B7-h3 minibodies, conjugates thereof, and uses thereof

Abstract

This document provides information on B7-H3 binding microproteins, their conjugates, and their uses. It discloses B7-H3 binding peptides and conjugates, including radionuclide conjugates, as well as one or more other proteins, such as decoy peptides. These peptides, conjugates, and / or decoys can be used in compositions and methods for the treatment, diagnosis, monitoring, and / or imaging of diseases, conditions, or disorders associated with the expression of one or more targets (e.g., B7-H3).

Description

Cross-references to related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 607,988, filed December 8, 2023; No. 63 / 636,076, filed April 18, 2024; No. 63 / 650,364, filed May 21, 2024; No. 63 / 650,365, filed May 21, 2024; and No. 63 / 721,990, filed November 18, 2024, the disclosure of each of which is incorporated herein by reference in its entirety for all purposes.

[0002] sequence list This application includes a sequence list, which is electronically submitted in XML format and is hereby incorporated in its entirety by reference. The XML file was created on December 6, 2024, named AKT-035WO_SL.xml, and has a size of 931,498 bytes. Background Technology

[0003] Cancer is a leading cause of death worldwide. Traditional cancer treatments, such as radiation therapy, chemotherapy, and surgery, can be accompanied by serious side effects, including those caused by killing healthy, non-cancerous cells. Newer therapeutics enhance the targeting of cytotoxic drugs to tumor cells compared to earlier therapies, including those using biological conjugates. Summary of the Invention

[0004] This disclosure provides techniques, such as compositions and methods of using and manufacturing thereof, to meet the needs of the cancer field. For example, targeting molecules can be engineered to improve the specificity of, for example, imaging or therapeutic modalities and reduce their toxicity compared to conventional cancer diagnostic agents or therapies. For instance, using peptides for specifically targeting therapeutic agents to deliver therapeutic agents, such as chelating agents and / or radionuclides (e.g., alpha emitters), to the tumor microenvironment can concentrate treatment on tumor cells and avoid or reduce the risk of toxicity to surrounding healthy tissues caused by therapeutic agents targeting, for example, tumors.

[0005] Compared to traditional cancer therapies, radionuclide therapy is more targeted and less toxic. For example, the specific delivery of radionuclides to the tumor microenvironment allows for selective targeting of radiation to tumor tissue, effectively killing malignant cells while protecting surrounding healthy tissue. For instance, radionuclides can employ targeting molecules that specifically bind to antigens expressed at increased levels and / or in increased density on the surface of tumor cells relative to non-tumor cells. The radionuclide binds to antigen-positive tumor cells, allowing radiation to target those cells without targeting healthy tissue. Full-length antibodies have previously been evaluated as targeting molecules; however, they may face several challenges due to considerations such as their large size, poor tumor tissue penetration, and long circulating half-life. Therefore, new methods for specifically targeting tumors, particularly solid tumors, remain needed. This disclosure provides techniques to meet this need, as well as other needs.

[0006] This disclosure specifically provides peptides (e.g., microproteins) that target the tumor microenvironment and / or tumor cells, compositions comprising said peptides, and conjugates comprising said peptides, said peptides being conjugated to one or more additional components (including, for example, linkers, chelating agents, and / or radionuclides (e.g., alpha emitters)). In some embodiments, the peptides specifically and strongly bind to B7-H3. In some embodiments, the peptides or conjugates of this disclosure can improve tumor penetration, reduce off-target toxicity and / or accumulation, improve stability (e.g., relative to comparative peptides), and improve affinity for B7-H3 (e.g., relative to prior art (e.g., antibodies, e.g., antibody-drug conjugates, peptides, etc.)). In some embodiments, such conjugates are used to treat cells expressing a target (e.g., B7-H3). In some such embodiments, the cells are cancer cells.

[0007] This disclosure also provides the insight that the compositions and conjugates provided herein can be further improved, for example by enhancing stability (e.g., thermal stability) while maintaining or enhancing the binding strength with B7-H3. For example, it is contemplated herein that, in some embodiments, conjugates, such as the radionuclide conjugates of this disclosure, can be improved, for example by improving one or more measurement criteria, including thermal stability, efficacy and / or toxicity grade and / or reduction of off-target effects (e.g., in cell-based assays, e.g., when administered to a subject in need), by modifying one or more amino acids in the polypeptide sequence of the conjugate (e.g., relative to a reference or starting sequence) and / or by adding one or more decoys.

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

[0009] In one aspect, this 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 position 3 corresponding to amino acid 3; lysine at position 5 corresponding to amino acid 5; isoleucine at position 6 corresponding to amino acid 6; tryptophan at position 14 corresponding to amino acid 14; at least one modified lysine residue at position 24 corresponding to amino acid 24; and alanine, arginine, or modified lysine at position 29, wherein each position is linear from the N-terminus to the C-terminus relative to SEQ ID NO: 267, starting from position 1; wherein the modification comprises at least one small alkyl group of nitrogen attached to the lysine side chain or guanidine nitrogen attached to the arginine side chain, wherein the at least one small alkyl group optionally comprises methyl, dimethyl, or trimethyl; a length of at least 48 amino acids; and a binding affinity for B7-H3 stronger than 100 nM as measured in a cell-based assay.

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

[0011] In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NO: 204 and 262-272. In some embodiments, the polypeptide is part of a compound selected from any one of C234-235 and C309-C332.

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

[0013] In another aspect, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula III (SEQ ID NO: 538): CAX3EKIAALSEIIWLPCLX19YAQIX24AFIX28X29LNX32DPCX36SX38X39ILSEAX45ELCS, wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme) or (Kme2); X28 is A or (Kme); X29 is (Kme) or A or R; X32 is (Kme) or D or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; and X45 is K or (Kme). In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NO: 204 and 262-272. In some embodiments, the polypeptide is part of a compound selected from C234-235 and C309-C332.

[0014] In one aspect, the composition comprises a polypeptide having an amino acid sequence comprising the amino acid sequence according to Formula IV (SEQ ID NO: 539): 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 amino acid sequence of the polypeptide comprises any one of SEQ ID NO: 204, 262, 265, 267, and 268.

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

[0016] In another aspect, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising the amino acid sequence of the following formula V (SEQ ID 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 amino acid sequence of the polypeptide comprises any one of SEQ ID NO: 265, 266, 267, 270, and 272. In some embodiments, the polypeptide is part of a compound selected from any one of C304-C309, C314, C315, C318, C319, C323, C324, C325, C328, C330, and C332.

[0017] In one aspect, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising the amino acid sequence of the following formula VI (SEQ ID 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 amino acid sequence of the polypeptide comprises any one of SEQ ID NO: 204, 262, 265, 266, 267, 270, and 272. In some embodiments, the polypeptide is part of a compound selected from any one of C234, C235, C298, C299, C304-C309, C314, C315, C318-C320, C323, C324, C325, C328, C330, and C332.

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

[0019] In one aspect, this disclosure provides a composition comprising a B7-H3-binding polypeptide having an amino acid sequence comprising: at least four cysteine ​​residues forming two disulfide bonds; at least one modified lysine residue at position X24 of SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group of nitrogen attached to the lysine side chain, the small alkyl group optionally comprising methyl, dimethyl, or trimethyl; a length of at least 48 amino acids; and a binding affinity to B7-H3 of greater than 100 nM as measured in a cell-based assay. In some embodiments, the polypeptide is a microprotein. In some embodiments, the polypeptide is at least 48 amino acids long but not exceeding 100 amino acids in length. In some embodiments, the polypeptide binds to B7-H3 with an affinity of greater than 100 nM as measured in a cell-based assay.

[0020] In some embodiments, the amino acid sequence of the disclosed polypeptide shares at least 90% identity with any one of SEQ ID NO: 204 and 262-537, and includes at least one lysine or arginine with at least one modification comprising at least one small alkyl group bonded to a nitrogen atom to a lysine side chain or to a guanidine nitrogen atom to a arginine side chain, the small alkyl group 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 a reference polypeptide longer than 47 amino acids, and binds to B7-H3 with a strength of at least 10 nM in cell-based assays, 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 NO: 198-537, provided that the 44 amino acids include at least four 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 a reference polypeptide, said reference polypeptide being longer than 47 amino acids. In some embodiments, the amino acid sequence shares at least 90% identity with at least 44 amino acids as described in any one of SEQ ID NO: 199, 204, 241, or 262-272. In some embodiments, the amino acid sequence shares 100% identity with at least 44 amino acids as described in any one of SEQ ID NO: 199, 204, 241, or 262-272.

[0021] In some embodiments, the amino acid sequence comprises at least four cysteine ​​residues having at least two disulfide bridges. In some embodiments, the polypeptide is at least 48 amino acids in length. In one aspect, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO: 267. In some embodiments, the composition comprises C234, C235, and C298-C333 as described in Table 2C.

[0022] In one aspect, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising any one of SEQ ID NO: 204 and 262-272.

[0023] In some embodiments, the compositions of this disclosure (e.g., B7-H3 peptides, such as B7-H3-binding microproteins) further comprise 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 some embodiments, the peptides of this disclosure include a C-terminus comprising -OH or -NH2. In some embodiments, the binding affinity for B7-H3 of the compositions provided herein is stronger than 100 nM. In some embodiments, the inhibition constant is not weaker than 100 nM.

[0024] In some embodiments, the compositions of this disclosure (e.g., comprising peptides) further comprise one or more of a linker, a chelating agent, and a radionuclide. In some embodiments, the linker comprises or consists of: polyethylene glycol (PEG) linkers (PEG4, PEG2, PEG, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4), ester linkers, amide linkers, maleimide linkers, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) linkers, propionic acid linkers, dTyr-Gly-Phe (yGF) linkers, decenoic acid linkers, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is 1 to 36, (Gly)1-10, or any fragment thereof or a combination thereof linked by covalent bonds. In some embodiments, the chelating agent comprises or is composed of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoate N-succinimide ester (BuSTB), or 3-trimethyltinylbenzoate N-succinimide ester (MeSTB). In some embodiments, the radionuclide is selected from Ac-225, Cu-64, Ga-68, Lu-177, Pb-212, In-111, Cu-67, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, Sm-153, Ra-225, Tb-165, or At-211.

[0025] In some embodiments, if the polypeptide comprises any of SEQ ID NO: 198-537, the polypeptide further comprises a linker, wherein the linker is PEG4, and optionally a chelating agent, wherein the chelating agent is DOTA. In some embodiments, the linker is attached to the N-terminus of the polypeptide when present. In some embodiments, the C-terminal amino acid of the polypeptide is not cysteine. In some embodiments, the chelating agent is attached to either the polypeptide or the linker when present. In some embodiments, a radionuclide is attached to the chelating agent when present.

[0026] In one aspect, this disclosure provides a composition comprising one or more of the formula (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 chelating agent (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NO: 198-537. 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 NO: 198-537. 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 NO: 198-537. In some embodiments, M has an amino acid sequence comprising an amino acid sequence having 100% identity with any of the amino acids in SEQ ID NO: 198-537. In some embodiments, the linker comprises or consists of: polyethylene glycol (PEG) linkers (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4), ester linkers, amide linkers, maleimide linkers, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) linkers, propionic acid linkers, dTyr-Gly-Phe(yGF) linkers, decenoic acid linkers, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is 1 to 36, (Gly)1-10, or any fragment thereof or a combination thereof linked by covalent bonds. In some embodiments, the chelating agent comprises or consists of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoate N-succinimide ester (BuSTB), or 3-trimethyltinylbenzoate N-succinimide ester (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.

[0027] In one aspect, this disclosure provides a composition comprising one or more of the formula (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 chelating agent (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NO: 204 and 262-272. In some 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 NO: 204 and 262-272. 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 NO: 204 and 262-272. In some embodiments, M has an amino acid sequence comprising an amino acid sequence that is 100% identical to any one of SEQ ID NO: 204 and 262-272. In some embodiments, L is present, and L comprises or consists of: polyethylene glycol (PEG) connectors (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36), ester connectors, amide connectors, maleimide connectors, valine-citrulline connectors, hydrazone connectors, 4-(2-pyridyldithio)butyrate N-succinimide ester (SPDB) connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylate succinimide ester (SMCC) connectors, vinyl sulfone-based connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is 1 to 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds. In some embodiments, when C is present, C comprises or is composed of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoate N-succinimide ester (BuSTB), or 3-trimethyltinylbenzoate N-succinimide ester (MeSTB). In some embodiments, when 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.In some embodiments, the linker is attached to the N-terminus of the peptide when present. In some embodiments, the C-terminal amino acid of the peptide is not cysteine. In some embodiments, a chelating agent is attached to the peptide or the linker when present. In some embodiments, a radionuclide is attached to the chelating agent when present. In some embodiments, the peptide contains at least one disulfide bridge. In some embodiments, the peptide contains at least two disulfide bridges. In some embodiments, the composition and / or its peptide selectively bind to B7-H3. In some embodiments, the binding affinity of the peptide to B7-H3 is 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM, in vivo, in vitro, or as measured in cell-based assays. In some embodiments, the binding inhibition constant of the peptide is not weaker than 100 nM.

[0028] In one aspect, this disclosure provides a composition comprising a polypeptide-drug conjugate, said polypeptide-drug conjugate comprising a polypeptide and at least one pharmaceutical 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 having any one of the amino acid sequences described in SEQ ID NO: 198-537. In some embodiments, the polypeptide comprises at least four cysteine ​​residues and two disulfide bridges. In some embodiments, the drug component is selected from topoisomerase inhibitors, orrisstatins (e.g., monomethylorrisstatin E), V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, saccharidin, maytansin-like substances, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, DHFR inhibitors, and immunotoxins.

[0029] In one aspect, this disclosure provides a composition comprising an isolated compound or a pharmaceutically acceptable salt or neutral molecule thereof, comprising an optional linker (L) and one or more of a polypeptide (M), a chelating agent (C), or a radionuclide (R), wherein 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 NO: 198-537 and 538-543 and 546-550, including amino acid substitutions as described in Table 1E.

[0030] In another aspect, this disclosure provides a composition comprising a compound designed to bind to B7-H3, said compound comprising or consisting of a polypeptide having an amino acid sequence comprising an amino acid sequence having at least 90% identity with an amino acid sequence of at least 44 amino acids of any one of SEQ ID NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E, and further comprising a modified N-terminus and / or C-terminus. In some embodiments, M has an amino acid sequence comprising an amino acid sequence comprising an amino acid sequence having at least 90% identity with an amino acid sequence of at least 44 amino acids of any one of SEQ ID NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E. In some embodiments, M has an amino acid sequence comprising an amino acid sequence comprising an amino acid sequence having at least 90% identity with an amino acid sequence of any one of SEQ ID NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E. 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 NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E. In some embodiments, M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E. In some embodiments, the modified N-terminus comprises one or more of the following: NH2-, acetyl-, PEGn (where n = 0-36), DOTA-, or biotin-. In some embodiments, the C-terminus comprises -NH2 or -OH. In some embodiments, the polypeptide selectively binds to B7-H3. In some embodiments, the polypeptide exhibits a binding affinity to B7-H3 greater than about 100 nM in vivo or in cell-based assays.

[0031] In one aspect, this disclosure provides a compound comprising a microprotein having an amino acid sequence comprising 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 formula MLCR, wherein M is the microprotein, L is a linker, C is a chelating agent, and R is a radionuclide. In some embodiments, M has an amino acid sequence comprising 100% identity with at least 44 amino acids of SEQ ID NO: 267. In some embodiments, M has an amino acid sequence comprising at least 90% identity with an amino acid sequence of SEQ ID NO: 267. In some embodiments, M has an amino acid sequence comprising 100% identity with an amino acid sequence of SEQ ID NO: 267. In some embodiments, L comprises or consists of: polyethylene glycol (PEG) connectors PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, any connectors described 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 thereof or combination thereof linked by covalent bonds. In some embodiments, C comprises or is composed of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoate N-succinimide ester (BuSTB), or 3-trimethyltinylbenzoate N-succinimide ester (MeSTB). In some embodiments, 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.

[0032] In one aspect, this disclosure provides a compound comprising a microprotein having at least 90% identity with at least 48 amino acids of the amino acid sequence of SEQ ID NO: 267, wherein the N-terminus and / or C-terminus contains one to thirty additional amino acids, and / or wherein the C-terminus contains at least one amino acid or at most 30 additional amino acids, provided that the length of the entire microprotein is not greater than about 100 amino acids.

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

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

[0035] In one aspect, this disclosure provides a method of treating cancer, the method comprising administering to a subject in need a composition comprising a conjugate, the 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 NO: 198-537, and a radionuclide. In some embodiments, the polypeptide has at least four cysteine ​​residues and two disulfide bridges. In some embodiments, the radionuclide is associated with the polypeptide using a linker and / or a chelating agent according to formula MLCR, wherein M is the polypeptide, L is the linker, C is the chelating agent, and R is the radionuclide. In some embodiments, the polypeptide has an amino acid sequence comprising or consisting of any one of SEQ ID NO: 204 or 262-272. In some embodiments, L comprises or consists of: polyethylene glycol (PEG) connectors PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, any connectors described in Table 2A, or (Gly)n-(gGlu)n- or (PEG)n, where n is 1 to 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds. In some embodiments, C comprises or consists of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoate N-succinimide ester (BuSTB), or 3-trimethyltinylbenzoate N-succinimide ester (MeSTB). In some 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 implementations, R is Cu-64, Ga-68, Lu-177, In-111, Cu-67, La-132, or F-18.

[0036] In some implementations, the length of the polypeptide does not exceed 100 amino acids.

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

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

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

[0040] In one aspect, this disclosure provides a method for improving the biodistribution of a pharmaceutical composition of a population of B7-H3-positive cancer cells in a subject suffering from B7-H3-positive cancer, comprising contacting the population with a polypeptide having a modified lysine at position X24 corresponding to SEQ ID NO: 241, wherein the lysine is modified by adding at least one small alkyl group to the lysine side chain, and wherein the biodistribution is improved compared to contacting the population with a polypeptide that does not contain the modified lysine at position X24 corresponding to SEQ ID NO: 241.

[0041] In one aspect, this disclosure provides a method for diagnosing the presence of a population of B7-H3 positive cancer cells, comprising: contacting the cell population 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 result in step (b) with a control signal; and diagnosing cancer if the composition, compound, or pharmaceutical composition of step (a) is detected at a higher level than the control signal. In some embodiments, contact is performed by administration to a subject in need. In some embodiments, administration is intravenous or subcutaneous. In some embodiments, contact is performed in vitro on a subject, optionally using a biopsy sample.

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

[0043] In one aspect, this disclosure provides for the use of the compositions, compounds, or pharmaceutical compositions disclosed herein.

[0044] In one aspect, this disclosure provides a method of treating a subject in need, comprising administering to the subject in need a composition, compound, or pharmaceutical composition of the disclosure. In some embodiments, the subject is diagnosed with cancer. In some embodiments, the subject's cancer cells express B7-H3. In some embodiments, B7-H3 expression in cancer cells is higher than in non-cancer cells, and this expression can be measured by protein and / or nucleic acid levels. In some embodiments, non-cancer cells are obtained from the subject. In some embodiments, the composition, compound, or pharmaceutical composition is internalized in cells expressing human B7-H3. In some implementation schemes, the cancer is selected from breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, urothelial carcinoma, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastoma, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma, Merkel cell carcinoma. Metastatic squamous cell carcinoma of the neck, multiple myeloma and other plasmacytomas, mycosis fungoides and Cezari syndrome, myelodyplasia syndrome, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small bowel 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.

[0045] In one aspect, this disclosure provides a method for targeting cancer cells expressing B7-H3, the method comprising: (i) determining or having determined the expression level of B7-H3 in a population of cancer cells; and (ii) administering to a subject in need a composition comprising a composition, compound, or pharmaceutical composition as provided herein, wherein a polypeptide of the composition, compound, or pharmaceutical composition is engineered to specifically bind to human B7-H3, wherein the composition, compound, or pharmaceutical composition is linked to the surface of one or more cancer cells expressing B7-H3 and / or internalized into the cancer cells.

[0046] In one aspect, this disclosure provides a method for targeting cancer cells expressing B7-H3, the method comprising administering to a subject in need a composition comprising a composition, compound, or pharmaceutical composition as provided herein, wherein a polypeptide of the composition, compound, or pharmaceutical composition is engineered to specifically bind to human B7-H3, wherein (i) the subject has cancer cells expressing B7-H3; and (ii) the composition, compound, or pharmaceutical composition is attached to the surface of one or more cancer cells expressing B7-H3 and / or internalized into the cancer cells. In some embodiments, the method further includes identifying or having identified cancer cells expressing B7-H3. In some embodiments, the subject receives treatment after administration compared to before administration.

[0047] In one aspect, this disclosure provides a method for targeting a population of cancer cells expressing B7-H3, an improvement comprising contacting the population with a composition according to a composition, compound, or pharmaceutical composition as provided herein, wherein the position corresponding to X24 (relative to SEQ ID NO: 241) contains a lysine having at least one additional small alkyl group having nitrogen attached to a side chain, wherein less of the composition is taken up by renal cells than a composition containing a polypeptide having a small alkyl group having nitrogen attached to the side chain at X24, wherein optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group.

[0048] In one aspect, this disclosure provides a conjugate comprising: (i) a polypeptide (M) specifically bound to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is actinium-225.

[0049] In one aspect, this disclosure provides a conjugate comprising: (i) a polypeptide (M) specifically bound to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is copper-64.

[0050] In one aspect, this disclosure provides a conjugate comprising: a polypeptide (M) specifically bound to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is gallium-68.

[0051] In one aspect, this disclosure provides a conjugate comprising: (i) a polypeptide (M) specifically bound to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is indium-111.

[0052] In one aspect, this disclosure provides a conjugate comprising: (i) a polypeptide (M) specifically bound to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is lead-212.

[0053] In one aspect, this disclosure provides a conjugate comprising: a polypeptide (M) specifically bound to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is lutetium-177.

[0054] A conjugate comprising: (i) a microprotein (M) specifically binding to B7-H3; (ii) an N-terminal modification conjugated to (M) via an optional linker (L), wherein (L) contains PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) an N-terminal modification containing biotin.

[0055] In some embodiments, M has an amino acid sequence comprising at least 44 amino acids having at least 90% identity with any of SEQ ID NO: 198-537. In some embodiments, M has an amino acid sequence comprising at least 90% identity with any of SEQ ID NO: 198-537. In some embodiments, M has an amino acid sequence comprising at least 44 amino acids having 100% identity with any of SEQ ID NO: 198-537. In some embodiments, M has an amino acid sequence comprising 100% identity with any of SEQ ID NO: 198-537. In some embodiments, the amino acid sequence has an amino acid sequence containing at least 90% identity with at least 44 amino acids of SEQ ID NO: 267, wherein the polypeptide has: at least four cysteine ​​residues forming two disulfide bonds; arginine, modified arginine, or modified lysine at position 3 corresponding to amino acid 3; lysine at position 5 corresponding to amino acid 5; isoleucine at position 6 corresponding to amino acid 6; tryptophan at position 14 corresponding to amino acid 14; at least one modified lysine residue at position 24 corresponding to amino acid 24; and alanine, arginine, or modified lysine at position 29, wherein each position is linear from the N-terminus to the C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group of a nitrogen or guanidine group attached to the lysine side chain, said small alkyl group optionally comprising methyl, dimethyl, or trimethyl; a length of at least 48 amino acids; and 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 any one of SEQ ID NO: 204 and 262-272. In some embodiments, the amino acid sequence comprises an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NO: 204 and 262-272. In some 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 some embodiments, the amino acid sequence comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 267. In some 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 262-272. In some embodiments, the amino acid sequence comprises or consists of SEQ ID NO: 267.

[0056] In one aspect, this disclosure provides an isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from any one of SEQ ID NO: 198-537; 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 NO: 198-573. In some embodiments, this disclosure provides a vector comprising the isolated polynucleotide as provided herein. In some embodiments, this disclosure provides a host cell transformed with the isolated polypeptide or vector as provided herein.

[0057] In one aspect, this disclosure provides a method for assessing the location of one or more cancer cell populations in a subject, the method comprising administering to the subject a composition, compound, pharmaceutical composition, or conjugate as provided herein, and detection to determine the location of the composition in the subject, wherein the composition, compound, or pharmaceutical composition comprises a detectable marker. In some embodiments, the detectable marker comprises a radionuclide.

[0058] In one aspect, this disclosure provides a method for reducing renal uptake of a composition administered to detect and / or treat one or more cancer cell populations, an improvement comprising administering to a subject in need a composition, compound, pharmaceutical composition, or conjugate as provided herein, wherein the position corresponding to X24 of SEQ ID NO: 241 contains a lysine residue having at least one additional small alkyl group linked to a nitrogen atom on a side chain, wherein less of the composition is taken up by renal cells than a composition containing a polypeptide having a small alkyl group on a side chain without a nitrogen atom linked to a lysine residue at the X24 position of SEQ ID NO: 241, wherein, optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group. In some embodiments, the detection includes an imaging procedure that allows selection of a subject, monitoring of the subject, and / or treatment of the subject with a therapeutic agent comprising a microprotein 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.

[0059] In one aspect, this disclosure provides an improved method for radionuclide delivery to a cancer cell population in a subject, the method comprising administering a composition, compound, conjugate, or pharmaceutical composition as provided herein, wherein the amino acid sequence of the polypeptide comprises an amino acid at position X24 corresponding to SEQ ID NO: 241, which contains an additional small alkyl group having at least one nitrogen atom linked to a side chain, and wherein renal cell uptake is less than that of a polypeptide having a small alkyl group without nitrogen atom linked to a lysine atom at position X24 corresponding to SEQ ID NO: 241. In some embodiments, the small alkyl group comprises monomethyl, dimethyl, or trimethyl.

[0060] In one aspect, this disclosure provides a method of treating an individual with cancer, an improvement comprising reducing one or more off-target effects or toxicity measures by administering a composition, compound, conjugate, or pharmaceutical composition as provided herein, wherein the amino acid sequence of the polypeptide comprises an amino acid at position X24 corresponding to SEQ ID NO: 241 containing an additional small alkyl group having at least one nitrogen linked to a side chain, wherein the uptake by renal cells is less than that of a polypeptide having a small alkyl group on a side chain without nitrogen linked to a lysine at position X24 corresponding to SEQ ID NO: 241.

[0061] In one aspect, this disclosure provides a method for treating an individual suffering from cancer, an improvement comprising reducing the concentration of R in kidney tissue in the presence of the composition, compound, pharmaceutical composition, or conjugate, compared to the concentration of R in kidney tissue in the absence of the composition, compound, pharmaceutical composition, or conjugate provided herein, wherein the amino acid sequence of the polypeptide comprises an amino acid corresponding to position X24 of SEQ ID NO: 241, and wherein X24 comprises a lysine having at least one additional small alkyl group attached to a nitrogen atom on a side chain, wherein the uptake by renal cells is less than that of a polypeptide having an amino acid sequence that does not contain a small alkyl group attached to a nitrogen atom on a side chain at position X24 of SEQ ID NO: 241. In some embodiments, the reduction in the concentration of R in kidney tissue is measured by means of: the amount of R excreted in urine as measured by the percentage of the applied radiation recovered, or by a detection result as measured by a cell-based in vitro assay or in vivo assay. In some embodiments, when a composition having 90% identity with at least 44 amino acids of SEQ ID NO: 241 and including modified lysine at position X24 of SEQ ID NO: 241 can be applied at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times more than when Q, V, L or K are present at position X24, the composition can be applied at least 2, 3, 4, 5, 6, 7, 8, 9 or more times.

[0062] In one aspect, this disclosure provides a method for reducing the uptake of a composition by renal tissue, an improvement comprising administering a composition comprising (a) 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 position X24 corresponding to SEQ ID NO: 241, such that in the presence of the modified lysine, the concentration of the radionuclide in the renal tissue is less than the concentration in the absence of the polypeptide.

[0063] In another aspect, this disclosure provides a method comprising administering to a subject in need a compound that binds to B7-H3 and comprises at least one modified lysine at position X24 corresponding to SEQ ID NO: 241, wherein administration of a compound having a microprotein containing at least one modified lysine reduces one or more off-target effects, toxicity levels, and / or uptake and / or retention in renal tissue compared to a compound that does not contain a modified lysine at position X24 (e.g., unmodified lysine, such as 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 262-272. 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 262-272. In some embodiments, the polypeptide has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NO: 204 and 262-272.

