Microproteins, conjugates thereof and uses thereof

Abstract

Provided herein are polypeptides and conjugates thereof, including radionuclide conjugates, that can be used in compositions and methods for treating, diagnosing, monitoring, and / or imaging diseases, disorders, or conditions associated with expression of one or more targets, including Nectin-4.

Description

Cross-references to related applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 587,042, filed September 29, 2023; No. 63 / 598,874, filed November 14, 2023; No. 63 / 618,228, filed January 5, 2024; and No. 63 / 636,078, filed April 18, 2024, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0002] sequence list This application includes a sequence list, which is electronically submitted in XML format and is hereby incorporated herein by reference in its entirety. The XML file was created on September 26, 2024, named AKT-031WO_SL.xml, and has a size of 488,567 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-cancer 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 targeted therapeutic agents to deliver therapeutic agents containing 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.

[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 irradiation of 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 components; however, full-length antibodies, and even antibody fragments (e.g., fragments larger than the peptides presented herein), may present several challenges due to factors such as poor tumor tissue penetration, long circulating half-lives leading to irradiation of normal tissue, and challenges in manufacturing and storage. Therefore, new methods remain needed to specifically target tumors, particularly solid tumors. This disclosure provides techniques to meet this need, as well as other needs. Among other things, this disclosure also provides compositions comprising peptides and conjugates thereof that, compared with prior art (e.g., antibodies, such as antibody-drug conjugates, peptides, etc.), have improved tumor penetration, reduced off-target toxicity and / or accumulation, and increased affinity for Nectin-4. Furthermore, this disclosure provides insights into even further improvements to the compositions and conjugates provided herein. It is contemplated herein that, in some embodiments, the conjugates of this disclosure (e.g., radionuclide conjugates) can be improved, for example, by modifying one or more amino acids in the peptide sequence of the conjugate and / or adding one or more decoys, for example, in terms of efficacy and / or reduction in toxicity level and / or one or more measures of off-target effects.

[0006] In some aspects, this disclosure provides polypeptides comprising certain amino acid sequences. In some embodiments, these polypeptides bind to Nectin-4 with a certain affinity.

[0007] In one aspect, this disclosure provides a composition comprising a polypeptide having a length of at least 44 amino acids and having an amino acid sequence comprising the sequence described in SEQ ID NO: 171, wherein X2 is E or D; X6 is E or Q; X17 is G or A; X21 is Q, Y, or E; X26 is Kme3, Kme2, Kme, K, Kipr, or S; X32 is A, G, or D; X41 is N or K; and X45 is S or absent.

[0008] In one aspect, this disclosure provides a composition comprising a polypeptide having a length of at least 44 amino acids and having an amino acid sequence comprising the sequence described in SEQ ID NO: 176, wherein X2 is E or D; X6 is E or Q; X9 is T or A; X10 is A or G; X12 is A, Kme3, Kme2, Kme, Kipr, or K; X13 is R or (Cit); X17 is G or A; X21 is Q, Y, or E; X24 is Q or K; X25 is A or K; X26 is Kme3, Kme2, Kme, K, Kipr, or S; X28 is Q or K; X29 is Y or K; X30 is L or V; X32 is A, G, or D; X41 is N or K; and X45 is S or absent.

[0009] In another aspect, this disclosure provides a composition comprising a Nectin-4 binding polypeptide having an amino acid sequence comprising at least 44 amino acids, wherein the amino acids include: (i) cysteine ​​at each of four positions corresponding to positions 1, 20, 34, and 44 of SEQ ID NO: 195; (ii) SEQ ID NO: 169 at positions 9-15 of SEQ ID NO: 195; (iii) QKKme3 at positions 24, 25, and 26 of SEQ ID NO: 195; and (iv) QYL at positions 28, 29, and 30 of SEQ ID NO: 195.

[0010] In one aspect, this disclosure provides a composition comprising a Nectin-4 binding polypeptide having an amino acid sequence comprising at least 44 amino acids, wherein said amino acids comprise: (i) cysteine ​​at each of four positions corresponding to positions 1, 20, 34, and 44 of SEQ ID NO: 200; (ii) SEQ ID NO: 247 at positions 9-15 of SEQ ID NO: 200; (iii) QKKme3 at positions 24, 25, and 26 of SEQ ID NO: 200; and (iv) QYL at positions 28, 29, and 30 of SEQ ID NO: 200.

[0011] In another aspect, this disclosure provides a composition comprising a Nectin-4 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at least four cysteine ​​residues forming two disulfide bonds; at least one modified lysine residue at position X12 and / or X26 of SEQ ID NO: 195, wherein the modification comprises at least one small alkyl group of nitrogen linked to the lysine side chain, optionally including methyl, dimethyl, trimethyl, or isopropyl; a length of at least 44 amino acids; and a binding affinity to Nectin-4 greater than 100 nM in a cell-based assay.

[0012] In some implementations, the polypeptide is at least 40 amino acids long, but no more than 100 amino acids long.

[0013] In some implementations, in cell-based assays, the peptide binds to Nectin-4 with an affinity greater than 10 nM.

[0014] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with any one of SEQ ID NO: 3-158, 161-168, 177-208 or 212-215, but includes at least one lysine with at least one modification, said modification including at least one small alkyl group bonded to a nitrogen atom in the side chain, optionally selected from: trimethyl, dimethyl, monomethyl and isopropyl.

[0015] 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, the reference polypeptide being longer than 44 amino acids, and binding to Nectin-4 with an intensity of at least 10 nM in cell-based assays, and / or having an inhibition constant of no more than 10 nM.

[0016] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with at least 40 amino acids of any one of SEQ ID NO: 3-158, 161-168, 177-208 or 212-215, provided that the 40 amino acids include at least four cysteine ​​residues that form two disulfide bridges.

[0017] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with at least 35 adjacent amino acids of any one of SEQ ID NO: 3-158, 161-168, 177-208 or 212-215, provided that the 40 amino acids include at least four cysteine ​​residues that form two disulfide bridges.

[0018] In some embodiments, the amino acid sequence of the polypeptide shares 100% identity with at least 44 amino acids of a reference polypeptide, the reference polypeptide being longer than 44 amino acids.

[0019] In some embodiments, the amino acid sequence shares 90% identity with at least 44 amino acids described in any one of SEQ ID NO: 78, 83, 85, 99, 103, 162-168, 195 or 200.

[0020] 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: 78, 83, 85, 99, 103, 162-168, 195 or 200.

[0021] In one aspect, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO: 195.

[0022] In one aspect, this disclosure provides a composition comprising a compound as described in C251 of Table 2A, the compound having an amino acid sequence comprising SEQ ID NO: 195.

[0023] In one aspect, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO: 200.

[0024] In one aspect, this disclosure provides a composition comprising a compound as described in C260 of Table 2A, the compound having an amino acid sequence comprising SEQ ID NO: 200.

[0025] In some embodiments, the composition also contains a radionuclide.

[0026] In some implementations, 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 a polypeptide having an amino acid sequence of at least 44 amino acids but having four amino acid substitutions at positions 12, 21, 26 and 32 corresponding to SEQ ID NO: 78, wherein the substitutions correspond to K12A, Y21Q, S26Kme3 and G32A.

[0028] In some implementations, the C-terminus has -OH or -NH2.

[0029] In some implementations, the binding affinity to Nectin-4 is stronger than 100 nM.

[0030] In some implementations, the suppression constant is no greater than 100 nM.

[0031] In some embodiments, the composition further comprises one or more of a connector, a chelating agent, and a radionuclide.

[0032] In some embodiments, the connector comprises or consists 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 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

[0033] 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).

[0034] 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.

[0035] In some embodiments, if the polypeptide comprises any one of SEQ ID NO: 83, 85, 93, 99, 134, 138, 145, 155, 162-168 or 195, the polypeptide further comprises a linker, wherein the linker is PEG4, and an optional chelating agent, wherein the chelating agent is DOTA.

[0036] 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, the 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.

[0037] 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 comprises an amino acid sequence of any one of SEQ ID NO: 162-176, 178-208, or 212-215.

[0038] 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- or (PEG)n, wherein n is 1 to 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

[0039] 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).

[0040] 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.

[0041] 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 any one of the sequences described in SEQ ID NO: 162-176, 178-208, or 212-215.

[0042] 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-carboxylic acid 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.

[0043] 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).

[0044] 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.

[0045] 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, the 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.

[0046] In some implementations, the polypeptide contains at least one disulfide bridge.

[0047] In some implementations, the polypeptide contains at least two disulfide bridges.

[0048] In some embodiments, the composition and / or its peptides selectively bind to Nectin-4 or a portion thereof.

[0049] In some embodiments, the binding affinity of the peptide to Nectin-4 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.

[0050] In some implementations, the binding inhibition constant of the peptide is no greater than 100 nM.

[0051] 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 any of the amino acid sequences described in SEQ ID NO: 3-158, 162-208 or 212-237.

[0052] In some embodiments, the pharmaceutical ingredient is selected from V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, orrisstatin, 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, topoisomerase inhibitors, orrisstatin (e.g., monomethylorrisstatin E), and immunotoxins.

[0053] In one aspect, this disclosure provides a composition comprising an isolated compound or a pharmaceutically acceptable salt 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: 3-158, 161-168, 171-208, 212-215, or 216-237, including amino acid substitutions as described in Tables 1C, 1D, 2C, 2D, 2E, 2F, or 2G.

[0054] In another aspect, this disclosure provides a composition comprising a compound designed to bind to Nectin-4, the compound comprising or consisting of a polypeptide having an amino acid sequence comprising any one of SEQ ID NO: 3-158, 161-168, 171-208, 212-215 or 216-237, including amino acid substitutions as described in Tables 1C, 1D, 2C, 2D, 2E, 2F or 2G, and further comprising a modified N-terminus and / or C-terminus.

[0055] In some embodiments, the modified N-terminus comprises one or more of the following: NH2-, acetyl-, PEGn- (where n = 0-10), DOTA-, or biotin-. In some embodiments, the C-terminus comprises -NH2 or -OH. In some embodiments, the peptide selectively binds to Nectin-4 or a portion thereof. In some embodiments, the peptide exhibits a binding affinity to Nectin-4 or a portion thereof greater than about 100 nM, either in vivo or in cell-based assays.

[0056] In one aspect, this disclosure provides a compound comprising a microprotein having an amino acid sequence having 90% identity with SEQ ID NO: 195, and further comprising one or more additional components according to formula MLCR, wherein L is a linker, C is a chelating agent, and R is a radionuclide.

[0057] 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 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.

[0058] In one aspect, this disclosure provides a compound comprising a microprotein comprising at least 40 amino acids that are 90% identical to the amino acid sequence of SEQ ID NO: 195, wherein the N-terminus and / or C-terminus comprises one to thirty additional amino acids, and / or wherein the C-terminus comprises 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.

[0059] In one aspect, this disclosure provides a method for improving the binding affinity strength of a peptide to Nectin-4, the improvement comprising modifying four amino acid residues of the peptide, the peptide being at least 44 amino acids in length and having substitutions at positions 12, 21, 26 and 32 corresponding to SEQ ID NO: 78, wherein the substitutions correspond to K12A, Y21Q, S26Kme3 and G32A.

[0060] In some embodiments, this disclosure provides a pharmaceutical composition comprising a polypeptide or compound as provided herein, and a pharmaceutically acceptable excipient.

[0061] 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 having at least 90% identity with at least 40 amino acids of an amino acid sequence as described in any one of SEQ ID NO: 3-158, 161-168, 171-208, 212-215 or 216-237 and a radionuclide.

[0062] In some embodiments, according to formula MLCR, a radionuclide associates with a 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. In some embodiments, the polypeptide has an amino acid sequence comprising or consisting of SEQ ID NO: 195 or SEQ ID NO: 200.

[0063] 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 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 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.

[0064] In some embodiments, R is a therapeutic agent and / or an imaging agent. In some such embodiments, R is Cu-64, Ga-68, Lu-177, In-111, Cu-67, La-132, or F-18.

[0065] In one aspect, this disclosure provides a method for reducing renal cell uptake of a composition, comprising administering to a subject a Nectin-4 binding protein having an amino acid sequence containing at least one modified lysine residue at positions X12 and / or X26 corresponding to SEQ ID NO: 195, wherein the modification comprises at least a small alkyl group of nitrogen linked to a lysine side chain, optionally comprising monomethyl, dimethyl, trimethyl, or isopropyl, and the reduction is compared to administering to the subject or a control subject a composition that is otherwise identical but does not contain the modified lysine residue at positions X12 and / or X26.

[0066] In another aspect, this disclosure provides a method of treating cancer, an improvement comprising administering a composition comprising a Nectin-4 binding protein having an amino acid sequence containing at least one modified lysine residue at positions X12 and / or X26 corresponding to SEQ ID NO: 195, wherein, compared to a composition not containing a modified lysine residue at positions X12 and / or X26, the modification comprises at least one carbon atom of nitrogen linked to a lysine side chain, optionally comprising methyl, dimethyl, trimethyl, or isopropyl.

[0067] In one aspect, this disclosure provides a method of treating a subject with refractory or recurrent cancer, comprising administering a composition, compound, or pharmaceutical composition as provided herein, wherein said treatment is therapeutic for cancer.

[0068] In one aspect, this disclosure provides a method for improving the biodistribution of a pharmaceutical composition of a population of Nectin-4 positive cancer cells in a subject suffering from Nectin-4 positive cancer, comprising contacting the population with a polypeptide having a modified lysine at positions X12 and / or X26 corresponding to SEQ ID NO: 195, 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 contact that does not contain the modified lysine at positions X12 and / or X26 corresponding to SEQ ID NO: 195.

[0069] On the other hand, this disclosure provides a method for diagnosing the presence of a Nectin-4 positive cancer cell population, comprising: The cell population is brought into contact with a composition, compound, or pharmaceutical composition as provided herein; the presence of the composition, compound, or pharmaceutical composition of step (a) is detected by measuring a signal; the detection result in step (b) is compared with a control signal; and if the detected composition, compound, or pharmaceutical composition of step (a) is higher than the control, cancer is diagnosed.

[0070] In some embodiments, contact is performed by administration to the desired subject. In some embodiments, administration is intravenous or subcutaneous. In some embodiments, contact is performed outside the subject, optionally using a biopsy sample.

[0071] In one aspect, this disclosure provides a method of treating a subject with cancer using immunotherapy, the method comprising administering to the subject a composition comprising a composition, compound, or pharmaceutical composition as provided herein.

[0072] On the other hand, this disclosure provides for the use of the compositions, compounds or pharmaceutical compositions provided herein for the treatment of a subject with cancer.

[0073] 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 as provided herein.

[0074] In some implementations, the subject has been diagnosed with cancer. In some implementations, cancer cells from the subject express Nectin-4 or a portion thereof. In some implementations, Nectin-4 expression is higher in cancer cells than in non-cancer cells, and this expression can be measured by protein and / or nucleic acid levels.

[0075] In some embodiments, the compositions, compounds, or pharmaceutical compositions are not taken up and / or retained by the kidneys compared to compounds that do not contain the compositions, compounds, or pharmaceutical compositions provided herein. In some embodiments, the compositions, compounds, or pharmaceutical compositions are internalized in cells expressing human Nectin-4.

[0076] 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 kidney tumors.

[0077] In some embodiments, the composition, compound, or pharmaceutical composition is administered intravenously or subcutaneously.

[0078] In one aspect, this disclosure provides a method for targeting cancer cells expressing Nectin-4, the method comprising: determining or having determined the expression level of Nectin-4 in a population of cancer cells; 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 Nectin-4; and (iii) wherein the composition, compound, or pharmaceutical composition is linked to the surface of one or more cancer cells expressing Nectin-4 and / or internalized into the cancer cells.

[0079] In some implementations, subjects receive treatment after administration, compared to receiving treatment before administration.

[0080] In one aspect, this disclosure provides a method for targeting a population of cancer cells expressing Nectin-4, an improvement comprising contacting the population with a composition, compound, or pharmaceutical composition as provided herein, wherein X12 and / or X26 comprises lysine residues having at least one additional small alkyl group attached to a nitrogen atom on a side chain, wherein the composition taken up by renal cells is less than a composition of a polypeptide containing a small alkyl group on the side chain without nitrogen atom attached to the lysine residue at position X12 and / or X26, wherein optionally, the small alkyl group is part of a monomethyl, dimethyl, trimethyl, or isopropyl group.

[0081] In one aspect, this disclosure provides a conjugate comprising: a polypeptide (M) specifically bound to Nectin-4; 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.

[0082] In one aspect, this disclosure provides a conjugate comprising: a polypeptide (M) specifically bound to Nectin-4; 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.

[0083] In one aspect, this disclosure provides a conjugate comprising: a polypeptide (M) specifically bound to Nectin-4; 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.

[0084] In another aspect, this disclosure provides a conjugate comprising: a polypeptide (M) specifically bound to Nectin-4; 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.

[0085] In one aspect, this disclosure provides a conjugate comprising: a polypeptide (M) specifically bound to Nectin-4; 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.

[0086] In another aspect, this disclosure provides a conjugate comprising: a polypeptide (M) specifically bound to Nectin-4; 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 ruthenium-177.

[0087] In one aspect, this disclosure provides a conjugate comprising: a microprotein (M) specifically bound to Nectin-4; 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 the N-terminal modification contains biotin.

[0088] In some embodiments, the M of the conjugate has an amino acid sequence comprising any one of the amino acid sequences described in SEQ ID NO:3-158, 161-168, 171-208, or 212-215. In some embodiments, the amino acid sequence has at least 90% identity with the sequence of at least 40 amino acids of SEQ ID NO: 176, wherein X2 is E or D; X6 is E or Q; X9 is T or A; X10 is A or G; X12 is A, Kme3, Kme2, Kme, Kipr, or K; X13 is R or (Cit); X17 is G or A; X21 is Q, Y, or E; X24 is Q or K; X25 is A or K; X26 is Kme3, Kme2, Kme, K, Kipr, or S; X28 is Q or K; X29 is Y or K; X30 is L or V; X32 is A, G, or D; X41 is N or K; and X45 is S or absent.

[0089] In some embodiments, M has an amino acid sequence that includes or consists of SEQ ID NO: 195.

[0090] In some embodiments, M has an amino acid sequence that includes or consists of SEQ ID NO: 200.

[0091] 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: 3-158, 161-208, and 212-215; 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: 3-158, 161-168, 171-208, or 212-215.

[0092] In some embodiments, this disclosure provides a vector comprising the isolated polynucleotides provided herein. In some embodiments, this disclosure provides a host cell transformed with the isolated polynucleotides provided herein or the vectors provided herein.

[0093] 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, or pharmaceutical composition as provided herein, and detection to determine the location of the composition in the subject.

[0094] 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, or pharmaceutical composition as provided herein, wherein X12 and / or X26 comprises lysine 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 of a polypeptide containing a small alkyl group on the side chain at position X12 and / or X26 that does not contain nitrogen atomized to a lysine atom on the side chain, wherein optionally, the small alkyl group is part of a monomethyl, dimethyl, trimethyl, or isopropyl group.

[0095] In some embodiments, the detection includes an imaging procedure that allows for the selection of subjects, monitoring of subjects, and / or treatment of subjects with a therapeutic agent comprising a microprotein designed to bind to Nectin-4 expressed on one or more cancer cells in one or more cancer cell populations. In some embodiments, the therapeutic agent comprises a composition, compound, pharmaceutical composition, or conjugate as provided herein.

[0096] In one aspect, this disclosure provides an improved method for delivering a radionuclide to a population of cancer cells, the method comprising administering a composition, compound, pharmaceutical composition, or conjugate as provided herein, wherein the amino acid sequence of the polypeptide comprises an amino acid corresponding to position X12 and / or X26 of SEQ ID NO: 195, and wherein X12 and / or X26 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 an additional small alkyl group attached to a nitrogen atom on a side chain at position X12 and / or X26.

[0097] In some implementations, the small alkyl group comprises monomethyl, dimethyl, trimethyl, or isopropyl.

[0098] 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, pharmaceutical composition or conjugate as provided herein, wherein the amino acid sequence of the polypeptide comprises an amino acid corresponding to position X12 and / or X26 of SEQ ID NO: 195, and wherein X12 and / or X26 comprises a lysine having at least one additional small alkyl group attached to a nitrogen atom on a side chain, wherein renal cell uptake is less than that of a polypeptide having an amino acid sequence that does not contain an additional small alkyl group attached to a nitrogen atom on a side chain at position X12 and / or X26.

[0099] On the other hand, this disclosure provides a method for treating an individual with cancer, an improvement comprising 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, compound, pharmaceutical composition, or conjugate provided herein, wherein the amino acid sequence of the polypeptide comprises amino acids corresponding to positions X12 and / or X26 of SEQ ID NO: 195, and wherein X12 and / or X26 comprises lysine having at least one additional small alkyl group attached to a nitrogen atom on a side chain, wherein the renal cells take up less of the polypeptide than a polypeptide having an amino acid sequence having a small alkyl group attached to a nitrogen atom on a side chain at positions X12 and / or X26.

[0100] In some implementations, the decrease in R concentration in kidney tissue is measured by means such as the amount of R excreted in urine as a percentage of the applied radiation recovered, or by means of a cell-based in vitro assay or in vivo assay.

