Polypeptide conjugate compositions and methods of use

Abstract

Provided herein are chemoselective modification conjugates of at least two payloads to at least two polypeptides.

Description

Cross-referencing

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 581,936, filed September 11, 2023; U.S. Provisional Patent Application No. 63 / 591,072, filed October 17, 2023; U.S. Provisional Patent Application No. 63 / 607,467, filed December 7, 2023; and U.S. Provisional Patent Application No. 63 / 631,357, filed April 8, 2024, the disclosures of which are hereby incorporated in their entirety by reference for all purposes. Background Technology

[0002] The conjugation of biomolecules (e.g., peptides such as antibodies) to payloads to create conjugates while preserving the function of the biomolecules has long been a goal of chemical biology and biomedical research. Examples of conjugates include protein-peptide conjugates for vaccine development, antibody-drug conjugates, and antibody-protein conjugates for immunotherapy.

[0003] While numerous techniques have been developed to allow payloads to be attached to peptides, developing methods for attaching payloads to any site on the peptide surface in a site-specific manner has been challenging. There is a need in the industry for improved conjugation procedures that can modify peptides or biomolecules in a simple yet site-specific manner. Summary of the Invention

[0004] This disclosure provides for the chemically selective modification of peptides or biomolecules.

[0005] On the one hand, this paper discloses conjugates of formula A, formula B, formula C, or formula D: Formula A, Formula B, Formula C, or Formula D, in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of the amino acids X1-X5 is tyrosine, and where m1 is an integer greater than or equal to 0; Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10For any amino acid, the condition is X6-X 10 At least one amino acid in it is tyrosine, and m2 is an integer greater than or equal to 0; n is an integer greater than 0; L1 is an optional first connector; L2 is an optional second connector; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0006] On the one hand, this paper discloses conjugates of formula A', formula B', formula C', or formula D': Formula A' Formula B' Formula C', or Formula D' in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid from X1-X5 contains tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid from X1-X3 is tyrosine or a portion thereof, where Y a Linked to "S" via the tyrosine or a portion thereof; Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the formula comprises tyrosine or a portion thereof, and wherein m2 is an integer greater than or equal to 0, or X1X2X3, wherein X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof, wherein Y b Linked to "S" via the tyrosine or a portion thereof; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0007] In some embodiments, the tyrosine or a portion thereof is selected from the group consisting of: , , and .

[0008] In some embodiments, the first terminal tag contains at least two amino acids selected from the group consisting of aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K).

[0009] In some embodiments, the second terminal tag contains at least two amino acids selected from the group consisting of aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K).

[0010] In some implementations, the first end tag includes at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

[0011] In some embodiments, the second end label includes at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

[0012] In some implementations, the first end label includes SGGY, SGY, SGGGY, ESY, EESY, ESSY, ESSSY, SESY, SGGGY, EESY, SSSY, SNNY, SSNY, EGGY, SESY, or SEGY.

[0013] In some implementations, the second end label includes EEEY, EEEEY, SSSEY, SSEEY, SSSSY, or SSNNY.

[0014] In some implementation schemes, Y a Y b Or Y a and Y b Both are glycosylated.

[0015] In some implementation schemes, Y a Y b Or Y a and Y b Both contain non-terminal tyrosine residues.

[0016] In some implementations, at least one of L3, L4, or L5 is a pyrolytic connector.

[0017] In some embodiments, the cleavable connector is an electrophilic cleavable connector, a nucleophilic cleavable connector, a photocleavable connector, a metallic cleavable connector, an electrolytic cleavable connector, an acid cleavable connector, or a protein hydrolytic cleavable connector.

[0018] In some embodiments, the pyrolytic connector further includes polyethylene glycol groups, glycosyl groups, or modifications that increase hydrophilicity.

[0019] In some embodiments, the pyrolytic connector is pyrolytic under reducing and / or oxidizing conditions.

[0020] In some embodiments, the pyrolytic connector is pyrolytic under acidic conditions.

[0021] In some embodiments, the pyrolytic linker comprises a disulfide bond.

[0022] In some embodiments, the cleavable adapter is a protein hydrolyzable adapter and contains a protease recognition sequence.

[0023] In some embodiments, the protease recognition sequence is recognized by a protease selected from the group consisting of metalloproteinases, cathepsin B, and tobacco etch virus (TEV).

[0024] In some embodiments, the cleavable linker comprises a dipeptide, a tripeptide, or a tetrapeptide. In some embodiments, the dipeptide is a valine-citrulline (Val-Cit) dipeptide, a valine-lysine dipeptide, or a valine-alanine dipeptide. In some embodiments, the tetrapeptide is a glycine-glycine-phenylalanine-glycine (GGFG) tetrapeptide.

[0025] In some embodiments, the cleavable connector is selected from the group consisting of PABC (p-aminobenzyl alcohol), glucuronic acid, and MABC (m-aminobenzyl alcohol).

[0026] In some implementations, the pyrolytic connector is Val-Cit-PABC.

[0027] In some implementations, “Y1-L4-S-” and / or “-S-L5-Y2” are selected from the group consisting of: S-VC-PAB-DXD, S-VC-PAB-MMAE, S-VC-PAB-camptothecin, and S-VC-PAB - Doxorubicin.

[0028] In some implementation schemes, Y a The variable region of the heavy chain (VH), variable region of the light chain (VL), heavy chain, light chain, constant chain of an antibody or antibody fragment; peptide; or cyclic peptide.

[0029] In some implementations, the peptide is a binding peptide.

[0030] In some implementation schemes, Y b The term refers to an antibody, a heavy chain variable region (VH) or light chain variable region (VL) of an antibody fragment, a heavy chain, a light chain, or a constant chain; a peptide; or a cyclic peptide. In some embodiments, the peptide is a binding peptide.

[0031] In some implementation schemes, Y a With Y b The linker is located via L3. In some embodiments, L3 comprises a peptide sequence, a dimerization and docking domain, a leucine zipper, or a mortise and tenon structure. In some embodiments, L3 comprises a peptide bond, a disulfide bond, a maleimide bond, a thioether bond, an azide-alkyne cycloaddition, a cystinyl-dopa bond, or a hydrogen bond. In some embodiments, L3 is a linker.

[0032] In some embodiments, the connector comprises a sequence selected from the group consisting of: (GS) n3 (G2S) n3 (G3S) n3 (G4S) n3 (G) n3 (GGSGGD) n3 (GGSGGE) n3 (GGGSGSGGGGS) n3 and (GGGGGPGGGGP) n3 , where n3 is an integer from 2 to 20.

[0033] In some implementations, L3 contains a terminal tyrosine or a portion thereof.

[0034] In some implementations, n is 1, 2, 3, 4, or 5. In some implementations, n is 1.

[0035] In some implementations, Y1 and Y2 are the same.

[0036] In some implementations, Y1 and Y2 are different.

[0037] In some implementations, Y1, Y2, or both Y1 and Y2 are small molecules.

[0038] In some embodiments, the small molecule is selected from the group consisting of: deruxtecan, exatecan, FL118, irinotecan, topotecan, SN-38, rubitecan, belototecan, lurototecan, gimatecan, diflomotecan, karenitecan, silatecan, namitecan, elomotecan, DRF-1042, delimotecan, NSC606985, chimmitecan, ZBH-1205, and auristatin. (MMAE, MMAF, MMAG, MMAH), calicheamicin, doxorubicin, paclitaxel and paclitaxel derivatives, maytansinoid (DM1-4), pyrrolodiazepine (PBD), tubulolysin, eribulin, antramycin, duocarmycin, anthracycline and camptothecin (CPT), including the lactone and carboxylic acid ester forms of CPT.

[0039] In some embodiments, the small molecule is selected from the group consisting of topoisomerase inhibitors and tubulin inhibitors.

[0040] In some embodiments, Y1, Y2, or both Y1 and Y2 comprise nucleic acids, immune agonists, peptides, cytokines, or binding domains.

[0041] In some implementations, Y1, Y2, or both Y1 and Y2 contain oligonucleotides.

[0042] In some implementations, Y1, Y2, or both Y1 and Y2 contain peptides.

[0043] In some implementations, Y1, Y2, or both Y1 and Y2 contain a binding peptide.

[0044] On the other hand, this paper provides conjugates of formulas AI, BI, CI, or DI: AI-style Formula BI, CI, or Formula DI; where: Y a' It is the first polypeptide. Each of X1, X2, X3, X4, and X5 is independently any amino acid, where m1, m2, and m3 are each integers greater than or equal to 0; Y b' It is the second polypeptide. X6, X7, X8, X9 and X 10 Each of them is independently any amino acid, where m4, m5, and m6 are each an integer greater than or equal to 0, and where -(X1) is an integer. m1 (X2) m2 X3X4 - Partially different from -(X6) m1 (X7) m2 X8X9 - Partial; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0045] In some implementations, the -(X1) m1 (X2) m2 The X3X4-part contains at least two amino acids selected from the following group: aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K).

[0046] In some implementations, the -(X6) m1 (X7) m2 The X8X9-part contains at least two amino acids selected from the following group: aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K).

[0047] In some implementations, the -(X1) m1 (X2) m2The X3 EDDD, EEDD, RRKK, KKGG, DDGGEES, ESSS, SSS, SNN, SSN, SEG, SSSE, EGG, SSEE, SES, ESS, ES, SR, SK, SN, ER, EK, EG, SE, SHRK, SARK, SPPE, SSEE, SEEE and SSSS.

[0048] In some implementations, the -(X6) m1 (X7) m2 The DDD, EEDD, RRKK, KKGG, DDGG, EES, ESSS, SSS, SNN, SSN, SEG, SSSE, EGG, SSEE, SES, ESS, ES, SR, SK, SN, ER, EK, EG, SE, SHRK, SARK, SPPE, SSEE, SEEE and SSSS.

[0049] In some implementations, the -(X1) m1 (X2) m2 X3X4 - Parts are SGG, SG, SGGG, ES, EES, ESS, ESSS, SES, SGGG, EES, SSS, SNN, SSN, EGG, SES, or SEG.

[0050] In some implementations, the -(X6) m1 (X7) m2 X8X9 - Parts are EEE, EEEE, SSSE, SSEE, SSSS, or SSNN.

[0051] In some implementation schemes, Y a' Y b' Or Y a' and Y b' Both are glycosylated.

[0052] In some embodiments, at least one of L3, L4, or L5 is a cleavable connector. In some embodiments, the cleavable connector is an electrophilic cleavable connector, a nucleophilic cleavable connector, a photocleavable connector, a metallic cleavable connector, an electrolytic cleavable connector, an acid-cleavable connector, or a protein hydrolytic cleavable connector.

[0053] In some embodiments, the cleavable linker further includes polyethylene glycol groups, glycosyl groups, or modifications to increase hydrophilicity. In some embodiments, the cleavable linker is cleavable under reducing and / or oxidizing conditions. In some embodiments, the cleavable linker is cleavable under acidic conditions. In some embodiments, the cleavable linker contains disulfide bonds.