[0064] In one aspect, this disclosure provides a method for treating an individual with or suspected of having B7-H3 positive cancer, the method comprising administering to the individual: means for blocking renal tissue uptake and / or retention of a radiotherapy agent, and a connector, chelating agent, and radionuclide. In some embodiments, the means for blocking renal tissue uptake and / or retention of the radiotherapy agent is bound to B7-H3 and includes a modified lysine at position X24 corresponding to 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 position X24 corresponding to SEQ ID NO: 267. In some embodiments, the means further includes a modified lysine at position X3 corresponding to SEQ ID NO: 267. In some embodiments, the means for blocking renal tissue uptake and / or retention of the radiotherapy agent has a greater effect on blocking renal tissue uptake and / or retention than means that exclude a modified lysine at position X24 of SEQ ID NO: 267 and / or have at least 90% identity with at least 44 amino acids of SEQ ID NO: 267 and / or have a modified lysine at position X24 of SEQ ID NO: 267. In some embodiments, the means for blocking renal tissue uptake and / or retention of the radiotherapy agent has a greater effect on blocking renal tissue uptake and / or retention than means that exclude a modified lysine at position X24 of SEQ ID NO: 267 and / or have at least 90% identity with at least 44 amino acids of SEQ ID NO: 267 and / or have a modified lysine at position X24 of SEQ ID NO: 267. In some embodiments, the means of blocking renal tissue uptake and / or retention of the radiotherapy agent is a radiotherapy agent. In some embodiments, the radiotherapy agent targets a tumor or cancer cell population. In some embodiments, the concentration of the radiotherapy agent targeting the tumor or cancer cell population is greater than the concentration in the absence of a means of binding to renal tissue. In some embodiments, the radiotherapy agent comprises a polypeptide targeting B7-H3. In some embodiments, the radiotherapy agent comprises or is composed of compounds selected from C227-C608 and C611. In some embodiments, the radionuclide of the radiotherapy agent is selected from Ac-225, Cu-64, Ga-68, In-111, Lu-177, or Pb-212.

[0065] In one aspect, this disclosure provides a kit comprising a polypeptide and instructions for use, wherein the polypeptide has the amino acid sequence described in the polypeptide of the compositions, compounds, conjugates, or pharmaceutical compositions provided herein. In some embodiments, the polypeptide further comprises one or more of a linker, a chelating agent, and a radionuclide. In some embodiments, the linker comprises or is composed of: polyethylene glycol (PEG) linkers (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4), ester linkers, amide linkers, maleimide linkers, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) linkers, propionic acid linkers, dTyr-Gly-Phe (yGF) linkers, decenoic acid linkers, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is 1 to 10, (Gly)1-10, or any fragment thereof or a combination thereof linked by covalent bonds. In some embodiments, the chelating agent comprises or consists of: DOTA, NOPO, Crown, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoate N-succinimide ester (BuSTB), or 3-trimethyltinylbenzoate N-succinimide ester (MeSTB). In some embodiments, prior to use, the compound is radiolabeled with a radionuclide, wherein the radionuclide chelates to the chelating agent to produce a composition having the 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.

[0066] In some embodiments, if the polypeptide has an amino acid comprising any of the sequences described in any of SEQ ID NO: 199, 204, 241, or 262-272, wherein the polypeptide further comprises a linker and / or a chelating agent, wherein the linker is PEG4 in the presence, and the chelating agent is DOTA in the presence. In some embodiments, when present, the linker is 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, when present, the chelating agent is attached to the polypeptide or the linker. In some embodiments, when present, a radionuclide is attached to the chelating agent. In some embodiments, when present, a radionuclide is attached to the N-terminal amino acid of the polypeptide. Attached Figure Description

[0067] Figure 1A The binding affinity of the exemplary microprotein (SEQ ID NO: 3) to B7-H3 is shown 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).

[0068] Figure 1B The 1:1 binding model analysis of the exemplary microprotein (SEQ ID NO: 3) is shown. The black lines are derived from the model fitting.

[0069] Figure 2A The binding affinity of the exemplary microprotein (SEQ ID NO: 4, run #1) to B7-H3 is shown at 50 nM (a), 25 nM (b), 12.5 nM (c), 6.25 nM (d) and 3.13 nM (e).

[0070] Figure 2B The 1:1 binding model analysis of the exemplary microprotein (SEQ ID NO: 4, run #1) is shown. The black lines are derived from the model fitting.

[0071] Figure 3A The binding affinity of the exemplary microprotein (SEQ ID NO: 4, run #2) to B7-H3 is shown at 50 nM (a), 25 nM (b), 12.5 nM (c), 6.25 nM (d), 3.13 nM (e) and 1.56 nM (f).

[0072] Figure 3B The 1:1 binding model analysis of the exemplary microprotein (SEQ ID NO: 4, run #2) is shown. The black lines are derived from the model fitting.

[0073] Figure 4 The 1:1 binding model analysis of the exemplary microprotein (SEQ ID NO: 6) is shown. The black lines are derived from the model fitting.

[0074] Figure 5A and Figure 5B The relative binding of an exemplary microprotein to cancer cells expressing B7-H3 (cancer cell line) as measured by flow cytometry is shown and compared with a control group (target-deficient cell line). Figure 5A The plotted median fluorescence intensity (MFI) is shown as the variation of microprotein concentration applied to cancer cells. Figure 5B The superposition of representative MFI histograms of cancer cells treated with different concentrations of microproteins compared to the medium control group is shown.

[0075] Figure 6 The internal localization of Cy5-labeled B7-H3-specific microproteins in B7-H3-expressing cancer cells is shown. Cell nuclei are stained with DAPI, and arrows indicate the localization of Cy5-labeled B7-H3 microproteins.

[0076] Figure 7 The quantitative uptake in proximal tubular cells (OK-PTC) of opossum kidneys is illustrated. Combined treatment with a 20-fold molar excess of the exemplary decoy peptide (compound ID NO: C10) (column 2, right) reduced the uptake of the biotinylated test reagent (compound ID NO: C8) compared to the use of the biotinylated test reagent alone (C8, left side).

[0077] Figure 8 The results of analysis using SPECT / CT scans to quantify the per-gram injection dose (%ID / g) in mouse kidney tissue are illustrated. Exemplary B7-H3 charge variant conjugates (compound ID NO: C5, C11-C14) exhibited reduced renal retention levels in mouse biodistribution.

[0078] Figure 9 The results of analysis using SPECT / CT scans to quantify the per-gram injection dose (%ID / g) in mouse kidney tissue are illustrated. Co-administration of the exemplary decoy peptide (compound ID NO: C10) reduced renal absorption of the exemplary B7-H3 targeting affinity conjugate (compound ID NO: C5).

[0079] Figures 10A and 10B plot the data, showing the reduction in cellular uptake of the exemplary target-targeted scaffold B microprotein conjugate when combined in vitro with the exemplary scaffold A decoy. Figure 10A is a bar chart showing the percentage of uptake (on the y-axis) of the exemplary B7-H3-targeted scaffold B microprotein conjugate alone / without decoy (20 mM; column 1, x-axis) or in combination with a 20-fold molar excess of the exemplary scaffold A decoy (C120) (column 2, x-axis). Figure 10B is a bar chart showing the percentage of uptake (on the y-axis) of 20 mM of the exemplary B7-H3-targeted scaffold B microprotein (C8) alone (20 mM; column 1, x-axis) or in combination with a 20-fold molar excess of the exemplary scaffold A decoy (C120) (column 2, x-axis). Error bars represent the standard error (SEM) of the mean.

[0080] Figure 11This chart plots data showing the reduction in cellular uptake when the exemplary B7-H3-targeted scaffold B microprotein conjugate is combined with one of the three exemplary scaffold A decoys in vitro. The bar chart shows the percentage of uptake (y-axis) when the exemplary B7-H3-targeted scaffold B microprotein conjugate is used alone (C8) / without decoy (20 mM; bar 1, x-axis) or combined with one of the three exemplary scaffold A decoys at a 20-fold molar excess (C118, bar 2; C119, bar 3; and C120, bar 4). Error bars represent the standard error (SEM) of the mean.

[0081] Figures 12A and 12B are graphs illustrating the data, showing the reduction in cellular uptake of the exemplary scaffold B target-binding microprotein compound when combined in vitro with the exemplary scaffold B decoy. Figure 12A is a bar graph showing the percentage of uptake (on the y-axis) when the exemplary B7-H3 targeting scaffold B microprotein conjugate (C8) alone / without decoy (20 mM; column 1, x-axis) or in combination with a 20-fold molar excess of the exemplary scaffold B decoy (C10) (column 2, x-axis). Figure 12B is a bar graph showing the percentage of uptake (on the y-axis) when the exemplary B7-H3 targeting scaffold B microprotein conjugate (C117) alone / without decoy (1 mM; column 1, x-axis) or in combination with a 100-fold molar excess of the exemplary scaffold B decoy (C10) (column 2, x-axis). Error bars represent the standard error (SEM) of the mean.

[0082] Figure 13A and Figure 13B It is a graph that plots the results of SPECT / CT scan analysis, used to analyze the results of individual injections. 111 In-labeled exemplary B7-H3 targeting scaffold B microprotein conjugates ( 111 In-C5) / No bait (C5); Figure 13A and Figure 13B (Solid line with a solid circle in the middle) after or with an illustrative support B decoy (C10) with an 850-fold molar excess; Figure 13A and Figure 13B After administration (4-24 hours post-injection) of the dotted line with hollow circles (x-axis), the kidneys of mice ( Figure 13A ) or liver ( Figure 13B The uptake and retention of the stent or stent plus decoy were quantified as a percentage of the injected dose per gram (%ID / g; y-axis). Error bars represent the standard deviation (SD).

[0083] Figure 14This is a graph showing the thermal stability of an exemplary compound containing a conjugate of an exemplary constrained B7-H3-bound microprotein. The remaining percentage (x-axis) of C309 (microprotein of SEQ ID NO: 267) over time (y-axis, hours (h)) was measured at 75 °C (solid line) and 90 °C (dashed line).

[0084] Figure 15 This is a graph showing the cellular binding of Ki used to measure the exemplary constrained B7-H3 microprotein conjugates (microproteins of C234 and SEQ ID NO:204).

[0085] Figures 16A-16B Line graph showing %ID / g of one of the three micro protein conjugates (C1B, C235, and C301) over 0–48 hours after administration. Figure 16A The percentage of %ID / g in the kidneys was shown at 1, 4, 24, and 48 hours after administration. Figure 16B The percentage of %ID / g in tumors is shown at 1, 4, 24, and 48 hours after application.

[0086] Figures 17A-17B Line graph showing %ID / g of one of the three micro protein conjugates (C1B, C235, and C309) over 0–48 hours after administration. Figure 17A The percentage of %ID / g in the kidneys was shown at 1, 4, 24, and 48 hours after administration. Figure 17B The percentage of %ID / g in tumors is shown at 1, 4, 24, and 48 hours after application.

[0087] Figures 18A-18B This is a bar chart, which shows, for example, the tumor ( Figure 18A ) and kidneys ( Figure 18B In this study, %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 microprotein conjugates indicated that the exemplary restricted B7-H3 microprotein conjugates had increased concentrations in tumors and decreased concentrations in the kidneys compared to the exemplary linear B7-H3 microprotein conjugates.

[0088] Figures 19A-19D Line graphs showing efficacy data, such as in cell lines with low (H1915) and high (H358) B7-H3 expression, show efficacy at 17 days (…). Figure 19A ,1915 cells) and 42 days ( Figure 19CFollowing administration of H358 cells, tumor volume was measured, and each assessment was performed after administration of a single dose of the mediator or one of two concentrations (500 or 1000 nCi) of the exemplary 225-Ac-labeled restricted B7-H3 microprotein conjugate (C332). Figure 19A The corresponding weight is as follows Figure 19C As shown, Figure 19B The corresponding weight is as follows Figure 19D As shown.

[0089] Figures 20A-20B The study demonstrated the effect of tumor volume control over approximately 0–6 weeks following administration of a single dose of the mediator or one of two concentrations (500 or 1000 nCi). Figure 20A ) and weight ( Figure 20B A line graph of the measured efficacy data. Detailed Implementation

[0090] Among other things, this disclosure also provides compositions and methods of using them. 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., chelating agents, radionuclides) wherein the therapeutic agents selectively target cells expressing B7-H3, thereby treating B7-H3-expressing cells without treating cells that do not express B7-H3. This disclosure acknowledges that the problem with treating cells expressing a target (e.g., cancer cells) stems from the insufficient selectivity of conventional therapies, which fail to specifically target cells (e.g., cancer cells) or deliver the therapeutic agent in a manner that minimizes damage to surrounding cells (e.g., non-cancer cells) (including, for example, cells surrounding cancer cells and cells in non-target organs (e.g., kidneys)). Surrounding cells (e.g., which may be present in one or more non-tumor tissues) may also express the target in amounts or at levels lower than those of the target cells. In some such embodiments, this expression may be undetectable. This disclosure provides the insight that a combination of selective targeting (e.g., B7-H3, e.g., by peptides, e.g., microproteins, e.g., microprotein targeting as provided herein) with specific therapeutic agents (e.g., chelators and / or radionuclides (e.g., alpha emitters)) offers advantages over previously and / or currently used therapeutic agents (e.g., antibodies, beta emitters, etc.).

[0091] Furthermore, this disclosure provides the insight that certain challenges may still arise even with more specific and targeted therapeutic agents (e.g., those disclosed herein). In addition, tumor uptake may be challenged by uptake, retention, and / or clearance from one or more non-target tissues (e.g., non-tumor tissues) (e.g., the kidneys), resulting in (1) accelerated clearance; (2) reduced tumor targeting (e.g., due to uptake and / or retention by non-target tissues); and / or (3) damage to non-target tissues (e.g., the kidneys). This disclosure acknowledges that any or all of these challenges can be mitigated or prevented by: (i) using microproteins as provided herein (e.g., as disclosed in Tables 2A and / or 2C), including microproteins having specific properties (e.g., limiting, e.g., as shown in Table 2C); and / or (ii) combining the administration of a therapeutic agent with the administration of a decoy (e.g., as described in Table 2D). In some embodiments, the decoy reduces or prevents renal uptake of a composition containing a radionuclide. Not wishing to be bound by theory, this disclosure takes into account the need to maintain at least the improvement in therapeutic efficacy while reducing damage to the kidneys, and in some embodiments, to improve therapeutic efficacy while reducing the risk or actual damage to kidney tissues and / or renal system tissues (e.g., ureters, bladder, etc.).

[0092] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature and techniques used in conjunction with those described herein are those well-known and commonly used in the art: biochemistry, enzymology, molecular and cell biology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization.

[0093] Unless otherwise indicated, the methods and techniques disclosed herein are generally carried out in accordance with conventional methods known in the art and set forth in the various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and 2002 supplemented editions); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); (1990); Wittrup and VanAntwerp, FineAffinity Discrimination by Yeast Surface Display and Flow Cytometry,Biotechnol. Prog. 2002, (16) 31-37; C. Queen et al., A humanized antibody thatbinds 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.

[0094] All publications, patents, and other references mentioned herein are hereby incorporated in their entirety by reference. In the event of any conflict, this specification, including its definitions, shall prevail. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0095] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature used in conjunction with the following and the following techniques are those well-known and commonly used in the art: biochemistry, enzymology, molecular and cell biology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization as described herein.

[0096] Throughout the specification and claims, the word “comprise” or variations such as “comprises” or “comprising” shall be understood to imply inclusion of the stated integer or group of integers, but not to exclude any other integer or group of integers.

[0097] As used herein, ranges and quantities may be expressed as “about” a specific value or range, such as “about” a specific value and / or “about” another specific value. “About” also includes precise quantities. Thus, “about 100 nucleotides” means both “about 100 nucleotides” and “100 nucleotides”. In the given context, the term “about” as used herein also includes quantities expected to be within the experimental error range. If “about” appears before a quantitative value, this disclosure also includes the specific quantitative value itself unless otherwise expressly stated. In such cases, “about” may also refer to a variation of ±10% from the nominal value unless otherwise indicated or inferred. When a value is expressed as an approximation using the antecedent “about”, it should be understood that this disclosure also considers embodiments that specify specific values ​​and ranges of values ​​without approximations.

[0098] Unless the context explicitly indicates otherwise, the singular forms “a / an” and “the” as used herein include a plural of indicators. Thus, for example, in some embodiments, references to “e.g.” decoys include multiple decoys, a single decoy, etc.

[0099] As used herein, the expression “and / or” relating to two or more described objects individually includes each of the described objects and various combinations of two or more described objects, unless otherwise understood from the context and usage.

[0100] Unless otherwise indicated, and as an example of all sequences set forth in the general format “SEQ ID NO:” herein, “nucleic acid containing SEQ ID NO: 1” means a nucleic acid having at least a portion of the sequence of SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 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 the probe is complementary to the desired target.

[0101] As used herein, the term "administration" means providing the composition to a subject or system. The composition may be administered to the subject via any appropriate route, dose, and / or dosing regimen.

[0102] As used herein, the term “affinity” refers to a subgenus of microproteins. Affinity is a molecule derived from the Z-domain of staphylococcal protein A, consisting of three α-helices with 58 amino acids and a molar mass of approximately 6 kDa. For illustrative details regarding the structure and use of affinity, see Orlova, A; Magnusson, M; Eriksson, TL; Nilsson, M; Larsson, B; Höidén-Guthenberg, I; Widström, C; Carlsson, J et al. (2006). “Tumor imaging using a picomolar affinity HER2 binding affibody molecule,” Cancer Res. 66 (8): 4339-48. Illustrative Affibody® molecules are available from Abcam Corp. Cambridge Mass. Affinity is stable at high temperatures and under acidic or alkaline conditions. Target specificity was obtained by randomizing 13 amino acids in two α-helices involved in the binding activity of the parent protein domain (Feldwisch J, Tolmachev V.; (2012) Methods Mol Biol. 899:103-26).

[0103] As used herein, the term “affinity maturation” generally refers to the process of making successive alterations (e.g., successive mutations) to a sequence and performing peptide sequence selection to select one or more sequences with higher affinity relative to the “starting” sequence, or to select another sequence with lower affinity compared to the sequence with higher affinity.

[0104] As used herein, the terms "amino acid sequence" and "peptide" refer to a polymer of amino acids linked by one or more peptide bonds. The polypeptides disclosed herein encompass naturally occurring and non-naturally occurring proteins, as well as any fragments, portions, peptides, mutants, derivatives, and analogs thereof. Polypeptides can be monomers or polymers. Furthermore, polypeptides may contain multiple distinct domains, each possessing one or more distinct activities. Polypeptides can be wholly synthetic or partially synthetic, or otherwise modified (i.e., containing one or more synthetically produced amino acids and / or their modifications). The term "peptide" may be used to refer to short polypeptides, such as those 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).

[0105] As used herein, a “compound” refers at least to a microprotein having an amino acid sequence. Compounds may include microproteins with different modifications, such as N-terminal or C-terminal modifications. In various embodiments, a “compound” may include a microprotein and one or more additional elements, examples of which include linkers, chelating agents, and / or radionuclides. For example, a compound may include a microprotein conjugated, for example, to a chelating agent and / or radionuclide via a linker. As indicated herein, compounds are identified with specific compound numbers, such as “C1”, “C2”, “C3”, etc. Different compounds may have different sequences. In various embodiments, different compounds may have the same sequence (e.g., assigned the same SEQ ID NO) but may have one or more different modifications (e.g., different N-terminal or C-terminal modifications), different linkers, different chelating agents, and / or different radionuclides. N-terminal and C-terminal modifications may include, but are not limited to, acetyl, acid, or amide (e.g., acetyl, NH2, OH / COOH), as provided in the exemplary compounds and microproteins in Table 2A or Table 2C. In some embodiments, the polypeptides of this disclosure may have various modifications to their N-terminus (e.g., as described in the exemplary compounds in Table 2A or Table 2C) and may have an acid or amide group at their 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 substantially altering the utility or function of the polypeptide, such as binding to B7-H3 (e.g., for the detection and / or treatment of cancer).

[0106] As used herein, the term "attenuation" generally refers to a loss of function, including mutations, partial or complete deletions, insertions, or other alterations to the gene sequence or the sequence controlling the transcription of the gene sequence that reduce or inhibit the production of the gene product or render the gene product nonfunctional. In some cases, loss of function is described as a knockout mutation. Attenuation also includes achieving amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, downregulating the expression of interfering RNA, ribozymes, or antisense sequences targeting the gene of interest, or by any other technique known in the art. In one instance, a particular enzyme is reduced in sensitivity to feedback inhibition or inhibition caused by a component that is not a product or reactant (non-path-specific feedback), such that the enzyme activity is unaffected by the presence of a compound. In other instances, an enzyme that has been altered to have lower activity may be referred to as an attenuated enzyme.

[0107] As used herein, the term "binding agent" refers to a subgenus of microproteins. A binding agent is characterized by comprising or consisting of a polypeptide (e.g., a peptide) capable of binding or known to bind and associate with a target. Binding agents typically contain a cysteine-containing peptide with one or more disulfide bonds, but some binding agents do not contain cysteine ​​residues and / or disulfide bonds. When administered systemically to mammalian subjects, it is preferable to rapidly remove the binding agent from circulation. It will be understood that, in a given context, reference to a binding agent may include or include its nucleic acid sequence or the amino acid sequence encoding it. Binding agents may be provided in the form of, for example, polynucleotides or polypeptides, using a carrier, host cell, etc., and / or any combination thereof. Binding agents may be derived or manufactured using any method known to those skilled in the art. For example, in some embodiments, the binding agent may be recombinant (i.e., generated using recombinant nucleic acids encoding a polypeptide). In some embodiments, the binding agent may be synthetic (e.g., synthesized using standard solid-phase synthesis methods, such as solid-phase peptide synthesis, as known to those skilled in the art (see, for example, Palomo, J.)). RSC Adv (., 2014, 4, 32658-32672), and described herein.

[0108] As used herein, the term "blocking" means preventing, slowing, inhibiting, or otherwise reducing or decreasing the uptake and / or retention of a compound by a tissue (e.g., non-tumor tissue, such as kidney tissue, or liver tissue). In some embodiments, the decoy blocks the uptake of the conjugates or compounds of this disclosure by non-tumor tissues (e.g., kidney tissue). In some embodiments, the decoy blocks the uptake of a compound (e.g., a radiotherapy compound, such as one containing a microprotein) by reducing the retention of the compound in non-tumor tissues (e.g., kidney, or liver).

[0109] As used herein, the term "chelating agent" refers to any molecule or portion capable of forming a complex (i.e., a "chelate") with a metal ion. Chelating agents typically have two or more unshared electron pairs that can be used to donate electrons to the metal ion. The metal ion is usually coordinated with the chelating agent via two or more electron pairs.

[0110] As used herein, the term "conjugate" refers to two compounds or agents joined together in a covalent or non-covalent manner.

[0111] As used herein, a “conservative amino acid substitution” is the substitution of one amino acid residue for another amino acid residue with a side chain (R group) that has similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. If two or more amino acid sequences are different from each other due to a conserved substitution, the conservation nature of the substitution can be corrected by adjusting the percentage of sequence identity or degree of homology upwards. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (incorporated herein by reference). The following six groups each contain amino acids that are conserved substitutes for each other: 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), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0112] As used herein, the terms "cysteine-dense peptide" and "CDP" are used interchangeably and refer to a subgenus of microproteins that typically contain at least two independent folded domains and a high density of cysteine ​​residues. In some embodiments, CDPs contain at least one, two, three, four, or more cysteine ​​residues in the range of about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues, see, for example, pubmed.ncbi.nlm.nih.gov / 29483648 / . In some embodiments, CDPs comprise a constrained distribution of cysteine ​​residues, 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).

[0113] As used in this article, the term "deletion" generally refers to the removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, where the regions on either side are joined together.

[0114] As used herein, the term “degenerate variant” of a reference nucleic acid sequence encompasses a nucleic acid sequence that can be translated according to the standard genetic code to provide the same amino acid sequence as that translated from the reference nucleic acid sequence. The terms “degenerate oligonucleotide” or “degenerate primer” are used to refer to oligonucleotides capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but are homologous to each other in one or more specific segments.

[0115] As used herein, the term "derived from" refers to a nucleic acid sequence that is generated using a reference nucleic acid sequence and has at least 85% sequence identity with the reference nucleic acid sequence. As used herein, the term "derived from" refers to an amino acid sequence that has at least 85% sequence identity with the reference natural amino acid sequence from which it 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, nucleic acid or amino acid sequences can be synthesized by chemical methods.

[0116] As used herein, the term “determined” refers to the process or action of requesting a third party (e.g., a laboratory, hospital, nurse, physician) to perform tests, procedures, experiments, measurements, analyses, etc., or to provide their results, in order to determine the presence of a given marker, such as a biomarker or gene mutation (e.g., the expression level of B7-H3 in a biological sample, such as a tumor sample).

[0117] As used herein, the term "domain" refers to a structure in a biomolecule that contributes to the known or suspected function of the biomolecule. A domain may extend along with its regions or parts; a domain may also include different, non-adjacent regions of the biomolecule. Examples of protein domains include, but are not limited to, Ig domains, extracellular domains, transmembrane domains, and cytoplasmic domains.

[0118] As used herein, the term "expression control sequence" refers to a polynucleotide sequence essential to the expression of a coding sequence operatively linked to it. Expression control sequences are sequences that control transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and sequences that enhance protein secretion when needed. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion chaperone sequences.

[0119] As used herein, the term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to a heterologous amino acid sequence. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. Fusion proteins contain 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. Fusion proteins comprising the entirety of the proteins disclosed herein have particular utility. The heterologous polypeptide contained within the fusion proteins of this disclosure is at least 6 amino acids in length, typically at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusion proteins comprising larger polypeptides (e.g., IgG Fc regions) or even entire proteins (e.g., proteins containing green fluorescent protein (“GFP”) chromophores) have particular utility. Fusion proteins can be generated recombinantly by constructing a nucleic acid sequence encoding a polypeptide or fragment thereof in the same frame as a nucleic acid sequence encoding a different protein or peptide, and then expressing the fusion protein. Alternatively, fusion proteins can also be produced chemically by crosslinking a polypeptide or fragment thereof with another protein.

[0120] As used herein, when referring to a protein, "homology" to a second protein may exist if the nucleic acid sequence encoding that protein has a similar sequence to that encoding a second protein. Alternatively, two proteins are homologous if they have a "similar" amino acid sequence. (Therefore, the term "homological protein" is defined as two proteins having similar amino acid sequences.) Homology between two regions of an amino acid sequence (especially in terms of predicted structural similarity) can be interpreted as implying functional similarity. Homologous proteins or peptides with different residue positions are generally considered distinct due to conserved amino acid substitutions.

[0121] As used herein, the term "identical" refers to nucleic acid sequences of at least two nucleic acids, or amino acid sequences of at least two amino acid sequences or subsequences, each having a specified percentage of nucleotides or amino acids, that are identical when performing maximum correspondence comparisons and alignments, as measured by sequence comparison algorithms or by visual inspection. For sequence comparisons, typically one sequence serves as a reference sequence to which the test sequence is compared. The length of a sequence identity comparison can be any number of nucleotide or amino acid extensions. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, the coordinates of the subsequences are specified (if necessary), and the sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. Several algorithms are known in the art. Non-limiting examples of algorithms suitable for determining sequence identity and percentage sequence similarity are the BLAST and BLAST 2.0 algorithms, described, for example, in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information. Alternatively or concurrently, sequences can be compared using FASTA, Gap, or Bestfit, programs in Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA provides alignment of the best overlapping region between the query and search sequences and the percentage of sequence identity. Pearson, Methods Enzymol. 183:63-98 (1990) (hereinafter incorporated herein by reference in its entirety). For example, the percentage of sequence identity can be determined using FASTA with its default parameters (word length of 6 and NOPAM factor for the scoring matrix) or using Gap, as provided in GCG version 6.1 (incorporated hereby by reference), with its default parameters.

[0122] As used herein, the term "isolated" polynucleotide or polypeptide is a polynucleotide or polypeptide substantially isolated from other cellular components (e.g., ribosomes, polymerases, and their naturally associated genomic sequences) that are naturally associated with the polynucleotide in the native host cell. For example, an isolated molecule is one that is characterized by its origin or derivation as follows: (1) not associating with its naturally associated components in their native state; (2) existing in a purity not found in nature, where purity can be determined by the presence of other cellular material (e.g., not containing other proteins from the same species); (3) expressed by cells from a different species; or (4) not existing in nature (e.g., it is a fragment of a polynucleotide or polypeptide found in nature, or it includes amino acid analogs or derivatives or bonds other than standard peptide bonds not found in nature). Thus, a polynucleotide or polypeptide synthesized chemically or in a cellular system different from the cell of its natural origin will be "isolated" from its naturally associated components. Polynucleotides or polypeptides can also be substantially free of their naturally associated components through isolation using protein purification techniques well known in the art. As defined herein, “isolated” does not necessarily require the physical removal of any molecule described herein from its natural environment. In some embodiments, as mentioned in references to the use of isolated constructs, “isolated” means the absence of pharmaceutically acceptable salts.