[0101] In some embodiments, when a composition having 90% identity with at least 40 amino acids of SEQ ID NO: 195 and including modified lysine at positions X12 and / or X26 of SEQ ID NO: 195 can be applied at least 2, 3, 4, 5, 6 or 7 times, compared to the presence of A or K at positions corresponding to X12 and / or X26.

[0102] In one aspect, this disclosure provides a method for reducing the uptake of a composition by renal tissue, the 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 40 amino acids of SEQ ID NO: 195 and / or has a modified lysine at positions corresponding to X12 and / or X26 of SEQ ID NO: 195, 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.

[0103] In another aspect, this disclosure provides a method comprising administering to a subject in need a compound that binds to Nectin-4 and comprises one or two modified lysines at positions X12 and / or X26, respectively, corresponding to SEQ ID NO: 195, wherein administration of a compound having a microprotein containing one or two modified lysines reduces one or more off-target effects, toxicity levels, and / or uptake and / or retention in renal tissue compared to a compound having alanine at position X12 or unmodified lysine at position X26.

[0104] In one aspect, this disclosure provides a method for treating an individual with or suspected of having Nectin-4 positive cancer, the method comprising administering to the individual: means for blocking the uptake and / or retention of a radiotherapy agent by renal tissue, and a connector, chelating agent, and radionuclide.

[0105] In some embodiments, the means for blocking renal tissue uptake and / or retention of radiotherapy agents is incorporated into Nectin-4 and includes one or two modified lysine residues at positions X12 and / or X26 of SEQ ID NO: 195, respectively, and / or has at least 90% identity with the 40 amino acids of SEQ ID NO: 195 and / or has modified lysine residues at positions X12 and / or X26 of SEQ ID NO: 195.

[0106] In some embodiments, the means for blocking renal tissue uptake and / or retention of radiotherapy agents is incorporated into Nectin-4 and includes one or two modified lysine residues at positions X12 and / or X26 of SEQ ID NO: 195, respectively, and / or has at least 90% identity with the 35 adjacent amino acids of SEQ ID NO: 195 and / or has modified lysine residues at positions X12 and / or X26 of SEQ ID NO: 195.

[0107] In some embodiments, the means for blocking the uptake and / or retention of the radiotherapy agent in the renal tissue have a greater effect on blocking the uptake and / or retention of the radiotherapy agent than means that do not include one or two modified lysines at positions X12 and / or X26 of SEQ ID NO: 195 and / or have at least 90% identity with the 40 amino acids of SEQ ID NO: 195 and / or have modified lysines at positions X12 and / or X26 of SEQ ID NO: 195.

[0108] In some embodiments, the means for blocking renal tissue uptake and / or retention of the radiotherapy agent have a greater effect on blocking renal tissue uptake and / or retention than means that do not include one or two modified lysines at positions X12 and / or X26 of SEQ ID NO: 195 and / or have at least 90% identity with the 35 adjacent amino acids of SEQ ID NO: 195 and / or have modified lysines at positions X12 and / or X26 of SEQ ID NO: 195.

[0109] In some embodiments, the means of blocking the uptake and / or retention of the radiotherapy agent by renal tissue 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 peptide targeting Nectin-4.

[0110] In some embodiments, the radiotherapy agent comprises or is composed of compounds selected from C3-C293 or C298-C307. In some embodiments, the radionuclide in the radiotherapy agent is selected from Ac-225, Cu-64, Ga-68, In-111, Lu-177, or Pb-212.

[0111] 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 any of the compositions, compounds, pharmaceutical compositions or conjugates provided herein.

[0112] In some embodiments, the kit further comprises 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, 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)benzoic acid N-succinimide ester (BuSTB) or 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB).

[0113] In some embodiments, the compound is labeled with a radionuclide prior to use, wherein the radionuclide is chelated to a chelating agent to produce a composition having the formula MLCR.

[0114] 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.

[0115] In some embodiments, if the polypeptide has an amino acid comprising any of the sequences described in any one of SEQ ID NO: 83, 85, 93, 99, 134, 138, 145, 155, 161-176, 195, or 200, the polypeptide further comprises a linker, wherein the linker is PEG4, and a chelating agent, wherein the chelating agent is DOTA. 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 either 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

[0116] Figure 1A and Figure 1B These are fitted sensor maps (SPRs) of exemplary Nectin-4 peptides that bind to mouse and human Nectin-4, respectively.

[0117] Figure 2A and Figure 2B These are fitted sensor maps (SPRs) of exemplary Nectin-4 peptides that bind to mouse and human Nectin-4, respectively.

[0118] Figure 3A and Figure 3B These are fitted sensor maps (SPRs) of exemplary conjugates containing exemplary Nectin-4 peptides that bind to mouse and human Nectin-4.

[0119] Figure 4A and Figure 4B This demonstrates the association of exemplary compounds containing the radioconjugated microproteins described herein with cancer cell lines expressing human Nectin-4. Figure 4A ) and dissociation Figure 4B A diagram of dynamics.

[0120] Figure 5A and Figure 5B This demonstrates the association of exemplary compounds containing the radioconjugated microproteins described herein with cancer cell lines expressing human Nectin-4. Figure 5A ) and dissociation Figure 5B A diagram of dynamics.

[0121] Figure 6A and 6B This shows an exemplary radioconjugate in HT-1376 ( Figure 6A ) and MCF7 ( Figure 6BA graph showing the internalization status of cell lines at 37°C and 4°C. TB = total binding, NSB = nonspecific binding.

[0122] Figure 7 It is a graph showing the thermal stability of exemplary compounds, expressed as the percentage of the parent compound remaining after 60 minutes of heating at 75°C.

[0123] Figure 8 This is a bar chart showing the percentage of uptake of an exemplary microprotein (tested at 20 μM) against a 20 μM control peptide (set to 100%) in a proximal tubular epithelial cell assay.

[0124] Figure 9A It shows that it accepts 500 nCi. 225 Ac-C251 (triangle), 1000 nCi 225 Tumor volume (mm) in mice treated with Ac-C251 (square) or a carrier (circular) 3 A graph showing how the drug changes over time. Mice were given the drug on day 0. Figure 9B Is it a display acceptance? Figure 9A A graph showing the percentage change in body weight relative to initial body weight of the mice treated as described above over time.

[0125] Figure 9C This is a graph showing the survival rate of a mouse xenograft model over an 8-week period. Xenografts were generated using HT-1376-parental Nectin-4 expressing cells (P) or HT1376-Nectin-4 overexpressing cells (OE). Mice received either the mediator (P, solid circles; OE, solid triangles) or 1000 nCi of [unclear - possibly a specific drug or treatment]. 225 Treatment of Ac-C251 (P, hollow triangle; OE, hollow square).

[0126] Figure 10A To display accepting 500 nCi 225 Ac-C244 (triangle), 1000 nCi 225 A graph showing the change in tumor volume over time in mice treated with Ac-C244 (square) or the medium (circle). Mice were administered the drug on day 0. Figure 10B Is it a display acceptance? Figure 10A A graph showing the percentage change in body weight relative to initial body weight of the mice treated as described above over time.

[0127] Figure 11A It shows that it accepts 500 nCi. 225 Ac-C260 (triangle), 1000 nCi 225A graph showing the change in tumor volume over time in mice treated with Ac-C260 (square) or the medium (circle). Mice were administered the drug on day 0. Figure 11B Is it a display acceptance? Figure 11A A graph showing the percentage change in body weight relative to initial body weight of the mice treated as described above over time.

[0128] Figure 12 This is an exemplary HPLC trace of the microprotein C251, showing the change in milli-absorbance units (mAU) over time (min).

[0129] Figure 13 This is an example mass spectrum of the microprotein C251.

[0130] Figures 14A-14C This is a chart displaying exemplary UPLC-MS data for C251.

[0131] Figure 15 It is a display 111 A graph comparing renal uptake (%ID / g) of radiolabeled microproteins (C109, C244, and C251).

[0132] Figure 16 This is a graph showing the reduction in cellular uptake of the exemplary target-binding microprotein compound C294 when combined with the exemplary bait C296 in vitro. The bars depict the percentage uptake of the exemplary Nectin-4-targeting microprotein (C294) alone / without the bait peptide (20 µM) or in combination with a 20-fold molar excess of the exemplary bait (C296). Error bars represent the standard error (SEM) of the mean.

[0133] Figures 17A-17D It is a chart showing, in vivo, the kidney (in an exemplary mouse xenograft model) Figure 17A and Figure 17C ) or tumor ( Figure 17B and Figure 17D The status of the stay is shown as follows: 111 In-labeled exemplary Nectin-4 targeting microprotein conjugates ( Figure 17A and Figure 17B In 111 In-C109; Figure 17C and Figure 17D In 111 In-C251) was injected alone or not with bait, or with an exemplary bait in 1000 molar excess. Figure 17A and Figure 17B C295 in the middle; Figure 17C and Figure 17DThe %ID / g of C296 was administered 0.25-32 h after injection. Figure 17A It is an exemplary Nectin-4 targeting microprotein conjugate ( 111 Line graph of %ID / g in the kidneys (In-C109) alone / not with a lure (round) or in combination with an exemplary lure (C295; square). Figure 17B It is an exemplary Nectin-4 targeting microprotein conjugate ( 111 Line graph of %ID / g in tumors (In-C109) alone / not with a decoy (circle) or in combination with an exemplary decoy (C295; square). Figure 17C It is an exemplary Nectin-4 targeting microprotein conjugate ( 111 Line graph of %ID / g in the kidneys (In-C251) alone / without a lure (round) or in combination with an exemplary lure (C296; square). Figure 17D It is an exemplary Nectin-4 targeting microprotein conjugate ( 111 Line graph of %ID / g in tumors (In-C251) alone / without a decoy (circle) or in combination with an exemplary decoy (C296; square).

[0134] Figure 18A and Figure 18B It is a chart showing the tumor volume (mm²) in an exemplary mouse xenograft model in vivo. 3 ; Figure 18A ) and weight (as a percentage of initial body weight) Figure 18B The exemplary mouse xenograft model received the following treatments on day 0: (i) a medium (solid circle); (ii) an exemplary decoy (C296; 1 mg; 1000 times the dose of C251, solid triangle); (iii) [other treatments]. 225 An example of Ac-labeled Nectin-4-targeted radionuclide conjugates ( 225 Ac-C251; 1 mg; 1,000 nCi; hollow circle); or (iv) exemplary decoy (C296; 1,000 times the dose of C251) and exemplary Nectin-4 targeted radionuclide conjugate ( 225 A combination of Ac-C251; 1,000 nCi) (hollow triangle). In Figure 18A Medium, 2,000 mm 3 The dashed line marks the predefined humane endpoint of the maximum tumor growth threshold in experimental mice. Figure 18B In the diagram, the dashed line at 80% of the initial body weight marks the predefined humane endpoint of weight loss for the experimental mice. Error bars represent the standard error (SEM) of the mean.

[0135] Figure 19A and 19B It is a chart showing the tumor volume (mm²) in an exemplary mouse xenograft model in vivo. 3 ; Figure 19A ) and weight (as a percentage of initial body weight) Figure 19B The results of the measurements, the exemplary mouse xenograft model received the following treatment on day 0: (i) a medium (round; single dose); 225 An example of Ac-labeled Nectin-4-targeted radionuclide conjugates ( 225 Ac-C251), two different doses ((ii) 0.5 mg; 1,000 nCi; single dose; triangle, or (iii) 1 mg; 2,000 nCi; single dose; inverted triangle); or (iv) anti-Nectin-4 antibody-drug conjugate (ADC) control (enfortumab vedotin, EV; 3 mg / kg; three doses; square). The black arrow below the X-axis indicates day 0. 225 Ac-C251 administration. The gray arrows below the x-axis indicate EV administration on days 0, 8, and 15. Figure 19A Medium, 1,500 mm 3 The dashed line marks the predefined humane endpoint of the maximum tumor growth threshold in experimental mice. Figure 19B In the diagram, the dashed line at 80% of the initial body weight marks the predefined humane endpoint of weight loss for the experimental mice. Error bars represent the standard error (SEM) of the mean.

[0136] Figure 20A and Figure 20B It is a chart showing the tumor volume (mm²) in an exemplary patient-derived mouse xenograft model in vivo. 3 ; Figure 20A ) and weight (as a percentage of initial body weight) Figure 20B The exemplary patient-derived mouse xenograft model received the following treatments on day 0: (i) a medium (circular); (ii) [other treatments]. 225 An example of Ac-labeled Nectin-4-targeted radionuclide conjugates ( 225 Ac-C251; 1 mg; 2,000 nCi; single dose; inverted triangle); or (iii) anti-Nectin-4 ADC control (Ventumumab (EV); 3 mg / kg; three doses; square). The black arrow below the X-axis indicates day 0. 225Ac-C251 dosing. The gray arrows below the x-axis indicate EV dosing on days 0, 8, and 15. Error bars represent the standard error (SEM) of the mean.

[0137] Figure 21 This is a graph showing the high binding affinity of biotin-labeled exemplary Nectin-4-targeting microprotein (C253) to endogenous Nectin-4 expressed on HT-1376 human urothelial carcinoma cells in the presence of a europium-labeled Nectin-4 binder (C307). The dose-response curves depict fluorescence units as a measure of binding of biotin-labeled exemplary Nectin-4 to microprotein C253, evaluating 12 concentrations ranging from 28 pM to 5 µM (x-axis; expressed in logM).

[0138] Figure 22 This is an example of an indium-labeled Nectin-4 targeting conjugate administered intravenously in rats at a glomerular filtration rate (GFR) of 0.03 mg / kg (round), 0.1 mg / kg (square), or 0.3 mg / kg (diamond) compared to FITC-glucose (FS; inverted triangle), a molecule known to be cleared by glomerular filtration rate (GFR) (62.4 mg / kg). nat. Plasma characteristics and clearance rate of In-C251. This figure shows the plasma characteristics and clearance rate over a 2-hour period. nat. Concentrations of In-C251 and FITC-glucose.

[0139] Figure 23A and Figure 23B This is a chart showing the selective target binding of biotin-tagged illustrative Nectin-4 targeting microprotein (C253) (as measured by flow cytometry). Figure 23A The dose-response curves were obtained from Nectin-4 expressing cells (HT-1376-parent or "HT-1376-P") or Nectin-4 knockout cells (HT-1376-KO). Figure 23B ). Figure 23A The percentage of cells identified as expressing Nectin-4 after incubation with 100 nM C253 (normalized to mode, y-axis) is shown as measured by mean fluorescence intensity (MFI, x-axis). Figure 23BThe difference in mean fluorescence intensity (ΔMFI; y-axis) of HT-1376-P (round) and HT-1376-KO (inverted triangle) cells incubated with C253 and cells incubated with the medium is shown as a measure of Nectin-4 binding at C253 (x-axis) at 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM and 1000 nM.

[0140] Figure 24A and Figure 24B The chart shows the results observed in vivo in an exemplary mouse allogeneic graft model, within 10 minutes to 22 hours after injection. 111 In-labeled exemplary Nectin-4 targeting microprotein conjugates 111 In-C109 ( Figure 24A )and 111 In-C251 ( Figure 24B Retention status in the kidney (square) and tumor (round). Detailed Implementation

[0141] Among other things, this disclosure also provides compositions and methods of using the same. In some embodiments, the composition selectively binds to a target (e.g., Nectin-4). In some embodiments, the target is on tumor cells. In some such embodiments, the tumor cells are part of a population of tumor cells (e.g., solid tumors). In some embodiments, the tumor cells are circulating (e.g., hematologic malignancies, circulating tumor cells, etc.). In some embodiments, the composition comprises one or more additional agents (e.g., chelating agents, radionuclides) that can be used as therapeutic agents (e.g., radionuclides used to kill cancer cells), wherein the therapeutic agent selectively targets cells, e.g., cells expressing Nectin-4, such as cancer cells expressing Nectin-4, as part of the composition. In some embodiments, the composition comprises one or more therapeutic agents (e.g., chelating agents, radionuclides), wherein the therapeutic agent selectively targets cells expressing Nectin-4, thereby treating Nectin-4-expressing cells without treating cells that do not express Nectin-4. 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., tumor cells) or deliver therapeutic agents in a manner that minimizes damage to surrounding cells (e.g., non-tumor cells). Surrounding cells (e.g., in one or more non-tumor tissues) may also express the target in amounts or at levels lower than those expressed by the target cells. This disclosure provides the insight that selective targeting combined with specific therapeutic agents (e.g., chelators and / or radionuclides (e.g., alpha emitters)) offers advantages over previously used therapeutic agents (e.g., antibodies, beta emitters, etc.).

[0142] Furthermore, this disclosure provides the insight that challenges may still arise even if therapeutic agents, such as those provided herein, are designed to be more specific to Nectin-4 and / or to tissues such as tumor tissue. Efficacy may be reduced and toxicity may be increased when Nectin-4 is expressed in non-tumor cells, and / or when a therapeutic agent (e.g., a radiotherapy agent) is taken up by an organ system (e.g., an organ system involved in clearing systemically administered agents, such as the kidneys). This disclosure takes into account that tumor uptake may be challenged by uptake, retention, and / or clearance by one or more non-target tissues (e.g., non-tumor tissues). For example, the kidneys may play a role in clearing administered therapeutic agents. In addition, such therapeutic agents may be taken up and / or retained by the kidneys. For example, a therapeutic agent intended to treat a tumor may also be taken up, retained, and / or cleared by the kidneys, resulting in (1) faster clearance of the therapeutic agent from the subject to which it has been administered; (2) reduced tumor targeting (including due to uptake and / or retention of the therapeutic agent by non-target tissues), and / or clearance; and / or (3) damage to non-target tissues (e.g., the kidneys, etc.).

[0143] This disclosure acknowledges that any or all of these challenges can be mitigated or prevented by combining a Nectin-4 targeted therapeutic agent with decoy administration. In some embodiments, the decoy can block renal uptake of peptide-containing compositions (e.g., radionuclide conjugates) as provided herein. Not wishing to be bound by theory, this disclosure contemplates improvements in maintaining therapeutic efficacy while reducing damage to one or more non-tumor tissues (e.g., the kidney), and in some embodiments, improvements in therapeutic efficacy while reducing the risk or actual damage to non-tumor tissues (e.g., kidney tissue and / or renal system tissues such as the ureter, bladder, etc.).

[0144] 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.

[0145] Unless otherwise indicated, the methods and techniques disclosed herein are generally performed according to conventional methods known in the art and set forth in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual , 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al. Current Protocols in Molecular Biology Greene Publishing Associates (1992, and 2002 supplement); Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Wittrup and VanAntwerp, Fine Affinity Discrimination by Yeast Surface Display and Flow Cytometry, Biotechnol. Prog. 2002, (16) 31-37; C. Queen et al., A humanized antibody 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] Unless otherwise understood from the context and use, 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 of the described objects.

[0152] 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.

[0153] 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.

[0154] As used herein, the term “affibody” refers to a subgenus of microproteins. An affibody 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 the affibody, 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. An illustrative Affibody® molecule is available from Abcam Corp. Cambridge Mass. The affibody 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).

[0155] 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.

[0156] 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).

[0157] As used herein, the term “anticalin” refers to a subgenus of microproteins. Anticalins are engineered proteins derived from lipid carriers (Beste G, Schmidt FS, Stibora T, Skerra A. (1999) Proc Natl Acad Sci US A. 96(5): 1898-903; Gebauer and Skerra (2009) CurrOpinion in Chemical Biology 13:245-255). Anticalins possess an eight-chain β-barrel structure, forming a highly conserved core unit within lipid carriers, and naturally forming ligand-binding sites through four structurally variable loops at the open ends. Although anticalins are not homologous to the IgG superfamily, they exhibit features that have so far been considered typical of antibody-binding sites: (i) high structural plasticity due to sequence variations, and (ii) increased conformational flexibility, allowing for induction of adaptation to targets of different shapes.

[0158] 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 less sensitive 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 cases, an enzyme that has been altered to have less activity may be referred to as an attenuated enzyme.

[0159] As used herein, the term “avimer” refers to a subgenus of microproteins. Avisers are a class of antibody mimics consisting of two or more peptide sequences, each preferably 30 to 35 amino acids long, derived from the A domain of various membrane receptors and linked by linker peptides. Target molecules bind via the A domain, and the domain having the desired binding specificity can be selected, for example, by phage display technology. The binding specificity of different A domains contained in an avimer may be the same, but not necessarily the same (Weidle UH et al., (2013), Cancer Genomics Proteomics; 10(4):155-68). For more details, see Nature Biotechnology 23(12), 1556–1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909–917 (June 2007). As used herein, the term “binding agent” refers to a subgenus of microproteins. The binder is characterized by comprising or consisting of a polypeptide capable of binding or known to be capable of binding and associating with a target or a portion thereof. Binders typically contain a cysteine-containing peptide with one or more disulfide bonds, but some binders do not contain cysteine ​​residues and / or disulfide bonds. When administered systemically to a mammalian subject, it is preferable that the binder be rapidly cleared from circulation. It is understood that, in a given context, reference to a binder may include or include its nucleic acid sequence or the amino acid sequence encoding it. The binder may be provided in the form of, for example, polynucleotides or polypeptides, using a carrier, host cell, etc., and / or any combination thereof. The binder may be derived or manufactured using any method known to those skilled in the art. For example, in some embodiments, the binder may be recombinant (i.e., generated using recombinant nucleic acids encoding a polypeptide). In some embodiments, the binder 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.

