B7-H3 miniprotein, conjugate and uses thereof

Abstract

B7-H3-binding miniproteins, conjugates, and uses thereof are provided herein. B7-H3-binding peptides and conjugates comprising radionuclide conjugates, as well as one or more additional proteins such as decoy peptides, are disclosed herein. These peptides, conjugates, and / or decoys may be used in compositions and methods for treating, diagnosing, monitoring, and / or imaging diseases, disorders, or pathological conditions associated with the expression of one or more targets (e.g., B7-H3).

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit of and priority to U.S. provisional patent applications No. 63 / 607,988 (filed December 8, 2023); 63 / 636,076 (filed April 18, 2024); 63 / 650,364 (filed May 21, 2024); 63 / 650,365 (filed May 21, 2024) and 63 / 721,990 (filed November 18, 2024), each of which the full text is incorporated herein by reference for all purposes.

[0003] Sequence list

[0004] The present application includes a list of sequences that are submitted electronically in XML format and whose entire text is referenced by reference in this specification. The XML filename created on December 6, 2024 is AKT-035WO_SL.xml and its size is 931,498 bytes. Background Technology

[0005] Cancer is a leading cause of death worldwide. Classic cancer therapies, such as radiation therapy, chemotherapy, and surgery, can be accompanied by serious side effects, including those resulting from the death of healthy, non-cancerous cells. Newer therapies improve the targeting of cytotoxic drugs against tumor cells compared to previous therapies, including those using biological conjugates.

[0006] The present disclosure provides technologies such as compositions, methods of use, and methods of preparation for addressing needs in the field of cancer. For example, in contrast to classical cancer diagnosis or therapy, targeted molecules may be designed to increase the specificity of imaging or therapeutic modes and reduce toxicity. For example, delivery of therapeutic agents using polypeptides to specifically target therapeutic agents, such as chelators and / or radionuclides (e.g., alpha emitters), to the tumor microenvironment provides treatment focused on tumor cells and, for example, avoids or reduces the risk of toxicity to surrounding healthy tissues caused by the therapeutic agent targeted to the tumor.

[0007] In contrast to classical cancer therapies, radionuclide therapy is more targeted and less toxic. For example, by specifically delivering radionuclides to the tumor microenvironment, radiation can be selectively directed toward tumor tissue, effectively killing malignant cells while preserving surrounding healthy tissue. For instance, radionuclides may utilize targeting molecules that specifically bind to antigens expressed at increased levels and / or densities on the surface of tumor cells compared to non-tumor cells. Binding of radionuclides to antigen-positive tumor cells targets radiation to those cells rather than targeting healthy tissue. Full-length antibodies have previously been evaluated as a targeting moiety; however, due to considerations such as their large size, full-length antibodies may present several challenges, such as poor tumor tissue penetration and long cyclic half-lives. Therefore, there remains a need for novel approaches to specifically target tumors, particularly solid tumors. The present disclosure provides a technology that meets these and other needs.

[0008] In particular, the present invention provides a conjugate comprising a polypeptide (e.g., a miniprotein) that targets the tumor microenvironment and / or tumor cells, conjugated to one or more additional components including a polypeptide, a composition comprising the polypeptide, and, for example, a linker, a chelator, and / or a radionuclide (e.g., an alpha emitter). In some embodiments, the polypeptide specifically and strongly binds to B7-H3. In certain embodiments, the polypeptide or conjugate of the present disclosure imparts improved tumor penetration, reduced off-target toxicity and / or accumulation, improved stability (e.g., compared to a comparative polypeptide), as well as improved affinity for B7-H3 (e.g., compared to currently existing technologies (e.g., antibodies, e.g., antibody-drug conjugates, polypeptides, etc.)). In some embodiments, such conjugates are used to treat cells expressing a target (e.g., B7-H3). In some of these embodiments, the cells are cancer cells.

[0009] The present disclosure also provides insight that the compositions and conjugates provided herein may be further improved by increasing stability (e.g., thermal stability) while maintaining or increasing binding strength to B7-H3. As considered herein, in some embodiments, the conjugate, e.g., the radionuclide conjugate of the present disclosure, may be improved, such as by one or more measures such as improvement in efficacy and / or toxicity class and / or reduction of off-target effects, by modifying one or more amino acids in the polypeptide sequence of the conjugate and / or adding one or more decoys.

[0010] These and other embodiments of the present disclosure are described in the following detailed description and claims.

[0011] In one embodiment, the present disclosure provides a composition comprising a polypeptide having an amino acid sequence, wherein the amino acid sequence comprises at least two constraints; arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3; lysine at a position corresponding to amino acid 5; isoleucine at a position corresponding to amino acid 6; tryptophan at a position corresponding to amino acid 14; at least one modified lysine residue at a position corresponding to amino acid 24; and alanine, arginine, or modified lysine at a position corresponding to 29 (wherein, each position is linear from the N-terminus to the C-terminus starting from position 1 with respect to SEQ ID NO. 267, and the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or the guanidino nitrogen of the arginine side chain, and at least one small alkyl group optionally comprises methyl, dimethyl, or trimethyl); and a length of at least 48 amino acids; It has a binding affinity for B7-H3 stronger than 100 nM when measured in cell-based assays.

[0012] In some embodiments, at least two constraints comprise at least two disulfide bridges. In some embodiments, the composition comprises at least one additional constraint. In certain embodiments, at least one additional constraint is a lactam bridge. In some embodiments, at least one additional constraint is a disulfide bridge.

[0013] In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs 204 and 262 to 272. In some embodiments, the polypeptide is part of a compound selected from any one of C234 to C235 and C309 to C332.

[0014] In one embodiment, the present disclosure provides a composition comprising a polypeptide having a length of at least 48 amino acids, an amino acid sequence according to Formula VI (SEQ ID NO. 541), and comprising at least 4 cysteine ​​and 2 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.

[0015] In another aspect, the present disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to the following formula III (SEQ No. 538): CAX3EKIAALSEIIWLPCLX19YAQIX24AFIX28X29LNX32DPCX36SX38X39ILSEAX45ELCS, where 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; X45 is K or (Kme). In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs 204 and 262 to 272. In some embodiments, the polypeptide is part of a compound selected from any one of C234 to C235 and C309 to C332.

[0016] In one embodiment, the composition comprises a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula IV (SEQ 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. In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ Nos. 204, 262, 265, 267 and 268.

[0017] In a specific embodiment, 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.

[0018] In another aspect, the present disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to the following formula V (SEQ No. 540): 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 Nos. 265, 266, 267, 270 and 272. In a specific embodiment, the polypeptide is part of a compound selected from any one of C304 to C309, C314, C315, C318, C319, C323, C324, C325, C328, C330 and C332.

[0019] In one embodiment, the present disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to the following formula VI (SEQ No. 541): 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 Nos. 204, 262, 265, 266, 267, 270 and 272. In a specific embodiment, the polypeptide is part of a compound selected from any one of C234, C235, C298, C299, C304 to C309, C314, C315, C318 to C320, C323, C324, C325, C328, C330 and C332.

[0020] In another aspect, the present disclosure provides a composition comprising a B7-H3 bonded polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acids include (i) cysteine ​​at each of the four positions corresponding to 1, 17, 35 and 48 of SEQ ID NO. 267, and 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.

[0021] In one embodiment, the present 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 forming 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 and optionally comprises methyl, dimethyl, or trimethyl); having a length of at least 48 amino acids; and having a binding affinity for B7-H3 stronger than 100 nM when measured in a cell-based assay. In some embodiments, the polypeptide is a miniprotein. In certain embodiments, the polypeptide has a length of at least 48 amino acids and no more than 100 amino acids. In certain embodiments, the polypeptide binds to B7-H3 with an affinity stronger than 100 nM when measured in a cell-based assay.

[0022] In some embodiments, the amino acid sequence of the polypeptide according to the present disclosure shares at least 90% identity with any one of SEQ ID NOs 204 and 262 to 537 and comprises at least one lysine or arginine having at least one modification that is bound to the nitrogen of the lysine side chain or the guanidino nitrogen of the arginine side chain and optionally comprises at least one small alkyl group selected from trimethyl, dimethyl, and monomethyl. In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with at least 44 amino acids of the reference polypeptide, wherein the reference polypeptide has an amino acid length longer than 47 and binds to B7-H3 with an intensity of at least 10 nM in a cell-based assay and / or has an inhibition constant not weaker than 10 nM. In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537, provided that the 44 amino acids include at least 4 cysteine ​​residues forming two disulfide bridges. In some embodiments, the amino acid sequence of the polypeptide shares 100% identity with at least 44 amino acids of the reference polypeptide, and the reference polypeptide has an amino acid length longer than 47. In some embodiments, the amino acid sequence shares at least 90% identity with at least 44 amino acids as presented in any one of SEQ ID NOs 199, 204, 241, or 262 to 272. In some embodiments, the amino acid sequence shares 100% identity with at least 44 amino acids as presented in any one of SEQ ID NOs 199, 204, 241, or 262 to 272.

[0023] In some embodiments, the amino acid sequence comprises at least four cysteines having at least two disulfide bridges. In some embodiments, the polypeptide is at least 48 amino acids long. In one aspect, the present disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO. 267. In certain embodiments, the composition comprises C234, C235, and C298 to C333 as presented in Table 2C.

[0024] In one embodiment, the present disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising any one of SEQ ID NOs 204 and 262 to 272.

[0025] In certain embodiments, the composition of the present disclosure (e.g., a B7-H3 polypeptide, e.g., a B7-H3 binding miniprotein) further comprises a radionuclide. Depending on the 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, the polypeptide of the present disclosure comprises a C-terminus comprising -OH or -NH2. In some embodiments, the binding affinity of the composition provided herein for B7-H3 is stronger than 100 nM. In certain embodiments, the inhibition constant is not weaker than 100 nM.

[0026] In some embodiments, the composition of the present disclosure (e.g., including polypeptide) further comprises one or more of a linker, a chelator, and a radionuclide. In some embodiments, the linker comprises or consists of any fragment or combination thereof through covalent bonding of (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 36), (Gly)1-10, or (Gly)n-(gGlu)n- or (PEG)n. 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.

[0027] In certain embodiments, where the polypeptide comprises any one of SEQ ID NOs 198 to 537, the polypeptide further comprises a linker, the linker is PEG4 and an optional chelator, and the chelator is DOTA. In some embodiments, the linker is attached to the N-terminus of the polypeptide, if present. In some embodiments, the C-terminal amino acid of the polypeptide is not cysteine. In some embodiments, the chelator is attached to the polypeptide or the linker, if present. In some embodiments, a radionuclide is attached to the chelator, if present.

[0028] In one embodiment, the present disclosure provides a composition comprising a formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a polypeptide (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), x is 1, 2, 3, or 4, and M comprises an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537. In some embodiments, M comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 198 to 537. In certain embodiments, M comprises an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537. In some embodiments, M has an amino acid sequence comprising an amino acid sequence having at least 100% identity with any one of SEQ ID NOs 198 to 537. In certain embodiments, the linker comprises or consists of any fragment or combination thereof through covalent bonding of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4 polyethylene glycol (PEG) linkers, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 36), (Gly)1-10, or (Gly)n-(gGlu)n- or (PEG)n.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 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.

[0029] In one embodiment, the present disclosure provides a composition comprising a formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a polypeptide (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), x is 1, 2, 3, or 4, and M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 204 and 262 to 272. In a specific embodiment, M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 204 and 262 to 272. In some embodiments, M has an amino acid sequence comprising an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 204 and 262 to 272. In certain embodiments, M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs 204 and 262 to 272. In some embodiments, where L is present, L is a polyethylene glycol (PEG) linker, ester linker, amide linker, maleimide linker, valine-citrulline linker, hydrazone linker, N-succinimidyl-4-(2-pyridylditeo)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, vinylsulfone-based linker, propanoic acid linker, dTyr-Gly-Phe(yGF) linker, caploreic acid linker, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 10), (Gly)1-10, or a combination thereof. It includes or consists of any fragment or combination through combination.In some embodiments, if 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, if 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 some embodiments, a linker is attached to the N-terminus of the polypeptide, if present. In certain embodiments, the C-terminal amino acid of the polypeptide is not cysteine. In some embodiments, a chelator is attached to the polypeptide or the linker, if present. In some embodiments, a radionuclide is attached to the chelator, if present. 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 the polypeptide thereof selectively bind 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 when measured in vivo, in vitro, in vitro, and / or in a cell-based assay. In some embodiments, the polypeptide has a binding inhibition constant not weaker than 100 nM.

[0030] In one embodiment, the present 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 with at least 48 amino acids of a polypeptide having an amino acid sequence presented in any one of SEQ ID NOs 198 to 537. In a specific embodiment, the polypeptide comprises at least four cysteine ​​groups and two disulfide bridges. In some embodiments, the drug moiety is a focal isomerase inhibitor, auristatin (e.g., monomethylauristatin E), V-ATPase inhibitor, apoptosis promoter, Bcl2 inhibitor, MCL1 inhibitor, HSP90 inhibitor, IAP inhibitor, mTor inhibitor, microtubule stabilizer, microtubule destabilizer, dolastine, metansinoid, MetAP (methionine aminopeptidase), inhibitor of extranuclear transport of protein CRMl, DPPIV inhibitor, proteasome inhibitor, inhibitor of phosphoryl transfer reaction in mitochondria, protein synthesis inhibitor, kinase inhibitor, CDK2 inhibitor, CDK9 inhibitor, kinesin inhibitor, HDAC inhibitor, DNA damaging agent, DNA alkylating agent, DNA intercalator, DNA minor groove binder, DHFR inhibitor, and It is selected from immunotoxins.

[0031] In one embodiment, the present disclosure provides a composition comprising a selective linker (L) and an isolated compound or a pharmaceutically acceptable salt thereof comprising one or more of a polypeptide (M), a chelator (C) or a radionuclide (R), wherein M has an amino acid sequence comprising at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537, 538 to 543, and 546 to 550, comprising amino acid substitutions as presented in Table 1E.

[0032] In another aspect, the present disclosure provides a composition comprising a compound designed to bind to B7-H3, wherein the compound comprises or consists of a polypeptide having an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, comprising amino acid substitutions as presented in Table 1E, and further comprises a modified N and / or C-terminus. In some embodiments, M has an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, comprising amino acid substitutions as presented in Table 1E. In some embodiments, M has an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, including amino acid substitutions as presented in Table 1E. In some embodiments, M has an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, including amino acid substitutions as presented in Table 1E. In some embodiments, M has an amino acid sequence having 100% identity with any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, including amino acid substitutions as presented in Table 1E. In some embodiments, the modified N-terminus comprises one or more of NH2-, acetyl-, PEGn- (wherein=0 to 36), DOTA-, or biotin-. In certain embodiments, the C-terminus comprises -NH2 or -OH. In certain embodiments, the polypeptide selectively binds to B7-H3.In some embodiments, the polypeptide has a binding affinity stronger than about 100 nM for B7-H3 in vivo or cell-based assays.

[0033] In one embodiment, the present disclosure provides a compound comprising a miniprotein having an amino acid sequence having at least 90% identity with at least 44 amino acids of SEQ ID NO. 267, and further comprising one or more additional components according to the formula MLCR, wherein M is the miniprotein, L is a linker, C is a chelator, and R is a radionuclide. In some embodiments, M has an amino acid sequence having 100% identity with at least 44 amino acids of SEQ ID NO. 267. In certain embodiments, M has an amino acid sequence having at least 90% identity with SEQ ID NO. 267. In some embodiments, M has an amino acid sequence having 100% identity with SEQ ID NO. 267. In some embodiments, L comprises or consists of polyethylene glycol (PEG) linkers of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, any linker presented in Table 2A or Table 2C, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 10), (Gly)1-10, or any fragment or combination thereof through covalent bonding. 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.

[0034] In one embodiment, the present disclosure provides a compound comprising a miniprotein that is at least 90% identical to at least 48 amino acids of the amino acid sequence of SEQ ID NO. 267, wherein the N and / or C-terminus has 1 to 30 additional amino acids and / or the C-terminus has less than 1 amino acid or up to 30 additional amino acids, provided that the total miniprotein has a length of about 100 amino acids or less.

[0035] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a polypeptide or compound as provided herein; and a pharmaceutically acceptable excipient.

[0036] In one embodiment, the present disclosure provides a method for improving the binding affinity strength of a polypeptide to B7-H3, wherein the improvement comprises modifying at least three amino acid residues of the polypeptide, the polypeptide having a length of at least 48 amino acids and having a cysteine ​​at positions corresponding to 1, 17, 35 and 48 of SEQ ID NO. 267, and a position corresponding to X24 being (Kme) or (Kme2); X29 being A or (Kme); X32 being D or (Kme) or (Cit); and X49 being S or absent.

[0037] In one embodiment, the present disclosure provides a method for treating cancer, wherein the method comprises the step of administering to a subject requiring treatment for cancer a composition comprising a conjugate comprising a polypeptide having an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537 and a radionuclide. In some embodiments, the polypeptide has at least four cysteine ​​and two disulfide bridges. In certain embodiments, the radionuclide is associated with the polypeptide by a linker and / or chelator according to the formula MLCR, wherein M is the polypeptide, L is the linker, C is the 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 to 272. In certain embodiments, L comprises or consists of any fragment or combination thereof through covalent bonding of polyethylene glycol (PEG) linkers of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, any linker presented in Table 2A, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 10), (Gly)1-10, or any of these. 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.

[0038] In certain embodiments, the polypeptide has a length of 100 or fewer amino acids.

[0039] In one embodiment, the present disclosure provides a method for reducing renal cell uptake of a composition and / or increasing tumor uptake, wherein the method comprises the step of 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 and optionally comprising methyl, dimethyl or trimethyl, and the reduction is compared with administering to said subject or control subject the same composition, but without the modified lysine residue at a position corresponding to X24.

[0040] In one embodiment, the present disclosure provides a method for treating cancer, wherein improvement comprises administering a composition comprising a B7-H3 binding protein having an amino acid sequence including at least one modified lysine residue at a position corresponding to X24 of SEQ ID NO. 241, modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain and optionally including methyl, dimethyl or trimethyl, and reduction is compared with administering the same composition to said subject or control subject, but not including a modified lysine residue at a position corresponding to X24.

[0041] In one embodiment, the present disclosure provides a method for treating a subject having intractable or recurrent cancer, wherein the method comprises the step of administering a composition, compound, or pharmaceutical composition as provided herein, thereby treating the cancer.

[0042] In one embodiment, the present disclosure provides a method for improving the biodistribution of a pharmaceutical composition to a population of B7-H3 positive cancer cells in a subject having B7-H3 positive cancer, wherein the method comprises the step of contacting the population with a polypeptide having a modified lysine at the position corresponding to X24 of SEQ ID NO. 241, wherein the lysine is modified by adding at least one small alkyl group to the lysine side chain, and the biodistribution is improved compared to contacting a population without the modified lysine at the position corresponding to X24 of SEQ ID NO. 241.

[0043] In one embodiment, the present disclosure provides a method for diagnosing the presence of a population of B7-H3 positive cancer cells, the method comprising: contacting the population of cells with a 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; comparing the detection in step (b) with a control signal; and diagnosing cancer if the composition, compound, or pharmaceutical composition of step (a) is detected in greater quantities than the control. In some embodiments, contact is performed by administration to a subject in need. In certain embodiments, administration is performed intravenously or subcutaneously. In some embodiments, contact is performed outside the subject, optionally in vitro, using a biopsy sample.

[0044] In one embodiment, the present disclosure provides a method for treating cancer of a subject, wherein the method comprises the step of administering to the subject a composition comprising a composition, a compound, or a pharmaceutical composition as provided herein.

[0045] In one embodiment, the present disclosure provides a use of a composition, compound, or pharmaceutical composition according to the present disclosure.

[0046] In one embodiment, the present disclosure provides a method for treating a subject in need of treatment, wherein the method comprises the step of administering a composition, compound, or pharmaceutical composition according to the present disclosure to the subject in need of treatment.

[0047] In certain embodiments, the subject is diagnosed as having cancer. In certain embodiments, cancer cells from the subject express B7-H3. In certain embodiments, the expression of B7-H3 is higher in cancer cells than in non-cancer cells, and the expression can be measured by protein and / or nucleic acid levels. In certain embodiments, non-cancer cells are obtained from the subject. In some embodiments, a composition, compound, or pharmaceutical composition is internalized in cells expressing human B7-H3. In some embodiments, cancer is breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-associated lymphoma, anal cancer, urothelial carcinoma, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic proliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, extracranial germ cell tumor, extrahepatic biliary cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, lip cancer, oral cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic cervical squamous cell carcinoma, multiple myeloma and other plasma cell neoplasms, mycosis fungoides and Sézary syndrome, It is selected from myelodysplastic syndrome, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrohistocytoma of bone, sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineal blastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor and other pediatric renal tumors. In some embodiments, the composition, compound, or pharmaceutical composition is administered intravenously or subcutaneously.

[0048] In one embodiment, the present disclosure provides a method for targeting cancer cells expressing B7-H3, wherein the method comprises: (i) determining or determining the level of expression of B7-H3 in a population of cancer cells; and (ii) administering to a subject in need a composition comprising a composition, a compound, or a pharmaceutical composition as provided herein, wherein the polypeptide of the composition, the compound, or the pharmaceutical composition is designed to specifically bind to human B7-H3, and the composition, the compound, or the pharmaceutical composition is attached to a surface and / or internalized in one or more B7-H3-expressing cancer cells.

[0049] In one embodiment, the present disclosure provides a method for targeting cancer cells expressing B7-H3, wherein the method comprises the step of administering a composition comprising a composition, compound, or pharmaceutical composition as provided herein to a subject in need, wherein the polypeptide of the composition, compound, or pharmaceutical composition is designed to specifically bind to human B7-H3; (i) the subject has cancer cells expressing B7-H3, and (ii) the composition, compound, or pharmaceutical composition is attached to a surface and / or internalized into one or more B7-H3-expressing cancer cells. In some embodiments, the method further comprises the step of determining or determining that the cancer cells express B7-H3. In certain embodiments, the subject is treated after administration compared to before administration.

[0050] In one embodiment, the present disclosure provides a method for treating a population of cancer cells expressing B7-H3, wherein the improvement comprises contacting the population with a composition, compound, or pharmaceutical composition as provided herein, wherein the position corresponding to X24 (based on SEQ ID NO. 241) comprises lysine having at least one additional small alkyl group attached to the nitrogen of the side chain, and the composition is less absorbed by kidney cells than in a composition comprising a polypeptide that does not have a small alkyl group attached to the nitrogen of the lysine on the side chain at X24, and optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group.

[0051] In one embodiment, the present disclosure comprises a conjugate comprising (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L) (wherein (C) comprises DOTA, (L) comprises PEG if present, and PEG is optionally PEG-4); and (iii) a radionuclide (R) chelated to (C) (wherein (R) is actinium-225).

[0052] In one embodiment, the present disclosure comprises a conjugate comprising (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L) (wherein (C) comprises DOTA, (L) comprises PEG if present, and PEG is optionally PEG-4); and (iii) a radionuclide (R) chelated to (C) (wherein (R) is copper-64).

[0053] In one embodiment, the present disclosure comprises a conjugate comprising (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L) (wherein (C) comprises DOTA, (L) comprises PEG if present, and PEG is optionally PEG-4); and (iii) a radionuclide (R) chelated to (C) (wherein (R) is Gallium-68).

[0054] In one embodiment, the present disclosure comprises a conjugate comprising (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L) (wherein (C) comprises DOTA, (L) comprises PEG if present, and PEG is optionally PEG-4); and (iii) a radionuclide (R) chelated to (C) (wherein (R) is indium-111).

[0055] In one embodiment, the present disclosure comprises a conjugate comprising (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L) (wherein (C) comprises DOTA, (L) comprises PEG if present, and PEG is optionally PEG-4); and (iii) a radionuclide (R) chelated to (C) (wherein (R) is lead-212).

[0056] In one embodiment, the present disclosure comprises a conjugate comprising (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L) (wherein (C) comprises DOTA, (L) comprises PEG if present, and PEG is optionally PEG-4); and (iii) a radionuclide (R) chelated to (C) (wherein (R) is lutetium-177).

[0057] The conjugate comprises (i) a miniprotein (M) that specifically binds to B7-H3; (ii) an N-terminal modification conjugated to (M) via a selective linker (L), wherein (L) comprises, if present, PEG, and the PEG is optionally PEG-4; and (iii) the N-terminal modification comprises biotin.

[0058] In certain embodiments, M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537. In some embodiments, M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 198 to 537. In some embodiments, M has an amino acid sequence comprising an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537. In certain embodiments, M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs 198 to 537. In certain embodiments, the amino acid sequence has an amino acid sequence having 90% identity with at least 44 amino acids of SEQ ID NO 267, wherein the polypeptide comprises at least 4 cysteines forming 2 disulfide bonds; arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3; Lysine at a position corresponding to amino acid 5; isoleucine at a position corresponding to amino acid 6; tryptophan at a position corresponding to amino acid 14; at least one modified lysine residue at a position corresponding to amino acid 24; and alanine, arginine, or modified lysine at a position corresponding to 29, wherein each position is linear from the N-terminus to the C-terminus with respect to SEQ ID NO. 267, and the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or the guanidino group of the arginine side chain, which optionally includes methyl, dimethyl, or trimethyl; having a length of at least 48 amino acids; and having a binding affinity for B7-H3 stronger than 100 nM in cell-based assays. In some embodiments, the amino acid sequence comprises an amino acid sequence having at least 90% identity with SEQ ID NO. 204 or any one of 262 to 272.In some embodiments, the amino acid sequence comprises an amino acid sequence having 100% identity with at least 44 amino acids of either SEQ ID NO. 204 or 262 to 272. In certain embodiments, the amino acid sequence comprises an amino acid sequence having 100% identity with at least 44 amino acids of SEQ ID NO. 267. In certain embodiments, the amino acid sequence comprises an amino acid sequence having at least 90% identity with SEQ ID NO. 267. In certain embodiments, the amino acid sequence comprises an amino acid sequence having 100% identity with SEQ ID NO. 267. In some embodiments, the amino acid sequence comprises or consists of any one of SEQ ID NO. 204 and 262 to 272. In some embodiments, the amino acid sequence comprises or consists of SEQ ID NO. 267.

[0059] In one embodiment, the present disclosure provides an isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from any one of SEQ ID NOs 198 to 537; or a nucleic acid sequence encoding a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% or greater identity with any one of SEQ ID NOs 198 to 573. In some embodiments, the present disclosure provides a vector comprising the isolated polynucleotide provided herein. In some embodiments, the present disclosure provides a host cell transformed with the isolated polynucleotide or vector as provided herein.

[0060] In one embodiment, the present disclosure provides a method for evaluating the location of one or more cancerous cell populations within a subject, wherein the method comprises the steps of administering to the subject a composition, compound, pharmaceutical composition, or conjugate as provided herein and detecting to determine the location of the composition within the subject, and the composition, compound, or pharmaceutical composition comprises a detectable label. In certain embodiments, the detectable label comprises a radionuclide.

[0061] In one embodiment, the present disclosure provides a method for reducing the renal uptake of a composition administered to detect and / or treat one or more cancer cell populations, wherein the improvement comprises administering a composition, compound, pharmaceutical composition, or conjugate as provided herein to a subject in need of improvement, wherein the composition comprises lysine having at least one additional small alkyl group attached to the nitrogen of the side chain at the position corresponding to X24 of SEQ ID NO. 241, and the composition is less uptaken by renal cells than in a composition comprising a polypeptide that does not have a small alkyl group attached to the nitrogen of the side chain of lysine at the position corresponding to X24 of SEQ ID NO. 241, optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group. In certain embodiments, detection comprises an imaging procedure that enables selecting a subject, monitoring the subject, and / or treating the subject with a therapeutic agent comprising a miniprotein designed to bind to B7-H3 expressed on one or more cancer cells in one or more cancer cell populations. In some embodiments, the therapeutic agent comprises a composition, compound, conjugate, or pharmaceutical composition as provided herein.

[0062] In one embodiment, the present disclosure provides a method for improving the delivery of a radionuclide to a population of cancer cells of a subject, wherein the method comprises the step of administering a composition, compound, conjugate, or pharmaceutical composition as provided in the specification, and the amino acid sequence of the polypeptide comprises an amino acid comprising lysine having at least one additional small alkyl group attached to the nitrogen of the side chain at the position corresponding to X24 of SEQ ID NO. 241, and uptake by kidney cells is less than that by a polypeptide not comprising a small alkyl group attached to the nitrogen of the side chain of lysine at the position corresponding to X24 of SEQ ID NO. 241. In some embodiments, the small alkyl group comprises a monomethyl, dimethyl, or trimethyl group.

[0063] In one embodiment, the present disclosure provides a method for treating an individual having cancer, wherein the improvement comprises reducing one or more non-target effects or toxicity measures by administering a composition, compound, conjugate or pharmaceutical composition as provided herein, wherein the amino acid sequence of the polypeptide comprises an amino acid comprising lysine having at least one additional small alkyl group attached to the nitrogen of the side chain corresponding to X24 of SEQ ID NO. 241, and the uptake by kidney cells is less than that of a polypeptide having an amino acid sequence that does not include a small alkyl group attached to the nitrogen of the side chain of lysine at the position corresponding to X24 of SEQ ID NO. 241.

[0064] In another aspect, the present disclosure provides a method for treating an individual with cancer, wherein the improvement comprises achieving a reduction in the concentration of R in 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, where X24 comprises lysine having at least one additional small alkyl group attached to the nitrogen of the side chain, and the uptake by kidney cells is less than that by a polypeptide having an amino acid sequence that does not include a small alkyl group attached to the nitrogen of the lysine side chain at the position X24 of SEQ ID NO. 241, compared to the concentration of R in kidney tissue in the absence of the composition, compound, pharmaceutical composition, or conjugate. In some embodiments, the reduction in the concentration of R in kidney tissue is measured by the urinary excretion of R measured by the percentage of administered radiation recovered, or by detection measured by a cell-based in vitro assay or an in vivo detection assay. In some embodiments, administration of the composition may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times in the presence of a composition having 90% identity with at least 44 amino acids of SEQ ID NO. 241, comprising lysine modified at the position corresponding to X24 of SEQ ID NO. 241, rather than in the presence of Q, V, L or K at the position corresponding to X24.