[0054] In some embodiments, the cleavable adapter is a proteolytically cleavable adapter and includes a protease recognition sequence. In some embodiments, the protease recognition sequence is recognized by a protease selected from the group consisting of metalloproteinases, cathepsin B, and tobacco etch virus (TEV).

[0055] In some embodiments, the cleavable linker comprises a dipeptide, a tripeptide, or a tetrapeptide. In some embodiments, the dipeptide is a valine-citrulline (Val-Cit) dipeptide, a valine-lysine dipeptide, or a valine-alanine dipeptide. In some embodiments, the tetrapeptide is a glycine-glycine-phenylalanine-glycine (GGFG) tetrapeptide.

[0056] In some embodiments, the cleavable connector is selected from the group consisting of PABC (p-aminobenzyl alcohol), glucuronic acid, and MABC (m-aminobenzyl alcohol).

[0057] In some implementation schemes, Y a' The peptide is a heavy chain variable region (VH), light chain variable region (VL), heavy chain, light chain, or constant chain of an antibody or antibody fragment; a peptide; or a cyclic peptide. In some embodiments, the peptide is a binding peptide.

[0058] In some implementation schemes, Y b' The term refers to an antibody, a heavy chain variable region (VH) or light chain variable region (VL) of an antibody fragment, a heavy chain, a light chain, or a constant chain; a peptide; or a cyclic peptide. In some embodiments, the peptide is a binding peptide.

[0059] In some implementation schemes, Y a' With Y b' Connected via L3.

[0060] In some implementations, L3 comprises a peptide sequence, dimerization and docking domains, a leucine zipper, or a pestle-and-mortar structure.

[0061] In some implementations, L3 comprises peptide bonds, disulfide bonds, maleimide bonds, thioether bonds, azide-alkyne cycloadditions, cystoyl-DOPA bonds, or hydrogen bonds.

[0062] In some embodiments, L3 is a connector. In some embodiments, the connector comprises a sequence selected from the group consisting of: (GS) n3 (G2S) n3 (G3S) n3 (G4S) n3 (G) n3 (GGSGGD) n3 (GGSGGE) n3 (GGGSGSGGGGS) n3 and (GGGGGPGGGGP) n3 And n3 is an integer from 2 to 20. In some implementations, n is 1, 2, 3, 4, or 5. In some implementations, n is 1.

[0063] In some implementations, Y1 and Y2 are the same.

[0064] In some implementations, Y1 and Y2 are different.

[0065] In some implementations, Y1, Y2, or both Y1 and Y2 are small molecules.

[0066] In some embodiments, the small molecule is selected from the group consisting of: delutec, ixanotecan, FL118, irinotecan, topotecan, SN-38, rubotecan, belototecan, letonotecan, gemmatotecan, diflutecan, calontecan, citranotecan, nanotecan, ileotecan, DRF-1042, delutec, NSC606985, cimenotecan, ZBH-1205, auristatin (MMAE, MMAF, MMAG, MMAH), chachiomycin, doxorubicin, paclitaxel and paclitaxel derivatives, maytansine (DM1-4), pyrrolodiazepine (PBD), tubulolysin, eribulin, atrazomycin, pyroxine, anthracyclines, and camptothecin (CPT), including the lactone and carboxylic acid ester forms of CPT.

[0067] In some embodiments, the small molecule is selected from the group consisting of topoisomerase inhibitors and tubulin inhibitors.

[0068] In some embodiments, Y1 and Y2, or both Y1 and Y2, comprise nucleic acids, immune agonists, peptides, cytokines, or binding domains. In some embodiments, Y1 and Y2, or both Y1 and Y2, comprise nucleic acids. In some embodiments, Y1 and Y2, or both Y1 and Y2, comprise oligonucleotides.

[0069] In some embodiments, Y1 and Y2, or both Y1 and Y2, comprise peptides. In some embodiments, Y1 and Y2, or both Y1 and Y2, comprise binding peptides.

[0070] On the other hand, this document provides a polypeptide comprising a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine or a portion thereof; and a second end tag different from the first end tag, the second end tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof.

[0071] In some embodiments, the first end is located at the N-terminus of the polypeptide, and the second end tag is located at the C-terminus of the polypeptide.

[0072] In some embodiments, the polypeptide comprises non-terminal tyrosine residues.

[0073] In some embodiments, the polypeptide is modified to expose the non-terminal tyrosine residues, making it accessible to the enzyme.

[0074] On the other hand, this document provides a composition of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine or a portion thereof; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine or a portion thereof; and where m1 is an integer greater than or equal to 0, and the second polypeptide contains a second end tag different from the first end tag, the second end tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10At least one amino acid in it is tyrosine or a part thereof, and m2 is an integer greater than or equal to 0.

[0075] In some embodiments, the first polypeptide and / or the second polypeptide contains non-terminal tyrosine residues.

[0076] In some embodiments, the first polypeptide and / or the second polypeptide are modified to expose the non-terminal tyrosine residues, making them accessible to the enzyme.

[0077] In some implementations, the first end tag includes at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

[0078] In some embodiments, the second end label includes at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

[0079] In some implementations, the first end label includes SGGY, SGY, SGGGY, ESY, EESY, ESSY, ESSSY, SESY, SGGGY, EESY, SSSY, SNNY, SSNY, EGGY, SESY, or SEGY.

[0080] In some implementations, the second end label includes EEEY, EEEEY, SSEEY, SSSEY, SSSSY, or SSNNY.

[0081] On the other hand, this document provides a method for covalently linking at least two peptides to at least two payloads, the method comprising: a) contacting a first peptide of the at least two peptides with a first payload of the at least two payloads using a first tyrosinase, wherein the first peptide comprises a first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine or a portion thereof; and b) using a second tyrosinase to contact a second polypeptide of the at least two polypeptides with a second payload of the at least two payloads, wherein the second polypeptide contains a second terminal tag, the second terminal tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof.

[0082] In some embodiments, the first tyrosinase, the second tyrosinase, or both the first tyrosinase and the second tyrosinase are Agaricus bisporus tyrosinase (abTYR).

[0083] In some embodiments, the first tyrosinase, the second tyrosinase, or both contain a sequence that is at least 80% identical to any one of SEQ ID NO: 1-6, 52, or 53.

[0084] In some embodiments, the first tyrosinase, the second tyrosinase, or both the first tyrosinase and the second tyrosinase are catenases.

[0085] In some embodiments, the first tyrosinase and the second tyrosinase are catenases.

[0086] In some embodiments, the first tyrosinase, the second tyrosinase, or both contain a sequence that is at least 90% identical to any of SEQ ID NO: 2-6.

[0087] In some embodiments, the first tyrosinase, the second tyrosinase, or both contain a sequence that is at least 90% identical to SEQ ID NO: 2.

[0088] In some embodiments, the first tyrosinase is Agaricus bisporus tyrosinase (abTYR) and the second tyrosinase is Catenase.

[0089] In some implementations, a first tyrosinase and a second tyrosinase are provided simultaneously.

[0090] In some implementations, a first tyrosinase is provided first, followed by a second tyrosinase.

[0091] On the other hand, this document provides a method for covalently linking at least two peptides to at least two payloads, the method comprising: a) contacting a first peptide of the at least two peptides with a first payload of the at least two payloads in the presence of a tyrosinase, wherein the first peptide comprises a first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine or a portion thereof; and b) contacting a second polypeptide of the at least two polypeptides with a second payload of the at least two payloads in the presence of the tyrosinase, wherein the second polypeptide comprises a second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof.

[0092] In some embodiments, the first polypeptide is contacted with the tyrosinase at a temperature between 0 and 15°C. In some embodiments, the first polypeptide is contacted with the tyrosinase at a temperature of 4°C.

[0093] In some embodiments, the second polypeptide is contacted with the tyrosinase at a temperature between 25 and 50°C. In some embodiments, the second polypeptide is contacted with the tyrosinase at a temperature of 30°C.

[0094] In some embodiments, the first polypeptide is contacted with the tyrosinase for 5-70 minutes. In some embodiments, the first polypeptide is contacted with the tyrosinase for 45 minutes. In some embodiments, the first polypeptide is contacted with the tyrosinase for 60 minutes.

[0095] In some embodiments, the second polypeptide is contacted with the tyrosinase for at least 15 minutes. In some embodiments, the second polypeptide is contacted with the tyrosinase for 60-160 minutes. In some embodiments, the second polypeptide is contacted with the tyrosinase for 90 minutes. In some embodiments, the second polypeptide is contacted with the tyrosinase for 120 minutes.

[0096] In some embodiments, the tyrosinase contains at least 80% identity with any of SEQ ID NO: 1-6, 52 or 53.

[0097] On the other hand, this paper provides conjugates of formula E', F', G', H', J', or K': E', Formula F', Formula G', Formula H', Formula J', or Formula K' in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5Y, where X1-X5 are any amino acids, provided that no more than two of X2, X3, X4 and X5 are aspartic acid or glutamic acid, and where m1 is an integer greater than or equal to 0; Y b It is the second polypeptide; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0098] On the other hand, this paper provides compounds of formula LI: Equation LI, where R is the effective load.

[0099] In some embodiments, the compound is selected from the group consisting of: SH-VC-PAB-DXD SH-VC-PAB-MMAE, SH-VC-PAB-camptothecin, and SH-VC-PAB-Dorothy Star.

[0100] On the other hand, this article provides polypeptide conjugates represented by formula S1, S2, S3 or S4. Formula S1, Formula S2, Formula S3, or Formula S4, Wherein: Pp is a polypeptide containing oxidized tyrosine; n is 1, 2 or 3; and the "-S-" of the compound is conjugated to the oxidized tyrosine on the polypeptide.

[0101] In some embodiments, the polypeptide is an enzyme, an antibody or a portion thereof, a structural polypeptide, a receptor ligand, or a receptor.

[0102] In some embodiments, the polypeptide is an antibody or a portion thereof.

[0103] In some embodiments, the polypeptide is an antibody or antibody fragment with a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain, a light chain, a constant chain; a peptide; or a cyclic peptide. Attached Figure Description

[0104] Figure 1A-Figure 1B This provides a feature overview of compositions and methods for conjugating peptides or biomolecules to one or more payloads disclosed herein. Figure 1A Bar graphs showing the selectivity of the methods disclosed herein for conjugating peptides engineered to have tyrosine tags. Figure 1BThe results of the stability assay are shown, which display the protein purification traces of peptides conjugated to the payload via maleimide bonds immediately after the conjugation reaction and 7 days after in serum at 37°C, compared to peptides conjugated to the payload via CysTyr bonds disclosed in the compositions and methods herein.