[0160] 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). In some embodiments, a decoy may block, inhibit, reduce, or otherwise decrease the uptake of the conjugates or compounds of this disclosure by non-tumor tissues (e.g., kidney tissue). In some embodiments, a decoy may reduce the retention of a compound (e.g., a radiotherapy compound, such as one containing a microprotein) in non-tumor tissues (e.g., kidney).

[0161] 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.

[0162] As used herein, the term "conjugate" refers to two compounds or agents joined together in a covalent or non-covalent manner.

[0163] 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).

[0164] As used herein, the terms "cysteine-dense peptide" and "CDP" are used interchangeably and refer to a subgenus of microproteins that typically contain a high density of cysteine ​​residues (e.g., at least one, two, three, four, or more cysteine ​​residues in a polypeptide ranging from about 10 to about 90 amino acids or from about 13 to 80 amino acids). In some embodiments, such CDPs may contain at least two separate folded domains. In some embodiments, CDPs contain at least one, two, three, four, or more cysteine ​​residues in a range of about 10 to about 90 amino acid residues, preferably 13 to 80 amino acid residues (pubmed.ncbi.nlm.nih.gov / 29483648 / ). In some embodiments, CDPs contain a restricted distribution of cysteine, Cys-X. [0–15] -Cys-X [0–15] -Cys-X [0–15] -Cys-X [0–15] -Cys-X [0–15]-Cys (where X represents any amino acid) (SEQ ID NO: 241).

[0165] 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), as provided in the exemplary compounds and microproteins in Table 2A. 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) and may have an acid or amide group at their C-terminus (e.g., see Table 2A). 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 Nectin-4 (e.g., for the detection and / or treatment of cancer).

[0166] 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.

[0167] 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 that are 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.

[0168] As used herein, the term "derived from," in the context of nucleic acid sequences, refers to a nucleic acid sequence that has at least 85% sequence identity with a reference natural nucleic acid sequence from which it is derived. The term "derived from," in the context of amino acid sequences, refers to an amino acid sequence that has at least 85% sequence identity with a reference natural amino acid sequence from which it is derived. As used herein, the term "derived from" does not imply any specific process or method for obtaining nucleic acid or amino acid sequences. For example, nucleic acid or amino acid sequences can be synthesized by chemical methods.

[0169] As used herein, the term “designed ankyrin repeat domain (DARPin)” refers to a subgenus of microproteins. DARPin is a peptide derived from ankyrins, a class of proteins that mediate the connection between integrated membrane proteins and the cytoskeleton. Individual ankyrin repeat sequences are preferably 33-residue motifs consisting of two α-helices and one β-turn. They can be engineered to bind different target antigens by randomizing the residues in the first α-helix and β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (an affinity maturation method). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003), J. Mol. Biol. 369, 1015-1028 (2007), and US20040132028A1. DARPin typically provides a rigid interface and lacks structural flexibility (Gebauer and Skerra, 2009).

[0170] 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.

[0171] As used herein, the term “engineered Kunitz domain” refers to a subgenus of microproteins. Engineered Kunitz domains are preferably peptides derived from Kunitz-type protease inhibitors (e.g., bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP), or tissue factor pathway inhibitor (TFPI)). Kunitz domains have a molecular weight of approximately 6 kDa, and domains with desired target specificity can be selected using display techniques such as phage display (Weidle et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).

[0172] 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.

[0173] As used herein, the term “functional variant” refers to a polypeptide that comprises or is composed of a portion of the polypeptide sequence provided herein, and still retains one or more functions of the polypeptide that comprises or is composed of the entire amino acid sequence provided herein (e.g., still binds to a target, such as Nectin-4).

[0174] 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.

[0175] 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.

[0176] As used herein, the term "identical" means at least two nucleic acid sequences or at least two amino acid sequences or subsequences that each have a specified percentage of nucleotides or amino acids and 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, and the test (i.e., query) sequence is compared to said reference sequence. 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 sequence and reference sequence are input into a computer, the subsequence coordinates 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. Various 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, 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 regions between the query and search sequences and the percentage of sequence identity. Pearson, MethodsEnzymol. 183:63-98 (1990) (included hereby 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 (included hereby by reference), with its default parameters. The percentage of identity is not necessarily determined over the entire length of the two sequences. For example, for a reference sequence and a query sequence, one may be longer or shorter than the other, and the percentage of identity is determined based on the matching of specific nucleic acid or amino acid extension lengths.For example, if a disclosed sequence is compared to a query sequence, and the query sequence is shorter, the identity percentage is determined by aligning the reference sequence and the query sequence and determining the percentage of identity between the query sequence and the aligned portion of the reference sequence. If the query sequence is longer than the disclosed sequence, the identity percentage refers to the identity at the aligned portion (e.g., 5, 10, 15, 20, 25, 30, 35 or more amino acids of a microprotein).

[0177] 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.

[0178] 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).

[0179] As used in this article, the term "k" d ” (s -1 The 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.

[0180] 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.

[0181] As used in this article, the term "K" D "(M) refers to the dissociation equilibrium constant of a given entity and its target (a specific interaction between the entity and its target, such as a peptide-target interaction)." D = k d / k a .

[0182] 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 .

[0183] 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) ® ).

[0184] In some embodiments, the dissociation equilibrium constant (Ki) of the peptide or microprotein of this disclosure (e.g., targeting microprotein, such as Nectin-4 binding microprotein) with Nectin-4 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 an SPR machine (e.g., a BIACORE T200 instrument).

[0185] 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.

[0186] As used in this article, the term "knotting protein" refers to the structural motif of a microprotein containing three disulfide bridges.

[0187] As used herein, the term "knotting peptide" refers to a subgenus of microproteins containing at least one knotting peptide.

[0188] As used in this article, the term "connector" refers to the portion used to conjugate microproteins to a chelating agent.

[0189] As used herein, the term "microprotein" refers to a short protein containing fewer than or equal to about 100 amino acids, having a well-defined folded structure comprising two or more secondary structural elements, an isolated hydrophobic core, and / or co-folding. Examples of microproteins include CDPs, knotting agents, affinity molecules, engineered Kunitz domains, adnectin monomers, anticalcitonins, designed ankyrin repeating domains (DARPin), and avimers disclosed herein. Furthermore, microproteins can refer to linear peptides, folded peptides (e.g., covalently linked peptides, non-covalently linked peptides, or peptides containing disulfide bonds), cysteine-dense peptides, knotting peptides, binding agents, affinity molecules, engineered Kunitz domains, adnectin monomers, anticalcitonins, designed ankyrin repeating domains (DARPin), or avimers.

[0190] 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. 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. Alterations may include, but are not limited to, changes to one or more atoms of a side chain, such as the addition of a methyl group (e.g., the methylated form of lysine). In some embodiments, natural amino acids are modified, for example, as described herein.

[0191] 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. For example, see Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and 2002 ed.).

[0192] 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.

[0193] As used herein, the terms “monomeric antibody” or “adnectin” are used interchangeably and refer to a subgenus of microproteins. Monomeric antibodies involve a molecule, preferably based on the 10th extracellular domain of human fibronectin III (10Fn3), which employs 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.

[0194] 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).

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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 or at a certain distance to control the expression of the gene of interest.

[0200] 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 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.

[0201] As used in this article, the term "radioactive nuclide" refers to an atom that is capable of undergoing radioactive decay.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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 of 2 and PAM250 score matrix) as provided in GCG version 6.1 (incorporated by reference).

[0208] As used herein, the term "specific activity" generally refers to the activity of a radionuclide per unit mass. Units of specific activity may include becquerels per kilogram (Bq / kg) or curies per gram (Ci / g).

[0209] 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 Nectin-4, or certain cell types, such as kidney cells, such as proximal tubular cells of the kidney, etc.).

[0210] 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. 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 two times, more commonly at least 10 times, often at least 100 times, and even 1000 times. Typically, the affinity or affinity (quantified by a dissociation constant) of a specific 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) be specific to a particular second entity (e.g., an antigen-binding sequence).

[0211] As used herein, the term "stabilizer" in the context of a pharmaceutical composition refers to an agent, molecule, or compound that can be used to influence one or more active pharmaceutical ingredients to maintain desired properties (e.g., therapeutic properties or properties that allow for therapeutic effects) until they are administered to a subject.

[0212] 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 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 a specific DNA hybrid. “Strict washing” is performed under a specific set of conditions at a temperature approximately 5°C lower than the Tm of a 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 h 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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., CDP, knotting agents, binders, affinity molecules, 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., Nectin-4). 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 Nectin-4, the target may also be a protein in a pathway essential for Nectin-4 activity. The target may be or contain a binding region, such as an epitope, to which the microprotein of this disclosure may 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., a non-tumor cell, such as a kidney cell, etc.) binds to a decoy rather than to a polypeptide (e.g., a microprotein) of a composition (e.g., a radiotherapy composition) provided herein.

[0217] As used herein, "thermal stability" refers to the ability of a microprotein to remain stable (e.g., folded, or structurally intact) over a period of time. In some embodiments, the microproteins of this disclosure retain at least 95% of their stability for at least one hour.

[0218] As used in this article, “tolerable” treatment means treatment administration and / or regimens that will not be discontinued due to dose-limiting toxicity.

[0219] 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.

[0220] 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 a replication origin that functions in the host cell). Other vectors, after being introduced 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").

[0221] As used herein, the term "decoy peptide" or "decoy" refers specifically to a subgenus of microproteins intended to: (i) reduce the accumulation of compounds (e.g., microproteins, such as radiolabeled microproteins, such as the radiotherapeutic agents provided herein) in non-tumor tissues (e.g., kidney tissue when the tumor is located elsewhere); and / or (ii) have minimal or no effect on tumor (e.g., tumors expressing targets such as Nectin-4) uptake of the compound; and / or (iii) reduce adverse (e.g., toxicity) accumulation in a subject's non-tumor-containing organs (e.g., kidneys, 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) to block the uptake and / or retention of the radioactive composition in kidney tissue, as compared to uptake and / or retention in kidney tissue in the absence of the decoy. Certain decoys, such as C295-C297 (SEQ ID NO: 209-211), are provided herein. The decoys of this disclosure may be approximately 10 -6 M, 10 -5 M, 10 -4 M, 10 -3M or higher (e.g., 10) -2 The decoy peptide (or bait) binds to the target protein (e.g., Nectin-4) with an affinity for M, or may have a binding that is undetectable using measures including those provided herein. Not wishing to be bound by theory, this disclosure describes, in some embodiments, a decoy peptide (or bait) that can lure a composition, such as a radiotherapy agent, meaning that the presence of the decoy peptide in non-tumor tissue (e.g., kidney) blocks the uptake and / or retention of the radiotherapy agent by the non-tumor tissue (e.g., kidney). For clarity, when a decoy peptide is mentioned as “luring” a composition (e.g., compound, e.g., microprotein) that binds to a target (e.g., Nectin-4), 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 tissue (e.g., kidney) if administered alone, even in the absence of the composition it lured.

[0222] As used herein, the term "scaffold" is used to describe microproteins that possess a common set of 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, such as those of the compounds listed in Table 2A, e.g., C1-C293, and C294.

[0223] Composition This document provides compositions comprising one or more of a polypeptide (e.g., a microprotein), a linker, a chelating agent, and / or a radionuclide. In some embodiments, the composition comprises a linker and a chelating agent. 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 a linker, a chelating agent, and a radionuclide. In some embodiments, the composition comprises or consists of a polypeptide (i.e., a microprotein), optionally a linker and a chelating agent, and / or a radionuclide. In some embodiments, the chelating agent and / or the radionuclide is conjugated to the microprotein via a linker. In some embodiments, the microprotein of this disclosure comprises or consists of: a linear polypeptide, a folded polypeptide (e.g., a covalently linked polypeptide, a non-covalently linked polypeptide, or a polypeptide containing disulfide bonds), a cysteine-dense peptide, a knotting peptide, a binding agent, an affinity compound, an engineered Kunitz domain, a monomeric antibody, an anticalcitonin, a designed ankyrin repeating domain (DARPin), or an avimer. 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 a microprotein via a linker.

[0224] 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-emitting radionuclides). Such microproteins or compositions comprising microproteins can be used to treat subjects in need, exhibiting improved target specificity, increased clearance rate, and reduced off-target effects (e.g., compared to conjugates of non-alpha-emitting radionuclides, such as, compared to compositions comprising antibodies or antibody-drug conjugates, etc.). For example, while microproteins (e.g., used in compositions as provided herein) possess several key properties of antibody-based therapeutics (e.g., affinity, potency, specificity, and ability to disrupt protein-protein interactions), they avoid some undesirable limitations, such as large size, high manufacturing cost, and the need for chimerism or humanization. For example, in some embodiments, the length of the microproteins disclosed herein (e.g., linear peptides, folded peptides (e.g., covalently linked peptides, non-covalently linked peptides, or peptides containing disulfide bonds), cysteine-dense peptides, knotting peptides, binding agents, affinity molecules, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankylosing spondylase repeating domains (DARPin), or avimers) is no more than about 100 amino acids. In some embodiments, such microproteins may be or contain cysteine-dense peptides. In some embodiments, microproteins contain one or more disulfide bridges. In some embodiments, microproteins contain at least two disulfide bridges. In some embodiments, microproteins contain no more than two disulfide bridges. In some embodiments, microproteins contain 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). Not wishing to be bound by theory, this disclosure considers that in some embodiments, microproteins may not contain multiple cysteine ​​residues; for example, microproteins may contain a single cysteine ​​residue. In some such embodiments, the microprotein can form a dimer, for example, by dimerizing with another microprotein (e.g., self-dimerization). In some embodiments, two microproteins are linked together to form a dimer. In other embodiments, two microproteins are each linked to a linker to form a dimer. In other embodiments, two distinct microproteins are each linked to a linker to form a dimer. This disclosure considers that the stability conferred by the cross-linked cysteine ​​helps to reduce the immunogenicity of the microprotein or containing such microproteins. In some embodiments, this stability can also confer resistance to more demanding conditions (e.g., high temperature, low pH incubation, etc.) required for effective chelation, while maintaining biological activity (e.g., the ability to bind to a target).

[0225] 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, without being bound by any particular theory, 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 reagents (e.g., visualization agents, such as metabolizable and visualization agents). 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.

[0226] In some embodiments, microproteins (e.g., linear peptides, folded peptides (e.g., covalently linked peptides, non-covalently linked peptides, or peptides including disulfide bonds), cysteine-dense peptides, knotting peptides, binding agents, affinity molecules, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankyrin repeating domains (DARPin), or 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).

[0227] In some embodiments, compositions comprising a connector, a chelating agent, and / or a radionuclide can effectively penetrate a tumor.

[0228] In some embodiments, microproteins have 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, the binder has excellent 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.

[0229] As described herein, microproteins are conjugated with 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 with the radionuclide. That is, without being bound by any particular theory, this disclosure provides a conjugate in which, in some embodiments, the microprotein targets cells expressing a target with the radioisotope to which it is conjugated. In some embodiments, the target is expressed on the cell surface. In some embodiments, the target is Nectin-4. In some embodiments, the cells are tumor cells. In some embodiments, the conjugate binds to Nectin-4 on the surface of the tumor cells. In some such embodiments, the radionuclide targets the tumor cells. In some embodiments, the radionuclide is an alpha emitter radionuclide and, when internalized, is used to specifically target (e.g., without damaging surrounding tissue / cells) the tumor cells.

[0230] target Any cell expressing the target can become a target for the microproteins provided in this article.

[0231] 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.

[0232] 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).

[0233] 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.

[0234] In some embodiments, the microprotein or a composition containing the microprotein specifically binds to a target expressed on the cell surface. In some embodiments, the target originates 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, the target is found at specific levels in, for example, blood, serum, or plasma. However, in some embodiments, 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 number of targets in a cell population (e.g., a tumor). In some embodiments, the target is a secreted protein. In some such embodiments, the target is found at specific levels in, for example, blood, serum, or plasma. In some such embodiments, the microprotein binds to a region of the target, such as 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.

[0235] In some embodiments, the microprotein or a composition comprising the microprotein specifically binds to Nectin-4. In some embodiments, the target comprises or is composed of Nectin-4. In some embodiments, the microprotein specifically binds to a target comprising the amino acid sequence described in Table 1A or a portion thereof.

[0236] In some embodiments, this disclosure provides a composition comprising a polypeptide having a length of at least 44 amino acids and having an amino acid sequence comprising the sequence described in SEQ ID NO: 171, wherein X2 is E or D; X6 is E or Q; X17 is G or A; X21 is Q, Y, or E; X26 is Kme3, Kme2, Kme, K, Kipr, or S; X32 is A, G, or D; X41 is N or K; and X45 is S or absent.

[0237] In some embodiments, this disclosure provides a composition comprising a polypeptide having a length of at least 44 amino acids and having an amino acid sequence comprising the sequence described in SEQ ID NO: 176, wherein X2 is E or D; X6 is E or Q; X9 is T or A; X10 is A or G; X12 is A, Kme3, Kme2, Kme, Kipr, or K; X13 is R or (Cit); X17 is G or A; X21 is Q, Y, or E; X24 is Q or K; X25 is A or K; X26 is Kme3, Kme2, Kme, K, Kipr, or S; X28 is Q or K; X29 is Y or K; X30 is L or V; X32 is A, G, or D; X41 is N or K; and X45 is S or absent.

[0238] In some embodiments, this disclosure provides a composition comprising a Nectin-4 binding polypeptide having an amino acid sequence comprising at least 44 amino acids, wherein the amino acids include: (i) cysteine ​​at each of four positions corresponding to positions 1, 20, 34, and 44 of SEQ ID NO: 195; (ii) TALARLR (SEQ ID NO: 169) at positions 9-15 of SEQ ID NO: 195; (iii) QKKme3 at positions 24, 25, and 26 of SEQ ID NO: 195; and (iv) QYL at positions 28, 29, and 30 of SEQ ID NO: 195.

[0239] In some embodiments, this disclosure provides a composition comprising a Nectin-4 binding polypeptide having an amino acid sequence comprising at least 44 amino acids, wherein the amino acids include: (i) cysteine ​​at each of four positions corresponding to positions 1, 20, 34, and 44 of SEQ ID NO: 200; (ii) TALA(Cit)LR (SEQ ID NO: 247) at positions 9-15 of SEQ ID NO: 200; (iii) QKKme3 at positions 24, 25, and 26 of SEQ ID NO: 200; and (iv) QYL at positions 28, 29, and 30 of SEQ ID NO: 200.

[0240] In some embodiments, this disclosure provides a composition comprising a Nectin-4 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: at least four cysteine ​​residues forming two disulfide bonds; at least one modified lysine residue at position X12 and / or X26 of SEQ ID NO: 195, wherein the modification comprises at least one small alkyl group of nitrogen linked to the lysine side chain, optionally including methyl, dimethyl, trimethyl, or isopropyl; a length of at least 44 amino acids; and a binding affinity to Nectin-4 greater than 100 nM in a cell-based assay.

[0241] In some embodiments, the peptide is at least 40 amino acids long but not more than 100 amino acids long. In some embodiments, in cell-based assays, the peptide binds to Nectin-4 with an affinity greater than 10 nM.

[0242] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with any one of SEQ ID NO: 3-158, 161-168, 177-208, or 212-215, but includes at least one lysine residue with at least one modification comprising at least one small alkyl group bonded to a nitrogen atom in the side chain, optionally selected from trimethyl, dimethyl, monomethyl, and isopropyl. 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, the reference polypeptide being longer than 44 amino acids, and binding to Nectin-4 with an intensity of at least 10 nM in cell-based assays, and / or having an inhibition constant of no more than 10 nM.

[0243] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with at least 40 amino acids of any one of SEQ ID NO: 3-158, 161-168, 177-208 or 212-215, provided that the 40 amino acids include at least four cysteine ​​residues that form two disulfide bridges.

[0244] In some embodiments, the amino acid sequence of the polypeptide shares at least 90% identity with at least 35 adjacent amino acids of any one of SEQ ID NO: 3-158, 161-168, 177-208, or 212-215, provided that the 40 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 44 amino acids.

[0245] In some embodiments, the amino acid sequence shares 90% identity with at least 44 amino acids described in any one of SEQ ID NO: 78, 83, 85, 99, 103, 162-168, 195 or 200.

[0246] 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: 78, 83, 85, 99, 103, 162-168, 195 or 200.

[0247] In some embodiments, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO: 195.

[0248] In some embodiments, this disclosure provides a composition comprising the compound described in C251 of Table 2A, the compound having an amino acid sequence comprising SEQ ID NO: 195.

[0249] In some embodiments, this disclosure provides a composition comprising a polypeptide having an amino acid sequence comprising SEQ ID NO: 200.

[0250] In some embodiments, this disclosure provides a composition comprising the compound described in C260 of Table 2A, the compound having an amino acid sequence comprising SEQ ID NO: 200.