[0123] As used in this article, the term "K" D "Kd" or "target" refers to the dissociation equilibrium constant between a specific entity and its target (e.g., antibody-antigen (or, for example, targeting microprotein-target protein), or a specific interaction between the entity and its target (e.g., peptide-target interaction, such as the peptide and B7-H3 described herein). Generally, the dissociation equilibrium constant (Kd) of the antibody (e.g., targeting microprotein, such as B7-H3-binding microprotein) of this disclosure bound to B7-H3 is... D Less than approximately 10 -7 M, for example, less than about 10 -8 M, 10 -9 M or 10 -10 M or lower, for example, as determined using surface plasmon resonance (SPR) techniques in the BIACORE instrument. D = k d / k a .

[0124] As used herein, the term “Ki” (M) refers to the binding inhibition constant of a given entity with a target (e.g., a specific peptide-target interaction).

[0125] As used in this article, the term "k" d (s) -1The dissociation rate constant (k) refers to the rate constant between a given entity and its target (e.g., a specific peptide-target interaction). This value is also known as k. off value.

[0126] As used in this article, the term "k" a (M) -1 ×s -1 The association rate constant (k) refers to the rate constant of association between a given entity and its target (e.g., a specific peptide-target interaction). This value is also known as k. on value.

[0127] As used in this article, the term "K" A (M) -1 K refers to the association equilibrium constant between a given entity and its target (e.g., a specific peptide-target interaction). A = k a / k d .

[0128] The affinity of molecule X for its target Y can be determined by the dissociation equilibrium constant (K). D The kinetic components that contribute to the dissociation equilibrium constant are as described above. For clarity, as is known in the art, the smaller K... D A value indicates a higher affinity interaction, while a larger K value indicates a lower affinity interaction. D The value indicates a lower affinity interaction. Affinity can be measured by commonly used methods known in the art, including those described herein, such as surface plasmon resonance (SPR) techniques (e.g., BIACORE). ® ) or biolayer interferometry (e.g., FORTEBIO) ® ).

[0129] As used herein, the term "knockout" generally refers to a gene whose expression or activity level has been reduced to zero. In some instances, a gene is knocked out by the partial or complete deletion of its coding sequence. In other instances, a gene is knocked out by introducing one or more nucleotides into its open reading frame, resulting in the translation of a nonsense, or in other words, nonfunctional, protein product.

[0130] As used in this article, the term "knotting protein" refers to the structural motif of a microprotein containing three disulfide bridges.

[0131] As used in this article, the term "knotting peptide" refers to a subgenus of microproteins containing at least one knotting peptide.

[0132] As used in this article, the term "connector" refers to the portion used to conjugate microproteins to a chelating agent.

[0133] As used herein, the term "microprotein" refers to a short protein containing 100 or fewer amino acids, having a well-defined folded structure, comprising two or more secondary structural elements, an isolated hydrophobic core, and / or co-folding. Affinities, CDPs, knotting agents, and binding agents disclosed herein are examples of microproteins.

[0134] As used herein, the term "modification" when referring to a nucleic acid sequence means that, compared to a reference nucleic acid sequence, the nucleic acid sequence contains at least one substitution, alteration, inversion, addition, or deletion of a nucleotide. Similarly, as used herein, the term "modification" when referring to an amino acid sequence means that, compared to a reference amino acid sequence, the amino acid sequence contains at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue.

[0135] As used herein, the term "modified derivative" refers to a polypeptide or fragment thereof that is substantially homologous in its primary structural sequence, but includes, for example, in vivo or in vitro chemical and biochemical modifications, or the incorporation of amino acids not present in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling (e.g., with radionuclides), and various enzymatic modifications, as readily apparent to those skilled in the art. Various methods for labeling polypeptides and a variety of substituents or labels that can be used for such purposes are well known in the art, and include radioisotopes (e.g., 125I, 32P, 35S, and 3H), ligands that bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands that can be used as specific binding pairs of labeled ligands. The choice of label depends on the required sensitivity, ease of primer conjugation, stability requirements, and available instruments. Methods for labeling polypeptides are well known in the art. See, for example, Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992 and 2002 eds). (Included hereby by reference).

[0136] As used herein, the term “molecule” means any compound, including but not limited to microproteins, small molecules, peptides, proteins, sugars, nucleotides, nucleic acids, lipids, etc., and such molecules (e.g., microproteins, compounds, etc.) can be natural or synthetic or a combination of natural and synthetic.

[0137] As used herein, the terms “monomeric antibody” or “adnectin” are used interchangeably and refer to a subgenus of microproteins. A monomeric antibody is a molecule, preferably based on the 10th extracellular domain of human fibronectin III (10Fn3), employing an Ig-like b sandwich fold with 2 to 3 exposed loops, preferably consisting of 94 residues, but lacking 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 into specific loops of the protein.

[0138] As used herein, the terms “mutant protein,” “mutant protein,” or “variant” refer to a protein whose amino acid sequence has at least one variation (e.g., insertion, deletion, or substitution, which may be conserved or non-conserved) compared to a reference sequence. “Mutant,” when applied to a sequence (e.g., a nucleic acid sequence, an amino acid sequence), means that nucleotides in the nucleic acid sequence or amino acids in the amino acid sequence may have been inserted, deleted, or altered compared to a reference sequence. A single change can be made at a locus (point mutation), or multiple nucleotides or amino acids can be inserted, deleted, or altered at a single locus. Additionally, one or more changes can be made at any number of loci within a nucleic acid or amino acid sequence. Nucleic acid or amino acid sequences can be mutated by any method known in the art, including but not limited to mutagenesis techniques such as “error-prone PCR” (a process of performing PCR under conditions of low replication fidelity of DNA polymerase, resulting in a high rate of point mutations along the full length of the PCR product; see, for example, Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCRMethods Applic. 2:28-33 (1992)); “oligonucleotide directed mutagenesis” (a process capable of generating site-specific mutations in the DNA segment of interest in any clone; see, for example, Reidhaar-Olson and Sauer, Science 241:53-57 (1988)); directed evolution (e.g., exposing a polypeptide to different sets of conditions, resulting in different polypeptides with one or more amino acid changes that may or may not confer greater fitness to the polypeptide); and site-directed mutagenesis (e.g., specific directed changes in a sequence).

[0139] As used herein, the term "peptide mutant" or "mutant protein" refers to a polypeptide whose sequence contains one or more amino acid insertions, repeats, deletions, rearrangements, or substitutions compared to the amino acid sequence of a native or wild-type protein. Mutant proteins may have one or more amino acid site substitutions, where a single amino acid at one position is changed to another amino acid; one or more insertions and / or deletions, where one or more amino acids are inserted or deleted in the sequence of the native protein; and / or truncation of the amino acid sequence at the N-terminus or C-terminus. Mutant proteins may have the same biological activity as the native protein, but preferably have different biological activities. The mutant protein has at least 85% overall sequence homology with its wild-type counterpart. Even more preferably, a mutant protein has at least 90% overall sequence homology with the wild-type protein. In even more preferred embodiments, the mutant protein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99%, and even more preferably 99.9% overall sequence identity. Sequence homology can be measured using any common sequence analysis algorithm, such as Gap or Bestfit. Amino acid substitution may include those that: (1) reduce sensitivity to protein hydrolysis, (2) reduce sensitivity to oxidation, (3) alter the binding affinity for forming protein complexes, (4) alter the binding affinity or enzyme activity, and (5) impart or alter other physicochemical or functional properties to such analogues.

[0140] As used herein, the term "non-disulfide bond sequence" refers to an amino acid sequence that encodes a polypeptide that does not contain more than one cysteine ​​residue and / or disulfide bond in its folded and active form. For example, in some embodiments, microproteins may contain or consist of non-disulfide bond sequences.

[0141] As used herein, the term "non-peptide analog" refers to a compound whose properties are similar to those of a reference peptide. Non-peptide compounds may also be referred to as "peptide mimetic" or "peptidomimetic". For example, see 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 the above-mentioned documents, which are incorporated herein by reference. Such compounds are typically developed using computer molecular modeling techniques. Peptide mimics that are structurally similar to the useful peptides of this disclosure may be used to produce equivalent effects and are therefore expected to be part of this disclosure.

[0142] As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably and refer to polymers 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 DNA or RNA analogs containing non-natural nucleotide analogs, non-natural nucleoside internucleotide bonds, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpin-shaped, circular, or padlock conformations. Nucleic acid sequences can contain natural, non-natural, or modified nucleotides; and can contain natural, non-natural, or modified internucleotide bonds, such as aminophosphate bonds or thiophosphate bonds, rather than phosphodiester bonds between nucleotides in an unmodified nucleic acid sequence. Nucleic acid sequences include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant methods, such as cloning nucleic acid sequences from self-recombinant libraries or cell genomes using conventional cloning techniques and polymerase chain reactions, as well as synthetic methods. The polynucleotides disclosed herein may include sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers as described above. As will be readily apparent to those skilled in the art, they may be chemically or biochemically modified or may contain non-natural or derived nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged bonds (e.g., methylphosphonates, triphosphates, aminophosphates, carbamates, etc.), charged bonds (e.g., thiophosphates, dithiophosphates, etc.), overhangs (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelating agents, alkylating agents, and modified bonds (e.g., α-anomeric nucleic acids, etc.). Synthetic molecules that mimic the polynucleotides in their ability to bind to a specified sequence via hydrogen bonding and other chemical interactions are also included. Such molecules are known in the art and include, for example, those in which peptide bonds replace phosphate ester bonds in the molecular backbone. Other modifications may include, for example, ribose rings containing bridging portions or analogues of other structures, such as those found in “locked” nucleic acids.

[0143] As used herein, the terms "operatively linked" or "operably linked" expression control sequences refer to expression control sequences that are adjacent to the gene of interest to control the linkage of the gene of interest, or that act in a trans manner or at a certain distance to control the expression of the gene of interest.

[0144] As used herein, the term "peptide fragment" refers to a polypeptide that has deletions (e.g., deletions of the N-terminus and / or C-terminus) compared to the full-length polypeptide. In a preferred embodiment, a polypeptide fragment is an adjacent sequence in which the amino acid sequence of the fragment is identical to the corresponding position in the native or parental sequence. Fragments are typically at least 5, 6, 7, 8, 9, or 10 amino acids in length, preferably at least 12, 14, 16, or 18 amino acids in length, more preferably at least 20 amino acids in length, even more preferably at least 25, 30, 35, 40, or 45 amino acids in length, even more preferably at least 50 or 60 amino acids in length, and even more preferably at least 70 amino acids in length.

[0145] As used in this article, the term "radioactive nuclide" refers to an atom that is capable of undergoing radioactive decay.

[0146] As used herein, the term "radiotherapy agent" refers to a radionuclide-labeled microprotein or compound containing a radionuclide, as provided herein. Radiotherapy agents can be administered to subjects, such as test subjects (e.g., mice or rats, e.g., non-human primates, e.g., healthy volunteers), and / or subjects requiring radiation therapy, such as cancer patients.

[0147] As used herein, the term “recombinant” refers to a biomolecule (e.g., a gene or protein) that: (1) has been removed from its naturally occurring environment; (2) is wholly or partially unrelated to the polynucleotides of the gene found in nature; (3) is operatively linked to a polynucleotide not linked to in nature; and / or (4) is not found in nature. The term “recombinant” can be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs or polynucleotide analogs biosynthesized via heterologous systems, and proteins and / or mRNAs encoded by such nucleic acids. As used herein, an endogenous nucleic acid sequence (or the protein product encoded by that sequence) in an organism’s genome is considered “recombinant” if a heterologous sequence is placed adjacent to an endogenous nucleic acid sequence, thereby altering the expression of that endogenous nucleic acid sequence. In this context, a heterologous sequence is a sequence that is not naturally adjacent to an endogenous nucleic acid sequence, whether the heterologous sequence itself is endogenous (derived from the same host cell or its descendants) or exogenous (derived from different host cells or their descendants). For example, the expression pattern of a gene can be altered by replacing its natural promoter in the host cell genome with a promoter sequence (e.g., through homologous recombination). Since the gene is separated from at least some of its natural flanking sequences, it is now considered "recombinant." A nucleic acid is also considered "recombinant" if it contains any naturally occurring modifications to the corresponding nucleic acid in the genome. For example, an endogenous coding sequence is considered "recombinant" if it contains, for example, an insertion, deletion, or point mutation introduced artificially through human intervention. "Recombinant nucleic acids" also include nucleic acids integrated into the host cell chromosome at heterologous sites and nucleic acid constructs existing as free organisms.

[0148] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell in which a recombinant vector has been introduced. It should be understood that such terms are intended not only to the specific subject cell but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may actually differ from the parent cells but are still included within the scope of the term "host cell" as used herein. Recombinant host cells can be isolated cells or cell lines grown in a culture or cells residing in living tissue or an organism.

[0149] As used herein, the term "region" refers to the actually adjacent portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by the adjacent portion of the protein's amino acid sequence.

[0150] As used herein, the term "secondary structural element" refers to a locally folded structure formed within a polypeptide due to interactions between atoms in the polypeptide backbone. Examples of secondary structural elements may include α-helices, β-sheets, 310 helices, π-helices, and random coils. The microproteins of this disclosure may contain one or more of these secondary structures (e.g., one or more α-helices, one or more α-helices, and one or more β-sheets). Those skilled in the art will understand that secondary structural elements can be joined by loop regions, which may or may not be modified to alter the interactions of polypeptide secondary structural elements. As those skilled in the art will appreciate, in some embodiments, a loop may be a secondary structural element. In some embodiments, a loop may be an interstructural element but is not necessarily considered a secondary structural element.

[0151] As used herein, peptide “sequence homology” (also known as “sequence identity percentage”) is typically measured using sequence analysis software. See, for example, the sequence analysis software package from the Genetic Computing Group (GCG) at the University of Wisconsin-Madison Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software uses homology measurements assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match similar sequences. 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 peptides (e.g., homologous peptides from organisms of different species) or between wild-type proteins and their mutant counterparts. See, for example, GCG version 6.1. When comparing a specific polypeptide sequence with a database containing a large number of sequences from different organisms, the 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 AcidsRes. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The preferred parameters for BLASTp are: Expected value: 10 (default); Filter: seg (default); Vacancy open penalty: 11 (default); Vacancy spread penalty: 1 (default); Maximum alignment: 100 (default); Word length: 11 (default); Number of descriptions: 100 (default); Penalty matrix: BLOSUM62. The length of the polypeptide sequence used for homology comparison will typically 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, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database retrieval using amino acid sequences can be measured using algorithms known in the art other than BLASTp. For example, polypeptide sequences can be compared using FASTA (a program in GCG version 6.1). FASTA provides the alignment of the best overlapping region between the query and search sequences and the percentage of sequence identity.Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated by reference). For example, the percentage of sequence identity between amino acid sequences can be determined using FASTA with its default parameters (word length 2, PAM250 score matrix) as provided in GCG version 6.1 (incorporated by reference).

[0152] As used herein, the term "specific activity" generally refers to the activity of a radionuclide per unit (e.g., mass, e.g., mole). Specific activity units may include megabecquerels per microgram (MBq / mg), microcuries per microgram (mCi / mg), microcuries per nanomolar (mCi / nmole), etc.

[0153] As used herein, the term "specific" generally refers to a sequence (e.g., a protein sequence, such as a microprotein sequence having certain amino acids) that, when in a binding conformation, selectively or "specifically" binds to a particular target (e.g., an antigen expressed on a tumor, such as B7-H3, or certain cell types, such as kidney cells, such as proximal tubular cells of the kidney, etc.).

[0154] As used herein, “specific binding” means that the binding of a polynucleotide, polypeptide, or protein is selective for a specific antigen (e.g., a target) and can be distinguished from unwanted or nonspecific interactions. Indicators of “specific binding” include saturation of binding to the target and a demonstrable ability to compete for the binding by introducing other specifically binding molecules targeting the same target. For example, the ability of a protein (e.g., a cysteine-rich peptide) to bind to a specific antigenic determinant can be measured using techniques familiar to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance. Between two molecules (e.g., entities of microproteins), “specific binding” refers to the ability of two molecules to preferentially bind to each other rather than to other molecules in the environment. Typically, “specific binding” is distinguished from accidental binding in a reaction by at least a two-fold increase, more commonly at least a ten-fold increase, often at least a hundred-fold increase, and even up to a thousand-fold increase. Typically, the affinity or affinity (quantified by dissociation constant) of a particular binding reaction is about 10⁻⁷ M or stronger (e.g., about 10⁻⁸ M, 10⁻⁹ M or even stronger). Specific binding requires that a particular first entity (e.g., a polypeptide) is specific to a particular second entity (e.g., an antigen-binding sequence).

[0155] As used herein, “strict hybridization conditions” and “strict washing conditions” in the context of nucleic acid hybridization experiments depend on many different physical parameters. Nucleic acid hybridization is affected by a variety of factors, such as salt concentration, temperature, solvent, base composition of the hybrid material, length of complementary regions, and the number of nucleotide base mismatches between hybrid nucleic acids, as will be readily understood by those skilled in the art. Those skilled in the art know how to modify these parameters to achieve a specific degree of hybridization strictness. Generally, “strict hybridization” is performed under a specific set of conditions at a temperature approximately 25°C lower than the thermal melting point (Tm) of the specific DNA hybrid. “Strict washing” is performed under a specific set of conditions at a temperature approximately 5°C lower than the Tm of the specific DNA hybrid. Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), p. 9.51, which is incorporated herein by reference. For the purposes of this article, "strict conditions" are defined for liquid-phase hybridization as aqueous hybridization (i.e., formamide-free) at 65°C for 8–12 hours in 6xSSC (of which 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate) and 1% SDS, followed by two washes at 65°C for 20 minutes each in 0.2xSSC and 0.1% SDS. Skilled operators will understand that the hybridization rate at 65°C will vary due to several factors, including the length of the hybridized sequences and the percentage of identity.

[0156] As used herein, the term "synthetic" refers to an entity manufactured in a laboratory, rather than an entity that arises or is isolated naturally from a natural source without modification. Recombinant polymers (e.g., recombinant polynucleotides or peptides) may be synthetic. Synthetic polymers (e.g., polynucleotides or peptides) can be produced by any method known to those skilled in the art, including but not limited to solid-phase synthesis, liquid-phase synthesis, and biosynthesis via, for example, host cell biosynthesis.

[0157] As used herein, the term "subject" refers to a mammal. A subject can be human or a non-human mammal. In the given context, subject may be used interchangeably with patient, individual, donor, etc. In some embodiments, a subject is a healthy subject without disease, contemplated for treatment with the compositions of this disclosure (e.g., administration of one or more compositions provided herein to a healthy volunteer). In some embodiments, a subject is a subject suspected of or diagnosed with a disease, condition, or disorder (e.g., cancer and / or tumor), as provided herein. In some such embodiments, the compositions of this disclosure are contemplated for treatment of such subjects. In some embodiments, an analysis of the results obtained using the techniques disclosed herein is performed in a population comprising multiple subjects.

[0158] As used herein, the terms "substantial homology" or "substantial similarity," when referring to a polynucleotide or polypeptide, mean that, when optimally aligned with another reference molecule (or its complementary strand, where appropriate) for the insertion or deletion of a suitable nucleotide or amino acid, sequence identity exists in at least about 70%, 75%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% or more of the nucleic acid or amino acid residues, as measured by any well-known sequence identity algorithm (e.g., FASTA, BLAST, Gap, etc.). Alternatively or additionally, substantial homology or similarity exists when, for example, a nucleic acid or a fragment thereof, hybridizes with another nucleic acid, a strand of another nucleic acid, or its complementary strand under stringent hybridization conditions.

[0159] As used herein, the term "target" refers to a protein or a functional portion or variant thereof. A target is a protein engineered to bind to another protein (e.g., a microprotein). A target may be or may contain a binding region, such as an epitope, to which microproteins of this disclosure (e.g., affinity molecules, CDPs, knotting agents, binding agents, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, engineered ankyrin repeat domains (DARPin), avimer) bind. Furthermore, the term "antigen" refers to a protein or a functional portion or variant thereof to which a polypeptide (e.g., a microprotein, etc.) or a variant thereof binds. A target may be or may contain an antigen. A target may be expressed on the surface of a specific cell ("target cell") or intracellularly (e.g., on the cell surface) within a cell population. A target may have a certain percentage of identity with a reference protein but is still referred to by a specific name (e.g., B7-H3). In some embodiments, as is apparent from the context, a target may also refer to a protein in a pathway associated with another protein. For example, if the target protein is B7-H3, the target may also be a protein in a pathway essential for B7-H3 activity. The target may be or may contain a binding region, such as an epitope, to which the microproteins of this disclosure (e.g., affinity molecules, CDPs, knotting agents, binding agents, engineered Kunitz domains, monomeric antibody-like substances, anticalcin, designed ankyrin repeat domains (DARPin), avimer) bind. In some embodiments, which will be clear in the given context, the target may also be a specific cell type (or localized to a specific cell type) characterized by expressing a specific surface entity, such as a receptor (e.g., cells in a tissue, such as proximal tubular cells in the kidney). Such targets may be different from or the same as those to which the microprotein (M) is designed to bind; in some embodiments, the target (e.g., non-tumor cells, such as kidney cells, liver cells, etc.) binds to a decoy rather than to a peptide (e.g., a microprotein) of a composition (e.g., a radiotherapy composition) provided herein.

[0160] As used herein, the term “treatment” (and “treat” or “treating”) means to partially or completely reduce, improve, alleviate, prevent, or eliminate a particular disease, condition, and / or ailment, reduce the risk of its onset, eliminate or suppress the particular disease, condition, and / or ailment, delay its onset, reduce its severity, or reduce the frequency or incidence of one or more causes, characteristics, and / or symptoms of or related to the particular disease, condition, and / or ailment.

[0161] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One class of vectors is the "plasmid," which generally refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked, but also includes linear double-stranded molecules, such as those obtained by polymerase chain reaction (PCR) amplification or by treating circular plasmids with restriction enzymes. Other vectors include granules, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another class of vectors is the viral vector, in which an additional DNA segment can be linked to a viral genome (discussed in more detail below). Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., vectors with an origin of replication that functions in the host cell). Other vectors, upon introduction into the host cell, can be integrated into the host cell's genome and thus replicated along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0162] As used herein, the term "decharged molecule" refers to a molecule that, compared to the parent molecule, has been modified to contain less positive or polar properties, more negative properties, and / or both.

[0163] As used herein, the term "surface charge" refers to the static charge present on the surface of a protein (e.g., a microprotein). In various embodiments, the surface charge of a microprotein can affect renal uptake of the microprotein (e.g., increase or decrease renal uptake of the microprotein).

[0164] As used herein, the term "surface plaque" refers to a region on the surface of a protein (e.g., a microprotein) that has surface characteristics. For example, surface plaques may be defined based on surface charge and / or surface hydrophobicity, which may affect the kidney's uptake of proteins (e.g., microproteins).

[0165] As used herein, the term "cleavable linker" refers to a linker that can be cleaved. A cleavable linker contains a cleavable bond that can be cleaved in vivo, for example, by acidic pH (pH less than 7, typically about 4 to 6), by glutathione, or in the presence of upregulated enzymes such as proteases or peptidases in the proximal tubule matrix. Examples of cleavable linkers are linkers containing hydrazine or disulfide bonds or enzyme-cleavable peptide sequences.

[0166] As used herein, the term "scaffold" refers to a specific set of structural features of a given protein (e.g., a microprotein, such as a binder, such as a decoy peptide). "Scaffold" is also used to describe microproteins that share a common set of general structural features (e.g., certain constraints, secondary structures, tertiary structures, etc.). Any single scaffold may contain varying numbers of α-helices, turns, and / or β-sheets, such as fully α-helical proteins ("a"), fully β-sheet proteins ("b"), mixed α-helical / β-sheet proteins ("a / b"), mixed α and β proteins ("a+b"), and small proteins. Examples and properties of certain scaffolds are provided herein. To cite just one example, a scaffold may be an "affinity" scaffold as provided herein, whereby an affinity can be a target-binding affinity (e.g., B7-H3 binding) or an affinity decoy, wherein each entity has an affinity scaffold containing, for example, certain secondary structural features (e.g., α-helices and / or β-sheets, disulfide bridges at specific and consistent cysteine ​​residues, etc.). Scaffolds of this disclosure may be described as "scaffold type A" and "scaffold type B". The exemplary scaffold A decoy comprises compounds C118-C120. The exemplary scaffold B decoy has an affinity scaffold and comprises compound C10; the B7-H3 targeting peptide of this disclosure is also an affinity of scaffold B, such as C1-C9, C11-C117.

[0167] As used herein, the term "decoy peptide" or "decoy" refers to a molecule specifically designed to mimic the action of a receptor protein and interact with certain target entities. One type of decoy peptide or decoy specifically relates to a subgenus of microproteins that has the following effects: (i) reducing the accumulation of compounds (e.g., microproteins, such as radiolabeled microproteins, such as the radiotherapy agents provided herein) in non-tumor tissues (e.g., kidney or liver tissue where the tumor is located elsewhere); and / or (ii) having minimal or no effect on the uptake of compounds by tumors (e.g., tumors expressing targets such as B7-H3); and / or (iii) reducing adverse (e.g., toxic) accumulation in a subject's non-tumor-containing organs (e.g., liver, kidney, etc.). To cite just one example, an exemplary decoy may be combined with a composition of this disclosure (e.g., comprising a microprotein and a radionuclide, such as a radiotherapy agent) to block the uptake and / or retention of the composition in kidney tissue, as compared to uptake and / or retention in kidney tissue in the absence of the decoy peptide. Not wishing to be bound by theory, this disclosure describes, in some embodiments, a decoy peptide (or decoy) that attracts, for example, a composition of a radiotherapy agent. This means that the presence of the decoy in non-tumor tissues (e.g., kidneys, for example, liver) blocks the uptake and / or retention of the radiotherapy agent by the non-tumor tissue. For clarity, when the decoy peptide is referred to as "attracting" a composition (e.g., compound, for example, microprotein) that binds to a target (e.g., B7-H3), the decoy does not act on the composition (or compound or microprotein), but rather on itself, and, for example, it will still be present in non-tumor tissues (e.g., kidneys, for example, liver) if administered alone, even in the absence of the composition it attracts.

[0168] Composition This document provides novel compositions comprising one or more of peptides, linkers, chelating agents, and / or radionuclides. In some embodiments, the composition comprises linkers and chelating agents. In some such embodiments, the composition is metallized (e.g., in the form of a cold metal labeled with an element, such as that provided herein). In some embodiments, the composition is radiolabeled (e.g., with a radionuclide as provided herein). In some embodiments, the composition comprises linkers, chelating agents, and radionuclides. In some embodiments, the composition comprises or consists of peptides (i.e., microproteins), optional linkers and chelating agents, and / or radionuclides. In some embodiments, the chelating agent and / or radionuclide is conjugated to the microprotein via a linker. In some embodiments, the microproteins of this disclosure comprise or consist of affinity molecules, CDPs, knotting agents, and / or binding agents. In some embodiments, the microproteins comprise or consist of affinity molecules. In some embodiments, the microproteins comprise or consist of CDPs. In some such embodiments, the microproteins comprise or consist of knotting agents. In some such embodiments, the microproteins comprise or consist of binding agents. In some embodiments, microproteins (e.g., affinity molecules, CDPs, knotting agents, binding agents, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankylosing spicule repeating domains (DARPin), avisers) are designed to be conjugated to one or more other components. For example, in some embodiments, microproteins (e.g., affinity molecules, CDPs, knotting agents, binding agents, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankylosing spicule repeating domains (DARPin), avisers) may be conjugated (attached) to another component, such as a chelating agent and / or a radionuclide. In some embodiments, the radionuclide disclosed herein is an alpha emitter. In some such embodiments, the chelating agent and / or the radionuclide is conjugated to the microprotein via a linker.