[0105] Figure 2 This is achieved via catenase conjugation, cysteine ​​residues (site 1), heavy and light chain ends (sites 2 and 4), and native cyclic tyrosine (site 3-Y). A diagram showing the different accessible reaction sites.

[0106] Figure 3 This displays in vitro activity data for the trastuzumab-based ADC. Figure 3 Figure A shows the chemical structure of an exemplary payload monomethylaurestatin E (MMAE) and an exemplary linker for conjugating to a trastuzumab-based antibody. Figure 3 The B-plot to G-plot is displayed using the CysTyr key. Figure 3 (Figures B to E) and maleimide bonds ( Figure 3 The results (Figures F to G) show the in vivo activity data of trastuzumab-based antibody-drug conjugates (ADCs). In HER2-expressing cell lines (Figures F to G), Figure 3 (Figures B, D, and F) and HER2-negative cell lines ( Figure 3 In vivo activity was tested in the C, E and G plots.

[0107] Figure 4A The gel shows Coomassie staining, which reveals the integrity and reduction conditions of antibodies modified with two protein cargoes (one cargo per heavy chain).

[0108] Figure 4B The results of mass spectrometry analysis are shown to observe the percentage of modification of natural tyrosine residues. Figure 4B Figure A shows the results for the control heavy chain, and Figure 4B Figure B shows the results of heavy chain reaction with the exemplary enzyme Catenase.

[0109] Figure 5 This displays the results of mass spectrometry analysis of multiple payloads conjugated with the same antibody. Figure 5 Figure A shows the results of the heavy chain control. Figure 5 Figure B shows the results for the light chain control. Figure 5 Figure C shows the modification results of the exemplary heavy chain, where one or two exemplary payloads are observed added to the exemplary heavy chain. Figure 5The D-plot shows the modification results of the exemplary light chain, where one or two exemplary payloads are observed added to the exemplary light chain.

[0110] Figure 6 This displays the mass spectrometry analysis results of an exemplary antibody heavy chain. The top plot is the mass spectrum of the control heavy chain. The second plot from the top is the mass spectrum of the heavy chain after undergoing a conjugation reaction with an exemplary payload (payload A) at 4°C. The third plot from the top is the mass spectrum of the heavy chain from the second plot from the top after the cleanup step. The fourth plot is the mass spectrum of the heavy chain from the third plot from the top after reacting with a second exemplary payload (payload B) at 37°C.

[0111] Figure 7 This displays the mass spectrometry analysis results of an exemplary antibody light chain. The top plot is the mass spectrum of the control light chain. The second plot from the top is the mass spectrum of the light chain after undergoing a conjugation reaction with an exemplary payload (payload A) at 4°C. The third plot from the top is the mass spectrum of the light chain from the second plot from the top after a cleanup step. The fourth plot is the mass spectrum of the light chain from the third plot from the top after reacting with a second exemplary payload (payload B) at 37°C.

[0112] Figure 8 The mass spectrometry analysis results of exemplary antibodies are shown. The top image shows the mass spectrum of the antibody and its natural sugar after conjugation with an enzyme (e.g., Catenase). The second image from the top shows the mass spectrum of the deglycosylated antibody after conjugation with an enzyme (e.g., Catenase) at 4°C. The third image from the top shows the mass spectrum of the exemplary deglycosylated antibody after conjugation with an enzyme (e.g., Catenase) at room temperature. The fourth image from the top shows the mass spectrum of the exemplary antibody without any sugar after conjugation with an enzyme (e.g., Catenase). The fifth image shows the mass spectrum of the exemplary deglycosylated antibody after conjugation with an enzyme (e.g., Catenase) under warm conditions (e.g., room temperature (22°C) or 37°C).

[0113] Figures 9A through 9C show the mass spectrometry analysis results of exemplary HER2 antibodies. Figure 9A shows the mass spectra of the unmodified HER2 antibody compared to HER2 antibodies conjugated with multiple payloads. Figure 9B shows close-up mass spectra of the light chain before and after conjugation with multiple payloads. Figure 9C shows close-up mass spectra of the heavy chain before and after conjugation with multiple payloads.

[0114] Figures 10A to 10G Shown in the high HER2 cell line BT474 ( Figure 10A ), SKBR3 ( Figure 10C ) and N87 ( Figure 10D ) and the low HER2 cell line JIMT-1 ( Figure 10BResults of in vitro activity data comparing the HER2 antibody conjugate (round) with DS8201a (T-DXd, triangular). One of DS8201a and the exemplary HER2 antibody conjugate showed high selectivity due to lack of inhibition in HER2-negative MDA-MB-468 cells. Figure 10E ). Figure 10F and Figure 10G Results showing in vitro activity data of the HER2 antibody conjugate (round) compared to DS8201a (T-DXd, triangular).

[0115] Figure 11 This study shows tumor growth volume in mice, comparing different doses of an exemplary HER2 antibody or Ds8201a (T-Dxd) conjugated with a topoisomerase inhibitor and a microtubule inhibitor to a control.

[0116] Figure 12A This image shows the mass spectrometry analysis results of an exemplary anti-HER2 antibody with a light chain modified to have a terminal tag. The top image shows the antibody before reaction with Catenase at 4°C, and the bottom image shows the antibody after reaction with Catenase at 4°C.

[0117] Figure 12B This image shows the mass spectrometry analysis results of an exemplary anti-HER2 antibody with a modified heavy chain and a terminal tag. The top image shows the antibody before reaction with Catenase at 4°C, and the bottom image shows the antibody after reaction with Catenase at 4°C.

[0118] Figure 13A This image shows the mass spectrometry analysis results of an exemplary anti-HER2 antibody with a light chain modified to have a terminal tag. The top image shows the modified antibody before reaction with Catenase at 30°C, and the bottom image shows the modified antibody after reaction with Catenase at 30°C.

[0119] Figure 13B This image shows the mass spectrometry analysis results of an exemplary anti-HER2 antibody, with both the heavy and light chains modified to have terminal tags. The top image shows the modified antibody before reaction with catenase at 30°C, and the bottom image shows the modified antibody after reaction with catenase at 30°C.

[0120] Figures 14A to 14C This displays the mass spectrometry analysis results of an exemplary anti-HER2 antibody, which is modified to have a terminal tag on the heavy chain, a terminal tag on the light chain, and to include the mutations S298G and T299A on the heavy chain before and after two reactions with Catenase and the exemplary payload ethathecan. Figure 14AThe mass spectrum of the modified antibody prior to the Catenase reaction is shown. Figure 14B The mass spectrum of the modified antibody after a 60-minute catenase reaction at 4°C is shown. Figure 14C show Figure 14B Mass spectrometry of the modified antibody after a second catenase reaction at 30°C for 90 min.

[0121] Figures 15A to 15D This displays the mass spectrometry analysis results of an exemplary anti-HER2 antibody, which is modified to have a terminal tag on the heavy chain, a terminal tag on the light chain, and to include the mutation S298G T299A on the heavy chain before and after two reactions with Catenase and two exemplary payloads, MMAE and essanotecan. Figure 15A The mass spectrum of the modified antibody prior to the Catenase reaction is shown. Figure 15B The mass spectra of the modified antibody after catenase reaction with MMAE at 4°C for 45 min are shown. Figure 15C show Figure 15B Mass spectrometry of the modified antibody after a second catenase reaction with essanotecan at 30°C for 120 min. Figure 15D show Figure 15C The modified antibody was analyzed by mass spectrometry after purification.

[0122] Figures 16A to 16D Displays the percentage of modification of the light or heavy chain with the indicated end label, which is attached to an exemplary payload via Catenase. Figure 16A The percentage of modification shown is for the exemplary No. 1 payload of the heavy chain sustained at 4°C for 60 min. Figure 16B The percentage of modification is shown for the exemplary No. 2 payload of the heavy chain sustained at 4°C for 60 min. Figure 16C The percentage of modification shown is for the exemplary No. 1 payload of the light chain at 30°C. Figure 16D The percentage of modification shown is for the exemplary No. 2 payload of the light chain at 30°C.

[0123] Figure 17 The percentage of modification of the heavy and light chains of the modified trastuzumab by the indicated payload of Catenase (SEQ ID NO: 2) at moderate temperatures is shown.

[0124] Figure 18 show Figure 17 The corresponding mass spectra of the heavy and light chains in the reaction.

[0125] Figure 19The results show the percentage of modification of the heavy and light chains of the modified trastuzumab by the indicated payload of the catenase at cold, low-temperature conditions.

[0126] Figure 20 show Figure 19 The corresponding mass spectra of the heavy and light chains in the reaction.

[0127] Figure 21 The NMR spectrum of the synthesized SH-VC-PAB-DXD compound is shown.

[0128] Figure 22 The NMR spectrum of the synthesized SH-VC-PAB-MMAE compound is shown. Detailed Implementation

[0129] For example, antibody-drug conjugates are among the fastest-growing drugs for diseases and conditions such as cancer. This approach typically involves conjugating a peptide (e.g., an antibody) to a cytotoxic payload via a chemical linker. Conjugates are complex molecules requiring careful attention to their various components. The selection of appropriate targets, peptides, cytotoxic payloads, and the linkage between the peptide and payload are key determinants of the safety and efficacy of such conjugates. The industry needs to improve conjugation procedures for peptides or biomolecules in a simple yet site-specific manner.

[0130] This article provides compositions and methods for conjugating peptides or biomolecules to one or more payloads.

[0131] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. 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 in biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are those well-known and commonly used in the art.

[0132] Throughout this specification and claims, the word “comprise” or variations thereof, such as “comprises” or “comprising”, should be understood to imply that the stated integer or group of integers is included, but does not exclude any other integer or group of integers.

[0133] Unless otherwise indicated, as an example of all sequences described herein in the general format “SEQ ID NO:”, “a nucleic acid containing SEQ ID NO: 1” means a nucleic acid that has 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 is determined by 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.

[0134] If a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range, and any other stated value or intermediate value within the stated range, is covered within this disclosure, and each intermediate value is accurate to one-tenth of the lower limit unit unless the context explicitly indicates otherwise. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also covered within this disclosure, subject to any explicitly excluded limits within the stated range. If a stated range includes one or both of the limits, this disclosure also includes ranges that exclude any or both of those included limits.

[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While similar or equivalent methods and materials to any methods and materials set forth herein may be used in practice or testing of this disclosure, preferred methods and materials are described herein. All publications referenced herein are incorporated by way of citation to disclose and illustrate methods and / or materials relating to the cited publications.

[0136] It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include a plural of indicators. Thus, for example, a reference to “a thiol group” includes a plurality of such thiol groups, and a reference to “the thiol group” includes a reference to one or more thiol groups and their equivalents known to those skilled in the art, and so on. Furthermore, it should be noted that claims may be designed to exclude any optional elements. Therefore, such a statement is intended to serve as a precondition for combining the enumeration of claim elements with exclusive terms such as “only,” “merely,” or with the use of a negative limitation.