[0251] 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.

[0252] In some embodiments, this disclosure provides a composition comprising a polypeptide having an amino acid sequence of at least 44 amino acids but having four amino acid substitutions at positions 12, 21, 26 and 32 corresponding to SEQ ID NO: 78, wherein the substitutions correspond to K12A, Y21Q, S26Kme3 and G32A.

[0253] In some implementations, the C-terminus has -OH or -NH2.

[0254] In some embodiments, the composition (e.g., a peptide) has a binding affinity for Nectin-4 greater than 100 nM.

[0255] In some implementations, the suppression constant is no greater than 100 nM.

[0256] In some embodiments, the composition further comprises one or more of a connector, a chelating agent, and a radionuclide.

[0257] In some embodiments, the connector comprises or consists 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 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

[0258] 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).

[0259] 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.

[0260] In some embodiments, if the polypeptide comprises any one of SEQ ID NO: 83, 85, 93, 99, 134, 138, 145, 155, 162-168 or 195, the polypeptide further comprises a linker, wherein the linker is PEG4, and an optional chelating agent, wherein the chelating agent is DOTA.

[0261] 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.

[0262] In some implementations, the C-terminal amino acid of the peptide is not cysteine.

[0263] In some implementations, the chelating agent is attached to the peptide or linker when present.

[0264] In some implementations, a radionuclide is attached to a chelating agent when present.

[0265] 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: 162-176, 178-208, or 212-215.

[0266] 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- or (PEG)n, wherein n is 1 to 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

[0267] 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).

[0268] 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.

[0269] 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 the sequences described in SEQ ID NO: 162-176, 178-208, or 212-215.

[0270] 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-carboxylic acid 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.

[0271] 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).

[0272] 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.

[0273] 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.

[0274] In some implementations, the C-terminal amino acid of the peptide is not cysteine.

[0275] In some implementations, the chelating agent is attached to the peptide or linker when present.

[0276] In some implementations, a radionuclide is attached to a chelating agent when present.

[0277] In some implementations, the polypeptide contains at least one disulfide bridge.

[0278] In some implementations, the polypeptide contains at least two disulfide bridges.

[0279] In some embodiments, the composition and / or its peptides selectively bind to Nectin-4 or a portion thereof.

[0280] In some embodiments, the binding affinity of the peptide to Nectin-4 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.

[0281] In some implementations, the binding inhibition constant of the peptide is no greater than 100 nM.

[0282] A composition comprising a polypeptide-drug conjugate, the polypeptide-drug conjugate comprising a polypeptide and at least one drug moiety, wherein the polypeptide comprises an amino acid sequence having at least 90% identity with at least 44 amino acids of a polypeptide having an amino acid sequence having any of the amino acid sequences described in SEQ ID NO: 3-158, 162-208 or 212-237.

[0283] In some embodiments, the drug portion is selected from V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, orrisstatin, sulphoside, 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, topoisomerase inhibitors, orrisstatin (e.g., monomethylorrisstatin E), and immunotoxins.

[0284] In some embodiments, this disclosure provides a composition comprising one or more isolated compounds or pharmaceutically acceptable salts thereof, including an optional linker (L) and 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: 3-158, 161-168, 171-208, 212-215, or 216-237, including amino acid substitutions as described in Tables 1C, 1D, 2C, 2D, 2E, 2F, or 2G.

[0285] In some embodiments, this disclosure provides a composition comprising a compound designed to bind to Nectin-4, the compound comprising or consisting of a polypeptide having an amino acid sequence comprising any one of SEQ ID NO: 3-158, 161-168, 171-208, 212-215 or 216-237, including amino acid substitutions as described in Tables 1C, 1D, 2C, 2D, 2E, 2F or 2G, and further comprising a modified N-terminus and / or C-terminus.

[0286] In some implementations, the modified N-terminus includes one or more of the following: NH2, acetyl, PEGn (where n = 0-10), DOTA, or biotin.

[0287] In some implementations, the C-terminus contains -NH2 or -OH.

[0288] In some implementations, the peptide selectively binds to Nectin-4 or a portion thereof.

[0289] In some implementations, the peptide exhibits a binding affinity for Nectin-4 or a portion thereof greater than about 100 nM, either in vivo or in cell-based assays.

[0290] Nectin-4 Nectin protein is involved in cell adhesion, migration, and polarization. Nectin-4 was found to be expressed uniformly at weak to moderate levels in most human organs, but was specifically overexpressed in most samples from metastatic urothelial, breast, lung, head and neck, and cervical tumors. An antibody-drug conjugate (ADC), namely veentumumab (“EV”; Padcev; Astellas; Tokyo, Japan; and Seattle Genetics; Bothell, WA, USA), specifically targets Nectin-4 overexpressed on the surface of bladder tumor cells. EV is conjugated with a microtubule inhibitor (monomethyloristatine E), which leads to G2 / M cell cycle arrest and apoptosis. In some embodiments, the microprotein of this disclosure targets Nectin-4 on tumor cells. (Bednova O. and Leyton JV. Int J Mol Sci. 2020 Oct 1; 21(19):7268).

[0291] Clinical trials of EV have demonstrated that, in some implementations, targeting Nectin-4 can be a whole or part of a successful treatment strategy for cancer, depending on the context (e.g., cancer type). For example, EV approval in the US followed results from Phase I and II clinical trials. In the Phase I study, the overall objective response rate (ORR) was 42% in patients who had previously received ICI therapy, and the ORR was 36% in patients with particularly high tumor burden (e.g., liver metastases). Furthermore, in the Phase II EV trial, patients with locally advanced or metastatic bladder cancer who had previously received platinum-based chemotherapy or ICI therapy received EV treatment, and all tumors were positive for Nectin-4 and described as having “strong” expression levels. PD-L1 expression was also evaluated, but EV treatment results showed an ORR of 44% and a complete response rate (CRR) of 12% at 10.2 months (median follow-up); PD-L1 status had no effect on ORR or CRR. This study demonstrates that Nectin-4 is a relevant target for bladder cancer, that PD-L1 status or therapy does not negatively affect the efficacy of Nectin-4-based therapies, and that it provides a targeted alternative or complementary therapy to ICI-based therapies.

[0292] EV is currently being explored in a Phase III study and is being developed in a Phase II study for use in combination with ICI therapy for patients who are not suitable for cisplatin therapy. (Bednova O. and Leyton JV. Int J Mol Sci. 2020 Oct 1;21(19): 7268).

[0293] In some implementations, Nectin-4 is an important target of the microproteins disclosed herein, whether used alone or in combination with one or more therapies.

[0294] In some embodiments, the compositions provided in this disclosure target cells overexpressing Nectin-4 (e.g., cancer cells) more specifically and effectively, while minimizing or eliminating damage to surrounding cells that do not express or overexpress Nectin-4 by providing a targeting composition that, in some embodiments, includes a chelating agent and / or an alpha emitter, which, when combined with the microproteins provided herein, provides a specific, efficient, and effective method for targeting target cells overexpressing Nectin-4.

[0295] Importantly, the novel compositions provided in this disclosure are able to specifically, efficiently, and effectively target Nectin-4 overexpressing cells, with reduced toxicity compared to currently available treatments. In other words, in some embodiments, the Nectin-4-targeting compositions provided in this disclosure offer improved treatment compared to currently available therapies.

[0296] In some embodiments, the target of the compositions of this disclosure comprises or consists of Nectin-4. In some embodiments, the basic and novel characteristics of the peptides provided herein are substantially altered to enable them to bind strongly and specifically to their intended targets (e.g., Nectin-4, e.g., Nectin-4 on cancer cells) with minimal or no off-target effects and minimal renal uptake (e.g., compared to renal uptake of previously developed Nectin-4 binding molecules). In some embodiments, Nectin-4 is expressed on the cell surface. 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 Nectin-4 expressed in tumor cells or a population of tumor cells is higher than the level of Nectin-4 expressed in non-tumor cells. In some embodiments, the microproteins provided of this disclosure or compositions containing microproteins target Nectin-4, which may specifically target the composition or one or more components thereof (e.g., chelating agents and / or radionuclides) to cancer cells or the tumor microenvironment (e.g., locations containing cancer cells or populations of cells at risk of becoming cancer cells).

[0297] In some embodiments, the microproteins according to this disclosure specifically bind to Nectin-4. Illustrative Nectin-4 microproteins are provided in Table 2A, and exemplary Nectin-4 sequences are provided in Table 1A, but are not limited thereto.

[0298] In some embodiments, the microproteins of this disclosure comprise or consist of polypeptide sequences corresponding to or bound to polypeptide sequences shown in Tables 1B, 1C, and / or 2A. In some embodiments, the microproteins comprise or consist of amino acid sequences having at least 85% identity with the polypeptide sequences shown in the tables. In some embodiments, the microproteins of this disclosure have at least 70%, 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 higher identity with the polypeptide sequences according to Tables 2A, 1B, 1C, and / or 1D.

[0299] In some embodiments, the compositions of this disclosure specifically bind to Nectin-4 (e.g., by specifically binding to a microprotein of Nectin-4). In some embodiments, the target is Nectin-4. In some such embodiments, Table 1B discloses a compound that targets Nectin-4.

[0300] In some embodiments, the composition comprising a microprotein comprises or consists of a protein containing a specific amino acid sequence that binds to Nectin-4 or a portion thereof. In some embodiments, certain exemplary Nectin-4 binding microproteins are... In some such embodiments, such Nectin-4 microproteins comprise or consist of an amino acid sequence selected from any one of SEQ ID NOs: 93, 99, 134, 138, 145, 155, and 178-215, or a functional variant or portion thereof (e.g., a functional fragment, such as a microprotein folded and bound to Nectin-4 or a portion thereof). In some embodiments, such Nectin-4 microproteins are binding proteins or part of conjugates comprising such binding proteins as described in Table 2A. In specific embodiments, such Nectin-4 microproteins comprise or consist of an amino acid sequence selected from any one of SEQ ID NOs: 99, 195, or 200, or a functional variant or portion thereof (e.g., a functional fragment, such as a microprotein folded and bound to Nectin-4 or a portion thereof). In some embodiments, the Nectin-4 microprotein comprises or consists of an amino acid sequence or a functional variant or portion thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or greater of any one of SEQ ID NO: 93, 99, 134, 138, 145, 155 or 178-215. In some embodiments, the Nectin-4 microprotein comprises or consists of an amino acid sequence or a functional variant or portion thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or greater of any one of SEQ ID NO: 99, 195 or 200.

[0301] In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 4. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 5. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 6. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 10. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 11. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 12. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 13. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 14. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 15. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 18. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 19. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 20. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 21. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 22.In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 23. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 24. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 25. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 26. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 27. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 28. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 29. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 30. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 31. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 32. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 33. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 34. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 35. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 36. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 37. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 38. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 39. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 40. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 41. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 42.In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 48. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 49. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 50. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 51. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 52. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 53. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 54. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 55. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 56. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 57. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 58. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 59. In some embodiments, the Nectin-4 microprotein comprises or is composed of the amino acid sequence according to SEQ ID NO: 60. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 61. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 62.In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 63. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 64. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 65. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 66. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 67. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 68. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 69. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 70. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 71. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 72. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 73. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 74. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 75. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 76. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 77. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 78. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 79. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 80. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 81. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 82.In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 83. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 84. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 85. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 86. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 87. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 88. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 89. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 90. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 91. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 92. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 93. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 94. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 95. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 96. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 97. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 98. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 99. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 100. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 101. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 102.In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 103. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 104. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 105. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 106. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 107. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 108. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 109. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 110. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 111. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 112. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 113. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 114. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 115. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 116. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 117. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 118. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 119. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 120. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 121. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 122.In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 123. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 124. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 125. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 126. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 127. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 128. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 129. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 130. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 131. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 132. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 133. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 134. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 135. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 136. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 137. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 138. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 139. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 140. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 141. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 142.In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 143. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 144. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 145. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 146. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 147. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 148. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 149. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 150. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 151. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 152. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 153. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 154. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 155. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 156. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 157. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 158. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 161. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 162. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 163. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 164.In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 165. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 166. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 167. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 168. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 177. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 178. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 179. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 180. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 181. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 182. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 183. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 184. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 185. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 186. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 187. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 188. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 189. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 190. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 191. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 192.In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 193. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 194. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 195. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 196. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 197. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 198. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 199. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 200. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 201. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 202. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 203. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 204. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 205. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 206. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 207. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 208. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 212. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 213. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 214. In some embodiments, the Nectin-4 microprotein comprises or consists of the amino acid sequence according to SEQ ID NO: 215.

[0302] In some embodiments, the Nectin-4 binding microprotein comprises or consists of an amino acid sequence according to any one of SEQ ID NO: 3-158, 161-168, 177-208, or 212-215, 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. N-terminal and / or C-terminal modifications of the Nectin-4 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, 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) 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, 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 Nectin-4 (e.g., for the detection and / or treatment of cancer).

[0303] In some embodiments, the decoy comprises or consists of the amino acid sequence according to SEQ ID NO: 209. In some embodiments, the decoy comprises or consists of the amino acid sequence according to SEQ ID NO: 210. In some embodiments, the decoy comprises or consists of the amino acid sequence according to SEQ ID NO: 211.

[0304] In some embodiments, the Nectin-4 microprotein comprises or consists of 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 of the same amino acid sequence or a functional variant or portion thereof as SEQ ID NO: 78, 93, 99, 134, 138, 145, 155, 161-168, 177-208 or 212-215. In some embodiments, the Nectin-4 microprotein comprises or consists of 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 of the same amino acid sequence or a functional variant or portion thereof as SEQ ID NO: 78, 93, 99, 134, 138, 145, 155, 194, 195, 200, 203 or 204. In some embodiments, the Nectin-4 microprotein comprises or consists of an amino acid sequence or a functional variant thereof that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or greater of any one of SEQ ID NOs: 99, 195, and 200. In some embodiments, the microproteins of SEQ ID NOs: 216-237 have one or more substitutions as described in Table 1C. In some embodiments, the microproteins of SEQ ID NOs: 170-176 or 243-246 have one or more substitutions as described in Table 1D.In some embodiments, the Nectin-4 microprotein comprises or consists of an amino acid sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or greater of any one of the amino acid sequences in SEQ ID NO: 216-237, or a functional variant or portion thereof having one or more substitutions as described in Tables 1C, 2C, 2D, 2E, 2F or 2G.

[0305] 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 a sequence encoding one or more components of the compositions provided herein. 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, e.g., microproteins comprising one or more of linkers, chelating agents, and radionuclides). In some embodiments, a therapeutic method includes administering a composition as provided herein to a subject in need.

[0306] 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.

[0307] In some embodiments, the polypeptide 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 polypeptide 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 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. In some embodiments, the polypeptide has an amino acid sequence that corresponds to the amino acid sequence of a given polypeptide, such as those described in Table 2A, in the sequence numbers 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. Or 45 adjacent amino acids 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.

[0308] In some embodiments, the peptide is a microprotein. In some such embodiments, the microprotein is capable of binding to targets such as those provided herein (e.g., Nectin-4 or a portion thereof).

[0309] In some embodiments, the microproteins of this disclosure comprise or consist of polypeptides capable of binding to targets as shown in Table 1A.

[0310] 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.

[0311] 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.

[0312] 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 any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246, 248 and / or 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% of the amino acid sequence described in Tables 1B and / or 1C, 1D, and / or 2A.

[0313] 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.

[0314] 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 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. 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 2A, 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, 4... 4 or 45 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.

[0315] In some embodiments, the polypeptide (e.g., a microprotein) has an amino acid sequence that has a certain percentage of identity at a certain coverage percentage (e.g., a reference sequence). That is, a Nectin-4 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%), where the identity percentage and coverage percentage may differ if the query molecule is shorter (e.g., 100% identity and 90% coverage). If the query molecule is longer, both the identity percentage and coverage can be 100% relative to the reference molecule, 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.

[0316] 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, ε-N-methyllysine, ε-N,N-dimethyllysine, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, ε-N-isopropyllysine, 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 with the left end corresponding to the amino terminus and the right end corresponding to the carboxyl terminus, conforming to standard usage and convention.

[0317] In some embodiments, the microproteins of this disclosure containing two or more cysteine ​​residues (such as those described in Table 2A) have cysteine ​​residues linked by disulfide bridges (e.g., by natural folding).

[0318] In some embodiments, the cysteine ​​linkage is located between positions corresponding to Cys1 and Cys34; and between positions corresponding to Cys20 and Cys44, such as those described in Table 2A (e.g., SEQ ID NO: 195). In some embodiments, the cysteine ​​linkage is between Cys1 and Cys20; and between Cys34 and Cys44. In some embodiments, the cysteine ​​linkage is between Cys1 and Cys44; and between Cys20 and Cys34.

[0319] In some embodiments, the one or more disulfide bridges comprise two or four cysteine ​​residues corresponding to positions 1, 20, 34, and 44 of SEQ ID NO: 195, wherein the cysteine ​​corresponding to position 1 can form a disulfide bridge with the cysteine ​​corresponding to positions 20, 34, or 44. In some embodiments, the cysteine ​​corresponding to position 20 can form a disulfide bridge with the cysteine ​​corresponding to positions 1, 34, or 44. In some embodiments, the cysteine ​​corresponding to position 34 can form a disulfide bridge with the cysteine ​​corresponding to positions 1, 20, or 44. In some embodiments, the cysteine ​​corresponding to position 44 can form a disulfide bridge with the cysteine ​​corresponding to positions 1, 20, or 44. In some embodiments, if four cysteines are present and correspond to positions 1, 20, 34, and 44 of SEQ ID NO: 195, the pairing may include pairing 1 with 34 and pairing 20 with 44; pairing 1 with 20 and pairing 34 with 44; or pairing 1 with 44 and pairing 20 with 34 (e.g., there is a disulfide bridge between the two paired cysteines).

[0320] In some embodiments, this disclosure provides a microprotein comprising or consisting of an amino acid sequence or a portion thereof or a functional variant thereof as described in SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246, 248 and / or as described in Tables 1B, 1C, 1D and / or 2A. 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 SEQ ID NO: 3-158 or 177-237 and / or as described in Tables 1B, 1C, 1D and / or 2A. 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 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: 3-158, 161-168, 170-208, 212-237, 243-246, 248 and / or as described in Tables 1B, 1C, 1D and / or 2A. 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 a difference of no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 amino acid residues compared to those described in Tables 1B, 1C, 1D, and / or 2A. In some embodiments, the microprotein comprising or consisting of a portion thereof or a functional variant thereof, as described in Tables 1B, 1C, 1D, and / or 2A, selectively binds to the target Nectin-4.

[0321] 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 document provides novel polypeptides (i.e., microproteins) and methods of using them. In some embodiments, the polypeptide comprises or is composed of a microprotein. In some such embodiments, the microprotein comprises or is composed of CDPs, knotting agents, and / or binding agents. In some embodiments, the microprotein is designed to be linked to one or more other components. For example, in some embodiments, the microprotein can 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.

[0322] 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.

[0323] 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 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 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.

[0324] 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.

[0325] In some embodiments, the microprotein comprises one or more disulfide bridges. In some embodiments, the microprotein comprises at least two disulfide bridges. In some embodiments, the microprotein comprises at least three cysteine ​​residues. In some embodiments, the microprotein comprises multiple (e.g., more than three) cysteine ​​residues. In some such embodiments, the cysteine ​​residues are cross-linked to maintain a highly stable folded state for peptides of their length (e.g., relative to peptides 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).

[0326] 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.

[0327] In some embodiments, microproteins (e.g., linear peptides, folded peptides (e.g., covalently linked peptides, non-covalently linked peptides, or peptides containing disulfide bonds), cysteine-dense peptides, knotting peptides, binders, affinity molecules, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankyrin repeating domains (DARPin), or avimers) have higher permeability than larger proteins. That is, in some embodiments, microproteins or compositions containing microproteins permeate 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.

[0328] 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.

[0329] In some embodiments, the microproteins or compositions comprising microproteins provided in this disclosure (e.g., conjugates, such as radionuclide conjugates) exhibit binding affinity for Nectin-4. In some embodiments, Nectin-4 is human Nectin-4. In some embodiments, human Nectin-4 is located on cells. In some embodiments, the cells are cell lines, primary cells, or cells within the human body (e.g., in tumors).

[0330] 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 Nectin-4. 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).

[0331] In some embodiments, the microprotein or its conjugates exhibit binding affinity (KD or Kd) for Nectin-4. In some embodiments, the binding affinity of the microprotein or its conjugates to human Nectin-4 is about 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, or stronger (e.g., 90 nM, 75 nM, 50 nM, 25 nM, 10 nM, 5 nM, etc.). In some embodiments, the 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 Nectin-4 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 nM, etc.). In some embodiments, the binding is selective for human Nectin-4 but not selective for, for example, non-human Nectin-4.

[0332] In some embodiments, the microprotein or its conjugates exhibit a binding inhibition constant. In some embodiments, the binding inhibition constant (Ki) with human Nectin-4 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, binding affinity to human Nectin-4 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 nM, etc.). In some embodiments, the binding is selective for human Nectin-4 but not selective for, for example, non-human Nectin-4.