[0065] In one embodiment, the present disclosure provides a method for reducing the uptake of a composition by renal tissue, wherein the improvement comprises (a) administering a composition comprising at least a polypeptide and a radionuclide therapeutic agent comprising a radionuclide (R); the polypeptide having at least 90% identity with at least 44 amino acids of SEQ ID NO. 241 and / or having a modified lysine at a position corresponding to X24 of SEQ ID NO. 241, such that in the presence of the modified lysine, the radionuclide is less concentrated in renal tissue than in the absence of the polypeptide.

[0066] In another aspect, the present disclosure provides a method comprising the step of administering to a subject in need of a compound that binds to B7-H3 and comprises 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 at least one modified lysine reduces one or more off-target effects, toxicity grades, and / or uptake and / or retention in renal tissue compared to a compound not having modified lysine at a position corresponding to X24 (e.g., unmodified lysine, e.g., L, V, or Q). In some embodiments, the polypeptide has an amino acid sequence comprising an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NO. 204 and 262 to 272. In some embodiments, the polypeptide has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NO. 204 and 262 to 272. In some embodiments, the polypeptide has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs 204 and 262 to 272.

[0067] In one embodiment, the present disclosure provides a method for treating an individual having or suspected of having a B7-H3-positive cancer, wherein the method comprises the step of administering to the individual means for blocking the absorption and / or retention of a radiotherapeutic agent in kidney tissue, a linker, a chelator, and a radionuclide. In some embodiments, the means for blocking the absorption and / or retention of a radiotherapeutic agent in kidney tissue binds to B7-H3 and comprises a modified lysine at a position corresponding to X24 of SEQ ID NO. 267 and / or has at least 90% identity with the 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. 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 the absorption and / or retention of a radiotherapeutic agent in kidney tissue blocks the absorption and / or retention in kidney tissue more significantly than the means for blocking the absorption and / or retention in kidney tissue by means that does not include lysine modified at the position corresponding to X24 of SEQ ID NO 267 and / or does not have at least 90% identity with at least 44 amino acids of SEQ ID NO 267 and / or does not have lysine modified at the position corresponding to X24 of SEQ ID NO 267. In some embodiments, the means for blocking the absorption and / or retention of a radiotherapeutic agent in kidney tissue blocks the absorption and / or retention in kidney tissue more significantly than the blocking of absorption and / or retention in kidney tissue by means that does not include lysine modified at the position corresponding to X24 of SEQ ID NO. 267 and / or does not have at least 90% identity with at least 44 amino acids of SEQ ID NO. 267 and / or does not have lysine modified at the position corresponding to X24 of SEQ ID NO. 267. In some embodiments, the means for blocking the absorption and / or retention of a radiotherapeutic agent in kidney tissue is a radiotherapeutic agent. In certain embodiments, the radiotherapeutic agent is targeted to a tumor or a population of cancer cells.In certain embodiments, the radiotherapeutic agent targeted to a tumor or population of cancer cells is present at a higher concentration than in the absence of means for binding to kidney tissue. In some embodiments, the radiotherapeutic agent comprises a polypeptide that targets B7-H3. In some embodiments, the radiotherapeutic agent comprises or consists of a compound selected from C227 to C608 and C611. In certain embodiments, the radionuclide of the radiotherapeutic agent is selected from Ac-225, Cu-64, Ga-68, In-111, Lu-177, or Pb-212.

[0068] In one embodiment, the present disclosure provides a kit comprising a polypeptide and instructions for use, wherein the polypeptide has an amino acid sequence as presented in the 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, a chelator, and a radionuclide. In some embodiments, the linker comprises or consists of any fragment or combination thereof through covalent bonding of polyethylene glycol (PEG) linkers of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 10), (Gly)1-10, or any fragment or combination 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, the compound is labeled with a radionuclide before use, wherein the radionuclide is chelated to the chelator to produce a composition having the chemical formula MLCR. 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.

[0069] In some embodiments, where the polypeptide has an amino acid comprising any of those presented in SEQ ID NOs 199, 204, 241, or 262 to 272, the polypeptide further comprises a linker and / or a chelator, wherein the linker is, if present, PEG4 and the chelator is, if present, DOTA. In some embodiments, the linker is, if present, attached to the N-terminal amino acid of the polypeptide. In some embodiments, the C-terminal amino acid of the polypeptide is not cysteine. In some embodiments, the chelator is, if present, attached to the polypeptide or the linker. In certain embodiments, a radionuclide is, if present, attached to the chelator. In certain embodiments, if present, the radionuclide is, if present, attached to the N-terminal amino acid of the polypeptide. Brief explanation of the drawing

[0070] Fig. 1a represents the binding affinity of an exemplary miniprotein (Sequence No. 3) to 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). Fig. 1b Figure 1 shows a 1:1 binding model analysis for an exemplary miniprotein (Sequence No. 3). The black line is fitted by the model. Fig. 2a This shows the binding affinity of an exemplary miniprotein (sequence number 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). Fig. 2b This represents a 1:1 binding model analysis for an exemplary miniprotein (Sequence No. 4, Run #1). The black line is fitted by the model. Fig. 3aThis shows the binding affinity of an exemplary miniprotein (sequence number 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). Fig. 3b represents a 1:1 binding model analysis for an exemplary miniprotein (Sequence No. 4, Run #2). The black line is fitted by the model. Fig. 4 Figure 1 shows a 1:1 binding model analysis for an exemplary miniprotein (Sequence No. 6). The black line is fitted by the model. Fig. 5a and Fig. 5b It shows the relative binding of an exemplary miniprotein to B7-H3-expressing cancer cells (cancer cell line) compared to a control (target-invalid line) when measured by flow cytometry. Fig. 5a represents the median fluorescence intensity (MFI) plotted as a function of the concentration of the miniprotein administered to cancer cells. Fig. 5b This shows an overlay of a representative histogram of MFI for cancer cells treated with different concentrations of miniproteins compared to a vehicle control. Fig. 6 silver This shows the internal localization of Cy5-labeled B7-H3-specific miniproteins in B7-H3-expressing cancer cells. Cell nuclei are labeled with DAPI staining, and arrows indicate the localization of Cy5-labeled B7-H3 miniproteins. Fig. 7 represents quantitative absorption in the OK-PTC (Opossum kidney proximal tubule cell) absorption assay. Co-treatment with an exemplary decoy peptide (compound ID number C10) at a 20X molar excess (bar 2, right) reduces the absorption of the biotinylated test agent (compound ID number C8) compared to the biotinylated test agent alone (C8, left). Fig. 8Figure [document] illustrates an analysis generated from SPECT / CT scans to quantify the injection dose (or injection dose) per gram (%ID / g) of kidney tissue in mice. Exemplary B7-H3 charge variant conjugates (compound ID numbers C5, C11 to C14) exhibit reduced kidney retention levels in mouse biodistribution. Fig. 9 Figure [document] illustrates the analysis generated from SPECT / CT scans to quantify the injection dose per gram (%ID / g) of kidney tissue in mice. Co-administration of an exemplary decoy peptide (compound ID number C10) reduces the renal uptake of an exemplary B7-H3-targeting affibody conjugate (compound ID number C5). Fig. 10a and Fig. 10b is an exemplary target-binding scaffold B miniprotein when combined with an exemplary scaffold A decoy in vitro composite This is a graph depicting data showing the reduction in cell absorption. Fig. 10a This is a bar graph showing the absorption percentage (y-axis) of an exemplary decoy (C120) in combination with a 20-fold molar excess (column 2, x-axis) or an exemplary B7-H3-targeting scaffold B miniprotein conjugate (C4) alone / without a decoy (20 μM; column 1, x-axis). Fig. 10b This is a bar graph showing the percentage of absorption (y-axis) of an exemplary decoy (C120) in combination with a 20-fold molar excess (column 2, x-axis) or an exemplary B7-H3-targeting scaffold B miniprotein (C8) alone / without a decoy (20 μM; column 1, x-axis). Error bars represent the standard error of the mean (SEM). Fig. 11This graph depicts data showing the 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 represents the uptake rate (on the y-axis) of the exemplary B7-H3-targeting scaffold B miniprotein conjugate (C8) alone / without a decoy (20 μM; column 1 on the x-axis) or combined with a 20-fold molar excess of one of the three exemplary scaffold A decoys (C118, column 2 on the x-axis; C119, column 3; and C120, column 4). Error bars represent the standard error of the mean (SEM). Fig. 12a and Fig. 12b This is a graph depicting data showing the reduction in cellular uptake of an exemplary scaffold B target-binding miniprotein compound when combined with an exemplary scaffold B decoy in vitro. Fig. 12a This is a bar graph showing the absorption percentage (y-axis) of an exemplary scaffold B decoy (C10) in combination with a 20-fold molar excess (column 2, x-axis) or an exemplary B7-H3-targeted scaffold B miniprotein conjugate (C8) alone / without decoy (20 μM; column 1, x-axis). Fig. 12b This is a bar graph showing the absorption percentage (y-axis) of an exemplary scaffold B decoy (C10) in combination with a 100-fold molar excess (Column 2, x-axis) or an exemplary B7-H3-targeted scaffold B miniprotein conjugate (C117) alone / without decoy (1 μM; Column 1, x-axis). Error bars represent the standard error of the mean (SEM). Fig. 13a and Fig. 13b is a graph showing the results from the analysis of SPECT / CT scans to quantify the in vivo renal (Fig. 13a) or liver (Fig. 13b) absorption and retention of the scaffold or scaffold plus decoy in mice, and 111 In-labeled exemplary B7-H3 targeting scaffold B miniprotein conjugate ( 111In-C5) was injected alone / without a decoy (C5; solid line containing black circles in Figs. 13a and 13b), or co-administered with an exemplary scaffold B decoy (C10; dashed line containing white circles in Figs. 13a and 13b) at an 850-fold molar excess, and is shown as the percentage of injection dose per gram (%ID / g; y-axis) for 4 to 24 hours after injection. Error bars represent the standard deviation (SD). Fig. 14 This is a graph showing the thermal stability of an exemplary compound containing a conjugate with an exemplary restricted B7-H3 binding miniprotein. The percentage of remaining C309 (miniprotein of sequence number 267) (x-axis) was measured over time (y-axis, time (h)) at 75 °C (solid line) and 90 °C (dotted line). Fig. 15 Is This is a graph showing on-cell binding used to measure Ki for an exemplary restricted B7-H3 miniprotein conjugate (C234 having the miniprotein of sequence number 204). FIGS. 16a to 16b is a line graph showing the % ID / g of one of the three miniprotein conjugates (C1B, C235 and C301) between 0 and 48 hours after administration. Fig. 16a %ID / g in the kidney at 1, 4, 24, and 48 hours after administration. Fig. 16b represents %ID / g in the tumor at 1, 4, 24, and 48 hours after administration. FIGS. 17a to 17b is a line graph showing the % ID / g of one of the three miniprotein conjugates (C1B, C235 and C309) between 0 and 48 hours after administration. Fig. 17a %ID / g in the kidney at 1, 4, 24, and 48 hours after administration. Fig. 17b represents %ID / g in the tumor at 1, 4, 24, and 48 hours after administration. FIGS. 18a to 18b is a tumor ( Fig. 18a ) and kidney ( Fig. 18bThis is a bar graph showing %ID / g measured 24 hours after administration of one of several exemplary 111-In labeled linear (C131 and C165) or restricted (C229, C235, C276, C301 and C333) B7-H3 miniprotein conjugates, showing an increase in intratumoral concentration and a decrease in renal concentration for the exemplary restricted B7-H3 miniprotein conjugate compared to the exemplary linear B7-H3 miniprotein conjugate. FIGS. 19a to 19d are on the 17th each ( Fig. 19a , 1915 cells) and 42 days ( Fig. 19c Line graphs of efficacy data measured by tumor volume in low (H1915) and high (H358) B7-H3 expressing cell lines are shown, each evaluated after administration of one of two concentrations (500 or 1000 nCi) of an exemplary 225-Ac labeled restricted B7-H3 miniprotein conjugate (C332) or a single dose of the vehicle. Fig. 19a The corresponding weight for is Fig. 19c Indicated in, Fig. 19b The weight for is Fig. 19d It is represented in. FIGS. 20a to 20b Tumor volume between about 0 and 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 restricted B7-H3 miniprotein conjugate (C331). Fig. 20a ) and body weight( Fig. 20b It shows a line graph of efficacy data measured by ). Specific details for implementing the invention

[0071] In particular, the present disclosure provides a composition and a method of using the same. In some embodiments, the composition selectively binds to a target (e.g., B7-H3). In some embodiments, the composition comprises one or more therapeutic agents (e.g., chelators, radionuclides), and the therapeutic agents are selectively targeted to B7-H3-expressing cells such that B7-H3-expressing cells are treated and cells that do not express B7-H3 are not treated. The present disclosure recognizes that the source of the problem in treating cells expressing a target (e.g., cancer cells) is that traditional therapies are not sufficiently selective to deliver the therapeutic agent in a manner that specifically targets the cells (e.g., tumor cells) and minimizes damage to surrounding cells (e.g., non-tumor cells), e.g., cells surrounding cancer cells and cells in non-target organs (e.g., kidneys). (e.g., as in one or more non-tumor tissues) surrounding cells may also express the target in lower amounts or levels than the target cells. In some of these embodiments, such expression may be undetectable. The present disclosure provides insight that a combination of selective targeting with a specific therapeutic agent, e.g., a chelator and / or a radionuclide (e.g., an alpha emitter) (e.g., by a polypeptide, e.g., a miniprotein, e.g., of B7-H3 as provided herein) offers advantages over previously used therapeutic agents (e.g., antibodies, beta emitters, etc.).

[0072] Furthermore, the present disclosure provides insight that certain problems may still arise even if therapeutic agents (e.g., those disclosed herein) are targeted more specifically. In particular, uptake by a tumor may be challenged by uptake, retention, and / or clearance by one or more non-target (e.g., non-tumor) tissues, e.g., the kidney, resulting in (1) faster clearance; (2) reduced tumor targeting (e.g., due to uptake and / or retention by non-target tissues); and / or (3) damage to the non-target tissue (e.g., kidney). The present disclosure recognizes that some or all of these problems may be mitigated or prevented by (i) with a miniprotein provided herein (e.g., as disclosed in Table 2A and / or 2C) comprising a miniprotein having a specific feature (e.g., as in Table 2C, e.g., pharmaceutical), and / or (ii) by combining the administration of a therapeutic agent with the administration of a decoy (e.g., as presented in Table 2D). In some embodiments, the decoy reduces or prevents the uptake by the kidney of a composition containing a radionuclide. Without wishing to be bound by theory, the present disclosure assumes that the improvement in therapeutic efficacy is at least maintained while reducing damage to the kidney, and in some embodiments, that the risk of harm or actual harm to kidney tissue and / or renal system tissues (e.g., ureter, bladder, etc.) is reduced while the therapeutic efficacy is improved.

[0073] Unless otherwise defined in this specification, scientific and technical terms used in connection with this disclosure shall have the same meaning as commonly understood by those skilled in the art. Additionally, unless otherwise required by the context, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0074] The methods and techniques of this disclosure generally follow ordinary methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification unless otherwise indicated. For example, the literature [Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, NY (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].

[0075] All publications, patents, and other literature cited herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification, including definitions, shall prevail. Materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. Additionally, unless otherwise required by the context, singular terms will include the plural, and plural terms will include the singular. In general, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0077] Throughout this specification and claims, variations of the word 'include', 'comprising', or 'comprising' will be understood to mean the inclusion of a specified integer or group of integers, but not the exclusion of any other integer or group of integers.

[0078] As used herein, ranges and quantities may be expressed as ‘about’ specific values ​​or ranges, for example, ‘about’ one specific value and / or ‘about’ another specific value. ‘About’ also includes exact amounts. Thus, ‘about 100 nucleotides’ means ‘about 100 nucleotides’ and also ‘100 nucleotides’. In a given context, the term ‘about’ as used herein also includes amounts expected to be within experimental error. Where ‘about’ appears before a quantitative value, the present disclosure also includes the specific quantitative value itself unless otherwise specifically stated. In such cases, ‘about’ also refers to a variation of ±10% from the nominal value unless otherwise indicated or inferred. It is understood that when values ​​are expressed as approximations by the use of the preceding word ‘about’, the present disclosure also presupposes embodiments that specify specific values ​​and ranges of values ​​without approximations.

[0079] As used herein, the singular forms 'one', 'one', and 'above' include multiple objects unless otherwise clearly indicated. Accordingly, for example, in some embodiments, the reference to a decoy includes multiple decoys, a single decoy, etc.

[0080] As used herein, with respect to two or more specified objects, the expression 'and / or' includes each specified object and various combinations of two or more specified objects individually, unless otherwise understood in context and usage.

[0081] Unless otherwise indicated, and as an example of all sequences described herein under the general form 'sequence number', 'nucleic acid comprising sequence number 1' refers to a nucleic acid, at least a portion of which has (i) the sequence of sequence number 1 or (ii) a sequence complementary to sequence number 1. The choice between the two depends on the context. For example, if the nucleic acid is used as a probe, the choice between the two depends on the requirement that it is complementary to the target.

[0082] Terms used in this specification ' administration ' refers to providing the composition to a subject or system. Administration to the subject may be by any appropriate route, dose, and / or dose schedule.

[0083] Terms used in this specification ' Affibody ' refers to a subgenus of miniproteins. Affibody is a molecule derived from the Z-domain of Staphylococcus protein A, consisting of three alpha helices with 58 amino acids and a molar mass of approximately 6 kDa. For exemplary details regarding the structure and uses of affibody, see the literature [Orlova, A; Magnusson, M; Eriksson, TL; Nilsson, M; Larsson, B; -Guthenberg, I; , C; Carlsson, J et al.See (2006). "Tumor imaging using a picomolar affinity HER2 binding affibody molecule," Cancer Res. 66 (8): 4339-48. An exemplary Affibody® molecule is commercially available from Abcam Corp., Cambridge, Massachusetts. Affibodies are stable under high temperatures and acidic or alkaline conditions. Target specificity is achieved by the randomization of 13 amino acids located on two alpha helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26).

[0084] Terms used in this specification ' Affinity Maturity ' generally refers to a process in which a successive change to a sequence (e.g., successive mutation) is made and the selection of a polypeptide sequence is performed to select one or more sequences with increased affinity compared to the 'start' sequence or another sequence with less affinity compared to the sequence with greater affinity.

[0085] Terms used in this specification ' amino acid sequence ' and ' polypeptide ' refers to a polymer of amino acids linked by one or more peptide bonds. Polypeptides of this disclosure include both naturally occurring proteins and non-naturally occurring proteins, and any fragments, portions, peptides, mutants, derivatives, and analogs thereof. Polypeptides may be monomeric or polymeric. Additionally, polypeptides may comprise a plurality of different domains, each having one or more distinct activities. Polypeptides may be wholly or partially synthetic or otherwise modified (i.e., include one or more synthetically generated amino acids and / or modifications thereof). The term ' peptide' can be used to refer to a short polypeptide containing fewer than about 70 amino acids (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids).

[0086] As used in this specification, ' compound' refers to a miniprotein having an amino acid sequence. The compound may include a miniprotein having different modifications, e.g., an N-terminal modification or a C-terminal modification. In various embodiments, the 'compound' may include a miniprotein and one or more additional elements, examples of which include a linker, a chelator, and / or a radionuclide. For example, the compound may include a miniprotein conjugated to a chelator and / or radionuclide, e.g., through a linker. As indicated herein, the compound is identified by 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 sequence number) 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 are not limited to, acetyl, acid, or amide (e.g., acetyl, NH2, NH2, OH / COOH) as provided in the exemplary compounds and miniproteins of Table 2A or Table 2C. In some embodiments, polypeptides according to the present disclosure may have various modifications at the N-terminus (e.g., as presented in the exemplary compounds in Table 2A or Table 2C) and may have an acid or amide group at the C-terminus (e.g., see Table 2A or Table 2C). A given polypeptide having a specific amino acid sequence may have one or more N-terminal and / or C-terminal differences without significant changes in the utility or function of the polypeptide, for example, for binding to B7-H3 (e.g., for the detection and / or treatment of cancer).

[0087] As used in this specification, the term 'as used in this specification attenuate'Functional deletion' generally refers to a functional deletion comprising a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of the gene sequence, which reduces or inhibits the production of a gene product or renders the gene product non-functional. In some cases, a functional deletion is described as a knockout mutation. Attenuation also includes changes in the amino acid sequence by altering the nucleic acid sequence, by positioning the gene under the control of a less active promoter, by downregulation, by expressing interfering RNA, ribozymes, or antisense sequences targeting the gene of interest, or through any other technique known in the art. As an example, the sensitivity of a specific enzyme to feedback inhibition or inhibition by a composition other than a product or reactant (pathway-non-specific feedback) is reduced so that enzyme activity is not affected by the presence of the corresponding compound. In other cases, an enzyme modified to be less active may be referred to as attenuated.

[0088] Terms used in this specification ' binder' refers to a subgenus of miniproteins. The binder is characterized by comprising or consisting of a polypeptide (e.g., a peptide) having a known ability to bind and associate with a target or capable of binding. The binder generally comprises a cysteine-containing peptide containing one or more disulfide bonds through some binders that do not contain cysteine ​​residues and / or disulfide bonds. The binder is preferably rapidly removed from circulation when administered systemically to a mammalian subject. As understood, in a given context, a reference to the binder may be its nucleic acid sequence or an amino acid sequence encoding it, or may comprise it. The binder may be provided as a polynucleotide or polypeptide using, for example, a vector, a host cell, etc. and / or any combination of such aspects. The binder may be derived or prepared using any method known to those skilled in the art. For example, in some embodiments, the binder may be recombinant (i.e., produced using recombinant nucleic acid encoding a polypeptide). In some embodiments, the binder is a synthetic (e.g., known to those skilled in the art (e.g., literature [Palomo, J. RSC Adv (See ., 2014, 4, 32658-32672) and can be synthesized using standard solid-phase synthesis methods such as the solid-phase peptide synthesis described in this specification.

[0089] Terms used in this specification ' block'...' refers to preventing, slowing, inhibiting, or otherwise reducing or decreasing the absorption and / or retention of a compound into a tissue (e.g., non-tumor tissue, e.g., kidney tissue, e.g., liver tissue). In some embodiments, the decoy blocks, inhibits, reduces, or otherwise decreases the absorption of the conjugate or compound of the present disclosure into a non-tumor tissue, e.g., kidney tissue. In some embodiments, the decoy reduces the retention of a compound (e.g., radiotherapeutic compound, e.g., miniprotein) in a non-tumor tissue (e.g., kidney, e.g., liver).

[0090] Terms used in this specification ' Chelicator ' refers to any molecule or moiety capable of forming a complex (i.e., a 'chelate') with a metal ion. Chelatators generally have two or more non-covalent electron pairs that can be used to donate to a metal ion. Metal ions are typically coordinated to the chelator by two or more pairs of electrons.

[0091] Terms used in this specification ' bonded ' refers to the combination of two types of compounds or preparations by means of covalent or non-covalent means.

[0092] As used in this specification, ' Conservative amino acid substitution' is the substitution of an amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein. Where two or more amino acid sequences differ from each other due to conservative substitutions, the sequence identity percentage or degree of homology may be upregulated to compensate for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. For example, refer to the literature [Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89] (referenced herein by reference). The following six groups each contain amino acids that are conservative substitutions with respect to 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).

[0093] Terms used in this specification ' Cysteine ​​- dense peptide ' and ' CDP ' is used interchangeably and generally refers to a subgenus of miniproteins comprising two or more independent folding domains and high density of cysteine. In some embodiments, the CDP comprises at least 1, 2, 3, 4, or more cysteine ​​residues within the range of about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues. For example, see pubmed.ncbi.nlm.nih.gov / 29483648 / . In some embodiments, the CDP comprises a restricted distribution of cysteine, Cys-X [0-15] -Cys-X [0-15] -Cys-X [0-15] -Cys-X [0-15] -Cys-X [0-15]It contains -Cys (where X represents any amino acid) (sequence number 553).

[0094] Terms used in this specification ' fruition ' generally refers to the removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, and the regions on both sides are connected together.

[0095] The phrase 'reference nucleic acid sequence' used in this specification degenerate variant ' includes a nucleic acid sequence that can be translated according to the standard genetic code and provide the same amino acid sequence as that translated from a reference nucleic acid sequence. The terms 'degenerate oligonucleotide' or 'degenerate primer' are used to refer to oligonucleotides that can hybridize with target nucleic acid sequences that are homologous to each other within one or more specific segments, although the sequences do not necessarily have to be identical.

[0096] As used herein, the term 'in relation to nucleic acid sequences Derived from 'Refers to a nucleic acid sequence developed using a reference nucleic acid sequence and having at least 85% sequence identity with said reference nucleic acid sequence. With respect to amino acid sequences, the term 'derived from' refers to an amino acid sequence having at least 85% sequence identity with a reference naturally occurring amino acid sequence from which said amino acid sequence is derived. The term 'derived from' as used herein does not imply any specific process or method for obtaining the derived nucleic acid or amino acid sequence. For example, the nucleic acid or amino acid sequence may be chemically synthesized.

[0097] Terms used in this specification ' determined' refers to a process or act requested by a third party (e.g., laboratory, hospital, nurse, physician) to perform or provide results from a test, procedure, experiment, assay, analysis, etc., which defines the presence (or absence) of a given marker from a patient, e.g., a biomarker or gene mutation (e.g., the expression level of B7-H3 in a biological sample, e.g., a sample from a tumor).

[0098] As used in this specification, the term 'as used in this specification' domain A domain refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. A domain may span across the same space as a region or part thereof; a domain may also include distinct, non-contiguous regions of the biomolecule. Examples of protein domains include, but are not limited to, Ig domains, extracellular domains, transmembrane domains, and cytoplasmic domains.

[0099] As used in this specification, the term 'as used in this specification' Expression control sequence'Control sequence' refers to a polynucleotide sequence necessary to influence the expression of an operantly linked coding sequence. An expression control sequence is a sequence that controls the transcription, post-transcriptional events, and translation of a nucleic acid sequence. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, e.g., splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (e.g., ribosome binding sites); sequences that increase protein stability; and sequences that increase protein secretion when desired. The properties of these control sequences vary depending on the host organism; in prokaryotes, these control sequences generally include promoter, ribosome binding site, and transcription termination sequences. The term 'control sequence' is intended to include, at a minimum, all components whose presence is essential for expression, and may also include additional components whose presence is advantageous, e.g., leader sequences and fusion partner sequences.

[0100] Terms used in this specification ' fusion protein' refers to a polypeptide comprising a polypeptide or fragment combined with a heterogeneous amino acid sequence. A fusion protein is useful because it can be configured to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 adjacent amino acids from the polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50, or 60 amino acids, and even more preferably at least 75, 100, or 125 amino acids. A fusion comprising the entire protein of the present disclosure has specific utility. The heterogeneous polypeptide included in the fusion protein of the present disclosure is at least 6 amino acid lengths, often at least 8 amino acid lengths, typically at least 15, 20, and 25 amino acid lengths. A fusion comprising a larger polypeptide, e.g., an IgG Fc region, and even the entire protein, e.g., a green fluorescent protein ('GFP') chromophore-containing protein, has specific utility. A fusion protein can be produced recombinantly by constructing a nucleic acid sequence encoding a polypeptide or a fragment thereof within a frame together 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 cross-linking a polypeptide or a fragment thereof to another protein.

[0101] As used herein, when referring to a protein, if the nucleic acid sequence encoding the protein has a sequence similar to the nucleic acid sequence encoding the second protein, 'for the second protein HomologyThis may exist. Alternatively, if two proteins have 'similar' amino acid sequences, the protein is homologous to the second protein. (Thus, the term 'homologous protein' is defined to mean that two proteins have similar amino acid sequences.) Homology between two regions of amino acid sequences (particularly in relation to predicted structural similarity) can be interpreted as implying similarity in function. Homologous proteins or peptides having non-identical residue positions are often recognized as different by conservative amino acid substitutions.

[0102] As used in this specification, the term ' Same 'Refers to at least two nucleic acid sequences or at least two amino acid sequences or subsequences having the same specified ratio of nucleotides or amino acids when compared and aligned for maximum correspondence when measured using a sequence comparison algorithm or via visual inspection. For sequence comparison, typically one sequence serves as the reference sequence against which the test sequence is compared. The length of the sequence identity comparison may span an extension of any number of nucleotides or amino acids. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and, if necessary, subsequence coordinates and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage of sequence identity for the test sequence(s) relative to the reference sequence based on the specified program parameters. Many algorithms are known in the art. Non-limiting examples of algorithms suitable for determining the percentages of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are, for example, [Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al.It is described in

[1977] Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. Additionally or alternatively, sequences may be compared using FASTA, Gap, or Bestfit, programs from the Wisconsin Package Version 10.0, Genetics Computer Group (GCG) (Madison, Wisconsin). FASTA provides the alignment of the region of best overlap between the query sequence and the search sequence and the percentage of sequence identity. Pearson, Methods Enzymol. 183:63-98 (1990) (the full text is incorporated herein by reference). For example, the percentage of sequence identity may be determined using FASTA with default parameters (word size of 6 and NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, incorporated herein by reference, or using Gap with default parameters.

[0103] Terms used in this specification ' isolatedPolynucleotides or polypeptides are substantially separated from other cellular components associated with natural polynucleotides within natural host cells, e.g., ribosomes, polymerases, and naturally associated genomic sequences. For example, the isolated molecule is, by its origin or source of derivatives, (1) not associated with the naturally associated components in its natural state, or (2) exists in a purity not found in nature, where the purity may be controlled by the presence of other cellular materials (e.g., the absence of other proteins from the same species), (3) expressed by cells from different species, or (4) not occurring in nature (e.g., being a fragment of a naturally occurring polynucleotide or polypeptide, or containing linkages other than standard peptide bonds, or amino acid analogs or derivatives not found in nature). Thus, a polynucleotide or polypeptide that is chemically synthesized, or synthesized in a cell system different from the cell of natural origin, will be 'isolated' from the naturally associated components. Polynucleotides or polypeptides may also be substantially free of naturally associated components by isolation using protein purification techniques well known in the art. As defined as such, 'isolated' does not necessarily require that any molecule described be physically removed from its natural environment. In some embodiments, as used in relation to isolated compositions, 'isolated' means in the absence of pharmaceutically acceptable salts.

[0104] Terms used in this specification ' K D ' or ' Kd' refers to a specific entity and target (e.g., dissociation equilibrium constant for antibody-antigen (or e.g., targeting miniprotein-target protein), e.g., a specific interaction between an entity and its target (e.g., polypeptide-target interaction, e.g., the polypeptide provided herein and B7-H3). Typically, the antibody of this disclosure (e.g., targeting miniprotein, e.g., B7-H3-binding miniprotein) is, when determined using surface plasmon resonance (SPR) technology on a BIACORE instrument, about 10 -7 Less than M, e.g., about 10 -8 M, 10 -9 Less than M or 10 -10 Dissociation equilibrium constant less than or equal to M (K D It binds to B7-H3 via ). K D = k d / k a .