[0137] It should be understood that certain features set forth in the context of individual embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features set forth in the context of a single embodiment for simplicity may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to this disclosure are specifically encompassed in this disclosure and disclosed herein, just as each combination is individually and explicitly disclosed herein. In addition, all sub-combinations of the various embodiments and their elements are also specifically encompassed in this disclosure and disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0138] The term "fluorophore" refers to any molecular entity capable of absorbing energy at a first wavelength and then re-emitting energy at a different second wavelength. In some embodiments, the subject biomolecule includes a fluorophore attached to one end of the biomolecule or attached to a central location. In some embodiments, the fluorophore may be attached to one end of the biomolecule. The fluorophore attached to the biomolecule is not necessarily a single molecule, but may include multiple molecules.

[0139] As is known to those skilled in the art, fluorophores can be synthetic or biological in nature. More generally, any fluorophore that is stable under coupling conditions and sufficiently suppressed in proximity to a quencher can be used, such that the fluorescence intensity of the fluorophore can be detected in response to a significant change in the target-specific binding of the probe. Examples of suitable fluorophores include (but are not limited to) Oregon Green 488 dye, rhodamine and rhodamine derivatives, fluorescein isothiocyanate, fluorescein, 6-carboxyfluorescein (6-FAM), coumarin and coumarin derivatives, anthocyanins and anthocyanin derivatives, Alexa Fluors, DyLight Fluors, etc.

[0140] In some embodiments, the biomolecule includes a metal chelating agent. As used herein with respect to complexes between metals and chelating ligands, a “chelate” refers to a combination of metal ions bonded to one or more ligands to form a heterocyclic structure. The formation of a chelate by neutralizing the positive charge of a metal ion may be via the formation of an ionic bond, a covalent bond, or a coordinate covalent bond. In some embodiments, the metal chelating agent includes (but is not limited to) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (also known as DOTA or tetraxetan).

[0141] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, this term includes (but is not limited to) single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0142] The terms “peptide” and “protein” are used interchangeably herein and refer to a polymeric amino acid form of any length, which may include coding and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and peptides having a modified peptide backbone. “Peptide” encompasses chemically modified peptides. For example, the term “peptide” may include monovalent or divalent groups of: i) the chemical structure of the peptide, or ii) the chemical structure of a peptide substituted with chemical functional groups. In some embodiments, the peptide is a peptide without a terminal tag. In some embodiments, the peptide is a small peptide, antibody, or immunoglobulin. The term “fusion protein” or its grammatical equivalent is intended to include proteins composed of multiple peptide components, although said peptide components are typically isolated or unconjugated in their native state, but are typically linked by their respective amino and carboxyl ends via peptide bonds to form a single continuous peptide. Fusion proteins may be combinations of two, three, or even four or more different proteins.

[0143] Generally, polypeptides can have any length, such as 2 or more amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, greater than about 50 amino acids, greater than about 100 amino acids, greater than about 300 amino acids, and typically up to about 500 or 1000 or more amino acids. The length of a “peptide” is typically 2 or more amino acids, such as greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, and typically up to about 50 amino acids. In some embodiments, the length of a peptide is 2 to 30 amino acids.

[0144] The terms “antibody” and “immunoglobulin” include any isotype antibody or immunoglobulin; antibody fragments that retain specific binding to antigens, including (but not limited to) Fab, Fv, scFv, and Fd fragments; chimeric antibodies; humanized antibodies; single-chain antibodies (scAbs); single-domain antibodies (dAbs); single-domain heavy-chain antibodies; single-domain light-chain antibodies; nanobodies; bispecific antibodies; multispecific antibodies; and fusion proteins comprising an antigen-binding (also referred to herein as antigen-binding) portion of both antibody and non-antibody proteins. Antibodies can be detectably labeled, for example, with radioactive isotopes, enzymes that generate detectable products, fluorescent proteins, etc. Antibodies can be further conjugated to other parts, such as members of specific binding pairs, such as biotin (a member of the biotin-antibiotin protein specific binding pair), etc. Antibodies can also be bound to solid supports, including (but not limited to) polystyrene plates or beads. The terminology also encompasses Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to the antigen, and monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which are generated from a single cell through repeated cell replication. That is, the cell clone produces only a single antibody species. Although hybridoma generation techniques can be used to produce monoclonal antibodies, other generation methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are “Y-shaped” molecules composed of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds.

[0145] As used herein, the term "humanized immunoglobulin" refers to an immunoglobulin comprising immunoglobulin motifs of different origins, wherein at least one motif contains an amino acid sequence of human origin. For example, a humanized antibody may comprise motifs derived from a non-human immunoglobulin (e.g., mouse) with essential specificity and from a human immunoglobulin sequence (e.g., chimeric immunoglobulin), chemically concatenated by conventional techniques (e.g., synthesis) or prepared as a neighboring polypeptide using genetic engineering techniques (e.g., DNA encoding the chimeric antibody protein motif may be expressed to generate a neighboring polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains comprising a complementarity-determining region (CDR) derived from a non-human antibody and a framework region derived from a human light chain and / or heavy chain (e.g., a CDR-grafted antibody with or without framework variations). The term humanized immunoglobulin also encompasses chimeric or CDR-grafted single-chain antibodies. See, for example, Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, MS et al., WO 86 / 01533; Neuberger, MS et al., European Patent No. 0,194,276 B1; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, EA et al., European Patent Application No. 0,519,596 A1. Regarding single-chain antibodies, see also Ladner et al., U.S. Patent No. 4,946,778; Huston, U.S. Patent No. 5,476,786; and Bird, RE et al., Science, 242: 423-426 (1988)).

[0146] As used herein, the term “nanobody” (Nb) refers to the smallest antigen-binding fragment or single variable domain (VHH) derived from naturally occurring heavy-chain antibodies, as is known to those skilled in the art. These are derived from heavy-chain-only antibodies, found in camels (Hamers-Casterman et al., (1993) Nature 363:446; Desmyter et al., (1996) Nature Struct. Biol. 3:803). Immunoglobulins lacking polypeptide light chains have been found within the “camelid” family. The “camelid” family includes Old World camels (Bactrian camel (Camelus bactrianus) and Dromedary camels (Camelus bactrianus)) and New World camels (e.g., alpaca (Llama paccos), llama (Llama glama), guanocamel (Llama guanicoe), and vicugna (Llama vicugna)). Single variable domain heavy chain antibodies are referred to as nanobodies or VHH antibodies in this paper.

[0147] An "antibody fragment" contains a portion of a complete antibody, such as the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bivalent antibodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); domain antibodies (dAb; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site; and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Pepsin treatment produces an F(ab')2 fragment with two antigen-binding sites and still capable of cross-linking the antigen.

[0148] As used herein, the term "Fv" refers to the smallest antibody fragment containing both an intact antigen recognition site and an antigen binding site. This region consists of a dimer of a tightly non-covalently associated heavy chain variable domain and a light chain variable domain. It is in this configuration that the three CDRS of each variable domain interact to achieve the desired effect at V. H -V L The surface of the dimer defines antigen-binding sites.

[0149] The six CDRs together confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv, which contains only three CDRs specific to the antigen) has the ability to recognize and bind to the antigen, but with lower affinity than the complete binding site.

[0150] The “Fab” fragment also contains a constant domain of the light chain and a first constant domain (Chi) of the heavy chain. The Fab fragment differs from the Fab' fragment in that several residues are added to the carboxyl terminus of the heavy chain Chi domain, including one or more cysteine ​​residues from the antibody hinge region. In this paper, Fab'-SH is the name of Fab' with one or more cysteine ​​residues in the constant domain bearing a free thiol group. The F(ab')2 antibody fragment was initially generated as a Fab' fragment pair with a hinge cysteine ​​residue between the fragments. Other chemical conjugations of antibody fragments are also known.

[0151] The "light chain" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domain. Depending on the amino acid sequence of the constant domain of the immunoglobulin heavy chain, it can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Subclasses can be further subdivided into types, such as IgG2a and IgG2b.

[0152] As used herein, a “single-chain Fv” or “sFv” or “scFv” antibody fragment contains the antibody’s V. H and V L Domains, wherein these domains are present within a single polypeptide chain. In some embodiments, the Fv polypeptide also contains V. H With V L The peptide linkers between the domains enable sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0153] As used herein, the term "bivalent antibody" refers to a small antibody fragment having two antigen-binding sites, said fragment comprising a polypeptide chain (V... H -V L The light chain variable structural domain (V) in ) L ) connected heavy chain variable structural domain (V HBy using a linker that is too short to allow pairing between two domains on the same strand, the domain is forced to pair with a complementary domain on another strand, resulting in two antigen-binding sites. Bivalent antibodies are described more fully in, for example, EP 404,097; WO93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0154] As used herein, the term "affinity" refers to the equilibrium constant of the reversible binding of two agents (e.g., antibody and antigen), and is expressed as the dissociation constant (K). D Affinity can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 1,000 times or more of the antibody's affinity for unrelated amino acid sequences. The antibody's affinity for the target protein can be, for example, from about 100 nanomolars (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term "affinity" refers to the resistance of a complex of two or more agents to dissociation upon dilution. Regarding antibody and / or antigen-binding fragments, the terms "immunoreactivity" and "preferential binding" are used interchangeably herein.

[0155] As used herein, the term "binding" refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding (including interactions such as salt bridges and water bridges). "Specific binding" refers to a binding affinity of at least about 10. 7 M or larger (e.g., 5 x 10) 7 M, 10 s M, 5 x 10 s The binding of M and larger. "Non-specific binding" refers to binding with an affinity of less than about 10. 7 The binding of M, for example, with an affinity of 10. 6 M, 10 5 M, 10 4 The combination of M, etc.

[0156] "Isolated" polypeptides are polypeptides that have been identified and isolated and / or recovered from their native environmental components. Contaminating components in the native environment of a polypeptide are materials that interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein- or non-protein-based solutes. In some embodiments, the polypeptide will be purified to the extent that: (1) it is purified to greater than 90% by weight, greater than 95% by weight, or greater than 98% by weight, for example, greater than 99% by weight, as determined by the Lowry method; (2) it is purified to the extent that at least 15 residues of the N-terminal or internal amino acid sequence can be obtained using a twist-cup sequencer; or (3) it is purified to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated polypeptides include recombinant intracellular in situ polypeptides, because at least one component in the native environment of the polypeptide will be absent. In some cases, isolated polypeptides will be prepared by at least one purification step.

[0157] The terms "HER2 positive" or "HER2 high expression" refer to HER2 overexpression in cancer. Cancer is classified as "HER2 positive" based on an immunohistochemical (IHC) score of 3+ or an IHC score of 2+ with HER2 amplification. The presence of at least four copies of HER2 per tumor cell can also be used to classify cancer as "HER2 positive," and this can be confirmed using in situ hybridization (ISH) assays.