[0169] Not wishing to be bound by any particular theory, this disclosure takes into account that the compositions of this disclosure are more effective than previously described compositions (e.g., compositions comprising antibodies and / or beta emitter radionuclides). For example, while microproteins (e.g., those used in the compositions provided herein) possess several key properties of antibody-based therapies (e.g., affinity, potency, specificity, and the ability to disrupt protein-protein interactions), they avoid undesirable limitations such as large size, high manufacturing costs, and the necessity of chimerism or humanization. For example, in some embodiments, the microproteins of this disclosure (e.g., affinity molecules, CDPs, knotting agents, binding agents, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankylosing repeat domains (DARPin), avimers) are no more than about 100 amino acids in length. In some embodiments, such a microprotein (e.g., affinity molecules, CDPs, knotting agents, binding agents, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankylosing repeat domains (DARPin), avimers) may be or comprise cysteine-dense peptides. In some embodiments, the microprotein (e.g., affinity protein, CDP, knotting agent, binding agent, engineered Kunitz domain, monomeric antibody, anticalcin, designed ankylosing spondylase repeating domain (DARPin), avimer) comprises one or more disulfide bridges. In some embodiments, the microprotein (e.g., affinity protein, CDP, knotting agent, binding agent, engineered Kunitz domain, monomeric antibody, anticalcin, designed ankylosing spondylase repeating domain (DARPin), avimer) comprises multiple cysteine ​​residues that are cross-linked to maintain a very stable folded state of the peptide having its length (e.g., relative to a peptide of the same length but without as many cysteine ​​residues).

[0170] Not wishing to be bound by theory, this disclosure considers that in some embodiments, the microprotein does not contain multiple cysteine ​​residues; for example, the microprotein contains a single cysteine ​​residue. In some such embodiments, the microprotein may form a dimer, for example, by dimerizing with other microproteins (e.g., self-dimerization). In some embodiments, the microprotein (e.g., a microprotein that binds to B7-H3) contains at least two cysteine ​​residues. In some embodiments, the microprotein contains at least four cysteine ​​residues. In some embodiments, when the microprotein contains at least two or at least four cysteine ​​residues, the microprotein also contains a disulfide bridge between paired cysteine ​​residues. In some embodiments, the microprotein must have at least one or two disulfide bridges. In some embodiments, the microprotein contains three disulfide bridges (and at least six cysteine ​​residues). In some embodiments, constraints include more than one type of constraint (e.g., disulfide bridges, lactam bridges, alkyl pinning bridges, and any combination thereof). For example, in some embodiments, this disclosure provides a polypeptide containing at least one or two disulfide bridges and one lactam bridge. Not wishing to be bound by theory, this disclosure takes into account that the stability conferred by cross-linked cysteine ​​helps to reduce the immunogenicity of microproteins or those containing such microproteins. In some embodiments, this stability may also confer resistance to more demanding conditions (e.g., high temperature, low pH incubation, etc.) provided for effective chelation, while maintaining biological activity (e.g., the ability to bind to a target) and drug-like physicochemical properties (e.g., stability, solubility, monomericity).

[0171] In some embodiments, the microproteins provided herein can act as targeting portions, for example, specifically binding to targets expressed on the surface of tumor cells. In some such embodiments, the microproteins are designed to bind to one or more additional components. For example, the microproteins of this disclosure can be formulated to combine with other components, such as therapeutic molecules (e.g., chelating agent compositions and / or radionuclides) and / or detectable agents (e.g., visualization agents, such as metabolizable and visualization agents, etc.). In some such embodiments, such microproteins conjugated to one or more additional components can be used, for example, for the diagnosis, prognosis, monitoring, and / or treatment of one or more diseases, conditions, or disorders, such as those expressing specific targets on specific cell populations.

[0172] In some embodiments, microproteins (e.g., affinity molecules, CDPs, knotting agents, conjugates, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankyrin repeating domains (DARPin), avimers) exhibit low immunogenicity relative to larger proteins. In some such embodiments, lower immunogenicity improves adaptability to more demanding environmental conditions (e.g., high temperature and low pH incubation) while preserving biological activity. Therefore, in some embodiments, conjugates containing microproteins have lower immunogenicity than compositions containing larger proteins or different targeting moieties (i.e., moieties other than microproteins).

[0173] In some embodiments, compositions comprising a connector, a chelating agent, and / or a radionuclide can effectively penetrate a tumor.

[0174] In some embodiments, microproteins (e.g., affinity molecules, CDPs, knotting agents, binding agents, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankyrin repeating domains (DARPin), avimer) exhibit superior penetration efficiency compared to larger proteins. That is, in some embodiments, microproteins or compositions containing microproteins penetrate solid tumors better than larger proteins or compositions containing proteins larger than microproteins. For example, in some embodiments, binding agents exhibit superior penetration efficiency with a hydrodynamic radius of approximately 1 nm–25 nm. In some embodiments, the hydrodynamic radius is approximately 1 nm–5 nm. In some embodiments, the hydrodynamic radius is approximately 1 nm–3 nm. In some embodiments, the hydrodynamic radius is approximately 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.

[0175] As described herein, microproteins (e.g., affinities, CDPs, knotting agents, binders) are conjugated to chelating agents. In some embodiments, the chelating agent binds to a radionuclide (e.g., an alpha emitter radionuclide, such as actinium). In some such embodiments, such radionuclide conjugates combine the specific binding ability and properties of the microprotein (e.g., affinities, CDPs, knotting agents, binders) with the radionuclide. That is, without being bound by any particular theory, this disclosure provides a conjugate in which, in some embodiments, the microprotein (e.g., affinities, CDPs, knotting agents, binders, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankyrin repeat domains (DARPin), avimer) targets cells expressing a target with its conjugated radioisotope. In some embodiments, the target is expressed on the cell surface. In some embodiments, the target is B7-H3. In some embodiments, the cells are tumor cells. In some embodiments, the conjugate binds to B7-H3 on the surface of tumor cells. In some such embodiments, the radionuclide targets the tumor cells. In some implementations, the radionuclide is an alpha emitter radionuclide and, when internalized, is used to specifically target (e.g., without damaging surrounding tissue / cells) tumor cells.

[0176] target Any cell expressing the target can be targeted by microproteins such as those provided in this article (e.g., affinity proteins, CDP, knotting proteins, binding agents, engineered Kunitz domains, monomeric antibody-like proteins, anticalcin, designed ankyrin repeat domains (DARPin), avimer).

[0177] In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are derived from cell lines. In some embodiments, the cells are primary cells. In some embodiments, the primary cells are derived from a sample of a subject, such as from a tumor or from a corresponding tumor-free tissue (e.g., from another region of an organ or from a healthy donor). In some embodiments, the cells are in vitro cells (e.g., primary cells, cell lines, etc.). In some embodiments, the cells are in vivo cells (e.g., in a subject's body, such as in a human subject's tumor). In some embodiments, the cells express or have been induced to express (e.g., via recombinant technologies) a target. In some embodiments, the target is expressed on the cell surface. In some embodiments, the cells are contacted by a composition that binds to the target expressed on its surface. In some embodiments, after binding (e.g., after binding to a microprotein provided in this disclosure), the target and any bound proteins and / or payloads are internalized into the cell. In some embodiments, the cells are killed by the payload (e.g., a radionuclide and / or a chelating agent, etc.) after internalization.

[0178] In some embodiments, the target is a protein or a portion thereof that is upregulated or overexpressed in cancer cells compared to non-cancer cells. That is, in some embodiments, the target is expressed or overexpressed in the tumor or in the tumor microenvironment relative to target levels in non-lesion tissue (e.g., tissue without a tumor or tumor microenvironment). In some such embodiments, the target is absent or undetectable in non-lesion (e.g., healthy) tissue. In some embodiments, the target is a biomarker of cancer (e.g., cancer cells, tumors).

[0179] In some embodiments, the target may be associated with a protein, such as a protein in a pathway activated or acted upon by another protein. For example, in some embodiments, the protein may be expressed on the surface of cancer cells, and the target may be a pathway acted upon by that surface cell protein. In some embodiments, the protein may be expressed on cancer cells, and the target may be a protein on different cells that causes cancer cell proliferation or, in other words, makes the cancer cells difficult to treat. In some embodiments, tumor-associated cell surface molecules or tumor-specific cell surface molecules may serve as targets for the microproteins or compositions comprising microproteins provided herein.

[0180] In some embodiments, the microprotein or a composition containing the microprotein specifically binds to a target (e.g., B7-H3) expressed on the cell surface. In some embodiments, the target is derived from cell surface lysis. In some such embodiments, if the target is located within an organism, the lysis of the target leads to its circulation throughout the organism's systems. In some such embodiments, specific levels of the target are found in, for example, blood, serum, plasma, etc. In some embodiments, however, a significant portion of the expressed target is localized to the cell surface; therefore, in some embodiments, measurements of target levels may not accurately reflect the amount of the target in a cell population (e.g., a tumor). In some embodiments, the target is a secreted protein. In some such embodiments, specific levels of the target are found in, for example, blood, serum, plasma, etc. In some such embodiments, the microprotein binds to a region of the target (e.g., an epitope). In some embodiments, the microprotein or a composition containing the microprotein specifically binds to a target expressed on the surface of cancer cells. In some embodiments, the cancer cells are located in, on, or near a solid tumor. In some embodiments, the cancer cells are circulating cancer cells. In some embodiments, the microprotein or a composition containing the microprotein specifically binds to a target or is expressed on cancer cells at a level higher than that of reference cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells.

[0181] In some embodiments, the microprotein or a composition comprising the microprotein specifically binds to B7-H3. In some embodiments, the target comprises or is composed of B7-H3. In some embodiments, the microprotein specifically binds to a target comprising an amino acid sequence as described in Table 1A or a portion thereof.

[0182] B7-H3 B7-H3, also known as CD276, is considered an immune checkpoint protein expressed by immune cells (such as APCs and macrophages) and tumor cells. B7-H3 has also been shown to suppress T cells and has been found to facilitate immune evasion by tumor cells. (Dong et al., Front Oncol. 2018 July 6; 8:264).

[0183] B7-H3 overexpression has been confirmed in a variety of cancers, including, for example, bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, glioma, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, intrahepatic bile duct cancer, hepatocellular carcinoma, oral squamous cell carcinoma, endometrial cancer, as well as squamous cell carcinoma and gastric cancer, glioma, and melanoma (Dong et al., Front Oncol. 2018 July 6; 8:264). Studies on B7-H3 overexpression have also shown that B7-H3 levels are associated with advanced tumor stage, high tumor grade, and poor clinical prognosis. Although not bound by any particular theory, such overexpression of B7-H3 is thought to promote metastasis in a variety of different cancer types through a variety of possible mechanisms. For example, in some implementations, B7-H3 may play a role in cancer cell proliferation and invasion (ibid.). In studies documenting B7-H3 knockdown, significant inhibition of cell migration and invasion was observed in cells of prostate cancer, breast cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, and melanoma. (Ibid.)

[0184] In some embodiments, B7-H3 overexpressed on tumor cells has been demonstrated to be successfully targeted, including via T-cell-mediated immunotherapy. For example, one study showed that the specific cytotoxic activity of activated T cells (ATCs) also possessing a novel anti-CD3 x anti-B7-H3 bispecific antibody exhibited increased cytotoxicity relative to ATCs alone, leading to inhibition of tumor growth and increased survival in xenograft models (Ma J. et al., Oncotarget (2016) 7(20):29480–91). In some embodiments, the binders of this disclosure bind to B7-H3 expressed on one or more cells (e.g., cancer cells). In some such embodiments, this disclosure contemplates the use of one or more binders of this disclosure to target B7-H3, wherein B7-H3 is expressed at higher levels on cancer cells than on non-cancer cells, and wherein the binding of the microprotein is specific to cancer cells.

[0185] In some embodiments, the compositions provided in this disclosure more specifically and effectively target cells overexpressing B7-H3, while minimizing or eliminating damage to surrounding cells that do not express or have low B7-H3 expression by providing a targeting composition that, in some embodiments, includes a chelating agent and / or an α-emitter, which, when combined with the microproteins provided herein, provides a specific, efficient, and effective method for targeting B7-H3-overexpressing target cells. As used herein, “low B7-H3-expressing” cells refer to cells that do not express B7-H3 and / or cells with B7-H3 expression levels less than 50% of the B7-H3 expression levels of B7-H3-overexpressing cells. In specific embodiments, “low B7-H3-expressing” cells refer to cells with B7-H3 expression levels less than 40%, 30%, 20%, 10%, or 5% of the B7-H3 expression levels of B7-H3-overexpressing cells.

[0186] In some implementations, B7-H3 levels are associated with metastasis. For example, some studies have documented the relationship between B7-H3 and metastasis, such as increased expression levels of metastasis-related proteins (e.g., MMP2, STAT3, and IL-8) (Tekle et al., Int J Cancer (2012) 130(10):2282–90), as well as increased CXCR4 levels and activation of the AKT, ERK, and JAK2 / STAT3 pathways (Wang et al., Tumour Biol (2016) 37(3):2961–71). In some implementations, changes in B7-H3 can affect cancer treatment. For example, studies have shown that changes in B7-H3 can affect the efficacy of some cancer treatments. In breast cancer, specifically, B7-H3 has been shown to reduce the efficacy of paclitaxel by activating JAK2 / STAT3, and silencing B7-H3 can both eliminate JAK2 / STAT3 phosphorylation and increase paclitaxel sensitivity. (Liu et al., Mol Cancer Ther (2011) 10(6):960–71). Similarly, in colorectal cancer, overexpression of B7-H3 has been shown to reduce 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) is an inhibitory receptor for certain immune cells and can upregulate B7-H3 expression in lung cancer cells through the PI3K / AKT / mTOR pathway (see, for example, Zhang P et al., FEBS Lett (2015) 589(17):2248–56). In some implementations, co-expression of ILT4 and B7-H3 may be positively correlated with lymph node metastasis, advanced tumor stage and poor clinical prognosis in NSCLC (see, for example, Zhang P et al., FEBS Lett (2015) 589(17):2248–56).

[0187] Without being bound by theory, B7-H3 can play a role in cellular transformation and potential. For example, in some embodiments, B7-H3 can increase cellular transformation and stemness. For example, B7-H3 has been shown to increase epithelial-mesenchymal transition and cancer “stemness” by increasing the expression of several proteins, including vimentin, CD133, CD44, and OCT4, and decreasing the 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.

[0188] By way of non-limiting examples, although it is currently unclear whether B7-H3 overexpression is due to, for example, genomic DNA amplification or increased transcription, chromatin immunoprecipitation has demonstrated that there is an androgen receptor binding site upstream of B7-H3, and that B7-H3 expression is reduced in the presence of androgens (Benzon B. et al., Prostate Cancer Prostatic Dis (2017) 20(1):28–35). Similarly, in some embodiments, B7-H3 can increase the expression of genes (e.g., thymidine synthase) through the PI3K / AKT pathway (see, for example, Jiang et al., Tumour Biol (2016) 37(7):9465–72). Furthermore, data show that inhibition can enhance the efficacy of oxaliplatin in the treatment of colorectal cancer. In some implementations, B7-H3 is thought to promote chemotherapy resistance by increasing XRCC1 expression through the PI3K / AKT pathway (see Zhang et al., Biochem Biophys Res Commun (2017) 490(3):1132–8). This may also increase BRCC3 expression in some implementations, thereby counteracting the DNA-damaging effects of 5-FU (see Sun et al., Oncol Rep (2016) 36(1):231–8).

[0189] In some embodiments, without limitation, B7-H3 may play a role in cancer metabolism. Specifically, it has been considered that B7-H3 promotes the "Warburg effect"—the preferential metabolism of glucose to lactate—under oxygen-rich conditions. This Warburg effect has been observed via HIF1α in breast cancer cells and mouse models of breast cancer (Lim et al., Cancer Res (2016) 76(8):2231–42). Similarly, in some embodiments, B7-H3-mediated reduction may affect proliferation and the Warburg effect. Specifically, data show that in metastatic melanoma, B7-H3-mediated reduction during treatment of metastatic melanoma has been shown to be overcome by using monoclonal B7-H3 antibodies, thereby reducing melanoma proliferation and the Warburg effect (Flem-Karlsen K et al., Pigment Cell Melanoma Res (2017) 30(5):467–76). Treatment with monoclonal antibody B7-H3 has also been shown to be effective for rare stage IV pediatric neuroblastoma, DIPG. (Zhou Z, J Neurooncol (2013) 111(3):257–64).

[0190] In some implementations, 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 considered to be associated with cancer (Dong et al., Front Oncol. 2018 July 6;8:264). For instance, the 3' UTR of the B7-H3 transcript has been observed to interact with miR-214 and miR-363 in breast cancer. ,miR-326,miR-940,miR-29c,miR-665,miR-34b The interaction of miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593 leads to decreased B7-H3 expression (Nygren MK et al., Br J Cancer (2014) 110(8):2072–80). In osteosarcoma, the 3' UTR of the B7-H3 transcript has been shown to interact with miR-124, and a study in colorectal cancer cells showed that iASPP-mediated p53 inhibition led to downregulation of miR-124, accompanied by increased B7-H3 expression (Dong et al., Front Oncol. 2018 July 6; 8:264). Furthermore, in a colorectal cancer study, the miR-155 / miR-143 axis promoted tumorigenesis (Zhou et al., Oncotarget (2016) 7(41):67196–211). Specifically, in colorectal cancer cells, elevated TGFβ-induced miR-155 expression (via SMAD3 and SMAD4) leads to decreased CEPBP expression, which in turn results in decreased miR-143 expression in colorectal cancer cells. B7-H3 is a target of miR-143, so decreased miR-143 expression leads to increased B7-H3 expression, which supports the possibility that TGFB may promote cancer immune escape in some cases by upregulating B7-H3. (Zhou et al., Oncotarget (2016) 7(41):67196–211; Dong et al., Front Oncol. 2018 July 6; 8:264).

[0191] Importantly, the novel compositions provided in this disclosure can specifically, efficiently, and effectively target B7-H3 overexpressing cells, with reduced toxicity compared to currently available treatments. In other words, in some embodiments, the B7-H3-targeting compositions provided in this disclosure offer improved treatment compared to currently available therapies.

[0192] In some embodiments, the target of the compositions disclosed herein comprises or consists of B7-H3 (e.g., human B7-H3).

[0193] In some embodiments, the microprotein is selected from any one of Tables 1B-1E, 2A, and 2C, or a portion or fragment thereof. In some embodiments, the microprotein binds to B7-H3 and is selected from any one of Tables 1B-1E, 2A, and 2C. In some embodiments, the microprotein binding to B7-H3 is not selected from any one of Tables 1B, 1C, or 2A.

[0194] In some embodiments, the amino acid sequence of the B7-H3 binding microprotein comprises or consists of the amino acid sequence according to any one of SEQ ID NO: 4-6, 8-94, or 100-537, and may have different N-termini and / or C-termini, for example, having an acetyl group, NH2, biotin-PEG4, DOTA-PEG4, radiolabeling, etc. at its N-terminus, and -OH / COOH or -NH2 at its C-terminus. The N-terminus and / or C-terminus of the B7-H3 binding microprotein of this disclosure may include, but are not limited to, acetyl groups, acids, or amides (e.g., acetyl, NH2, OH / COOH), such as those provided in the exemplary compounds and microproteins of Table 2A or Table 2C.

[0195] In some embodiments, the B7-H3 binding microprotein does not have an amino acid sequence comprising or consisting of an amino acid sequence according to any one of SEQ ID NO: 4-6, 8-94 and 100-197.

[0196] In some embodiments, the polypeptides of this disclosure may have various modifications at their N-terminus (e.g., a linker, chelating agent, and / or radionuclide, as described in the exemplary compounds in Table 2A or Table 2C) or their C-terminus (e.g., a linker, chelating agent, and / or radionuclide). In some embodiments, the C-terminus of a given polypeptide may have an acid or amide group at its C-terminus (see Table 2A, e.g., Table 2C). A given polypeptide having a specific amino acid sequence may have one or more N-terminal and / or C-terminal differences without substantially altering the utility or function of the polypeptide, such as binding to B7-H3 (e.g., for the detection and / or treatment of cancer).

[0197] In some embodiments, the target of the compositions disclosed herein comprises or consists of B7-H3. In some embodiments, a substantial change in the fundamental and novel characteristics of the peptides provided herein is their ability to bind strongly and specifically to their intended target (e.g., B7-H3, e.g., B7-H3 on cancer cells) with minimal or no off-target effects and minimal renal uptake (e.g., compared to renal uptake of previously developed B7-H3 binding molecules). In some embodiments, B7-H3 is expressed on the cell surface. Alternatively, in some embodiments, B7-H3 is released from tumor-associated cells and is present in the tumor microenvironment (see, for example, Zhang et al., Immunology. April 2008; 123(4): 538–546). In some embodiments, the cells are cancer cells. In some such embodiments, the cancer cells are tumor cells and the tumor is a solid tumor. In some embodiments, the level of B7-H3 expressed in tumor cells or a population of tumor cells is higher than the level of B7-H3 expressed in non-tumor cells. In some embodiments, the microproteins or compositions containing microproteins provided in this disclosure target B7-H3, which can specifically target the composition or one or more components thereof (e.g., chelating agents and / or radionuclides) to cancer cells or tumor microenvironments (e.g., locations containing cancer cells or populations of cells at risk of becoming cancer cells).

[0198] In some embodiments, the compositions of this disclosure specifically bind to B7-H3 (e.g., via peptides that bind to B7-H3, such as via microproteins that specifically bind to B7-H3). In some embodiments, compounds targeting B7-H3 are disclosed in Table 2A or Table 2C.

[0199] In some embodiments, the composition comprising a microprotein comprises or is composed of a protein containing a specific amino acid sequence that binds to B7-H3 or a portion thereof. In some embodiments, certain exemplary B7-H3 binding microproteins are engineered to have an amino acid sequence comprising one or more non-natural amino acids (e.g., as described herein, such as, as described in Tables 2A and / or 2C, such as amino acids with small alkyl groups on their side chains, etc.). In some such embodiments, such B7-H3 microproteins comprise or are composed of an amino acid sequence selected from any one of SEQ ID NOs: 4-6, 8-94, and 100-537, or a functional variant or portion thereof (e.g., a functional fragment, such as a microprotein folded and bound to B7-H3 or a portion thereof). In some embodiments, such B7-H3 microproteins are a binding protein or part of a conjugate comprising such a binding protein as described in Tables 2A or 2C. In some embodiments, the B7-H3 microprotein has an amino acid sequence comprising the sequence described in any one of Tables 1B-1E.

[0200] In some embodiments, the composition containing the microprotein comprises or consists of a protein containing a specific amino acid sequence that binds to B7-H3 or a portion thereof.

[0201] In some embodiments, this disclosure provides a polynucleotide encoding a polypeptide comprising or composed of one or more portions of the compositions provided herein. In some embodiments, this disclosure provides a vector and / or host cell comprising sequences encoding one or more components of the compositions provided herein.

[0202] In some embodiments, this disclosure provides methods for detecting a target. In some embodiments, methods as provided herein include detecting the presence of a target for purposes such as imaging (e.g., diagnostic, prognostic, and / or monitoring) (e.g., treatment). In some embodiments, this disclosure provides therapeutic methods and / or manufacturing methods using compositions as provided herein (e.g., microproteins, such as linker-chelating agents, such as microproteins comprising one or more of linkers, chelating agents, and radionuclides). In some embodiments, the therapeutic method includes administering a composition as provided herein to a subject in need.

[0203] In some embodiments, when present, the linker is attached to the N-terminus of the peptide (e.g., a B7-H3 binding microprotein). In some embodiments, when present, the linker is attached to the C-terminus of the peptide (e.g., a B7-H3 binding microprotein).

[0204] In some implementations, the C-terminal amino acid of the peptide is not cysteine.

[0205] In some embodiments, the chelating agent is attached to the peptide or a linker when present. In some embodiments, when the chelating agent is present in the absence of a linker, the chelating agent is attached to the N-terminus of the peptide. In some embodiments, when the chelating agent is present in the absence of a linker, the chelating agent is attached to the C-terminus of the peptide.

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

[0207] In some embodiments, this disclosure provides a composition comprising one or more of the formula (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 chelating agent (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M comprises an amino acid sequence of any one of SEQ ID NO: 4-6, 8-94, and 100-537.

[0208] In some embodiments, the connector comprises or consists of: polyethylene glycol (PEG) connectors (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4), ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment thereof or a combination thereof linked by covalent bonds.

[0209] In some embodiments, the chelating agent comprises or is composed of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

[0210] In some implementations, the radionuclides are 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.

[0211] In some embodiments, this disclosure provides a composition comprising one or more of the formula selected from (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 chelating agent (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising any of the sequences described in SEQ ID NO: 198-537.

[0212] In some embodiments, when L is present, L comprises or consists of: polyethylene glycol (PEG) connectors (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36), ester connectors, amide connectors, maleimide connectors, valine-citrulline connectors, hydrazone connectors, 4-(2-pyridyldithio)butyrate N-succinimide ester (SPDB) connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylate succinimide ester (SMCC) connectors, vinyl sulfone-based connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment thereof or a combination thereof linked by covalent bonds.

[0213] In some embodiments, when C is present, C comprises or consists of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

[0214] In some implementations, when R is present, R comprises or consists of the following: 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.

[0215] In some embodiments, when present, the linker is attached to the N-terminus of the peptide (e.g., a B7-H3 binding microprotein). In some embodiments, when present, the linker is attached to the C-terminus of the peptide (e.g., a B7-H3 binding microprotein).

[0216] In some implementations, the C-terminal amino acid of the peptide is not cysteine.

[0217] In some embodiments, the chelating agent is attached to the peptide or a linker when present. In some embodiments, when the chelating agent is present in the absence of a linker, the chelating agent is attached to the N-terminus of the peptide. In some embodiments, when the chelating agent is present in the absence of a linker, the chelating agent is attached to the C-terminus of the peptide.

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

[0219] In some embodiments, the polypeptides provided herein (e.g., microproteins that bind to B7-H3) contain at least one disulfide bridge.

[0220] In some embodiments, the polypeptides provided herein (e.g., microproteins that bind to B7-H3) contain at least two disulfide bridges.

[0221] In some embodiments, the composition and / or its polypeptides selectively bind to B7-H3 or a portion thereof.

[0222] In some embodiments, the binding affinity of the peptide to B7-H3 or a portion thereof is 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM, whether in vivo, in vitro, or in vitro and / or as measured in cell-based assays.

[0223] In some implementations, the binding inhibition constant of the peptide is no greater than 100 nM.

[0224] A composition comprising a polypeptide-drug conjugate, said polypeptide-drug conjugate comprising a polypeptide and at least one pharmaceutical moiety, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with at least 44 amino acids of a polypeptide having the amino acid sequence described in any one of SEQ ID NO: 198-537. 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 such embodiments, the modified amino acid is an amino acid containing a small alkyl group on its side chain (e.g., methylated lysine, such as monomethyl lysine, dimethyl lysine, trimethyl lysine, etc.). For example, in some embodiments, a small alkyl group (e.g., methyl, such as monomethyl, dimethyl, trimethyl, etc.) may be present on the nitrogen atom of the lysine side chain. In some embodiments, a small alkyl group may be present on the guanidine group of the arginine side chain.

[0225] In some embodiments, the B7-H3 binding microprotein comprises or consists of an amino acid sequence according to any one of SEQ ID NO: 4-6, 8-94, and 100-537, and may have different N-termini and / or C-termini, for example, having an acetyl group, NH2, biotin-PEG4, DOTA-PEG4, radiolabeling, etc. at its N-terminus, and -OH or -NH2 at its C-terminus. The N-terminus and / or C-terminus of the B7-H3 binding microprotein of this disclosure may include, but are not limited to, acetyl groups, acids, or amides (e.g., acetyl, NH2, OH), as provided in the exemplary compounds and microproteins of Table 2A or Table 2C, for example. In some embodiments, the polypeptide of this disclosure may have various modifications at its N-terminus (e.g., a linker, chelating agent, and / or radionuclide, as described in the exemplary compounds of Table 2A and / or Table 2C) or its C-terminus (e.g., a linker, chelating agent, and / or radionuclide). In some embodiments, the C-terminus of a given polypeptide may have an acid or amide group at its C-terminus (see Table 2A and / or Table 2C for example). A given polypeptide having a specific amino acid sequence may have one or more N-terminal and / or C-terminal differences without substantially altering the utility or function of the polypeptide, such as binding to B7-H3 (e.g., for the detection and / or treatment of cancer).