[0105] Terms used in this specification 'Ki' (M) refers to the inhibition constant of binding between a given entity and a target (e.g., a specific polypeptide-target interaction).

[0106] Terms used in this specification 'k d ' (s -1 ) refers to the dissociation rate constant between a given entity and a target (e.g., of a specific polypeptide-target interaction). This value is k off It is also referred to as a value.

[0107] Terms used in this specification 'k a ' (M -1 ×s -1 ) is a given entity and target (e.g., , It refers to the binding rate constant of a specific polypeptide-target interaction. This value is k on It is also referred to as a value.

[0108] Terms used in this specification 'K A ' (M -1 ) refers to the binding equilibrium constant between a given entity and a target (e.g., a specific polypeptide-target interaction). K A =k a / k d .

[0109] The affinity of molecule X for its target Y is the dissociation equilibrium constant (K D It can be expressed by ). The kinetic components contributing to the dissociation equilibrium constant are as described above. For clarity, as is known in the art, the smaller K D While the value indicates a higher affinity interaction, a larger K D The value indicates a lower affinity interaction. Affinity refers to those described herein, e.g., surface plasmon resonance (SPR) techniques (e.g., BIACORE). ® ) or biolayer interferometer (e.g., FORTEBIO ® It can be measured by conventional methods known in the art, including ).

[0110] Terms used in this specification ' knockout ' generally refers to a gene whose level of expression or activity has been reduced to zero. In some examples, a gene is knocked out through the deletion of part or all of its coding sequence. In other examples, a gene is knocked out by introducing one or more nucleotides into its open reading frame, resulting in the translation of nonsense or other non-functional protein products.

[0111] Terms as used in this specification ' Knottin ' refers to a structural motif of a miniprotein containing three disulfide bridges.

[0112] Terms as used in this specification ' Notine peptide' refers to a subgenus of miniproteins containing at least one notine.

[0113] As used in this specification, the term ' Linker ' refers to the moiety used to conjugate miniproteins to a chelator.

[0114] Terms used in this specification ' miniproteins ' refers to a short protein of 100 or fewer amino acids having a well-defined fold including two or more secondary structural elements, an isolated hydrophobic core, and / or a cooperative fold. Affibodies, CDPs, notins, and binders as disclosed herein are all examples of miniproteins.

[0115] As used herein, the term 'in relation to nucleic acid sequences strain ' refers to a nucleic acid sequence comprising at least one substitution, alteration, inversion, addition, or deletion of a nucleotide relative to a reference nucleic acid sequence. As used herein, with respect to an amino acid sequence, the term 'modification' refers to an amino acid sequence comprising at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue relative to a reference amino acid sequence.

[0116] Terms used in this specification ' modified derivative' refers to a polypeptide or a fragment thereof that is substantially homologous in its primary structural sequence but includes, for example, chemical and biochemical modifications in vivo or in vitro, or contains amino acids not found in the natural polypeptide. Such modifications include, for example, as readily understood by those skilled in the art, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling using radionuclides, for example, and various enzymatic modifications. Various methods of substituents or labels useful for labeling polypeptides and for such purposes are well known in the art and include radioisotopes such as 125I, 32P, 35S, and 3H, ligands that bind to a labeled antiligand (e.g., antibody), fluorescent dyes, chemiluminescent agents, enzymes, and antiligands that can act as members of a specific binding pair with the labeled ligand. The choice of label depends on the required sensitivity, ease of conjugation with primers, stability requirements, and available instruments. Methods for targeting polypeptides are well known in the art. For example, the literature [Ausubel et al. [See , Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002)] (refer to by reference in this specification).

[0117] Terms used in this specification ' molecule ' means any compound including, but not limited to, miniproteins, small molecules, peptides, proteins, sugars, nucleotides, nucleic acids, lipids, etc., and such molecules (e.g., miniproteins, compounds, etc.) may be natural or synthetic or a combination of natural and synthetic.

[0118] Terms used in this specification ' Monobody ' or ' Adnectin' is used interchangeably and refers to a subgenus of miniproteins. The monobody is a molecule based on the 10th extracellular domain (10Fn3) of human fibronectin III, which preferably adopts an Ig-like β-sandwich fold of 94 residues having 2 to 3 exposed loops but lacks a central disulfide bridge (Gebauer and Skerra (2009) Curr Opinion in Chemical Biology 13:245-255). Adnectin with desired target specificity can be genetically engineered by introducing modifications to specific loops of the protein.

[0119] Terms used in this specification ' Mutane ' or ' Mutant protein ' or ' variant ' refers to a protein comprising an amino acid sequence having at least one variation (e.g., insertion, deletion, or substitution that may be conservative or non-conservative) compared to a reference sequence. When applied to a sequence (e.g., nucleic acid sequence, amino acid sequence), ' mutated ' implies that nucleotides within a nucleic acid sequence or amino acids within an amino acid sequence may be inserted, deleted, or altered relative to a reference sequence. A single alteration may occur at a single locus (point mutation), or multiple nucleotides or amino acids may be inserted, deleted, or altered at a single locus. Additionally, one or more alterations may occur at any number of loci within the nucleic acid or amino acid sequence. The nucleic acid or amino acid sequence may be subject to mutagenic techniques, e.g., 'error-induced PCR' (a process of performing PCR under conditions where the copy fidelity of the DNA polymerase is low, resulting in a high proportion of point mutations along the entire length of the PCR product); e.g., literature [Leung et al.[See , Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)]); 'oligonucleotide-designated mutagenesis' (a process that enables the generation of site-specific mutations in any cloned DNA segment of interest; e.g., see literature [Reidhaar-Olson and Sauer, Science 241:53-57 (1988)]); designation evolution (e.g., exposing a polypeptide to a different set of conditions that cause the generation of different polypeptides having one or more amino acid changes that may or may not confer greater fitness to the polypeptide); and site-designated mutagenesis (e.g., specific designation changes in a sequence) may be mutated by any method including, but not limited to, these.

[0120] Terms used in this specification ' polypeptide mutant ' or ' Mutane'Mutein' refers to a polypeptide having a sequence comprising one or more insertions, duplications, deletions, rearrangements, or substitutions of amino acids compared to the amino acid sequence of a natural or wild-type protein. The mutain may have one or more amino acid point substitutions in which a single amino acid at one position is changed to another amino acid, one or more insertions and / or deletions in which one or more amino acids are inserted or deleted, respectively, from the sequence of the naturally occurring protein, and / or cleavages of the amino acid sequence at either or both of the amino or carboxy terminus. The mutain may be identical but preferably has different biological activity compared to the naturally occurring protein. The mutain has at least 85% total sequence homology with its wild-type counterpart. A mutain having at least 90% total sequence homology with the wild-type protein is more preferred. In a more preferred embodiment, the mutain exhibits at least 95% sequence identity, more preferably 98%, even more preferably 99%, and even more preferably 99.9% total sequence identity. Sequence homology can be measured by any conventional sequence analysis algorithm such as Gap or Bestfit. Amino acid substitutions may include (1) reducing susceptibility to protein degradation, (2) reducing susceptibility to oxidation, (3) changing binding affinity for protein complex formation, (4) changing binding affinity or enzyme activity, and (5) conferring or modifying other physicochemical or functional properties of such analogs.

[0121] Terms used in this specification ' Non-disulfide sequence ' refers to an amino acid sequence encoding a polypeptide that does not contain more than one cysteine ​​residue and / or disulfide bond in its folded active form. For example, in some embodiments, the miniprotein may include or be composed of a non-disulfide sequence.

[0122] Terms used in this specification ' Non-peptide analogues ' refers to a compound having properties similar to those of a reference polypeptide. Nonpeptide compounds may also be referred to as 'peptide mimics' or 'peptide imitators'. For example, the literature [J. 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., WH 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 the references cited in each of the above, which are incorporated herein by reference. These compounds are often developed with the help of computerized molecular modeling. Peptide mimics structurally similar to the useful peptides of this disclosure may be used to produce equivalent effects and are therefore conceived as part of this disclosure.

[0123] Terms used in this specification ' nucleic acid sequence ' and ' polynucleotideThe term is used interchangeably and refers to a polymer of nucleotides. This term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as non-natural nucleotide analogs, DNA or RNA analogs containing non-natural nucleoside linkages or both. Nucleic acids may have any topological conformation. For example, nucleic acids may be single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin-shaped, circular, or lock-shaped. Nucleic acid sequences may contain natural, non-natural, or modified nucleotides; and may contain natural, non-natural, or modified nucleotide linkages, e.g., phosphoramidate linkages or phosphorothioate linkages, instead of phosphodiesters found between nucleotides in unmodified nucleic acid sequences. Nucleic acid sequences include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, such as recombinant means, e.g., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and polymerase chain reaction, etc., and by synthetic means. The polynucleotides of this disclosure may include RNA, cDNA, sense and antisense strands of genomic DNA, and synthetic forms and mixed polymers thereof. They may be chemically or biochemically modified or may include non-natural or derivatized nucleotide bases, which will be readily understood by those skilled in the art.These modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, e.g., uncharged linkages (e.g., methylphosphonate, phospholith ester, phosphoramidate, carbamate, etc.), charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), pendant moiety (e.g., polypeptide), intercalation (e.g., acridine, psoralen, etc.), chelators, alkylation, and modified linkages (e.g., alpha-anomer nucleic acid, etc.). Additionally, synthetic molecules that mimic polynucleotides in their ability to bind to a specified sequence through hydrogen bonding and other chemical interactions are included. Such molecules are known in the art and include, for example, molecules in which peptide linkages replace phosphate linkages within the molecular backbone. Other modifications may include analogs containing modifications found in ribose rings, for example, in bridge moiety or other structures, such as 'lock' nucleic acids.

[0124] Terms used in this specification ' Operablely connected ' or ' Operablely connected An expression control sequence refers to a linkage that regulates the gene of interest by being adjacent to the gene of interest, and refers to an expression control sequence that controls the gene of interest by acting trans- or at a distance.

[0125] Terms used in this specification ' polypeptide fragment'As used herein,' refers to a polypeptide having deletions, e.g., amino-terminal and / or carboxy-terminal deletions relative to the full-length polypeptide. In a preferred embodiment, the polypeptide fragment is an adjacent sequence in which the amino acid sequence of the fragment is identical to a naturally occurring or corresponding position within the parent sequence. The fragment is typically at least 5, 6, 7, 8, 9, or 10 amino acid lengths, preferably at least 12, 14, 16, or 18 amino acid lengths, more preferably at least 20 amino acid lengths, even more preferably at least 25, 30, 35, 40, or 45 amino acids, even more preferably at least 50 or 60 amino acid lengths, and even more preferably at least 70 amino acid lengths.

[0126] Terms used in this specification ' radioactive nuclides ' refers to an atom capable of undergoing radioactive decay.

[0127] Terms used in this specification ' radiotherapeutic agents ' refers to a radionuclide-labeled miniprotein or compound as provided herein, comprising a radionuclide. The radiotherapeutic agent may be administered to a subject, e.g., a test subject (e.g., a mouse or rat, e.g., a non-human primate, e.g., a healthy volunteer) and / or a subject requiring radiotherapy, e.g., a subject having cancer.

[0128] Terms used in this specification ' recombination'Recombines' refers to (1) removed from a naturally occurring environment, (2) a gene that is not associated with all or part of a polynucleotide found in nature, (3) operably linked to a polynucleotide that is not linked in nature, and / or (4) a biomolecule that does not occur in nature, e.g., a gene or a protein. The term 'recombination' may be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by a heterologous system, as well as proteins and / or mRNA encoded by such nucleic acids. As used herein, an endogenous nucleic acid sequence (or protein product encoded by such sequence) within the genome of an organism is considered 'recombined' in this specification when a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence and the expression of such endogenous nucleic acid sequence is altered. In this context, a heterogeneous sequence is a sequence that is not naturally adjacent to an endogenous nucleic acid sequence, whether the heterogeneous sequence itself is endogenous (originating from the same host cell or its offspring) or exogenous (originating from a different host cell or its offspring). For example, a promoter sequence may be substituted for the gene's native promoter in the host cell's genome (e.g., by homologous recombination), and thus the gene has an altered expression pattern. Because this gene is separated from at least some of its naturally adjacent sequences, it will now be considered 'recombined'. Nucleic acids are also considered 'recombined' if they contain any modifications that do not naturally occur in the corresponding nucleic acid in the genome. For example, an endogenous coding sequence is considered 'recombined' if it contains insertions, deletions, or point mutations introduced artificially, for example, by human intervention. 'Recombined nucleic acids' also include nucleic acids incorporated into the host cell chromosome from a heterogeneous site and nucleic acid constructs existing as episomes.

[0129] Terms used in this specification ' 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 this term is intended to refer not only to specific target cells but also to the offspring of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such offspring may not actually be identical to the parent cells, but are 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 a culture, or a cell present in a living tissue or organism."

[0130] Terms used in this specification ' area 'Region' refers to a physically adjacent portion of the primary structure of a biomolecule as used herein. In the case of a protein, the region is defined by an adjacent portion of the amino acid sequence of the corresponding protein.

[0131] Phrase ' used in this specification Secondary structural elements ' refers to a locally folded structure formed within a polypeptide due to interactions between atoms of its backbone. Examples of secondary structural elements may include alpha helices, beta sheets, 310 helices, pi helices, and random coils. The miniproteins of the present disclosure have these secondary structures (e.g., It may include any one or more of one or more alpha helices, one or more alpha helices, and one or more beta sheets. It will be understood by those skilled in the art that secondary structural elements may be connected by loop regions that may or may not be modified to change the interaction of secondary structural elements of the polypeptide. As understood by those skilled in the art, in some embodiments, the loop may be a secondary structural element. In some embodiments, the loop may be an interstructural element that is not necessarily considered a secondary structural element.

[0132] As used in this specification, ' Sequence identity percentage Regarding polypeptides also referred to as ' Sequence homology ' is typically measured using sequence analysis software. For example, refer to the Sequence Analysis Software Package of the Genetics Computer Group (GCG) at the University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin, 53705. Protein analysis software matches similar sequences using measurements of homology assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG includes programs, such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, e.g., homologous polypeptides from different species of organisms, or between a wild-type protein and its mutates. For example, refer to GCG Version 6.1. When comparing a specific polypeptide sequence with a database containing multiple sequences from different organisms, a preferred algorithm 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)), 특히 blastp 또는 tblastn(Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The preferred parameters for BLASTp are Expected Value: 10 (default); Filter: seg (default); Gap Opening Cost: 11 (default); Gap Extension Cost: 1 (default); Max Alignment: 100 (default); Word Size: 11 (default); Description Number: 100 (default); Penalty Matrix: BLOSUM62. The length of the polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, typically at least about 20 residues, more typically at least about 24 residues, typically at least about 28 residues, preferably more than about 35 residues. When searching a database containing sequences from many different organisms, it is desirable to compare amino acid sequences. Database searches using amino acid sequences may be measured by algorithms other than blastp known in the art. For example, polypeptide sequences may be compared using FASTA, a program within GCG Version 6.1. FASTA provides alignment of the region of best overlap between the query sequence and the search sequence and the percentage of sequence identity. Pearson, Methods Enzymol. 183:63-98 (1990) (referenced by reference herein). For example, the percentage of sequence identity between amino acid sequences may be determined using FASTA with default parameters (word size 2 and PAM250 scoring matrix) as provided in GCG Version 6.1, referenced by reference herein.

[0133] Terms used in this specification ' specific activity' generally refers to the activity per unit (e.g., mass, e.g., mole) of a radionuclide. Units of inactivity may include megabecquerel units per microgram (MBq / μg), microcurie units per microgram (μCi / μg), microcurie units per nanomolar (μCi / nmol), etc.

[0134] Terms used in this specification ' Specificity 'Generally refers to a sequence (e.g., of a protein, e.g., a miniprotein, e.g., a miniprotein having a specific amino acid) that can selectively bind to a specific target (e.g., an antigen such as one expressed on a tumor, e.g., B7-H3, e.g., a specific cell type, e.g., kidney cells, e.g., renal proximal tubular cells, etc.) or bind 'specifically' when in a form that can bind generally.

[0135] As used in this specification, ' It binds specifically.'Specific binding' means that the binding of a polynucleotide, polypeptide, or protein to a specified antigen (e.g., target) is selective and distinguishable from unwanted or non-specific interactions. The characteristics of 'specific binding' include not only the saturation of binding to the target but also the verifiable ability of that binding to be competed for by the introduction of additional specific binding molecules to the same target. For example, the ability of a protein (e.g., a cysteine-tight peptide) to bind to a specific epitope can be measured by techniques familiar to those skilled in the art, such as enzyme-linked immunosorbent assays (ELISA) or surface plasmon resonance. Between two molecules (e.g., entities such as miniproteins), 'specific binding' refers to the ability of the two molecules to bind to each other preferentially over binding to other molecules in the environment. Typically, 'specific binding' distinguishes itself from accidental binding in a reaction by at least twofold, more typically at least tenfold, often at least one hundredfold, or even one thousandfold. Typically, the affinity or binding strength of a specific binding reaction, as quantified by the dissociation constant, is about 10⁻⁷ M or greater (e.g., about 10⁻⁸ M, 10⁻⁹ M, or even greater). Specific binding requires specificity of a specific first entity (e.g., polypeptide) to a specific second entity (e.g., antigen-binding sequence).

[0136] As used herein in the context of nucleic acid hybridization experiments, ' Strict hybridization conditions ' and ' Strict cleaning conditions' depends on a number of different physical parameters. As will be readily understood by those skilled in the art, nucleic acid hybridization will be influenced by conditions such as salt concentration, temperature, solvent, base composition of the hybridization species, length of the complementary region, and the number of nucleotide base mismatches between the hybridized nucleic acids. Those skilled in the art know how to achieve a specific degree of hybridization by changing these parameters. Generally, 'strict hybridization' is performed at a temperature about 25 °C below the thermal melting point (Tm) for a specific DNA hybrid under a specific set of conditions. 'Strict washing' is performed at a temperature about 5 °C below the Tm for a specific DNA hybrid under a specific set of conditions. Tm is the temperature at which 50% of the target sequence hybridizes to a probe that is perfectly matched. Literature [Sambrook] by reference incorporated herein et al. Refer to [Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), page 9.51]. For the purposes of this specification, 'strict conditions' are defined for solution-phase hybridization as aqueous hybridization (i.e., formamide-free) of 6xSSC (wherein 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS for 8 to 12 hours at 65 °C, followed by two washes with 0.2xSSC and 0.1% SDS for 20 minutes at 65 °C. It will be understood by a skilled operator that hybridization at 65 °C will occur at different rates depending on a number of factors, including the length and percent identity of the sequence being hybridized.

[0137] Terms used in this specification ' synthesis' is used to refer to an entity that is laboratory-generated and is not naturally generated or isolated from a naturally occurring source without modification. Recombinant polymers, e.g., recombinant polynucleotides or polypeptides, may be synthetic. Synthetic polymers, such as polynucleotides or polypeptides, may be produced by any method known to those skilled in the art, including but not limited to solid-phase synthesis, solution-phase synthesis, and biological synthesis, e.g., by host cells, etc.

[0138] Terms used in this specification ' object ' is a mammal. The subject may be a human or non-human mammal. In a given context, the subject may be interchangeably used as a patient, individual, donor, etc. In some embodiments, the subject is a healthy subject without a disease intended for treatment with the composition of the present disclosure (e.g., a healthy volunteer to whom one or more of the compositions provided herein are administered). In some embodiments, the subject is a subject suspected of or diagnosed with a disease, disorder, or pathological condition as provided herein, e.g., cancer and / or tumor. In some such embodiments, such a subject is considered for treatment with the composition of the present disclosure. In some embodiments, the analysis of results achieved by the technology disclosed herein is evaluated in a group comprising a plurality of subjects.

[0139] As used herein, when referring to polynucleotides or polypeptides, the term ' Substantial homology ' or ' Substantial similarity' indicates that when optimally aligned by appropriate nucleotide or amino acid insertion or deletion by another reference molecule (or its complementary strand, where appropriate), sequence identity exists in at least about 70%, 75%, 80%, 85%, preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% or more of nucleic acid amino acid residues when measured by any well-known algorithm of sequence identity, e.g., FASTA, BLAST, Gap, etc. Alternatively or additionally, substantial homology or similarity exists, for example, when a nucleic acid or a fragment thereof is hybridized to another nucleic acid, a strand of another nucleic acid, or its complementary strand under strict hybridization conditions.

[0140] Terms used in this specification ' Target ' refers to a protein or a functional part or variant thereof. The target is another protein (e.g., , It is a protein designed to bind to a miniprotein. The target may be a binding region, e.g., an epitope, to which the miniprotein of the present disclosure (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) binds, or may include such a region. Additionally, the term ' antigen' refers to a protein to which a polypeptide (e.g., a miniprotein, etc.) or a variant thereof is bound, or a functional part or variant thereof. The target may be an antigen or may include it. The target may be expressed on the surface of a specific cell ('target cell') or within a cell within a population of cells (e.g., on the surface of the cell). The target may have a certain percentage of identity with respect to a reference protein and may also be referred to as a target by a specific designation (e.g., B7-H3). In certain embodiments that will become clear from the context, the target may also refer to a protein in a pathway related to another protein. For example, if the target is B7-H3, the target may also be a protein in a pathway required for B7-H3 activity. The target may be or may include a binding region, e.g., an epitope, to which the miniprotein of the present disclosure (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) is bound. In certain embodiments that will become apparent from the given context, the target may be a specific cell type characterized by the expression of a specific surface entity, such as a receptor (e.g., cells within a tissue, e.g., proximal tubular cells within a kidney) (or may be limited to a specific cell type). Such a target may be different from or identical to the target designed to be bound to the miniprotein (M); and in some embodiments, the target (e.g., non-tumor cells, e.g., kidney cells, liver cells, etc.) is bound by a decoy rather than by a polypeptide (e.g., miniprotein) of the composition provided herein (e.g., radiotherapeutic composition).

[0141] As used herein, the term '(as well as 'to treat' or 'treating') therapy' refers to the partial or complete alleviation, improvement, reduction, prevention, reduction of risk of onset, relief, inhibition, delay of onset, reduction of severity, reduction of frequency or incidence of one or more causes, characteristics and / or symptoms of a specific disease, disorder and / or condition or related matters.

[0142] As used in this specification, the term 'as used in this specification' vector ' is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. One type of vector is a 'plasmid,' which generally refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated, but also includes linear double-stranded molecules such as those produced from amplification by polymerase chain reaction (PCR) or by treating a circular plasmid with restriction enzymes. Other vectors include cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, to which additional DNA segments can be ligated into the viral genome (discussed in more detail below). A specific vector (e.g., a vector having a replication origin that functions in a host cell) can replicate autonomously in the host cell into which it is introduced. Other vectors can be incorporated into the host cell's genome upon introduction into the host cell and thus replicate along with the host genome. Furthermore, a specific desirable vector can direct the expression of an operantly linked gene. These vectors are referred to herein as 'recombinant expression vectors' (or simply 'expression vectors').

[0143] Terms used in this specification ' Decharged molecules ' refers to a modified molecule that has a smaller positive charge or polar characteristics compared to the parent molecule, has a larger negative charge or / or both.

[0144] Terms used in this specification ' surface charge' refers to an electrostatic charge present on the surface of a protein (e.g., a miniprotein). In various embodiments, the surface charge of the miniprotein may affect the elongation absorption of the miniprotein (e.g., may increase or decrease the elongation absorption of the miniprotein).

[0145] Terms used in this specification ' Surface patch ' refers to a region on the surface of a protein (e.g., a miniprotein) having surface characteristics. For example, a surface patch may be defined by surface charge and / or surface hydrophobicity that may affect the elongation uptake of the protein (e.g., a miniprotein).

[0146] Terms used in this specification ' Cutable linker ' refers to a cleavable linker. A cleavable linker contains a cleavable bond that is cleaved in vivo, for example, by acidic pH (pH less than 7, typically about 4 to 6), by glutathione, or by up-regulation by an enzyme such as a substrate protease or peptidase from the proximal tubule. Examples of cleavable linkers are linkers containing a hydrazine or disulfide bond or an enzymatically cleavable peptide sequence.

[0147] Terms used in this specification ' scaffold' refers to a specific set of structural features of a given protein (e.g., miniprotein, e.g., binder, e.g., decoy peptide). A scaffold is also used to describe a miniprotein that shares a general set of structural features (e.g., a specific constraint, secondary structure, tertiary structure, etc.). Any individual scaffold may comprise varying amounts of alpha helices, turns, and / or beta sheets, e.g., all alpha helice proteins ('a'), all beta sheet proteins ('b'), combined alpha helice / beta sheet proteins ('a / b'), combined alpha and beta proteins ('a+b'), and small proteins. Examples and features of specific scaffolds are provided herein. However, to provide an example, the scaffold may be an 'affibody' scaffold as provided herein, and the affibody may be a target-bonding affibody (e.g., B7-H3 bond) or an affibody decoy, wherein each entity has an affibody scaffold comprising, for example, specific secondary structural features (e.g., alpha helices and / or beta sheets, disulfide bridges, particularly, coherent cysteine ​​residues, etc.). The scaffolds of the present disclosure may be characterized as types 'Scaffold A' and 'Scaffold B'. An exemplary scaffold A decoy comprises compounds C118 to C120. An exemplary scaffold B decoy has an affibody scaffold and comprises compound C10; The B7-H3-targeting polypeptide of the present disclosure is also a scaffold B affibody, e.g., C1 to C9, C11 to C117.

[0148] Terms used in this specification ' Decoy peptide ' or ' decoy' refers to a molecule specifically designed to mimic the role of a specific receptor protein and to interact with a specific target entity. A decoy peptide or one such type of decoy is a subgenus of miniproteins specifically designed to (i) reduce the accumulation of a compound (e.g., miniproteins, e.g., radiolabeled miniproteins, e.g., radiotherapeutic agents as provided herein) in non-tumor tissues (e.g., kidney tissue or liver tissue where the tumor is located elsewhere); (ii) have no substantial effect on the uptake of the compound by a tumor (e.g., a target, e.g., a tumor expressing B7-H3); and (iii) reduce adverse (e.g., toxic) accumulation in a non-tumor-containing organ of the subject (e.g., kidney, etc.). However, to provide an example, an exemplary decoy may be combined with the composition of the present disclosure (e.g., including a miniprotein and a radionuclide, e.g., a radiotherapeutic agent) to block the composition from kidney tissue compared to uptake and / or retention in kidney tissue in the absence of the decoy peptide. Without wishing to be bound by theory, the present disclosure describes, in some embodiments, a decoy peptide (or decoy) that decoys a composition such as a radiotherapeutic agent, which means that the presence of the decoy in non-tumor tissue blocks the uptake and / or retention of the radiotherapeutic agent into non-tumor tissue (e.g., kidney, e.g., liver). For clarity, when a decoy peptide is referred to as 'decoying' a composition, for example, a compound, for example, a miniprotein that binds to a target (e.g., B7-H3), the decoy does not act on the composition (or compound or miniprotein), but rather acts on itself, and will still be present in non-tumor tissue (e.g., kidney, e.g., liver) when administered alone, for example, even in the absence of the composition being decoyed.

[0149] composition

[0150] Novel compositions comprising one or more of a polypeptide, a linker, a chelator, and / or a radionuclide are provided herein. In some embodiments, the composition comprises a linker and a chelator. In some of these embodiments, the composition is metallized (e.g., in the form of an elementally labeled cold metal as provided herein). In some embodiments, the composition is radiolabeled (e.g., with a radionuclide as provided herein). In some embodiments, the composition comprises a linker, a chelator, and a radionuclide. In some embodiments, the composition comprises or consists of a polypeptide (i.e., a miniprotein), a selective linker, and a chelator and / or radionuclide. In some embodiments, the chelator and / or radionuclide is conjugated to the miniprotein through the linker. In some embodiments, the miniprotein of the disclosure comprises or consists of an affibody, CDP, notin, and / or a binder. In some embodiments, the miniprotein comprises or consists of an affibody. In some embodiments, the miniprotein comprises or consists of CDP. In some of these embodiments, the miniprotein comprises or consists of notin. In some of these embodiments, the miniprotein comprises or consists of a binder. In some of these embodiments, the miniprotein (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) is designed to be linked to one or more other components. For example, in some embodiments, the miniprotein (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) may be linked (conjugated) to another component, such as a chelator and / or a radionuclide. In some embodiments, the radionuclide of the disclosure is an alpha emitter.In some of these embodiments, a chelator and / or radionuclide is conjugated to a miniprotein through a linker.

[0151] While not wishing to be bound by any particular theory, the present disclosure assumes that the compositions of the present disclosure are more effective than previously described compositions (e.g., those comprising antibodies and / or beta-emitted radionuclides). For example, miniproteins (to be used in compositions such as those provided herein) possess some of the main characteristics of antibody-based therapeutics (e.g., affinity, potency, specificity, and the ability to interfere with protein-protein interactions), but can avoid undesirable limitations such as, for example, large size, expensive manufacturing, and the need for chimerization or humanization. For example, in some embodiments, the miniproteins of the present disclosure (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) are about 100 amino acids or less in length. In some embodiments, these miniproteins (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) may be or comprise cysteine ​​dense peptides. In some embodiments, the miniproteins (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) comprise one or more disulfide bridges. In some embodiments, the miniprotein (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) comprises a number of cysteine ​​residues that crosslink to maintain a very stable folded state for a peptide of its length (e.g., compared to a peptide of the same length that does not have many cysteine ​​residues).

[0152] In particular, the present disclosure assumes that, in some embodiments, the miniprotein does not contain multiple cysteine ​​residues, such as a miniprotein containing a single cysteine ​​residue. In some such embodiments, the miniprotein may form a dimer, such as another miniprotein (e.g., auto-dimerization). In certain embodiments, the miniprotein (e.g., binding to B7-H3) contains at least two cysteine ​​residues. In some embodiments, the miniprotein contains at least four cysteine ​​residues. In certain embodiments, where the miniprotein contains at least two or at least four cysteine ​​residues, the miniprotein also contains a disulfide bridge between pairs of cysteine ​​residues. In certain embodiments, the miniprotein has at least one or two disulfide bridges. In some embodiments, the miniprotein contains three disulfide bridges (and at least six cysteine ​​residues). In certain embodiments, the compound comprises more than one type of compound (e.g., disulfide bridges, lactam bridges, alkyl stapled bridges, and any combination thereof). For example, in some embodiments, the present disclosure provides a polypeptide comprising at least one or two disulfide bridges and lactam bridges. Without being bound by theory, the present disclosure assumes that stability imparted by cross-linked cysteine ​​contributes to reduced immunogenicity of the miniprotein or compositions comprising such miniprotein. In some embodiments, such stability may also impart resistance to harsher conditions (e.g., high temperature, low pH incubation, etc.) that provide efficient chelation while maintaining biological activity (e.g., ability to bind to a target) and drug-like molar chemical properties (e.g., stability, solubility, monomericity).