[0158] The term "HER2 low expression" refers to low HER2 expression in cancer. Cancers are classified as "HER2 low expression" based on an immunohistochemical (IHC) score of 0, 1+, or 2+ with no HER2 amplification. "HER2 low expression" cancers can refer to HER2-negative cancers and HER2 triple-negative cancers (estrogen receptor (ER) negative, progesterone receptor (PR) negative, and HER2 negative). Each tumor cell has up to four (e.g., up to three, two, or one) HER2 copies. This can also be used to classify cancers as "HER2 low expression" and can be determined using in situ hybridization (ISH) assays.

[0159] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments set forth and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of this disclosure. Any of the enumerated methods may be implemented in the order of the enumerated events or in any other logically possible order.

[0160] If a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range, and any other stated value or intermediate value within the stated range, is covered within this disclosure, and each intermediate value is accurate to one-tenth of the lower limit unit unless the context explicitly indicates otherwise. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also covered within this disclosure, subject to any explicitly excluded limits within the stated range. If a stated range includes one or both of the limits, this disclosure also includes ranges that exclude any or both of those included limits.

[0161] The disclosed polypeptide In some embodiments, this document describes conjugates of formula A', formula B', formula C', or formula D': Formula A' Formula B' Formula C' Formula D' in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid from X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid from X1-X3 is tyrosine or a portion thereof, where Y a Linked to "S" via the tyrosine or a portion thereof; Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the formula is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof, where Y b Linked to "S" via the tyrosine or a portion thereof; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0162] In some implementations, this document describes conjugates of formula (A), formula (B), formula (C), or formula (D): Formula A Formula B Formula C Formula D in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of the amino acids X1-X5 is tyrosine, and where m1 is an integer greater than or equal to 0; Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in it is tyrosine, and m2 is an integer greater than or equal to 0; n is an integer greater than 0; L1 is an optional first connector; L2 is an optional second connector; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0163] In some implementations, this document describes conjugates of formula A'', formula B'', formula C'', or formula D'': Formula A'' Formula B'' Formula C'' Formula D'' in: Y aThe first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid from X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid from X1-X3 is tyrosine or a portion thereof, where Y a Linked to "S" via the tyrosine or a portion thereof; Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the formula is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof, where Y b Linked to "S" via the tyrosine or a portion thereof; r is 1, 2, or 3; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0164] In some implementations, this document describes conjugates of formula (A), formula (B), formula (C), or formula (D): Formula A Formula B Formula C Formula D in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of the amino acids X1-X5 is tyrosine, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of the amino acids X1-X3 is tyrosine. Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in is tyrosine, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine. n is an integer greater than 0; L1 is an optional first connector; L2 is an optional second connector; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0165] In some embodiments, this document describes conjugates of formula A', formula B', formula C', or formula D': Formula A' Formula B' Formula C' Formula D' in: Y a The group is derived from a first biomolecule, the first biomolecule comprising a first terminal tag, the first terminal tag comprising (X1). m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine, wherein the phenol of said tyrosine is formed by oxidation. or ; Y b The second biomolecule contains a second end tag different from the first end tag, the second end tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10At least one amino acid in the form is tyrosine, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine, wherein the phenol of the tyrosine is formed by oxidation: or ; n is an integer greater than 0; L3 is the optional first connector; L4 is an optional second connector; L5 is an optional third connector; Y1 is the first effective payload; and Y2 is the second effective load. In some implementation schemes, Y a Y b Or Y a and Y b These can be nanoparticles, polymers, nucleic acid sequences, or aptamers.

[0166] In some embodiments, this document describes conjugates comprising a phenolic or catechol moiety conjugated to one or more loads containing a thiol moiety.

[0167] In some embodiments, conjugates of formula (E), formula (F), formula (G), or formula (H) are described herein: Formula E Formula F Formula G Formula H in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid from X1-X5 is tyrosine or a subset thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid from X1-X3 is tyrosine, where Y a Linked to "S" via the tyrosine or a portion thereof; Y b This is the second polypeptide; n is an integer greater than 0. L1 is an optional first connector; L2 is an optional second connector; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0168] In some implementations, this document describes conjugates of formula E', F', G', H', J', or K': E', Formula F', Formula G', Formula H', in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid from X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid from X1-X3 is tyrosine or a portion thereof, where Y a Linked to "S" via the tyrosine or a portion thereof; Y b It is the second polypeptide; n is an integer greater than 0; L3 is the optional first connector; L4 is an optional second connector; L5 is the third connector that can be selected from Y2; Y1 is the first effective payload; and Y2 is a group derived from a second payload containing phenol, wherein the phenol is attached to "S".

[0169] In some implementations, this document describes conjugates of formula E', F', G', H', J', or K': E', Formula F', Formula G', Formula H', in: Y a The group is derived from a first polypeptide, the first polypeptide comprising a first terminal tag, the first terminal tag comprising (X1) m1X2X3X4X5Y, where X1-X5 are any amino acids, provided that no more than two of X2, X3, X4, and X5 are aspartic acid or glutamic acid, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine, wherein the phenol of said tyrosine is formed by oxidation. or ; Y b It is the second polypeptide; n is an integer greater than 0; L3 is the optional first connector; L4 is an optional second connector; L5 is the third connector that can be selected from Y2; Y1 is the first effective payload; and Y2 is a group derived from the second payload, which contains phenol, wherein the phenol is formed by oxidation: or .

[0170] In some implementations, this document describes conjugates of formula (J) or formula (K): Formula J Formula K in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of the amino acids X1-X5 is tyrosine, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of the amino acids X1-X3 is tyrosine. Y b It is the second polypeptide; n is an integer greater than 0; L1 is an optional first connector; L2 is an optional second connector; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0171] In some implementations, this document describes conjugates of formula J' or formula K': Formula J', or Formula K' in: Y a The group is derived from a first polypeptide, the first polypeptide comprising a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine, wherein the phenol of said tyrosine is formed by oxidation. or ; Y b It is the second polypeptide; n is an integer greater than 0; L3 is the optional first connector; L4 is an optional second connector; L5 is an optional third connector; Y1 is the first effective payload; and Y2 is the second payload.

[0172] In some embodiments, this document describes conjugates of formulas L, M, N, and O: Formula L Formula M Formula N Formula O in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid from X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid from X1-X3 is tyrosine or a portion thereof, where Y a Linked to "S" via the tyrosine or a portion thereof; Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10For any amino acid, the condition is X6-X 10 At least one amino acid in the formula is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof, where Y b Linked to "S" via the tyrosine or a portion thereof; n is an integer greater than 0; L1 is an optional third connector; L2 is an optional fourth connector; L3 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0173] In some embodiments, this document describes conjugates of formulas L, M, N, and O: Formula L Formula M Formula N Formula O in: Y a The group is derived from a first polypeptide, the first polypeptide comprising a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine, wherein the phenol of said tyrosine is formed by oxidation. or ; Y b The second polypeptide contains a group derived from the first terminal tag, which includes a second terminal tag different from the first terminal tag, and the second terminal tag includes (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the form is tyrosine, and m2 is an integer greater than or equal to 0, wherein the phenol of the tyrosine is formed by oxidation: or ; n is an integer greater than 0; L1 is an optional first connector; L2 is an optional second connector; L3 is an optional third connector; Y1 is the first effective payload; and Y2 is the second payload.

[0174] In some implementations, this document describes conjugates of formulas LI, MI, NI, and OI: Formula LI, MI, Formula NI, Formula OI, Y a' It is the first polypeptide. Each of X1, X2, X3, X4, and X5 is independently any amino acid, where m1, m2, and m3 are each integers greater than or equal to 0; Y b' It is the second polypeptide. X6, X7, X8, X9 and X 10 Each of them is independently any amino acid, where m4, m5, and m6 are each an integer greater than or equal to 0, and where -(X1) is an integer. m1 (X2) m2 X3X4 - Partially different from -(X6) m1 (X7) m2 X8X9 - Partial; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0175] In some implementations, this document describes conjugates of formulas P, Q, R, and S: Formula P Formula Q Formula R Formula S in: Ya The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid from X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid from X1-X3 is tyrosine or a portion thereof, where Y a Linked to "S" via the tyrosine or a portion thereof; Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the formula is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof, where Y b Linked to "S" via the tyrosine or a portion thereof; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0176] In some implementations, this document describes conjugates of formulas P, Q, R, and S: Formula P Formula Q Formula R Formula S in: Y a The group is derived from a first polypeptide, the first polypeptide comprising a first terminal tag, the first terminal tag comprising (X1) m1X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine, wherein the phenol of said tyrosine is formed by oxidation: or Y b The second polypeptide contains a group derived from the first terminal tag, which includes a second terminal tag different from the first terminal tag, and the second terminal tag includes (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the form is tyrosine, and m2 is an integer greater than or equal to 0, wherein the phenol of the tyrosine is formed by oxidation: or ; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0177] In some implementations, this document describes conjugates of formulas PI, QI, RI, and SI: Formula PI, Formula QI, Formula RI, Formula SI, Y a' It is the first polypeptide. Each of X1, X2, X3, X4, and X5 is independently any amino acid, where m1, m2, and m3 are each integers greater than or equal to 0; Y b' It is the second polypeptide. X6, X7, X8, X9 and X 10Each of them is independently any amino acid, where m4, m5, and m6 are each an integer greater than or equal to 0, and where -(X1) is an integer. m1 (X2) m2 X3X4 - Partially different from -(X6) m1 (X7) m2 X8X9 - Partial; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

[0178] In some implementations, this document describes conjugates of formulas T, U, V, and W: Formula T Formula U Formula V Formula W in: Y a It is the first polypeptide; Y b It is the second polypeptide; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is the fifth connector that can be selected from Y2; Y1 is a first payload containing phenol, wherein the phenol is linked to imidazole; and Y2 is a second payload containing phenol, wherein the phenol is linked to imidazole.

[0179] In some implementations, this document describes conjugates of formulas X, Y, Z, and AA: Formula X Formula Y Formula Z Formula AA in: Y a It is the first polypeptide; Y b It is the second polypeptide; n is an integer greater than 0; L1 is an optional first connector; L2 is an optional second connector; L3 is an optional third connector; Y1 is a first payload containing phenol, wherein the phenol is connected to "N", and Y2 is a second payload containing phenol, wherein the phenol is connected to "N".

[0180] In some embodiments, the tyrosine or a portion thereof is selected from the group consisting of: , , and .

[0181] In some implementation schemes, Y a It includes a first end label. In some embodiments, the first end label includes (X1). m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is a tyrosine, and where m1 is an integer greater than or equal to 0. In some embodiments, the first terminal tag includes (X1). m1 X2X3X4X5Y, where m1 is an integer greater than or equal to 0. In some embodiments, the first end label contains Y(X1). m1 X2X3X4X5, where m1 is an integer greater than or equal to 0. In some embodiments, the first end tag contains X1X2X3, where X1-X3 are any amino acids, provided that at least one of the amino acids in X1-X3 is tyrosine.

