDDPCQSX38EILSEAX45ELC S, wherein X3 is (Kme3) or R; X7 is A or N; X10 is S or G; X21 is A or D; X24 is (Kme2) or (Kme); X29 is A or (Kme); X38 is S or A; and X45 is K or (Kme). In some embodiments, X49 is absent.

[0423] In certain embodiments, the polypeptide (e.g., a B7-H3-binding miniprotein) has an amino acid sequence comprising an amino acid sequence of any one of SEQ ID NOs: 204, 238, 241, 262, 263, 265, 292, 295, 297, 302, 304, 305, 307, 309, 313-319, 321-323, 326, 328, 335, 337, 338, 352, 367, 384, 392, 396, 402, 404, 414, 415, 417, 426, 437, 439, 502, 503, 505, and 506.

[0424] In one aspect, the disclosure provides a composition, comprising a polypeptide of at least 48 amino acids in length and having an amino sequence comprising that set forth in SEQ ID NO: 541, comprising at least four cysteines and two disulfide bonds, wherein X24 is (Kme) or (Kme2); X29 is (Kme) or A or R; X32 is D or (Kme) or (Cit), and X45 is (Kme) or K. In some embodiments, X49 is absent.

[0425] In certain embodiments, the disclosure provides a composition comprising a polypeptide that binds to B7-H3, has at least 48 amino acids, two disulfide bonds, and a modified amino acid at positions corresponding to 3, 24, and 29 relative to SEQ ID NO: 267, wherein the modification is a small alkyl group on the side chain of the amino acid.

[0426] In one aspect, the disclosure provides a composition with one or more modified amino acids. In some such embodiments, the one or more modifications comprises a small alkyl group. In one aspect, the disclosure provides a composition comprising a B7-H3 Page 99 of 344 IPTS / 128790492.1binding polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acids include (i) a cysteine at each of four positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267 and wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; X45 is K or (Kme), and X49 is S or absent.

[0427] In one aspect, the disclosure provides a composition, comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at least four cysteines, which form two disulfide bonds; an arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3, a lysine at a position corresponding to amino acid 5, an isoleucine at a position corresponding to amino acid 6, a tryptophan at a position corresponding to amino acid 14, at least one modified lysine residue at a position corresponding to amino acid 24, and an alanine, arginine, or modified lysine at a position corresponding to 29, where each position is linear, from N-to-C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or to the Guanidino group of the arginine side chain, optionally comprising a methyl, dimethyl, or trimethyl group; at least 48 amino acids in length; and the miniprotein has a binding affinity for B7-H3 stronger than 100 nM as measured in a cell-based assay.

[0428] In one aspect, the disclosure provides a composition, comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at least four cysteines, which form two disulfide bonds; at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, optionally comprising a methyl, dimethyl, or trimethyl group; at least 48 amino acids in length; and has a binding affinity for B7-H3 stronger than 100 nM as measured in a cell-based assay. CDPs

[0429] 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 or antigen 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 Page 100 of 344 IPTS / 128790492.1one, 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). 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: 553). In some embodiments, a CDP comprises one or more cysteine dense regions comprising at least one cysteine residue, preferably at least two, three, four, or more cysteine residues in a span of from about 10 to 80 amino acid residues. In some embodiments, a CDP can be further engineered to modify binding, folding, and / or related properties.

[0430] In some embodiments, a CDP specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the CDP specifically binds to B7-H3 or a fragment thereof. In some embodiments, a CDP 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 CDP employed in a conjugate of the present disclosure may vary depending on the target protein or antigen of interest.

[0431] In some embodiments, in miniproteins having cysteine residues, to ensure proper folding and connectivity, selected cysteine pairs can be replaced with selenocysteines. In some embodiments, diselenide crosslinks may form more readily than disulfide crosslinks due to their lower redox potential. In some such embodiments, such replacement can lead to cross-coupling of remaining cysteines. Knottins

[0432] In some 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 an antigen 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 some embodiments, a knottin can be further engineered to modify binding, folding, and / or related properties. Page 101 of 344 IPTS / 128790492.1

[0433] 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 B7-H3 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 or antigen of interest.

[0434] In some embodiments, folded structures of miniproteins (e.g., affibodies, CDPs, knottins, binders) 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., affibody, CDP, knottin, binder, engineered Kunitz domain, monobody, anticalin, designed ankyrin repeat domain (DARPin), avimer) exhibits extraordinary stability with resistance to heat, peptidase cleavage, and pH. Binders

[0435] 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. In some embodiments a binder that binds to B7-H3 is provided herein.

[0436] 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 may comprise an alpha helix, a beta sheet, or a combination of one or more of each. 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.

[0437] 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 miniprotein specifically binds to B7- Page 102 of 344 IPTS / 128790492.1H3 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

[0438] In some embodiments, miniproteins of the present disclosure comprise or consist of affibodies. In some embodiments, the affibody functions as a targeting moiety, e.g., specifically binding to a target expressed on the surface of a tumor cell. In some embodiments an affibody that binds to B7-H3 is provided herein. In some embodiments, a miniprotein (e.g., an affibody) comprises part or all of any of the compounds (e.g., comprising or consisting of miniproteins) set forth in Table 2A and / or Table 2C. In some embodiments, the miniprotein (e.g., affibody) comprises, consists essentially of, or consists of an amino acid sequence of any of SEQ ID NOs: 4-6, 8-94 and 100-537 or portions thereof. Table 2B shows certain characteristics of selected exemplary compounds / miniproteins of Table 2A. 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.

[0439] The present disclosure also provides the surprising finding that by truncating (e.g., removing residues from N- and / or C-termini of an affibody with three alpha helices spanning 58 amino acids) an affibody, such as by removing some or all of, for example, amino acids corresponding to positions 1-6 beginning at the N-terminus and positions 54-58 at the C- terminus with reference to N-to-C terminal positions along the length of SEQ ID NO: 3, and / or removing and replacing such amino acids with for example, a cysteine, the binding Page 103 of 344 IPTS / 128790492.1affinity and / or specificity to B7-H3 improves. The disclosure further contemplates that in addition to truncations, one or more constraints may be introduced. In some embodiments, the constraints comprise at least one, two, or three disulfide bridges and / or at least one additional constraint such as disclosed herein (e.g., a lactam bridge, e.g., a staple). In some embodiments, an affibody of a particular scaffold comprises three alpha helices with at least four cysteines (between which disulfides can be introduced), spanning 49 amino acids. Thus, in some such embodiments, the scaffold, relative to affibodies of 58 amino acids, differs in its length (e.g., is truncated) and constraint pattern. In some such embodiments, such truncated and constrained affibodies may have one or more desirable characteristics, such as, for example, improved affinity and / or specificity for B7-H3. In certain embodiments, such affibodies may be further modified to introduce one or more non-natural amino acids which can, in certain embodiments, impact toxicity by decreasing kidney cell uptake (e.g., in vitro, e.g., in vivo).

[0440] 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 B7-H3 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

[0441] 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 or antigen 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) 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.

[0442] 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 B7-H3 or a fragment thereof. In some Page 104 of 344 IPTS / 128790492.1embodiments, 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 or antigen of interest. Monobodies

[0443] In some embodiments, miniproteins of the present disclosure comprise or consist of monobodies. In some embodiments, conjugates provided herein comprise a monobody. In some embodiments, a monobody 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, a 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, a monobody can be further engineered to modify binding, folding, and / or related properties.