[0153] In some embodiments, the miniproteins provided herein function as targeting moiety that specifically binds to targets expressed on the surface of tumor cells, for example. In some of these embodiments, the miniproteins are designed to be conjugated to one or more additional components. For example, the miniproteins of this disclosure may be formulated to be combined with other components, such as therapeutic molecules (e.g., chelator compositions and / or radionuclides) and / or detectable agents (e.g., visualizable agents, e.g., metabolizable and visualizable agents, etc.). In some of these embodiments, these miniproteins conjugated to one or more additional components may be used for the diagnosis, prognosis, monitoring, and / or treatment of one or more diseases, disorders, or conditions, such as having the expression of specific targets on a specific population of cells.

[0154] In some embodiments, miniproteins (e.g., affibodies, CDPs, notin, binders, engineered Kunitz domains, monobodies, anticalins, engineered ankyrin repeat domains (DARPin), avimers) have lower immunogenicity compared to larger proteins. In some of these embodiments, lower immunogenicity increases tolerance to harsher environmental conditions (e.g., high temperature and low pH incubation) while maintaining biological activity. Thus, in some embodiments, a conjugate containing a miniprotein has lower immunogenicity than a composition containing a larger protein or a different targeting moiety (i.e., other than the miniprotein).

[0155] In some embodiments, a composition comprising a linker, a chelator, and / or a radionuclide can efficiently penetrate a tumor.

[0156] In some embodiments, the miniprotein (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) has superior penetration efficiency compared to larger proteins. That is, in some embodiments, the miniprotein or a composition containing the miniprotein can penetrate solid tumors better than a larger protein or composition containing a protein larger than the miniprotein. For example, in some embodiments, the binder has superior tumor penetration efficiency with a hydrodynamic radius of about 1 nm to 25 nm. In some embodiments, the hydrodynamic radius is about 1 nm to 5 nm. In some embodiments, the hydrodynamic radius is about 1 nm to 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.

[0157] As described herein, a miniprotein (e.g., affibody, CDP, notin, binder) is conjugated to a chelator. In some embodiments, the chelator binds to a radionuclide (e.g., an alpha-emitting radionuclide, e.g., actinium). In some of these embodiments, the radionuclide conjugate combines the specific binding ability and characteristics of a miniprotein (e.g., affibody, CDP, notin, binder) having a radionuclide. That is, without being bound by any particular theory, the present disclosure provides, in some embodiments, a conjugate targeting a radioisotope in which a miniprotein (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) is conjugated to a cell expressing a target. In some embodiments, the target is expressed on the surface of the cell. In some embodiments, the target is B7-H3. In some embodiments, the cell is a tumor cell. In some embodiments, the zygote binds to B7-H3 on the surface of the tumor cell. In some of these embodiments, the radionuclide is targeted to the tumor cell. In some embodiments, the radionuclide is an alpha-emitting radionuclide and, when internalized, serves to specifically target the tumor cell (e.g., without damaging surrounding tissue / cells).

[0158] Target

[0159] Any cell expressing a target can be targeted by miniproteins as provided herein (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer).

[0160] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is derived from a cell line. In some embodiments, the cell is a primary cell. In some embodiments, the primary cell is derived from a sample from a subject, such as from a tumor or from a corresponding tissue without a tumor (e.g., from another area of ​​an organ or from a healthy donor). In some embodiments, the cell is in vitro (e.g., primary cell, cell line, etc.). In some embodiments, the cell is in vivo (e.g., within a subject, e.g., within a human subject, e.g., within a tumor of a human subject). In some embodiments, the cell expresses a target (e.g., through recombinant technology) or is intended to express it. In some embodiments, the target is expressed on the surface of the cell. In some embodiments, the cell is contacted by a composition that binds to the target expressed on the surface. In some embodiments, upon binding (e.g., upon binding of a miniprotein provided by the present disclosure), the target and any bound protein and / or payload are internalized into the cell. In some embodiments, the cell is killed by the payload (e.g., radionuclides and / or chelators, etc.) after internalization.

[0161] In some embodiments, the target is a protein or a part thereof that is upregulated or overexpressed on cancer cells compared to non-cancer cells. That is, in some embodiments, the target is expressed or overexpressed in the tumor or tumor microenvironment compared to the level of the target in non-diseased tissue (e.g., a tumor or tissue without a tumor microenvironment). In some of these embodiments, the target is not detectable or is not present in non-diseased (e.g., healthy) tissue. In some embodiments, the target is a biomarker for cancer (e.g., for cancer cells, for tumors).

[0162] In some embodiments, the target may be associated with a protein, for example, in a pathway activated or acted upon by another protein. For example, in some embodiments, the protein may be expressed on the surface of a cancer cell, and the target may be a pathway in which the surface-cell protein acts. In some embodiments, the protein may be expressed on a cancer cell, and the target may be a protein on a different cell that causes the cancer cell to proliferate or otherwise become resistant to treatment. In some embodiments, the tumor-associated cell surface molecule or the tumor-specific cell surface molecule may be targeted by a miniprotein or a composition comprising a miniprotein as provided herein.

[0163] In some embodiments, the miniprotein or the composition containing the miniprotein specifically binds to a target (e.g., B7-H3) expressed on the surface of a cell. In some embodiments, the target is cleaved from the cell surface. In some of these embodiments, if the target is an organism, the cleaving of the target results in the circulation of the target throughout the organism's system. In some of these embodiments, the target is found at specific levels in, for example, blood, serum, plasma, etc. However, in some embodiments, since a substantial portion of the expressed target is confined to the cell surface; in some embodiments, measurements of the target level may not accurately reflect the amount of the target (e.g., tumor) in a population of cells. In some embodiments, the target is a secreted protein. In some of these embodiments, the target is found at specific levels in, for example, blood, serum, plasma, etc. In some of these embodiments, the miniprotein binds to a region of the target, such as an epitope, for example. In some embodiments, the miniprotein or the composition containing the miniprotein specifically binds to a target expressed on the surface of a cancer cell. In some embodiments, the cancer cells are within, on, or near a solid tumor. In some embodiments, the cancer cells are circulating cancer cells. In some embodiments, the miniprotein or the composition containing the miniprotein specifically binds to the target or is expressed at a higher level on the cancer cells than on the reference cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0164] In some embodiments, the miniprotein or the composition containing the miniprotein specifically binds to B7-H3. In some embodiments, the target comprises or consists of B7-H3. In some embodiments, the miniprotein is Table 1A It specifically binds to a target containing an amino acid sequence or a part thereof as presented in [the document].

[0165] B7-H3

[0166] 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 against T cells and has been identified as contributing to tumor cell immune evasion. (Dong et al. , Front Oncol. 2018 Jul 6;8:264).

[0167] B7-H3 overexpression has been recorded 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 on B7-H3 overexpression have also shown associations between B7-H3 levels and advanced tumor stage, high tumor grade, and poor clinical prognosis. Without being bound by any specific theory, this overexpression of B7-H3 is believed to contribute to metastasis in numerous different cancer types and through multiple 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 inhibition of cell migration and invasion was observed in cells from prostate, breast, gastric, liver, pancreatic, colorectal, and melanoma cancers. Id. ).

[0168] In some embodiments, B7-H3 overexpressed on tumor cells has been shown to be successfully targeted, including by T-cell-mediated immunotherapy. For example, one study indicated that the specific cytotoxic activity of activated T cells (ATCs) with a novel anti-CD3 x anti-B7-H3 bispecific antibody inhibited tumor growth and increased survival in xenograft models, with increased cytotoxicity compared to ATCs alone (Ma J., et al, Oncotarget (2016) 7(20):29480-91). In some embodiments, the binder of the present disclosure binds to B7-H3 expressed on one or more cells (e.g., cancer cells). In these specific embodiments, the present disclosure envisions targeting B7-H3 with one or more binders of the present disclosure, wherein B7-H3 is expressed at higher levels on cancer cells compared to non-cancer cells, and the binding of the miniprotein is specific to cancer cells.

[0169] In some embodiments, the composition provided by the present disclosure more specifically and effectively targets B7-H3 overexpressing cells while minimizing or eliminating damage to surrounding cells that do not express or low-express B7-H3 by providing a targeted composition comprising a chelator and / or alpha-emitter that, in some embodiments, when combined with a miniprotein as provided herein, provides a specific, efficient, and effective approach to target cells that overexpress B7-H3. As used herein, "cells that low-express B7-H3" refers to cells that do not express B7-H3 and / or cells that express B7-H3 at a level of less than 50% of the B7-H3 expression level of cells that overexpress B7-H3. In certain embodiments, cells ‘expressing B7-H3 at low concentrations’ refer to cells that express B7-H3 at a level of less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the expression level of B7-H3 in cells that overexpress B7-H3.

[0170] In some embodiments, there is a relationship between B7-H3 levels and metastasis. For example, some studies have found that B7-H3 and metastasis, such as increased expression levels of metastasis-associated proteins like MMP2, STAT3, and IL-8 (Tekle et al. , Int J Cancer (2012) 130(10):2282-90) and documented the relationship between increased levels of CXCR4 and activation of the AKT, ERK, and JAK2 / STAT3 pathways. (Wang et al., Tumour Biol (2016) 37(3):2961-71). In some embodiments, changes in B7-H3 may affect cancer treatment. For example, there are studies suggesting that changes in B7-H3 may affect the efficacy of some cancer treatments. Specifically, in breast cancer, B7-H3 has been shown to reduce the efficacy of paclitaxel through the activation of JAK2 / STAT3 and the silencing of B7-H3, both of which disrupt JAK2 / STAT3 phosphorylation and improve paclitaxel sensitivity. (Liu et al. , Mol Cancer Ther (2011) 10(6):960-71). Similarly, in colorectal cancer, overexpression of B7-H3 was shown to reduce the 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 specific immune cells, can upregulate B7-H3 expression in lung cancer cells via the PI3K / AKT / mTOR pathway (e.g., the literature [Zhang P et al. [ , see 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 (e.g., the literature [Zhang P et al. , see FEBS Lett (2015) 589(17):2248-56]).

[0171] While I do not wish to be bound by theory, B7-H3 may play a role in cell metastasis and translocation. For example, in some embodiments, B7-H3 may increase cell metastasis and stemness. For instance, 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 reduced degradation.

[0172] As a non-limiting example, although it is not currently known whether B7-H3 overexpression is due to, for example, genomic DNA amplification or increased transcription, chromatin immunoprecipitation demonstrated not only the reduction of B7-H3 expression in the presence of androgens but also the upstream of the androgen receptor-binding site of B7-H3 (Benzon B. et al. , Prostate Cancer Prostatic Dis (2017) 20(1):28-35). Along a similar lineage, in some embodiments, B7-H3 can increase the expression of a gene (e.g., thymidylate synthase) via the PI3K / AKT pathway. (e.g., Literature [Jiang et al. [ , see Tumour Biol (2016) 37(7):9465-72]). Additionally, data have shown that inhibition can make oxaliplatin treatment in colorectal cancer more effective. In some embodiments, B7-H3 is considered to contribute to chemo-resistant properties by increasing the expression of XRCC1 via the PI3K / AKT pathway (e.g., Literature [Zhang et al.[See Biochem Biophys Res Commun (2017) 490(3):1132-8]), this may increase the expression of BRCC3, which is also opposite to the DNA damaging effect of 5-FU in some embodiments (e.g., Literature [Sun et al. , see Oncol Rep (2016) 36(1):231-8]).

[0173] Without limitation, in some embodiments, B7-H3 may play a role in cancer metabolism. Specifically, B7-H3 has been considered to promote the 'Warburg Effect' of the preferential metabolism of glucose to lactate in the presence of abundant oxygen. This Warburg Effect phenomenon has been observed via HIF1α in breast cancer cells and breast cancer mouse models. (Lim et al. , Cancer Res (2016) 76(8):2231-42). Similarly, in some embodiments, B7-H3-mediated reduction may affect proliferation and Warburg effects. Specifically, data indicated that in metastatic melanoma, B7-H3-mediated reduction during the treatment of metastatic melanoma is overcome through the use of a monoclonal B7-H3 antibody that reduces proliferation and Warburg effects 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 rare stage IV pediatric neuroblastoma, DIPG. (Zhou Z, J Neurooncol (2013) 111(3):257-64).

[0174] In some embodiments, B7-H3 interacts with other cellular components or units, such as miRNAs. For example, B7-H3 has also been found to interact with many miRNAs believed to be involved in cancer. (Dong et al., Front Oncol. 2018 Jul 6;8:264). For example, the 3' UTR of the B7-H3 transcript was observed to reduce B7-H3 expression in breast cancer by interacting 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 (Nygren MK, et al. , Br J Cancer (2014) 110(8):2072-80). In osteosarcoma, the 3' UTR of the B7-H3 transcript was shown to interact with miR-124, and studies in colorectal cancer cells indicated that iASPP-mediated p53 inhibition induces the downregulation of miR-124 accompanied by increased B7-H3 expression. (Dong et al. , Front Oncol. 2018 Jul 6;8:264). Also, in a colorectal cancer study, tumorigenesis was promoted through the miR-155 / miR-143 axis (Zhou et al. , Oncotarget (2016) 7(41):67196-211). Specifically, in colorectal cancer cells, TGFβ-elevated miR-155 expression (via SMAD3 and SMAD4) resulted in reduced CEPBP expression, which in turn resulted in reduced miR-143 expression in colorectal cancer cells. Since B7-H3 is a target of miR-143, reduced miR-143 expression increased B7-H3 expression; this supports the possibility that, in some contexts, TGFβ may contribute to cancer immune evasion by upregulating B7-H3. (Zhou et al. , Oncotarget (2016) 7(41):67196-211; Dong et al. , Front Oncol. 2018 Jul 6;8:264).

[0175] Importantly, the novel compositions provided by the present disclosure can specifically, efficiently, and effectively target B7-H3 overexpressing cells with reduced toxicity compared to currently available treatments. That is, in some embodiments, compositions targeting B7-H3 as provided by the present disclosure provide an improved treatment compared to currently available treatments.

[0176] In some embodiments, the target of the composition of the present disclosure comprises or consists of B7-H3 (e.g., human B7-H3).

[0177] In certain embodiments, the miniprotein is selected from any of Tables 1B through 1E, Table 2A, and Table 2C, or a part or fragment thereof. In certain embodiments, the miniprotein binds to B7-H3 and is selected from any of Tables 1B through 1E, Table 2A, and Table 2C. In certain embodiments, the miniprotein binding to B7-H3 is not selected from any of Tables 1B, 1C, or 2A.

[0178] In some embodiments, the B7-H3 binding miniprotein comprises or is composed of an amino acid sequence according to any one of SEQ ID NOs 4 to 6, 8 to 94 and 100 to 537, and may have different N- and / or C-terminal ends, e.g., acetyl, NH2, biotin-PEG4, DOTA-PEG4, radiolabels, etc. on the N-terminus and -OH / COOH or -NH2 on the C-terminus. The N- and / or C-terminus of the B7-H3 binding miniprotein of the present disclosure may comprise, but is not limited to, acetyl, acid, or amide (e.g., acetyl, NH2, OH / COOH) as provided in the exemplary compounds and miniproteins of Table 2A or Table 2C.

[0179] In some embodiments, the B7-H3 binding miniprotein comprises an amino acid sequence according to any one of SEQ ID NOs 4 to 6, 8 to 94 and 100 to 197, or does not have an amino acid sequence composed thereof.

[0180] In some embodiments, polypeptides according to the present disclosure may have various modifications at the N-terminus (e.g., linkers, chelators, and / or radionuclides, e.g., those presented in the exemplary compounds of Table 2A or Table 2C) or the C-terminus (e.g., linkers, chelators, and / or radionuclides). In some embodiments, the C-terminus of a given polypeptide may have an acid or amide group on the C-terminus (e.g., see Table 2A, e.g., Table 2C). A given polypeptide having a specific amino acid sequence may have one or more N-terminus and / or C-terminus differences without significant changes in the utility or function of the polypeptide, e.g., for binding to B7-H3 (e.g., for the detection and / or treatment of cancer).

[0181] In some embodiments, the target of the composition of the present disclosure comprises or consists of B7-H3. In some embodiments, a major change in the fundamental and novel feature of the polypeptide (e.g., miniprotein) provided herein is the ability to bind strongly and specifically to an intended target (e.g., B7-H3, e.g., B7-H3 on cancer cells) with minimal or no non-target effect and minimal elongation uptake (e.g., compared to the elongation uptake of already developed B7-H3 binding molecules). In some embodiments, B7-H3 is expressed on the surface of cells. Alternatively, in some embodiments, B7-H3 is released from tumor-associated cells and is present in the tumor microenvironment. (e.g., Literature [Zhang et al.[See , Immunology. 2008 Apr; 123(4): 538-546]). In some embodiments, the cell is a cancer cell. In some of these embodiments, the cancer cell is a tumor cell, and the tumor is a solid tumor. In some embodiments, the level of B7-H3 expressed in the tumor cell or a population of tumor cells is higher than that expressed in non-tumor cells. In some embodiments, the targeting of B7-H3 by a miniprotein or a composition comprising a miniprotein as provided by the present disclosure specifically targets the composition or one or more components thereof (e.g., a chelator and / or a radionuclide) to the cancer cell or the tumor microenvironment (e.g., a location containing a population of cancer cells or cells at risk of becoming cancer cells).

[0182] In some embodiments, the composition according to the present disclosure specifically binds 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, compounds that target B7-H3 are disclosed in Table 2A or Table 2C.

[0183] In some embodiments, the composition comprising the miniprotein comprises or consists of a protein comprising a specific amino acid sequence that binds to B7-H3 or a portion thereof. In some embodiments, a specific exemplary B7-H3 binding miniprotein is engineered to have an amino acid sequence comprising one or more non-natural amino acids (e.g., those as presented herein, e.g., those presented in Table 2A and / or Table 2C, e.g., amino acids having a small alkyl group on their side chain, etc.). In some of these embodiments, the B7-H3 miniprotein comprises or consists of an amino acid sequence selected from any of SEQ ID NOs 4-6, 8 to 94 and 100 to 537, or a functional variant thereof or a portion thereof (e.g., a functional fragment, e.g., a miniprotein that folds B7-H3 or a portion thereof and binds to it). In some embodiments, these B7-H3 miniproteins are part of a binding protein as presented in Table 2A or Table 2C, or a conjugate comprising such binding protein. In certain embodiments, the B7-H3 miniproteins have an amino acid sequence comprising that presented according to any of Tables 1B through 1E.

[0184] In some embodiments, the composition containing the miniprotein comprises or consists of a protein comprising a specific amino acid sequence that binds to B7-H3 or a part thereof.

[0185] In some embodiments, the present disclosure provides a polynucleotide encoding a polypeptide comprising or consisting of one or more parts of a composition as provided herein. In some embodiments, the present disclosure provides a vector and / or a host cell comprising a sequence encoding one or more components of a composition as provided herein.

[0186] In some embodiments, the present disclosure provides a method for detecting a target. In some embodiments, the method as provided herein includes the step of detecting the presence of a target for, for example, imaging, for example, diagnosis, prognosis and / or monitoring purposes, for example, treatment. In some embodiments, the present disclosure provides a treatment method and / or a method of preparation using a composition as provided herein (e.g., a miniprotein, for example, a linker-chelator, for example, a miniprotein comprising one or more of a linker, a chelator, and a radionuclide). In some embodiments, the treatment method includes the step of administering a composition as provided herein to a subject in need thereof.

[0187] In some embodiments, a linker is attached to the N-terminus of a polypeptide (e.g., a B7-H3 binding miniprotein), if present. In some embodiments, a linker is attached to the C-terminus of a polypeptide (e.g., a B7-H3 binding miniprotein), if present.

[0188] In some embodiments, the C-terminal amino acid of the polypeptide is not cysteine.

[0189] In some embodiments, the chelator is attached to the polypeptide or linker, if present. In some embodiments, if the chelator is present without a linker, the chelator is attached to the N-terminus of the polypeptide. In some embodiments, if the chelator is present without a linker, the chelator is attached to the C-terminus of the polypeptide.

[0190] In some embodiments, the radionuclide is attached to the chelator, if present. In some embodiments, if the radionuclide is present without a linker or chelator, the radionuclide is attached to the N-terminus of the polypeptide. In some embodiments, if the radionuclide is present without a linker or chelator, the radionuclide is attached to the C-terminus of the polypeptide.

[0191] In some embodiments, the present disclosure provides a composition comprising a formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a polypeptide (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), x is 1, 2, 3, or 4, and M comprises any one of the amino acid sequences of SEQ ID NOs 4 to 6, 8 to 94, and 100 to 537.

[0192] In some embodiments, the linker comprises or consists of any fragment or combination thereof through covalent bonding of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4 polyethylene glycol (PEG) linkers, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, or (Gly)n-(gGlu)n-(SEQ No. 551) or (PEG)n (where n is 1 to 36), (Gly)1-10 (SEQ No. 552).

[0193] 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).

[0194] 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.

[0195] In some embodiments, the present disclosure provides a composition comprising a formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a polypeptide (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), x is 1, 2, 3, or 4, and M has an amino acid sequence comprising any one of those presented in SEQ ID NOs 198 to 537.

[0196] In some embodiments, where L is present, L is a polyethylene glycol (PEG) linker, ester linker, amide linker, maleimide linker, valine-citrulline linker, hydrazone linker, N-succinimidyl-4-(2-pyridylditeo)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, vinylsulfone-based linker, propanoic acid linker, dTyr-Gly-Phe(yGF) linker, caploreic acid linker or (Gly)n-(gGlu)n-(SEQ No. 551) or (PEG)n (where n is 1 to 36), (Gly)1-10 (SEQ ID 552), or any fragment or combination thereof formed by covalent bonding.

[0197] In some embodiments, where 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).

[0198] In some embodiments, where 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.

[0199] In some embodiments, a linker is attached to the N-terminus of a polypeptide (e.g., a B7-H3 binding miniprotein), if present. In some embodiments, a linker is attached to the C-terminus of a polypeptide (e.g., a B7-H3 binding miniprotein), if present.

[0200] In some embodiments, the C-terminal amino acid of the polypeptide is not cysteine.

[0201] In some embodiments, the chelator is attached to the polypeptide or linker, if present. In some embodiments, the chelator is attached to the N-terminus of the polypeptide, whether the linker or the chelator is present. In some embodiments, the chelator is attached to the C-terminus of the polypeptide, whether the linker or the chelator is present.

[0202] In some embodiments, the radionuclide is attached to the chelator, if present. In some embodiments, if the radionuclide is present without a linker or chelator, the radionuclide is attached to the N-terminus of the polypeptide. In some embodiments, if the radionuclide is present without a linker or chelator, the radionuclide is attached to the C-terminus of the polypeptide.

[0203] In some embodiments, the polypeptide provided herein (e.g., a miniprotein binding to B7-H3) comprises at least one disulfide bridge.

[0204] In some embodiments, the polypeptide provided herein (e.g., a miniprotein binding to B7-H3) comprises at least two disulfide bridges.

[0205] In some embodiments, the composition and / or polypeptide thereof selectively bind to B7-H3 or a part thereof.

[0206] In some embodiments, the polypeptide has a binding affinity for B7-H3 or a portion thereof, when measured in vivo, in vitro, or in vitro and / or in a cell-based assay, of 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM.

[0207] In some embodiments, the polypeptide has a binding inhibition constant of 100 nM or less.

[0208] 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 with at least 44 amino acids of a polypeptide having an amino acid sequence presented in any one of SEQ ID NOs 198 to 537. In some embodiments, the polypeptide comprises at least one disulfide bridge. In some embodiments, the polypeptide comprises at least two disulfide bridges. In some embodiments, the polypeptide comprises one or two disulfide bridges. In some embodiments, the polypeptide comprises at least one non-natural amino acid (e.g., methylated lysine, citrulline, etc.). In some embodiments, the polypeptide comprises at least one modified amino acid. In some of these embodiments, the modified amino acid is an amino acid comprising a small alkyl group on the side chain of the amino acid (e.g., Methylated lysine, for example, It is mono, di, tri-methyllysine, etc.). For example, in some embodiments, lysine has a small alkyl group on the nitrogen of the side chain (e.g., Methyl group, for example, It may have mono, di, tri-methyl, etc. In some embodiments, arginine may have a small alkyl group on the guanidino group of its side chain.

[0209] In some embodiments, the B7-H3 binding miniprotein comprises or is composed of an amino acid sequence according to any one of SEQ ID NOs 4 to 6, 8 to 94 and 100 to 537, and may have different N- and / or C-terminal ends, e.g., acetyl, NH2, biotin-PEG4, DOTA-PEG4, radiolabels, etc. on the N-terminus and -OH or -NH2 on the C-terminus. The N- and / or C-terminus of the B7-H3 binding miniprotein of the present disclosure may comprise, but is not limited to, acetyl, acid, or amide (e.g., acetyl, NH2, OH) as provided in the exemplary compounds and miniproteins of Table 2A or Table 2C. In some embodiments, polypeptides according to the present disclosure may have various modifications at the N-terminus (e.g., linkers, chelators, and / or radionuclides, e.g., those presented in the exemplary compounds of Table 2A and / or Table 2C) or the C-terminus (e.g., linkers, chelators, and / or radionuclides). In some embodiments, the C-terminus of a given polypeptide may have an acid or amide group on the C-terminus (e.g., see Table 2A and / or Table 2C). A given polypeptide having a specific amino acid sequence may have one or more N-terminus and / or C-terminus differences without significant changes in the utility or function of the polypeptide, e.g., for binding to B7-H3 (e.g., for the detection and / or treatment of cancer).

[0210] In some embodiments, the B7-H3 binding miniprotein comprises or is composed of an amino acid sequence according to any one of SEQ ID NOs 198 to 537 and may have different N- and / or C-terminal ends, e.g., acetyl, NH2, biotin-PEG4, DOTA-PEG4, radiolabels, etc. on the N-terminus and -OH or -NH2 on the C-terminus. The N- and / or C-terminus of the B7-H3 binding miniprotein of the present disclosure may comprise, but is not limited to, acetyl, acid, or amide (e.g., acetyl, NH2, OH) as provided in the exemplary compounds and miniproteins of Table 2A or Table 2C. In some embodiments, the polypeptide according to the present disclosure has an N-terminus (e.g., Linkers, chelators and / or radionuclides, e.g., Those presented in the exemplary compounds of Table 2C) or C-terminals (e.g., Various modifications may be made to the linker, chelator, and / or radionuclide. In some embodiments, the C-terminus of a given polypeptide may have an acid or amide group on the C-terminus (e.g., (See Table 2C). A given polypeptide having a specific amino acid sequence is for binding to, for example, B7-H3 without significant changes in the polypeptide's utility or function (e.g., For the detection and / or treatment of cancer) it may have one or more N-terminal and / or C-terminal differences.

[0211] In certain embodiments, the present disclosure provides a composition comprising a polypeptide having at least 48 amino acids, 2 disulfide bonds, and amino acids modified at positions corresponding to 3, 24, and 29 for SEQ ID NO. 267, wherein the modification is on the side chain of the amino acids (e.g., On the nitrogen phase of the lysine side chain, for example, The guanidino group of the arginine side chain is a small alkyl group.

[0212] In one aspect, the present disclosure provides a composition having one or more modified amino acids. In some of these embodiments, one or more modifications include small alkyl groups. In one aspect, the present disclosure provides a composition comprising a B7-H3 bonded polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acids include (i) cysteine ​​at each of the four positions corresponding to 1, 17, 35 and 48 of SEQ ID NO. 267, and 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.

[0213] In one embodiment, the present disclosure provides a composition comprising a B7-H3 bonded polypeptide having an amino acid sequence, wherein the amino acid sequence comprises at least four cysteines forming two disulfide bonds; arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3; lysine at a position corresponding to amino acid 5; isoleucine at a position corresponding to amino acid 6; tryptophan at a position corresponding to amino acid 14; at least one modified lysine residue at a position corresponding to amino acid 24; and alanine, arginine, or modified lysine at a position corresponding to 29, wherein each position is linear from the N-terminus to the C-terminus with respect to SEQ ID NO. 267, and the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or the guanidino group of the arginine side chain, which optionally comprises a methyl, dimethyl, or trimethyl group; and having a length of at least 48 amino acids; It has a binding affinity for B7-H3 stronger than 100 nM when measured in cell-based assays.

[0214] In one embodiment, the present 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 forming 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 and optionally comprising a methyl, dimethyl, or trimethyl group); having a length of at least 48 amino acids; and having a binding affinity for B7-H3 stronger than 100 nM when measured in a cell-based assay.

[0215] In some embodiments, the polypeptide has a length of at least 48 amino acids and no more than 100 amino acids. In some embodiments, the polypeptide binds to B7-H3 with an affinity stronger than 10 nM in a cell-based assay.

[0216] In some embodiments, B7-H3 overexpressed on tumor cells has been shown to be successfully targeted, including by T-cell-mediated immunotherapy. For example, one study indicated that the specific cytotoxic activity of activated T cells (ATCs) with a novel anti-CD3 x anti-B7-H3 bispecific antibody inhibited tumor growth and increased survival rates in xenograft models, with increased cytotoxicity compared to ATCs alone. (Ma J., et al ., Oncotarget (2016) 7(20):29480-91). In some embodiments, the binder of the present disclosure is one or more cells (e.g., , It binds to B7-H3 expressed on cancer cells. In some of these embodiments, the present disclosure considers targeting B7-H3 with one or more of the binders of the present disclosure, wherein B7-H3 is expressed at higher levels in cancer cells compared to non-cancer cells, and the binding of the miniprotein is specific to cancer cells. The amino acid sequence of the polypeptide shares at least 90% identity with any one of SEQ ID NOs 198 to 537, but comprises at least one lysine and / or arginine having at least one modification comprising at least one small alkyl group attached 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 with at least 44 amino acids of the reference polypeptide, wherein the reference polypeptide has a length of more than 48 amino acids and binds to B7-H3 with an intensity of at least 10 nM in a cell-based assay and / or has an inhibition constant of 10 nM or less.