[0226] In some embodiments, the B7-H3 binding microprotein comprises or consists of an amino acid sequence according to any one of SEQ ID NO: 198-537, and may have different N-termini and / or C-termini, for example, having an acetyl group, NH2, biotin-PEG4, DOTA-PEG4, radiolabeling, etc. at its N-terminus, and -OH or -NH2 at its C-terminus. The N-terminus and / or C-terminus of the B7-H3 binding microprotein of this disclosure may include, but are not limited to, acetyl groups, acids, or amides (e.g., acetyl, NH2, OH), as provided in the exemplary compounds and microproteins of Table 2A or Table 2C. In some embodiments, the polypeptide of this disclosure may have various modifications at its N-terminus (e.g., a linker, chelating agent, and / or radionuclide, as described in the exemplary compounds of Table 2C) or its C-terminus (e.g., a linker, chelating agent, and / or radionuclide). In some embodiments, the C-terminus of a given polypeptide may have an acid or amide group at its C-terminus (see Table 2C for example). A given polypeptide with a specific amino acid sequence may have one or more N-terminal and / or C-terminal differences without substantially altering the polypeptide’s utility or function, such as binding to B7-H3 (e.g., for the detection and / or treatment of cancer).

[0227] In some embodiments, this disclosure provides a composition comprising a polypeptide bound to B7-H3 having at least 48 amino acids, two disulfide bonds, and modified amino acids at positions corresponding to 3, 24, and 29 relative to SEQ ID NO: 267, wherein the modification is a small alkyl group on the side chain of the amino acid (e.g., on the nitrogen of the lysine side chain, or, for example, on the guanidinium of the arginine side chain).

[0228] In one aspect, this disclosure provides a composition comprising one or more modified amino acids. In some such embodiments, the one or more modifications include small alkyl groups. In one aspect, this disclosure provides a composition comprising a B7-H3 binding polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acid comprises (i) a cysteine ​​at one of four positions corresponding to positions 1, 17, 35, and 48 of SEQ ID NO: 267, and wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; X45 is K or (Kme), and X49 is S or absent.

[0229] In one aspect, this disclosure provides a composition comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: At least four cysteine ​​residues forming two disulfide bonds; arginine, modified arginine, or modified lysine at position 3, lysine at position 5, isoleucine at position 6, tryptophan at position 14, at least one modified lysine residue at position 24, and alanine, arginine, or modified lysine at position 29, wherein each position is linear from the N-terminus to the C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group of a nitrogen or guanidine group attached to the lysine side chain, optionally including methyl, dimethyl, or trimethyl; a length of at least 48 amino acids; and a binding affinity for B7-H3 greater than 100 nM as measured in a cell-based assay.

[0230] In one aspect, this disclosure provides a composition comprising a B7-H3 binding polypeptide having an amino acid sequence comprising: at least four cysteine ​​residues forming two disulfide bonds; at least one modified lysine residue at position X24 of SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group of nitrogen linked to the lysine side chain, the small alkyl group optionally comprising methyl, dimethyl, or trimethyl; a length of at least 48 amino acids; and a binding affinity for B7-H3 greater than 100 nM as measured in a cell-based assay.

[0231] In some embodiments, the peptide is at least 48 amino acids long but not more than 100 amino acids long. In some embodiments, in cell-based assays, the peptide binds to B7-H3 with an affinity greater than 10 nM.

[0232] In some embodiments, B7-H3 overexpressed on tumor cells has been demonstrated to be successfully targeted, including via T-cell-mediated immunotherapy. For example, one study showed that the specific cytotoxic activity of activated T cells (ATCs) also possessing a novel anti-CD3 x anti-B7-H3 bispecific antibody exhibited increased cytotoxicity relative to ATCs alone, leading to inhibition of tumor growth and increased survival in xenograft models (Ma J. et al., Oncotarget (2016) 7(20):29480–91). In some embodiments, the binders of this disclosure bind to B7-H3 expressed on one or more cells (e.g., cancer cells). In some such embodiments, this disclosure is contemplated for targeting B7-H3 using one or more binders of this disclosure, wherein B7-H3 is expressed at higher levels on cancer cells than on non-cancer cells, and wherein the binding of the microprotein is specific to cancer cells. The amino acid sequence of the polypeptide shares at least 90% identity with any of SEQ ID NO: 198-537, but includes at least one lysine and / or arginine with at least one modification comprising at least one small alkyl group bonded to a nitrogen- or guanidine-based side chain of the lysine side chain, said small alkyl group 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 a reference polypeptide longer than 48 amino acids, and binds to B7-H3 with an intensity of at least 10 nM in cell-based assays, and / or has an inhibition constant of no more than 10 nM.

[0233] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with at least 44 amino acids of any of SEQ ID NO: 198-537, provided that the 44 amino acids include at least two cysteine ​​residues that form a disulfide bridge.

[0234] In some embodiments, the amino acid sequence shares 90% identity with at least 44 amino acids as described in any one of SEQ ID NO: 199, 204, 241 or 262-272.

[0235] In some embodiments, the amino acid sequence shares 100% identity with at least 44 amino acids as described in any one of SEQ ID NO: 199, 204, 241 or 262-272.

[0236] In some embodiments, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO: 267.

[0237] In some embodiments, this disclosure provides a composition comprising a compound as described 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.

[0238] 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.

[0239] In some embodiments, this disclosure provides a composition comprising a polypeptide having an amino acid sequence of at least 48 amino acids, but having four cysteine ​​positions corresponding to SEQ ID NO: 267 at positions 1, 17, 35, and 48, wherein the following amino acids are present: arginine, modified arginine, or modified lysine at position 3; lysine at position 5; isoleucine at position 6; tryptophan at position 14; at least one modified lysine residue at position 24; and alanine, arginine, or modified lysine at position 29, wherein each position is linear from the N-terminus to the C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group of a nitrogen or guanidine group attached to a lysine side chain, optionally including methyl, dimethyl, or trimethyl.

[0240] In some implementations, the C-terminus has -OH or -NH2.

[0241] In some embodiments, the composition (e.g., a peptide) has a binding affinity for B7-H3 greater than 100 nM.

[0242] In some implementations, the suppression constant is no greater than 100 nM.

[0243] In some embodiments, the composition further comprises one or more of a connector, a chelating agent, and a radionuclide.

[0244] In some embodiments, the connector comprises or consists of: polyethylene glycol (PEG) connectors (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4), ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment thereof or a combination thereof linked by covalent bonds.

[0245] In some embodiments, the chelating agent comprises or is composed of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

[0246] In some implementations, 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.

[0247] In some embodiments, the polypeptide has an amino acid sequence comprising any one of SEQ ID NO: 198-537. In some embodiments, the polypeptide sequence comprises any one of SEQ ID NO: 199, 204, 241 or 262-272 and further comprises a linker, wherein the linker is PEG4, and optionally a chelating agent, wherein the chelating agent is DOTA.

[0248] In some embodiments, the adapter attaches to the N-terminus of the peptide when present. In some embodiments, the adapter attaches to the C-terminus of the peptide when present.

[0249] In some implementations, the C-terminal amino acid of the peptide is not cysteine.

[0250] In some implementations, the chelating agent is attached to the peptide or linker when present.

[0251] In some implementations, a radionuclide is attached to a chelating agent when present.

[0252] In some embodiments, this disclosure provides a composition comprising one or more of the formula (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 chelating agent (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M comprises any one of SEQ ID NOs: 4-6, 8-94, and 100-537 or any one of SEQ ID NOs: 538-543 and 546-550 and Table 1E. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 204 and 262-272.

[0253] In some embodiments, the connector comprises or consists of: polyethylene glycol (PEG) connectors (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4), ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment thereof or a combination thereof linked by covalent bonds.

[0254] In some embodiments, the chelating agent comprises or is composed of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

[0255] In some implementations, the radionuclides are 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.

[0256] In some embodiments, this disclosure provides a composition comprising one or more of the formula (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 chelating agent (C), R comprises a radionuclide (R), and x is 1, 2, 3, or 4, wherein M has an amino acid sequence comprising any one of those sequences described in SEQ ID NO: 4-6 and 8-94 and 100-537 or according to SEQ ID NO: 95 and 96 and Tables 1B and 1C, and SEQ ID NO: 538-543 and 546-550 (Table 1D) and Table 1E. In some embodiments, the amino acid sequence comprises any one of SEQ ID NO: 204 and 262-272.

[0257] In some embodiments, when L is present, L comprises or consists of: polyethylene glycol (PEG) connectors (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36), ester connectors, amide connectors, maleimide connectors, valine-citrulline connectors, hydrazone connectors, 4-(2-pyridyldithio)butyrate N-succinimide ester (SPDB) connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylate succinimide ester (SMCC) connectors, vinyl sulfone-based connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- (SEQ ID NO: 551) or (PEG)n, wherein n is 1 to 36, (Gly)1-10 (SEQ ID NO: 552), or any fragment thereof or a combination thereof linked by covalent bonds.

[0258] In some embodiments, when C is present, C comprises or consists of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

[0259] In some implementations, when R is present, R comprises or consists of the following: 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.

[0260] In some embodiments, the adapter attaches to the N-terminus of the peptide when present. In some embodiments, the adapter attaches to the C-terminus of the peptide when present.

[0261] In some implementations, the C-terminal amino acid of the peptide is not cysteine.

[0262] In some implementations, the chelating agent is attached to the peptide or linker when present.

[0263] In some implementations, a radionuclide is attached to a chelating agent when present.

[0264] In some implementations, the polypeptide contains at least one disulfide bridge.

[0265] In some implementations, the polypeptide contains at least two disulfide bridges.

[0266] In some embodiments, the composition and / or its polypeptides selectively bind to B7-H3 or a portion thereof.

[0267] In some embodiments, the binding affinity of the peptide to B7-H3 or a portion thereof is 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM, whether in vivo, in vitro, or in vitro and / or as measured in cell-based assays.

[0268] In some implementations, the binding inhibition constant of the peptide is no greater than 100 nM.

[0269] In one aspect, this disclosure provides a composition comprising a polypeptide-drug conjugate, the polypeptide-drug conjugate comprising a polypeptide and at least one pharmaceutical 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 having an amino acid sequence according to SEQ ID NO: 4-6, 8-94 and 100-197 or according to SEQ ID NO: 95 or 96 and any one of Tables 1B and 1C.

[0270] A composition comprising a polypeptide-drug conjugate comprising a polypeptide and at least one pharmaceutical 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 having an amino acid sequence according to any one of SEQ ID NO: 198-537, Table 2C, or according to any one of SEQ ID NO: 538-543 and 546-550 (Table 1D) and Table 1E.

[0271] In some embodiments, the drug component is selected from topoisomerase inhibitors, orrisstatins (e.g., monomethylorrisstatin E), V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, saccharidin, maytansin-like substances, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinase inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, DHFR inhibitors, and immunotoxins.

[0272] In some embodiments, this disclosure provides a composition comprising an isolated compound or a pharmaceutically acceptable salt or neutral molecule thereof, comprising an optional linker (L) and one or more of a polypeptide (M), a chelating agent (C), or a radionuclide (R), wherein M has an amino acid sequence comprising any one of SEQ ID NO: 4-6, 8-94, and 100-197 or SEQ ID NO: 95 and 96 and Tables 1B and 1C, and / or wherein M has an amino acid sequence comprising any one of SEQ ID NO: 198-537, Table 2C, or any one of SEQ ID NO: 538-543 and 546-550 (Table 1D) and Table 1E.

[0273] In some embodiments, this disclosure provides a composition comprising a compound designed to bind to B7-H3, the compound comprising or consisting of a polypeptide having an amino acid sequence comprising any one of SEQ ID NO: 4-6, 8-94 and 100-197 or SEQ ID NO: 95 and 96 and Tables 1B and 1C, or comprising any one of SEQ ID NO: 198-537, Table 2C or according to any one of SEQ ID NO: 538-543 and 546-550 (Table 1D) and Table 1E.

[0274] In some embodiments, the modified N-terminus includes one or more of the following: NH2, acetyl, PEGn (where n=1-36), DOTA, or biotin.

[0275] In some implementations, the C-terminus contains -NH2 or -OH.

[0276] In some implementations, the peptide selectively binds to B7-H3 or a portion thereof.

[0277] In some implementations, the peptide exhibits a binding affinity for B7-H3 or a portion thereof greater than about 100 nM, either in vivo or in cell-based assays.

[0278] polypeptide Among other things, this disclosure provides polypeptides. In some embodiments, the polypeptides are assembled using solid-phase synthesis methods. In some embodiments, the polypeptides are recombinant polypeptides. In some embodiments, the polypeptides comprise or consist of microproteins. In some such embodiments, the microproteins comprise or consist of binding agents. In some embodiments, the polypeptides of this disclosure (including mutants, allelic variants, fragments, derivatives, and analogs) are encoded by polynucleotides as described and provided herein.

[0279] In some embodiments, the microproteins of this disclosure comprise or consist of polypeptides capable of binding to targets as shown in Table 1A.

[0280] In some embodiments, this disclosure provides a binding agent comprising or composed of a polypeptide fragment 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.

[0281] In some embodiments, the microproteins of this disclosure may also comprise fusions or conjugates formed with one or more other components, such as heterologous peptides. For example, in some embodiments, the heterologous sequence may comprise or consist of sequences designed to facilitate purification (e.g., histidine tags) and / or visualization of the recombinant expressed protein. Other non-limiting examples of such fusions or conjugates include those that allow the display of protein-encoding components on the surface of bacteriophages or cells, including any detectable or visible component, such as green fluorescent protein (GFP), and fusions with the Fc region of IgG.

[0282] In some embodiments, the microprotein comprises or is composed of a specific amino acid sequence. In some embodiments, the microprotein has an amino acid sequence that is identical to 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% of the amino acid sequence described in any of 1B, 1C, 1D, 1E, 2A, 2C, or 2D.

[0283] In some embodiments, the microprotein 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 any of the amino acid sequences described in Table 2A.

[0284] In some embodiments, the microprotein does not have an amino acid sequence selected from any of those sequences according to Table 1B, Table 1C and / or Table 2A.

[0285] In some embodiments, the microprotein has an amino acid sequence that corresponds to the amino acid sequence 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 of a given polypeptide, such as those described in Table 2A or Table 2C. The 46, 47, 48, or 49 adjacent amino acids are 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. In some embodiments, the microprotein does not have the amino acid sequence as described in Table 2A.

[0286] In some embodiments, the microprotein 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 any of the amino acid sequences described in Table 2A or Table 2C.

[0287] In some embodiments, the microprotein has an amino acid sequence that is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to a portion of the amino acid sequence described in Table 2A. For example, in some embodiments, the microprotein has an amino acid sequence that is identical to 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%, 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% of the 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids of a given polypeptide, such as those described in Table 2A.

[0288] In some embodiments, the microprotein has an amino acid sequence that is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to a portion of the amino acid sequence described in Table 2C. For example, in some embodiments, the microprotein has an amino acid sequence that is identical to 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%, 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% of the 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acids of a given polypeptide, such as those described in Table 2C.

[0289] In some embodiments, the microprotein has an amino acid sequence that corresponds to the amino acid sequence of a given polypeptide, such as those described in Table 2C, at positions 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, 4 6, 47, 48, or 49 adjacent amino acids are 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.

[0290] In some embodiments, the polypeptide (e.g., a microprotein) has an amino acid sequence that has a certain percentage of identity with respect to a coverage percentage (e.g., a reference sequence). Unless otherwise stated, the percentage of identity refers to the maximum percentage of identity measured according to any of the methods described herein. That is, a B7-H3 binding polypeptide (e.g., a reference molecule) as provided herein may have 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with a given query molecule but at a coverage percentage (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), where if the query molecule is shorter, its percentage of identity and coverage percentage may differ (e.g., 100% identity and 90% coverage). If the query molecule is longer, both the percentage of identity and coverage may be 100% relative to the reference molecule, and the reference molecule may have a certain percentage of identity with respect to a certain length of the query molecule (e.g., at least 20, 25, or 30 amino acids). For example, in some embodiments, if a reference sequence (e.g., a microprotein provided herein) is shorter than a query sequence, then such a query sequence is considered within the scope of this disclosure if it has the length of a reference sequence that aligns with the query sequence, wherein the percentage of identity is determined between the two sequences (the query sequence and the reference sequence) at at least a minimum length of alignment. That is, if the polypeptide disclosed herein is longer than the query sequence, the percentage of identity is determined by aligning the reference sequence and the query sequence and determining the percentage of identity between the query sequence and the portion of the reference sequence that it aligns with. Conversely, when the query sequence is longer than the reference sequence, the percentage of identity is equal to the identity of the portion aligned with the reference sequence. That is, if the reference sequence is shorter, then the query sequence may fall within the scope of the reference sequence if it aligns between the two polypeptides (the reference sequence and the query sequence) at the aligned portion with the claimed percentage of identity.

[0291] As used herein and as is known to those skilled in the art, the twenty common amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (eds. Golub and Gren, Sinauer Associates, Sunderland, Mass., 2nd edition, 1991), which is incorporated herein by reference. In some embodiments, the amino acids of this disclosure may be stereoisomers of the twenty common amino acids (e.g., D-amino acids). In some embodiments, the amino acids in the polypeptides of this disclosure may be non-natural amino acids. For example, amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components of the polypeptides of this disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The polypeptide sequence representation used in this article is arranged such that the left end corresponds to the amino terminus and the right end corresponds to the carboxyl terminus, which conforms to standard usage and convention.

[0292] In some embodiments, the microproteins of this disclosure comprising two or more cysteine ​​residues (e.g., those described in Table 2C) have cysteine ​​residues linked by disulfide bridges (e.g., by natural folding). In some embodiments, the microprotein (e.g., bound to B7-H3) comprises one disulfide bridge. In some embodiments, the microprotein comprises two disulfide bridges.

[0293] In some embodiments, a disulfide bridge may be located between cysteine ​​residues, which correspond to, for example, Cys4 and Cys37; Cys5 and Cys34; Cys5 and Cys37; Cys12 and Cys26; Cys12 and Cys44; Cys17 and Cys48, wherein the position is a linear position relative to the N-terminus to the C-terminus of reference SEQ ID NO: 267.

[0294] In some embodiments, cysteine ​​linkages may occur between two different pairs of cysteine ​​residues. For example, in some embodiments, referring to the microprotein of SEQ ID NO: 213, two disulfide bridges may be located between positions corresponding to cysteine ​​pairs (e.g., Cys1 and Cys35, and Cys17 and Cys52). For example, in some embodiments, disulfide bridges may be located between Cys1 and Cys35, and Cys17 and Cys48, corresponding to reference sequences such as those described in Table 2C (e.g., SEQ ID NO: 267). In some embodiments, cysteine ​​linkages are between Cys1 and Cys17; and between Cys35 and Cys48. In some embodiments, cysteine ​​linkages are between Cys1 and Cys48; and between Cys17 and Cys35.

[0295] In some embodiments, one or more disulfide bridges comprise two or four cysteine ​​residues corresponding to positions 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, if four cysteines are present and correspond to positions 1, 17, 35 and 48 of SEQ ID NO: 267, the pairing may include pairing 1 with 35 and pairing 17 with 48; pairing 1 with 17 and pairing 35 with 48; or pairing 1 with 48 and pairing 17 with 35 (e.g., there is a disulfide bridge between the two paired cysteines).

[0296] In some embodiments, this disclosure provides a microprotein or a portion thereof or a functional variant thereof as described in Tables 1B, 1C, 1D, 1E, 2A, 2C and / or 2D, and / or the microprotein comprises or consists of the amino acid sequence described in any one of SEQ ID NO: 4-6, 8-94 and 100-537.

[0297] In some embodiments, the microprotein comprises or consists of an amino acid sequence or a portion thereof or a functional variant thereof, said amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or greater sequence identity with any of the sequences described in SEQ ID NO: 4-6, 8-94 and 100-537 and / or Tables 1B, 1C, 1D, 1E, 2A, 2C or 2D.

[0298] In some embodiments, the microprotein comprises or consists of an amino acid sequence or a portion thereof or a functional variant thereof, said amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more amino acid residues different from those described in SEQ ID NO: 4-6, 8-94 and 100-537 and / or as described in Tables 1B, 1C, 1D, 1E, 2A, 2C, and 2D.

[0299] In some embodiments, the microprotein comprises or consists of an amino acid sequence or a portion thereof or a functional variant thereof, said amino acid sequence having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 100-537 and / or as described in Tables 1B, 1C, 1D, 1E, 2A, 2C, and 2D having no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 amino acid residues different from those described in Tables 1B, 1C, 1D, 1E, 2A, 2C, and 2D.

[0300] In some embodiments, the microprotein has an amino acid sequence or a portion thereof or a functional variant thereof, said amino acid sequence comprising or consisting of any one of SEQ ID NO: 4-6, SEQ ID NO: 12-94 or SEQ ID NO: 100-537 and / or as described in Tables 1B, 1C, 1D, 1E, 2A, 2C, and 2D, selectively binding to the target B7-H3.

[0301] The polypeptides disclosed herein may have one or more modifications. A modification may refer to a substitution, alteration, inversion, addition, or deletion of at least one amino acid residue compared to a reference amino acid sequence. Alterations may include, but are not limited to, alterations of one or more atoms in the side chain, such as the addition of a methyl group (e.g., a methylated form of lysine). In some embodiments, natural amino acids are modified, for example, as described herein. In some embodiments, the modification includes the addition of at least one small alkyl group attached to a nitrogen atom of an amino acid side chain (e.g., the lysine side chain). As used herein, "small alkyl group" refers to an alkyl group with a short carbon chain, typically containing 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 may be added to the nitrogen atom of the lysine side chain to produce monomethyllysine, dimethyllysine, or trimethyllysine. In some embodiments, one, two, three, four, or more small alkyl groups may be added to a given amino acid (e.g., by attaching to a nitrogen atom of the side chain). In some embodiments, no more than five, four, three, two, or one small alkyl group may be added. Microproteins This disclosure provides a polypeptide comprising: an amino acid sequence, wherein the amino acid sequence comprises formula I: X1X2X3X4YX6X7EX9X11ALX14EIIWLPNX22X23X24X25QIX28AFIAALNX36DPSQSSELLSEAX49X50LX52DSX55X56X57X58 (SEQ ID NO: 95), where 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, (hyperleucine), 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, (hyperleucine), 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 not present; X56 is A or not present; X57 is P or not present; and X58 is G, K, (KAc), or not present; wherein if X28 is A, (hyperleucine), M(O2), S, T, or V, then X24 is Y; or wherein if X28 is M, then X7 is A, E, (Kme3), L, Q, or S.

[0302] In some implementations, if X4 is K or (KAc), then X24 is Y.

[0303] In some embodiments, the amino acid sequence includes formula II: X1X2X3X4YAX7EKIAALSEIIWLPNX22TX24X25QIX28AFIAALNX36DPSQSSELLSEAX49X50LNDSQAP (SEQ ID NO: 96), wherein X1 is A or absent; X2 is E or absent; X3 is A or absent; X4 is L or absent; X7 is K or Q; X22 is D or L; X24 is H or Y; X25 is A or G; X28 is ((homoleucine)) or M; X36 is D or N; X49 is E or K; and X50 is E.

[0304] In some embodiments, the amino acid sequence comprises Formula III (SEQ ID NO:538) as described in Tables 1D and 1E.

[0305] In some embodiments, the amino acid sequence comprises Formula IV (SEQ ID NO:539) as described in Tables 1D and 1E.

[0306] In some embodiments, the amino acid sequence comprises formula V (SEQ ID NO:540) as described in Tables 1D and 1E: In some embodiments, the amino acid sequence comprises formula VI (SEQ ID NO:541) as described in Tables 1D and 1E.

[0307] In some embodiments, the amino acid sequence comprises Formula VII (SEQ ID NO:542) as described in Tables 1D and 1E.

[0308] In some embodiments, the amino acid sequence comprises formula VIII (SEQ ID NO:543) as described in Tables 1D and 1E.

[0309] In some embodiments, the amino acid sequence comprises Formula IX (SEQ ID NO:546) as described in Tables 1D and 1E.

[0310] In some embodiments, the amino acid sequence comprises formula X (SEQ ID NO:547) as described in Tables 1D and 1E.

[0311] In some embodiments, the amino acid sequence comprises formula XI (SEQ ID NO:548) as described in Tables 1D and 1E.

[0312] In some embodiments, the amino acid sequence comprises formula XII (SEQ ID NO:549) as described in Tables 1D and 1E.

[0313] In some embodiments, the amino acid sequence comprises formula XIII (SEQ ID NO:550) as described in Tables 1D and 1E.

[0314] In some embodiments, the amino acid sequence shares at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identity with any one of SEQ ID NO: 4-6 or SEQ ID NO: 12-94.

[0315] In some embodiments, the amino acid sequence shares 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identity with any of SEQ ID NO: 6, 21 or 30.

[0316] In some implementations, the amino acid sequence shares 100% identity with any one of SEQ ID NO: 4-6 or SEQ ID NO: 12-94.

[0317] In some implementations, the amino acid sequence shares 100% identity with any one of SEQ ID NO: 6, 21 or 30.

[0318] In some implementations, the amino acid sequence shares 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identity with SEQ ID NO: 6.

[0319] In some implementations, the amino acid sequence shares 100% identity with SEQ ID NO: 6.

[0320] This disclosure also provides a polypeptide whose amino acid sequence comprises or consists of any one of SEQ ID NO: 4-6, 8-94 or 100-537.

[0321] This disclosure also provides a polypeptide whose amino acid sequence comprises or consists of any one of SEQ ID NO: 4-6, 8-94 or 100-197.

[0322] This disclosure also provides a polypeptide whose amino acid sequence comprises or consists of any one of SEQ ID NO: 198-537. In some embodiments, the microprotein includes at least one constraint, wherein the constraint is a disulfide bridge.

[0323] In some implementations, the polypeptide also includes one or more of a linker, a chelating agent, and a radionuclide.

[0324] In some embodiments, when present, the connector comprises or consists of: polyethylene glycol (PEG) connectors (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4), ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, decenoic acid connectors, aminohexanoic acid (Ahx) connectors, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is 1 to 36, (Gly)n-(gGlu)n-(PEG)n-( ... 1-10 , or any fragment thereof or a combination thereof connected by covalent bonds.

[0325] In some implementations, when present, the chelating agent comprises or consists of the following: i) NOPO ii) Crown iii) DOTA or iv) Macropa .

[0326] In some embodiments, the chelating agent comprises or consists of a derivative of NOPO, Crown, Macropa, or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0327] In some implementations, the radionuclide comprises or consists of the following: 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.

[0328] In some implementations, the linker is attached to the N-terminus or C-terminus of the peptide when present.

[0329] In some implementations, the chelating agent is attached to the peptide or linker in the presence of the chelating agent.

[0330] In some implementations, the radionuclide is attached to the chelating agent in the presence of the radionuclide.

[0331] In some embodiments, the polypeptide comprises or is composed of: linear polypeptides, folded polypeptides (e.g., covalently linked polypeptides, non-covalently linked polypeptides, or polypeptides containing disulfide bonds), cysteine-dense peptides, knotting peptides, binding agents, affinity molecules, engineered Kunitz domains, monomeric antibody-like molecules, anticalcitonins, engineered ankyrin repeating domains (DARPin), or avimers.

[0332] In some implementations, the polypeptide contains at least one disulfide bridge.

[0333] In some implementations, the peptide selectively binds to B7-H3 or a portion thereof.

[0334] In some embodiments, the peptide exhibits a binding affinity of 10 pM to 200 nM, 10 pM to 100 nM, or 10 nM to 100 nM in vivo or in cell-based assays.

[0335] In some embodiments, in cell-based assays, the peptide exhibits a binding affinity for B7-H3 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).

[0336] Microproteins This document provides novel peptides (e.g., microproteins) and methods of using them. In some embodiments, the peptide comprises or is composed of microproteins. This disclosure recognizes that one problem with therapeutic agents (e.g., microproteins) is their binding to B7-H3 with sufficient specificity and affinity. Therefore, this document provides microproteins that bind strongly, efficiently, and specifically to B7-H3 (e.g., on cells, such as cancer cells).

[0337] Another source of concern includes toxicity (e.g., nephrotoxicity). In some implementations, the binding specificity and strength of B7-H3 in cancer cells reduces renal uptake.

[0338] In some such embodiments, the microprotein comprises or is composed of: an affinity compound, CDP, knotting agent, binding agent, monomeric antibody, anticalcin, designed ankyrin repeat, engineered Kunitz domain, and / or avimer. In some embodiments, the microprotein is designed to be linked to one or more other components. For example, in some embodiments, the microprotein may be linked (conjugated) to another component, such as a chelating agent and / or a radionuclide. In some embodiments, conjugation is performed via lysine or cysteine ​​residues. For example, in some embodiments, the microprotein is engineered to remove all lysine residues except for one lysine residue, which in some embodiments is used for conjugation. In some embodiments, conjugation is performed via an optional linker. In some embodiments, the conjugation between the microprotein and the chelating agent and / or radionuclide is direct.