[0444] 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 B7-H3 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 or antigen of interest. Anticalins

[0445] 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 or antigen expressed on the surface of a target tumor cell. In some embodiments, an anticalin comprises or consists of 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. In some embodiments, an anticalin can be further engineered to modify binding, folding, and / or related properties. Page 105 of 344 IPTS / 128790492.1

[0446] 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 B7-H3 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 or antigen of interest. Designed Ankyrin Repeat Domains

[0447] 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 or antigen 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.

[0448] 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 B7-H3 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 or antigen of interest. Avimers

[0449] 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 or antigen expressed on the surface of a target tumor cell. In some embodiments, an Page 106 of 344 IPTS / 128790492.1avimer 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(–2), 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.

[0450] 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 B7-H3 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 or antigen of interest. Constraints

[0451] Constraints can be designed (e.g., from an initial polypeptide sequence) or introduced (e.g., engineered, e.g., into an existing miniprotein) into proteins, such as any miniprotein provided herein. Constraints may be introduced by modifications to an existing miniprotein sequence such as, for example, by substituting and / or adding two or more cysteine residues to form at least one disulfide bond.

[0452] A constraint can alter function (e.g., binding, e.g., binding affinity, etc.) and / or structure (e.g., folding) of a protein. In certain embodiments, constraints can assist in maintaining secondary structure of a given protein (e.g., a miniprotein as provided herein, e.g., a conjugate as provided herein). For example, a common protein secondary structure is an α-helix. Alpha helices can play key roles in both structure and function of a protein, such as impacting how a particular protein can interact with a binding partner. Alpha helices can mediate protein–protein interactions (PPIs) by serving as recognition motifs. In proteins comprising alpha helical structures, introducing a constraint can change one or more features of a protein such as affinity of a protein for a target (e.g., increase affinity), cell penetration (e.g., increased cell penetration), resistance to proteolysis (e.g., increased resistance to proteolytic degradation). Page 107 of 344 IPTS / 128790492.1

[0453] Examples of constraints (e.g.,α-helix constraints) can include, but are not limited to disulfide bridges / bonds, staples (e.g., hydrocarbon staples), salt bridges between charged amino acid side chain residues, lactam bridges, hydrogen bond surrogates, hydrophobic interactions, metal ligation, triazole staples synthesized from alkenyl and azido side chain residues, photocontrollable macrocycles, and introduction amino acids, such as, e.g., α,α- disubstituted amino acids. As will be known to those of skill in the art, a staple can refer to a synthetic constraint (e.g., a brace) between two previously independent entities. For example, a staple can be formed via covalent linkage between two previously independent entities such as, for example, amino acid side-chains (e.g., forming, for example, a peptide macrocycle).

[0454] The present disclosure provides the insight that engineering a B7-H3 binding protein to have constraints (e.g., one, two, three). In some embodiments, the constraints are the same (e.g., the same type, e.g., disulfide bonds). In certain embodiments, constraints are combinations of different types of constraints (e.g., disulfide bridge and lactam bridge, a disulfide bridge and a stapled alkyl bridge, two disulfide bridges and a lactam bridge, etc.). In certain embodiments, constraints can be added. For example, in certain embodiments, a miniprotein can have one, two, or three disulfide bridges; one or two disulfide bridges and another constraint such as a lactam bridge; one or two constraints such as a lactam bridge or a stapled alkyl bridge and one disulfide bridge, etc. In certain embodiments, a miniprotein of the disclosure has one, two, or three disulfide bridges and / or one or more additional constraints (e.g., one disulfide bridge and one additional constraint, e.g., two disulfide bridges and one additional constraint, wherein, for example, the one additional constraint is a lactam bridge), creates a miniprotein that not only binds with strong affinity to B7-H3, but is also taken up by cancerous cells (e.g., in a tumor), in higher concentrations than as compared to an unconstrained B7-H3 miniprotein (e.g., such as disclosed in Table 2A). The disclosure contemplates that features such as size (e.g., folded size, e.g., linear size) and stability (e.g., thermal stability) are important to efficacy of a B7-H3-binding conjugate and also important to limiting or preventing toxicity (e.g., renal toxicity) due to off-target effects. Without wishing to be bound by theory, the disclosure provides the insight that engineering B7-H3 binding proteins to add constraints, and, in some embodiments, adding such constraints after truncation (e.g., such as set forth in Table 2C relative to Table 2A) provides miniproteins with stronger affinity (e.g., for B7-H3), better efficacy (e.g., of accessing cancer cells), greater stability (e.g., thermal stability), and less toxicity (e.g., renal toxicity) as compared to molecules that are neither truncated nor constrained (e.g., as set forth in Table 2A). In some embodiments, miniproteins provided herein comprise one or more constraints such as, for Page 108 of 344 IPTS / 128790492.1example, a disulfide bridge or a staple. Exemplary constrained miniproteins are provided in Table 2C. In certain embodiments, a constraint confers an increase in thermal stability of a miniprotein (e.g., such as compared to a miniprotein having an amino acid sequence comprising or consisting of that of SEQ ID NO: 6, or as compared to compound C8; e.g., such as compared to a miniprotein with the same or similar primary sequence, but no constraints). In some embodiments, a miniprotein of the present disclosure comprises one or more disulfide bridges. In some embodiments, a miniprotein provided by the present disclosure comprises two or more disulfide bridges. In some embodiments, a miniprotein of the disclosure comprises one disulfide bridge. In some embodiments, a miniprotein of the disclosure comprises two disulfide bridges. For example, miniproteins of the present disclosure herein may contain a set of amino acids that together support formation of or are part of a constraint (e.g., a disulfide bridge, e.g., a staple). In some embodiments, a structural feature of a miniprotein is having at least two cysteine residues, positioned relative to one another so that disulfide bridge can be formed (e.g., as provided herein, see, e.g., Table 2C). In some embodiments, a miniprotein provided by the present disclosure has at least three, or at least four cysteine residues, which form one or two disulfide bridges. In certain embodiments, a miniprotein has three disulfide bridges (from three pairs of cysteines).

[0455] In some embodiments, miniproteins of the present disclosure comprising two or more cysteine residues, such as those set forth in Table 2C, have cysteine residues connected via disulfide bridges (e.g., via natural folding).

[0456] In some embodiments, a miniprotein has an amino acid sequence comprising two cysteines and a single disulfide bridge. In some embodiments, a miniprotein has an amino acid sequence comprising four cysteines and two disulfide bridges. In some embodiments, a miniprotein has an amino acid sequence comprising six cysteines and three disulfide bridges.

[0457] In some embodiments, miniproteins of the present disclosure comprising two or more cysteine residues, such as those set forth in Table 2C, have cysteine residues connected via disulfide bridges (e.g., via natural folding). In some embodiments a miniprotein (e.g., that binds to B7-H3) comprises one disulfide bridge. In some embodiments, a miniprotein comprises two disulfide bridges. In some embodiments, a miniprotein comprises three disulfide bridges.