[0182] In some embodiments, the first terminal tag comprises at least two amino acids selected from aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K). In some implementations, the first end tag includes at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

[0183] In some implementation schemes, Y b Includes a second end label. In some implementations, Y b A second end label, different from the first end label, is included. In some embodiments, the second end label includes (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the form is tyrosine, and m2 is an integer greater than or equal to 0. In some embodiments, the second terminal tag includes (X6). m2 X7 X8 X9 X 10 Y, where m2 is an integer greater than or equal to 0. In some implementations, the second end label contains Y(X6). m2 X7 X8 X9 X 10 , where m2 is an integer greater than or equal to 0. In some embodiments, the second end tag comprises X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is a tyrosine.

[0184] In some embodiments, the second terminal tag comprises at least two amino acids selected from aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K). In some embodiments, the second end label includes at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

[0185] In some implementations, the first end label includes SGGY, SGY, SGGGY, ESY, EESY, ESSY, ESSSY, SESY, SGGGY, EESY, SSSY, SNNY, SSNY, EGGY, SESY, or SEGY.

[0186] In some implementations, the second end label includes EEEY, EEEEY, SSEEY, SSSEY, SSSSY, or SSNNY.

[0187] In some implementations, the first end label contains SGGY or SGY, and the second end label contains EEEY.

[0188] In some implementations, the first end label contains SEY, and the second end label contains SSEEY or SSSEY.

[0189] In some implementations, the first end label contains SGGGY, and the second end label contains EEEEY.

[0190] In some implementations, the first end label contains ESSY, and the second end label contains SSEEY.

[0191] In some implementations, the first end label contains ESSY, and the second end label contains SSSEY.

[0192] In some implementation schemes, Y a Y b Or Y a and Y b Both are glycosylated. In some implementations, Y a Y b Or Y a and Y b Both contain non-terminal tyrosine residues.

[0193] In some implementation schemes, Y a The variable region (VH) of the heavy chain of an antibody or antibody fragment, the variable region (VL) of the light chain of an antibody or antibody fragment, the heavy chain of an antibody or antibody fragment, the light chain of an antibody or antibody fragment, the constant chain of an antibody or antibody fragment, a peptide, or a cyclic peptide.

[0194] In some implementation schemes, Y b The variable region (VH) of the heavy chain of an antibody or antibody fragment, the variable region (VL) of the light chain of an antibody or antibody fragment, the heavy chain of an antibody or antibody fragment, the light chain of an antibody or antibody fragment, the constant chain of an antibody or antibody fragment, a peptide, or a cyclic peptide.

[0195] In some implementation schemes, Y a With Y bThe linker is connected via L3. In some embodiments, L3 comprises a peptide sequence, a dimerization and docking domain, a leucine zipper, or a mortise and tenon structure. In some embodiments, L3 comprises a peptide bond, a disulfide bond, a maleimide bond, a thioether bond, an azide-alkyne cycloaddition, a cysteine-DOPA bond, or a hydrogen bond. In some embodiments, L3 is a linker. In some embodiments, the linker comprises a sequence selected from the group consisting of (GS)n3, (G2S)n3, (G3S)n3, (G4S)n3, (G)n3, (GGSGGD)n3, (GGSGGE)n3, (GGGSGSGGGGS)n3, and (GGGGGPGGGGP)n3, wherein n3 is an integer from 2 to 20.

[0196] In some implementations, L3 contains a terminal tyrosine residue.

[0197] In some implementations, n is 1, 2, 3, 4, or 5. In some implementations, n is 1.

[0198] In some embodiments, Y1 and Y2 are the same payload. In some embodiments, Y1 and Y2 are different payloads. In some embodiments, Y1 and Y2, or both Y1 and Y2, are small molecules.

[0199] In some embodiments, the small molecule is selected from the group consisting of: delutec, ixanotecan, FL118, irinotecan, topotecan, SN-38, rubotecan, belototecan, letonotecan, gemmatotecan, diflutecan, calontecan, citranotecan, nanotecan, ileotecan, DRF-1042, delutec, NSC606985, cimenotecan, ZBH-1205, auristatin (MMAE, MMAF, MMAG, MMAH), chachiomycin, doxorubicin, paclitaxel and paclitaxel derivatives, maytansine (DM1-4), pyrrolodiazepine (PBD), tubulolysin, eribulin, atrazomycin, pyroxine, anthracyclines, and camptothecin (CPT), including the lactone and carboxylic acid ester forms of CPT.

[0200] In some implementations, Y1 and Y2, or both Y1 and Y2, contain nucleic acids, immune agonists, peptides, cytokines, or binding domains.

[0201] On the other hand, this document provides a polypeptide comprising a first terminal tag, the first terminal tag comprising (X1) m1X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is a tyrosine; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is a tyrosine; and where m1 is an integer greater than or equal to 0; and a second polypeptide, the second polypeptide containing a second end tag different from the first end tag, the second end tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the polypeptide is a tyrosine, and m2 is an integer greater than or equal to 0. In some embodiments, the polypeptide contains non-terminal tyrosine residues. In some embodiments, the polypeptide is modified to expose non-terminal tyrosine residues, making it accessible to the enzyme. In some embodiments, the modification includes mutating the sequence of the polypeptide to produce a glycosylated polypeptide. In some embodiments, the polypeptide is deglycosylated using conventional methods known in the art.

[0202] On the other hand, this document discloses a composition of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is a tyrosine; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is a tyrosine; and where m1 is an integer greater than or equal to 0, and the second polypeptide contains a second end tag different from the first end tag, the second end tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in it is tyrosine, and m2 is an integer greater than or equal to 0.

[0203] In some embodiments, the first polypeptide and / or the second polypeptide contains non-terminal tyrosine residues.

[0204] In some embodiments, the first polypeptide and / or the second polypeptide are modified to expose non-terminal tyrosine residues, making them accessible to the enzyme.

[0205] connector Connector sequences can be used to separate the different components of the conjugates described herein. In some embodiments, the conjugates described herein include connectors L1, L2, L3, L4 and / or L5.

[0206] In some embodiments, any of the linkers comprises at least 5 to about 50 amino acids. In some embodiments, the linker comprises about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 10 to about 50 amino acids, about 15 to about 50 amino acids, about 20 to about 50 amino acids, about 25 to about 50 amino acids, about 30 to about 50 amino acids, about 35 to about 50 amino acids, about 40 to about 50 amino acids, or about 45 to about 50 amino acids.

[0207] In some embodiments, any of the connectors comprises a sequence selected from the group consisting of (GS)n, (G2S)n, (G3S)n, (G4S)n, and (G)n, where n is an integer from 2 to 20. In some embodiments, n is an integer from 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20.

[0208] In some embodiments, any of the connectors comprises a sequence selected from the group consisting of (GGSGGD)n or (GGSGGE)n, where n is an integer from 2 to 6.

[0209] In some embodiments, any of the connectors comprises a sequence selected from the group consisting of (GGGSGSGGGGS)n and (GGGGGPGGGGP)n, where n is an integer from 1 to 3.

[0210] In some embodiments, any of the connectors comprises a sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, where z is between 1 and 20, and where n is at least 8. In some embodiments, z is between 2 and 18, 2 and 16, 2 and 14, 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 20, 6 and 20, 8 and 20, 10 and 20, 12 and 20, 14 and 20, 16 and 20, or 18 and 20. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0211] In some embodiments, the linker in the conjugates disclosed herein is a cleavable linker. In some embodiments, at least one of L1, L2, L3, L4, or L5 is a cleavable linker. In some embodiments, the cleavable linker is an electrophilic cleavable linker, a nucleophilic cleavable linker, a photocleavable linker, a metal cleavable linker, an electrolytic cleavable linker, an acid cleavable linker, or a proteolytic cleavable linker. In some embodiments, the cleavable linker is cleavable under reducing and / or oxidizing conditions. In some embodiments, the cleavable linker is cleavable under acidic conditions. In some embodiments, the cleavable linker is cleaved by an enzyme. In some cases, the cleavable linker is a linker that cleaves under reducing conditions. In some embodiments, the cleavable linker is rapidly cleaved by glutathione reduction. In some embodiments, the cleavable linker contains a disulfide bond. In some embodiments, the cleavable linker is cleaved by physical stimulation. In some embodiments, the cleavable linker is photocleavable.

[0212] In some embodiments, L1, L2, L3, L4, or L5 are acid-instability connectors. In some embodiments, the connectors cleave at pH 6 or lower, such as 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, 4.9, 4.85, 4.80, 4.75, 4.7, 4.65, 4.6, 4.55, 4.5, or even lower.

[0213] In some implementations, L1, L2, L3, L4, or L5 are photolytically degradable connectors. Suitable photolytically degradable connectors include o-nitrobenzyl connectors, benzoylmethyl connectors, alkoxybenzoin connectors, chromium aromatic complex connectors, NpSSMpact connectors, and pivaloyl diol connectors, as described by Guillier et al. (Chem. Rev. 2000 1000:2091-2157).

[0214] In some implementations, L1, L2, L3, L4, or L5 are protein hydrolyzable linkers. The proteolytically cleavable linker may include a protease recognition sequence recognized by a protease selected from the group consisting of: alanine carboxypeptidase, Armillaria mellea astaxanthin, bacterial leucylaminopeptidase, cancer procoagulant, cathepsin B, clostridium protease, cytoplasmic alanylaminopeptidase, elastase, intracellular protease Arg-C, enterokinase, gastric protease, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, ferroin C, IgA-specific serine endopeptidase, leucylaminopeptidase, leucylaminopeptidase, lysC, lysosomal pre-X carboxypeptidase, lysylaminopeptidase, methionylaminopeptidase, myxobacteria, nardilysin, pancreatic endopeptidase E, and picornain. 2A) Microribonucleic acid viral endopeptidase 3C, endopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, Russelllysin, saccharopepsin, semenogelase 3, T-fibrinolysin activator, thrombin, tissue kallikrein, tobacco etching virus (TEV), togavirin, tryptophan-acyl aminopeptidase, U-fibrinolysin activator, V8, venombin A, venombin AB, and Xaa-proaminopeptidase.

[0215] In some embodiments, the proteolytically cleavable linker includes matrix metalloproteinase cleavage sites, such as cleavage sites selected from the following MMPs: collagenase-1, -2 and -3 (MMP-1, -8 and -13), gelatinase A and B (MMP-2 and -9), matrix lysin 1, 2 and 3 (MMP-3, -10 and -11), matrix lysin (MMP-7), and membrane metalloproteinases (MT1-MMP and MT2-MMP).