[0217] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537, provided that the 44 amino acids include at least two cysteine ​​residues that form one disulfide bridge.

[0218] In some embodiments, the amino acid sequence shares 90% identity with at least 44 amino acids as presented in any one of SEQ ID NOs 199, 204, 241 or 262 to 272.

[0219] I In some embodiments, the amino acid sequence shares 100% identity with at least 44 amino acids as presented in any one of SEQ ID NOs 199, 204, 241 or 262 to 272.

[0220] In some embodiments, the present disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO. 267.

[0221] In some embodiments, the present disclosure provides a composition comprising a compound as presented 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.

[0222] 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.

[0223] In some embodiments, the present disclosure provides a composition comprising a polypeptide having four cysteines corresponding to 1, 17, 35 and 48 of SEQ ID NO. 267, having an amino acid sequence of at least 48 amino acids in length, wherein the following amino acids are present: arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3; lysine at a position corresponding to amino acid 5; isoleucine at a position corresponding to amino acid 6; tryptophan at a position corresponding to amino acid 14; at least one modified lysine residue at a position corresponding to amino acid 24; and alanine, arginine, or modified lysine at a position corresponding to 29, wherein each position is linear from the N-terminus to the C-terminus with respect to SEQ ID NO. 267, and the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain, which optionally comprises methyl, dimethyl, or trimethyl, or to the guanidino group of the arginine side chain.

[0224] In some embodiments, the C-terminus has -OH or -NH2.

[0225] In some embodiments, the binding affinity of the composition (e.g., polypeptide) to B7-H3 is stronger than 100 nM.

[0226] In some embodiments, the inhibition constant is 100 nM or less.

[0227] In some embodiments, the composition further comprises one or more of a linker, a chelator, and a radionuclide.

[0228] In some embodiments, the linker comprises or consists of any fragment or combination thereof through covalent bonding of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4 polyethylene glycol (PEG) linkers, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, or (Gly)n-(gGlu)n-(SEQ No. 551) or (PEG)n (where n is 1 to 36), (Gly)1-10 (SEQ No. 552).

[0229] 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).

[0230] 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.

[0231] In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs 198 to 537. In some embodiments, the polypeptide sequence comprises any one of SEQ ID NOs 199, 204, 241, or 262 to 272, and further comprises a linker, the linker is PEG4 and an optional chelator, and the chelator is DOTA.

[0232] In some embodiments, a linker is attached to the N-terminus of the polypeptide, if present. In some embodiments, a linker is attached to the C-terminus of the polypeptide, if present.

[0233] In some embodiments, the C-terminal amino acid of the polypeptide is not cysteine.

[0234] In some embodiments, the chelator is attached to the polypeptide or linker, if present.

[0235] In some embodiments, a radionuclide is attached to the chelator if present.

[0236] In some embodiments, the present disclosure provides a composition comprising a formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a polypeptide (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), x is 1, 2, 3, or 4, and M comprises an amino acid sequence according to any one of SEQ ID NOs 4 to 6, 8 to 94, and 100 to 537 or SEQ ID NOs 538 to 543 and 546 to 550 and Table 1E. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs 204 and 262 to 272.

[0237] In some embodiments, the linker comprises or consists of any fragment or combination thereof through covalent bonding of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4 polyethylene glycol (PEG) linkers, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, or (Gly)n-(gGlu)n-(SEQ No. 551) or (PEG)n (where n is 1 to 36), (Gly)1-10 (SEQ No. 552).

[0238] 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).

[0239] 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 present disclosure provides a composition comprising a formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a polypeptide (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), x is 1, 2, 3, or 4, and M has an amino acid sequence comprising any one of those presented in SEQ ID NOs 4 to 6, 8 to 94, and 100 to 537, or SEQ ID NOs 95 and 96 and Table 1B and Table 1C, and SEQ ID NOs 538 to 543 and 546 to 550 (Table 1D) and Table 1E. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs 204 and 262 to 272.

[0241] In some embodiments, where L is present, L is a polyethylene glycol (PEG) linker, ester linker, amide linker, maleimide linker, valine-citrulline linker, hydrazone linker, N-succinimidyl-4-(2-pyridylditeo)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, vinylsulfone-based linker, propanoic acid linker, dTyr-Gly-Phe(yGF) linker, caploreic acid linker or (Gly)n-(gGlu)n-(SEQ No. 551) or (PEG)n (where n is 1 to 36), (Gly)1-10 (SEQ ID 552), or any fragment or combination thereof formed by covalent bonding.

[0242] In some embodiments, where 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).

[0243] In some embodiments, where 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.

[0244] In some embodiments, a linker is attached to the N-terminus of the polypeptide, if present. In some embodiments, a linker is attached to the C-terminus of the polypeptide, if present.

[0245] In some embodiments, the C-terminal amino acid of the polypeptide is not cysteine.

[0246] In some embodiments, the chelator is attached to the polypeptide or linker, if present.

[0247] In some embodiments, a radionuclide is attached to the chelator if present.

[0248] In some embodiments, the polypeptide comprises at least one disulfide bridge.

[0249] In some embodiments, the polypeptide comprises at least two disulfide bridges.

[0250] In some embodiments, the composition and / or polypeptide thereof selectively bind to B7-H3 or a part thereof.

[0251] In some embodiments, the polypeptide has a binding affinity for B7-H3 or a portion thereof, when measured in vivo, in vitro, or in vitro and / or in a cell-based assay, of 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM.

[0252] In some embodiments, the polypeptide has a binding inhibition constant of 100 nM or less.

[0253] In one embodiment, the present 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 with at least 44 amino acids of a polypeptide having an amino acid sequence according to any one of SEQ ID NOs 4 to 6, 8 to 94 and 100 to 197 or SEQ ID NOs 95 or 96 and Tables 1B and 1C.

[0254] The composition comprises 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 with at least 44 amino acids of a polypeptide having an amino acid sequence according to any one of SEQ ID NOs 198 to 537 and Table 2C or any one of SEQ ID NOs 538 to 543 and 546 to 550 (Table 1D) and Table 1E.

[0255] In some embodiments, the drug moiety is selected from focal isomerase inhibitors, auristatin (e.g., monomethylauristatin E), V-ATPase inhibitors, apoptosis promoters, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, dolastine, metansinoids, MetAP (methionine aminopeptidase), inhibitors of extranuclear transport of protein CRMl, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, and immunotoxins.

[0256] In one embodiment, the present disclosure provides a composition comprising a selective linker (L) and an isolated compound comprising one or more of a polypeptide (M), a chelator (C), or a radionuclide (R), or a pharmaceutically acceptable salt or neutral molecule thereof, wherein M has an amino acid sequence comprising SEQ ID NOs 4 to 6, 8 to 94 and 100 to 197 or SEQ ID NOs 95 or 96 and any one of Table 1B and Table 1C, or M has an amino acid sequence according to any one of SEQ ID NOs 198 to 537 and Table 2C or SEQ ID NOs 538 to 543 and 546 to 550 (Table 1D) and Table 1E.

[0257] In some embodiments, the present disclosure provides a composition comprising a compound designed to bind to B7-H3, wherein the compound comprises or consists of a polypeptide having an amino acid sequence comprising any one of SEQ ID NOs 4 to 6, 8 to 94 and 100 to 197 or SEQ ID NOs 95 and 96 and Table 1B and Table 1C or any one of SEQ ID NOs 198 to 537 and Table 2C or any one of SEQ ID NOs 538 to 543 and 546 to 550 (Table 1D) and Table 1E.

[0258] In some embodiments, the modified N-terminus comprises one or more of NH2, acetyl, PEGn (wherein=1 to 36), DOTA, or biotin.

[0259] In some embodiments, the C terminal includes -NH2 or -OH.

[0260] In some embodiments, the polypeptide selectively binds to B7-H3 or a part thereof.

[0261] In some embodiments, the polypeptide has a binding affinity stronger than about 100 nM for B7-H3 or a portion thereof in vivo or cell-based assays.

[0262] polypeptide

[0263] In particular, the present disclosure provides polypeptides. In some embodiments, polypeptides are assembled using a solid-state synthesis method. In some embodiments, polypeptides are recombinants. In some embodiments, polypeptides comprise or consist of miniproteins. In some of these embodiments, miniproteins comprise or consist of binders. In some embodiments, polypeptides of the present disclosure (including mutains, allelic variants, fragments, derivatives, and analogs) are encoded by polynucleotides as described and provided herein.

[0264] In some embodiments, the miniproteins of the present disclosure are Table 1A It comprises or consists of a polypeptide capable of binding to a target as shown in [figure].

[0265] In some embodiments, the present disclosure provides a binder comprising or made of a fragment of a polypeptide as provided herein. In some such embodiments, the fragment comprises at least 20 adjacent amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more adjacent amino acids.

[0266] In some embodiments, the miniproteins of the present disclosure may also comprise a fusion or conjugation with one or more other components, such as a heterogeneous polypeptide. For example, in some embodiments, the heterogeneous sequence may comprise or consist of a sequence designed to facilitate purification, for example, a histidine tag and / or visualization of the recombinantly expressed protein. Other non-limiting examples of such fusions or conjugations include any detectable or visualizable component that allows for the display of the protein encoded on the surface of a phage or cell, such as a fusion to a green fluorescent protein (GFP) and an IgG Fc region.

[0267] In some embodiments, the miniprotein includes or consists of a specific amino acid sequence. In some embodiments, the miniprotein comprises an amino acid sequence according to any of SEQ ID NOs. 4 to 6, SEQ ID NOs. 12 to 94, or SEQ ID NOs. 100 to 537 and / or any of Table 1B, Table 1C, Table 1D, Table 1E, Table 2A, Table 2C, or Table 2D and 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%, It has 99.7%, 99.8%, 99.9%, or 100% identical amino acid sequences.

[0268] In some embodiments, the 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 presented in any of the amino acid sequences in Table 2A.

[0269] In some embodiments, the miniprotein does not have an amino acid sequence selected from any one of those according to Table 1B, Table 1C and / or Table 2A.

[0270] In some embodiments, the miniprotein comprises 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 adjacent amino acids of a given polypeptide, such as those presented in Table 2A or Table 2C, and 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% have the same amino acid sequence. In some embodiments, the miniprotein does not have the amino acid sequence as presented in Table 2A.

[0271] In some embodiments, the 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 presented in any amino acid sequence presented in Table 2A or Table 2C.

[0272] In some embodiments, the 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 the amino acid sequence presented in Table 2A. For example, in some embodiments, the miniprotein comprises 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 as presented in Table 2A and 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%, It has 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical amino acid sequences.

[0273] In some embodiments, the 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 the amino acid sequence presented in Table 2C. For example, in some embodiments, the miniprotein comprises 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 as shown in Table 2C and 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%, It has 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical amino acid sequences.

[0274] In some embodiments, the miniprotein comprises 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 adjacent amino acids of a given polypeptide such as those presented in Table 2C, and 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% have the same amino acid sequence.

[0275] In some embodiments, a polypeptide (e.g., a miniprotein) has an amino acid sequence with a certain percentage of identity relative to a certain percentage of coverage (e.g., of a reference sequence). Unless otherwise noted, % identity refers to the maximum % identity measured according to any of the approaches presented herein. That is, a B7-H3 bonded polypeptide (e.g., a reference molecule) as provided herein may have a percentage of identity of 70, 75, 80, 85, 90, 95, 99, or 100% across the percentage of coverage of the molecule (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%) but with a given query, and if the query molecule is shorter, it may have different percentages of identity and coverage (e.g., 100% identity and 90% coverage). If the query molecule is longer, the identity percentage and coverage percentage may each be 100% with respect to the reference molecule, and the reference molecule will have an identity percentage over the length of the query molecule (e.g., at least 20, 25, or 30 amino acids). For example, in some embodiments, if the reference sequence (e.g., a miniprotein as provided herein) is shorter than the query sequence, such query sequence is within the scope of the disclosure if it has the length of the reference sequence aligned with the query sequence, wherein the identity percentage is determined over at least the minimum length of the alignment between the two sequences (query and reference). That is, if the polypeptide disclosed herein is longer than the query sequence, the identity percentage is determined by aligning the reference and the query and determining the identity percentage as being between the query and the portion of the reference sequence aligned thereon. Conversely, if the query sequence is longer than the reference sequence, the identity percentage is equivalent to the identity over the portion aligned with the reference sequence.That is, if the reference sequence is shorter, the query sequence may fall within the range of the reference sequence when aligned in the claimed percentage of identity across the aligned portion between the two polypeptides (reference and query).

[0276] As used herein and known to those skilled in the art, the 20 common amino acids and their abbreviations follow common usage. Reference is made to the literature [Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991)], which is incorporated herein by reference. In some embodiments, the amino acids of the present disclosure may be stereoisomers of the 20 common amino acids (e.g., D-amino acids). In some embodiments, the amino acids of the polypeptide of the present disclosure may be non-natural amino acids. For example, amino acids, such as α,α-disubstituted amino acids, N-alkyl amino acids, and other non-conventional amino acids may also be suitable components of the polypeptide of the present disclosure. Examples of non-conventional 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). The arrangement of polypeptide sequence notations used herein has a left end corresponding to the amino terminus and a right end corresponding to the carboxy terminus, in accordance with standard use and convention.

[0277] In some embodiments, the table 2CA miniprotein of the present disclosure comprising two or more cysteine ​​residues such as those presented herein has cysteine ​​residues connected via a disulfide bridge (e.g., through natural folding). In some embodiments, the miniprotein (e.g., binding to B7-H3) comprises one disulfide bridge. In some embodiments, the miniprotein comprises two disulfide bridges.

[0278] In some embodiments, one disulfide bridge may be located between cysteine ​​residues corresponding, for example, Cys4 and Cys37; Cys5 and Cys34; Cys5 and Cys37; Cys12 and Cys26; Cys12 and Cys44; Cys17 and Cys48, and the position is based on a linear position from the N-terminus to the C-terminus with respect to SEQ ID NO. 267.

[0279] In some embodiments, the cysteine ​​linkage may be between two different pairs of cysteine ​​residues. For example, in some embodiments, two disulfide bridges may be between positions corresponding to cysteine ​​pairs, e.g., Cys1 and Cys35 and Cys17 and Cys52, in relation to the miniprotein of SEQ ID NO. 213. For example, in some embodiments, the disulfide bridges are Cys1 and Cys35 of the reference sequence (e.g., SEQ ID NO. 267) as presented in Table 2C; and Cys17 and It may be located between the positions corresponding to Cys48. In some embodiments, the cysteine ​​linkage is between Cys1 and Cys17; and between Cys35 and Cys48. In some embodiments, the cysteine ​​linkage is between Cys1 and Cys48; and between Cys17 and Cys35.

[0280] In some embodiments, the disulfide bridge or bridges comprise two or four cystes at positions corresponding to 1, 17, 35, and 48 of SEQ ID NO. 267, wherein the cysteine ​​corresponding to position 1 may form a disulfide bridge with the cysteine ​​corresponding to positions 17, 35, or 48. In some embodiments, the cysteine ​​corresponding to position 17 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 35, or 48. In some embodiments, the cysteine ​​corresponding to position 35 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 17, or 48. In some embodiments, the cysteine ​​corresponding to position 48 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 17, or 35. In some embodiments, where four cystes are present and correspond to positions 1, 17, 35, and 48 of SEQ ID NO 267, the pairing may include 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., a disulfide bridge between two cystes of the pair).

[0281] In some embodiments, the present disclosure provides a miniprotein or a part or functional variant thereof comprising or composed of an amino acid sequence as presented in Table 1B, Table 1C, Table 1D, Table 1E, Table 2A, Table 2C and / or Table 2D and / or as presented in SEQ ID NOs 4 to 6, 8 to 94 and 100 to 537.

[0282] In some embodiments, the miniprotein is 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%, 99.7%, 99.8%, 99.8%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.8%, 99.5%, 99.6%, 99.7%, 99.8%, 99.8%, 99.5%, 99.6%, 99.7%, 99.8%, 99.8%, 99.5%, 99.6%, 99.7%, 99.8%, 99.8%, 99.5%, 99.5%, 99.8 It comprises or consists of an amino acid sequence having 99.9% or more of sequence identity, or a part thereof or a functional variant thereof.

[0283] In some embodiments, the miniprotein comprises or consists of an amino acid sequence or a part thereof or a functional variant thereof 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, or at least 20 amino acid residue differences from SEQ ID NOs 4 to 6, 8 to 94 and 100 to 537 and / or Table 1B, Table 1C, Table 1D, Table 1E, Table 2A, Table 2C, and Table 2D.

[0284] In some embodiments, the miniprotein comprises or consists of an amino acid sequence or a part thereof or a functional variant thereof having a difference of 1 or fewer, 2 or fewer, 3 or fewer, 4 or fewer, 5 or fewer, 6 or fewer, 7 or fewer, 8 or fewer, 9 or fewer, 10 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, or 35 or fewer amino acid residues as presented in SEQ ID NOs 4 to 6, 8 to 94 and / or Table 1B, Table 1C, Table 1D, Table 1E, Table 2A, Table 2C, and Table 2D.

[0285] In some embodiments, the miniprotein comprises any of SEQ ID NOs 4 to 6, SEQ ID NOs 12 to 94 or SEQ ID NOs 100 to 537 and / or any of those presented in Tables 1B, 1C, 1D, 1E, 2A, 2C, and 2D, or has an amino acid sequence or part thereof or part of a functional variant thereof and selectively binds to target B7-H3.

[0286] Polypeptides of the present disclosure may have one or more modifications. A modification may refer to an amino acid sequence comprising at least one substitution, alteration, reversal, addition, or deletion of an amino acid residue relative to a reference amino acid sequence. An alteration may include, but is not limited to, changes to one or more side chains, such as, for example, the addition of a methyl group (e.g., a methylated version of lysine). In some embodiments, natural amino acids are modified as set forth in this specification. In some embodiments, a modification includes the addition of at least one small alkyl group attached to the nitrogen of an amino acid side chain, for example, a lysine side chain. As used herein, 'small alkyl group' typically refers to an alkyl group having a short carbon chain having one to four carbon atoms, such as methyl, ethyl, propyl, or butyl, and also includes, 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, five, four, three, two, or fewer than one small alkyl groups are added. Miniprotein

[0287] The present disclosure provides a polypeptide comprising an amino acid sequence, wherein the amino acid sequence comprises the following formula I: X1X2X3X4YX6X7EX9IX11ALX14EIIWLPNX22X23X24X25QIX28AFIAALNX36DPSQSSELLSEAX49X50LX52DSX55X56X57X58 (SEQ 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; X58 is G, K, (KAc) or absent; if X28 is A, (homo-leucine), M(O2), S, T, or V, X24 is Y; if X28 is M, X7 is A, E, (Kme3), L, Q, or S.

[0288] In some embodiments, if X4 is K or (KAc), X24 is Y.

[0289] In some embodiments, the amino acid sequence comprises the following formula II: X1X2X3X4YAX7EKIAALSEIIWLPNX22TX24X25QIX28AFIAALNX36DPSQSSELLSEAX49X50LNDSQAP (SEQ No. 96), where 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; X50 is E.

[0290] In some embodiments, the amino acid sequence comprises Formula III (Sequence No. 538) as presented in Tables 1D and 1E.

[0291] In some embodiments, the amino acid sequence comprises Formula IV (Sequence No. 539) as presented in Tables 1D and 1E.

[0292] In some embodiments, the amino acid sequence comprises formula V (sequence number 540) as presented in Tables 1D and 1E.

[0293] In some embodiments, the amino acid sequence comprises formula VI (sequence number 541) as presented in Tables 1D and 1E.

[0294] In some embodiments, the amino acid sequence comprises Formula VII (Sequence No. 542) as presented in Tables 1D and 1E.

[0295] In some embodiments, the amino acid sequence comprises Formula VIII (Sequence No. 543) as presented in Tables 1D and 1E.

[0296] In some embodiments, the amino acid sequence comprises Formula IX (Sequence No. 546) as presented in Tables 1D and 1E.

[0297] In some embodiments, the amino acid sequence comprises formula X (sequence number 547) as presented in Tables 1D and 1E.

[0298] In some embodiments, the amino acid sequence comprises formula XI (sequence number 548) as presented in Tables 1D and 1E.

[0299] In some embodiments, the amino acid sequence comprises formula XII (sequence number 549) as presented in Tables 1D and 1E.

[0300] In some embodiments, the amino acid sequence comprises formula XIII (sequence number 550) as presented in Tables 1D and 1E.

[0301] In some embodiments, the amino acid sequence is at least 90% (e.g., any one of SEQ ID NOs 4 to 6 or SEQ ID NOs 12 to 94) 91, 92, 93, 94, 95, 96, 97, 98, 99 or more %) share identity.

[0302] In some embodiments, the amino acid sequence is 90% (e.g., any one of SEQ ID NOs 6, 21, or 30) 91, 92, 93, 94, 95, 96, 97, 98, 99 or more %) share identity.

[0303] In some embodiments, the amino acid sequence shares 100% identity with any one of SEQ ID NOs 4 to 6 or SEQ ID NOs 12 to 94.

[0304] In some embodiments, the amino acid sequence shares 100% identity with any one of SEQ ID NOs 6, 21, or 30.

[0305] In some embodiments, the amino acid sequence shares 90% identity (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99 or more) with SEQ ID NO. 6.

[0306] In some embodiments, the amino acid sequence shares 100% identity with SEQ ID NO. 6.

[0307] The present disclosure also provides a polypeptide having an amino acid sequence comprising or consisting of any one of SEQ ID NOs 4 to 6, 8 to 94 or 100 to 537.

[0308] The present disclosure also provides a polypeptide having an amino acid sequence comprising or consisting of any one of SEQ ID NOs 4 to 6, 8 to 94, or 100 to 197.

[0309] The present disclosure also provides a polypeptide having an amino acid sequence comprising any one of SEQ ID NOs 198 to 537 or consisting thereof. In some embodiments, the miniprotein comprises at least one binding agent, wherein the binding agent is a disulfide bridge.

[0310] In some embodiments, the polypeptide further comprises one or more of a linker, a chelator, and a radionuclide.

[0311] In some embodiments, the linker is a polyethylene glycol (PEG) linker of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester linker, amide linker, maleimide linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, propanoic acid linker, caploreic acid linker, aminocaproic acid (Ahx) linker or (Gly)n-(γGlu)n- or (PEG)n (where n is 1 to 36), (Gly) 1-10 or includes or is composed of any fragment or combination through the covalent combination of these.

[0312] In some embodiments, the chelator comprises or consists of, if present:

[0313] i) NOPO

[0314]

[0315] ii) Crown

[0316]

[0317] iii) DOTA or

[0318]

[0319] iv) Macropa

[0320] .

[0321] In some embodiments, the chelator comprises or consists of NOPO, Crown, Macropa, or a derivative of tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0322] In some embodiments, the radionuclides include or consist 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.

[0323] In some embodiments, a linker is attached to the N-terminal or C-terminal end of the polypeptide, if present.

[0324] In some embodiments, the chelator is attached to the polypeptide or linker, if present.

[0325] In some embodiments, a radionuclide is attached to the chelator if present.

[0326] In some embodiments, the polypeptide comprises or consists of a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide comprising a disulfide linkage), a cysteine-tight peptide, a notine peptide, a binder, an affibody, a engineered Kunitz domain, a monobody, an anticalin, an engineered ankyrin repeat domain (DARPin), or an abimer.

[0327] In some embodiments, the polypeptide comprises at least one disulfide bridge.

[0328] In some embodiments, the polypeptide selectively binds to B7-H3 or a part thereof.

[0329] 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 for B7-H3 or a portion thereof in vivo or cell-based assays.

[0330] 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) for B7-H3 in a cell-based assay.

[0331] miniproteins

[0332] This specification provides a novel polypeptide (i.e., a miniprotein) and a method of using the same. In some embodiments, the polypeptide comprises or consists of a miniprotein. The present disclosure recognizes that the cause of the problem in therapeutic agents (e.g., miniprotein therapeutic agents) is binding to B7-H3 with sufficient specificity and affinity. Accordingly, this specification provides a miniprotein that binds strongly, efficiently, and specifically to B7-H3 (e.g., on cells, e.g., on cancer cells).

[0333] Another cause of the problem includes toxicity (e.g., nephrotoxicity). In some embodiments, the specificity and strength of binding to B7-H3 in cancer cells reduce absorption into the kidneys.

[0334] In some of these embodiments, the miniprotein comprises or consists of an affibody, CDP, notin, binder, monobody, anticalin, designed ankyrin repeat, engineered Kunitz domain and / or avimer. In some embodiments, the miniprotein is designed to be linked to one or more other components. For example, in some embodiments, the miniprotein may be linked (conjugated) to another component, such as a chelator and / or radionuclide. In some embodiments, conjugation occurs through lysine or cysteine ​​residues. For example, in some embodiments, the miniprotein is engineered to remove all lysine residues except those used for conjugation. In some embodiments, conjugation occurs through a selective linker. In some embodiments, conjugation between the miniprotein and the chelator and / or radionuclide is direct.

[0335] While not wishing to be bound by any particular theory, the present disclosure assumes that a therapeutic agent comprising the composition provided by the present disclosure is characterized by several properties compared to other (e.g., antibody-based) therapeutic agents. For example, in some embodiments, the miniprotein not only exhibits several major properties of antibody-based therapeutic agents (e.g., affinity, potency, specificity, and the ability to disrupt protein-protein interactions) but also has several advantages over antibody-based therapeutic agents, such as a smaller size, lower manufacturing costs, and the elimination of the need to chimeric or humanize the protein. Additionally, the size and specificity of the binding increase tumor penetration and uptake into cells expressing the target of the composition (e.g., conjugate) comprising the miniprotein.

[0336] In some embodiments, the miniprotein of the present disclosure has a length of about 100 amino acids or less. In some embodiments, the miniprotein has a length of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 amino acids or more, up to about 100 amino acids. However, in some of these embodiments, the miniprotein of the present disclosure does not exceed a length of about 100 amino acids. In some embodiments, the miniprotein has a length of 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, but does not exceed a length of about 100 amino acids. In some preferred embodiments, the miniprotein has about 65 or fewer amino acids. In some preferred embodiments, the miniprotein has about 50 or fewer amino acids.

[0337] In some embodiments, the miniprotein of the present disclosure is about 12 kDa or less. In some embodiments, the 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 greater than kDa. However, in some of these embodiments, the miniprotein of the present disclosure does not exceed about 12 kDa.

[0338] In some embodiments, the miniprotein of the present disclosure comprises or consists of a cysteine-tight peptide, notine and / or a binder.

[0339] In some embodiments, the miniprotein comprises one or more disulfide bridges. In some embodiments, the miniprotein comprises a plurality of cysteine ​​residues. In some of these embodiments, the cysteine ​​residues are crosslinked so that they can maintain a very stable folded state for a peptide of that length (e.g., compared to a peptide of the same length without many cysteine ​​residues). The present disclosure assumes that such crosslinking imparts improved stability with reduced (i.e., very low to none) immunogenicity and / or sustains or improves the ability to maintain biological activity under harsh but efficient chelation conditions (e.g., high temperature and low pH).

[0340] In some embodiments, the miniprotein or the composition containing the miniprotein (e.g., a radionuclide conjugate) has lower immunogenicity compared to the larger protein (e.g., an antibody) or the larger protein or composition made therefrom.

[0341] In some embodiments, miniproteins (e.g., affibodies, CDPs, notin, binders, engineered Kunitz domains, monobodies, anticalins, engineered ankyrin repeat domains (DARPin), avimers) have superior penetration efficiency compared to larger proteins. That is, in some embodiments, a miniprotein or a composition containing a miniprotein can penetrate a solid tumor better than a larger protein or composition containing a protein larger than the miniprotein. For example, in some of these embodiments, the miniprotein or a composition containing a miniprotein has a hydrodynamic radius of about 1 to about 25 nm. In some embodiments, the hydrodynamic radius is in the range of about 1 to 25 nm, 10 to 20 nm, 5 to 15 nm, 1 to 5 nm, 2 to 4 nm, or 1 to 3 nm. In some embodiments, the hydrodynamic radius is measured using a light scattering method known to those skilled in the art.

[0342] In some embodiments, the miniprotein of the present disclosure is characterized by having one or more properties compared to proteins having more than 100 amino acids, such as antibodies, antibody fragments, VHH domains, single-strand antibodies, or other proteins or binders greater than 12 kDa. In some embodiments, the properties are selected from increased protein expression, increased thermal activity, increased thermal stability, increased pH activity, increased stability, increased activity, increased receptor binding specificity and / or affinity, increased inactivity, 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 hepatic absorption, increased renal absorption or increased resistance to healthy tissue binding, increased tumor infiltration and / or increased volume distribution.

[0343] In some embodiments, the miniprotein or composition comprising the miniprotein provided by the present disclosure (e.g., a conjugate, e.g., a radionuclide conjugate) exhibits binding affinity for B7-H3. In some embodiments, B7-H3 is human B7-H3. In some embodiments, human B7-H3 is on a cell. In some embodiments, the cell is a cell line, primary cell, or cell in humans (e.g., in a tumor).

[0344] In some embodiments, the binding affinity of the miniprotein or its conjugate to human B7-H3 is about 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, or greater (e.g., 90 nM, 75 nM, 50 nM, 25 nM, 10 nM, 5 nM, etc.). In some embodiments, the miniprotein has a picomolar binding affinity. In some embodiments, the miniprotein or its conjugate comprises a binding affinity for human B7-H3 characterized by a dissociation constant in the range of 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 greater than (e.g., 0.3, 0.2, 0.1, 0.09 nM, nM, etc.). In some embodiments, the binding is selective for human B7-H3 and, for example, not selective for non-human B7-H3.

[0345] In some embodiments, the miniprotein or its conjugate exhibits a binding inhibition constant. In some embodiments, the binding inhibition constant (Ki) for 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 has a picomolar binding affinity. In some embodiments, the miniprotein or its conjugate comprises a binding affinity for human B7-H3 characterized by a dissociation constant in the range of 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 greater than (e.g., 0.3, 0.2, 0.1, 0.09 nM, etc.). In certain embodiments, the binding inhibition constant of the B7-H3 bonded polypeptide of the present disclosure is about 100 pM to about 50 nM (e.g. , Cell-based assays, for example , DELFIA). In some embodiments, the binding is selective for human B7-H3 and, for example, not selective for non-human B7-H3.