[0339] Not wishing to be bound by any particular theory, this disclosure takes into account that therapeutic agents comprising the compositions provided herein are characterized by several properties relative to other (e.g., antibody-based) therapeutic agents. For example, in some embodiments, microproteins exhibit several key properties of antibody-based therapeutic agents (e.g., affinity, potency, specificity, and ability to disrupt protein-protein interactions), but also have several advantages compared to antibody-based therapeutic agents, such as smaller size, lower manufacturing cost, and no need for protein chimerism or humanization. Furthermore, the size and specificity of the binding increase tumor penetration and uptake of the microprotein or a composition containing the microprotein (e.g., a conjugate) by cells expressing the target.

[0340] In some embodiments, the length of the microprotein of this disclosure does not exceed about 100 amino acids. In some embodiments, the length of the microprotein is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more amino acids, up to a maximum of about 100 amino acids. In some such embodiments, however, the length of the microprotein of this disclosure does not exceed about 100 amino acids. In some embodiments, the length of the microprotein is 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 about 100 amino acids. In some preferred embodiments, the microprotein is about 65 amino acids or less. In some preferred embodiments, the microprotein is about 50 amino acids or less.

[0341] In some embodiments, the microproteins of this disclosure are no larger than about 12 kDa. In some embodiments, the microproteins of this disclosure are about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or more kDa. In some such embodiments, however, the microproteins of this disclosure do not exceed about 12 kDa.

[0342] In some embodiments, the microproteins of this disclosure comprise or consist of cysteine-dense peptides, knotting agents, and / or binding agents.

[0343] In some embodiments, the microprotein comprises one or more disulfide bridges. In some embodiments, the microprotein comprises multiple cysteine ​​residues. In some such embodiments, the cysteine ​​residues are cross-linked to maintain a highly stable folded state for the peptide of its length (e.g., relative to a peptide of the same length without so many cysteine ​​residues). This disclosure contemplates that such cross-linking confers increased stability and reduced (i.e., very low or no) immunogenicity, and / or maintains or enhances the ability to maintain biological activity under harsh but effective chelating conditions (e.g., high temperature and low pH).

[0344] In some embodiments, microproteins or compositions containing microproteins (e.g., radionuclide conjugates) have lower immunogenicity than larger proteins (e.g., antibodies) or compositions containing or composed of larger proteins.

[0345] In some embodiments, microproteins (e.g., affinity molecules, CDPs, knotting agents, binding agents, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankyrin repeating domains (DARPin), avimer) exhibit superior penetration efficiency compared to larger proteins. That is, in some embodiments, microproteins or compositions containing microproteins penetrate solid tumors better than larger proteins or compositions containing proteins larger than microproteins. For example, in some such embodiments, the hydrodynamic radius of the microprotein or composition containing microproteins is from about 1 nm to about 25 nm. In some embodiments, the hydrodynamic radius ranges from about 1-25 nm, 10-20 nm, 5-15 nm, 1-5 nm, 2-4 nm, or 1-3 nm. In some embodiments, the hydrodynamic radius is measured using light scattering methods known to those skilled in the art.

[0346] In some embodiments, the microprotein of this disclosure is characterized by having one or more properties relative to proteins of more than 100 amino acids (such as antibodies, antibody fragments, VHH domains, single-chain 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 specific activity, increased resistance to substrate and / or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., decreased sensitivity to proteolysis), reduced aggregation, increased solubility, decreased immunogenicity, and altered temperature profile, increased resistance to hepatic uptake, renal uptake, or binding to healthy tissue, reduced binding to macroproteins and / or cuboproteins, increased tumor penetration, and / or increased volume of distribution.

[0347] In some embodiments, the microproteins or compositions comprising microproteins provided in this disclosure (e.g., conjugates, such as radionuclide conjugates) exhibit binding affinity for B7-H3. In some embodiments, B7-H3 is human B7-H3. In some embodiments, human B7-H3 is on cells. In some embodiments, the cells are cell lines, primary cells, or cells within the human body (e.g., in tumors).

[0348] In some embodiments, the microprotein or its conjugates have a binding affinity for human B7-H3 of about 500 nM, 400 nM, 300 nM, 200 nM, 100 nM or stronger (e.g., 90 nM, 75 nM, 50 nM, 25 nM, 10 nM, 5 nM, etc.). In some embodiments, the microprotein has a picomolar binding affinity. In some embodiments, the microprotein or its conjugates are characterized by binding affinity in the range of about 900 nM to about 1 nM, for example, binding affinity to human B7-H3 of 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 nM or stronger (e.g., 0.3, 0.2, 0.1, 0.09 nM, etc.). In some embodiments, the binding is selective for human B7-H3 but not for, for example, non-human B7-H3.

[0349] In some embodiments, the microprotein or its conjugates exhibit a binding inhibition constant. In some embodiments, the binding inhibition constant (Ki) with human B7-H3 is about 300 nM, 200 nM, 100 nM, 50 nM, 25 nM, 10 nM, 5 nM or lower (e.g., 1 nM, etc.). In some embodiments, the microprotein has a picomolar binding affinity. In some embodiments, the microprotein or its conjugates are characterized by binding affinity 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 stronger (e.g., 0.3, 0.2, 0.1, 0.09 nM, etc.) to human B7-H3. In some embodiments, the binding inhibition constant of the B7-H3 binding peptide of this disclosure is between about 100 pM and about 50 nM (e.g., in cell-based assays such as DELFIA). In some implementations, the binding is selective for human B7-H3 but not for, for example, non-human B7-H3.

[0350] In some embodiments, the microproteins or conjugates thereof provided in this disclosure have a high affinity for B7-H3 (e.g., as measured by binding affinity and / or inhibition constant, etc.). In some such embodiments, B7-H3 is human B7-H3. In some embodiments, the microproteins of this disclosure are stable, including in the presence of one or more additional molecules (e.g., cytotoxic molecules, such as radionuclides).

[0351] In some embodiments, the microproteins or compositions comprising microproteins provided in this disclosure (e.g., conjugates, such as radionuclide conjugates) exhibit nm or sub-nm binding affinity for B7-H3. In some embodiments, affinity is measured in an in vitro assay. In some embodiments, the in vitro assay is a cell-based assay. In some embodiments, affinity is measured in an in vivo assay (e.g., a PET scan) or using a sample from a subject (e.g., an in vitro assay using a biological specimen such as blood or a cell biopsy from a subject).

[0352] In some embodiments, the microprotein or its conjugates exhibit binding affinity for B7-H3. In some embodiments, the microprotein or its conjugates have a binding affinity of about 500 nM for human B7-H3. In some embodiments, the microprotein has a binding affinity in the picomolar range. In some embodiments, the microprotein or its conjugates contain binding kinases characterized by a dissociation constant ranging from about 900 nM to about 1 nM, for example, binding kinases of 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 for human B7-H3. In some implementations, the binding is selective for human B7-H3 but not for, for example, non-human B7-H3.

[0353] In some embodiments, the microproteins or conjugates thereof provided in this disclosure have a high affinity for B7-H3. In some such embodiments, B7-H3 is human B7-H3. In some embodiments, the microproteins of this disclosure are stable, including in the presence of one or more additional molecules (e.g., cytotoxic molecules, such as radioactivity).

[0354] In some embodiments, the binding affinity of the microprotein or its conjugate to the target is enhanced by one or more modifications. For example, in some embodiments, the use of chemical cross-linking can enhance the binding affinity of the microprotein or its conjugate, as provided herein, to B7-H3. In some embodiments, binding can be enhanced by using one or more of lysine residues, fusion proteins, non-natural amino acids, or other chemical motifs used to enhance binding and / or functional activity.

[0355] In some embodiments, to ensure proper folding and linkage, selected cysteine ​​pairs may be replaced with selenocysteine. Considering that in some embodiments, diselenide crosslinks are more easily formed than disulfide crosslinks due to their lower redox potential, and that such substitutions may lead to cross-coupling of the remaining cysteine ​​residues.

[0356] In some embodiments, the microproteins or conjugates thereof provided in this disclosure comprise or consist of monomers constituting dimers, polymers, or polymers. In some such embodiments, the monomers all bind to the same target. For example, in some embodiments, when more than one microprotein is present, each microprotein is no longer than about 30-40 amino acids, or its total size is no greater than about 8 kDa (in the case of two microproteins). In some embodiments, the monomers each bind to different targets. In some embodiments, some monomers bind to one target, while other monomers bind to one or more additional targets.

[0357] In some embodiments, the microproteins of this disclosure comprise or consist of an antigen for generating antibodies that specifically bind to at least one epitope on B7-H3. In some embodiments, such antibodies can be used for, for example, diagnostic purposes, blocking (e.g., antagonism), etc.

[0358] In some implementations, the microprotein contains one or more disulfide bridges.

[0359] In some embodiments, the microproteins or their conjugates provided herein do not contain one or more cysteine ​​residues. In some embodiments, the microproteins do not contain one or more disulfide bridges.

[0360] In some embodiments, the microproteins or their conjugates, as provided herein, are specific to the target. In some embodiments, the binder is specific to B7-H3 or fragments thereof.

[0361] In some embodiments, such as the microproteins or their conjugates provided herein, the protein comprises or is composed of a specific amino acid sequence.

[0362] In some embodiments, a microprotein or a composition comprising a microprotein (e.g., a radionuclide conjugate) is conjugated to a chelating agent, which optionally binds a radionuclide (e.g., actinium). In some embodiments, the conjugation is performed via a linker. In some embodiments, the conjugation is a direct conjugation. In some embodiments, such radionuclide conjugates combine and produce a synergistic effect to provide target specificity (e.g., via a microprotein) and superior therapeutic effect (e.g., by directing the radioisotope to cells expressing the target).

[0363] In some embodiments, the microprotein or a composition containing the microprotein is conjugated to a chelating agent, which optionally binds a cold metal substitute. In some embodiments, the cold metal substitute is a non-radioactive natural isotope of an element. In some embodiments, the element may have more than one non-radioactive natural isotope. In some embodiments, "cold" is used to refer to a non-radioactive isotype of the element. In some embodiments, "hot" refers to a radioactive isotype of the element.

[0364] As used herein and as is known to those skilled in the art, the twenty common amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (eds. Golub and Gren, Sinauer Associates, Sunderland, Mass., 2nd ed., 1991), which is incorporated herein by reference. In some embodiments, the amino acids of this disclosure may be stereoisomers of the twenty common amino acids (e.g., D-amino acids). In some embodiments, the amino acids in the polypeptides of this disclosure may be non-natural amino acids. For example, amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components of the polypeptides of this 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, leucine, citrulline, L-citrulline, methylated arginine (Rme, Rme2), symmetrically dimethylated arginine (sRme2, Rme2s, or SDMA), nitroarginine (Arg(NO2)), Leu-13C6,15N (a stable isotope-rich form of leucine), and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The polypeptide sequence representation used herein is arranged with the left end corresponding to the amino terminus and the right end corresponding to the carboxyl terminus, conforming to standard usage and convention.

[0365] In some embodiments, the microproteins disclosed herein have one or more of the following unconventional amino acids: trimethyllysine, dimethyllysine, monomethyllysine, isopropyllysine, Lys(Ac), leucine, citrulline, L-citrulline, methylated arginine (Rme, Rme2), symmetrically dimethylated arginine (sRme2, Rme2s, or SDMA), nitroarginine (Arg(NO2)) or Leu-13C6,15N, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). The polypeptide sequence representation used herein is arranged such that the left end corresponds to the amino terminus and the right end corresponds to the carboxyl terminus, conforming to standard usage and convention.

[0366] In some embodiments, the microprotein comprises or is composed of a specific amino acid sequence. In some embodiments, the microprotein 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 described in any one of SEQ ID NO: 4-6, 8-94, and 100-537. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in any one of SEQ ID NO: 4-6, 8-94, and 100-537. In each embodiment, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in any one of SEQ ID NO: 4-6, 8-94 and 100-537.

[0367] In some embodiments, the microprotein comprises or is composed of a specific amino acid sequence. In some embodiments, the microprotein 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 described in any one of SEQ ID NO: 4-6, 8-94, or 100-537. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in any one of SEQ ID NO: 4-6, 8-94, or 100-537. In each embodiment, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in any one of SEQ ID NO: 4-6, 8-94 or 100-537.

[0368] The polypeptides (e.g., microproteins) disclosed herein may have an amino acid sequence that has a certain percentage of identity at a certain coverage percentage (e.g., a reference sequence). That is, for example, a B7-H3 binding polypeptide (e.g., a reference molecule) as provided herein may have 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with a given query molecule at a coverage percentage (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), wherein if the query molecule is shorter, its identity percentage and coverage percentage may differ (e.g., 100% identity and 90% coverage). If the query molecule is longer, both the identity percentage and coverage relative to the reference molecule may be 100%, and the reference molecule may have a certain percentage of identity at a certain length of the query molecule (e.g., at least 20, 25, or 30 amino acids). For example, in some embodiments, if a reference sequence (e.g., a microprotein provided herein) is shorter than a query sequence, then such a query sequence is considered within the scope of this disclosure if it has the length of a reference sequence that aligns with the query sequence, wherein the percentage of identity is determined between the two sequences (the query sequence and the reference sequence) at at least a minimum length of alignment. That is, if the polypeptide disclosed herein is longer than the query sequence, the percentage of identity is determined by aligning the reference sequence and the query sequence and determining the percentage of identity between the query sequence and the portion of the reference sequence that it aligns with. Conversely, when the query sequence is longer than the reference sequence, the percentage of identity is equal to the identity of the portion aligned with the reference sequence. That is, if the reference sequence is shorter, then the query sequence may fall within the scope of the reference sequence if it aligns between the two polypeptides (the reference sequence and the query sequence) at the aligned portion with the claimed percentage of identity.

[0369] In some embodiments, the microprotein 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 described in any one of SEQ ID NO: 4-6, SEQ ID NO: 12-94, or SEQ ID NO: 100-537. In each embodiment, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in any one of SEQ ID NO: 4-6, SEQ ID NO: 12-94, or SEQ ID NO: 100-537. In each embodiment, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in any one of SEQ ID NO: 4-6, SEQ ID NO: 12-94, or SEQ ID NO: 100-537.

[0370] In some embodiments, the microprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or higher) identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in any one of SEQ ID NO: 4-6, SEQ ID NO: 12-94 or SEQ ID NO: 100-537. In some embodiments, the microprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in any one of SEQ ID NO: 4-6, SEQ ID NO: 12-94 or SEQ ID NO: 100-537.

[0371] In some embodiments, the microprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or higher) identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in any one of SEQ ID NO: 198-537. In some embodiments, the microprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in any one of SEQ ID NO: 198-537.

[0372] In some embodiments, the microprotein has an amino acid sequence that is at least 90% (91, 92, 93, 94, 95, 96, 97, 98, 99% or higher) identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in any one of SEQ ID NO: 204 and 262-272. In some embodiments, the microprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in any one of SEQ ID NO: 204 and 262-272.

[0373] In some embodiments, the microprotein 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%, 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 described in any one of SEQ ID NO: 6, 21, or 30. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in any one of SEQ ID NO: 6, 21, or 30. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in any one of SEQ ID NO: 6, 21, 30, 130-131, 183, 199, 204, 241 or 267.

[0374] In some embodiments, the microprotein 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 described in SEQ ID NO: 6. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 6. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 6.

[0375] In some embodiments, the microprotein 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 described in SEQ ID NO: 21. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 21. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 21.

[0376] In some embodiments, the microprotein 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 described in SEQ ID NO: 30. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 30. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 30.

[0377] In some embodiments, the microprotein 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 described in SEQ ID NO: 130. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 130. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 130.

[0378] In some embodiments, the microprotein 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 described in SEQ ID NO: 131. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 131. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 131.

[0379] In some embodiments, the microprotein 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 described in SEQ ID NO: 183. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 183. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 183.

[0380] In some embodiments, the microprotein 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 described in SEQ ID NO: 199. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 199. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 199. In some embodiments, the microprotein has an amino acid sequence that is at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in SEQ ID NO: 199. In some embodiments, the microprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in SEQ ID NO: 199.

[0381] In some embodiments, the microprotein 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 described in SEQ ID NO: 204. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 204. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 204. In some embodiments, the microprotein has an amino acid sequence that is at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in SEQ ID NO: 204. In some embodiments, the microprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in SEQ ID NO: 204.

[0382] In some embodiments, the microprotein 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 described in SEQ ID NO: 241. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 241. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 241. In some embodiments, the microprotein has an amino acid sequence that is at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in SEQ ID NO: 241. In some embodiments, the microprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in SEQ ID NO: 241.

[0383] In some embodiments, the microprotein 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 described in SEQ ID NO: 267. In various embodiments, the microprotein has an amino acid sequence that is 90% identical to the amino acid sequence described in SEQ ID NO: 267. In various embodiments, the microprotein has an amino acid sequence that is 100% identical to the amino acid sequence described in SEQ ID NO: 267. In some embodiments, the microprotein has an amino acid sequence that is at least 90% (91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater) identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in SEQ ID NO: 267. In some embodiments, the microprotein has an amino acid sequence that is 100% identical to at least 40, 41, 42, 43, 44, 45, 46, 47 or 48 amino acids as described in SEQ ID NO: 267.

[0384] In some implementations, the microprotein may also have one or more additional amino acids added to its N-terminus and / or C-terminus.

[0385] In some embodiments, the B7-H3 binding microprotein of this disclosure may have different N-termini and / or C-termini, for example, having an acetyl group, NH2, biotin-PEG4, DOTA-PEG4, radiolabeling, etc. at its N-terminus, and -OH or -NH2 at its C-terminus. The N-terminus and / or C-terminus of the B7-H3 binding microprotein of this disclosure may include, but are not limited to, acetyl groups, acids, or amides (e.g., acetyl, NH2, OH), as provided in the exemplary compounds and microproteins of Table 2A or Table 2C.

[0386] In some embodiments, the polypeptides of this disclosure may have various modifications at their N-terminus (e.g., a linker, chelating agent, and / or radionuclide, as described in the exemplary compounds in Table 2A or Table 2C) or their C-terminus (e.g., a linker, chelating agent, and / or radionuclide). In some embodiments, the C-terminus of a given polypeptide may have an acid or amide group at its C-terminus (see Table 2A, e.g., Table 2C). A given polypeptide having a specific amino acid sequence may have one or more N-terminal and / or C-terminal differences without substantially altering the utility or function of the polypeptide, such as binding to B7-H3 (e.g., for the detection and / or treatment of cancer).

[0387] In some embodiments, the addition of the N-terminus and / or C-terminus may include one or more of a linker and / or a chelating agent, such as N-terminal DOTA-PEG4. In some embodiments, the C-terminus of the microprotein contains NH2, OH, and / or COOH.

[0388] In some embodiments, the N-terminus and / or C-terminus further comprise an added microprotein having 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 described in SEQ ID NO: 4-6, 8-94, and 100-537. In some such embodiments, the microprotein is contained within the compound. In some embodiments, the compound is selected from any of compound numbers C1-C608 and C611.

[0389] In some embodiments, the N-terminus and / or C-terminus further comprise an added microprotein having 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 described in SEQ ID NO: 4-6, 8-94, or 100-537. In some such embodiments, the microprotein is contained within the compound. In some embodiments, the compound is selected from compound numbers C1-C9, C11-C117, or C121-C608 and C611.

[0390] In some embodiments, the microprotein does not contain one or more constraints (e.g., disulfide bridges). In some embodiments, the microprotein 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 described in SEQ ID NO: 4-6, 8-94, or 100-197. In some embodiments, the microprotein is part of a compound. In some embodiments, the compound is selected from any of compound numbers C1-C226.

[0391] In some embodiments, the microprotein does not contain one or more constraints (e.g., disulfide bridges). In some embodiments, the microprotein 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 described in SEQ ID NO: 198-537. In some embodiments, the microprotein is part of a compound. In some embodiments, the compound is selected from any of compound numbers C227-C608 and C611.

[0392] In some embodiments, the microprotein includes other variations such as, for example, one or more constraints (e.g., as disclosed herein). In some embodiments, the constraint is a disulfide bridge. In some embodiments, the microprotein has at least one, two, or three disulfide bridges. In some embodiments, the microprotein has one disulfide bridge. In some embodiments, the microprotein has two disulfide bridges. In some such embodiments, this microprotein may be referred to as a constrained microprotein. In some embodiments, constrained microproteins are more stable than, for example, microproteins without one or more constraints, characterized by a higher melting temperature, e.g., microproteins having the same or similar amino acid sequence but without one or more constraints (e.g., disulfide bridges). In some embodiments, the constrained microprotein 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 described in SEQ ID NO: 198-537. In some embodiments, the microprotein is part of a compound. In some embodiments, the compound is selected from any of compound numbers C227-C608 and C611.

[0393] In some embodiments, the microproteins provided herein are specific to or bind to polypeptides having or a portion thereof the amino acid sequence described in Table 1A.

[0394] Table 1A. Exemplary target protein amino acid sequences

[0395] Common sequences and substitutions In some embodiments, the microproteins provided in this disclosure are described with one or more common sequences provided in Table 1B. In some embodiments, microproteins having the sequences described in Table 1B have amino acid substitutions as provided in Table 1C.

[0396] Table 1B. Common sequences of illustrative B7-H3 microproteins Table 1C. Common Sequence Substitution

[0397] "(KAc)" refers to acetylated lysine, "((Kme3)" refers to trimethyllysine, "(MO2)" refers to thiosulfinate sulfone, "Cit" refers to citrulline, and "hSer" refers to homoserine.

[0398] In some embodiments, the microproteins provided in this disclosure have an amino acid sequence comprising the amino acid sequence according to Formula I: X1X2X3X4YX6X7EX9IX11ALX14EIIWLPNX22X23X24X25QIX28AFIAALNX36DPSQSSELLSEAX49X50LX52DSX55X56X57X58 (SEQ ID NO: 95), Where 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, (hyperleucine), 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, (high 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 not present; X56 is A or not present; X57 is P or not present; and X58 is G, K, (KAc), or not present. If X28 is A, (hyperleucine), (MO2), S, T, or V, then X24 is Y; or If X28 is M, then X7 is A, E, (Kme3), L, Q, or S.

[0399] In each implementation scheme, if X4 is K or (KAc), then X24 is Y.

[0400] In various embodiments, the microproteins provided in this disclosure have an amino acid sequence comprising the amino acid sequence according to Formula II: X1X2X3X4YAX7EKIAALSEIIWLPNX22TX24X25QIX28AFIAALNX36DPSQSSELLSEAX49X50LNDSQAP (SEQ ID NO: 96), Where X1 is A or not present; X2 is E or not present; X3 is A or not present; X4 is L or not present; X7 is K or Q; X22 is D or L; X24 is H or Y; X25 is A or G; X28 is ((high leucine)) or M; X36 is D or N; X49 is E or K; and X50 is E.

[0401] In some embodiments, the microproteins provided in this disclosure are described with one or more common sequences provided in Table 1D. In some embodiments, microproteins having the sequences described in Table 1D have amino acid substitutions as provided in Table 1E.

[0402] Table 1D. Common sequences of illustrative constrained B7-H3 miniature proteins Table 1E. Constraint Common Sequence Replacement

[0403] "(Kme)" refers to monomethyllysine; "(Kme2)" refers to dimethyllysine; "(Kme3)" refers to trimethyllysine; "Cit" refers to citrulline; and "(Rme)" refers to methylated arginine. On the one hand, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) whose amino acid sequence comprises the amino acid sequence according to Formula III (SEQ ID NO: 538): 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; and X45 is K or (Kme). In some embodiments, X49 is absent.

[0404] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising any one of the amino acid sequences of SEQ ID NO: 204, 238, 239, 241, 247, 262-273, 292, 295, 297, 298, 307, 309, 310, 314-317, 335, 336, 338, 352, 374, 389, 392, 396, 401, 460-462, 466-468, 470, 471, 497, and 528.

[0405] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) having an amino acid sequence comprising the amino acid sequence according to Formula IV (SEQ ID NO: 539): CAX3EKIAALSEIIWLPCLX19YAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme2) or (Kme); X29 is A or (Kme); X32 is D or (Kme); and X45 is K or (Kme). In some embodiments, X49 is absent.

[0406] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising any one of SEQ ID NO: 204, 241, 262, 265, 267, 268, 292, 307, 314-317, 338, 352, 392, 396, and 401.

[0407] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) having an amino acid sequence comprising the amino acid sequence according to formula V (SEQ ID NO: 540): CAX3EKIAALSEIIWLPCLTYAQIX24AFIX28X29LNX32DPCQSSEILSEAX45ELCS, wherein X3 is (Kme3) or R or (Rme); X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit); and X45 is K or (Kme). In some embodiments, X49 is absent.

[0408] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising any one of SEQ ID NO: 204, 241, 262, 265-267, 270, 272, 292, 307, 314-317, 338, 352, 374, 389, 392, 396, 401, 460-462, 466, 467, 468, 470, 471, and 497.

[0409] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) having an amino acid sequence comprising the amino acid sequence according to formula VI (SEQ ID NO: 541): CA(Kme3)EKIAALSEIIWLPCLTYAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, wherein X24 is (Kme2) or (Kme); X29 is (Kme) or A or R; X32 is D or (Kme) or (Cit); and X45 is K or (Kme). In some embodiments, X49 is absent.

[0410] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising any one of SEQ ID NO: 204, 262, 265, 267, 270, 272, 314-317, 338, 352, 389, 392, 396, 401, 462, and 471.

[0411] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) having an amino acid sequence comprising an amino acid sequence according to formula VII (SEQ ID NO: 542): CAX3EKIX7ALX10EIIWLPX17LTYX21QIX24AFIX28X29LNX32DPCQSX38X39X40LX42EAX45ELCS, where X3 is (Kme3) or R or K; X7 is A or N; X10 is S or G; X17 is C or N; X21 is A or D; X24 is (Kme3) or (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme); X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; and X45 is K or (Kme) or (Kme3) or Q. In some embodiments, X49 is absent.

[0412] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising SEQ ID NO: 204, 206, 217, 219, 221, 238, 239, 241, 247, 250, 262-267, 273, 278-280, 287, 288, 292, 295, 297-302, 304, 305, 307, 309-319, 321-326, 328-331, 335-338, 352, 356, 366, 367. The amino acid sequence of any one of the following: 374-377, 383-385, 389, 392, 394-396, 401, 402, 404-409, 414-418, 426, 437-439, 449-451, 460-462, 465-468, 470, 471, 489, 497, 502-508, 518-521, 523, and 527.

[0413] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) having an amino acid sequence comprising the amino acid sequence according to formula VIII (SEQ ID NO: 543): CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIX28X29LNX32DPCQSX38X39X40LX42EAX45ELCS, where X3 is (Kme3) or R; X7 is A or N; X10 is S or G; X21 is A or D; X24 is (Kme3) or (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme); X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; and X45 is K or (Kme) or (Kme3) or Q. In some embodiments, X49 is absent.

[0414] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising SEQ ID NO: 204, 206, 238, 239, 241, 247, 250, 262-267, 273, 278-280, 287, 288, 292, 295, 297-302, 304, 305, 307, 309-319, 321-326, 328, 329, 330, 331, 335-338, 352, 356, 366, 367, 374. The amino acid sequence of any one of the following: 375-377, 383-385, 389, 392, 394-396, 401, 402, 404-409, 414-418, 426, 437-439, 449-451, 460-462, 465-468, 470, 471, 489, 497, 502-508, 518-521, 523, and 527.

[0415] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) having an amino acid sequence comprising an amino acid sequence according to the following formula IX (SEQ ID NO: 546): 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 (Kme3) or K or Q or R; X24 is (Kme3) or K or Q or R; X25 is (Kme3) or K or Q or R; X26 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 (Kme3) or K or Q or R; X25 is (Kme3) or K or Q or R; X26 is (Kme3) or K or Q or R; X27 is A or Q or R; X28 is (Kme3) or K or Q or R; X29 is A or Q or R; X21 is A or D; X24 is (Kme3) or K ... X25 is A or E; X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit) or A; X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; X45 is K or (Kme) or (Kme3) or Q or R; X46 is E or A; and X48 is C or N. In some embodiments, X49 is absent.