[0216] In some embodiments, the cleavable linker comprises a disulfide bond and is cleavable under reducing conditions, for example, using β-mercaptoethanol, cysteine-HCl, tris(2-carboxyethyl)phosphonic acid hydrochloride, or another reducing agent.

[0217] In some embodiments, the cleavable linker comprises a dipeptide. In some embodiments, the dipeptide is a valine-citrulline (Val-Cit) dipeptide or a valine-lysine dipeptide.

[0218] In some embodiments, the cleavable linker comprises a tetrapeptide. In some embodiments, the tetrapeptide is glycine-glycine-phenylalanine-glycine tetrapeptide (GGFG).

[0219] In some embodiments, the cleavable linker is PABC (p-aminobenzyl alcohol), glucuronide, or MABC (m-aminobenzyl alcohol).

[0220] In some embodiments, the cleavable linker is a valine-citrulline (Val-Cit) PABC (p-aminobenzyl alcohol) (VC-PABC) linker. In some embodiments, the VC-PABC linker has the following structure: The connection point of the effective payload is " ".

[0221] In some embodiments, this disclosure provides compounds of formula L1: , where R is the effective payload.

[0222] In some embodiments, the compound is selected from the group consisting of: SH-VC-PAB-DXD SH-VC-PAB-MMAE, SH-VC-PAB-camptothecin, and SH-VC-PAB-Dorothy Star.

[0223] In some embodiments, the cleavable linker further includes a polyethylene glycol group (i.e., a PEGylated group), a glycosyl group, or modifications to increase hydrophilicity. In some embodiments, the cleavable linker including the glycosyl group is a glucuronide linker. In some embodiments, modifications to increase hydrophilicity include longer peptide chains, incorporation of charged residues, additional PEG spacers, branched PEG substituents, peptide-like bonds, sulfonate bonds, glucuronide linkers, and methylation of tertiary amines to generate cationic compounds.

[0224] peptides and biomolecules In some embodiments, this document describes conjugates comprising one or more polypeptides or biomolecules.

[0225] In some embodiments, the polypeptide is an enzyme, antibody, structural polypeptide, receptor ligand, or receptor.

[0226] In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a single-chain Fv (scFv). In some embodiments, the polypeptide is an antibody or an antibody fragment with a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain, a light chain, and a constant chain. Other antibody-based recognition domains (cAb VFiFi (cameloid antibody variable domain) and humanized forms, IgNAR VFi (shark antibody variable domain) and humanized forms, sdAb VFi (single-domain antibody variable domain) and "camelized" antibody variable domains) are applicable. In some embodiments, T-cell receptor (TCR)-based recognition domains (e.g., single-chain TCRs (scTv, nanobodies containing single-chain dual-domain TCRs)) are also applicable.

[0227] Biomolecules applicable to the methods or conjugates of this disclosure include polypeptides, peptides, cyclic peptides, polynucleotides, nucleic acids, aptamers, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, fatty acids, lipopolysaccharides, sugars, amino acids, organic dyes, synthetic polymers, steroids, nanoparticles, polymers, purines, pyrimidines, their derivatives, structural analogs, and combinations thereof.

[0228] In some embodiments, the biomolecule suitable for conjugation with the payload is a peptide. In some embodiments, the peptide is a peptide ligand or a binding peptide. In some embodiments, the binding peptide may have a different origin, such as synthetic, human, mouse, or rat. In some embodiments, the binding peptide may be or has been engineered to include one or more (e.g., two, three, four, or five) cysteine ​​or lysine residues exposed to a solvent, which can provide conjugation sites. In some embodiments, the binding peptide may include only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues. In some embodiments, the binding peptide may be linear or cyclic. In some embodiments, the peptide ligand or binding peptide is a bicyclic peptide. In some embodiments, the binding peptide may be a single-specific peptide or a multi-specific peptide (e.g., a bispecific peptide or a trispecific peptide). A bispecific peptide ligand or a dual-specific peptide ligand may bind one target at a time or bind two targets simultaneously. In some embodiments, the binding peptide is a cysteine ​​motif-binding peptide.

[0229] As used herein, “binding peptide” refers to a peptide that has the potential to bind to other compounds and / or structures (such as polypeptides and proteins).

[0230] Suitable lipids may include, for example, 3-N-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-di-linoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, dipalmitoylphosphatidylcholine, and 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-di-myristyloxy-propylamine (PEG-C-DMA), 1,2-di-myristyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-di-myristyloxy ... Myristyloxypropylamine, 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane, 1,2-distearate-sn-glycerol-3-phosphocholine, PEG-cDMA, 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxacyclopentane (DLin-KC2-DMA), etc.

[0231] Suitable biomolecules may include affinity moieties. Suitable affinity moieties may include His5 (HHHHH) (SEQ ID NO:34); HisX6 (HHHHHH) (SEQ ID NO:35); c-myc (EQKLISEEDL) (SEQ ID NO:36); Flag (DYKDDDDK) (SEQ ID NO:37); StrepTag (WSHPQFEK) (SEQ ID NO:38); hemagglutinins, such as HA tags (YPYDVPDYA) (SEQ ID NO:39); glutathione S-transferase (GST); thioredoxin; cellulose-binding domain, RYIRS (SEQ ID NO:40); Phe-His-His-Thr (SEQ ID NO:41); chitin-binding domain; S-peptide; T7 peptide; SH2 domain; C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO:38). NO:42); metal-binding domains, such as zinc-binding or calcium-binding domains, such as those from calcium-binding proteins, such as calmodulin, troponin C, calmoneurin B, myosin light chain, recovery protein, S-regulatory protein, cone protein, VILIP, neurotrophin, hippocampal calcium-binding protein, neuronal calcium-sensing protein (frequenin), calcium-binding protein, and calpain large subunit. SI 00 proteins, including parvalbumin, calcium-binding protein D9K, calcium-binding protein D28K, and calreticulin; biotin; streptavidin; MyoD; leucine zipper polypeptide; and maltose-binding protein. In some embodiments, the suitable biomolecule is biotin.

[0232] In some embodiments, the biomolecule suitable for conjugation with the payload is a dimerizing domain. Non-limiting examples of suitable dimerizing domains include peptides with the following dimerizing pairs: a) FK506-binding protein (FKBP) and FKBP; b) FKBP and catalytic subunit A of calmotropic phosphatase (CnA); c) FKBP and cyclophilin; d) FKBP and FKBP-rapamycin-associated protein (FRB); e) Gyrase B (GyrB) and GyrB; f) Dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB; h) PYL and ABI; i) Cry2 and CIB 1; and j) GAI and GID1.

[0233] In some cases, the biomolecules suitable for binding with the payload are members of specific binding pairs. Specific binding pairs include, for example: i) antibody-antigen; ii) cell adhesion molecule-extracellular matrix; iii) ligand-receptor; iv) biotin-avidin; etc.

[0234] Suitable synthetic polymers include (but are not limited to) polyolefins, such as polyethylene and polypropylene and polyethylene glycol (PEG); polychloroprene; polyethylene ethers, such as poly(vinyl acetate); polyhalogenated vinyl groups, such as poly(vinyl chloride); polysiloxanes; polystyrene; polyurethanes; polyacrylates, such as poly((meth)acrylate), poly(((meth)acrylate), poly(((meth)acrylate) n-butyl acrylate), poly((((meth)acrylate) isobutyl acrylate), poly((((meth)acrylate) tert-butyl acrylate), and poly((((meth)acrylate) hexyl acrylate). Poly((meth)acrylate isodecyl), poly((meth)acrylate lauryl), poly((meth)acrylate phenyl), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate) and poly(octadecyl acrylate); polyacrylamide, such as poly(acrylamide), poly(methacrylamide), poly(ethylacrylamide), poly(ethyl methacrylamide), poly(N-isopropylacrylamide), poly(n-butylacrylamide, isobutylacrylamide and tert-butylacrylamide); and copolymers and mixtures thereof.

[0235] In some embodiments, the biomolecule is a fluorescent protein. Suitable fluorescent proteins include (but are not limited to) green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer 2, t-dimer 2 (12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, phycobiliproteins and phycobiliprotein conjugates, including B-phycoerythrin, R-phycoerythrin and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909). As illustrated, for example, in Matz et al. (1999) Nature Biotechnol. 17:969-973, any of the various fluorescent and colored proteins from coral species are applicable.

[0236] In some embodiments, the biomolecule is a nucleic acid. In some embodiments, the nucleic acid is a DNA molecule. In some embodiments, the nucleic acid is an RNA molecule. In some embodiments, the nucleic acid comprises both deoxyribonucleotides and ribonucleotides. In some embodiments, the nucleic acid is a single-stranded DNA molecule. In some embodiments, the nucleic acid is a double-stranded DNA molecule. In some embodiments, the nucleic acid is a single-stranded RNA molecule. Suitable nucleic acids include, for example, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, aptamers, etc. Suitable nucleic acids include those that are or function as: siRNA or other RNA interference agents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, self-cleaving RNA, ribozymes, fragments thereof and / or variants thereof (e.g., peptidyl transferase 23S rRNA, RNase P, group I and group II introns, GIR1 branched ribozymes, leadzymes, hairpin ribozymes, hammerhead ribozymes, HDV ribozymes, mammalian CPEB3 ribozymes, VS ribozymes, glmS ribozymes, CoTC ribozymes, etc.), microRNAs, microRNA mimics, supermir, aptamers, antimir, antagomir, U1 aptamers, triplet-forming oligonucleotides, RNA activators, long non-coding RNAs, and short non-coding RNAs. Nucleic acids can be of any length and may include one or more of the following: modified ribonucleotide bases, modified deoxyribonucleotide bases, modified deoxyribose, modified ribose, and modified backbone bonds (e.g., phosphate thioester bonds). Nucleic acids can be of any length and may include one or more of the following: modified ribonucleotide bases, modified deoxyribonucleotide bases, modified deoxyribose, modified ribose, and modified backbone bonds (e.g., phosphate thioester bonds).

[0237] In some embodiments, the biomolecule is an oligonucleotide. In some embodiments, the oligonucleotide may include an oligonucleotide complementary to a gene-meaning sequence, a pre-mRNA-meaning sequence, and / or a portion of a mRNA-meaning sequence, or thereof. In some embodiments, the oligonucleotide may include an oligonucleotide of a gene-meaning sequence, a pre-mRNA-meaning sequence, and / or a portion of a mRNA-meaning sequence, or thereof. In some embodiments, the oligonucleotide described herein may also be a nucleotide-based chemical analog compound capable of binding to a gene-meaning sequence, a pre-mRNA-meaning sequence, and / or a portion of a mRNA-meaning sequence, or thereof. In some embodiments, the oligonucleotide is a meaning oligonucleotide. In some embodiments, the oligonucleotide is an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide may be a single-stranded nucleic acid molecule.