[0333] In some embodiments, the microproteins or conjugates thereof provided in this disclosure have a high affinity for Nectin-4 (e.g., as measured by binding affinity and / or inhibition constant, etc.). In some such embodiments, Nectin-4 is human Nectin-4. 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).

[0334] 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 Nectin-4. 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.

[0335] In some embodiments, to ensure proper folding and linkage, selected cysteine ​​pairs may be replaced with selenocysteine. It can be considered that in some embodiments, diselenide crosslinks are more easily formed than disulfide crosslinks due to their lower redox potential, and such substitutions may allow for cross-linking of the remaining cysteine ​​residues.

[0336] 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.

[0337] In some embodiments, the microproteins of this disclosure comprise or consist of an antigen for generating an antibody that specifically binds to at least one epitope on Nectin-4. In some embodiments, such antibodies can be used for, for example, diagnostic purposes, blocking (e.g., antagonism), etc.

[0338] In some embodiments, the microprotein contains one or more disulfide bridges. In some embodiments, the microprotein contains at least two disulfide bridges.

[0339] 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.

[0340] In some embodiments, the microproteins or their conjugates provided herein are specific to the target. In some embodiments, the microproteins are specific to Nectin-4 or fragments thereof.

[0341] In some embodiments, such as the microproteins or their conjugates provided herein, the protein comprises or is composed of a specific amino acid sequence.

[0342] 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).

[0343] 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.

[0344] 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, symmetric dimethylarginine (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. 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, symmetrically dimethylated arginine (sRme2, Rme2s, or SDMA), nitroarginine (Arg(NO2)), or Leu-13C6,15N.

[0345] In some embodiments, the microproteins provided herein are specific to polypeptides or portions thereof having the amino acid sequences or functional variants described in Table 1A.

[0346] Table 1A: Exemplary target protein amino acid sequences

[0347] Certain Nectin-4 binding microproteins are known in the art. See, for example, SEQ ID NO: 1 and SEQ ID NO: 2 described below. Molecular weights (calculated and observed) are also provided herein.

[0348] This disclosure acknowledges that one problem with therapeutics that bind to Nectin-4 is the lack of sufficient specificity and affinity. Therefore, this article presents a microprotein capable of binding to Nectin-4 (e.g., on cells, such as cancer cells) strongly, efficiently, and specifically.

[0349] Another source of concern includes toxicity (e.g., nephrotoxicity). In some implementations, the binding specificity and strength of Nectin-4 in cancer cells reduces renal uptake.

[0350] 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: 3-158, 161-168, 170-208, 212-237, 243-246, and 248.

[0351] In some embodiments, the microprotein has an amino acid sequence that is identical to at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids of any one of SEQ ID NO 3-158, 161-168, 170-208, 212-237, 243-246, and 248, representing 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% of the amino acid sequence. In some embodiments, the microprotein has an amino acid sequence that is at least 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 of any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246, and 248. Alternatively, the 45 adjacent amino acids may be 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. 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%, 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: 170-176, 216-237, or 243-246.

[0352] In some embodiments, the microprotein has an amino acid sequence that is identical to at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids of any one of SEQ ID NO: 170-176, 216-237, or 243-246, representing 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% of the amino acid sequence.

[0353] In some embodiments, the microprotein has an amino acid sequence that has at least 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, or 45 adjacent amino groups to any one of SEQ ID NO: 170-176, 216-237, or 243-246. The acidity 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%.

[0354] 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%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to the amino acid sequences described in any one of Tables 1B, 1C, 1D, and / or 2A.

[0355] In some embodiments, the microprotein has an amino acid sequence that is identical to at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids of any one of the amino acid sequences described in any one of Tables 1B, 1C, 1D, and / or 2A, representing 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% of the amino acid sequences described in any one of Tables 1B, 1C, 1D, and / or 2A.

[0356] In some embodiments, the microprotein has an amino acid sequence that is at least 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, or 41 different from any of the amino acid sequences described in any one of Tables 1B, 1C, 1D, and / or Table 2A. 42, 43, 44 or 45 adjacent amino acids are 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.

[0357] 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.

[0358] In some embodiments, the microproteins provided in this disclosure are described using one or more common sequences provided in Table 1B.

[0359] In some embodiments, the microprotein having the sequences described in Table 1B has amino acid substitutions as provided in Table 1C.

[0360] Table 1B: Common sequences of illustrative Nectin-4 miniature proteins

[0361] Table 1C: Common Sequence Substitution

[0362] "Kme3" refers to trimethyllysine, "sRme2" refers to symmetrically dimethylated arginine, "Cit" refers to citrulline, "Arg(NO2)" refers to nitroarginine, "Norleu" refers to ortholeucine, and "OH-Norleu" refers to OH-ortholeucine.

[0363] Table 1D: Common sequences of exemplary Nectin-4 miniature proteins

[0364] “dD” refers to D-aspartic acid; “hR” refers to arginine; “hyP” refers to hydroxyproline; “1Nal” refers to 1-naphthylamine; “sRme2” refers to symmetrical dimethylarginine; “Cit” refers to citrulline; “Kme” refers to methyllysine; “Kme2” refers to dimethyllysine; “Kme3” refers to trimethyllysine; “Kipr” refers to Nε-isopropyl-L-lysine; “Dap” refers to diaminopimelic acid; “K(Ac)” refers to acetylated lysine; “RNO2” or “Arg(NO2)” refers to nitroarginine; “LCN” refers to Leu-13C6,15N; “NI” refers to hydroxyleucine; “Nle” refers to ortholeucine.

[0365] In some embodiments, this disclosure provides microproteins. In some embodiments, the microprotein is or comprises a polypeptide comprising: an amino acid sequence wherein the amino acid sequence comprises Formula III: CX2YX4X5X6FFTX10LX12X13LX15GX17DICX21YIX24X25X26FX28X29X30X31X32X33CIX36EIX39X40X41LGCX45 (SEQ ID NO: 216) Where X45 is an optional amino acid or carboxyl terminus containing -OH, and X2 is D or E; X4 is D or K; X5 is E or G; X6 is Q or E; X10 is A or E; X12 is A, K, E, or S; X13 is A, R, Q, K, S, or Cit; X15 is Y, R, or K; X17 is A, D, G, or S; X21 is D, Q, E, L, S, or Y; X24 is Q, L, K, or S; X25 is A, Q, E, K; X26 is A, Q, K, S, Y, T, D, R, (Kme3), (sRme2), Cit, Arg(NO2), OH-Norleu or Norleu; X28 is A, N, Q, D, K or S; X29 is N, T or Y; X30 is L, Y or V; X31 is P or E; X32 is A, D, Q, G or K; X33 is D, Q, E, I or L; X36 is Q, K or E; X39 is L or R; X40 is D, Q or E; X41 is N, K or Q; and X45 is S when present as an amino acid.

[0366] In some embodiments, the amino acid sequence satisfies one or more of the following: wherein if X4 is K, then X26 is K and X15 is Y; wherein if X4 is D, then X26 is one of N, T, D, R, (Kme3), (sRme2), Cit, or Arg(NO2); or X26 is K, X10 is E, X36 is K, and X39 is R; or wherein if the amino acid is leucine, then leucine may be L-leucine.

[0367] In some embodiments, the polypeptide according to any one of Formula III or Formulas IIIA-IIIP further comprises one or more of a linker, a chelating agent, and / or a radionuclide. In some embodiments, the linker may be or comprise a linker comprising or consisting of: polyethylene glycol (PEG) linkers (PEG4, PEG, PEG2, PEG6, PEG8, PEG12, PEG24, lys(MPB)-PEG4, PEG36), 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- (SEQ ID NO: 239) or (PEG)n, wherein n is 1 to 10, (Gly)1-10 (SEQ ID NO: 240), or any fragment thereof or a combination thereof linked by covalent bonds. In some embodiments, the chelating agent comprises or consists of: DOTA, DOPA, Macropa, PSC, 3-(tri-n-butyltinyl)benzoic acid N-succinimide (BuSTB), 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB), and Crown. 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.

[0368] In some embodiments, a linker is attached to the C-terminus of the peptide when present. 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 one or more additional N-terminal amino acids. In some embodiments, one or more amino acids are present at the N-terminus and / or C-terminus of the peptide.

[0369] In some embodiments, the microprotein containing any one of SEQ ID NO: 170-176, 216-237 or 243-246 and / or an amino acid sequence according to any one of Table 1B, Table 1C, Table 1D and / or Table 2A has at least one disulfide bridge.

[0370] In some embodiments, the microproteins comprising any one of SEQ ID NO: 170-176, 216-237 or 243-246 and / or the amino acid sequences according to any one of Table 1B, Table 1C, Table 1D and / or Table 2A have at least two disulfide bridges.

[0371] In some implementations, the microprotein has at least two disulfide bridges.

[0372] In some embodiments, disulfide bridges are formed between cysteine ​​1 and 34, and between cysteine ​​20 and 44. In some embodiments, the miniature protein containing four cysteines has two disulfide bridges, with Cys 1 linked to Cys 34 and Cys 20 linked to Cys 44. In some embodiments, the positions of the cysteines are relative to a reference sequence of a certain length (e.g., SEQ ID NO: 195 with 45 amino acids). For example, in some embodiments, the cysteine ​​corresponding to position 1 is linked to 20, 34 to 44, and so on, as provided herein.

[0373] In some embodiments, the microprotein inserts one or more additional amino acids into the amino acids within the CDP loop region (amino acids in the secondary structure loop between linked cysteine ​​residues, for example, amino acids in the structure formed between Cys 1 and Cys 34 and between Cys 20 and Cys 44). As those skilled in the art will understand, conformational preservation is crucial for binding affinity and behavior; therefore, in some embodiments, any addition within the CDP loop region can be made in a manner that maintains appropriate conformation and stability, such that binding affinity is not reduced and / or conformation or stability is not impaired or disrupted.

[0374] In some embodiments, the polypeptide is a monomer. In some embodiments, the polypeptide is a dimer, trimer, or tetramer. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 78. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 3-158 or 177-215. In some embodiments, the polypeptide comprises a common sequence according to any one of formulas IIIA-IIIP (SEQ ID NO: 216-237) in Table 1B, and also comprises substitutions according to those described in Table 1C. In some embodiments, the polypeptide has an amino acid sequence conforming to the definition described in Table 1D. In some embodiments, the polypeptide has an amino acid sequence conforming to the definition of any one or more of SEQ ID NO: 170-176, 216-237, or 243-246.

[0375] In some embodiments, sequences containing variable positions (e.g., as shown in Table 1B) may also include substitutions for any homologous amino acids. As used herein, a homologous amino acid is an amino acid that has one or more similar characteristics to another amino acid, such as a similar charge (e.g., a negatively charged amino acid, referring to the charge at physiological pH), or in some embodiments, a set of substitutions (e.g., more than one amino acid) that produces a charge distribution similar to that of the polypeptide preceding the substitution or the set of substitutions, and so on.

[0376] Table 2A. Illustrative microprotein sequences and compound structures

[0377]

[0378] #Note: In all the sequences shown, Cys1 is linked to Cys34, and Cys20 is linked to Cys44.

[0379] The compounds marked with In in Table 2A are cold metal labeled, using "natural abundance" indium (also known as...) nat The indium may be labeled with 113-In and 115-In, and is different from 111-In, which can be used as a radioactive label for the conjugates provided herein.

[0380] DOTA-PEG4: α-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ester)-4(ethylene glycol) DOTA-PEG8: α-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ester)-8(ethylene glycol) DOTA-PEG12: α-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ester)-12(ethylene glycol) Biotin-PEG4: FITCI-PEG4: Fluorescein isothiocyanate-4 (ethylene glycol) Table 2B. Binding affinity of illustrative compounds to Nectin-4

[0381] In some embodiments, the microproteins of this disclosure exhibit binding specificity for human Nectin-4. For example, in some embodiments, the microproteins provided in this disclosure, such as those represented by any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246, and 248, have been shown to bind when expressed on a yeast surface and to bind to Nectin-4 by flow cytometry. In some embodiments, the microproteins provided in this disclosure, such as any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248 or those represented according to Tables 1B, 1C, 1D and / or 2A, are demonstrated by flow cytometry to have binding specificity, for example when such Nectin-4 microproteins (e.g., such as any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248 or those represented according to Tables 1B, 1C, 1D and / or 2A) bind only to Nectin-4 and not to other antigens.

[0382] In some embodiments, the microproteins of this disclosure, such as any of those represented by SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248, or according to Tables 1B, 1C, 1D, 2A, 2B and / or 2C, exhibit potency greater than 10 nM. In some embodiments, the microproteins exhibit potency greater than 1, 2, 3, 4, 5, 6, 7, 8, 9 nM or higher.

[0383] In some embodiments, the microprotein is part of a conjugate containing one or more modifications or components (e.g., as provided herein (see, for example, Table 2A)).

[0384] In some embodiments, the microproteins provided in this disclosure are stated using a common sequence as provided in Formula IV: CX2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19CX21X22X23X24X25X26X27X28X29X30X31X32X33CX35X36X37X38X39X40X41X42X43CS (SEQ ID NO: 233) in X2 is E, G, A, S, T, D, N, K, R, Y, F, V, I, or W; X3 is Y, P, G, A, S, T, Q, K, H, F, W, V, M, D, E, L, or I; X4 is D, S, N, R, Y, L, M, G, T, or I; X5 is E, G, D, Q, F, W, V, L, I, M, K, or R; X6 is E, P, G, D, Q, N, K, Y, F, W, or V; X7 is F, Y, or W; X8 is F, Y, W, I, or M; X9 is T, P, A, S, D, Q, N, K, Y, F, V, M, E, R, or L; X10 is A, S, E, N, K, F, W, V, L, I, G, T, D, Q, R, or H; X11 is L, T, I, or M. X12 is K, L, G, A, S, T, N, R, H, F, W, V, I, or M; X13 is R, G, A, S, E, N, K, Y, W, L, I, or M; X14 is L, T, V, I, or M; X15 is R, T, Q, K, L, I, M, or V; X16 is G, D, N, K, R, H, Y, W, V, L, I, or M X17 is G, P, A, S, T, D, Q, N, R, H, Y, F, W, V, L, or M; X18 is D, P, G, A, T, E, Q, N, K, Y, F, W, V, I, M, or S; X19 is I, P, A, T, D, N, Y, V, M, G, or Q; X21 is Y, S, T, D, E, K, F, W, L, I, or V; X22 is Y or E; X23 is I, A, T, Y, V, L, M or F; X24 is Q, P, G, D, K, R, H, F, V, L, I, M or N; X25 is A, P, S, D, E, Q, K, R, H, Y, F, W, V, I, M or T; X26 is S, P, G, A, D, Q, N, R, F, V, L, I, T, E or W; X27 is F, P, A, S, K, H, Y, W, M, E or V; X28 is Q, P, G, A, S, D, E, K, R, H, Y, W, V, L, F or I; X29 is Y, P, A, S, E, Q, N, K, F, W, L, I, M, T or V; X30 is L, P, G, A, S, T, E, N , R, H, F, W, V, I, M, Q or K; X31 is P, G, A, T, D, E, Q, N, K, R, H, Y, V, S, W, L or I; X32 is G, A, S, D, N, R, H, Y, L, I or V; X33 is L, P, G, A, S, D, E, N, K, R, H, Y, F, W, V, I or M; 35 represents I, P, S, D, E, N, K, R, Y, F, W, L, or M; X36 represents E, G, A, S, T, D, Q, K, R, H, Y, W, L, I, or M; X37 represents E, P, G, A, S, T, N, K, R, H, Y, F, W, V, or D; X38 represents I, A, S, E, Y, F, W, V, L, T, or H.X39 is L, S, T, E, N, R, H, Y, F, W, V, M, D, or I; X40 is D, G, A, S, E, Q, R, H, F, W, V, L, I, M, N, or K; X41 is N, G, A, S, D, Q, R, H, Y, F, V, L, I, M, P, or K; X42 is I, L, P, G, Q, N, H, F, W, I, M, or S; and X43 is G, P, A, T, D, N, K, R, Y, F, W, V, I, or Q.

[0385] In some embodiments, the common sequence of Formula IV is summarized in Table 2C. In some embodiments, the microproteins provided in this disclosure are stated with the common sequence as provided in Formula IV, wherein X2-X43 comprises any of the amino acid residues of SEQ ID NO: 78 or any of the permitted substitutions in Table 2C, and / or any of the preferred substitutions in Table 2C. In some embodiments, the microproteins provided in this disclosure are stated with the common sequence as provided in Formula IV, wherein X2-X43 comprises any of the amino acid residues of SEQ ID NO: 78 and / or any of the preferred substitutions in Table 2C. In some embodiments, the microproteins provided in this disclosure comprise SEQ ID NO: 78, which comprises a single permitted substitution shown in Table 2C. In some embodiments, the microproteins provided in this disclosure comprise SEQ ID NO: 78, which comprises a single preferred substitution shown in Table 2C. In some embodiments, the microproteins provided in this disclosure comprise SEQ ID NO: 78, which comprises two or more permitted substitutions shown in Table 2C. In some embodiments, the microproteins provided in this disclosure comprise SEQ ID NO: 78, which includes 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, as shown in Table 2C. 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, or 42 or more permitted substitutions. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 78, which comprises a single preferred substitution shown in Table 2C. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 78, which comprises two, three, four, or five preferred substitutions shown in Table 2C.

[0386] Table 2C. Common sequences and substitutions of Formula IV

[0387] In some embodiments, the microproteins provided in this disclosure are stated using a common sequence as provided in Formula V: CX2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19CX21X22X23X24X25X26X27X28X29X30X31X32X33CX35X36X37X38X39X40X41X42X43CS (SEQ ID NO: 234) Where X2 is E, P, G, A, S, T, Q, N, K, R, H, Y, F, W, V, L, I, or M; X3 is Y, P, G, A, S, T, D, E, Q, N, K, R, H, F, W, V, L, I, or M; X4 is D, P, G, A, S, T, E, Q, N, K, R, H, Y, F, W, V, L, I, or M; X 5 is E, P, G, S, T, D, Q, N, K, R, H, F, W, V, L, I, M, or Y; X6 is E, P, G, A, S, T, D, Q, N, K, R, H, F, W, V, L, I, M, or Y; X7 is F, Y, or W; X8 is F, T, Y, W, V, L, I, or M; X9 is T, P, G, S, D, E, N, K, R, H, Y, F, V, L, I, M, A, Q, or W; X10 is A, P, G, S, T, D, E, Q, N, K, R, H, Y, F, W, V, L, I, or M; X11 is L, A, V, I, or M; X12 is A, G, S, D, E, Q, N, K, R, H, Y, F, W, V, L, I, M, or T; X13 is... R, P, S, T, D, E, Q, K, H, Y, F, V, L, M, G, A, N, W, or I; X14 is L, A, T, F, V, I, or M; X15 is R, Q, Y, F, W, V, L, I, M, or K; X16 is G, P, A, S, T, D, E, Q, N, K, R, H, Y, F, W, V, L, M, or I; X1 7 is G, P, A, S, T, D, E, Q, N, K, R, H, Y, W, V, L, I, M or F; X18 is D, P, G, A, S, T, E, Q, N, K, R, H, Y, W, V, I, M, F or L; X19 is I, P, G, A, S, T, D, E, Q, N, K, R, Y, F, W, V, L, M or H; X21 is Y, P, G, A, S, T, D, E, Q, N, K, R, H, F, W, V, L, I, or M; X22 is Y, H, or F; X23 is I, G, A, S, T, Y, W, V, L, M, or F; X24 is Q, G, S, T, D, E, N, K, R, H, F, W, V, L, I, M, P, A, or Y; X25 is... A, G, S, T, D, E, Q, N, K, R, H, Y, F, W, V, M, P, L or I; X26 is K, P, G, A, S, T, D, E, Q, N, R, H, Y, F, W, V, L, I or M; X27 is F, P, G, A, S, T, D, E, Q, N, K, R, H, Y, W, V, L, I or M; X2 8 is Q, P, G, A, S, T, D, E, N, K, R, H, Y, F, W, V, L, I or M; X29 is Y, P, G, A, S, T, D, E, N, K, R, H, F, W, V, L, I, M or Q; X30 is L, P, G, A, S, T, D, E, Q, N, K, R, H, Y, F, W, V, I or M;X31 is P, G, A, S, T, D, Q, N, K, R, H, Y, F, V, I, M, E, W or L; X32 is G, P, A, S, T, D, E, Q, N, K, R, H, Y, F, W, V, L, I or M; X33 is L, P, G, A, S, T, D, E, Q, N, K, R, H, Y, F, W, V, I or M; X35 is I, P, G, A, S, T, D, E, Q, N, K, R, Y, F, W, V, L, M or H; N, R, H, F, V, L, I, M, T, K, Y or W; X37 is E, P, G, A, S, T, D, N, K, H, Y, F, W, V, L, I, M, Q or R; X38 is I, P, G, A, S, T, E, Q, N, K, H, Y, F, W, V, L or M; X39 is L, G, A, S, T, D, E, Q, N, K, R, H, Y, W, V, I, M or F; X40 is D, P, G, A, S, E, Q, N, K, R, H, Y, F, W, V, L, I, M or T; X41 is N, P, G, A, S, T, D, E, Q, K, R, H, Y, F, W, V, L, I, or M; X42 is L, G, A, S, T, E, Q, N, K, R, H, Y, F, W, V, I, or M; and X43 is G, P, A, S, T, D, E, Q, N, K, R, H, Y, F, W, V, L, M, or I.