[0416] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising SEQ ID NO: 203-205, 209-213, 216-220, 225, 226, 230-233, 235, 238, 239, 241, 242, 247, 248, 250, 262-267, 270, 272, 273, 278-280, 287, 288, 292, 294-302, 304, 305, 307, 309-319, 321-326, 328, 329, 330, 331, 335-338, 352, 356, 358, 366, 367, 374 The amino acid sequence of any one of -377, 380, 383-385, 389, 392, 394-396, 399, 401, 402, 404-409, 414-418, 426, 434, 437-439, 447, 449, 450, 451, 460-462, 465-468, 470, 471, 474, 475, 489, 497, 499, 502-508, 515, 518-521, 523, 524, 527, 532, 533, 535 and 537.

[0417] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) having an amino acid sequence comprising an amino acid sequence according to formula X (SEQ ID 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 (homoleucine); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme); X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; X45 is K or (Kme) or (Kme3) or Q; and X48 is C or N. In some embodiments, X49 is absent.

[0418] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising SEQ ID NO: 204, 209-213, 217-220, 225, 226, 238, 239, 241, 242, 247, 250, 262-267, 273, 278-280, 287, 288, 292, 295, 297-302, 304, 305, 307, 309-319, 321-326, 328, 329, 330, 331, 335-338, 352, 3 The amino acid sequence of any one of the following: 56, 366, 367, 374-377, 383-385, 389, 392, 394-396, 401, 402, 404-409, 414-418, 426, 437-439, 449-451, 460-462, 465-468, 470, 471, 489, 497, 502-508, 518-521, 523, and 527.

[0419] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) whose amino acid sequence comprises an amino acid sequence according to the following formula XI (SEQ ID 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; and X48 is C or N. In some embodiments, X49 is absent.

[0420] In some embodiments, the polypeptide (e.g., a B7-H3 binding microprotein) has an amino acid sequence comprising SEQ ID NO: 204, 217-220, 238, 239, 241, 242, 247, 250, 262-266, 273, 278, 279, 280, 287, 288, 292, 295, 297-302, 304, 305, 307, 309-319, 321-326, 328, 329, 330, 331, 335. The amino acid sequence of any one of -338, 352, 356, 366, 367, 374-377, 383-385, 389, 392, 394-396, 402, 404-409, 414-418, 426, 437-439, 449, 460, 466-468, 471, 489, 497 and 502-508.

[0421] In one aspect, this disclosure provides a polypeptide (e.g., a B7-H3 binding microprotein) having an amino acid sequence comprising the amino acid sequence according to formula XII (SEQ ID NO: 549): CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIX28X29LNDDPCQSX38X39X40LX42EAX45ELX48S, where X3 is (Kme3) or K or R; X7 is A or N; X10 is S or G; X21 is A or D; X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme); X38 is S or A; X39 is E or N; X40 is I or L; X42 is S or A; X45 is K or (Kme); and X48 is C or N. In some embodiments, X49 is absent.

[0422] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising SEQ ID NO: The amino acid sequence of any one of the following: 204, 217-220, 238, 239, 241, 242, 247, 262-266, 273, 278, 279, 280, 287, 292, 295, 297-302, 304, 305, 307, 309-319, 321-326, 328-330, 335-338, 352, 356, 366, 367, 374, 383, 384, 392, 395, 396, 402, 404-406, 414, 415, 417, 426, 437, 439, 460, 466, 468, 502, 503, 505, and 506.

[0423] In one aspect, this disclosure provides a polypeptide (e.g. ， B7-H3 binding microprotein has an amino acid sequence comprising the amino acid sequence according to formula XIII (SEQ ID NO: 550): CAX3EKIX7ALX10EIIWLPCLTYX21QIX24AFIAX29LNDDPCQSX38EILSEAX45ELCS, wherein X3 is (Kme3) or R; X7 is A or N; X10 is S or G; X21 is A or D; X24 is (Kme2) or (Kme); X29 is A or (Kme); X38 is S or A; and X45 is K or (Kme). In some embodiments, X49 is absent.

[0424] In some embodiments, the polypeptide (e.g., B7-H3 binding microprotein) has an amino acid sequence comprising any one of the amino acid sequences of SEQ ID NO: 204, 238, 241, 262, 263, 265, 292, 295, 297, 302, 304, 305, 307, 309, 313-319, 321-323, 326, 328, 335, 337, 338, 352, 367, 384, 392, 396, 402, 404, 414, 415, 417, 426, 437, 439, 502, 503, 505, and 506.

[0425] In one aspect, this disclosure provides a composition comprising a polypeptide having a length of at least 48 amino acids and having an amino acid sequence comprising at least four cysteine ​​residues and two disulfide bonds, wherein X24 is (Kme) or (Kme2); X29 is (Kme) or A or R; X32 is D or (Kme) or (Cit); and X45 is (Kme) or K. In some embodiments, X49 is absent.

[0426] In some embodiments, this disclosure provides a composition comprising a polypeptide bound to B7-H3 having at least 48 amino acids, two disulfide bonds, and modified amino acids at positions corresponding to 3, 24, and 29 relative to SEQ ID NO: 267, wherein the modification is a small alkyl group on the side chain of the amino acid.

[0427] In one aspect, this disclosure provides a composition comprising one or more modified amino acids. In some such embodiments, the one or more modifications include small alkyl groups. In one aspect, this disclosure provides a composition comprising a B7-H3 binding polypeptide having an amino acid sequence comprising at least 48 amino acids, wherein the amino acid comprises (i) a cysteine ​​at one of four positions corresponding to positions 1, 17, 35, and 48 of SEQ ID NO: 267, and wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme2) or (Kme); X28 is A or (Kme); X29 is A or (Kme) or R; X32 is D or (Kme) or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; X45 is K or (Kme), and X49 is S or absent.

[0428] In one aspect, this disclosure provides a composition comprising a B7-H3 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at least four cysteine ​​residues forming two disulfide bonds; arginine, modified arginine, or modified lysine at position 3; lysine at position 5; isoleucine at position 6; tryptophan at position 14; at least one modified lysine residue at position 24; and alanine, arginine, or modified lysine at position 29, wherein each position is linear from the N-terminus to the C-terminus relative to SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group, optionally including methyl, dimethyl, or trimethyl, attached to a nitrogen or guanidine group of a lysine side chain; a length of at least 48 amino acids; and a binding affinity of the microprotein B7-H3 greater than 100 nM as measured in a cell-based assay.

[0429] In one aspect, this disclosure provides a composition comprising a B7-H3 binding polypeptide having an amino acid sequence comprising: at least four cysteine ​​residues forming two disulfide bonds; at least one modified lysine residue at position X24 of SEQ ID NO: 267, wherein the modification comprises at least one small alkyl group of nitrogen linked to the lysine side chain, the small alkyl group optionally comprising methyl, dimethyl, or trimethyl; a length of at least 48 amino acids; and a binding affinity for B7-H3 greater than 100 nM as measured in a cell-based assay.

[0430] CDP In some embodiments, the microproteins of this disclosure comprise or consist of cysteine-dense peptides (CDPs). In some embodiments, the conjugates provided herein comprise CDPs. In some embodiments, the CDPs act as targeting moieties, for example, specifically binding to protein targets or antigens expressed on the surface of target tumor cells. In some embodiments, the CDPs comprise or consist of at least two independent folded domains and a high density of cysteine ​​residues. In some embodiments, the CDPs contain at least one, two, three, four, five, six, or more than six cysteine ​​residues in a span of about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues. (For illustrative CDPs and their characteristics, see, for example, Correnti et al., Nat Struct Mol Biol. 2018 Mar; 25(3):270-278). In some embodiments, the CDP comprises a constrained distribution of cysteine ​​residues, 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 containing at least one cysteine ​​residue across a span of about 10 to 80 amino acid residues, preferably at least two, three, four, or more cysteine ​​residues. In some embodiments, the CDP may be further engineered to modify binding, folding, and / or related properties.

[0431] In some embodiments, the CDP specifically binds to the target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the CDP specifically binds to B7-H3 or a fragment thereof. In some embodiments, the CDP conjugates to a chelating agent and / or a radionuclide. In some embodiments, conjugation occurs via a linker. Those skilled in the art will understand that, in some embodiments, the specific CDP used in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0432] In some embodiments, in microproteins containing cysteine ​​residues, selected cysteine ​​pairs may be replaced with selenocysteine ​​to ensure proper folding and linkage. In some embodiments, diselenide crosslinks may be more readily formed than disulfide crosslinks due to their lower redox potential. In some such embodiments, such substitution may result in cross-coupling of the remaining cysteine ​​residues.

[0433] Knotting agent In some embodiments, the microproteins of this disclosure comprise or consist of knotting peptides. In some embodiments, the conjugates provided herein comprise knotting peptides. In some embodiments, the knotting peptides act as targeting portions, for example, specifically binding to antigens expressed on the surface of target tumor cells. In some embodiments, knotting peptides comprise at least three disulfide bonds linked in an arrangement forming so-called “cysteine ​​knots,” hence the name knotting peptide. (See, for example, Kintzing and Cochran et al., Curr Opin Chem Biol. 2016 Oct; 34:143-150.) In some embodiments, knotting peptides exhibit high stability (e.g., thermal stability, proteolytic stability, chemical stability, etc.). In some embodiments, knotting peptides can be further engineered to modify binding, folding, and / or related properties.

[0434] In some embodiments, the given knotting agent is highly specific to a given target. In some embodiments, the knotting agent specifically binds to the target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the knotting agent specifically binds to B7-H3 or a fragment thereof. In some embodiments, the knotting agent conjugates to a chelating agent 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 knotting agent used in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0435] In some embodiments, the folded structure of microproteins (e.g., affinity molecules, CDPs, knotting agents, binders) makes them rigid, enabling them to bind very tightly and efficiently to target proteins or antigens (as opposed to weakly structured peptides). In some such embodiments, microproteins (e.g., affinity molecules, CDPs, knotting agents, binders, engineered Kunitz domains, monomeric antibody mimics, anticalcitonins, designed ankyrin repeating domains (DARPin), avimer) exhibit remarkable stability against heat, peptidase cleavage, and pH.

[0436] binder In some embodiments, the microproteins of this disclosure comprise or consist of a binding agent. In some embodiments, the binding agent acts as a targeting portion, for example, specifically binding to a target expressed on the surface of tumor cells. In some embodiments, this document provides a binding agent that binds to B7-H3.

[0437] In some embodiments, the binder has certain structural properties; for example, in some embodiments, the binder may be rich in α-helices, such as a helical-helical-helical structure (see, for example, Crook et al., Nat Commun. (2017) 8, 2244; Berger et al., Elife (2016) 5, e20352; and Procko et al., Cell (2014), 157, 1644-1656). In some embodiments, the binder may comprise α-helices, β-sheets, or one or more combinations thereof. In some embodiments, the binder comprises a surface sufficient to functionalize molecules on different surfaces to the binding surface. In some embodiments, the binder comprises an isolated hydrophobic core. In some embodiments, the binder exhibits co-folding. In some embodiments, the binder has two or more of the following properties: (i) represented by an amino acid sequence of 100 amino acids or less; (ii) at least two secondary structural elements; (iii) an isolated hydrophobic core; and / or (iv) co-folding.

[0438] In some embodiments, the given binder is highly specific to a given target. In some embodiments, the binder specifically binds to the target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the microprotein specifically binds to B7-H3 or a fragment thereof. In some embodiments, the binder conjugates to a chelating agent and / or a radionuclide. In some embodiments, conjugation occurs via a linker. Those skilled in the art will understand that, in some embodiments, the specific binder used in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0439] Affinity In some embodiments, the microproteins of this disclosure comprise or consist of an affinity echelon. In some embodiments, the affinity echelon acts as a targeting moiety, for example, specifically binding to a target expressed on the surface of tumor cells. In some embodiments, an affinity echelon that binds to B7-H3 is provided herein. In some embodiments, the microprotein (e.g., the affinity echelon) comprises part or all of any of the compounds described in Tables 2A and / or 2C (e.g., comprising or consisting of a microprotein). In some embodiments, the microprotein (e.g., the affinity echelon) comprises, is primarily composed of, or is composed of any of the amino acid sequences or portions thereof of SEQ ID NOs: 4-6, 8-94, and 100-537. Table 2B shows certain characteristics of the exemplary compounds / microproteins selected in Table 2A. In some embodiments, the conjugates provided herein comprise an affinity echelon. In some embodiments, the affinity echelon acts as a targeting moiety, for example, specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, the affinity compound comprises or consists of no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acids. In some embodiments, the affinity compound comprises or consists of at least three α-helices containing 58 amino acids. In some embodiments, the affinity compound exhibits target specificity obtained by randomizing 13 amino acids located in the two α-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 affinity compound may be further engineered to modify binding, folding, and / or related properties.

[0440] This disclosure also provides the surprising finding that binding affinity and / or specificity to B7-H3 can be improved by truncating the affinity (e.g., removing residues from the N-terminus and / or C-terminus of an affinity having three α-helices spanning 58 amino acids), for example by removing part or all of the amino acids corresponding to positions 1-6 from the N-terminus and positions 54-58 from the C-terminus (refer to the N-terminus to C-terminus positions along the length of SEQ ID NO:3), and / or removing and replacing such amino acids with, for example, cysteine. This disclosure also contemplates that, in addition to truncation, one or more constraints may be introduced. In some embodiments, the constraints comprise at least one, two, or three disulfide bridges and / or at least one additional constraint, such as those disclosed herein (e.g., lactam bridges, e.g., pinning compounds). In some embodiments, the affinity of a particular scaffold comprises three α-helices containing at least four cysteine ​​residues (with disulfide bonds potentially introduced therebetween), spanning 49 amino acids. Thus, in some such embodiments, the length (e.g., truncated) and constraint pattern of the scaffold differ relative to the 58-amino acid affinity. In some of these embodiments, such truncated and constrained affinities may have one or more desirable characteristics, such as enhanced affinity and / or specificity for B7-H3. In some embodiments, such affinities may be further modified to introduce one or more non-natural amino acids, which in some embodiments may affect toxicity by reducing renal cell uptake (e.g., in vitro, for example, in vivo).

[0441] In some embodiments, the affinity specifically binds to the target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the affinity specifically binds to B7-H3 or a fragment thereof. In some embodiments, the affinity conjugates to a chelating agent and / or a radionuclide. In some embodiments, conjugation occurs via a linker. Those skilled in the art will understand that, in some embodiments, the specific affinity used in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0442] Engineered Kunitz domains In some embodiments, the microproteins of this disclosure comprise or consist of engineered Kunitz domains. In some embodiments, the conjugates provided herein comprise engineered Kunitz domains. In some embodiments, the engineered Kunitz domains act as targeting portions, for example, specifically binding to protein targets or antigens expressed on the surface of target tumor cells. In some embodiments, the engineered Kunitz domains comprise or consist of a peptide of at least one Kunitz domain derived from a Kunitz-type protease inhibitor (e.g., bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP), or tissue factor pathway inhibitor (TFPI)). In some embodiments, the engineered Kunitz domains may be further engineered to alter binding, folding, and / or related properties.

[0443] In some embodiments, the engineered Kunitz domain specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the engineered Kunitz domain specifically binds to B7-H3 or a fragment thereof. In some embodiments, the engineered Kunitz domain is conjugated to a chelating agent and / or a radionuclide. In some embodiments, conjugation occurs via a linker. Those skilled in the art will understand that, in some embodiments, the specific engineered Kunitz domain used in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0444] Monomeric Antibody In some embodiments, the microproteins of this disclosure comprise or consist of monomeric antibody-like substances. In some embodiments, the conjugates provided herein comprise monomeric antibody-like substances. In some embodiments, the monomeric antibody-like substances act as a targeting moiety, for example, specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, the monomeric antibody-like substances comprise or consist of a molecule or composition based on the 10th extracellular domain (10Fn3) of human fibronectin III, which employs an Ig-like b-sandwich fold of approximately 94 residues, having 2 to 3 exposed loops but lacking a central disulfide bridge. In some embodiments, the monomeric antibody-like substances may be further engineered to modify binding, folding, and / or related properties.

[0445] In some embodiments, the monomeric antibody-like substance specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the monomeric antibody-like substance specifically binds to B7-H3 or a fragment thereof. In some embodiments, the monomeric antibody-like substance is conjugated to a chelating agent 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 monomeric antibody-like substance used in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0446] Anticalcin In some embodiments, the microproteins of this disclosure comprise or consist of anticalcin. In some embodiments, the conjugates provided herein comprise anticalcin. In some embodiments, the anticalcin acts as a targeting moiety, for example, specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, the anticalcin comprises or consists of an eight-chain b-barrel structure that forms a highly conserved core unit in a lipid transport protein and naturally forms a ligand binding site by means of four structurally variable loops at the open ends. In some embodiments, the anticalcin may be further engineered to alter its binding, folding, and / or related properties.

[0447] In some embodiments, the anticalcitonin specifically binds to a target. In some embodiments, the target is located within, on, or near a cell. In some embodiments, the anticalcitonin specifically binds to B7-H3 or a fragment thereof. In some embodiments, the anticalcitonin is conjugated to a chelating agent and / or a radionuclide. In some embodiments, conjugation occurs via a linker. Those skilled in the art will understand that, in some embodiments, the specific anticalciton used in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0448] Designed ankyrin repeating domain In some embodiments, the microproteins of this disclosure comprise or consist of designed ankyrin repeat domains. In some embodiments, the conjugates provided herein comprise designed ankyrin repeat domains. In some embodiments, the designed ankyrin repeat domains act as targeting portions, for example, specifically binding to protein targets or antigens expressed on the surface of target tumor cells. In some embodiments, the designed ankyrin repeat domains comprise peptides derived from ankyrin. In some embodiments, the designed ankyrin repeat domains comprise a single ankyrin repeat structure, preferably comprising a 33-residue motif comprising two α-helices and one β-turn. In some embodiments, the designed ankyrin repeat domains provide a rigid interface and lack structural flexibility. In some embodiments, the designed ankyrin repeat domains may be further engineered to modify binding, folding, and / or related properties.

[0449] In some embodiments, the designed ankyrin repeat domain specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the designed ankyrin repeat domain specifically binds to B7-H3 or a fragment thereof. In some embodiments, the designed ankyrin repeat domain is conjugated to a chelating agent 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 conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0450] Avimer In some embodiments, the microproteins of this disclosure comprise or consist of Avimer. In some embodiments, the conjugates provided herein comprise Avimer. In some embodiments, Avimer acts as a targeting moiety, for example, specifically binding to a protein target or antigen expressed on the surface of a target tumor cell. In some embodiments, Avimer comprises a peptide of about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. In some embodiments, Avimer comprises at least one peptide sequence having about 30 to 35 amino acids. In some embodiments, Avimer comprises at least two or more peptide sequences having about 30 to 35 amino acids. In some embodiments, 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, see Nature Biotechnology 23(–2), 1556–1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909–917 (June 2007). In some embodiments, avimer can be further engineered to alter binding, folding, and / or related properties.

[0451] In some embodiments, the aviser specifically binds to a target. In some embodiments, the target is located in, on, or near a cell. In some embodiments, the aviser specifically binds to B7-H3 or a fragment thereof. In some embodiments, the aviser conjugates to a chelating agent and / or a radionuclide. In some embodiments, conjugation occurs via a linker. Those skilled in the art will understand that, in some embodiments, the specific aviser used in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0452] constraint Constraints can be designed (e.g., from an initial polypeptide sequence) or introduced (e.g., engineered, for example, into an existing microprotein) into a protein, such as any microprotein provided herein. Constraints can be introduced by modifying an existing microprotein sequence, for example by substituting and / or adding two or more cysteine ​​residues to form at least one disulfide bond.

[0453] Constraints can alter a protein's function (e.g., binding, such as binding affinity) and / or structure (e.g., folding). In some embodiments, constraints can help maintain the secondary structure of a given protein (e.g., the microproteins provided herein, such as the conjugates provided herein). For example, a common secondary structure of proteins is the α-helix. α-helices can play a crucial role in both the structure and function of a protein, such as influencing how a particular protein can interact with its binding conjugate. α-helices can mediate protein-protein interactions (PPIs) by acting as recognition motifs. In proteins containing α-helical structures, introducing constraints can alter one or more properties of the protein, such as protein affinity for a target (e.g., increased affinity), cell permeability (e.g., increased cell permeability), and resistance to proteolytic degradation (e.g., increased resistance to proteolytic degradation).

[0454] Examples of constraints (e.g., α-helical constraints) include, but are not limited to, disulfide bridges / bonds, pinning compounds (e.g., hydrocarbon pinning compounds), salt bridges between charged amino acid side chain residues, lactam bridges, hydrogen bond substitutions, hydrophobic interactions, metal coordination, triazole pinning compounds synthesized from alkenyl and azide side chain residues, photocontrolled macrocycles, and the introduction of amino acids (e.g., α,α-disubstituted amino acids). Those skilled in the art will understand that a pinning compound can refer to a synthetic constraint (e.g., a support) between two previously independent entities. For example, a pinning compound can be formed by a covalent bond (e.g., the formation of, for example, a peptide macrocycle) between two previously independent entities (e.g., amino acid side chains).

[0455] This disclosure provides insights into engineering B7-H3 binding proteins with constraints (e.g., one, two, or three). In some embodiments, the constraints are identical (e.g., of the same type, such as disulfide bonds). In some embodiments, the constraints are combinations of different types of constraints (e.g., disulfide bridges and lactam bridges, disulfide bridges and pinned alkyl bridges, two disulfide bridges and one lactam bridge, etc.). In some embodiments, constraints may be added. For example, in some embodiments, the microprotein may have one, two, or three disulfide bridges; one or two disulfide bridges and another constraint (e.g., a lactam bridge); one or two constraints (e.g., a lactam bridge or a pinned alkyl bridge) and one disulfide bridge, etc. In some embodiments, the microproteins of this disclosure have one, two, or three disulfide bridges and / or one or more additional constraints (e.g., one disulfide bridge and one additional constraint, or two disulfide bridges and one additional constraint, wherein, for example, the additional constraint is a lactam bridge), forming a microprotein that not only binds to B7-H3 with strong affinity but is also taken up by cancer cells (e.g., in tumors) at concentrations higher than unconstrained B7-H3 microproteins (e.g., microproteins disclosed in Table 2A). This disclosure recognizes that properties such as size (e.g., fold size, e.g., linear size) and stability (e.g., thermal stability) are important for the efficacy of B7-H3 binding conjugates and are also important for limiting or preventing toxicity (e.g., nephrotoxicity) caused by off-target effects. Not wishing to be bound by theory, this disclosure provides the insight that engineering B7-H3-binding proteins to add constraints, and in some embodiments, adding such constraints after truncation (e.g., as described in Table 2C relative to Table 2A), yields microproteins with stronger affinity (e.g., for B7-H3), better efficacy (e.g., proximity to cancer cells), higher stability (e.g., thermal stability), and lower toxicity (e.g., nephrotoxicity) than molecules that are neither truncated nor constrained (e.g., as described in Table 2A). In some embodiments, the microproteins provided herein comprise one or more constraints, such as disulfide bridges or pinned compounds. Exemplary constrained microproteins are provided in Table 2C. In some embodiments, constraints can improve the thermal stability of the microprotein (e.g., compared to microproteins whose amino acid sequence comprises or consists of SEQ ID NO: 6, or compared to compound C8; e.g., compared to microproteins having the same or similar primary sequence but without constraints). In some embodiments, the microproteins of this disclosure comprise one or more disulfide bridges. In some embodiments, the microproteins of this disclosure comprise two or more disulfide bridges. In some embodiments, the microprotein of this disclosure comprises a disulfide bridge. In some embodiments, the microprotein of this disclosure comprises two disulfide bridges.For example, the microproteins of this disclosure may contain a group of amino acids that collectively support the formation of constraints (e.g., disulfide bridges, e.g., pinned compounds) or constitute part of a constraint. In some embodiments, the microprotein is structurally characterized by having at least two cysteine ​​residues positioned relative to each other to form disulfide bridges (e.g., see Table 2C, for example, as provided herein). In some embodiments, the microproteins provided in this disclosure have at least three or at least four cysteine ​​residues, thereby forming one or two disulfide bridges. In some embodiments, the microprotein has three disulfide bridges (from three pairs of cysteine ​​residues).

[0456] In some embodiments, the microproteins of this disclosure containing two or more cysteine ​​residues (such as those described in Table 2C) have cysteine ​​residues linked by disulfide bridges (e.g., by natural folding).

[0457] In some embodiments, the microprotein has an amino acid sequence comprising two cysteine ​​residues and a single disulfide bridge. In some embodiments, the microprotein has an amino acid sequence comprising four cysteine ​​residues and two disulfide bridges. In some embodiments, the microprotein has an amino acid sequence comprising six cysteine ​​residues and three disulfide bridges.

[0458] In some embodiments, the microproteins of this disclosure comprising two or more cysteine ​​residues (e.g., those described in Table 2C) have cysteine ​​residues linked by disulfide bridges (e.g., by spontaneous folding). In some embodiments, the microprotein (e.g., bound to B7-H3) comprises one disulfide bridge. In some embodiments, the microprotein comprises two disulfide bridges. In some embodiments, the microprotein comprises three disulfide bridges.

[0459] In some embodiments, a disulfide bridge may be located between cysteine ​​residues, which correspond to, for example, Cys4 and Cys37; Cys5 and Cys34; Cys5 and Cys37; Cys12 and Cys26; Cys12 and Cys44; Cys17 and Cys48, wherein the position is a linear position relative to the N-terminus to the C-terminus of reference SEQ ID NO: 267.

[0460] In some embodiments, cysteine ​​linkages may occur between two different pairs of cysteine ​​residues. For example, in some embodiments, referring to the microprotein of SEQ ID NO: 213, two disulfide bridges may be located between positions corresponding to cysteine ​​pairs (e.g., Cys1 and Cys35, and Cys17 and Cys52). For example, in some embodiments, disulfide bridges may be located between Cys1 and Cys35, and Cys17 and Cys48, corresponding to reference sequences such as those described in Table 2C (e.g., SEQ ID NO: 267). In some embodiments, cysteine ​​linkages are between Cys1 and Cys17; and between Cys35 and Cys48. In some embodiments, cysteine ​​linkages are between Cys1 and Cys48; and between Cys17 and Cys35.

[0461] In some embodiments, the one or more disulfide bridges comprise two or four cysteine ​​residues corresponding to positions 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, if four cysteines are present and correspond to positions 1, 17, 35 and 48 of SEQ ID NO: 267, the pairing may include pairing 1 with 35 and pairing 17 with 48; pairing 1 with 17 and pairing 35 with 48; or pairing 1 with 48 and pairing 17 with 35 (e.g., there is a disulfide bridge between the two paired cysteines).

[0462] In some embodiments, the cysteine ​​link (disulfide bridge) is located between positions corresponding to Cys1 and Cys35; and between positions corresponding to Cys17 and Cys48, such as those described in Table 2C (e.g., SEQ ID NO: 267). In some embodiments, the cysteine ​​link is between Cys1 and Cys17; and between Cys35 and Cys48 (refer to the reference sequence, e.g., SEQ ID NO: 267). In some embodiments, the cysteine ​​link is between Cys1 and Cys48; and between Cys17 and Cys35.

[0463] In some embodiments, the one or more disulfide bridges comprise two or four cysteine ​​residues corresponding to positions 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, if four cysteines are present and correspond to positions 1, 17, 35 and 48 of SEQ ID NO: 267, the pairing may include pairing 1 with 35 and pairing 17 with 48; pairing 1 with 17 and pairing 35 with 48; or pairing 1 with 48 and pairing 17 with 35 (e.g., there is a disulfide bridge between the two paired cysteines).

[0464] In some embodiments, the microprotein having two disulfide bridges (e.g., cysteine ​​residues from positions corresponding to, for example, Cys1, Cys17, Cys35, and Cys48 in SEQ ID NO: 267) also includes a third disulfide bridge introduced between a third pair of cysteine ​​residues. In some embodiments, cysteine ​​residues may also be introduced at positions corresponding to Cys11 and Cys45.

[0465] In some embodiments, such microproteins also include one or more additional constraints (e.g., pinning compounds, such as lactam bridges). For example, in some embodiments, a combination of constraint types (e.g., disulfide bonds and lactam bridges) may be used in a single microprotein. For example, in some embodiments, the microprotein may include one, two, or three disulfide bridges, and may also include at least one additional constraint (e.g., pinning compounds, such as lactam bridges). In some embodiments, the lactam bridge may be located between positions 11 and 45 corresponding to SEQ ID NO: 544.

[0466] In some embodiments, the microproteins of this disclosure that include changes such as, for example, one or more constraints (e.g., as disclosed herein) also comprise 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 sequences described in any of SEQ ID NO: 198-537. In each embodiment, the amino acid sequence is 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence described in any of SEQ ID NO: 198-537. In each embodiment, the amino acid sequence is 100% identical to the amino acid sequence described in any of SEQ ID NO: 198-537.