[0458] In some embodiments, the one disulfide bridge can be between cysteine residues corresponding to, for example, Cys4 and Cys37; Cys5 and Cys34; Cys5 and Cys37; Cys12 and Cys26; Cys12 and Cys44; Cys17 and Cys48, with positions relative to linear position from N-to-C-terminus with reference to SEQ ID NO: 267. Page 109 of 344 IPTS / 128790492.1

[0459] In some embodiments, cysteine connections can be between two different pairs of cysteine residues. For example, in some embodiments, two disulfide bridges can be between positions corresponding to pairs of cysteines such as Cys1 and Cys35 and Cys17 and Cys52, with reference to the miniprotein of SEQ ID NO: 213. For example, in some embodiments, disulfide bridges can be between positions corresponding to Cys1 and Cys35; and Cys17 and Cys48 of a reference sequence such as set forth in Table 2C (e.g., SEQ ID NO: 267). In some embodiments, cysteine connections are between Cys1 and Cys17; and Cys35 and Cys48. In some embodiments, cysteine connections are between Cys1 and Cys48; and Cys17 and Cys35.

[0460] In some embodiments, the disulfide bridge or bridges comprise two or four cysteines at positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267, wherein the cysteine corresponding to position 1 can form a disulfide bridge with the cysteine corresponding to position 17, 35, or 48. In some embodiments, the cysteine corresponding to position 17 can form a disulfide bridge with the cysteine corresponding to position 1, 35, or 48. In some embodiments, the cysteine corresponding to position 35 can form a disulfide bridge with the cysteine corresponding to position 1, 17, or 48. In some embodiments, the cysteine corresponding to position 48 can form a disulfide bridge with the cysteine corresponding to position 1, 17, or 35. In some embodiments, where four cysteines are present and correspond to positions 1, 17, 35, and 48 of SEQ ID NO: 267, pairings can comprise 1 paired with 35 and 17 paired with 48, 1 paired with 17 and 35 paired with 48, or 1 paired with 48 and 17 paired with 35 (e.g., disulfide bridges between the two cysteines of the pair).

[0461] In certain embodiments, cysteine connections (disulfide bridges) are between positions corresponding to Cys1 and Cys35; and Cys17 and Cys48 of a reference sequence such as set forth in Table 2C, for example, SEQ ID NO: 267. In some embodiments, cysteine connections are between Cys1 and Cys17; and Cys35 and Cys48 (with reference to a reference sequence, e.g., SEQ ID NO: 267). In some embodiments, cysteine connections are between Cys1 and Cys48; and Cys17 and Cys35.

[0462] In some embodiments, the disulfide bridge or bridges comprise two or four cysteines at positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267, wherein the cysteine corresponding to position 1 can form a disulfide bridge with the cysteine corresponding to position 17, 35, or 48. In some embodiments, the cysteine corresponding to position 17 can form a disulfide bridge with the cysteine corresponding to position 1, 35, or 48. In some embodiments, the cysteine corresponding to position 35 can form a disulfide Page 110 of 344 IPTS / 128790492.1bridge with the cysteine corresponding to position 1, 17, or 48. In some embodiments, the cysteine corresponding to position 48 can form a disulfide bridge with the cysteine corresponding to position 1, 17, or 34. In some embodiments, where four cysteines are present and correspond to positions 1, 17, 35, and 48 of SEQ ID NO: 267, pairings can comprise 1 paired with 35 and 17 paired with 48, 1 paired with 17 and 35 paired with 48, or 1 paired with 48 and 17 paired with 35 (e.g., disulfide bridges between the two cysteines of the pair).

[0463] In some embodiments, a miniprotein with two disulfide bridges (e.g., from cysteines at positions corresponding to Cys1, Cys17, Cys35, and Cys48, e.g., of SEQ ID NO: 267) further comprises a third disulfide bridge, introduced between a third pair of cysteines. In some embodiments, cysteines can also be introduced at positions corresponding to Cys11 and Cys45.

[0464] In some embodiments, such a miniprotein further comprises one or more additional constraints (e.g., staples, e.g., lactam bridges). For example, in certain embodiments, a combination of constraint types (e.g., disulfide bond and lactam bridges) can be used in a single miniprotein. For example, in some embodiments, a miniprotein can comprise one, two, or three disulfide bridges and, further comprise at least one additional constraint (e.g., a staple, e.g., a lactam bridge). In some embodiments, a lactam bridge can be between positions corresponding to 11 and 45 of SEQ ID NO: 544.

[0465] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein) further comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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: 198-537. In various embodiments, the amino acid sequence is 90% (e.g., 91%, 92%, 93%, 94%, 95, 96, 97, 98, 99 or more percent) identical to the amino acid sequence set forth in any of SEQ ID NOs: 198-537. In various embodiments, the amino acid sequence that is 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 198-537.

[0466] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), is part of a compound. In some embodiments, the compound is selected from any of C227-C608 and C611, and / or has an amino acid sequence selected from any of SEQ ID NOs: 198-537. Page 111 of 344 IPTS / 128790492.1

[0467] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein) further comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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: 204 and 262-272. In various embodiments, the amino acid sequence is 90% (e.g., 91%, 92%, 93%, 94%, 95, 96, 97, 98, 99 or more percent) identical to the amino acid sequence set forth in any of SEQ ID NOs: 204 and 262-272. In various embodiments, the amino acid sequence that is 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 204 and 262-272.

[0468] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), is part of a compound. In some embodiments, the compound is selected from any of C234, C235, and C298-C332, and / or has an amino acid sequence selected from any of SEQ ID NOs: 204 or 262-272.

[0469] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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: 199, 204, 241, or 267. In various embodiments, the amino acid sequence is 90% (e.g., 95, 96, 97, 98, 99 or more percent) identical to the amino acid sequence set forth in any of SEQ ID NOs: 199, 204, 241, or 267. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence set forth in any of SEQ ID NOs: 199, 204, 241, or 267.

[0470] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), is part of a compound. In some embodiments, the compound is selected from any of C228, C234, C235, C275, C309, C325, C332 or any other disclosed compound in Table 2C having an amino acid sequence comprising any of SEQ ID NOs; 199, 204, 241, or 267, and / or has an amino acid sequence selected from any of SEQ ID NOs: 199, 204, 241 or 267.

[0471] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, Page 112 of 344 IPTS / 128790492.198.2%, 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 SEQ ID NO: 199. In various embodiments, the amino acid sequence is 90% (e.g., 95, 96, 97, 98, 99 or more percent) identical to the amino acid sequence set forth in SEQ ID NO: 199. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence set forth in SEQ ID NO: 199.

[0472] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein) is part of a compound. In some embodiments, the compound is C228 and / or has an amino acid sequence of SEQ ID NO: 199.

[0473] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 204. In various embodiments, the amino acid sequence is 90% (e.g., 95, 96, 97, 98, 99 or more percent) identical to the amino acid sequence set forth in SEQ ID NO: 204. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence set forth in SEQ ID NO: 204.

[0474] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein) is part of a compound. In some embodiments, the compound is C234 or C235 and / or has an amino acid sequence of SEQ ID NO: 204.

[0475] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 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 SEQ ID NO: 241. In various embodiments, the amino acid sequence is 90% (e.g., 95, 96, 97, 98, 99 or more percent) identical to the amino acid sequence set forth in SEQ ID NO: 241. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence set forth in SEQ ID NO: 241.