[0388] In some embodiments, the common sequence of Formula V is summarized in Table 2D. In some embodiments, the microprotein provided in this disclosure is stated with the common sequence as provided in Formula V, wherein X2-X43 comprises the amino acid residue of SEQ ID NO: 83, any one of the permitted substituted amino acids in Table 2D, and / or any one of the preferred substitutions in Table 2D. In some embodiments, the microprotein provided in this disclosure is stated with the common sequence as provided in Formula V, wherein X2-X43 comprises the amino acid residue of SEQ ID NO: 83 or any one of the preferred substitutions in Table 2D. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 83, which comprises a single permitted substitution shown in Table 2D. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 83, which comprises a single preferred substitution shown in Table 2D. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 83, which comprises two or more permitted substitutions shown in Table 2D. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 83, which includes 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, as shown in Table 2D. 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, or 42 or more permitted substitutions. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 83, which comprises a single preferred substitution shown in Table 2D. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 83, which comprises two, three, four, or five preferred substitutions shown in Table 2D.

[0389] Table 2D. Common sequences and substitutions in Equation V

[0390] In some embodiments, the microproteins provided in this disclosure are stated using a common sequence as provided in Formula VI: CX2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19CX21X22X23X24X25X26X27X28X29X30X31X32X33CX35X36X37X38X39X40X41X42X43CS (SEQ ID NO: 235) Where X2 is E, P, G, A, S, T, D, Q, N, K, R, H, Y, W, V, L, I, M, or F; X3 is Y, P, G, A, S, T, D, E, Q, K, R, H, F, W, V, L, I, M, or N; X4 is D, P, A, S, E, Q, N, or H; X5 is E, P, G, A, T, Q, N, K, R , H, Y, F, W, V, L, I, M, S or D; X6 is Q, P, G, A, S, T, D, E, N, K, R, H, Y, F, W, V, L, I or M; X7 is F, Y or W; X8 is F, Y, W, V, I or M; X9 is T, P, G, A, S, D, Q, N, R, H, Y, F, V, L, I, M, E, K Or W; X10 is A, G, T, D, E, Q, N, K, R, H, W, L, I, M, S, Y, F, or V; X11 is L, V, I, or M; X12 is A, G, S, T, D, K, R, Y, F, W, V, L, I, M, E, Q, N, or H; X13 is R, G, A, S, T, D, Q, N, K, H, Y, F, W X14 is L, T, V, I, or M; X15 is R, Q, K, I, or M; X16 is G, A, T, D, E, Q, N, K, R, H, Y, W, V, L, I, M, P, S, or F; X17 is G, P, A, S, T, D, E, Q, N, K, R, H, Y, F, W, V, L, I, or M; X X18 is D, P, G, A, S, T, E, N, K, H, Y, F, L, M, Q, or K; X19 is I, G, A, S, T, E, Q, N, K, R, H, Y, F, W, V, L, or M; X21 is Y, P, G, A, S, D, E, Q, N, K, R, H, F, W, V, L, I, M, or T; X22 is Y; X2 3 is I, A, T, Y, F, W, V, L or M; X24 is Q, P, A, S, D, E, N, K, H, Y, F, W, V, L, I, G, T, R or M; X25 is E, P, G, A, S, T, D, Q, N, R, H, Y, F, W, V, L, I, M or K; X26 is Q, G, A, S, T, E, N, K, R, H, F, W, V, L, I, M, D, or Y; X27 is F, Y, W, L, I, M, or V; X28 is A, P, G, S, T, D, E, Q, N, K, R, H, Y, W, V, L, I, M, or F; X29 is T, P, G, A, S, D, E, Q, K, R, H, Y, F, W, V, L, I, M, or N; X3 0 is V, P, G, A, T, D, E, N, K, R, H, Y, F, W, I, M, S, Q or L; X31 is P, G, A, S, T, D, E, Q, N, K, R, H, Y, F, W, V, L, I or M; X32 is G, P, A, T, D, E, Q, N, K, R, H, Y, F, W, V, L, I, M or S;X33 is L, P, G, A, S, T, D, E, Q, K, R, H, Y, F, W, V, I, M, or N; X35 is I, P, G, A, S, T, D, E, Q, N, K, R, H, Y, F, W, L, M, or V; X36 is E, P, G, A, S, T, D, N, R, H, F, W, V, L, I, M, K, or Y; X37 is E, P, G, A, S, T, D, N, R, H, Y, W, V, L, I, M, Q, K, or F; X38 is I, G, A, S, E, Q, H, Y, W, V, L, M, P, or F; X3 9 is L, G, A, S, T, D, E, Q, N, K, H, Y, F, W, V, I, M or R; X40 is D, P, G, A, S, T, E, Q, N, R, H, Y, F, W, L, I, M or K; K, R, H, Y, F, W, V, L, I, or M; X42 is L, G, A, S, T, E, Q, R, H, Y, F, V, I, M, N, or W; and ;

[0391] In some embodiments, the common sequence of Formula VI is summarized in Table 2E. In some embodiments, the microproteins provided in this disclosure are stated with the common sequence of Formula VI, wherein X2-X43 comprises the amino acid residue of SEQ ID NO: 85, any one of the permitted substituted amino acids in Table 2E, and / or any one of the preferred substitutions in Table 2E. In some embodiments, the microproteins provided in this disclosure are stated with the common sequence of Formula VI, wherein X2-X43 comprises the amino acid residue of SEQ ID NO: 85 and / or any one of the preferred substitutions in Table 2E. In some embodiments, the microproteins provided in this disclosure comprise SEQ ID NO: 85, which comprises a single permitted substitution shown in Table 2E. In some embodiments, the microproteins provided in this disclosure comprise SEQ ID NO: 85, which comprises a single preferred substitution shown in Table 2E. In some embodiments, the microproteins provided in this disclosure comprise SEQ ID NO: 85, which comprises two or more permitted substitutions shown in Table 2E. In some embodiments, the microproteins provided in this disclosure comprise SEQ ID NO: 85, which includes 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, as shown in Table 2E. 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, or 42 or more permitted substitutions. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 85, which comprises a single preferred substitution shown in Table 2E. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 85, which comprises two, three, four, or five preferred substitutions shown in Table 2E.

[0392] Table 2E. Formula VI Common Sequences and Substitutions

[0393] In some embodiments, the common sequence of Formula VII is summarized in Table 2F. In some embodiments, the microprotein provided by this disclosure is stated with the common sequence of Formula VII, wherein X2-X43 comprises the amino acid residue of SEQ ID NO: 195, any one of the permitted substituted amino acids in Table 2F, and / or any one of the preferred substitutions in Table 2F. In some embodiments, the microprotein provided by this disclosure is stated with the common sequence of Formula VII, wherein X2-X43 comprises the amino acid residue of SEQ ID NO: 195 and / or any one of the preferred substitutions in Table 2F. In some embodiments, the microprotein provided by this disclosure comprises SEQ ID NO: 195, which comprises a single permitted substitution shown in Table 2F. In some embodiments, the microprotein provided by this disclosure comprises SEQ ID NO: 195, which comprises a single preferred substitution shown in Table 2F. In some embodiments, the microprotein provided by this disclosure comprises SEQ ID NO: 195, which comprises two or more permitted substitutions shown in Table 2F. In some embodiments, the microprotein provided by this disclosure comprises SEQ ID NO: 195, which includes 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more as shown in Table 2F The permitted substitutions include 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, or 42 or more. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 195, which includes a single preferred substitution shown in Table 2F. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 195, which includes two, three, four, or five preferred substitutions shown in Table 2F.

[0394] Table 2F. Common sequences and substitutions in Equation VII

[0395] In some implementations, the common sequence of formula VIII is summarized in Table 2G. Here, the difference between Table 2G and Table 2F is that in Table 2G, the substituted amino acid at position X24 of formula VIII is trimethyllysine (Kme3), while in Table 2F, the substituted amino acid at position X26 of formula VII is lysine (K).

[0396] In some embodiments, the microprotein provided in this disclosure is stated with a common sequence as provided in Formula VIII, wherein X2-X43 comprises any of the amino acid residues of SEQ ID NO: 103, any of the permitted substituted amino acids in Table 2G, and / or any of the preferred substitutions in Table 2G. In some embodiments, the microprotein provided in this disclosure is stated with a common sequence as provided in Formula VIII, wherein X2-X43 comprises any of the amino acid residues of SEQ ID NO: 103 and / or any of the preferred substitutions in Table 2G. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 103, which comprises a single permitted substitution shown in Table 2G. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 103, which comprises a single preferred substitution shown in Table 2G. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 103, which comprises two or more permitted substitutions shown in Table 2G. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 103, which includes 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more as shown in Table 2G. The permitted substitutions include 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, or 42 or more. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 103, which includes a single preferred substitution shown in Table 2G. In some embodiments, the microprotein provided in this disclosure comprises SEQ ID NO: 103, which includes two, three, four, or five preferred substitutions shown in Table 2G.

[0397] Table 2. Common sequences and substitutions of Formula VIII.

[0398] In some embodiments, the microproteins of this disclosure exhibit binding specificity for human Nectin-4. In some embodiments, the microproteins are characterized by binding affinity in the range of about 500 nM to about 1 pM, for example, binding affinity to human Nectin-4 of 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nM, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM. Not bound by theory, this disclosure takes into account that, in some embodiments, the preferred dissociation constant of the microprotein is about 10 nM or less, about 7.5 nM or less, about 5 nM or less, about 2.5 nM or less, or about 1 nM or less (i.e., in the picomolar range). For clarity, a stronger binding agent may be one with a binding affinity of 2.5 nM for Nectin 4 rather than one with a binding affinity of 5 nM, and a weaker binding agent may be one with a binding affinity of 5 nM for Nectin-4 rather than one with a binding affinity of 2.5 nM for Nectin-4.

[0399] In some embodiments, the microproteins of this disclosure bind to Nectin-4 with a binding affinity of about 1 pM to 100 nM.

[0400] In some embodiments, the microproteins of this disclosure bind to Nectin-4 with the following binding affinities: about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 pM; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nM. In some embodiments, the microproteins of this disclosure bind to Nectin-4 with binding affinity of about 1 pM to 100 pM, 10 pM to 1 nM, 100 pM to 10 nM, or 1 nM to 100 nM.

[0401] In some embodiments, the binding affinity (K0) of the polypeptides provided herein (or compositions comprising such polypeptides, such as microproteins) is... D (or Kd) in the range of approximately 1 pM to 100 nM.

[0402] In some implementations, the binding affinity is about 1 pM to 100 nM, 1 pM to 100 pM, 10 pM to 1 nM, 100 pM to 10 nM, or 1 nM to 100 nM. In some implementations, the binding affinity is approximately 1 pM to 5 pM, 1 pM to 10 pM, 1 pM to 15 pM, 1 pM to 20 pM, 1 pM to 25 pM, 1 pM to 30 pM, 1 pM to 35 pM, 1 pM to 40 pM, 1 pM to 45 pM, 1 pM to 50 pM, 1 pM to 55 pM, 1 pM to 60 pM, 1 pM to 65 pM, 1 pM to 70 pM, 1 pM to 75 pM, 1 pM to 80 pM, 1 pM to 85 pM, 1 pM to 90 pM, 1 pM to 95 pM, or 1 pM to 100 pM. In some implementations, the binding affinity is approximately 25 pM to 30 pM, 25 pM to 35 pM, 25 pM to 40 pM, 25 pM to 45 pM, 25 pM to 50 pM, 25 pM to 55 pM, 25 pM to 60 pM, 25 pM to 65 pM, 25 pM to 70 pM, 25 pM to 75 pM, 25 pM to 80 pM, 25 pM to 85 pM, 25 pM to 90 pM, 25 pM to 95 pM, or 25 pM to 100 pM. In some embodiments, the binding affinity is about 50 pM to 55 pM, 50 pM to 60 pM, 50 pM to 65 pM, 50 pM to 70 pM, 50 pM to 75 pM, 50 pM to 80 pM, 50 pM to 85 pM, 50 pM to 90 pM, 50 pM to 95 pM, or 50 pM to 100 pM. In some embodiments, the binding affinity is about 75 pM to 80 pM, 75 pM to 85 pM, 75 pM to 90 pM, 75 pM to 95 pM, or 75 pM to 100 pM. In some embodiments, the binding affinity is about 10 pM to 1 nM, 100 pM to 1 nM, 200 pM to 1 nM, 300 pM to 1 nM, 400 pM to 1 nM, 500 pM to 1 nM, 600 pM to 1 nM, 700 pM to 1 nM, 800 pM to 1 nM, or 900 pM to 1 nM. In some embodiments, the binding affinity is about 1 nM to 100 nM, 10 nM to 100 nM, 20 nM to 100 nM, 30 nM to 100 nM, 40 nM to 100 nM, 50 nM to 100 nM, 60 nM to 100 nM, 70 nM to 100 nM, 80 nM to 100 nM, or 90 nM to 100 nM.In some implementations, the binding affinity is about 5 nM to 100 nM, 10 nM to 100 nM, 15 nM to 100 nM, 20 nM to 100 nM, 25 nM to 100 nM, 30 nM to 100 nM, 35 nM to 100 nM, 40 nM to 100 nM, 45 nM to 100 nM, 50 nM to 100 nM, 55 nM to 100 nM, 60 nM to 100 nM, 65 nM to 100 nM, 70 nM to 100 nM, 75 nM to 100 nM, 80 nM to 100 nM, 85 nM to 100 nM, 90 nM to 100 nM, or 95 nM to 100 nM.

[0403] In some embodiments, the binding affinity is about 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, or 100 pM. In some embodiments, the binding affinity is about 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM, 950 pM, or 1 nM. In some embodiments, the binding affinity is about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or 10 nM. In some embodiments, the binding affinity is about 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, or 100 nM.

[0404] In some embodiments, the binding affinity is about 1 pM. In some embodiments, the binding affinity is about 2 pM. In some embodiments, the binding affinity is about 3 pM. In some embodiments, the binding affinity is about 4 pM. In some embodiments, the binding affinity is about 5 pM. In some embodiments, the binding affinity is about 10 pM. In some embodiments, the binding affinity is about 15 pM. In some embodiments, the binding affinity is about 20 pM. In some embodiments, the binding affinity is about 25 pM. In some embodiments, the binding affinity is about 30 pM. In some embodiments, the binding affinity is about 35 pM. In some embodiments, the binding affinity is about 40 pM. In some embodiments, the binding affinity is about 45 pM. In some embodiments, the binding affinity is about 50 pM. In some embodiments, the binding affinity is about 55 pM. In some embodiments, the binding affinity is about 60 pM. In some embodiments, the binding affinity is about 65 pM. In some embodiments, the binding affinity is about 70 pM. In some embodiments, the binding affinity is about 75 pM. In some embodiments, the binding affinity is about 80 pM. In some embodiments, the binding affinity is about 85 pM. In some embodiments, the binding affinity is about 90 pM. In some embodiments, the binding affinity is about 95 pM. In some embodiments, the binding affinity is about 100 pM. In some embodiments, the binding affinity is about 150 pM. In some embodiments, the binding affinity is about 200 pM. In some embodiments, the binding affinity is about 250 pM. In some embodiments, the binding affinity is about 300 pM. In some embodiments, the binding affinity is about 350 pM. In some embodiments, the binding affinity is about 400 pM. In some embodiments, the binding affinity is about 450 pM. In some embodiments, the binding affinity is about 500 pM. In some embodiments, the binding affinity is about 550 pM. In some embodiments, the binding affinity is about 600 pM. In some embodiments, the binding affinity is about 650 pM. In some embodiments, the binding affinity is about 700 pM. In some embodiments, the binding affinity is about 750 pM. In some embodiments, the binding affinity is about 800 pM. In some embodiments, the binding affinity is about 850 pM. In some embodiments, the binding affinity is about 900 pM. In some embodiments, the binding affinity is about 950 pM. In some embodiments, the binding affinity is about 1 nM. In some embodiments, the binding affinity is about 2 nM. In some embodiments, the binding affinity is about 3 nM. In some embodiments, the binding affinity is about 4 nM.In some embodiments, the binding affinity is about 5 nM. In some embodiments, the binding affinity is about 6 nM. In some embodiments, the binding affinity is about 7 nM. In some embodiments, the binding affinity is about 8 nM. In some embodiments, the binding affinity is about 9 nM. In some embodiments, the binding affinity is about 10 nM. In some embodiments, the binding affinity is about 15 nM. In some embodiments, the binding affinity is about 20 nM. In some embodiments, the binding affinity is about 25 nM. In some embodiments, the binding affinity is about 30 nM. In some embodiments, the binding affinity is about 35 nM. In some embodiments, the binding affinity is about 40 nM. In some embodiments, the binding affinity is about 45 nM. In some embodiments, the binding affinity is about 50 nM. In some embodiments, the binding affinity is about 55 nM. In some embodiments, the binding affinity is about 60 nM. In some embodiments, the binding affinity is about 65 nM. In some embodiments, the binding affinity is about 70 nM. In some embodiments, the binding affinity is about 75 nM. In some embodiments, the binding affinity is about 80 nM. In some embodiments, the binding affinity is about 85 nM. In some embodiments, the binding affinity is about 90 nM. In some embodiments, the binding affinity is about 95 nM. In some embodiments, the binding affinity is about 100 nM.

[0405] In some embodiments, the binding affinity is stronger than about 100 nM (e.g., 90 nM, 80 nM, etc.). In some embodiments, the binding affinity is not weaker than about 0.5 nM, 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, or 100 nM. In some embodiments, the binding affinity is not weaker than about 0.5 nM. In some embodiments, the binding affinity is not greater than about 1 nM. In some embodiments, the binding affinity is not weaker than about 5 nM. In some embodiments, the binding affinity is not weaker than about 10 nM. In some embodiments, the binding affinity is not weaker than about 15 nM. In some embodiments, the binding affinity is not weaker than about 20 nM. In some embodiments, the binding affinity is not weaker than about 25 nM. In some embodiments, the binding affinity is not weaker than about 30 nM. In some embodiments, the binding affinity is not weaker than about 35 nM. In some embodiments, the binding affinity is not weaker than about 40 nM. In some embodiments, the binding affinity is not weaker than about 45 nM. In some embodiments, the binding affinity is not weaker than about 50 nM. In some embodiments, the binding affinity is not weaker than about 55 nM. In some embodiments, the binding affinity is not weaker than about 60 nM. In some embodiments, the binding affinity is not weaker than about 65 nM. In some embodiments, the binding affinity is not weaker than about 70 nM. In some embodiments, the binding affinity is not weaker than about 75 nM. In some embodiments, the binding affinity is not weaker than about 80 nM. In some embodiments, the binding affinity is not weaker than about 85 nM. In some embodiments, the binding affinity is not weaker than about 90 nM. In some embodiments, the binding affinity is not weaker than about 95 nM. In some embodiments, the binding affinity is not weaker than 100 nM.

[0406] In some embodiments, the inhibition constant (Ki) of the peptide (or composition comprising such peptide) provided herein is in the range of about 1 pM to about 100 nM.

[0407] In some implementations, the suppression constant (Ki) is about 1 pM to 100 nM, 1 pM to 100 pM, 10 pM to 1 nM, 100 pM to 10 nM, or 1 nM to 100 nM. In some implementations, the suppression constant is approximately 1 pM to 5 pM, 1 pM to 10 pM, 1 pM to 15 pM, 1 pM to 20 pM, 1 pM to 25 pM, 1 pM to 30 pM, 1 pM to 35 pM, 1 pM to 40 pM, 1 pM to 45 pM, 1 pM to 50 pM, 1 pM to 55 pM, 1 pM to 60 pM, 1 pM to 65 pM, 1 pM to 70 pM, 1 pM to 75 pM, 1 pM to 80 pM, 1 pM to 85 pM, 1 pM to 90 pM, 1 pM to 95 pM, or 1 pM to 100 pM. In some implementations, the suppression constant is approximately 25 pM to 30 pM, 25 pM to 35 pM, 25 pM to 40 pM, 25 pM to 45 pM, 25 pM to 50 pM, 25 pM to 55 pM, 25 pM to 60 pM, 25 pM to 65 pM, 25 pM to 70 pM, 25 pM to 75 pM, 25 pM to 80 pM, 25 pM to 85 pM, 25 pM to 90 pM, 25 pM to 95 pM, or 25 pM to 100 pM. In some embodiments, the suppression constant is about 50 pM to 55 pM, 50 pM to 60 pM, 50 pM to 65 pM, 50 pM to 70 pM, 50 pM to 75 pM, 50 pM to 80 pM, 50 pM to 85 pM, 50 pM to 90 pM, 50 pM to 95 pM, or 50 pM to 100 pM. In some embodiments, the suppression constant is about 75 pM to 80 pM, 75 pM to 85 pM, 75 pM to 90 pM, 75 pM to 95 pM, or 75 pM to 100 pM. In some embodiments, the inhibition constant is about 10 pM to 1 nM, 100 pM to 1 nM, 200 pM to 1 nM, 300 pM to 1 nM, 400 pM to 1 nM, 500 pM to 1 nM, 600 pM to 1 nM, 700 pM to 1 nM, 800 pM to 1 nM, or 900 pM to 1 nM. In some embodiments, the inhibition constant is about 1 nM to 100 nM, 10 nM to 100 nM, 20 nM to 100 nM, 30 nM to 100 nM, 40 nM to 100 nM, 50 nM to 100 nM, 60 nM to 100 nM, 70 nM to 100 nM, 80 nM to 100 nM, or 90 nM to 100 nM.In some implementations, the suppression constant is approximately 5 nM to 100 nM, 10 nM to 100 nM, 15 nM to 100 nM, 20 nM to 100 nM, 25 nM to 100 nM, 30 nM to 100 nM, 35 nM to 100 nM, 40 nM to 100 nM, 45 nM to 100 nM, 50 nM to 100 nM, 55 nM to 100 nM, 60 nM to 100 nM, 65 nM to 100 nM, 70 nM to 100 nM, 75 nM to 100 nM, 80 nM to 100 nM, 85 nM to 100 nM, 90 nM to 100 nM, or 95 nM to 100 nM.