[0467] In some embodiments, the microprotein of this disclosure that includes, for example, changes to one or more constraints (e.g., as disclosed herein) is part of the compound. In some embodiments, the compound is selected from any of C227-C608 and C611, and / or has an amino acid sequence selected from any of SEQ ID NO: 198-537.

[0468] In some embodiments, the microproteins of this disclosure that include changes such as, for example, one or more constraints (e.g., as disclosed herein) also comprise 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 sequences described in any one of SEQ ID NO: 204 and 262-272. In each embodiment, the amino acid sequence is 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence described in any one of SEQ ID NO: 204 and 262-272. In each embodiment, the amino acid sequence is 100% identical to the amino acid sequence described in any one of SEQ ID NO: 204 and 262-272.

[0469] In some embodiments, the microprotein of this disclosure, which includes, for example, changes to one or more constraints (e.g., as disclosed herein), is part of the compound. In some embodiments, the compound is selected from any one of C234, C235, and C298-C332, and / or has an amino acid sequence selected from any one of SEQ ID NO: 204 or 262-272.

[0470] In some embodiments, the microprotein of this disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein) comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to the amino acid sequence described in any of SEQ ID NO: 199, 204, 241, or 267. In each embodiment, the amino acid sequence is 90% (e.g., 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence described in any one of SEQ ID NO: 199, 204, 241 or 267. In each embodiment, the amino acid sequence is 100% identical to the amino acid sequence described in any one of SEQ ID NO: 199, 204, 241 or 267.

[0471] In some embodiments, the microprotein of this disclosure, which includes, for example, changes to one or more constraints (e.g., as disclosed herein), is part of the compound. In some embodiments, the compound is selected from any of C228, C234, C235, C275, C309, C325, C332, or any other compound disclosed in Table 2C, having an amino acid sequence comprising any one of SEQ ID NO 199, 204, 241, or 267, and / or having an amino acid sequence selected from any one of SEQ ID NO 199, 204, 241, or 267.

[0472] In some embodiments, the microprotein of this disclosure, comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to the amino acid sequence described in SEQ ID NO: 199. In various embodiments, the amino acid sequence is 90% (e.g., 95%, 96%, 97%, 98%, 99%, or a greater percentage) identical to the amino acid sequence described in SEQ ID NO: 199. In each embodiment, the amino acid sequence is 100% identical to the amino acid sequence described in SEQ ID NO 199.

[0473] In some implementations, it includes, for example, one or more constraints (e.g.) ， The microprotein of this disclosure (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.

[0474] In some embodiments, the microprotein of this disclosure comprising changes such as, for example, one or more constraints (e.g., as disclosed herein) comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to the amino acid sequence described in SEQ ID NO: 204. In various embodiments, the amino acid sequence is 90% (e.g., 95%, 96%, 97%, 98%, 99%, or a greater percentage) identical to the amino acid sequence described in SEQ ID NO: 204. In each embodiment, the amino acid sequence is 100% identical to the amino acid sequence described in SEQ ID NO 204.

[0475] In some embodiments, the microprotein of this disclosure, which includes, for example, changes to one or more constraints (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.

[0476] In some embodiments, the microprotein of this disclosure, comprising changes such as, for example, one or more constraints (e.g., as disclosed herein), comprises an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to the amino acid sequence described in SEQ ID NO: 241. In various embodiments, the amino acid sequence is 90% (e.g., 95%, 96%, 97%, 98%, 99%, or a greater percentage) identical to the amino acid sequence described in SEQ ID NO: 241. In each embodiment, the amino acid sequence is 100% identical to the amino acid sequence described in SEQ ID NO 241.

[0477] In som...

Claims

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 position 3 corresponding to amino acid 3, lysine at position 5 corresponding to amino acid 5, isoleucine at position 6 corresponding to amino acid 6, tryptophan at position 14 corresponding to amino acid 14, at least one modified lysine residue at position 24 corresponding to amino acid 24, and alanine, arginine, or modified lysine at position 29, wherein each position is linear from the N-terminus to the C-terminus relative to SEQ ID NO: 267, starting from position 1, wherein the modification comprises at least one small alkyl group of nitrogen attached to the lysine side chain or of guanidine nitrogen attached to the arginine side chain, wherein the at least one small alkyl group optionally comprises methyl, dimethyl, or trimethyl. (c) The length is at least 48 amino acids; and (d) As measured in cell-based assays, it has a binding affinity for B7-H3 stronger than 100 nM.

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

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

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

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

6. The composition of any one of claims 1-5, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NO: 204 and 262-272.

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

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

9. A composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to Formula III (SEQ ID NO: 538): CAX3EKIAALSEIIWLPCLX19YAQIX24AFIX28X29LNX32DPCX36SX38X39ILSEAX45ELCS, wherein X3 is (Kme3) or R or (Rme); X19 is T or N; X24 is (Kme) or (Kme2); X28 is A or (Kme); X29 is (Kme) or A or R; X32 is (Kme) or D or (Cit); X36 is Q or N; X38 is S or A; X39 is E or N; and X45 is K or (Kme).

10. The composition of any one of claims 1-9, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NO: 204 and 262-272.

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

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

13. The composition of claim 12, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NO: 204, 262, 265, 267 and 268.

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

15. A composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to the following formula V (SEQ ID 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.

16. The composition of claim 15, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NO: 204, 262, 265, 266, 267, 270 and 272.

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

18. A composition comprising a polypeptide having an amino acid sequence comprising an amino acid sequence according to formula VI (SEQ ID NO: 541): CA(Kme3)EKIAALSEIIWLPCLTYAQIX24AFIAX29LNX32DPCQSSEILSEAX45ELCS, wherein X24 is (Kme) or (Kme2); X29 is (Kme), A or R; X32 is (Kme) or D or (Cit); and X45 is (Kme) or K.

19. The composition of claim 18, wherein the polypeptide has an amino acid sequence comprising any one of SEQ ID NO: 204, 262, 265, 267, 270 and 272.

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

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

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

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

24. The composition of any of the preceding claims, wherein the polypeptide has a length of at least 48 amino acids but not more than 100 amino acids.

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

26. The composition of any of the preceding claims, wherein the amino acid sequence of the polypeptide shares at least 90% identity with any one of SEQ ID NO: 204 and 262-537, and comprises at least one lysine or arginine having at least one modification, the modification comprising at least one small alkyl group of nitrogen bonded to the lysine side chain or of guanidine nitrogen bonded to the arginine side chain, the small alkyl group optionally selected from: trimethyl, dimethyl and monomethyl.

27. The composition of any of the preceding claims, wherein the amino acid sequence of the polypeptide shares at least 90% identity with at least 44 amino acids of a reference polypeptide, the reference polypeptide being longer than 47 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 not less than 10 nM.

28. The composition of any of the preceding claims, 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 NO: 198-537, provided that the 44 amino acids include at least four cysteine ​​residues forming at least two disulfide bridges.

29. The composition of any of the preceding claims, wherein the amino acid sequence of the polypeptide has 100% identity with at least 44 amino acids of a reference polypeptide, the reference polypeptide being longer than 47 amino acids.

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

31. The composition of any of the preceding claims, wherein the amino acid sequence is 100% identical with at least 44 amino acids as described in any one of SEQ ID NO: 199, 204, 241 or 262-272.

32. The composition of claim 30 or 31, wherein the amino acid sequence comprises at least four cysteine ​​residues having at least two disulfide bridges.

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

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

267.

35. A composition comprising compounds described in Table 2C, namely C234, C235 and C298-C333.

36. A composition comprising a polypeptide having an amino acid sequence comprising any one of SEQ ID NO: 204 and 262-272.

37. The composition according to any one of claims 1-36, further comprising a radionuclide.

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

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

40. The composition as claimed in any of the preceding claims, wherein the binding affinity for B7-H3 is greater than 100 nM.

41. The composition of any of the preceding claims, wherein the inhibition constant is not weaker than 100 nM.

42. The composition as described in any of the preceding claims further comprises one or more of a connector, a chelating agent, and a radionuclide.

43. The composition of claim 42, wherein the connector comprises or is composed of: Polyethylene glycol (PEG) connectors PEG4, PEG2, PEG, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is 1 to 36, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

44. The composition of 42 or 43, wherein the chelating agent comprises or is composed of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

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

46. ​​The composition of any one of claims 42-45, wherein if the polypeptide comprises any one of SEQ ID NO: 198-537, the polypeptide further comprises a linker, wherein the linker is PEG4, and optionally a chelating agent, wherein the chelating agent is DOTA.

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

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

49. The composition according to any one of claims 42-48, wherein, When present, the chelating agent is attached to the polypeptide or the linker.

50. The composition according to any one of claims 41-48, wherein, In the presence of the radionuclide, the radionuclide is attached to the chelating agent.

51. A composition comprising one or more of the formula selected from (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 chelating agent (C), R comprises a radionuclide (R), and x is 1, 2, 3 or 4, wherein M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NO: 198-537.

52. The composition of claim 51, wherein M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 198-537.

53. The composition of claim 51 or 52, wherein 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 NO: 198-537.

54. The composition of any one of claims 51-53, wherein M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NO: 198-537.

55. The composition of any one of claims 51-54, wherein the connector comprises or is composed of: Polyethylene glycol (PEG) connectors PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- or (PEG)n, wherein n is 1 to 36, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

56. The composition of any one of claims 51-55, wherein the chelating agent comprises or is composed of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

57. The composition according to any one of claims 51-56, 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.

58. A composition comprising one or more of the formula selected from (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 chelating agent (C), R comprises a radionuclide (R), and x is 1, 2, 3 or 4, wherein M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with at least 44 amino acids of any one of SEQ ID NO: 204 and 262-272.

59. The composition of claim 58, wherein M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 204 and 262-272.

60. The composition of claim 58 or 59, wherein 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 NO: 204 and 262-272.

61. The composition of any one of claims 58-60, wherein M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NO: 204 and 262-272.

62. The composition according to any one of claims 58-61, wherein, When L is present, L comprises or consists of: polyethylene glycol (PEG) connectors PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36, ester connectors, amide connectors, maleimide connectors, valine-citrulline connectors, hydrazone connectors, 4-(2-pyridyldithio)butyrate N-succinimide ester (SPDB) connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylate succinimide ester (SMCC) connectors, vinyl sulfone-based connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- or (PEG)n, where n is 1 to 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

63. The composition according to any one of claims 58-62, wherein, When C is present, C contains or is composed of the following: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

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

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

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

67. The composition according to any one of claims 58-66, wherein, When present, the chelating agent is attached to the polypeptide or the linker.

68. The composition according to any one of claims 58-67, wherein, In the presence of the radionuclide, the radionuclide is attached to the chelating agent.

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

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

71. The composition as claimed in any of the preceding claims, wherein the composition and / or its polypeptides selectively bind to B7-H3.

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

73. The composition of any one of the preceding claims, wherein the binding inhibition constant of the polypeptide is not less than 100 nM.

74. A composition comprising a polypeptide-drug conjugate, the polypeptide-drug conjugate comprising a polypeptide and at least one pharmaceutical 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 having any one of the amino acid sequences described in SEQ ID NO: 198-537.

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

76. The composition of claim 74 or 75, wherein the pharmaceutical portion is selected from topoisomerase inhibitors, orrisstatin (e.g., monomethylorrisstatin E), V-ATPase inhibitors, pro-apoptotic agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, saccharidin, maytansin-like substances, MetAP (methionine aminopeptidase), protein CRM1 nuclear export inhibitors, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphoryl transfer reactions in mitochondria, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binding agents, DHFR inhibitors, and immunotoxins.

77. A composition comprising an isolated compound or a pharmaceutically acceptable salt or neutral molecule thereof, comprising optionally a linker (L) and one or more of a polypeptide (M), a chelating agent (C) or a radionuclide (R), wherein M has an amino acid sequence comprising at least 90% identity of an amino acid sequence comprising at least 44 amino acids of any one of SEQ ID NO: 198-537 and 538-543 and 546-550, including amino acid substitutions as described in Table 1E.

78. A composition comprising a compound designed to bind to B7-H3, said compound comprising or consisting of a polypeptide having an amino acid sequence comprising an amino acid sequence having at least 90% identity with an amino acid sequence of at least 44 amino acids of any one of SEQ ID NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E, and further comprising a modified N-terminus and / or C-terminus.

79. The composition of claim 77 or 78, wherein 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 NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E.

80. The composition of any one of claims 77-79, wherein M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E.

81. The composition of any one of claims 77-80, wherein M has an amino acid sequence comprising an amino acid sequence having 100% identity with at least 44 amino acids of any one of SEQ ID NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E.

82. The composition of any one of claims 77-81, wherein M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NO: 198-537 or 538-543 and 546-550, including amino acid substitutions as described in Table 1E.

83. The composition of any one of claims 78-82, wherein the modified N-terminus comprises one or more of the following: NH2-, acetyl-, PEGn- where n=0-36, DOTA-, or biotin-.

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

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

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

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

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

267.

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

267.

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

267.

91. The compound according to any one of claims 87-90, wherein L comprises or consists of: Polyethylene glycol (PEG) connectors PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, any connectors described 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 thereof or combination thereof linked by covalent bonds.

92. The compound according to any one of claims 87-91, wherein C comprises or consists of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

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

94. A compound comprising a microprotein having at least 90% identity with at least 48 amino acids of the amino acid sequence of SEQ ID NO: 267, wherein the N-terminus and / or C-terminus contains one to thirty additional amino acids, and / or wherein the C-terminus contains at least one amino acid or at most 30 additional amino acids, provided that the length of the entire microprotein is not greater than about 100 amino acids.

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

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

97. A method of treating cancer, the method comprising administering to a subject in need a composition comprising a conjugate, the 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 NO: 198-537; and a radionuclide.

98. The method of claim 97, wherein the polypeptide has at least four cysteine ​​residues and two disulfide bridges.

99. The method of claim 97 or 98, wherein, according to formula MLCR, the radionuclide is associated with the polypeptide using a linker and / or a chelating agent, wherein M is the polypeptide, L is the linker, C is the chelating agent, and R is the radionuclide.

100. The method of any one of claims 97-99, wherein the polypeptide has an amino acid sequence comprising or consisting of any one of SEQ ID NO: 204 or 262-272.

101. The method of claim 99 or 100, wherein L comprises or consists of: Polyethylene glycol (PEG) connectors PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, any connectors described in Table 2A or (Gly)n-(gGlu)n- or (PEG)n, where n is 1 to 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

102. The method of any one of claims 99-101, wherein C comprises or consists of: DOTA, Crown, NOPO, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

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

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

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

106. The method of any one of claims 96-105, wherein the length of the polypeptide does not exceed 100 amino acids.

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

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

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

110. A method for improving the biodistribution of a pharmaceutical composition of a population of B7-H3 positive cancer cells in a subject with B7-H3 positive cancer, comprising contacting the population with a polypeptide having a modified lysine at position X24 corresponding to SEQ ID NO: 241, wherein the lysine is modified by adding at least one small alkyl group to the lysine side chain, and wherein the biodistribution is improved compared to contacting the population with a polypeptide that does not contain the modified lysine at position X24 corresponding to SEQ ID NO:

241.

111. A method for diagnosing the presence of a B7-H3 positive cancer cell population, comprising: (a) Contacting a cell population with the composition of any one of claims 1-86, the compound of any one of claims 87-94, or the pharmaceutical composition of claim 95; (b) Detect the presence of the composition, compound, or pharmaceutical composition of step (a) by measuring the signal; as well as (c) Compare the detection result from step (b) with the control signal; and (d) If the composition, compound, or pharmaceutical composition of step (a) is detected to be higher than the control signal, cancer is diagnosed.

112. The method of claim 111, wherein the contact is performed by applying it to a subject in need.

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

114. The method of claim 111, wherein the contact is performed in vitro on the subject, optionally using a biopsy sample.

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

116. Use of the composition of any one of claims 1-86, the compound of any one of claims 87-94, or the pharmaceutical composition of claim 95 for the treatment of a subject with cancer.

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

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

119. The method of claim 118, wherein the cancer cells from the subject express B7-H3.

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

121. The method of claim 120, wherein the non-cancer cells are obtained from the subject.

122. The method of any one of claims 117-122, wherein the composition, compound, or pharmaceutical composition is internalized in cells expressing human B7-H3.

123. The method of any one of claims 117-122, wherein the cancer is selected from breast cancer, ovarian cancer, melanoma, pancreatic cancer, peripheral neuroma, glioblastoma, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, urothelial carcinoma, bladder cancer, meningioma, glioma, astrocytoma, cervical cancer, chronic myeloproliferative disorder, colon cancer, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gestational trophoblastoma, hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip cancer, oral cancer, liver cancer, male breast cancer, malignant mesothelioma, medulloblastoma. Plasma cell carcinoma, Merkel cell carcinoma, metastatic squamous cell carcinoma of the neck, multiple myeloma and other plasmacytomas, mycosis fungoides and Cezari syndrome, myelodyplasia syndrome, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, skin cancer, oropharyngeal cancer, bone cancer including osteosarcoma and malignant fibrous histiocytoma of bone, paranasal sinus cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary adenoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small bowel 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 kidney tumors.

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

125. A method for treating cancer cells expressing B7-H3, the method comprising: (i) The expression level of B7-H3 in cancer cell populations has been determined or is known; and (ii) administering to a subject in need a composition comprising any one of claims 1-86, any one of claims 87-94, 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, wherein the composition, compound, or pharmaceutical composition is linked to the surface of one or more B7-H3-expressing cancer cells and / or internalized into the cancer cells.

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

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

128. The method of any one of claims 125-127, wherein the subject receives treatment after the administration, compared to receiving treatment before the administration.

129. In a method of targeting a population of cancer cells expressing B7-H3, the improvement comprises contacting the population with the composition of any one of claims 1-86, the compound of any one of claims 87-94, or the pharmaceutical composition of claim 95, wherein the position corresponding to X24 (relative to SEQ ID NO: 241) contains a lysine residue having at least one nitrogen-linked side chain, wherein less of the composition is taken up by renal cells than a composition containing a polypeptide having a small alkyl group on the side chain without nitrogen linked to the lysine residue at X24, wherein, Optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group.

130. A conjugate comprising: (i) A polypeptide (M) that specifically binds to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) A radionuclide (R) chelated with (C), wherein (R) is actinium-225.

131. A conjugate comprising: (i) A polypeptide (M) that specifically binds to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) A radionuclide (R) chelated with (C), wherein (R) is copper-64.

132. A conjugate comprising: (i) A polypeptide (M) that specifically binds to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) A radionuclide (R) chelated with (C), wherein (R) is gallium-68.

133. A conjugate comprising: (i) A polypeptide (M) that specifically binds to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) A radionuclide (R) chelated with (C), wherein (R) is indium-111.

134. A conjugate comprising: (i) A polypeptide (M) that specifically binds to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) A radionuclide (R) chelated with (C), wherein (R) is lead-212.

135. A conjugate comprising: (i) A polypeptide (M) that specifically binds to B7-H3; (ii) a chelating agent (C) conjugated to (M) via an optional linker (L), wherein (C) comprises DOTA and (L) comprises PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) A radionuclide (R) chelated with (C), wherein (R) is lutetium-177.

136. A conjugate comprising: (i) A microprotein (M) that specifically binds to B7-H3; (ii) N-terminal modification, which is attached to (M) via an optional connector (L), wherein (L) contains PEG when present, wherein the PEG is optionally PEG-4; and (iii) N-terminal modification containing biotin.

137. The conjugate according to any one of claims 130-136, wherein 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 NO: 198-537.

138. The conjugate according to any one of claims 130-137, wherein M has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 198-537.

139. The conjugate according to any one of claims 130-138, wherein 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 NO: 198-537.

140. The conjugate according to any one of claims 130-139, wherein M has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NO: 198-537.

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

142. The conjugate of claim 141, wherein the amino acid sequence comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 204 or 262-272.

143. The conjugate of 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 NO: 204 or 262-272.

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

267.

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

267.

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

267.

147. The conjugate according to any one of claims 130-141, wherein the amino acid sequence comprises or consists of any one of SEQ ID NO: 204 and 262-272.

148. The conjugate according to any one of claims 130-142, wherein the amino acid sequence comprises or consists of SEQ ID NO:

267.

149. An isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from any one of SEQ ID NO: 198-537; 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 NO: 198-573.

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

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

152. A method for assessing the location of one or more cancer cell populations in a subject, the method comprising administering to the subject a composition of any one of claims 1-86, a compound of any one of claims 87-94, a pharmaceutical composition of claim 95, or a conjugate of any one of claims 130-148, and detection to determine the location of the composition in the subject, wherein the composition, compound, or pharmaceutical composition comprises a detectable marker.

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

154. In a method of reducing renal uptake of a composition administered to detect and / or treat one or more cancer cell populations, the improvement comprises administering to a subject in need the composition of any one of claims 1-86, the compound of any one of claims 87-94, the pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148, wherein the position corresponding to X24 of SEQ ID NO: 241 contains a lysine having at least one additional small alkyl group attached to a nitrogen atom on a side chain, wherein less of the composition is taken up by renal cells than a composition containing a polypeptide having a small alkyl group on the side chain without a nitrogen atom attached to a lysine at the position corresponding to X24 of SEQ ID NO: 241, wherein, Optionally, the small alkyl group is part of a monomethyl, dimethyl, or trimethyl group.

155. The method of claim 152 or 154, wherein the detection includes an imaging procedure that allows selection of subjects, monitoring of subjects, and / or treatment of subjects with a therapeutic agent comprising a microprotein, said microprotein being designed to bind to B7-H3 expressed on one or more cancer cells in one or more cancer cell populations.

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

157. An improved method of radionuclide delivery to a cancer cell population in a subject, the method comprising administering a composition of any one of claims 1-86, a compound of any one of claims 87-94, a pharmaceutical composition of claim 95, or a conjugate of any one of claims 130-148, wherein the amino acid sequence of the polypeptide comprises an amino acid at position X24 corresponding to SEQ ID NO: 241, which comprises an additional small alkyl group having at least one nitrogen attached to a side chain, and wherein renal cell uptake is less than that of a polypeptide having a small alkyl group on the side chain without nitrogen attached to the lysine at position X24 corresponding to SEQ ID NO:

241.

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

159. In a method of treating an individual suffering from cancer, the improvement comprises reducing one or more off-target effects or toxicity measures by administering the composition of any one of claims 1-86, the compound of any one of claims 87-94, the pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148, wherein the amino acid sequence of the polypeptide comprises an amino acid at position X24 corresponding to SEQ ID NO: 241 containing an additional small alkyl group having at least one nitrogen attached to a side chain, wherein the renal cells take up less of the polypeptide than a polypeptide without a small alkyl group having nitrogen attached to the side chain at position X24 corresponding to SEQ ID NO:

241.

160. In a method of treating an individual suffering from cancer, the improvement comprises reducing the concentration of R in the renal tissue in the presence of the composition, compound, pharmaceutical composition, or conjugate, compared to the concentration of R in the renal tissue in the absence of the composition of any one of claims 1-86, the compound of any one of claims 87-94, the pharmaceutical composition of claim 95, or the conjugate of any one of claims 130-148, wherein the amino acid sequence of the polypeptide comprises an amino acid corresponding to position X24 of SEQ ID NO: 241, and wherein X24 comprises a lysine having at least one additional small alkyl group attached to a nitrogen atom on the side chain, wherein the renal cells take up less of the polypeptide than a polypeptide having an amino acid sequence that does not contain a small alkyl group attached to a nitrogen atom on the side chain at position X24 of SEQ ID NO:

241.

161. The method of claim 160, wherein the decrease in the concentration of R in the kidney tissue is measured by means of: the amount of R excreted in urine as measured by the percentage of the applied radiation recovered, or by means of a detection result measured by a cell-based in vitro assay or in vivo assay.

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

163. In a method for reducing the uptake of a composition by renal tissue, the improvement includes administering a composition comprising (a) 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 position X24 corresponding to SEQ ID NO: 241, such that in the presence of the modified lysine, the concentration of the radionuclide in the renal tissue is less than the concentration in the absence of the polypeptide.

164. A method comprising administering to a subject in need a compound that binds to B7-H3 and comprises at least one modified lysine at position X24 corresponding to SEQ ID NO:241, wherein administration of the compound having a microprotein containing said at least one modified lysine reduces one or more off-target effects, toxicity levels, and / or uptake and / or retention in renal tissue compared to a compound that does not contain a modified lysine at position X24 (e.g., unmodified lysine, such as L, V, or Q).

165. The method of 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 NO: 204 and 262-272.

166. The method of any one of claims 163-165, wherein the polypeptide has an amino acid sequence comprising an amino acid sequence having at least 90% identity with any one of SEQ ID NO:204 and 262-272.

167. The method of any one of claims 163-166, wherein the polypeptide has an amino acid sequence comprising an amino acid sequence having 100% identity with any one of SEQ ID NO:204 and 262-272.

168. A method of treating an individual who has or is suspected of having B7-H3 positive cancer, the method comprising administering to the individual: (a) Means for blocking the uptake and / or retention of radiotherapy agents in renal tissue, and (b) Connectors, chelating agents and radionuclides.

169. The method of claim 168, wherein the means for blocking renal tissue uptake and / or retention of the radiotherapy agent is bound to B7-H3 and includes a modified lysine at position X24 corresponding to 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 position X24 corresponding to SEQ ID NO:

267.

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

267.

171. The method of any one of claims 168-170, wherein the means for blocking the uptake and / or retention of the radiotherapy agent in the renal tissue has a greater effect on the blocking of the uptake and / or retention of the renal tissue than means for not including a modified lysine at position X24 of SEQ ID NO: 267 and / or having at least 90% identity with at least 44 amino acids of SEQ ID NO: 267 and / or having a modified lysine at position X24 of SEQ ID NO:

267.

172. The method of any one of claims 168-171, wherein the means for blocking the uptake and / or retention of the radiotherapy agent in the renal tissue has a greater effect on the blocking of the uptake and / or retention of the renal tissue than means for omitting a modified lysine at position X24 of SEQ ID NO: 267 and / or having at least 90% identity with at least 44 amino acids of SEQ ID NO: 267 and / or having a modified lysine at position X24 of SEQ ID NO:

267.

173. The method of any one of claims 168-172, wherein the means for blocking the uptake and / or retention of the radiotherapy agent by the renal tissue is a radiotherapy agent.

174. The method of claim 173, wherein the radiotherapy agent targets a tumor or a population of cancer cells.

175. The method of claim 174, wherein the concentration of the radiotherapy agent targeting the tumor or the cancer cell population is greater than the concentration when the means of binding to the kidney tissue are absent.

176. The method of claim 175, wherein the radiotherapy agent comprises a polypeptide targeting B7-H3.

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

178. The method of claim 177, wherein the radionuclide of the radiotherapy agent is selected from Ac-225, Cu-64, Ga-68, In-111, Lu-177 or Pb-212.

179. A kit comprising a polypeptide and instructions for use, wherein the polypeptide has the amino acid sequence described in any one of the compositions of any one of claims 1-86, the compounds of any one of claims 87-94, the pharmaceutical compositions of claim 95, or the conjugates of any one of claims 130-148.

180. The kit of claim 179, further comprising one or more of a connector, a chelating agent, and a radionuclide.

181. The kit of claim 180, wherein the connector comprises or is composed of: Polyethylene glycol (PEG) connectors PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, PEG36, lys(MPB)-PEG4, ester connectors, amide connectors, maleimide connectors, 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or (Gly)n-(gGlu)n- or (PEG)n, where n is 1 to 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

182. The kit of claim 180 or 181, wherein the chelating agent comprises or is composed of: DOTA, NOPO, Crown, Macropa, lead-specific chelating agent (PSC), 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

183. The kit according to any one of claims 179-182, wherein the compound is labeled with a radionuclide prior to use, wherein the radionuclide is chelated to the chelating agent to produce a composition having the formula MLCR.

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

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

186. The kit of any one of claims 179-185, wherein the polypeptide has an amino acid comprising any one of the sequences described in any one of SEQ ID NO: 199, 204, 241 or 262-272, and wherein the polypeptide further comprises a linker and / or a chelating agent, wherein the linker is PEG4 in the presence, and the chelating agent is DOTA in the presence.

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

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

189. The kit according to any one of claims 180-188, wherein, in the presence, the chelating agent is linked to the polypeptide or the linker.

190. The kit according to any one of claims 180-189, wherein, in the presence, the radionuclide is linked to the chelating agent.

191. The kit according to any one of claims 180-190, wherein, in the presence, the radionuclide is linked to the N-terminal amino acid of the polypeptide.

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