[0476] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein) is part of a Page 113 of 344 IPTS / 128790492.1compound. In some embodiments, the compound is C275 and / or has an amino acid sequence of SEQ ID NO: 241.

[0477] In some embodiments, a miniprotein of the present disclosure comprising changes such as, for example, ...

Claims

CLAIMS What is claimed is:

1. A composition, comprising a polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: (a) at least two constraints; (b) an arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3, a lysine at a position corresponding to amino acid 5, an isoleucine at a position corresponding to amino acid 6, a tryptophan at a position corresponding to amino acid 14, at least one modified lysine residue at a position corresponding to amino acid 24, and an alanine, arginine, or modified lysine at a position corresponding to 29, where each position is linear, from N-to-C-terminus, beginning at position 1, relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or to the Guanidino group nitrogen of the arginine side chain, wherein the at least one small alkyl group optionally comprises a methyl, dimethyl, or trimethyl; (c) at least 48 amino acids in length; and (d) has a binding affinity for B7-H3 stronger than 100 nM as measured in a cell-based assay.

2. The composition of claim 1, wherein the at least two constraints comprises at least two disulfide bridges.

3. The composition of claim 1 or 2, further comprising at least one additional constraint.

4. The composition of claim 3, wherein the at least one additional constraint is a lactam bridge.

5. The composition of claim 3, wherein the at least one additional constraint is a disulfide bridge.

6. The composition of any one of claims 1-5, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204 and 262-272. Page 316 of 344 IPTS / 128790492.

17. The composition of any one of claims 1-6, wherein the polypeptide is part of a compound selected from any one of C234-235 and C309-C332.

8. A composition comprising a polypeptide of at least 48 amino acids in length and having an amino sequence according to Formula VI (SEQ ID NO: 541), comprising at least four cysteines and two disulfide bonds, wherein X24 is (Kme) or (Kme2); X29 is (Kme) or A or R; X32 is (Kme), D, or (Cit); and X45 is (Kme) or K.

9. A composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula III (SEQ ID NO: 538) as follows: CAX3EKIAALSEIIWLPCLX19YAQIX24AFIX28X29LNX32DPCX36SX38X39ILSEA X45ELCS, wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme) or (Kme2); X28 is A or (Kme); X29 is (Kme) or A or R; X32 is (Kme) or D or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; and X45 is K or (Kme).

10. The composition of any one of claims 1-9, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204 and 262-272.

11. The composition of any one of claims 1-10, wherein the polypeptide is part of a compound selected from any one of C234-235 and C309-C332.

12. A composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula IV (SEQ ID NO: 539) as follows: CAX3EKIAALSEIIWLPCLX19YAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS , wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme) or (Kme2); X29 is (Kme) or A; X32 is (Kme) or D; and X45 is (Kme) or K.

13. The composition of claim 12, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204, 262, 265, 267, and 268.

14. The composition of claim 12 or 13, wherein the polypeptide is part of a compound selected from any one of C234, C235, C298, C299, C304, C305, C308-C311, C320, C323, C325, C326 and C332. Page 317 of 344 IPTS / 128790492.

115. A composition comprising a polypeptide having an amino acid sequence comprising an amino sequence according to Formula V (SEQ ID NO: 540) as follows: CAX3EKIAALSEIIWLPCLTYAQIX24AFIX28X29LNX32DPCQSSEILSEAX45ELCS , wherein X3 is (Kme3) or (Rme) or R; X24 is (Kme) or (Kme2); X28 is A or (Kme); X29 is (Kme) or A or R; X32 is (Kme) or D or (Cit); and X45 is (Kme) or K.

16. The composition of claim 15, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204, 262, 265, 266, 267, 270, and 272.

17. The composition of claim 15 or 16, wherein the polypeptide is part of a compound selected from any one of C275, C276, C298, C299, C304-C309, C314, C315, C318, C319, C323-C325, C328, C330, and C332.

18. A composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula VI (SEQ ID NO: 541) as follows: CA(Kme3)EKIAALSEIIWLPCLTYAQIX24AFIAX29LNX32DPCQSSEILSEAX45EL CS, wherein X24 is (Kme) or (Kme2); X29 is (Kme), A, or R; X32 is (Kme) or D or (Cit); and X45 is (Kme) or K.

19. The composition of claim 18, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs: 204, 262, 265, 267, 270, and 272.

20. The composition of claim 18 or 19, wherein the polypeptide is part of a compound selected from any one of C234, C235, C298, C299, C304, C305, C308, C314, C315, C318-C320, C323, C325, C328, C330, and C332.

21. A composition, comprising a B7-H3 binding polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acids include (i) a cysteine at each of four positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267 and wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme) or (Kme2); X28 is (Kme) or A; X29 is (Kme) or A or R; X32 is (Kme) or D or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; X45 is (Kme) or K, and X49 is S or absent. Page 318 of 344 IPTS / 128790492.

122. A composition, comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: (a) at least four cysteines, which form two disulfide bonds; (b) at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, optionally comprising a methyl, dimethyl, or trimethyl; (c) at least 48 amino acids in length; and (d) has a binding affinity for B7-H3 stronger than 100 nM in a cell-based assay.

23. The composition of any one of the preceding claims, wherein the polypeptide is a miniprotein.

24. The composition of any one of the preceding claims, wherein the polypeptide is at least 48 amino acids in length, but no greater than 100 amino acids in length.

25. The composition of any one of the preceding claims, wherein the polypeptide binds to B7-H3 with an affinity of stronger than 100 nM in a cell-based assay.

26. The composition of any one of the preceding claims, wherein the amino acid sequence of the polypeptide shares at least 90% identity to any one of SEQ ID NOs: 204 and 262-537, and includes at least one lysine or arginine with at least one modification comprising at least one small alkyl group bonded to the nitrogen of the lysine side chain or to the Guanidino group nitrogen of the arginine side chain, optionally selected from: trimethyl, dimethyl, and monomethyl.

27. The composition of any one of the preceding claims, wherein the amino acid sequence of the polypeptide shares at least 90% identity to at least 44 amino acids of a reference polypeptide, which reference polypeptide is longer than 47 amino acids in length and binds to B7-H3 with a strength of at least 10 nM on a cell-based assay, and / or has an inhibition constant of no weaker than 10 nM. Page 319 of 344 IPTS / 128790492.

128. The composition of any one of the preceding claims, wherein the amino acid sequence of the polypeptide shares at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537, provided that the 44 amino acids include at least four cysteine residues that form at least two disulfide bridges.

29. The composition of any one of the preceding claims, wherein the amino acid sequence of the polypeptide shares 100% identity to at least 44 amino acids of a reference polypeptide, which reference polypeptide is longer than 47 amino acids in length.

30. The composition of any one of the preceding claims, wherein the amino acid sequence shares at least 90% identity to at least 44 amino acids as set forth in any one of SEQ ID NO: 199, 204, 241, or 262-272.

31. The composition of any one of the preceding claims, wherein the amino acid sequence shares 100% identity to at least 44 amino acids as set forth in any one of SEQ ID NO: 199, 204, 241, or 262-272.