[0408] In some embodiments, the inhibition constant is about 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 55 pM, 60 pM, 65 pM, 70 pM, 75 pM, 80 pM, 85 pM, 90 pM, 95 pM, or 100 pM. In some embodiments, the inhibition constant is about 100 pM, 150 pM, 200 pM, 250 pM, 300 pM, 350 pM, 400 pM, 450 pM, 500 pM, 550 pM, 600 pM, 650 pM, 700 pM, 750 pM, 800 pM, 850 pM, 900 pM, 950 pM, or 1 nM. In some embodiments, the inhibition constant is about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, or 10 nM. In some embodiments, the inhibition constant is about 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, or 100 nM.

[0409] In some embodiments, the inhibition constant is about 1 pM. In some embodiments, the inhibition constant is about 2 pM. In some embodiments, the inhibition constant is about 3 pM. In some embodiments, the inhibition constant is about 4 pM. In some embodiments, the inhibition constant is about 5 pM. In some embodiments, the inhibition constant is about 10 pM. In some embodiments, the inhibition constant is about 15 pM. In some embodiments, the inhibition constant is about 20 pM. In some embodiments, the inhibition constant is about 25 pM. In some embodiments, the inhibition constant is about 30 pM. In some embodiments, the inhibition constant is about 35 pM. In some embodiments, the inhibition constant is about 40 pM. In some embodiments, the inhibition constant is about 45 pM. In some embodiments, the inhibition constant is about 50 pM. In some embodiments, the inhibition constant is about 55 pM. In some embodiments, the inhibition constant is about 60 pM. In some embodiments, the inhibition constant is about 65 pM. In some embodiments, the inhibition constant is about 70 pM. In some embodiments, the inhibition constant is about 75 pM. In some embodiments, the inhibition constant is about 80 pM. In some embodiments, the inhibition constant is about 85 pM. In some embodiments, the inhibition constant is about 90 pM. In some embodiments, the inhibition constant is about 95 pM. In some embodiments, the inhibition constant is about 100 pM. In some embodiments, the inhibition constant is about 150 pM. In some embodiments, the inhibition constant is about 200 pM. In some embodiments, the inhibition constant is about 250 pM. In some embodiments, the inhibition constant is about 300 pM. In some embodiments, the inhibition constant is about 350 pM. In some embodiments, the inhibition constant is about 400 pM. In some embodiments, the inhibition constant is about 450 pM. In some embodiments, the inhibition constant is about 500 pM. In some embodiments, the inhibition constant is about 550 pM. In some embodiments, the inhibition constant is about 600 pM. In some embodiments, the inhibition constant is about 650 pM. In some embodiments, the inhibition constant is about 700 pM. In some embodiments, the inhibition constant is about 750 pM. In some embodiments, the inhibition constant is about 800 pM. In some embodiments, the inhibition constant is about 850 pM. In some embodiments, the inhibition constant is about 900 pM. In some embodiments, the inhibition constant is about 950 pM. In some embodiments, the inhibition constant is about 1 nM. In some embodiments, the inhibition constant is about 2 nM. In some embodiments, the inhibition constant is about 3 nM. In some embodiments, the inhibition constant is about 4 nM. In some embodiments, the inhibition constant is about 5 nM. In some embodiments, the inhibition constant is about 6 nM.In some embodiments, the inhibition constant is about 7 nM. In some embodiments, the inhibition constant is about 8 nM. In some embodiments, the inhibition constant is about 9 nM. In some embodiments, the inhibition constant is about 10 nM. In some embodiments, the inhibition constant is about 15 nM. In some embodiments, the inhibition constant is about 20 nM. In some embodiments, the inhibition constant is about 25 nM. In some embodiments, the inhibition constant is about 30 nM. In some embodiments, the inhibition constant is about 35 nM. In some embodiments, the inhibition constant is about 40 nM. In some embodiments, the inhibition constant is about 45 nM. In some embodiments, the inhibition constant is about 50 nM. In some embodiments, the inhibition constant is about 55 nM. In some embodiments, the inhibition constant is about 60 nM. In some embodiments, the inhibition constant is about 65 nM. In some embodiments, the inhibition constant is about 70 nM. In some embodiments, the inhibition constant is about 75 nM. In some embodiments, the inhibition constant is about 80 nM. In some embodiments, the inhibition constant is about 85 nM. In some embodiments, the inhibition constant is about 90 nM. In some embodiments, the inhibition constant is about 95 nM. In some embodiments, the inhibition constant is about 100 nM.

[0410] In some implementations, the suppression constant is no greater than about 0.5 nM, 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, or 100 nM.

[0411] In some embodiments, the inhibition constant is no greater than about 0.5 nM. In some embodiments, the inhibition constant is no greater than about 1 nM. In some embodiments, the inhibition constant is no greater than about 5 nM. In some embodiments, the inhibition constant is no greater than about 10 nM. In some embodiments, the inhibition constant is no greater than about 15 nM. In some embodiments, the inhibition constant is no greater than about 20 nM. In some embodiments, the inhibition constant is no greater than about 25 nM. In some embodiments, the inhibition constant is no greater than about 30 nM. In some embodiments, the inhibition constant is no greater than about 35 nM. In some embodiments, the inhibition constant is no greater than about 40 nM. In some embodiments, the inhibition constant is no greater than about 45 nM. In some embodiments, the inhibition constant is no greater than about 50 nM. In some embodiments, the inhibition constant is no greater than about 55 nM. In some embodiments, the inhibition constant is no greater than about 60 nM. In some embodiments, the inhibition constant is no greater than about 65 nM. In some embodiments, the inhibition constant is no greater than about 70 nM. In some embodiments, the inhibition constant is no greater than about 75 nM. In some embodiments, the suppression constant is no greater than about 80 nM. In some embodiments, the suppression constant is no greater than about 85 nM. In some embodiments, the suppression constant is no greater than about 90 nM. In some embodiments, the suppression constant is no greater than about 95 nM. In some embodiments, the suppression constant is no greater than about 100 nM.

[0412] 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. (See, for example, Correnti et al., Nat Struct MolBiol. 2018 Mar; 25(3):270-278, which contains exemplary CDPs and their characteristics). In some embodiments, the CDP comprises a restricted distribution of cysteine, Cys-X[0–15]-Cys-X[0–15]-Cys-X[0–15]-Cys-X[0–15]-Cys (where X represents any amino acid) (SEQ ID NO: 241). 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.

[0413] 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 Nectin-4 or a fragment thereof. In some embodiments, the CDP is conjugated to a chelating agent and / or a radionuclide. In some embodiments, the conjugation is performed 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.

[0414] 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.

[0415] 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.

[0416] 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 Nectin-4 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.

[0417] In some embodiments, the folded structure of microproteins (e.g., linear peptides, folded peptides (e.g., covalently linked peptides, non-covalently linked peptides, or peptides including disulfide bonds), cysteine-dense peptides, knottin peptides, binding agents, affinity molecules, engineered Kunitz domains, monomeric antibody-like substances, anticalcitonins, designed ankyrin repeating domains (DARPin), or avimers) makes them rigid, thereby providing a very tight and potent binding to target proteins or antigens (compared to less structured peptides). In some such embodiments, microproteins exhibit exceptional resistance to heat, peptidase cleavage, and pH stability.

[0418] 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.

[0419] 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 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.

[0420] 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 binder specifically binds to Nectin-4 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.

[0421] Affinity In some embodiments, the microproteins of this disclosure comprise or consist of an affinity echelon. 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 echelon comprises or consists of no more than 100 amino acids, 90 amino acids, 80 amino acids, 70 amino acids, 60 amino acids, 50 amino acids, 40 amino acids, 30 amino acids, 20 amino acids, or 10 amino acids. In some embodiments, the affinity echelon comprises or consists of at least three α-helices containing 58 amino acids. In some embodiments, the affinity echelon contains 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 implementations, the affinity can be further engineered to alter binding, folding, and / or related properties.

[0422] 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 Nectin-4 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.

[0423] 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.

[0424] 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 Nectin-4 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 is performed 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.

[0425] 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.

[0426] 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 Nectin-4 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.

[0427] 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.

[0428] 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 Nectin-4 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.

[0429] 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.

[0430] 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 Nectin-4 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 employed in the conjugates of this disclosure may vary depending on the target protein or the antigen of interest.

[0431] 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.

[0432] 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 Nectin-4 or a fragment thereof. In some embodiments, the aviser is conjugated to a chelating agent and / or a radionuclide. In some embodiments, the conjugation is performed 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.

[0433] connector In some embodiments, this disclosure provides a connector for one or more conjugates. In some embodiments, the connector is attached to the N-terminus of the microprotein provided herein. In some embodiments, the connector is attached to the C-terminus of the microprotein provided herein. For example, in some embodiments, the connector is attached to a chelating agent. In some embodiments, the connector is attached to a chelating agent that is itself coupled to a radionuclide. In some embodiments, the microprotein is conjugated to a chelating agent and / or a radionuclide. In some embodiments, the microprotein is conjugated to a chelating agent, optionally via a connector. In some embodiments, the compositions provided herein comprise one or more connectors. In some embodiments, the microprotein is conjugated to a chelating agent and / or a cold metal substitute.

[0434] As described herein, in some embodiments, the microprotein conjugate includes a linker. In some embodiments, the linker is used to link a chelating agent to the microprotein. In some embodiments, the linker is non-cleavable. In some embodiments, the peptide is cleavable. In some embodiments, selecting and placing one or more linkers and chelating agents onto the microprotein helps maintain the desired potency and receptor binding profile, enhances binding affinity, and optimizes the physicochemical and pharmacokinetic properties of the microprotein or its conjugate. Any suitable linker known in the art can be used. Exemplary connectors include, but are not limited to, polyethylene glycol (PEG) connectors, 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-carboxylic acid succinimide ester (SMCC) connectors, vinyl sulfone-based connectors, propionic acid connectors, dTyr-Gly-Phe (yGF) connectors, decenoic acid connectors, or connectors comprising any combination thereof. One or more connectors may be considered, as is known to those skilled in the art, and may be selected depending on the context and composition of a given composition. In some embodiments, the connector is a PEG connector. In some embodiments, the connector is a non-pyrolytic PEG connector. In some embodiments, the PEG connector is any of PEG (2-24). In some embodiments, the connector is a PEG4, PEG, PEG2, PEG6, or PEG8 connector. In some embodiments, the connector is a PEG4 connector.

[0435] In some embodiments, the adapter is used to evaluate the binding of the lead peptide sequence to the target, the target expressed on the cell, and the target selectivity and / or affinity. For example, in some embodiments, an SPR machine (e.g., Biacore) can be used to evaluate the in vitro target binding and affinity of the lead peptide sequence and the lead peptide sequence-adaptor-fluorophore reagent. In some embodiments, other adapters, such as fast clear adapters or halogen adapters, may also be considered.

[0436] Chelating agents In some embodiments, the compositions (e.g., conjugates) provided herein comprise a linker. In some embodiments, the composition comprises a linker and a chelating agent. In some embodiments, the composition comprises a linker, a chelating agent, and a radionuclide. In some embodiments, the composition comprises a microprotein, optionally a linker, a chelating agent, and / or a radionuclide. In some embodiments, the chelating agent is covalently linked to the microprotein. In some embodiments, the chelating agent binds to a radionuclide. In some embodiments, the chelating agent binds to a cold metal substitute. In some embodiments, a chelating agent refers to any molecule or portion that is “bound” to a metal ion in solution (effectively collecting / binding the metal ion so that it may, for example, no longer participate in one or more cellular activities or processes). In some embodiments, the chelating agent chelates one or more components in a cellular metabolic pathway (e.g., metal ions, such as copper, iron, zinc, etc.). In some such embodiments, the chelating agent disrupts the life cycle of cancer cells and, in some embodiments, may reduce their viability, function, and / or growth or proliferation capacity. In some embodiments, the chelating agent chelates one or more toxins generated by targeted radiotherapy (e.g., to reduce the toxicity of the therapy).

[0437] In some embodiments, the chelating agent comprises, but is not limited to, the following or consists of, but is not limited to, the following: tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), ({4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[1,4,7]triazacyclononane-1-yl}acetic acid (NETA), Macropa, lead-specific chelating agent (PSC) (e.g., lead-specific chelating agent based on Cyclin), 3-(tri-n-butyltinyl)benzyl The chelating agent is N-succinimide ester (BuSTB), 3-trimethyltinylbenzoate N-succinimide ester (MeSTB), p-bromoacetamidobenzyl-tetraethylaminetetraacetic acid (TETA), porphyrin, polyamine, crown ether, dithiocarbazone, or polyoxime. In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelating agent is a lead-specific chelating agent (PSC). In some embodiments, the chelating agent is 3-(tri-n-butyltinyl)benzoate N-succinimide ester (BuSTB). In some embodiments, the chelating agent is 3-trimethyltinylbenzoate N-succinimide ester (MeSTB); in some embodiments, the chelating agent is Macropa. In some embodiments, the chelating agent comprises or consists of the following: (i) DOTA: (ii) CROWN (iii) NOPO (iv) Macropa or (v) Lead-specific chelating agent (PSC) .

[0438] In some embodiments, the chelating agent comprises, but is not limited to, the following or consists of, but is not limited to, the following: diethylenetriaminepentaacetic acid (DTPA), tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), ({4-[2-(bis-carboxymethyl-amino)-ethyl]-7-carboxymethyl-[ [1,4,7]triazacyclononane-1-yl]acetic acid (NETA), Macropa, p-bromoacetamoxybenzyl-tetraethylaminetetraacetic acid (TETA), PSC, 3-(tri-n-butyltinyl)benzoic acid N-succinimide ester (BuSTB), 3-trimethyltinylbenzoic acid N-succinimide ester (MeSTB), porphyrin, polyamine, crown ether, dithiohexacarbazone, or polyoxime. In some embodiments, the chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In some embodiments, the chelating agent is Macropa.

[0439] In other embodiments, the chelation conditions are optimized using methods known to those skilled in the art (see, for example, J Nucl Med. December 1998; 39(12):2105-10). In some embodiments, the chelation efficiency is approximately 99%, >98%, >97%, >96%, >95%, >94%, >93%, >92%, >91%, >90%, >89%, >88%, >87%, >86%, >85%, >84%, >83%, >82%, >81%, or >80%.

[0440] In some embodiments, the chelating agent used in the compositions described herein is selected based on the presence and type of radionuclide. As provided herein, in some embodiments, the chelating agent is DOTA, NOPO, Crown, PSC, 3-(tri-n-butyltinyl)benzoate N-succinimide ester (BuSTB), 3-trimethyltinylbenzoate N-succinimide ester (MeSTB), or Macropa. In some embodiments, DOTA is the chelating agent, and the radionuclide is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, or At-211. In some embodiments, Crown is the chelating agent, and the radionuclide is Ac-225, In-111, Ga-68, Pb-212, Lu-177, Cu-67, Cu-64, La-132, La-135, Ce-134, F-18, I-131, I-124, Pb-203, Th-232, Bi-123, or At-2...

Claims

1. A composition comprising a polypeptide having a length of at least 44 amino acids and having an amino sequence comprising the sequence described in SEQ ID NO: 171, wherein X2 is E or D; X6 is E or Q; X17 is G or A; X21 is Q, Y or E; X26 is Kme3, Kme2, Kme, K, Kipr or S; X32 is A, G or D; X41 is N or K; and X45 is S or absent.

2. A composition comprising a polypeptide having a length of at least 44 amino acids and having an amino acid sequence comprising the sequence described in SEQ ID NO: 176, wherein X2 is E or D; X6 is E or Q; X9 is T or A; X10 is A or G; X12 is A, Kme3, Kme2, Kme, Kipr, or K; X13 is R or (Cit); X17 is G or A; X21 is Q, Y, or E; X24 is Q or K; X25 is A or K; X26 is Kme3, Kme2, Kme, K, Kipr, or S; X28 is Q or K; X29 is Y or K; X30 is L or V; X32 is A, G, or D; X41 is N or K; and X45 is S or absent.

3. A composition comprising a Nectin-4 binding polypeptide having an amino acid sequence comprising at least 44 amino acids, wherein said amino acids include: (i) Cysteine ​​at each of the four positions 1, 20, 34 and 44 of SEQ ID NO: 195; (ii) SEQ ID NO: 169 at positions 9-15 of SEQ ID NO: 195; (iii) QKKme3 at positions 24, 25 and 26 of SEQ ID NO: 195; and (iv) QYL at positions 28, 29 and 30 of SEQ ID NO:

195.

4. A composition comprising a Nectin-4 binding polypeptide having an amino acid sequence comprising at least 44 amino acids, wherein the amino acids comprise (i) cysteine ​​at each of four positions corresponding to positions 1, 20, 34, and 44 of SEQ ID NO: 200; (ii) SEQ ID NO: 247 at positions 9-15 of SEQ ID NO: 200; (iii) QKKme3 at positions 24, 25, and 26 of SEQ ID NO: 200; and (iv) QYL at positions 28, 29, and 30 of SEQ ID NO:

200.

5. A composition comprising a Nectin-4 binding polypeptide having an amino acid sequence, wherein the amino acid sequence comprises: a. At least four cysteine ​​residues, forming two disulfide bonds; b. At least one modified lysine residue, the position of which corresponds to X12 and / or X26 of SEQ ID NO: 195, wherein the modification comprises at least one small alkyl group of nitrogen attached to the lysine side chain, optionally comprising methyl, dimethyl, trimethyl or isopropyl; c. A length of at least 44 amino acids; and d. It exhibits a binding affinity for Nectin-4 stronger than 100 nM in cell-based assays.

6. The composition of any of the preceding claims, wherein the polypeptide has a length of at least 40 amino acids but not more than 100 amino acids.

7. The composition as claimed in any of the preceding claims, wherein the polypeptide binds to Nectin-4 with an affinity greater than 10 nM in cell-based assays.

8. 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: 3-158, 161-168, 177-208 or 212-215, but includes at least one lysine having at least one modification, said modification comprising at least one small alkyl group of the nitrogen bonded to the side chain, optionally selected from: trimethyl, dimethyl, monomethyl and isopropyl.

9. 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 44 amino acids, and binds to Nectin-4 with an intensity of at least 10 nM in a cell-based assay, and / or has an inhibition constant of not more than 10 nM.

10. The composition of any of the preceding claims, wherein the amino acid sequence of the polypeptide shares at least 90% identity with at least 40 amino acids of any one of SEQ ID NO: 3-158, 161-168, 177-208 or 212-215, provided that the 40 amino acids include at least four cysteine ​​residues forming two disulfide bridges.

11. The composition of any of the preceding claims, wherein the amino acid sequence of the polypeptide shares at least 90% identity with at least 35 adjacent amino acids of any one of SEQ ID NO: 3-158, 161-168, 177-208 or 212-215, provided that the 40 amino acids include at least four cysteine ​​residues forming two disulfide bridges.

12. 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 44 amino acids.

13. The composition of any of the preceding claims, wherein the amino acid sequence shares 90% identity with at least 44 amino acids as described in any one of SEQ ID NO: 78, 83, 85, 99, 103, 162-168, 195 or 200.

14. 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: 78, 83, 85, 99, 103, 162-168, 195 or 200.

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

195.

16. A composition comprising a compound as described in C251 of Table 2A, said compound having an amino acid sequence comprising SEQ ID NO:

195.

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

200.

18. A composition comprising a compound as described in C260 of Table 2A, said compound having an amino acid sequence comprising SEQ ID NO:

200.

19. The composition according to any one of claims 13-18, further comprising a radionuclide.

20. The composition of claim 19, 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.

21. A composition comprising a polypeptide having an amino acid sequence of at least 44 amino acids but having four amino acid substitutions at positions 12, 21, 26 and 32 corresponding to SEQ ID NO: 78, wherein the substitutions correspond to K12A, Y21Q, S26Kme3 and G32A.

22. The composition as claimed in any of the preceding claims, wherein the C-terminus has -OH or -NH2.

23. The composition as claimed in any of the preceding claims, wherein the binding affinity for Nectin-4 is greater than 100 nM.