[0346] In some embodiments, the miniprotein or conjugate provided by the present disclosure has a high affinity for B7-H3 (as measured, e.g., by binding affinity and / or inhibition constant, etc.). In some of these embodiments, B7-H3 is human B7-H3. In some embodiments, the miniprotein of the present disclosure is stable in the presence of one or more additional molecules (e.g., cytotoxic molecules, e.g., radionuclides).

[0347] In some embodiments, the miniprotein or the composition containing the miniprotein (e.g., a conjugate, e.g., a radionuclide conjugate) exhibits nm or sub-nm binding affinity for 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, the affinity is measured in vivo (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).

[0348] In some embodiments, the miniprotein or its conjugate exhibits a binding affinity for B7-H3. In some embodiments, the binding affinity of the miniprotein or its conjugate for human B7-H3 is about 500 nM. In some embodiments, the miniprotein has a picomolar binding affinity. In some embodiments, the miniprotein or its conjugate comprises a binding affinity for human B7-H3 in the range of about 900 nM to about 1 nM, for example, 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, characterized by a dissociation constant. In some embodiments, binding is selective for human B7-H3 and, for example, not selective for non-human B7-H3.

[0349] In some embodiments, the miniprotein or conjugate thereof provided by the present disclosure has a high affinity for B7-H3. In some of these embodiments, B7-H3 is human B7-H3. In some embodiments, the miniprotein of the present disclosure is stable in the presence of one or more additional molecules (e.g., cytotoxic molecules, e.g., radiation).

[0350] In some embodiments, the binding ability of the miniprotein or its conjugate to the target is improved by one or more modifications. For example, in some embodiments, the binding ability of the miniprotein or its conjugate to B7-H3, as provided herein, is improved using chemical crosslinking. In some embodiments, binding may be enhanced using one or more lysine residues, fusion proteins, non-natural amino acids, or other chemical moieties to increase binding and / or functional activity.

[0351] In some embodiments, to ensure proper folding and connectivity, the selected cysteine ​​pair may be replaced with selenocysteine. In some embodiments, diselenide crosslinks are formed more easily than disulfide bridges due to their lower redox potential, and this replacement may crosslink the remaining cysteine ​​residues.

[0352] In some embodiments, the miniprotein or conjugate provided by the present disclosure comprises or consists of monomers constituting a dimer, polymer, or multimer. In some of these embodiments, the monomers all bind to the same target. For example, in some embodiments, where more than one miniprotein is present, each miniprotein is about 30 to 40 amino acid lengths or less, or a total size of about 8 kDa (including both miniproteins). In some embodiments, the monomers each bind to different targets. In some embodiments, some monomers bind to one target, and others bind to one or more additional targets.

[0353] In some embodiments, the miniprotein of the present disclosure comprises or consists of an antigen for use in generating an antibody that specifically binds to at least one epotope on B7-H3. In some embodiments, such an antibody may be used, for example, for diagnostic purposes, blocking (e.g., antagonism), etc.

[0354] In some embodiments, the miniprotein comprises one or more disulfide bridges.

[0355] In some embodiments, the miniprotein or conjugate thereof as provided herein does not contain one or more cysteine ​​residues. In some embodiments, the miniprotein does not contain one or more disulfide bridges.

[0356] In some embodiments, the miniprotein or its conjugate as provided herein is specific to the target. In some embodiments, the miniprotein is specific to B7-H3 or a fragment thereof.

[0357] In some embodiments, a miniprotein or a conjugate thereof as provided herein comprises or is composed of a specific amino acid sequence.

[0358] In some embodiments, a miniprotein or a composition containing a miniprotein (e.g., a radionuclide conjugate) is conjugated to a chelator that selectively binds to a radionuclide (e.g., actinium). In some embodiments, conjugation is performed via a linker. In some embodiments, conjugation is direct conjugation. In some embodiments, such radionuclide conjugates are combined and synergistic to provide target specificity (e.g., via the miniprotein) and superior treatment (e.g., via directed radioisotope delivery to cells expressing the target).

[0359] In some embodiments, a miniprotein or a composition containing a miniprotein is conjugated to a chelator that selectively binds to a cold metal substitute. In some embodiments, the cold metal substitute is a natural isotope of a non-radioactive element. In some embodiments, the element may have more than one natural isotope of a non-radioactive element. In some embodiments, 'cold' is used to refer to an isotype of a non-radioactive element. In some embodiments, 'hot' refers to an isotype of a non-radioactive element.

[0360] As used herein and known to those skilled in the art, the 20 common amino acids and their abbreviations follow common usage. Reference is made to the literature [Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991)], which is incorporated herein by reference. In some embodiments, the amino acids of the present disclosure may be stereoisomers of the 20 common amino acids (e.g., D-amino acids). In some embodiments, the amino acids of the polypeptide of the present disclosure may be non-natural amino acids. For example, amino acids, such as α,α-disubstituted amino acids, N-alkyl amino acids, and other non-conventional amino acids may also be suitable components of the polypeptide 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 abundant stable isotope version of leucine), and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The arrangement of polypeptide sequence notations used in this specification has a left end corresponding to an amino terminus and a right end corresponding to a carboxy terminus, according to standard use and convention.

[0361] In some embodiments, the miniprotein disclosed herein has one or more of the following non-conventional 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 (Lys(Ac)), O-phosphorescent, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The arrangement of polypeptide sequence notations used in this specification has a left end corresponding to an amino terminus and a right end corresponding to a carboxy terminus, according to standard use and convention.

[0362] In some embodiments, the miniprotein comprises or consists of a specific amino acid sequence. In some embodiments, the 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%, 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 presented in any of SEQ ID NOs 4 to 6, 8 to 94, and 100 to 537. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in any of SEQ ID NOs 4 to 6, 8 to 94, and 100 to 537. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in any of SEQ ID NOs 4 to 6, 8 to 94, and 100 to 537.

[0363] In some embodiments, the miniprotein includes or consists of a specific amino acid sequence. 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%, 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 presented in any of SEQ ID NOs 4 to 6, 8 to 94, or 100 to 537 In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in any of SEQ ID NOs 4 to 6, 8 to 94, or 100 to 537. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in any of SEQ ID NOs 4 to 6, 8 to 94, or 100 to 537.

[0364] A polypeptide according to the present disclosure (e.g., a miniprotein) has an amino acid sequence with a certain percentage of identity relative to a certain percentage of coverage (e.g., of a reference sequence). That is, for example, a B7-H3 bonded polypeptide as provided herein (e.g., a reference molecule) may have a percentage of identity of 70, 75, 80, 85, 90, 95, 99, or 100 percent over a percentage of coverage of the molecule (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%) but with a given query, and if the query molecule is shorter, it may have different percentages of identity and coverage (e.g., 100% identity and 90% coverage). If the query molecule is longer, the identity percentage and coverage percentage may each be 100% with respect to the reference molecule, and the reference molecule will have an identity percentage over the length of the query molecule (e.g., at least 20, 25, or 30 amino acids). For example, in some embodiments, the reference sequence (e.g., If a miniprotein as provided in this specification is shorter than a query sequence, such query sequence is within the scope of this disclosure if it has the length of a reference sequence aligned with the query sequence, wherein the identity percentage is determined over at least the minimum length of the alignment between the two sequences (query and reference). That is, if the polypeptide disclosed in this specification is longer than the query sequence, the identity percentage is determined by aligning the reference and the query and determining the identity percentage as being between the query and the portion of the reference sequence aligned thereon. Conversely, if the query sequence is longer than the reference sequence, the identity percentage is equivalent to the identity over the portion aligned with the reference sequence. That is, if the reference sequence is shorter, the query sequence may fall within the scope of the reference sequence if aligned at the claimed identity percentage over the aligned portion between the two polypeptides (reference and query).

[0365] In some embodiments, the miniprotein 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%, 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 presented in any of SEQ ID NOs. 4 to 6, 12 to 94, or SEQ ID NOs. 100 to 537. It has an amino acid sequence. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in any of SEQ ID NOs 4 to 6, SEQ ID NOs 12 to 94, or SEQ ID NOs 100 to 537. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in any of SEQ ID NOs 4 to 6, SEQ ID NOs 12 to 94, or SEQ ID NOs 100 to 537.

[0366] In some embodiments, the miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 99% or more) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids presented in any one of SEQ ID NOs 4 to 6, SEQ ID NOs 12 to 94, or SEQ ID NOs 100 to 537. In some embodiments, the 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 presented in any one of SEQ ID NOs 4 to 6, SEQ ID NOs 12 to 94, or SEQ ID NOs 100 to 537.

[0367] In some embodiments, the miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or more) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids presented in any one of SEQ ID NOs 198 to 537. In some embodiments, the 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 presented in any one of SEQ ID NOs 198 to 537.

[0368] In some embodiments, the miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or more) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids presented in any one of SEQ ID NOs 204 and 262 to 272. In some embodiments, the 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 presented in any one of SEQ ID NOs 204 and 262 to 272.

[0369] In some embodiments, the miniprotein comprises the amino acid sequence presented in any of SEQ ID NOs. 6, 21, 30, 130 to 131, 183, 199, 204, 241, or 267 and 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%, It has an amino acid sequence that is 99.8%, 99.9%, or 100% identical. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in any of SEQ ID NOs 6, 21, or 30. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in any of SEQ ID NOs 6, 21, 30, 130 to 131, 183, 199, 204, 241, or 267.

[0370] In some embodiments, the 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 presented in SEQ ID NO. 6. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 6. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 6.

[0371] In some embodiments, the 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 presented in SEQ ID NO. 21. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 21. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 21.

[0372] In some embodiments, the 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 presented in SEQ ID NO. 30. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 30. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 30.

[0373] 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 presented in SEQ ID NO. 130. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 130. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 130.

[0374] 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 presented in SEQ ID NO. 131. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 131. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 131.

[0375] 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 presented in SEQ ID NO. 183. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 183. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 183.

[0376] 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 presented in SEQ ID NO. 199. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 199. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 199. In some embodiments, the miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or more) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids presented in SEQ ID NO. 199. In some embodiments, the 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 presented in SEQ ID NO. 199.

[0377] 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 presented in SEQ ID NO. 204. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 204. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 204. In some embodiments, the miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or more) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids presented in SEQ ID NO. 204. In some embodiments, the 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 presented in SEQ ID NO. 204.

[0378] 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 presented in SEQ ID NO. 241. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 241. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 241. In some embodiments, the miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or more) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids presented in SEQ ID NO. 241. In some embodiments, the 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 presented in SEQ ID NO. 241.

[0379] 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 presented in SEQ ID NO. 267. In various embodiments, the miniprotein has an amino acid sequence that is 90% identical to the amino acid sequence presented in SEQ ID NO. 267. In various embodiments, the miniprotein has an amino acid sequence that is 100% identical to the amino acid sequence presented in SEQ ID NO. 267. In some embodiments, the miniprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or more) identical to at least 40, 41, 42, 43, 44, 45, 46, 47, or 48 amino acids presented in SEQ ID NO. 267. In some embodiments, the 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 presented in SEQ ID NO. 267.

[0380] In some embodiments, the miniprotein may further comprise an adduct on one or more additional amino acids at its N- and / or C-terminus, for example.

[0381] In some embodiments, the B7-H3 binding miniprotein according to the present disclosure may have different N- and / or C-terminal ends, e.g., acetyl, NH2, biotin-PEG4, DOTA-PEG4, radiolabels, etc. on the N-terminus and -OH or -NH2 on the C-terminus. The N- and / or C-terminus of the B7-H3 binding miniprotein of the present disclosure may comprise, but are not limited to, acetyl, acid, or amide (e.g., acetyl, NH2, OH) as provided in the exemplary compounds and miniproteins of Table 2A or Table 2C.

[0382] In some embodiments, polypeptides according to the present disclosure may have various modifications at the N-terminus (e.g., linkers, chelators, and / or radionuclides, e.g., those presented in the exemplary compounds of Table 2A or Table 2C) or the C-terminus (e.g., linkers, chelators, and / or radionuclides). In some embodiments, the C-terminus of a given polypeptide may have an acid or amide group on the C-terminus (e.g., see Table 2A, e.g., Table 2C). A given polypeptide having a specific amino acid sequence may, for example, bind to B7-H3 (e.g., without significant changes in the utility or function of the polypeptide) For the detection and / or treatment of cancer) it may have one or more N-terminal and / or C-terminal differences.

[0383] In some embodiments, addition on the N and / or C-terminus may include one or more of a linker and / or chelator, e.g., N-terminal DOTA-PEG4. In some embodiments, the C-terminus of the miniprotein includes NH2, OH and / or COOH.

[0384] 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 sequences presented in SEQ ID NOs 4 to 6, 8 to 94 and 100 to 537. In some of these embodiments, the miniprotein is included in the compound. In some embodiments, the compound is selected from any of compound numbers C1 to C608 and C611.

[0385] 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 presented in SEQ ID NOs 4 to 6, 8 to 94, or 100 to 537. In some of these embodiments, the miniprotein is included in the compound. In some embodiments, the compound is selected from any of compound numbers C1 to C9, C11 to C117, or C121 to C608 and C611.

[0386] In some embodiments, the miniprotein comprises one or more restrictions (e.g., It does not include disulfide bridges. 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 presented in SEQ ID NOs 4 to 6, 8 to 94, or 100 to 197. In some embodiments, the miniprotein is part of the compound. In some embodiments, the compound is selected from any of compound numbers C1 to C226.

[0387] In some embodiments, the miniprotein comprises one or more restrictions (e.g., It does not include 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 sequences presented in SEQ ID NOs 198 to 537. In some embodiments, the miniprotein is part of the compound. In some embodiments, the compound is selected from any of compound numbers C227 to C608 and C611.

[0388] In some embodiments, the miniprotein is, for example, one or more restrictions (e.g., It includes additional variations such as those disclosed herein. In some embodiments, the 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 of these embodiments, the miniprotein may be referred to as a constrained miniprotein. In some embodiments, the constrained miniprotein is, for example, one or more constraints (e.g., It is more stable than, for example, miniproteins without one or more constraints, such as having a higher melting point, having the same or similar amino acid sequence without such disulfide bridges. In some embodiments, the restricted 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 sequences presented in SEQ ID NOs 198 to 537. In some embodiments, the miniprotein is part of the compound. In some embodiments, the compound is selected from any of compound numbers C227 to C608 and C611.

[0389] In some embodiments, the miniprotein as provided herein is Table 1A It is specific to or binds to a polypeptide having an amino acid sequence or a part thereof as presented in [the example].

[0390] Table 1A. Exemplary target protein amino acid sequences

[0391]

[0392] Common sequences and substitutions

[0393] In some embodiments, the miniproteins provided by the present disclosure are Table 1B It is presented in one or more common sequences provided in. In some embodiments, Table 1B The miniprotein having the sequence presented in is Table 1C It has amino acid substitutions as provided in.

[0394] Table 1B. Common sequences of exemplary B7-H3 miniproteins

[0395]

[0396] Table 1C. Common sequence substitutions

[0397]

[0398] In some embodiments, the miniprotein provided by the present disclosure has an amino acid sequence comprising that presented according to the following chemical formula I:

[0399] X1X2

[0400] In the formula, 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; X58 is G, K, (KAc), or absent;

[0401] If X28 is A, (homo-leucine), (MO2), S, T, or V, then X24 is Y or;

[0402] If X28 is M, then X7 is A, E, (Kme3), L, Q, or S.

[0403] In various embodiments, if X4 is K or (KAc), X24 is Y.

[0404] In various embodiments, the miniprotein provided by the present disclosure has an amino acid sequence comprising that presented according to the following chemical formula II:

[0405] X1X2X3

[0406] Here, 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.

[0407] In some embodiments, the miniproteins provided by the present disclosure are Table 1D It is presented in one or more common sequences provided in. In some embodiments, Table 1D The miniprotein having the sequence presented in is Table 1E It has amino acid substitutions as provided in.

[0408] Table 1D. Common sequences of exemplary restricted B7-H3 miniproteins

[0409]

[0410] Table 1E. Restricted common sequence substitutions

[0411]

[0412]

[0413]

[0414]

[0415] In one embodiment, the present disclosure relates to a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula III (SEQ ID NO. 538) as follows (e.g., Provides B7-H3-binding miniprotein:

[0416] CAX3EKIAALSEIIWLPCLX19YAQIX24AFIX28X29LNX32DPCX36SX38X39ILSEAX45ELCS, where 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). In some embodiments, X49 is absent.

[0417] In certain embodiments, polypeptide (e.g., B7-H3-binding miniprotein) has an amino acid sequence comprising any one of the amino acid sequences of SEQ ID NOs 204, 238, 239, 241, 247, 262 to 273, 292, 295, 297, 298, 307, 309, 310, 314 to 317, 335, 336, 338, 352, 374, 389, 392, 396, 401, 460 to 462, 466 to 468, 470, 471, 497, and 528.

[0418] In one embodiment, the present disclosure relates to a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula IV (SEQ ID NO. 539) as follows (e.g., Provides a B7-H3-binding miniprotein: CAX3EKIAALSEIIWLPCLX19YAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, where 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.

[0419] In a specific embodiment, the polypeptide (e.g., B7-H3-binding miniprotein) has an amino acid sequence comprising any one of the amino acid sequences of SEQ ID NOs 204, 241, 262, 265, 267, 268, 292, 307, 314 to 317, 338, 352, 392, 396 and 401.

[0420] In one embodiment, the present disclosure relates to a polypeptide having an amino acid sequence comprising an amino acid sequence according to the following formula V (SEQ ID NO. 540) (e.g., Provides a B7-H3-binding miniprotein: CAX3EKIAALSEIIWLPCLTYAQIX24AFIX28X29LNX32DPCQSSEILSEAX45ELCS, where X3 is (Kme3) or R or (Rme); X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme); X32 is D or (Kme) or (Cit); and X45 is K or (Kme). In some embodiments, X49 is absent.

[0421] In a specific embodiment, the polypeptide (e.g., B7-H3-binding miniprotein) has an amino acid sequence comprising any one of the amino acid sequences of SEQ ID NOs 204, 241, 262, 265 to 267, 270, 272, 292, 307, 314 to 317, 338, 352, 374, 389, 392, 396, 401, 460 to 462, 466, 467, 468, 470, 471, and 497.

[0422] In one embodiment, the present disclosure relates to a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula VI (SEQ ID NO. 541) as follows (e.g., Provides a B7-H3-binding miniprotein: CA(Kme3)EKIAALSEIIWLPCLTYAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, where 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.

[0423] In a specific embodiment, the polypeptide (e.g., B7-H3-binding miniprotein) has an amino acid sequence comprising any one of the amino acid sequences of SEQ ID NOs 204, 262, 265, 267, 270, 272, 314 to 317, 338, 352, 389, 392, 396, 401, 462 and 471.

[0424] In one embodiment, the present disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) having an amino acid sequence comprising an amino acid sequence according to Formula VII (SEQ No. 542) as follows: CAX3EKIX7ALX10EIIWLPX17LTYX21QIX24AFIX28X29LNX32DPCQSX38X39X40LX42EAX45ELCS, 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); 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. In some embodiments, X49 is absent.

[0425] In certain embodiments, the polypeptide (e.g., B7-H3-binding miniprotein) is SEQ ID NOs 204, 206, 217, 219, 221, 238, 239, 241, 247, 250, 262 to 267, 273, 278 to 280, 287, 288, 292, 295, 297 to 302, 304, 305, 307, 309 to 319, 321 to 326, 328 to 331, 335 to 338, 352, 356, 366, 367, 374 to 377, 383 to 385, 389, 392, 394 It has an amino acid sequence comprising any one of the amino acid sequences of 396, 401, 402, 404 to 409, 414 to 418, 426, 437 to 439, 449 to 451, 460 to 462, 465 to 468, 470, 471, 489, 497, 502 to 508, 518 to 521, 523, and 527.

[0426] In one embodiment, the present disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) having an amino acid sequence comprising an amino acid sequence according to Formula VIII (SEQ No. 543) as follows: CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIX28X29LNX32DPCQSX38X39X40LX42EAX45ELCS, 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); 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. In some embodiments, X49 is absent.

[0427] In certain embodiments, the polypeptide (e.g., B7-H3-binding miniprotein) is SEQ ID NOs 204, 206, 238, 239, 241, 247, 250, 262 to 267, 273, 278 to 280, 287, 288, 292, 295, 297 to 302, 304, 305, 307, 309 to 319, 321 to 326, 328, 329, 330, 331, 335 to 338, 352, 356, 366, 367, 374, 375 to 377, 383 to 385, 389, 392, 394 It has an amino acid sequence comprising any one of the amino acid sequences of 396, 401, 402, 404 to 409, 414 to 418, 426, 437 to 439, 449 to 451, 460 to 462, 465 to 468, 470, 471, 489, 497, 502 to 508, 518 to 521, 523, and 527.

[0428] In one embodiment, the present disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) having an amino acid sequence comprising an amino acid sequence according to Formula IX (SEQ No. 546) as follows: CAX3EKIX7ALX10EIIWLPX17LTYX21QIX24X25FIX28X29LNX32DPCQSX38X39X40LX42EAX45X46LX48S, 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; X48 is C or N. In some embodiments, X49 is absent.

[0429] In certain embodiments, polypeptides (e.g., B7-H3-binding miniproteins) are SEQ ID NOs 203 to 205, 209 to 213, 216 to 220, 225, 226, 230 to 233, 235, 238, 239, 241, 242, 247, 248, 250, 262 to 267, 270, 272, 273, 278 to 280, 287, 288, 292, 294 to 302, 304, 305, 307, 309 to 319, 321 to 326, 328, 329, 330, 331, 335 to 338, 352, 356, 358, 366, 367, 374 to 377, 380, 383 to 385, 389, 392, 394 to 396, 399, 401, 402, 404 to 409, 414 to 418, 426, 434, 437 to 439, 447, 449, 450, 451, 460 to 462, 465 to 468, 470, 471, 474, 475, 489, 497, 499, 502 to 508, 515, 518 to 521, 523, 524, 527, 532, It has an amino acid sequence containing any one of the amino acid sequences of 533, 535, and 537.

[0430] In one embodiment, the present disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) having an amino acid sequence comprising an amino acid sequence according to the following formula X (SEQ No. 547): CAX3EKIX7ALX10EIIWLPX17LTYX21QIX24AFIX28X29LNX32DPCQSX38X39X40LX42EAX45ELX48S, 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; X48 is C or N. In some embodiments, X49 is absent.

[0431] In certain embodiments, the polypeptide (e.g., B7-H3-binding miniprotein) is SEQ ID NOs 204, 209 to 213, 217 to 220, 225, 226, 238, 239, 241, 242, 247, 250, 262 to 267, 273, 278 to 280, 287, 288, 292, 295, 297 to 302, 304, 305, 307, 309 to 319, 321 to 326, 328, 329, 330, 331, 335 to 338, 352, 356, 366, 367, 374 to 377, 383 It has an amino acid sequence comprising any one of the amino acid sequences of 385, 389, 392, 394 to 396, 401, 402, 404 to 409, 414 to 418, 426, 437 to 439, 449 to 451, 460 to 462, 465 to 468, 470, 471, 489, 497, 502 to 508, 518 to 521, 523, and 527.

[0432] In one embodiment, the present disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) having an amino acid sequence comprising an amino acid sequence according to the following formula XI (SEQ No. 548): CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIX28X29LNDDPCQSX38X39X40LX42EAX45ELX48S, 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; X48 is C or N. In some embodiments, X49 is a part.

[0433] In certain embodiments, a polypeptide (e.g., a B7-H3-binding miniprotein) is SEQ ID NOs 204, 217 to 220, 238, 239, 241, 242, 247, 250, 262 to 266, 273, 278, 279, 280, 287, 288, 292, 295, 297 to 302, 304, 305, 307, 309 to 319, 321 to 326, 328, 329, 330, 331, 335 to 338, 352, 356, 366, 367, 374 to 377, 383 to 385, 389, It has an amino acid sequence comprising any one of the amino acid sequences of 392, 394 to 396, 402, 404 to 409, 414 to 418, 426, 437 to 439, 449, 460, 466 to 468, 471, 489, 497, and 502 to 508.

[0434] In one embodiment, the present disclosure provides a polypeptide (e.g., a B7-H3-binding miniprotein) having an amino acid sequence comprising an amino acid sequence according to the following formula XII (SEQ No. 549): CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIX28X29LNDDPCQSX38X39X40LX42EAX45ELX48S, 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); X48 is C or N. In some embodiments, X49 is a component.

[0435] In certain embodiments, the polypeptide (e.g., B7-H3-binding miniprotein) is SEQ ID NOs 204, 217 to 220, 238, 239, 241, 242, 247, 262 to 266, 273, 278, 279, 280, 287, 292, 295, 297 to 302, 304, 305, 307, 309 to 319, 321 to 326, 328 to 330, 335 to 338, 352, 356, 366, 367, 374, 383, 384, 392, 395, 396, 402, 404 to 406, It has an amino acid sequence including any one of the amino acid sequences of 414, 415, 417, 426, 437, 439, 460, 466, 468, 502, 503, 505, and 506.

[0436] In one embodiment, the present disclosure relates to a polypeptide having an amino acid sequence comprising an amino acid sequence according to the following chemical formula XIII (SEQ ID NO. 550) (e.g., Provides a B7-H3-binding miniprotein: CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIAX29LNDDPCQSX38EILSEAX45ELCS, where 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; X45 is K or (Kme). In some embodiments, X49 is absent.

[0437] In a specific embodiment, the polypeptide (e.g., B7-H3-binding miniprotein) has an amino acid sequence comprising any one of the amino acid sequences of SEQ ID NOs 204, 238, 241, 262, 263, 265, 292, 295, 297, 302, 304, 305, 307, 309, 313 to 319, 321 to 323, 326, 328, 335, 337, 338, 352, 367, 384, 392, 396, 402, 404, 414, 415, 417, 426, 437, 439, 502, 503, 505, and 506.

[0438] In one embodiment, the present disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising that presented in SEQ ID NO. 541, having a length of at least 48 amino acids and comprising at least 4 cysteine ​​and 2 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.

[0439] In certain embodiments, the present disclosure provides a composition comprising a polypeptide having at least 48 amino acids, two disulfide bonds, and amino acids modified at positions corresponding to 3, 24, and 29 for SEQ ID NO. 267, wherein the modification is a small alkyl group on the side chain of the amino acid.

[0440] In one aspect, the present disclosure provides a composition having one or more modified amino acids. In some of these embodiments, one or more modifications include small alkyl groups. In one aspect, the present disclosure provides a composition comprising a B7-H3 bonded polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acids include (i) cysteine ​​at each of the four positions corresponding to 1, 17, 35 and 48 of SEQ ID NO. 267, and 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.

[0441] In one embodiment, the present disclosure provides a composition comprising a B7-H3 bonded polypeptide having an amino acid sequence, wherein the amino acid sequence comprises at least four cysteines forming two disulfide bonds; arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3; lysine at a position corresponding to amino acid 5; isoleucine at a position corresponding to amino acid 6; tryptophan at a position corresponding to amino acid 14; at least one modified lysine residue at a position corresponding to amino acid 24; and alanine, arginine, or modified lysine at a position corresponding to 29, wherein each position is linear from the N-terminus to the C-terminus with respect to SEQ ID NO. 267, and the modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or the guanidino group of the arginine side chain, which optionally comprises a methyl, dimethyl, or trimethyl group; and having a length of at least 48 amino acids; The miniprotein has a binding affinity for B7-H3 stronger than 100 nM when measured in cell-based assays.

[0442] In one embodiment, the present 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 forming 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 and optionally a methyl, dimethyl, or trimethyl group); having a length of at least 48 amino acids; and having a binding affinity for B7-H3 stronger than 100 nM when measured in a cell-based assay.

[0443] CDP

[0444] In some embodiments, the miniprotein of the present disclosure comprises or consists of a cysteine-tight peptide (CDP). In some embodiments, the conjugate provided herein comprises a CDP. In some embodiments, the CDP functions as a targeting moiety that specifically binds to a protein target or antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the CDP comprises or consists of at least two independent folding domains and a high density of cysteine. In some embodiments, the CDP comprises at least 1, 2, 3, 4, 5, 6, or more than 6 cysteine ​​residues within a range of about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues. (For example, regarding exemplary CDPs and their characteristics, see the literature [Correnti et al. (Refer to Nat Struct Mol Biol. 2018 Mar; 25(3):270-278). In some embodiments, the CDP comprises a restricted distribution of cysteine, Cys-X[0-15]-Cys-X[0-15]-Cys-X[0-15]-Cys-X[0-15]-Cys-X[0-15]-Cys (where X represents any amino acid) (SEQ ID NO. 553). In some embodiments, the 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 the range of about 10 to 80 amino acid residues. In some embodiments, the CDP may be further manipulated to modify binding, folding, and / or related properties.

[0445] In some embodiments, CDP specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, CDP specifically binds to B7-H3 or a fragment thereof. In some embodiments, CDP is conjugated to a chelator and / or radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that, in some embodiments, the specific CDP used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0446] In some embodiments, in a miniprotein having cysteine ​​residues, selected cysteine ​​pairs may be replaced with selenocysteine ​​to ensure proper folding and connectivity. In some embodiments, diselenoid crosslinks may be formed more easily because they have a lower redox potential than disulfide crosslinks. In some of these embodiments, this replacement may lead to crosslinking of the remaining cysteine.

[0447] Notin

[0448] In some embodiments, the miniprotein of the present disclosure comprises or is composed of a notin peptide. In some embodiments, the conjugate provided herein comprises a notin peptide. In some embodiments, the notin peptide functions as a targeting moiety that specifically binds to an antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the notin comprises at least three disulfide bonds connected in an arrangement that generates the so-called 'cysteine-knot' named notin. (e.g., the literature [Kintzing & Cochran et al.[See , Curr Opin Chem Biol. 2016 Oct;34:143-150.]). In some embodiments, notin has high stability (e.g., thermal, proteolytic, chemical). In some embodiments, notin may be further manipulated to modify binding, folding and / or related properties.

[0449] In some embodiments, a given notin is highly specific for a given target. In some embodiments, notin specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, notin specifically binds to B7-H3 or a fragment thereof. In some embodiments, notin is conjugated to a chelator and / or a radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that, in some embodiments, the specific notin used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0450] In some embodiments, the folded structure of the miniprotein (e.g., affibody, CDP, notin, binder) makes them rigid, providing a very tight and strong binding to a target protein or antigen (compared to less structured peptides). In some of these embodiments, the miniprotein (e.g., affibody, CDP, notin, binder, engineered Kunitz domain, monobody, anticalin, engineered ankyrin repeat domain (DARPin), avimer) exhibits excellent stability with resistance to heat, peptidase cleavage, and pH.