32. The composition of claim 30 or 31, wherein the amino acid sequence comprises at least four cysteines with at least two disulfide bridges.

33. The composition of claim 32, wherein the polypeptide is at least 48 amino acids in length.

34. A composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO:

267.

35. A composition comprising a compound as set forth in C234, C235, and C298-C333 as set forth in Table 2C.

36. A composition comprising a polypeptide having an amino acid sequence comprising any one of SEQ ID NOs: 204 and 262-272.

37. The composition of any one of claims 1-36, further comprising a radionuclide. Page 320 of 344 IPTS / 128790492.

138. The composition of claim 37, wherein the radionuclide is Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi- 123, Sm-153, Ra-225, Tb-165, or At-211.

39. The composition of any one of the preceding claims, wherein the C-terminus comprises an -OH or an -NH2.

40. The composition of any one of the preceding claims, wherein the binding affinity for B7- H3 is stronger than 100 nM.

41. The composition of any one of the preceding claims, wherein the inhibition constant is no weaker than 100 nM.

42. The composition of any one of the preceding claims, further comprising one or more of a linker, chelator, and radionuclide.

43. The composition of claim 42, wherein the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG2, PEG, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 36, (Gly)1-10, or any fragment or combination via covalent bond thereof.

44. The composition of 42 or 43, wherein the chelator comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n- butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

45. The composition of any one of claims 42-44, wherein the radionuclide is selected from Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I- 131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211. Page 321 of 344 IPTS / 128790492.

146. The composition of any one of claims 42-45, wherein if the polypeptide comprises any one of SEQ ID NOs: 198-537 the polypeptide further comprises a linker, wherein the linker is PEG4, and an optional chelator, wherein the chelator is DOTA.

47. The composition of any one of claims 42-46, wherein, when present, the linker is attached to the N-terminus of the polypeptide.

48. The composition of any one of the preceding claims, wherein the C-terminal amino acid of the polypeptide is not a cysteine.

49. The composition of any one of claims 42-48, wherein, when present, the chelator is attached to either the polypeptide or the linker.

50. The composition of any one of claims 41-48, wherein, when present, the radionuclide is attached to the chelator.

51. A composition comprising a formula selected from one or more of (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 (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537.

52. The composition of claim 51, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 198-537.

53. The composition of claim 51 or 52, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537.

54. The composition of any one of claims 51-53, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 198- 537. Page 322 of 344 IPTS / 128790492.

155. The composition of any one of claims 51-54, wherein the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n- (gGlu)n- or (PEG)n, wherein n is from 1 to 36, (Gly)1-10, or any fragment or combination via covalent bond thereof.

56. The composition of any one of claims 51-55, wherein the chelator comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri- n-butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

57. The composition of any one of claims 51-56, wherein the radionuclide Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb- 203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

58. A composition comprising a formula selected from one or more of (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 (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 204 and 262-272.

59. The composition of claim 58, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 204 and 262- 272.

60. The composition of claim 58 or 59, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 204 and 262-272. Page 323 of 344 IPTS / 128790492.

161. The composition of any one of claims 58-60, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 204 and 262-272.

62. The composition of any one of claims 58-61, wherein, when L is present, L comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36, 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 dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 10, (Gly)1-10, or any fragment or combination via covalent bond thereof.

63. The composition of any one of claims 58-62, wherein, when C is present, C comprises or consists of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N- succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB), or N-succinimidyl 3- trimethylstannylbenzoate (MeSTB).

64. The composition of any one of claims 58-63, wherein when R is present, R comprises or consists of Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce- 134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

65. The composition of any one of claims 58-64, wherein, when present, the linker is attached to the N-terminus of the polypeptide.

66. The composition of claim 65, wherein the C-terminal amino acid of the polypeptide is not a cysteine.

67. The composition of any one of claims 58-66, wherein, when present, the chelator is attached to either the polypeptide or the linker.

68. The composition of any one of claims 58-67, wherein, when present, the radionuclide is attached to the chelator. Page 324 of 344 IPTS / 128790492.

169. The composition of any one of claims 58-68, wherein the polypeptide comprises at least one disulfide bridge.

70. The composition of claim 69, wherein the polypeptide comprises at least two disulfide bridges.

71. The composition of any one of the preceding claims, which composition and / or polypeptide thereof selectively binds to B7-H3.

72. The composition of any one of the preceding claims, wherein the polypeptide has a binding affinity for B7-H3 of 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM, in vivo, ex vivo, or in vitro and / or as measured in a cell-based assay.

73. The composition of any one of the preceding claims, wherein the polypeptide has a binding inhibition constant of no weaker than 100 nM.

74. A composition comprising a polypeptide-drug conjugate, comprising a polypeptide and at least one drug moiety, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to at least 48 amino acids a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 198-537.

75. The composition of claim 74, wherein the polypeptide comprises at least four cysteines and two disulfide bridges.

76. The composition of claim 74 or 75, wherein the drug moiety is selected from a topoisomerase inhibitor, an auristatin (e.g., monomethyl auristatin E), 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, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), an inhibitor of nuclear export of proteins CRMl, a DPPIV 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 Page 325 of 344 IPTS / 128790492.1DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder, a DHFR inhibitor, and an immunotoxin.

77. A composition comprising an isolated compound or pharmaceutically acceptable salt thereof, or a neutral molecule, comprising an optional linker (L), and one or more of a polypeptide (M), chelator (C) or radionuclide (R), wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids any one of SEQ ID NOs: 198-537 and 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E.

78. A composition comprising, a compound designed to bind to B7-H3, which compound comprises or consists of a polypeptide having an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E, and further comprises a modified N and / or C-terminus.

79. The composition of claim 77 or 78, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E.

80. The composition of any one of claims 77-79, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E.

81. The composition of any one of claims 77-80, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E.

82. The composition of any one of claims 77-81, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 198- 537 or 538-543 and 546-550, including amino acid substitutions as set forth in Table 1E. Page 326 of 344 IPTS / 128790492.

183. The composition of claim any one of claims 78-82, wherein the modified N-terminus comprises one or more of an NH2-, Acetyl-, PEGn-, wherein n=0-36, DOTA-, or Biotin-.

84. The composition of any one of the preceding claims, wherein the C terminus comprises an -NH2 or an -OH.

85. The composition of any one of the preceding claims, wherein the polypeptide selectively binds to B7-H3.

86. The composition of any one of the preceding claims, wherein the polypeptide has a binding affinity of stronger than about 100 nM to B7-H3, in vivo or in a cell-based assay.

87. A compound comprising a miniprotein having an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of SEQ ID NO: 267, and further comprising one or more additional components according to a formula M-L-C-R, wherein M is the miniprotein, L is a linker, C is a chelator, and R is a radionuclide.

88. The compound of claim 87, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of SEQ ID NO:

267.

89. The compound of claim 87, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to SEQ ID NO:

267.

90. The compound of claim 87, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to SEQ ID NO:

267.

91. The compound of any one of claims 87-90, wherein L comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, any linker set forth in Table 2A or Table 2C, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 10, (Gly)1-10, or any fragment or combination via covalent bond thereof. Page 327 of 344 IPTS / 128790492.