24. The composition as claimed in any of the preceding claims, wherein the inhibition constant is not greater than 100 nM.

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

26. The composition of claim 25, 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, where n is 1 to 10, (Gly)1-10, or any fragment thereof or combination thereof linked by covalent bonds.

27. The composition of claim 25 or 26, 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).

28. The composition according to any one of claims 25-27, 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.

29. The composition of any one of claims 25-28, wherein if the polypeptide comprises any one of SEQ ID NO: 83, 85, 93, 99, 134, 138, 145, 155, 162-168 or 195, the polypeptide further comprises a linker, wherein the linker is PEG4, and optionally a chelating agent, wherein the chelating agent is DOTA.

30. The composition according to any one of claims 25-29, wherein, When present, the adapter is attached to the N-terminus of the polypeptide.

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

32. The composition according to any one of claims 25-31, wherein, When present, the chelating agent is attached to the polypeptide or the linker.

33. The composition according to any one of claims 25-32, wherein, In the presence of the radionuclide, the radionuclide is attached to the chelating agent.

34. 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: 3-158, 161-168, 170-208, 212-237, 243-246 or 248.

35. The composition of claim 34, 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.

36. The composition of claim 34 or 35, 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).

37. The composition according to any one of claims 34-36, 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.

38. 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 one of the sequences described in SEQ ID NO: 162-176, 178-208 or 212-215.

39. The composition of claim 38, 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.

40. The composition of claim 38 or 39, 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).

41. The composition of any one of claims 38-40, 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.

42. The composition according to any one of claims 38-41, wherein, When present, the adapter is attached to the N-terminus of the polypeptide.

43. The composition of claim 42, wherein the C-terminal amino acid of the polypeptide is not cysteine.

44. The composition according to any one of claims 38-43, wherein, When present, the chelating agent is attached to the polypeptide or the linker.

45. The composition according to any one of claims 38-44, wherein, In the presence of the radionuclide, the radionuclide is attached to the chelating agent.

46. ​​The composition of any one of claims 38-45, wherein the polypeptide comprises at least one disulfide bridge.

47. The composition of claim 46, wherein the polypeptide comprises at least two disulfide bridges.

48. The composition as claimed in any of the preceding claims, wherein the composition and / or its polypeptides selectively bind to Nectin-4 or a portion thereof.

49. The composition of any of the preceding claims, wherein the binding affinity of the polypeptide to Nectin-4 or a portion thereof 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.

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

51. 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 the amino acid sequence described in any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248.

52. The composition of claim 51, wherein the pharmaceutical portion is selected from V-ATPase inhibitors, apoptosis-promoting agents, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, orrisstatin, sulphoside, 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, topoisomerase inhibitors, orrisstatin (e.g., monomethylorrisstatin E), and immunotoxins.

53. A composition comprising an isolated compound or a pharmaceutically acceptable salt 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: 3-158, 161-168, 170-208, 212-237, 243-246, and 248, including amino acid substitutions as described in Tables 1C, 1D, 2C, 2D, 2E, 2F, or 2G.

54. A composition comprising a compound designed to bind to Nectin-4, said compound comprising or consisting of a polypeptide having an amino acid sequence comprising any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248, including amino acid substitutions as described in Table 1C, Table 1D, Table 2C, Table 2D, Table 2E, Table 2F or Table 2G, and further comprising a modified N-terminus and / or C-terminus.

55. The composition of claim 54, wherein the modified N-terminus comprises one or more of the following: NH2-, acetyl-, PEGn- where n = 0-10, DOTA-, or biotin-.

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

57. The composition of any of the preceding claims, wherein the polypeptide selectively binds to Nectin-4 or a portion thereof.

58. The composition of any of the preceding claims, wherein the polypeptide has a binding affinity for Nectin-4 or a portion thereof greater than about 100 nM in vivo or in a cell-based assay.

59. A compound comprising a microprotein having an amino acid sequence having 90% identity with SEQ ID NO: 195, and further comprising one or more additional components according to formula MLCR, wherein L is a linker, C is a chelating agent, and R is a radionuclide.

60. The compound of claim 59, 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.

61. The compound of claim 59 or 60, 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).

62. The compound according to any one of claims 59-61, 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.

63. A compound comprising a microprotein comprising at least 40 amino acids that are 90% identical to the amino acid sequence of SEQ ID NO: 195, wherein the N-terminus and / or C-terminus comprises one to thirty additional amino acids, and / or wherein the C-terminus comprises 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.

64. In a method for improving the binding affinity strength of a peptide to Nectin-4, the improvement comprises modifying four amino acid residues of the peptide, the peptide being at least 44 amino acids in length and having substitutions at positions 12, 21, 26 and 32 corresponding to SEQ ID NO: 78, wherein the substitutions correspond to K12A, Y21Q, S26Kme3 and G32A.

65. A pharmaceutical composition comprising a polypeptide of any one of claims 1-58 or a compound of any one of claims 59-63; and a pharmaceutically acceptable excipient.

66. A method of treating cancer, the method comprising administering to a subject in need a composition comprising a conjugate having at least 90% identity with at least 40 amino acids of an amino acid sequence as described in any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248 and a radionuclide.

67. The method of claim 66, 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.

68. The method of claim 66 or 67, wherein the polypeptide has an amino acid sequence comprising or consisting of SEQ ID NO: 195 or SEQ ID NO:

200.

69. The method of claim 67 or 68, 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.

70. The method of any one of claims 67-69, 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).

71. The method of any one of claims 67-70, 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.

72. The method of claim 71, wherein R is a therapeutic agent and / or an imaging agent.

73. The method of claim 71 or 72, wherein R is Cu-64, Ga-68, Lu-177, In-111, Cu-67, La-132 or F-18.

74. A method for reducing renal cell uptake of a composition, comprising administering to a subject a Nectin-4 binding protein having an amino acid sequence containing at least one modified lysine residue at positions X12 and / or X26 corresponding to SEQ ID NO: 195, wherein the modification comprises at least a small alkyl group of nitrogen linked to a lysine side chain, optionally comprising monomethyl, dimethyl, trimethyl, or isopropyl, and the reduction is compared to administration to the subject or a control subject of a composition that is otherwise identical but does not contain the modified lysine residue at positions X12 and / or X26.

75. In a method of treating cancer, an improvement includes administering a composition comprising a Nectin-4 binding protein having an amino acid sequence containing at least one modified lysine residue at positions X12 and / or X26 corresponding to SEQ ID NO: 195, wherein, compared to a composition not containing a modified lysine residue at positions X12 and / or X26, the modification comprises at least one carbon atom of a nitrogen linked to a lysine side chain, optionally comprising a methyl, dimethyl, trimethyl, or isopropyl group.

76. A method of treating a subject with refractory or recurrent cancer, comprising administering a composition of any one of claims 1-58, a compound of any one of claims 59-63, or a pharmaceutical composition of claim 65, wherein the treatment is therapeutic to the cancer.

77. A method for improving the biodistribution of a pharmaceutical composition of a population of Nectin-4 positive cancer cells in a subject with Nectin-4 positive cancer, comprising contacting the population with a polypeptide having a modified lysine at positions X12 and / or X26 corresponding to SEQ ID NO: 195, 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 positions X12 and / or X26 corresponding to SEQ ID NO:

195.

78. A method for diagnosing the presence of a population of Nectin-4 positive cancer cells, comprising: a. Contacting a cell population with the composition of any one of claims 1-58, the compound of any one of claims 59-63, or the pharmaceutical composition of claim 65; 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 concentration of the composition, compound, or pharmaceutical composition from step (a) is higher than that of the control, cancer is diagnosed.

79. The method of claim 78, wherein the contact is performed by application to a subject in need.

80. The method of claim 79, wherein the administration is intravenous or subcutaneous.

81. The method of claim 78, wherein the contact is performed in vitro on the subject, optionally using a biopsy sample.

82. A method of treating a subject with cancer using immunotherapy, the method comprising administering to the subject a composition comprising the composition of any one of claims 1-58, the compound of any one of claims 59-63, or the pharmaceutical composition of claim 65.

83. Use of the composition of any one of claims 1-58, the compound of any one of claims 59-63, or the pharmaceutical composition of claim 65 for the treatment of a subject with cancer.

84. A method of treating a subject in need, comprising administering to the subject in need the composition of any one of claims 1-58, the compound of any one of claims 59-63, or the pharmaceutical composition of claim 65.

85. The method of claim 84, wherein the subject is diagnosed with cancer.

86. The method of claim 85, wherein the cancer cells from the subject express Nectin-4 or a portion thereof.

87. The method of claim 86, wherein the expression of Nectin-4 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.

88. The method of any one of claims 84-87, wherein the composition, compound, or pharmaceutical composition is not taken up and / or retained by the kidneys compared to a composition not comprising any one of claims 1-58, a compound comprising any one of claims 59-63, or a pharmaceutical composition comprising any one of claims 65.

89. The method of any one of claims 84-88, wherein the composition, compound, or pharmaceutical composition is internalized in cells expressing human Nectin-4.

90. The method of any one of claims 84-89, 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, glioblastoma, 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. Tumors, 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.

91. The method of any one of claims 84-90, wherein the composition, compound, or pharmaceutical composition is administered intravenously or subcutaneously.

92. A method for treating cancer cells expressing Nectin-4, the method comprising: (i) The expression level of Nectin-4 in cancer cell populations has been determined or has been determined; (ii) administering to a subject in need a composition comprising any one of claims 1-58, any one of claims 59-63, or a pharmaceutical composition of claim 65, wherein the polypeptide of the composition, compound, or pharmaceutical composition is designed to specifically bind to human Nectin-4, and (iii) wherein the composition, compound, or pharmaceutical composition is attached to the surface of one or more cancer cells expressing Nectin-4 and / or internalized into the cancer cells.

93. The method of claim 92, wherein the subject receives treatment after the administration, compared to receiving treatment before the administration.

94. In a method for targeting a population of cancer cells expressing Nectin-4, an improvement comprises contacting the population with the composition of any one of claims 1-58, the compound of any one of claims 59-63, or the pharmaceutical composition of claim 65, wherein X12 and / or X26 comprise lysine residues 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 residue at position X12 and / or X26, wherein, Optionally, the small alkyl group is part of a monomethyl, dimethyl, trimethyl, or isopropyl group.

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

96. A conjugate comprising: (i) A polypeptide (M) that specifically binds to Nectin-4; (ii) a chelating agent (C) chelated to (M) via an optional connector (L), wherein (C) contains DOTA and (L) contains PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is copper-64.

97. A conjugate comprising: (i) A polypeptide (M) that specifically binds to Nectin-4; (ii) a chelating agent (C) chelated to (M) via an optional connector (L), wherein (C) contains DOTA and (L) contains PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is gallium-68.

98. A conjugate comprising: (i) A polypeptide (M) that specifically binds to Nectin-4; (ii) a chelating agent (C) chelated to (M) via an optional connector (L), wherein (C) contains DOTA and (L) contains PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is indium-111.

99. A conjugate comprising: (i) A polypeptide (M) that specifically binds to Nectin-4; (ii) a chelating agent (C) chelated to (M) via an optional connector (L), wherein (C) contains DOTA and (L) contains PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is lead-212.

100. A conjugate comprising: (i) A polypeptide (M) that specifically binds to Nectin-4; (ii) a chelating agent (C) chelated to (M) via an optional linker (L), wherein (C) contains DOTA and (L) contains PEG in the presence, wherein the PEG is optionally PEG-4; and (iii) a radionuclide (R) chelated to (C), wherein (R) is ruthenium-177.

101. A conjugate comprising: (i) A microprotein (M) that specifically binds to Nectin-4; (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.

102. The conjugate according to any one of claims 95-100, wherein M has an amino acid sequence comprising any one of the amino acid sequences described in SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248.

103. The conjugate according to any one of claims 95-102, wherein the amino acid sequence has at least 90% identity with the sequence of at least 40 amino acids of SEQ ID NO:176, wherein X2 is E or D; X6 is E or Q; X9 is T or A; X10 is A or G; X12 is A, Kme3, Kme2, Kme, Kipr, or K; X13 is R or (Cit); X17 is G or A; X21 is Q, Y, or E; X24 is Q or K; X25 is A or K; X26 is Kme3, Kme2, Kme, K, Kipr, or S; X28 is Q or K; X29 is Y or K; X30 is L or V; X32 is A, G, or D; X41 is N or K; and X45 is S or absent.

104. The conjugate according to any one of claims 95-103, wherein M has an amino acid sequence comprising or consisting of SEQ ID NO:

195.

105. The conjugate according to any one of claims 95-104, wherein M has an amino acid sequence comprising or consisting of SEQ ID NO:

200.

106. An isolated polynucleotide comprising one or more nucleic acid sequences encoding a polypeptide selected from any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248; or a nucleic acid sequence encoding a polypeptide having at least 90%, 95%, 96%, 97%, 98%, 99% or greater identity to any one of SEQ ID NO: 3-158, 161-168, 170-208, 212-237, 243-246 and 248.

107. A vector comprising the isolated polynucleotide of claim 106.

108. A host cell transformed with the isolated polynucleotide of claim 106 or the vector of claim 107.

109. 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 1-58, a compound of any one of claims 59-63, or a pharmaceutical composition of claim 65, and detection to determine the location of the composition in the subject.

110. In a method of reducing renal uptake of a composition administered to detect and / or treat one or more cancer cell populations, an improvement comprises administering to a subject in need the composition of any one of claims 1-58, the compound of any one of claims 59-63, or the pharmaceutical composition of claim 65, wherein X12 and / or X26 comprise lysine having at least one additional small alkyl group of nitrogen linked 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 of nitrogen not linked to the side chain at position X12 and / or X26, wherein, Optionally, the small alkyl group is part of a monomethyl, dimethyl, trimethyl, or isopropyl group.

111. The method of claim 109 or 110, wherein the detection includes an imaging procedure to allow selection of subjects, monitoring of subjects, and / or treatment of subjects with a therapeutic agent comprising a microprotein, the microprotein being programmed to bind to Nectin-4 expressed on one or more cancer cells in one or more cancer cell populations.

112. The method of claim 111, wherein the therapeutic agent comprises a composition of any one of claims 1-58, a compound of any one of claims 59-63, a pharmaceutical composition of claim 65, or a conjugate of any one of claims 95-105.

113. An improved method for radionuclide delivery to a cancer cell population in a subject, the method comprising administering a composition of any one of claims 1-58, a compound of any one of claims 59-63, a pharmaceutical composition of claim 65, or a conjugate of any one of claims 95-105, wherein the amino acid sequence of the polypeptide comprises an amino acid corresponding to position X12 and / or X26 of SEQ ID NO: 195, and wherein X12 and / or X26 comprises a lysine having at least one additional small alkyl group of nitrogen attached to a side chain, wherein renal cell uptake is less than that of a polypeptide having an amino acid sequence having no additional small alkyl group of nitrogen attached to the side chain of lysine at position X12 and / or X26.

114. The method of claim 113, wherein the small alkyl group comprises monomethyl, dimethyl, trimethyl, or isopropyl.

115. In a method of treating an individual with cancer, an improvement includes reducing one or more off-target effects or toxicity measures by administering the composition of any one of claims 1-58, the compound of any one of claims 59-63, the pharmaceutical composition of claim 65, or the conjugate of any one of claims 95-105, wherein the amino acid sequence of the polypeptide comprises amino acids corresponding to positions X12 and / or X26 of SEQ ID NO: 195, and wherein X12 and / or X26 comprises lysine having at least one additional small alkyl group of nitrogen attached to a side chain, wherein renal cell uptake is less than that of a polypeptide having an amino acid sequence that does not contain an additional small alkyl group of nitrogen attached to the side chain of lysine at positions X12 and / or X26.

116. In a method of treating an individual with 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 1-58, the compound of any one of claims 59-63, the pharmaceutical composition of claim 65, or the conjugate of any one of claims 95-105, wherein the amino acid sequence of the polypeptide comprises amino acids corresponding to positions X12 and / or X26 of SEQ ID NO: 195, and wherein X12 and / or X26 comprises lysine having at least one additional small alkyl group attached to a nitrogen atom on a side chain, wherein the renal cells take up less of the polypeptide than a polypeptide having an amino acid sequence having a small alkyl group attached to a nitrogen atom on the side chain at positions X12 and / or X26.

117. The method of claim 116, 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.

118. The method of claim 117, wherein, compared to the presence of A or K at positions corresponding to X12 and / or X26, the application of the composition can be repeated at least 2, 3, 4, 5, 6, or 7 times when a composition having 90% identity with at least 40 amino acids of SEQ ID NO: 195 and including modified lysine at positions corresponding to X12 and / or X26 of SEQ ID NO: 195 is present.

119. In a method of reducing the uptake of a composition by renal tissue, an 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 40 amino acids of SEQ ID NO: 195 and / or has a modified lysine at positions corresponding to X12 and / or X26 of SEQ ID NO: 195, 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.

120. A method comprising administering to a subject in need a compound that binds to Nectin-4 and comprises one or two modified lysine residues at positions X12 and / or X26, respectively, corresponding to SEQ ID NO: 195, wherein administration of the compound having the microprotein containing the one or two modified lysine residues reduces one or more off-target effects, toxicity levels, and / or uptake and / or retention in renal tissue compared to a compound having alanine at position X12 or unmodified lysine at position X26.

121. A method of treating an individual who has or is suspected of having Nectin-4 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.

122. The method of claim 121, wherein the means for blocking renal tissue uptake and / or retention of the radiotherapy agent is bound to Nectin-4 and comprises one or two modified lysines at positions X12 and / or X26 of SEQ ID NO: 195 and / or has at least 90% identity with the 40 amino acids of SEQ ID NO: 195 and / or has modified lysines at positions X12 and / or X26 of SEQ ID NO:

195.

123. The method of claim 122, wherein the means for blocking renal tissue uptake and / or retention of the radiotherapy agent is bound to Nectin-4 and comprises one or two modified lysines at positions X12 and / or X26 of SEQ ID NO: 195 and / or has at least 90% identity with the 35 adjacent amino acids of SEQ ID NO: 195 and / or has modified lysines at positions X12 and / or X26 of SEQ ID NO:

195.

124. The method of any one of claims 121-123, 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 one or two modified lysines at positions X12 and / or X26 of SEQ ID NO: 195 and / or having at least 90% identity with the 40 amino acids of SEQ ID NO: 195 and / or having modified lysines at positions X12 and / or X26 of SEQ ID NO:

195.

125. The method of any one of claims 121-124, 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 one or two modified lysines at positions X12 and / or X26 of SEQ ID NO: 195 and / or having at least 90% identity with the 35 adjacent amino acids of SEQ ID NO: 195 and / or having modified lysines at positions X12 and / or X26 of SEQ ID NO:

195.

126. The method of any one of claims 121-125, wherein the means for blocking the uptake and / or retention of the radiotherapy agent by the renal tissue is a radiotherapy agent.

127. The method of claim 126, wherein the radiotherapy agent targets a tumor or a population of cancer cells.

128. The method of claim 127, wherein the concentration of the radiotherapy agent targeting the tumor or the cancer cell population is greater than the concentration in the absence of means of binding to kidney tissue.

129. The method of claim 128, wherein the radiotherapy agent comprises a polypeptide targeting Nectin-4.

130. The method of any one of claims 121-129, wherein the radiotherapy agent comprises or is composed of a compound selected from C3-C293 or C298-C307.

131. The method of claim 130, wherein the radionuclide of the radiotherapy agent is selected from Ac-225, Cu-64, Ga-68, In-111, Lu-177 or Pb-212.

132. 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 1-58, the compounds of any one of claims 59-63, the pharmaceutical compositions of claim 65, or the conjugates of any one of claims 95-105.

133. The kit of claim 132, further comprising one or more of a connector, a chelating agent, and a radionuclide.

134. The kit of claim 133, 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.

135. The kit of claim 133 or 134, 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).

136. The kit according to any one of claims 132-135, wherein, prior to use, the compound is labeled with a radionuclide, wherein the radionuclide is chelated to the chelating agent to produce a composition having the formula MLCR.

137. The kit according to any one of claims 133-136, 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.

138. The kit of claim 137, wherein the radionuclide is Ac-225, Cu-64, Ga-68, In-111, Lu-177 or Pb-212.

139. The kit of any one of claims 132-138, wherein if the polypeptide has an amino acid comprising any one of the sequences described in any one of SEQ ID NO: 83, 85, 93, 99, 134, 138, 145, 155, 161-176, 195 or 200, the polypeptide further comprises a linker, wherein the linker is PEG4, and a chelating agent, wherein the chelating agent is DOTA.

140. The kit according to any one of claims 133-139, wherein, in the presence, the linker is attached to the N-terminal amino acid of the polypeptide.

141. The kit according to any one of claims 132-140, wherein the C-terminal amino acid of the polypeptide is not cysteine.

142. The kit according to any one of claims 133-141, wherein, in the presence, the chelating agent is linked to the polypeptide or the linker.

143. The kit according to any one of claims 133-142, wherein, in the presence, the radionuclide is linked to the chelating agent.

144. The kit according to any one of claims 133-143, wherein, in the presence, the radionuclide is linked to the N-terminal amino acid of the polypeptide.

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