[0451] binder

[0452] In some embodiments, the miniprotein of the present disclosure comprises or is composed of a binder. In some embodiments, the binder functions as a targeting moiety that specifically binds to a target expressed on the surface of a target tumor cell, for example. In some embodiments, a binder that binds to B7-H3 is provided herein.

[0453] In some embodiments, the binder has a certain structural feature; for example, in some embodiments, the binder may be rich in alpha-helical, e.g., helix-helical-helical structures (e.g., literature [Crook et al. , Nat Commun. (2017) 8, 2244; Berger et al, Elife (2016) 5, e20352; and Procko et al. [See Cell (2014), 157, 1644-1656]). In some embodiments, the binder may comprise an alpha helix, a beta sheet, or a combination of one or more of each. In some embodiments, the binder comprises a surface sufficient to functionalize a molecule on a heterogeneous surface as a binding surface. In some embodiments, the binder comprises an isolated hydrophobic core. In some embodiments, the binder exhibits cooperative folding. In some embodiments, the binder has two or more of the following features: (i) represented by an amino acid sequence of 100 or fewer amino acids; (ii) at least two secondary structural elements; (iii) an isolated hydrophobic core; and / or (iv) cooperative folding.

[0454] In some embodiments, a given binder is highly specific for a given target. In some embodiments, the binder specifically binds to the target. In some embodiments, the target is located within, on, or near the cell. In some embodiments, the miniprotein specifically binds to B7-H3 or a fragment thereof. In some embodiments, the binder is conjugated to a chelator and / or a radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that, in some embodiments, the specific binder used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0455] Affibody

[0456] In some embodiments, the miniprotein of the present disclosure comprises or is composed of an affibody. In some embodiments, the affibody functions as a targeting moiety that specifically binds to a target expressed on the surface of a target tumor cell, for example. In some embodiments, an affibody that binds to B7-H3 is provided herein. In some embodiments, the miniprotein (e.g., affibody) is Table 2A and / or Table 2C It comprises all or part of any compound presented in (e.g., including or composed of a miniprotein). In some embodiments, the miniprotein (e.g., affibody) comprises, is essentially composed of, or is composed of any of SEQ ID NOs 4 to 6, 8 to 94 and 100 to 537 or any part thereof. Table 2B Is Table 2ASpecific characteristics of selected exemplary compounds / miniproteins are indicated. In some embodiments, the conjugate provided herein comprises an affibody. In some embodiments, the affibody functions as a targeting moiety that specifically binds to a protein target or antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the affibody comprises or consists of 100 or fewer amino acids, 90 or fewer amino acids, 80 or fewer amino acids, 70 or fewer amino acids, 60 or fewer amino acids, 50 or fewer amino acids, 40 or fewer amino acids, 30 or fewer amino acids, 20 or fewer amino acids, or 10 amino acids. In some embodiments, the affibody comprises or consists of at least three alpha helices having 58 amino acids. In some embodiments, the affibody comprises target specificity obtained by randomizing 13 amino acids located on 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, the affibody may be further manipulated to modify binding, folding and / or related properties.

[0457] The present disclosure also describes cutting the aphibody (e.g., The present disclosure provides a surprising finding that binding affinity and / or specificity for B7-H3 is improved by removing residues from the N- and / or C-terminus of an affibody having three alpha helices spanning 58 amino acids), for example, by removing some or all of the amino acids corresponding to positions 1 to 6 starting from the N-terminus and positions 54 to 58 at the C-terminus based on the N-to-C-terminal position along the length of SEQ ID NO. 3, and / or by removing and replacing these amino acids, for example, with cysteine. The present disclosure further considers that, in addition to cleavage, one or more restraints may be introduced. In some embodiments, the restraint comprises at least one, two, or three disulfide bridges and / or at least one additional restraint as disclosed herein (e.g., lactam bridges, e.g., staples). In some embodiments, the affibody of a particular scaffold comprises three alpha helices having at least four cysteines (disulfides may be introduced between them) spanning 49 amino acids. Thus, in some of these embodiments, compared to a 58-amino acid affibody, the scaffold has a length (e.g., The truncated and pharmaceutical patterns differ. In some of these embodiments, these truncated and pharmaceutical affibodies may have one or more desirable properties, such as, for example, improved affinity and / or specificity for B7-H3. In certain embodiments, these affibodies may be further modified to introduce one or more non-natural amino acids that may affect toxicity by reducing renal cell uptake (e.g., in vitro, e.g., in vivo).

[0458] In some embodiments, the affibody specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, the affibody specifically binds to B7-H3 or a fragment thereof. In some embodiments, the affibody is conjugated to a chelator and / or a radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that in some embodiments, the specific affibody used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0459] Manipulated Kunitz domain

[0460] In some embodiments, the miniprotein of the present disclosure comprises or consists of a engineered Kunitz domain. In some embodiments, the conjugate provided herein comprises a engineered Kunitz domain. In some embodiments, the engineered Kunitz domain functions as a targeting moiety that specifically binds to a protein target or antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the 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, the engineered Kunitz domain may be further engineered to modify binding, folding, and / or related properties.

[0461] In some embodiments, the engineered Kunitz domain specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, the engineered Kunitz domain specifically binds to B7-H3 or a fragment thereof. In some embodiments, the engineered Kunitz domain is conjugated to a chelator and / or a radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that, in some embodiments, the specific engineered Kunitz domain used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0462] Monobody

[0463] In some embodiments, the miniprotein of the present disclosure comprises or consists of a monobody. In some embodiments, the conjugate provided herein comprises a monobody. In some embodiments, the monobody functions as a targeting moiety that specifically binds to a protein target or antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the monobody comprises or consists of a molecule based on the 10th extracellular domain (10Fn3) of human fibronectin III, which adopts an Ig-like β-sandwich fold of about 94 residues having 2 to 3 exposed loops but lacks a central disulfide bridge. In some embodiments, the monobody may be further manipulated to modify binding, folding, and / or related properties.

[0464] In some embodiments, the monobody specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, the monobody specifically binds to B7-H3 or a fragment thereof. In some embodiments, the monobody is conjugated to a chelator and / or a radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that, in some embodiments, the specific monobody used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0465] Anticalin

[0466] In some embodiments, the miniprotein of the present disclosure comprises or is composed of anticalin. In some embodiments, the conjugate provided herein comprises anticalin. In some embodiments, the anticalin functions as a targeting moiety that specifically binds to a protein target or antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the anticalin comprises or is composed of an 8-strand b-barrel that forms a highly conserved core unit between lipocalins and naturally forms a binding site for a ligand by four structurally variable loops at the open end. In some embodiments, the anticalin may be further manipulated to modify binding, folding, and / or related properties.

[0467] In some embodiments, the anticalin specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, the anticalin specifically binds to B7-H3 or a fragment thereof. In some embodiments, the anticalin is conjugated to a chelator and / or a radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that, in some embodiments, the specific anticalin used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0468] Designed Ankyrin repetitive domain

[0469] In some embodiments, the miniprotein of the present disclosure comprises or is composed of an engineered ankyrin repeat domain. In some embodiments, the conjugate provided herein comprises an engineered ankyrin repeat domain. In some embodiments, the engineered ankyrin repeat domain functions as a targeting moiety that specifically binds to a protein target or antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the engineered ankyrin repeat domain comprises a peptide derived from ankyrin. In some embodiments, the engineered ankyrin repeat domain comprises a single ankyrin repeat comprising a 33-residue motif, preferably comprising two alpha-helices and beta-turns. In some embodiments, the engineered ankyrin repeat domain provides a rigid interface and lacks structural flexibility. In some embodiments, the engineered ankyrin repeat domain may be further manipulated to modify binding, folding, and / or related properties.

[0470] In some embodiments, the designed ankyrin repeat domain specifically binds to a target. In some embodiments, the target is located within, 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, the designed ankyrin repeat domain is conjugated to a chelator and / or a radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that, in some embodiments, the specific designed ankyrin repeat domain used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0471] Abimer

[0472] In some embodiments, the miniprotein of the present disclosure comprises or is composed of an avimer. In some embodiments, the conjugate provided herein comprises an avimer. In some embodiments, the avimer functions as a targeting moiety that specifically binds to a protein target or antigen expressed on the surface of a target tumor cell, for example. In some embodiments, the avimer comprises a peptide of about 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, 80 amino acids, 90 amino acids, or 100 amino acids. In some embodiments, the avimer comprises at least one peptide sequence of about 30 to 35 amino acids. In some embodiments, the avimer comprises two or more than two peptide sequences of about 30 to 35 amino acids. In some embodiments, the avimer comprises one or more peptide sequences derived from the A-domain of various membrane receptors. (Weidle UH, et al. (2013), Cancer Genomics Proteomics; 10(4): 155-68). For further details, refer to [Nature Biotechnology 23(-2), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007)]. In some embodiments, the avimer may be further manipulated to modify binding, folding, and / or related properties.

[0473] In some embodiments, the avimer specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, the avimer specifically binds to B7-H3 or a fragment thereof. In some embodiments, the avimer is conjugated to a chelator and / or a radionuclide. In some embodiments, conjugation is performed via a linker. Those skilled in the art will understand that, in some embodiments, the specific avimer used in the conjugate of this disclosure may vary depending on the target protein or antigen of interest.

[0474] Pharmaceuticals

[0475] The restriction may be designed (e.g., from an initial polypeptide sequence) or introduced (e.g., into an engineered, e.g., existing miniprotein) into a protein such as any miniprotein provided herein. The restriction may be introduced by modification 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.

[0476] Constraints can alter the function of a protein (e.g., binding, e.g., binding affinity, etc.) and / or its structure (e.g., folding). In certain embodiments, constraints can assist in maintaining the secondary structure of a given protein (e.g., a miniprotein as provided herein, e.g., a conjugate as provided herein). For example, a typical protein secondary structure is an alpha helix. Alpha helices can play a key role in both the structure and function of a protein, such as influencing how a particular protein interacts with a binding partner. Alpha helices can mediate protein-protein interactions (PPIs) by acting as recognition motifs. In proteins containing an alpha helix structure, introducing constraints can alter one or more properties of the protein, such as the protein's affinity for a target (e.g., increased affinity), cell penetration (e.g., increased cell penetration), and resistance to proteolysis (e.g., increased resistance to proteolysis).

[0477] Examples of constraints (e.g., α-helical constraints) may 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, disulfide bridges, hydrogen bond substitutes, hydrophobic interactions, metal ligations, triazole staples synthesized from alkenyl and azido side chain residues, phototunable macrocyclic rings, and introduced amino acids, e.g., α,α-disubstituted amino acids. As is known to those skilled in the art, a staple may refer to a synthetic constraint (e.g., a brace) between two previously independent entities. For example, a staple may be formed through a covalent linkage (e.g., peptide macrocyclic formation) between two previously independent entities, such as, for example, amino acid side chains.

[0478] The present disclosure provides insights into manipulating a B7-H3 binding protein to have constraints (e.g., one, two, or three). In some embodiments, the constraints are identical (e.g., of the same type, e.g., disulfide bonds). In certain embodiments, the constraints are a combination of different types of constraints (e.g., a disulfide bridge and a lactam bridge, a disulfide bridge and a stapled alkyl bridge, two disulfide bridges and a lactam bridge, etc.). In certain embodiments, constraints may be added. For example, in certain embodiments, the miniprotein may have one, two, or three disulfide bridges; other constraints such as one or two disulfide bridges and 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, the miniprotein of the present invention may have one, two, or three disulfide bridges and / or one or more additional restraints (e.g., one disulfide bridge and one additional restraint, e.g., two disulfide bridges and one additional restraint, wherein, e.g., one additional restraint is a lactam bridge) and produce a miniprotein that not only binds to B7-H3 with strong affinity but is also absorbed by cancer cells (e.g., in tumors) at higher concentrations compared to an unrestrained B7-H3 miniprotein (e.g., as disclosed in Table 2A). The present disclosure assumes that features such as size (e.g., folded size, e.g., linear size) and stability (e.g., thermal stability) are important for the efficacy of the B7-H3-binding conjugate and also for limiting or preventing toxicity (e.g., nephrotoxicity) due to off-target effects. While not intended to be confined to theory, the present disclosure describes manipulating a B7-H3 binding protein to add a restriction, and in some embodiments, adding such restriction (e.g., ,It provides insight that the miniproteins provided (as presented in Table 2C compared to Table 2A) have a stronger affinity (e.g., for B7-H3), better efficacy (e.g., for approaching cancer cells), greater stability (e.g., thermal stability), and less toxicity (e.g., nephrotoxicity) compared to molecules that are not cleaved or unrestricted (e.g., as presented in Table 2A). In some embodiments, the miniproteins provided herein include one or more restraints, such as disulfide bridges or staples. An exemplary restricted miniprotein is provided in Table 2C. In certain embodiments, the restraint imparts an increase in the thermal stability of the miniprotein (e.g., compared to a miniprotein having an amino acid sequence comprising or consisting of that of SEQ ID NO. 6 or compound C8; e.g. compared to an unrestricted miniprotein having the same or similar primary sequence). In some embodiments, the miniprotein of the present disclosure comprises one or more disulfide bridges. In some embodiments, the miniprotein provided by the present disclosure comprises two or more disulfide bridges. In some embodiments, the miniprotein of the present disclosure comprises one disulfide bridge. In some embodiments, the miniprotein of the present disclosure comprises two disulfide bridges. For example, the miniprotein of the present disclosure herein may contain a set of amino acids that together support or are part of the formation of a pharmaceutical (e.g., a disulfide bridge, e.g., a staple). In some embodiments, the structural characteristic of the miniprotein is having at least two cysteine ​​residues positioned relative to each other so that a disulfide bridge can be formed (e.g., as provided herein, e.g., see Table 2A).In some embodiments, the 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, the miniprotein has three disulfide bridges (from three pairs of cysteine).

[0479] In some embodiments, the miniproteins of the present disclosure comprising two or more cysteine ​​residues such as those presented in Table 2C have cysteine ​​residues connected via a disulfide bridge (e.g., through natural folding).

[0480] In some embodiments, the miniprotein has an amino acid sequence comprising two cysteine ​​and a single disulfide bridge. In some embodiments, the miniprotein has an amino acid sequence comprising four cysteine ​​and two disulfide bridges. In some embodiments, the miniprotein has an amino acid sequence comprising six cysteine ​​and three disulfide bridges.

[0481] In some embodiments, Table 2C Miniproteins of the present disclosure comprising two or more cysteine ​​residues such as those presented herein have cysteine ​​residues connected via disulfide bridges (e.g., through natural folding). In some embodiments, (e.g., The miniprotein (binding to B7-H3) comprises one disulfide bridge. In some embodiments, the miniprotein comprises two disulfide bridges. In some embodiments, the miniprotein comprises three disulfide bridges.

[0482] In some embodiments, one disulfide bridge may be located between cysteine ​​residues corresponding, for example, Cys4 and Cys37; Cys5 and Cys34; Cys5 and Cys37; Cys12 and Cys26; Cys12 and Cys44; Cys17 and Cys48, and the position is based on a linear position from the N-terminus to the C-terminus with respect to SEQ ID NO. 267.

[0483] In some embodiments, the cysteine ​​linkage may be between two different pairs of cysteine ​​residues. For example, in some embodiments, two disulfide bridges may be between positions corresponding to cysteine ​​pairs, e.g., Cys1 and Cys35 and Cys17 and Cys52, in relation to the miniprotein of SEQ ID NO. 213. In some embodiments, the disulfide bridges may be between positions corresponding to Cys1 and Cys35; and Cys17 and Cys48 of the reference sequence (e.g., SEQ ID NO. 267) as presented in Table 2C. In some embodiments, the cysteine ​​linkage is between Cys1 and Cys17; and between Cys35 and Cys48. In some embodiments, the cysteine ​​linkage is between Cys1 and Cys48; and between Cys17 and Cys35.

[0484] In some embodiments, the disulfide bridge or bridges comprise two or four cystes at positions corresponding to 1, 17, 35, and 48 of SEQ ID NO. 267, wherein the cysteine ​​corresponding to position 1 may form a disulfide bridge with the cysteine ​​corresponding to positions 17, 35, or 48. In some embodiments, the cysteine ​​corresponding to position 17 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 35, or 48. In some embodiments, the cysteine ​​corresponding to position 35 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 17, or 48. In some embodiments, the cysteine ​​corresponding to position 48 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 17, or 35. In some embodiments, where four cystes are present and correspond to positions 1, 17, 35, and 48 of SEQ ID NO 267, the pairing may include 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., a disulfide bridge between two cystes of the pair).

[0485] In some embodiments, the cysteine ​​linkage (disulfide bridge) is between Cys1 and Cys35 of the reference sequence as presented in Table 2C, e.g., SEQ ID NO. 267; and between Cys17 and Cys48. In some embodiments, the cysteine ​​linkage is between Cys1 and Cys17 (in relation to the reference sequence, e.g., SEQ ID NO. 267); and between Cys35 and Cys48. In some embodiments, the cysteine ​​linkage is between Cys1 and Cys48; and between Cys17 and Cys35.

[0486] In some embodiments, the disulfide bridge or bridges comprise two or four cystes at positions corresponding to 1, 17, 35, and 48 of SEQ ID NO. 267, wherein the cysteine ​​corresponding to position 1 may form a disulfide bridge with the cysteine ​​corresponding to positions 17, 35, or 48. In some embodiments, the cysteine ​​corresponding to position 17 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 35, or 48. In some embodiments, the cysteine ​​corresponding to position 35 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 17, or 48. In some embodiments, the cysteine ​​corresponding to position 48 may form a disulfide bridge with the cysteine ​​corresponding to positions 1, 17, or 34. In some embodiments, where four cystes are present and correspond to positions 1, 17, 35, and 48 of SEQ ID NO 267, the pairing may include 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., a disulfide bridge between two cystes of the pair).

[0487] In some embodiments, a miniprotein having two disulfide bridges (e.g., from cysteine ​​at positions corresponding to Cys1, Cys17, Cys35, and Cys48, for example of SEQ ID NO. 267) further comprises a third disulfide bridge introduced between the third pair of cysteine. In some embodiments, cysteine ​​may also be introduced at positions corresponding to Cys11 and Cys45.

[0488] In some of these embodiments, these miniproteins further comprise one or more additional restraints (e.g., staples). For example, in certain embodiments, a combination of restraint types (e.g., disulfide bonds and lactam bridges) may be used in a single miniprotein. For example, in some embodiments, the miniprotein may comprise one, two, or three disulfide bridges and at least one additional restraint (e.g., Staples, for example, It may additionally include a lactam bridge. In some embodiments, the lactam bridge may be located between positions corresponding to 11 to 45 of SEQ ID NO. 544.

[0489] In some embodiments, for example, (for example, A miniprotein of the present disclosure comprising one or more modifications such as restrictions (as disclosed in this specification) 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 presented in any of SEQ ID NOs 198 to 537. In various embodiments, the amino acid sequence is 90% of the amino acid sequence presented in any of any sequences 198 to 537 (e.g., 91%, 92%, 93%, 94%, 95, 96, 97, 98, 99% or greater than that are identical. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence presented in any of SEQ ID NOs 198 to 537.

[0490] In some embodiments, for example, (for example, A miniprotein of the present disclosure comprising one or more modifications such as restrictions (as disclosed in this specification) is part of a compound. In some embodiments, the compound has an amino acid sequence selected from any of C227 to C608 and C611 and / or selected from any of SEQ ID NOs 198 to 537.

[0491] In some embodiments, for example, (for example, A miniprotein of the present disclosure comprising one or more modifications such as a restriction (as disclosed in this specification) 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 presented in any of SEQ ID NOs 204 and 262 to 272. In various embodiments, the amino acid sequence is 90% (e.g., the amino acids presented in any of SEQ ID NOs 204 and 262 to 272 and the amino acids (e.g., 91%, 92%, 93%, 94%, 95, 96, 97, 98, 99% or more are identical. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence presented in any of SEQ ID NOs 204 and 262 to 272.

[0492] In some embodiments, the miniprotein of the present disclosure, comprising one or more modifications such as restrictions (e.g., as disclosed herein), is part of the compound. In some embodiments, the compound has an amino acid sequence selected from any of C234, C235, and C298 to C332 and / or selected from any of SEQ ID NOs 204 or 262 to 272.

[0493] In some embodiments, for example, (for example, A miniprotein of the present disclosure comprising one or more modifications such as restrictions (as disclosed herein) 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%, 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 presented in any of SEQ ID NOs 199, 204, 241, or 267. The sequence comprises a sequence. In various embodiments, the amino acid sequence comprises 90% of the amino acids presented in any of SEQ ID NOs 199, 204, 241, or 267 (e.g., 95, 96, 97, 98, 99 or greater than %) is identical. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence presented in any of SEQ ID NOs 199, 204, 241 or 267.

[0494] In some embodiments, the miniprotein of the present disclosure, comprising one or more modifications such as restraints (e.g., as disclosed herein), is part of the compound. In some embodiments, the compound is selected from any other disclosed compound in Table 2C having an amino acid sequence comprising any of C228, C234, C235, C275, C309, C325, C332 or 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.

[0495] In some embodiments, for example, (for example, A miniprotein of the present disclosure comprising one or more modifications such as a restriction (as disclosed in this specification) 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 presented in SEQ ID NO. 199. In various embodiments, the amino acid sequence is the amino acid presented in SEQ ID NO. 199 and 90% (e.g., 95, 96, 97, 98, 99 or greater than %) are identical. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence presented in SEQ ID NO. 199.

[0496] In some embodiments, the miniprotein of the present disclosure, comprising one or more modifications such as restraints (e.g., as disclosed herein), is part of the compound. In some embodiments, the compound is C228 and / or has the amino acid sequence of SEQ ID NO. 199.

[0497] In some embodiments, for example, (for example, A miniprotein of the present disclosure comprising one or more modifications such as a restriction (as disclosed in this specification) 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 presented in SEQ ID NO. 204. In various embodiments, the amino acid sequence is the amino acid presented in SEQ ID NO. 204 and 90% (e.g., 95, 96, 97, 98, 99 or greater than %) are identical. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence presented in SEQ ID NO. 204.

[0498] In some embodiments, the miniprotein of the present disclosure, comprising one or more modifications such as restraints (e.g., as disclosed herein), is part of the compound. In some embodiments, the compound is C234 or C235 and / or has the amino acid sequence of SEQ ID NO. 204.

[0499] In some embodiments, for example, (for example, A miniprotein of the present disclosure comprising one or more modifications such as a restriction (as disclosed in this specification) 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 presented in SEQ ID NO. 241. In various embodiments, the amino acid sequence is the amino acid presented in SEQ ID NO. 241 and 90% (e.g., 95, 96, 97, 98, 99 or greater than %) are identical. In various embodiments, the amino acid sequence is 100% identical to the amino acid sequence presented in SEQ ID NO. 241.

[0500] In some embodiments, the miniprotein of the present disclosure, comprising one or more modifications such as restraints (e.g., as disclosed herein), is part of the compound. In some embodiments, the compound is C275 and / or has the amino acid sequence of SEQ ID NO. 241.

[0501] In some embodiments, ...

Claims

Claim 1 A composition comprising a polypeptide having an amino acid sequence, wherein the amino acid sequence comprises (a) at least two constraints; (b) arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3; lysine at a position corresponding to amino acid 5; isoleucine at a position corresponding to amino acid 6; and tryptophan at a position corresponding to amino acid 14. (c) at least one modified lysine residue at a position corresponding to amino acid 24, and alanine, arginine, or modified lysine at a position corresponding to 29 (wherein each position is linear from N-terminus to C-terminus starting from position 1 with respect to SEQ ID NO. 267, said modification comprises at least one small alkyl group attached to the nitrogen of the lysine side chain or the guanidino nitrogen of the arginine side chain, said at least one small alkyl group optionally comprises a methyl, dimethyl, or trimethyl group); (c) comprising a length of at least 48 amino acids; and (d) having a binding affinity for B7-H3 stronger than 100 nM when measured in a cell-based assay. Claim 2 In claim 1, the composition wherein the at least two constraints have at least two disulfide bridges. Claim 3 A composition according to claim 1 or 2, further comprising at least one additional restriction. Claim 4 A composition according to paragraph 3, wherein at least one additional constraint is a lactam bridge. Claim 5 In paragraph 3, the composition wherein at least one additional constraint is a disulfide bridge. Claim 6 A composition according to any one of claims 1 to 5, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs 204 and 262 to 272. Claim 7 A composition according to any one of claims 1 to 6, wherein the polypeptide is part of a compound selected from any one of C234 to C235 and C309 to C332. Claim 8 A composition comprising a polypeptide having a length of at least 48 amino acids, an amino acid sequence according to Formula VI (SEQ ID NO. 541), and comprising at least 4 cysteine ​​and 2 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. Claim 9 A composition comprising a polypeptide having an amino acid sequence including an amino acid sequence according to the following chemical formula III (SEQ No. 538): CAX3EKIAALSEIIWLPCLX19YAQIX24AFIX28X29LNX32DPCX36SX38X39ILSEAX45ELCS, where 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; X45 is K or (Kme). Claim 10 A composition according to any one of claims 1 to 9, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs 204 and 262 to 272. Claim 11 A composition according to any one of claims 1 to 10, wherein the polypeptide is part of a compound selected from any one of C234 to C235 and C309 to C332. Claim 12 Composition comprising a polypeptide having an amino acid sequence including an amino acid sequence according to the following chemical formula IV (SEQ No. 539): CAX3EKIAALSEIIWLPCLX19YAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, where 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; X45 is (Kme) or K. Claim 13 A composition according to claim 12, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs 204, 262, 265, 267 and 268. Claim 14 A composition according to claim 12 or 13, wherein the polypeptide is part of a compound selected from any one of C234, C235, C298, C299, C304, C305, C308 to C311, C320, C323, C325, C326 and C332. Claim 15 Composition comprising a polypeptide having an amino acid sequence including an amino acid sequence according to the following chemical formula V (SEVENTEEN NO. 540): CAX3EKIAALSEIIWLPCLTYAQIX24AFIX28X29LNX32DPCQSSEILSEAX45ELCS, where 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. Claim 16 A composition according to 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. Claim 17 A composition according to claim 15 or 16, wherein the polypeptide is part of a compound selected from any one of C275, C276, C298, C299, C304 to C309, C314, C315, C318, C319, C323 to C325, C328, C330 and C332. Claim 18 Composition comprising a polypeptide having an amino acid sequence including an amino acid sequence according to the following chemical formula VI (SEQ No. 541): CA(Kme3)EKIAALSEIIWLPCLTYAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, where 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. Claim 19 A composition according to claim 18, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NOs 204, 262, 265, 267, 270 and 272. Claim 20 A composition according to 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 to C320, C323, C325, C328, C330 and C332. Claim 21 A composition comprising a B7-H3 bonded polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acids include (i) cysteine ​​at each of the four positions corresponding to 1, 17, 35 and 48 of SEQ ID NO. 267, and 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. Claim 22 A composition comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: (a) at least four cysteines forming 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 and optionally comprising a methyl, dimethyl, trimethyl, or isopropyl group); (c) having a length of at least 48 amino acids; and (d) having a binding affinity for B7-H3 stronger than 100 nM in a cell-based assay. Claim 23 A composition according to any one of claims 1 to 22, wherein the polypeptide is a miniprotein. Claim 24 A composition according to any one of claims 1 to 23, wherein the polypeptide has a length of at least 48 amino acids to 100 amino acids or less. Claim 25 A composition according to any one of claims 1 to 24, wherein the polypeptide binds to B7-H3 with an affinity stronger than 100 nM in a cell-based assay. Claim 26 A composition according to any one of claims 1 to 25, wherein the amino acid sequence of the polypeptide shares at least 90% identity with any one of SEQ ID NOs 204 and 262 to 537 and comprises at least one lysine or arginine having at least one variant bonded to the nitrogen of the lysine side chain or the guanidino group nitrogen of the arginine side chain and optionally comprising at least one small alkyl group selected from trimethyl, dimethyl, and monomethyl. Claim 27 A composition according to any one of claims 1 to 26, wherein the amino acid sequence of the polypeptide shares at least 90% identity with at least 44 amino acids of a reference polypeptide, wherein the reference polypeptide has an amino acid length longer than 47 and binds to B7-H3 with an intensity of at least 10 nM in a cell-based assay and / or has an inhibition constant not weaker than 10 nM. Claim 28 A composition according to any one of claims 1 to 27, wherein the amino acid sequence of the polypeptide shares at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537, provided that the 44 amino acids comprise at least 4 cysteine ​​residues forming at least 2 disulfide bridges. Claim 29 A composition according to any one of claims 1 to 28, wherein the amino acid sequence of the polypeptide shares 100% identity with at least 44 amino acids of the reference polypeptide, and the reference polypeptide has an amino acid length longer than 47. Claim 30 A composition according to any one of claims 1 to 29, wherein the amino acid sequence shares at least 90% identity with at least 44 amino acids as presented in any one of SEQ ID NOs 199, 204, 241 or 262 to 272. Claim 31 A composition according to any one of claims 1 to 30, wherein the amino acid sequence shares 100% identity with at least 44 amino acids as presented in any one of SEQ ID NOs 199, 204, 241 or 262 to 272. Claim 32 A composition according to claim 30 or 31, wherein the amino acid sequence comprises four or more cystes having at least two disulfide bridges. Claim 33 In claim 32, the polypeptide is a composition having a length of at least 48 amino acids. Claim 34 A composition comprising a polypeptide having an amino acid sequence including SEQ ID NO.