192. The compound of any one of claims 87-91, wherein C comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n- butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

93. The compound of any one of claims 87-92, wherein R comprises or consists of Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I- 124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

94. A compound comprising a miniprotein with at least 90% identity to at least 48 amino acids of the amino acid sequence of SEQ ID NO: 267, wherein the N and / or C-terminus comprise between one and thirty additional amino acids, and / or wherein the C-terminus comprises one fewer amino acids or up to 30 additional amino acids, provided that the entire miniprotein is no greater than about 100 amino acids in length.

95. A pharmaceutical composition comprising a polypeptide of any one of claims 1-86 or compound as set forth in any one of claims 87-94; and a pharmaceutically acceptable excipient.

96. In a method of improving binding affinity strength of a polypeptide to B7-H3, the improvement comprising modifying at least three amino acid residues of a polypeptide, which polypeptide has at least 48 amino acids in length and has cysteines at positions corresponding to 1, 17, 35, and 48 of SEQ ID NO: 267, wherein a position corresponding to X24 is (Kme) or (Kme2); X29 is A or (Kme); and X32 is D or (Kme) or (Cit), and wherein X49 is S or absent.

97. A method of treating cancer, the method comprising administering to a subject in need thereof, a composition comprising a conjugate comprising a polypeptide having an amino acid sequence, which comprises an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537 and a radionuclide.

98. The method of claim 97, wherein the polypeptide has at least four cysteines and two disulfide bridges. Page 328 of 344 IPTS / 128790492.

199. The method of claim 97 or 98, wherein the radionuclide is associated with the polypeptide with a linker and / or chelator according to a formula M-L-C-R, wherein M is the polypeptide, L is a linker, C is a chelator, and R is the radionuclide.

100. The method of any one of claims 97-99, wherein the polypeptide has an amino acid sequence comprising or consisting of any of SEQ ID NOs: 204 or 262-272.

101. The method of claim 99 or 100, wherein L comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, any linker set forth in Table 2A, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 10, (Gly)1-10, or any fragment or combination via covalent bond thereof.

102. The method of any one of claims 99-101, wherein C comprises or consists of DOTA, Crown, NOPO, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n- butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

103. The method of any one of claims 99-102, wherein R is Ac-225, Cu-64, Ga-68, Lu- 177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

104. The method of claim 103, wherein R is a therapeutic agent and / or an imaging agent.

105. The method of claim 103 or 104, wherein R is Cu-64, Ga-68, Lu-177, In-111, Cu-67, La-132, or F-18.

106. The method of any one of claims 96-105, wherein the polypeptide is no longer than 100 amino acids in length.

107. A method of reducing kidney cell uptake and / or increasing tumor uptake of a composition comprising administering to a subject a B7-H3 binding protein having an Page 329 of 344 IPTS / 128790492.1amino acid sequence comprising at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO: 241, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, optionally comprising a methyl, dimethyl, or trimethyl, and the reduction is as compared to administering to the subject or a control subject an otherwise identical composition but not comprising the modified lysine residue at the position corresponding to X24.

108. In a method of treating cancer, the improvement comprising administering a composition comprising a B7-H3 binding protein having an amino acid sequence comprising at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO: 241, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, optionally comprising a methyl, dimethyl, or trimethyl, and the reduction is as compared to administering to the subject or a control subject an otherwise identical composition but not comprising the modified lysine residue at the position corresponding to X24.

109. A method of treating a subject with refractory or recurrent cancer comprising administering the composition according to any one of claims 1-86, the compound of any one of claims 87-94, or the pharmaceutical composition of claim 95, thereby treating the cancer.

110. A method of improving biodistribution of a pharmaceutical composition for a B7-H3 positive population of cancer cells in a subject having a B7-H3-positive cancer, comprising contacting the population with a polypeptide that has a modified lysine at a position corresponding to X24 of SEQ ID NO: 241, wherein the lysine is modified by adding at least one small alkyl group to a lysine side chain and wherein the biodistribution is improved as compared to contacting the population without the modified lysine at a position corresponding to X24 of SEQ ID NO:

241.

111. A method of diagnosing presence of a B7-H3 positive population of cancer cells comprising: (a) contacting a population of cells with the composition according to any one of claims 1-86, the compound of any one of claims 87-94, or the pharmaceutical composition of claim 95; Page 330 of 344 IPTS / 128790492.1(b) detecting the presence of the composition, compound, or pharmaceutical composition of step (a) by measuring a signal; and (c) comparing the detection in step (b) to a control signal; and (d) diagnosing cancer if the composition, compound, or pharmaceutical composition of step (a) is detected above the control signal.

112. The method of claim 111, wherein the contacting is performed by administering to a subject in need thereof.

113. The method of claim 112, wherein the administering is intravenous or subcutaneous.

114. The method of claim 111, wherein the contacting is outside of the subject, optionally in vitro with a biopsy sample.

115. A method of treating a cancer in a subject, the method comprising administering to the subject a composition comprising the composition as set forth in claims 1-86, the compound of any one of claims 87-94, or the pharmaceutical composition of claim 95.

116. Use of the composition of any one of claims 1-86, the compound of any one of claims 87-94, or the pharmaceutical composition of claim 95 to treat cancer in a subject.

117. A method of treating a subject in need thereof comprising administering to the subject in need thereof the composition of any one of claims 1-86, the compound of any one of claims 87-94, or the pharmaceutical composition of claim 95.

118. The method of claim 117, wherein the subject is diagnosed as having cancer.

119. The method of claim 118, wherein a cancer cell from the subject expresses B7-H3.

120. The method of claim 119, wherein the expression of B7-H3 is higher in the cancer cell than in a non-cancer cell, which expression can be measured by protein and / or nucleic acid levels.

121. The method of claim 120, wherein the non-cancer cell is obtained from the subject. Page 331 of 344 IPTS / 128790492.1122. The method of any one of claims 117-122, wherein the composition, compound, or pharmaceutical composition is internalized in a cell expressing human B7-H3.

123. The method of any one of claims 117-122, wherein the cancer is selected from breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, urothelial 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 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.

124. The method of any one of claims 117-123, wherein the composition, compound, or pharmaceutical composition is administered intravenously or subcutaneously.

125. A method of targeting cancer cells expressing B7-H3, the method comprising: (i) determining or having determined a level of expression of B7-H3 in a population of cancer cells; and Page 332 of 344 IPTS / 128790492.1(ii) administering to a subject in need thereof a composition comprising the composition according to any one of claims 1-86, the compound according to any one of claims 87-94, or the pharmaceutical composition of claim 95, wherein the polypeptide of the composition, compound, or pharmaceutical composition is designed to specifically bind to human B7-H3, wherein the composition, compound, or pharmaceutical composition is attached to the surface and / or internalized into one or more B7-H3 expressing cancer cells.

126. A method of targeting cancer cells expressing B7-H3, the method comprising: administering to a subject in need thereof a composition comprising the composition according to any one of claims 1-86, the compound according to any one of claims 87-94, or the pharmaceutical composition of claim 95, wherein the polypeptide of the composition, compound, or pharmaceutical composition is designed to specifically bind to human B7-H3; wherein (i) the subject has cancer cells that express B7-H3; and (ii) the composition, compound, or pharmaceutical composition is attached to the surface and / or internalized into one or more B7-H3 expressing cancer cells.

127. The method of claim 126, further comprising determining or having determined that the cancer cells express B7-H3.

128. The method of any one of claims 125-127, wherein the subject is treated after the administering as compared to prior to the administering.

129. In a method of targeting a population of cancer cells expressing B7-H3, the improvement comprising contacting the population with the composition according to any one of claims 1-86, the compound according to any one of claims 87-94, or the pharmaceutical composition of claim 95, wherein a position corresponding to X24 (relative to SEQ ID NO: 241) comprises a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein the composition is taken up less by kidney cells than in a composition comprising a polypeptide that does not have a small alkyl group attached to a nitrogen on the side chain of a lysine at X24, wherein, optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group.

130. A conjugate comprising: Page 333 of 344 IPTS / 128790492.1(i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Actinium-225.

131. A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Copper-64.

132. A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Gallium-68.

133. A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Indium-111.

134. A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Lead-212. Page 334 of 344 IPTS / 128790492.1135. A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) through an optional linker (L), wherein (C) comprises DOTA, and (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is Lutetium-177.

136. A conjugate comprising: (i) a miniprotein (M) that specifically binds to B7-H3; (ii) an N-terminal modification, conjugated to (M) through an optional linker (L), wherein (L), when present, comprises PEG, wherein the PEG is optionally PEG-4; and (iii) the N-terminal modification comprises a biotin.

137. The conjugate of any one of claims 130-136, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537.

138. The conjugate of any one of claims 130-137, wherein M has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 198-537.

139. The conjugate of any one of claims 130-138, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 198-537.

140. The conjugate of any one of claims 130-139, wherein M has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 198- 537.

141. The conjugate of any one of claims 130-140 wherein the amino acid sequence has an amino acid sequence comprising at least 90% identity to at least 44 amino acids of SEQ ID NO: 267, wherein the polypeptide has: (a) at least four cysteines, which form two disulfide bonds; Page 335 of 344 IPTS / 128790492.1(b) an arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3, a lysine at a position corresponding to amino acid 5, an isoleucine at a position corresponding to amino acid 6, a tryptophan at a position corresponding to amino acid 14, at least one modified lysine residue at a position corresponding to amino acid 24, and an alanine, arginine, or modified lysine at a position corresponding to 29, where each position is linear, from N-to-C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or to the Guanidino group of the arginine side chain, optionally comprising a methyl, dimethyl, or trimethyl; (c) at least 48 amino acids in length; and (d) a binding affinity for B7-H3 stronger than 100 nM as measured in a cell-based assay.

142. The conjugate of claim 141, wherein the amino acid sequence comprises an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 204 or 262-272.

143. The conjugate of claim 141 or 142, wherein the amino acid sequence comprises an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 204 or 262-272.

144. The compound of any one of claims 141-143, wherein the an amino acid sequence comprises an amino acid sequence with 100% identity to at least 44 amino acids of SEQ ID NO:

267.

145. The compound of any one of claims 141-144, wherein the amino acid sequence comprises an amino acid sequence with at least 90% identity to SEQ ID NO:

267.

146. The compound of any one of claims 141-145, wherein the amino acid sequence comprises an amino acid sequence with 100% identity to SEQ ID NO:

267.

147. The conjugate of any one of claims 130-141, wherein the amino acid sequence comprises or consists of any one of SEQ ID NOs: 204 and 262-272. Page 336 of 344 IPTS / 128790492.1148. The conjugate of any one of claims 130-142, wherein the amino acid sequence comprises or consists of SEQ ID NO:

267.

149. An isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from any one of SEQ ID NOs: 198-537; or a nucleic acid sequence encoding a polypeptide comprising at least 90%, 95%, 96%, 97%, 98%, 99% or greater identity to any one of SEQ ID NOs: 198-573.

150. A vector comprising the isolated polynucleotide of claim 149.

151. A host cell transformed with the isolated polynucleotide of claim 149 or the vector of claim 150.

152. A method of evaluating locations of one or more populations of cancerous cells in a subject, the method comprising administering to the subject the composition of any one of claims 1-86, the compound according to any one of claims 87-94, the pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148, and detecting to determine location of the composition in the subject, wherein the composition, compound, or pharmaceutical composition comprises a detectable label.

153. The method of claim 152, wherein the detectable label comprises a radionuclide.

154. In a method of decreasing kidney uptake of a composition administered to detect and / or treat one or more populations of cancer cells, the improvement comprising administering to a subject in need thereof the composition according to any one of claims 1-86, the compound according to any one of claims 87-94, the pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148, wherein a position corresponding to X24 of SEQ ID NO: 241 comprises a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein the composition is taken up less by kidney cells than in a composition comprising a polypeptide that does not have a small alkyl group attached to a nitrogen on the side chain of a lysine at a position corresponding to X24 of SEQ ID NO: 241, wherein, optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group. Page 337 of 344 IPTS / 128790492.1155. The method of claim 152 or 154, wherein the detecting comprises an imaging procedure that allows for selecting subjects, monitoring subjects, and / or treating subjects with a therapeutic comprising a miniprotein designed to bind to B7-H3 expressed on one or more cancer cells in the one or more populations of cancer cells.

156. The method of claim 155, wherein the therapeutic comprises the composition of any one of claims 1-86, the compound according to any one of claims 87-94, the pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148.

157. A method of improving delivery of a radionuclide to a population of cancer cells in a subject, the method comprising administering the composition of any one of claims 1-86, the compound according to any one of claims 87-94, or pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148, wherein the amino acid sequence of the polypeptide comprises an amino acid at a position corresponding to X24 of SEQ ID NO: 241 comprising a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein uptake by kidney cells is less than with a polypeptide that does not have a small alkyl group attached to a nitrogen on the side chain of a lysine at a position corresponding to X24 of SEQ ID NO:

241.

158. The method of claim 157, wherein the small alkyl group comprises a monomethyl, dimethyl, or trimethyl group.

159. In a method of treating an individual with cancer, the improvement comprising reducing one or more off-target effects or toxicity measures by administering the composition of any one of claims 1-86, the compound according to any one of claims 87- 94, the pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148, wherein the amino acid sequences of the polypeptide comprises an amino acid at a position corresponding to X24 of SEQ ID NO: 241 comprising a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein uptake by kidney cells is less than with a polypeptide that does not have a small alkyl group attached to a nitrogen on the side chain of a lysine at a position corresponding to X24 of SEQ ID NO:

241. Page 338 of 344 IPTS / 128790492.1160. In a method of treating an individual with cancer, the improvement comprising achieving a reduction in concentration of R in a kidney tissue in the presence of the composition of any one of claims 1-86, the compound according to any one of claims 87- 94, the pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148, wherein the amino acid sequence of the polypeptide comprises an amino acid corresponding to position X24 of SEQ ID NO: 241, and wherein X24 comprises a lysine with at least one additional small alkyl group attached to the nitrogen in the side chain, wherein uptake by kidney cells is less than with a polypeptide having an amino acid sequence that does not comprise a small alkyl group attached to a nitrogen on the side chain of a lysine at a position corresponding to X24 of SEQ ID NO: 241, as compared to the concentration of R in the kidney tissue in the absence the composition, compound, pharmaceutical composition, or conjugate.