267. Claim 35 A composition comprising the compounds C234, C235 and C298 to C333 presented in Table 2C. Claim 36 A composition comprising a polypeptide having an amino acid sequence comprising any one of SEQ ID NOs 204 and 262 to 272. Claim 37 A composition according to any one of claims 1 to 36, further comprising a radioactive nuclide. Claim 38 A composition according to 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. Claim 39 A composition according to any one of claims 1 to 38, wherein the C-terminus comprises -OH or -NH2. Claim 40 A composition according to any one of claims 1 to 39, wherein the binding affinity for B7-H3 is stronger than 100 nM. Claim 41 A composition according to any one of claims 1 to 40, wherein the inhibition constant is 100 nM or less. Claim 42 A composition according to any one of claims 1 to 41, further comprising one or more of a linker, a chelator, and a radionuclide. Claim 43 A composition according to claim 42, wherein the linker comprises or is composed of any fragment or combination thereof through covalent bonding of PEG4, PEG2, PEG, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4 polyethylene glycol (PEG) linkers, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 36), (Gly)1-10, or any fragment or combination thereof. Claim 44 A composition according to claim 42 or 43, wherein the chelator comprises or is composed of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB). Claim 45 A composition according to any one of claims 42 to 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. Claim 46 A composition according to any one of claims 42 to 45, wherein the polypeptide comprises any one of SEQ ID NOs 198 to 537, the polypeptide further comprises a linker, said linker is PEG4 and a selective chelator, and said chelator is DOTA. Claim 47 A composition according to any one of claims 42 to 46, wherein the linker, if present, is attached to the N-terminus of the polypeptide. Claim 48 A composition according to any one of claims 1 to 47, wherein the C-terminal amino acid of the polypeptide is not cysteine. Claim 49 A composition according to any one of claims 42 to 48, wherein the chelator, if present, is attached to either the polypeptide or the linker. Claim 50 A composition according to any one of claims 41 to 48, wherein the radioactive nuclide, when present, is attached to the chelator. Claim 51 A composition comprising a chemical formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a polypeptide (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), x is 1, 2, 3, or 4, and M comprises an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537. Claim 52 In claim 51, M is a composition having an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 198 to 537. Claim 53 A composition according to claim 51 or 52, wherein M comprises an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537. Claim 54 A composition according to any one of claims 51 to 53, wherein M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs 198 to 537. Claim 55 A composition according to any one of claims 51 to 54, wherein the linker comprises or is composed of any fragment or combination thereof through covalent bonding of PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4 polyethylene glycol (PEG) linkers, ester linkers, amide linkers, maleimide linkers, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linkers, propanoic acid linkers, dTyr-Gly-Phe(yGF) linkers, caproleic acid linkers, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 36), (Gly)1-10, or any fragment or combination thereof. Claim 56 A composition according to any one of claims 51 to 55, wherein the chelator comprises or is composed of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB). Claim 57 A composition according to any one of claims 51 to 56, 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. Claim 58 A composition comprising a chemical formula selected from one or more of (M)xLCR, (M)xLC, (M)xCR, (M)xLR, (M)xC, (M)xL, and (M)xR, wherein M comprises a polypeptide (M), L comprises a linker (L), C comprises a chelator (C), R comprises a radionuclide (R), x is 1, 2, 3, or 4, and M comprises an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 204 and 262 to 272. Claim 59 In claim 58, M is a composition having an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 204 and 262 to 272. Claim 60 A composition according to claim 58 or 59, wherein M comprises an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 204 and 262 to 272. Claim 61 A composition according to any one of claims 58 to 60, wherein M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs 204 and 262 to 272. Claim 62 In any one of claims 58 to 61, where L is present, L is a polyethylene glycol (PEG) linker, ester linker, amide linker, maleimide linker, valine-citrulline linker, hydrazone linker, N-succinimidyl-4-(2-pyridylditeo)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, vinylsulfone-based linker, propanoic acid linker, dTyr-Gly-Phe(yGF) linker, caploreic acid linker, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 10). A composition comprising (Gly)1-10 or any fragment or combination thereof through covalent bonding. Claim 63 A composition according to any one of claims 58 to 62, wherein C is present, C comprises or is composed of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB). Claim 64 A composition according to any one of claims 58 to 63, wherein if R is present, R comprises or is composed 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. Claim 65 A composition according to any one of claims 58 to 64, wherein the linker, if present, is attached to the N-terminus of the polypeptide. Claim 66 In paragraph 65, the composition wherein the C-terminal amino acid of the polypeptide is not cysteine. Claim 67 A composition according to any one of claims 58 to 66, wherein the chelator, if present, is attached to either the polypeptide or the linker. Claim 68 A composition according to any one of claims 58 to 67, wherein the radioactive nuclide, when present, is attached to the chelator. Claim 69 A composition according to any one of claims 58 to 68, wherein the polypeptide comprises at least one disulfide bridge. Claim 70 In claim 69, the polypeptide comprises at least two disulfide bridges. Claim 71 A composition according to any one of claims 1 to 70, wherein the composition and / or the polypeptide thereof selectively binds to B7-H3. Claim 72 A composition according to any one of claims 1 to 71, 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 when measured in vivo, in vitro, in vitro, and / or in a cell-based assay. Claim 73 A composition according to any one of claims 1 to 72, wherein the polypeptide has a binding inhibition constant of 100 nM or more. Claim 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 with at least 48 amino acids of a polypeptide having an amino acid sequence presented in any one of SEQ ID NOs 198 to 537. Claim 75 In claim 74, the polypeptide is a composition having at least four cysteine ​​groups and two disulfide bridges. Claim 76 In claim 74 or 75, the drug moiety is a focal isomerase inhibitor, auristatin (e.g., monomethylauristatin E), V-ATPase inhibitor, apoptosis promoter, Bcl2 inhibitor, MCL1 inhibitor, HSP90 inhibitor, IAP inhibitor, mTor inhibitor, microtubule stabilizer, microtubule destabilizer, dolastine, metansinoid, MetAP (methionine aminopeptidase), inhibitor of extranuclear transport of protein CRMl, DPPIV inhibitor, proteasome inhibitor, inhibitor of phosphoryl transfer reaction in mitochondria, protein synthesis inhibitor, kinase inhibitor, CDK2 inhibitor, CDK9 inhibitor, kinesin inhibitor, HDAC inhibitor, DNA damaging agent, DNA alkylating agent, DNA intercalator, DNA minor groove binder [DNA minor groove A composition selected from binder, DHFR inhibitor, and immunotoxin. Claim 77 A composition comprising an isolated compound or a pharmaceutically acceptable salt or neutral molecule thereof, comprising a selective linker (L) and one or more of a polypeptide (M), a chelator (C), or a radionuclide (R), wherein M comprises amino acid substitutions as presented in Table 1E, and having an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537, 538 to 543, and 546 to 550. Claim 78 A composition comprising a compound designed to bind to B7-H3, wherein the compound comprises or consists of a polypeptide having an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, comprising amino acid substitutions as presented in Table 1E, and further comprising a modified N and / or C-terminus. Claim 79 A composition according to claim 77 or 78, wherein M comprises an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, comprising amino acid substitutions presented in Table 1E. Claim 80 A composition according to any one of claims 77 to 79, wherein M comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, comprising amino acid substitutions presented in Table 1E. Claim 81 A composition according to any one of claims 77 to 80, wherein M comprises an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, including amino acid substitutions presented in Table 1E. Claim 82 A composition according to any one of claims 77 to 81, wherein M comprises an amino acid sequence having 100% identity with any one of SEQ ID NOs 198 to 537 or 538 to 543 and 546 to 550, comprising amino acid substitutions presented in Table 1E. Claim 83 A composition according to any one of claims 78 to 82, wherein the modified N-terminus comprises one or more of NH2-, acetyl-, PEGn- (wherein=0 to 36), DOTA-, or biotin-. Claim 84 A composition according to any one of claims 1 to 83, wherein the C terminal comprises -NH2 or -OH. Claim 85 A composition according to any one of claims 1 to 84, wherein the polypeptide selectively binds to B7-H3. Claim 86 A composition according to any one of claims 1 to 85, wherein the polypeptide has a binding affinity stronger than about 100 nM for B7-H3 in vivo or cell-based assays. Claim 87 A compound comprising a miniprotein having an amino acid sequence having at least 90% identity with at least 44 amino acids of SEQ ID NO. 267, wherein the compound further comprises one or more additional components according to the chemical formula MLCR, wherein M is a miniprotein, L is a linker, C is a chelator, and R is a radionuclide. Claim 88 A compound according to claim 87, wherein M has an amino acid sequence comprising an amino acid sequence having 100% identity with respect to at least 44 amino acids of SEQ ID NO.

267. Claim 89 In claim 87, M is a compound having an amino acid sequence comprising an amino acid sequence having at least 90% identity with SEQ ID NO 267. Claim 90 In claim 87, M is a compound having an amino acid sequence comprising an amino acid sequence having 100% identity with SEQ ID NO.

267. Claim 91 A compound according to any one of claims 87 to 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 presented in Table 2A or Table 2C, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 10), (Gly)1-10, or any fragment or combination thereof through covalent bonding. Claim 92 A method according to any one of claims 87 to 91, wherein C comprises or is composed of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB). Claim 93 A compound according to any one of claims 87 to 92, wherein R comprises or is composed 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. Claim 94 A compound comprising a miniprotein having at least 90% identity with at least 48 amino acids of the amino acid sequence of SEQ ID NO. 267, wherein the N and / or C-terminus has 1 to 30 additional amino acids and / or the C-terminus has less than 1 amino acid or up to 30 additional amino acids, provided that the total miniprotein has a length of about 100 amino acids or less. Claim 95 A pharmaceutical composition comprising a polypeptide of any one of claims 1 to 86 or a compound of any one of claims 87 to 94 and a pharmaceutically acceptable excipient. Claim 96 A method for improving the binding affinity strength of a polypeptide to B7-H3, wherein the improvement comprises modifying at least three amino acid residues of the polypeptide, wherein the polypeptide has a length of at least 48 amino acids and has cysteine ​​at positions corresponding to 1, 17, 35 and 48 of SEQ ID NO. 267, and the position corresponding to X24 is (Kme) or (Kme2); X29 is A or (Kme); X32 is D or (Kme) or (Cit); and X49 is S or absent. Claim 97 A method for treating cancer, comprising the step of administering to a subject requiring treatment for cancer a composition comprising a conjugate comprising a polypeptide having an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537 and a radionuclide. Claim 98 In claim 97, the polypeptide has at least four cysteine ​​and two disulfide bridges. Claim 99 A method according to claim 97 or 98, wherein the radionuclide is associated with a polypeptide by a linker and / or chelator according to the chemical formula MLCR, wherein M is the polypeptide, L is the linker, C is the chelator, and R is the radionuclide. Claim 100 A method according to claim 97 or 99, wherein the polypeptide comprises SEQ ID NO. 204 or 262 to 272 or has an amino acid sequence composed thereof. Claim 101 A method according to claim 99 or 100, wherein L comprises or is composed 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 as presented in Table 2A, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 10), (Gly)1-10, or any fragment or combination thereof through covalent bonding. Claim 102 A method according to any one of claims 99 to 101, wherein C comprises or is composed of DOTA, Crown, NOPO, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB). Claim 103 A method according to any one of claims 99 to 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. Claim 104 In paragraph 103, R is a method that is a therapeutic agent and / or an imaging agent. Claim 105 A method according to claim 103 or 104, wherein R is Cu-64, Ga-68, Lu-177, In-111, Cu-67, La-132 or F-18. Claim 106 A method according to any one of claims 96 to 105, wherein the polypeptide has a length of 100 or fewer amino acids. Claim 107 A method for reducing renal cell uptake of a composition and / or increasing tumor uptake, comprising the step of 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 and optionally comprising methyl, dimethyl, or trimethyl, and the reduction is compared with administering to the subject or a control subject the same composition except that the modified lysine residue at the position corresponding to X24 is not included. Claim 108 A method for treating cancer, wherein improvement comprises administering a composition comprising a B7-H3 binding protein having an amino acid sequence including 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 and optionally including methyl, dimethyl, or trimethyl, and reduction comprises administering the same composition to a subject or a control subject, wherein the modified lysine residue at a position corresponding to X24 is not included but otherwise identical. Claim 109 A method for treating a subject having intractable or recurrent cancer, comprising the step of administering a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, or a pharmaceutical composition according to claim 95, wherein the treatment is said to treat the cancer. Claim 110 A method for improving the biodistribution of a pharmaceutical composition to a B7-H3-positive population of cancer cells in a subject having B7-H3-positive cancer, comprising the step of contacting said population with a polypeptide having a modified lysine at the position corresponding to X24 of SEQ ID NO. 241, wherein said lysine is modified by adding at least one small alkyl group to the lysine side chain, and said biodistribution is improved compared to contacting a population without said modified lysine at the position corresponding to X24 of SEQ ID NO.

241. Claim 111 A method for diagnosing the presence of a B7-H3 positive population of cancer cells, comprising: (a) contacting a population of cells with a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, or a pharmaceutical composition according to claim 95; (b) detecting the presence of said composition, compound, or pharmaceutical composition of step (a) by measuring a signal; and (c) comparing the detection in step (b) with a control signal; and (d) diagnosing cancer when said composition, compound, or pharmaceutical composition of step (a) is detected in greater quantities than the control. Claim 112 In paragraph 111, the method wherein the above contact is performed by administering to a subject in need. Claim 113 In paragraph 112, the above administration is performed intravenously or subcutaneously. Claim 114 In paragraph 111, the method wherein the contact is performed outside the subject using a biopsy sample, optionally in vitro. Claim 115 A method for treating cancer of a subject, comprising the step of administering to the subject a composition as presented in any one of claims 1 to 86, a compound of any one of claims 87 to 94, or a pharmaceutical composition of claim 95. Claim 116 Use of a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, or a pharmaceutical composition of claim 95 for treating cancer of a subject. Claim 117 A method for treating a subject requiring treatment, comprising the step of administering to the subject requiring treatment a composition of any one of claims 1 to 86, a compound of any one of claims 87 to 94, or a pharmaceutical composition of claim 95. Claim 118 In paragraph 117, the above-mentioned subject is diagnosed as having cancer, a method. Claim 119 In claim 118, the cancer cells from the subject express B7-H3. Claim 120 A method according to claim 119, wherein the expression of B7-H3 is higher in cancer cells than in non-cancer cells, and the expression can be measured by protein and / or nucleic acid levels. Claim 121 In paragraph 120, the above non-cancerous cells are obtained from the above subject, in a method. Claim 122 A method according to any one of claims 117 to 122, wherein the composition, compound, or pharmaceutical composition is internalized into human B7-H3 expressing cells. Claim 123 In any one of claims 117 to 122, the cancer is breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-associated lymphoma, anal cancer, urothelial carcinoma, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic proliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, extracranial germ cell tumor, extrahepatic biliary cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastic tumor, hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukemia, lip cancer, oral cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma, metastatic cervical squamous cell carcinoma, multiple myeloma and other plasma cell neoplasms. A method selected from mycosis fungoides and Sézary syndrome, myelodysplastic syndrome, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrohistocytoma of bone, sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small intestine cancer, soft tissue sarcoma, supratentorial primitive neuroectodermal tumor, pineal blastoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor and other pediatric renal tumors. Claim 124 A method according to any one of claims 117 to 123, wherein the composition, compound, or pharmaceutical composition is administered intravenously or subcutaneously. Claim 125 A method for targeting cancer cells expressing B7-H3, comprising: (i) determining or determining the expression level of B7-H3 in a population of cancer cells; (ii) administering to a subject in need a composition comprising a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, or a pharmaceutical composition of claim 95, wherein the polypeptide of the composition, compound, or pharmaceutical composition is designed to specifically bind to human B7-H3, and the composition, compound, or pharmaceutical composition is attached to a surface and / or internalized in one or more B7-H3-expressing cancer cells. Claim 126 A method for targeting cancer cells expressing B7-H3, comprising the step of administering to a subject in need a composition comprising a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, or a pharmaceutical composition of claim 95, wherein the polypeptide of the composition, compound, or pharmaceutical composition is designed to specifically bind to human B7-H3; (i) the subject has cancer cells expressing B7-H3; and (ii) the composition, compound, or pharmaceutical composition is attached to a surface and / or internalized in one or more B7-H3-expressing cancer cells. Claim 127 A method according to claim 126, further comprising the step of determining that the cancer cells express B7-H3 or determining that such step. Claim 128 A method according to any one of claims 125 to 127, wherein the subject is treated after administration compared to before administration. Claim 129 A method for treating a population of cancer cells expressing B7-H3, wherein the improvement comprises contacting said population with a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, or a pharmaceutical composition of claim 95, wherein (for SEQ ID NO 241) the position corresponding to X24 comprises lysine having at least one additional small alkyl group attached to the nitrogen of the side chain, said composition is less absorbed by kidney cells than in a composition comprising a polypeptide that does not have a small alkyl group attached to the nitrogen of the lysine side chain at position X24, and optionally, said small alkyl group is a portion of monomethyl, dimethyl, or trimethyl. Claim 130 A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA, (L) comprises PEG if present, and said PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), said (R) comprising actinium-225. Claim 131 A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG, where present, and said PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), said (R) comprising copper-64. Claim 132 A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA, (L) comprises PEG if present, and said PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), said (R) comprising Gallium-68. Claim 133 A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA, (L) comprises PEG if present, and said PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), said (R) comprising indium-111. Claim 134 A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG, where present, and said PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), said (R) comprising lead-212. Claim 135 A conjugate comprising: (i) a polypeptide (M) that specifically binds to B7-H3; (ii) a chelator (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA, (L) comprises PEG if present, and said PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), said (R) comprising lutetium-177. Claim 136 A conjugate comprising: (i) a miniprotein (M) that specifically binds to B7-H3; (ii) an N-terminal modification conjugated to (M) via a selective linker (L), wherein (L) comprises, if present, PEG, and said PEG is optionally PEG-4; and (iii) said N-terminal modification comprising biotin. Claim 137 A conjugate according to any one of claims 130 to 136, wherein M is a conjugate having an amino acid sequence comprising at least 90% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537. Claim 138 A conjugate having an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 198 to 537, in any one of 130 to 137. Claim 139 A conjugate according to any one of claims 130 to 138, wherein M is a conjugate having an amino acid sequence comprising an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 198 to 537. Claim 140 A conjugate having an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs 198 to 537, in any one of 130 to 139. Claim 141 In any one of claims 130 to 140, the amino acid sequence has an amino acid sequence having at least 90% identity with at least 44 amino acids of SEQ ID NO. 267, and the polypeptide comprises (a) at least four cysteines forming two disulfide bonds; (b) arginine, modified arginine, or modified lysine at a position corresponding to amino acid 3; lysine at a position corresponding to amino acid 5; isoleucine at a position corresponding to amino acid 6; and tryptophan at a position corresponding to amino acid 14; (c) having at least one modified lysine residue at a position corresponding to amino acid 24, and alanine, arginine, or modified lysine at a position corresponding to 29 (wherein each position is linear from N-terminus to C-terminus based on SEQ ID NO. 267, said modification comprising at least one small alkyl group attached to the nitrogen of the lysine side chain or the guanidino nitrogen of the arginine side chain and optionally comprising a methyl, dimethyl, or trimethyl group); (d) having a length of at least 48 amino acids; and (d) having a binding affinity for B7-H3 stronger than 100 nM when measured in a cell-based assay. Claim 142 A conjugate according to claim 141, wherein the amino acid sequence comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 204 or 262 to 272. Claim 143 A conjugate according to claim 141 or 142, wherein the amino acid sequence comprises an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 204 or 262 to 272. Claim 144 A compound according to any one of claims 141 to 143, wherein the amino acid sequence comprises an amino acid sequence having 100% identity with at least 44 amino acids of SEQ ID NO.

267. Claim 145 A compound according to any one of claims 141 to 144, wherein the amino acid sequence comprises an amino acid sequence having at least 90% identity with SEQ ID NO.

267. Claim 146 A compound according to any one of claims 141 to 145, wherein the amino acid sequence comprises an amino acid sequence having 100% identity with SEQ ID NO.

267. Claim 147 A conjugate according to any one of claims 130 to 141, wherein the amino acid sequence comprises any one of SEQ ID NOs 204 and 262 to 272 or is composed of these. Claim 148 A conjugate according to any one of claims 130 to 142, wherein the amino acid sequence comprises or is composed of SEQ ID NO.

267. Claim 149 An isolated polynucleotide comprising: one or more nucleic acid sequences encoding a polypeptide selected from any one of SEQ ID NOs 198 to 537; or a nucleic acid sequence encoding a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% or more identity with any one of SEQ ID NOs 198 to 573. Claim 150 A vector comprising the isolated polynucleotide of claim 149. Claim 151 A host cell transformed with the isolated polynucleotide of claim 149 or the vector of claim 150. Claim 152 A method for evaluating the location of one or more cancerous cell populations within a subject, comprising the steps of administering to the subject a composition of any one of claims 1 to 86, a compound according to any one of claims 87 to 94, a pharmaceutical composition of claim 95, or a conjugate of any one of claims 130 to 148, and detecting to determine the location of said composition within the subject, wherein said composition, compound, or pharmaceutical composition comprises a detectable label. Claim 153 In paragraph 152, the method wherein the detectable label comprises a radioactive nuclide. Claim 154 A method for reducing renal uptake of a composition administered to detect and / or treat one or more cancer cell populations, comprising administering to a subject requiring improvement a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, a pharmaceutical composition of claim 95, or a conjugate of any one of claims 130 to 148, wherein the composition comprises lysine having at least one additional small alkyl group attached to the nitrogen of the side chain at the position corresponding to X24 of SEQ ID NO. 241, said composition is less absorbed by renal cells than a composition comprising a polypeptide that does not have a small alkyl group attached to the nitrogen of the side chain of lysine at the position corresponding to X24 of SEQ ID NO. 241, and optionally, said small alkyl group is part of a monomethyl, dimethyl, trimethyl, or isopropyl group. Claim 155 A method according to claim 152 or 154, wherein the detecting comprises an imaging procedure that enables selecting a target, monitoring the target, and / or treating the target with a therapeutic agent comprising a miniprotein designed to bind to B7-H3 expressed on one or more cancer cells in one or more cancer cell populations. Claim 156 A method according to claim 155, wherein the therapeutic agent comprises a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, a pharmaceutical composition according to claim 95, or a conjugate according to any one of claims 130 to 148. Claim 157 A method for improving the delivery of a radionuclide to a population of cancer cells of a subject, comprising the step of administering a composition of any one of claims 1 to 86, a compound according to any one of claims 87 to 94, a pharmaceutical composition of claim 95, or a conjugate of any one of claims 130 to 148, wherein the amino acid sequence of the polypeptide comprises an amino acid including lysine having at least one additional small alkyl group attached to the nitrogen of the side chain at a position corresponding to position X24 of SEQ ID NO. 241, and the uptake by kidney cells is less than that by a polypeptide not comprising a small alkyl group attached to the nitrogen of the side chain of lysine at a position corresponding to position X24 of SEQ ID NO.

241. Claim 158 In claim 157, the method wherein the small alkyl group comprises monomethyl, dimethyl, or trimethyl. Claim 159 A method for treating an individual having cancer, wherein improvement comprises reducing one or more non-target effects or toxicity measures by administering a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, a pharmaceutical composition according to claim 95, or a conjugate according to any one of claims 130 to 148, wherein the amino acid sequence of the polypeptide comprises an amino acid comprising lysine having at least one additional small alkyl group attached to the nitrogen of the side chain at a position corresponding to position X24 of SEQ ID NO. 241, and absorption by kidney cells is less than that by a polypeptide not comprising a small alkyl group attached to the nitrogen of the side chain of lysine at a position corresponding to position X24 of SEQ ID NO.

241. Claim 160 A method for treating an individual having cancer, wherein the improvement comprises achieving a reduction in the concentration of R in kidney tissue in the presence of a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, a pharmaceutical composition according to claim 95, or a conjugate according to any one of claims 130 to 148, wherein the amino acid sequence of the polypeptide comprises an amino acid corresponding to position X24 of SEQ ID NO. 241, where X24 comprises lysine having at least one additional small alkyl group attached to the nitrogen of a side chain, and the absorption by kidney cells is less than that by a polypeptide having an amino acid sequence that does not include a small alkyl group attached to the nitrogen of a lysine side chain at a position corresponding to position X24 of SEQ ID NO. 241, compared to the concentration of R in kidney tissue in the absence of the composition, compound, pharmaceutical composition, or conjugate. Claim 161 A method according to claim 160, wherein the reduction in the concentration of R in the kidney tissue is measured by the urinary excretion of R measured by the percentage of recovered administered radiation, or by detection measured by a cell-based in vitro assay or an in vivo detection assay. Claim 162 A method according to claim 161, wherein the administration of the composition, compound, pharmaceutical composition, or conjugate may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times in the presence of Q, V, L, or K at the position corresponding to position X24 of SEQ ID NO. 241, or in the presence of a composition, compound, pharmaceutical composition, or conjugate having 90% identity with at least 44 amino acids of SEQ ID NO. 241, comprising a modified lysine at the position corresponding to position X24 of SEQ ID NO.

241. Claim 163 A method for reducing the absorption of a composition by renal tissue, wherein the improvement comprises (a) administering a composition comprising a radionuclide therapeutic agent comprising at least a polypeptide and a radionuclide (R); wherein the polypeptide has at least 90% identity with at least 44 amino acids of SEQ ID NO. 241 and / or has a modified lysine at a position corresponding to X24 of SEQ ID NO. 241, such that in the presence of the modified lysine, the radionuclide is less concentrated in renal tissue than in the absence of the polypeptide. Claim 164 A method comprising the step of administering to a subject in need of a compound having at least one modified lysine at a position corresponding to X24 of SEQ ID NO. 241 that binds to B7-H3, wherein the administration of said compound having a miniprotein together with said at least one modified lysine reduces one or more off-target effects, toxicity grades and / or absorption and / or retention in kidney tissue compared to a compound not having modified lysine at a position corresponding to X24 (e.g., unmodified lysine, e.g., L, V, or Q). Claim 165 A method according to claim 163 or 164, wherein the polypeptide has an amino acid sequence comprising an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NOs 204 and 262 to 272. Claim 166 A method according to any one of claims 163 to 165, wherein the polypeptide has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NOs 204 and 262 to 272. Claim 167 A method according to any one of claims 163 to 166, wherein the polypeptide has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NOs 204 and 262 to 272. Claim 168 A method for treating an individual having or suspected of having B7-H3-positive cancer, comprising the steps of: (a) a means for blocking the absorption and / or retention of a radiotherapeutic agent in kidney tissue and (b) administering a linker, a chelator, and a radionuclide to the individual. Claim 169 In claim 168, the means for blocking the absorption and / or maintenance of a radiotherapeutic agent in the kidney tissue comprises a lysine modified at a position corresponding to X24 of SEQ ID NO. 267 that binds to B7-H3 and / or has at least 90% identity with the 40 amino acids of SEQ ID NO. 267 and / or has a lysine modified at a position corresponding to X24 of SEQ ID NO.

267. Claim 170 In claim 169, the method further comprises a modified lysine at a position corresponding to X3 of sequence number 267. Claim 171 A method according to any one of claims 168 to 170, wherein the means for blocking the absorption and / or retention of a radiotherapeutic agent in the kidney tissue blocks the absorption and / or retention in the kidney tissue more significantly than the blocking of absorption and / or retention in the kidney tissue by means that does not include lysine modified at the position corresponding to X24 of SEQ ID NO 267 and / or does not have at least 90% identity with at least 44 amino acids of SEQ ID NO 267 and / or does not have lysine modified at the position corresponding to X24 of SEQ ID NO 267. Claim 172 A method according to any one of claims 168 to 171, wherein the means for blocking the absorption and / or retention of a radiotherapeutic agent in kidney tissue blocks the absorption and / or retention in kidney tissue more significantly than the blocking of absorption and / or retention in kidney tissue by means that does not include lysine modified at the position corresponding to X24 of SEQ ID NO 267 and / or does not have at least 90% identity with at least 44 amino acids of SEQ ID NO 267 and / or does not have lysine modified at the position corresponding to X24 of SEQ ID NO 267. Claim 173 A method according to any one of claims 168 to 172, wherein the means for blocking the absorption and / or maintenance of the radiotherapeutic agent in the kidney tissue is the radiotherapeutic agent. Claim 174 In paragraph 173, the radiotherapeutic agent is targeted to a tumor or a population of cancer cells. Claim 175 In paragraph 174, the radiotherapeutic agent targeted to the tumor or the cancer cell population is present at a higher concentration than in the absence of means for binding to kidney tissue. Claim 176 In paragraph 175, the method comprises a radiotherapeutic agent comprising a polypeptide that targets B7-H3. Claim 177 A method according to any one of claims 168 to 176, wherein the radiotherapeutic agent comprises or is composed of a compound selected from C227 to C608 and C611. Claim 178 In claim 177, the radionuclide of the radiotherapeutic agent is selected from Ac-225, Cu-64, Ga-68, In-111, Lu-177, or Pb-212. Claim 179 A kit comprising a polypeptide and instructions for use, wherein the polypeptide has an amino acid sequence as presented in any one of the polypeptides: a composition according to any one of claims 1 to 86, a compound according to any one of claims 87 to 94, a pharmaceutical composition according to claim 95, or a conjugate according to any one of claims 130 to 148. Claim 180 A kit according to paragraph 179, further comprising one or more of a linker, a chelator, and a radionuclide. Claim 181 In claim 180, the linker comprises or consists of any fragment or combination thereof, wherein the linker comprises PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4 polyethylene glycol (PEG) linker ester linker, amide linker, maleimide linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, propanoic acid linker, dTyr-Gly-Phe(yGF) linker, caproleic acid linker, or (Gly)n-(gGlu)n- or (PEG)n (where n is 1 to 10), (Gly)1-10, or any fragment or combination thereof through covalent bonding. Claim 182 A kit according to claim 180 or 181, wherein the chelator comprises or is composed of DOTA, NOPO, Crown, Macropa, lead-specific chelator (PSC), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (BuSTB) or N-succinimidyl 3-trimethylstannylbenzoate (MeSTB). Claim 183 A kit according to any one of claims 179 to 182, wherein the compound is labeled with a radionuclide before use, said radionuclide is chelated with said chelator to produce a composition having the chemical formula MLCR. Claim 184 A kit according to any one of claims 180 to 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. Claim 185 In paragraph 184, the above radionuclide is Ac-225, Cu-64, Ga-68, In-111, Lu-177, or Pb-212, Kit. Claim 186 A kit according to any one of claims 179 to 185, wherein the polypeptide has an amino acid comprising any of those presented in SEQ ID NOs 199, 204, 241 or 262 to 272, the polypeptide further comprises a linker and / or a chelator, wherein the linker is PEG4 if present and the chelator is DOTA if present. Claim 187 A kit according to any one of claims 180 to 186, wherein the linker, if present, is attached to the N-terminal amino acid of the polypeptide. Claim 188 In any one of claims 179 to 187, the C-terminal amino acid of the polypeptide is not cysteine, Kit. Claim 189 A kit according to any one of claims 180 to 188, wherein the chelator, if present, is attached to either the polypeptide or the linker. Claim 190 In any one of claims 180 to 189, the above radionuclide is attached to the chelator if present. Claim 191 A kit according to any one of claims 180 to 190, wherein the radioactive nuclide, if present, is attached to the N-terminal amino acid of the polypeptide.

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