161. The method of claim 160, wherein the reduction in concentration of R in the kidney tissue is measured by urine output of R as measured by percent of administered radiation recovered or by detection as measured by a cell-based in vitro assay, or an in vivo detection assay.

162. The method of claim 161, wherein the administration of the composition, compound, pharmaceutical composition, or conjugate can be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more in the presence of the composition, compound, pharmaceutical composition, or conjugate having 90% identity to at least 44 amino acids of SEQ ID NO: 241 including a modified lysine at positions corresponding to X24 of SEQ ID NO: 241 than in the presence of an Q, V, L, or K at positions corresponding to X24.

163. In a method of reducing uptake by a kidney tissue of a composition, the improvement comprising administering a composition comprising (a) a radionuclide therapeutic comprising at least a polypeptide and a radionuclide (R); wherein the polypeptide has at least 90% identity to at least 44 amino acids of SEQ ID NO: 241 and / or has a modified lysine at positions corresponding X24 of SEQ ID NO: 241, such that in the presence of the modified lysine, the radionuclide is less concentrated in the kidney tissue than in the absence of the polypeptide. Page 339 of 344 IPTS / 128790492.1164. A method comprising administering to a subject in need thereof a compound that binds to B7-H3 and includes at least one modified lysine at a position corresponding to X24 of SEQ ID NO: 241, wherein administration of the compound having a miniprotein with the at least one modified lysine reduces one or more off target effects, toxicity grades, and / or uptake and / or retention in a kidney tissue as compared to a compound not having a modified lysine (e.g., an unmodified lysine, e.g., an L, V, or Q) at a position corresponding to X24.

165. The method of claim 163 or 164, wherein the polypeptide has an amino acid sequence comprising an amino acid sequence with 100% identity to at least 44 amino acids of any one of SEQ ID NOs: 204 and 262-272.

166. The method of any one of claims 163-165, wherein the polypeptide has an amino acid sequence comprising an amino acid sequence with at least 90% identity to any one of SEQ ID NOs: 204 and 262-272.

167. The method of any one of claims 163-166, wherein the polypeptide has an amino acid sequence comprising an amino acid sequence with 100% identity to any one of SEQ ID NOs: 204 and 262-272.

168. A method of treating an individual having or suspected of having a B7-H3-positive cancer, the method comprising administering to the individual: (a) a means for blocking uptake and / or retention of a radiotherapeutic to kidney tissue, and (b) a linker, a chelator, and a radionuclide.

169. The method of claim 168, wherein the means for blocking uptake and / or retention of a radiotherapeutic to kidney tissue binds to B7-H3 and includes a modified lysine at a position corresponding to X24 of SEQ ID NO: 267 and / or has at least 90% identity to 40 amino acids of SEQ ID NO: 267 and / or has a modified lysine at a position corresponding to X24 of SEQ ID NO:

267.

170. The method of claim 169, wherein the means further comprises a modified lysine at a position corresponding to X3 of SEQ ID NO:

267. Page 340 of 344 IPTS / 128790492.1171. The method of any one of claims 168-170, wherein the means for blocking uptake and / or retention of a radiotherapeutic to the kidney tissue blocks uptake and / or retention to the kidney tissue greater than as compared to the blocking of uptake and / or retention to the kidney tissue by a means that does not include a modified lysine at a position corresponding to X24 of SEQ ID NO: 267 and / or have at least 90% identity to at least 44 amino acids of SEQ ID NO: 267 and / or have a modified lysine at a position corresponding to X24 of SEQ ID NO:

267.

172. The method of any one of claims 168-171, wherein the means for blocking uptake and / or retention of a radiotherapeutic to the kidney tissue blocks uptake and / or retention to the kidney tissue greater than as compared to the blocking of uptake and / or retention to the kidney tissue by a means that does not include a modified lysine at a position corresponding to X24 of SEQ ID NO: 267 and / or have at least 90% identity to at least 44 amino acids of SEQ ID NO: 267 and / or have a modified lysine at a position corresponding to X24 of SEQ ID NO:

267.

173. The method of any one of claims 168-172, wherein the means for blocking uptake and / or retention of a radiotherapeutic to the kidney tissue is a radiotherapeutic.

174. The method of claim 173, wherein the radiotherapeutic is targeted to a tumor or a population of cancer cells.

175. The method of claim 174, wherein the radiotherapeutic targeted to the tumor or the population of cancer cells is at a greater concentration than in the absence of the means for binding to kidney tissue.

176. The method of claim 175, wherein the radiotherapeutic comprises a polypeptide that targets B7-H3.

177. The method of any one of claims 168-176, wherein the radiotherapeutic comprises or consists of a compound selected from C227-C608 and C611.

178. The method of claim 177, wherein the radionuclide of the radiotherapeutic is selected from Ac-225, Cu-64, Ga-68, In-111, Lu-177, or Pb-212. Page 341 of 344 IPTS / 128790492.1179. A kit comprising a polypeptide and instructions for use, wherein the polypeptide has an amino acid sequence as set forth in a polypeptide of any one of the composition of any one of claims 1-86, the compound according to any one of claims 87-94, or the pharmaceutical composition of claim 95 or the conjugate of any one of claims 130-148.

180. The kit of claim 179, further comprising one or more of a linker, chelator, and radionuclide.

181. The kit of claim 180, wherein the linker comprises or consists of a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, an ester linker, an amide linker, a maleimide linker, a succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, a propanoic acid linker, a dTyr-Gly-Phe (yGF) linker, a caproleic acid linker, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is from 1 to 10, (Gly)1-10, or any fragment or combination via covalent bond thereof.

182. The kit of claim 180 or 181, wherein the chelator comprises or consists of DOTA, NOPO, Crown, Macropa, lead specific chelator (PSC), N-succinimidyl 3-(tri-n- butylstannyl)benzoate (BuSTB), or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB).

183. The kit of any one of claims 179-182, wherein prior to use, the compound is labeled with a radionuclide, wherein the radionuclide is chelated to the chelator to produce a composition with a formula M-L-C-R.

184. The kit of any one of claims 180-183, wherein the radionuclide is selected from Ac- 225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

185. The kit of claim 184, wherein the radionuclide is Ac-225, Cu-64, Ga-68, In-111, Lu- 177, or Pb-212.

186. The kit of any one of claims 179-185, wherein if the polypeptide has an amino acid comprising any of those set forth in any one of SEQ ID NOs: 199, 204, 241, or 262-272 Page 342 of 344 IPTS / 128790492.1and wherein the polypeptide further comprises a linker and / or a chelator, wherein the linker, when present, is PEG4, and the chelator, when present, is DOTA.

187. The kit of any one of claims 180-186, wherein, when present, the linker is attached to the N-terminus amino acid of the polypeptide.

188. The kit of any one of claims 179-187, wherein the C-terminal amino acid of the polypeptide is not cysteine.

189. The kit of any one of claims 180-188, wherein, when present, the chelator is attached to either the polypeptide or the linker.

190. The kit of any one of claims 180-189, wherein, when present, the radionuclide is attached to the chelator.

191. The kit of any one of claims 180-190, wherein, when present, the radionuclide is attached to the N-terminus amino acid of the polypeptide. Page 343 of 344 IPTS / 128790492.1