[0238] In some embodiments, the oligonucleotide may be an oligonucleotide sequence of 5 to 100 nucleotides in length, for example, 10 to 40 nucleotides in length, for example, 14 to 40 nucleotides in length, for example, 10 to 30 nucleotides in length, for example, 14 to 30 nucleotides in length, for example, 14 to 25 nucleotides in length, for example, 15 to 22 nucleotides in length, for example, 16 to 40 nucleotides in length, for example, 18 to 24 nucleotides in length, for example, 20 to 40 nucleotides in length, or for example, 20 to 24 nucleotides in length. The oligonucleotide may contain an oligonucleotide sequence complementary to one or more portions of the mRNA sequence.

[0239] In some embodiments, the oligonucleotide comprises one or more ribonucleotides, one or more deoxyribonucleotides, or a mixture of ribonucleotides and deoxyribonucleotides.

[0240] In some embodiments, the oligonucleotide comprises one or more modified nucleosides, such as 5-methylcytidine, 5-methyl-2'-deoxycytidine, deoxycytidine, 5-methyl-2'-deoxycytidine 5'-monophosphate, or 5-methyl-2'-deoxycytidine-5'-monothiophosphate. In some embodiments, the oligonucleotide comprises one or more modified nucleosides, such as 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methylthymidine, 2'-O-methyluridine, or 2'-O-methyladenosine. In some embodiments, the oligonucleotide comprises one or more modified nucleotides, such as 5-methylcytosine or 5-methylguanine. In some embodiments, the oligonucleotide comprises one or more modified nucleotides, such as 2'-O-(2-methoxyethyl)nucleoside, 2'-deoxy-2'-fluoronucleoside, or 2'-fluoro-β-D-arabinonucleotide.

[0241] In some implementations, the oligonucleotide comprises bridging nucleic acid, locked nucleic acid (LNA), restricted ethyl (cET) nucleic acid, tricyclic DNA (tcDNA), 2'-O,4'-C-ethylene linked nucleic acid (ENA), or peptide nucleic acid (PNA).

[0242] In some embodiments, the oligonucleotide may have modified linkages, such as phosphorothioate linkages, dithiothioate linkages, phosphate triester linkages, alkylphosphonate linkages, aminoalkyl phosphate triester linkages, alkylene phosphonate linkages, hypophosphonate linkages, aminophosphonate linkages, morpholino phosphate linkages, piperazine phosphate linkages, and aminoalkylamino phosphate linkages, thioamino phosphate linkages, thionylalkylphosphonate linkages, thionylalkyl phosphate triester linkages, thiophosphate linkages, selenophosphate linkages, and / or borane phosphate linkages.

[0243] The payload of this disclosure In some implementations, this document describes conjugates comprising one or more payloads.

[0244] In some embodiments, the conjugate comprises multiple payloads. In some embodiments, the conjugate comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more payloads. In some embodiments, the conjugate comprises one payload. In some embodiments, the conjugate comprises two payloads. In some embodiments, the conjugate comprises three payloads. In some embodiments, the conjugate comprises four payloads. In some embodiments, the conjugate comprises five payloads. In some embodiments, the conjugate comprises six payloads. In some embodiments, the conjugate comprises seven payloads. In some embodiments, the conjugate comprises eight payloads. In some embodiments, the conjugate comprises nine payloads. In some embodiments, the conjugate comprises ten payloads. In some embodiments, the conjugate comprises multiple payloads conjugated to different chains of the antibody (e.g., light and heavy chains). In some embodiments, the conjugate comprises multiple identical payloads. In some embodiments, the conjugate comprises multiple different payloads. In some embodiments, the conjugate comprises multiple different payloads, wherein more than one of the different payloads is identical. For example, the conjugate comprises two tubulin inhibitor payloads and two topoisomerase inhibitor payloads.

[0245] The payload may be naturally occurring or can be produced synthetically or recombinantly, and may be isolated, substantially purified, or present in the natural environment of the unmodified molecule upon which the payload based (e.g., on the cell surface or within the cell, including within a host animal, such as mammals, like rodent hosts (e.g., rats, mice), hamsters, dogs, cats, cattle, pigs, etc.). In some embodiments, the payload is present in vitro in a cell-free reaction. In other embodiments, the payload is present in cells and / or displayed on the cell surface. In many embodiments of interest, the payload is in living cells; on the surface of living cells; in a living organism, such as in a living multicellular organism. Suitable living cells include cells that are part of a living multicellular organism; cells isolated from a multicellular organism; immortalized cell lines; etc.

[0246] The payload may consist of D-amino acids, L-amino acids, or both, and may be further modified naturally, synthetically, or recombinantly to include other portions. For example, the payload may be a lipoprotein, glycoprotein, or other such modified protein.

[0247] In some embodiments, the payload comprises at least one thiol moiety for reacting with at least one polypeptide comprising a reactive moiety, but may comprise two or more, three or more, five or more, ten or more thiol moieties. The number of thiol moieties that may be present in the target molecule will vary depending on the intended application of the modified target molecule to the reaction, the properties of the target molecule itself, and other considerations that will readily become apparent to those skilled in the art when practicing the methods disclosed herein.

[0248] The payload may be modified to include a thiol moiety at the site where it is desired to bond with a polypeptide containing a reactive moiety. For example, when the payload is a peptide or polypeptide, it may be modified to contain an N-terminal thiol moiety, thereby producing a target peptide or polypeptide containing a thiol moiety. It should be understood that any convenient site on the peptide or polypeptide substrate may be modified to contain a thiol moiety, thereby producing a target peptide or polypeptide for use in the subject method.

[0249] In some implementations, the payload is a small molecule.

[0250] In some embodiments, the payload containing the reactive portion will contain a small molecule drug, toxin, or other molecule for delivery to cells. In some embodiments, the small molecule drug, toxin, or other molecule will provide pharmacological activity. In some embodiments, the small molecule drug, toxin, or other molecule will serve as a target for the delivery of other molecules.

[0251] Small molecule drugs can be small organic or inorganic compounds with a molecular weight greater than 50 Daltons and less than about 2,500 Daltons. Small molecule drugs may contain functional groups necessary for interaction with protein structure (specifically hydrogen bonding) and may include at least amine, carbonyl, hydroxyl, or carboxyl groups, and may contain at least two functional chemical groups. Drugs may contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Small molecule drugs are also found in biomolecules, including peptides, sugars, fatty acids, steroids, purines, pyrimidines, their derivatives, structural analogs, or combinations thereof.

[0252] In some embodiments, the payload is a cancer chemotherapeutic agent, which may include small molecules as described herein.

[0253] Suitable cancer chemotherapy agents include, for example, alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan); nitrosoureas (e.g., carmustine, formmustine, lomustine, and streptozocin); and platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3). 464); busulfan; dacarbazine; mechlorethamine; procarbazine; temozolomide; thiotepa; uramustine; antimetabolites, such as folic acid (e.g., methotrexate, pemetrexed, and raltitrexed); purines (e.g., cladribine, clofarabine, fludara). Fludarabine, mercaptopurine, and thioguanine; pyrimidines (e.g., capecitabine); cytarabine; fluorouracil; gemcitabine; plant alkaloids, such as those from the Podophyllum genus (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), and periwinkle alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine). (vinorelbine); cytotoxic / antitumor antibiotics, such as anthracycline family members (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin;Other agents, such as atranovin, hexamethylmelamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, and denileukin-2. The drugs include diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, emurafenib, vandetanib, and tretinoin. In some embodiments, the cancer chemotherapeutic agent is a topoisomerase inhibitor. In some embodiments, the cancer chemotherapeutic agent is a tubulin inhibitor. In some embodiments, the conjugate comprises two cancer chemotherapeutic agents. In some embodiments, the two cancer chemotherapeutic agents are a topoisomerase inhibitor and a tubulin inhibitor.

[0254] In some embodiments, the payload containing the reactive portion is selected from the group consisting of: delutec, ixanotecan, FL118, irinotecan, topotecan, SN-38, rubotecan, belototecan, letopotecan, gemmatotecan, diflutecan, calontecan, citratecan, nanotecan, ileotecan, DRF-1042, delutec, NSC606985, cimenotecan, ZBH-1205, auristatin (MMAE, MMAF, MMAG, MMAH), chachiomycin, doxorubicin, paclitaxel and paclitaxel derivatives, maytansine (DM1-4), pyrrolodiazepine (PBD), tubulolysin, eribulin, atrazomycin, pyroxine, anthracyclines, and camptothecin (CPT), including lactone and carboxylic acid ester forms of CPT. In some embodiments, the payload containing the reactive portion is selected from the group consisting of: ethatecan, auristatin (MMAE, MMAF, MMAG, MMAH), doxorubicin, and camptothecin (CPT). In some embodiments, the payload containing the reactive portion comprises an immunostimulatory compound. In some embodiments, the payload containing the reactive portion comprises a DNA repair inhibitor.

[0255] In some implementations, the payload containing the reactive portion is selected from the group consisting of: STING agonists, including cyclic bis-GMP, diABZI and its derivatives, TLR7 agonists, TLR8 agonists and other immune receptor agonists.

[0256] In some embodiments, the payload containing the reactive portion includes one of a pair of binding partners (e.g., a ligand; a ligand-binding portion of a receptor; an antibody; an antigen-binding fragment of an antibody; an antigen; a hapten; a lectin; a lectin-binding carbohydrate). For example, the payload may contain a polypeptide that acts as a viral receptor and, upon binding to a viral envelope protein or viral capsid protein, facilitates viral attachment to the cell surface displaying the biomolecule.

[0257] In some embodiments, the payload is a nucleic acid. In some embodiments, the nucleic acid is a DNA molecule. In some embodiments, the nucleic acid is an RNA molecule. In some embodiments, the nucleic acid comprises both deoxyribonucleotides and ribonucleotides. In some embodiments, the nucleic acid is a single-stranded DNA molecule. In some embodiments, the nucleic acid is a double-stranded DNA molecule. In some embodiments, the nucleic acid is a single-stranded RNA molecule. Suitable nucleic acids include, for example, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, aptamers, etc. Suitable nucleic acids include those that are or function as: siRNA or other RNA interference agents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, self-cleaving RNA, ribozymes, fragments thereof and / or variants thereof (e.g., peptidyl transferase 23S rRNA, RNase P, group I and group II introns, GIR1 branched ribozymes, leadzymes, hairpin ribozymes, hammerhead ribozymes, HDV ribozymes, mammalian CPEB3 ribozymes, VS ribozymes, glmS ribozymes, CoTC ribozymes, etc.), microRNAs, microRNA mimics, supermir, aptamers, antimir, antagomir, U1 aptamers, triplet-forming oligonucleotides, RNA activators, long non-coding RNAs, and short non-coding RNAs. Nucleic acids can be of any length and may include one or more of the following: modified ribonucleotide bases, modified deoxyribonucleotide bases, modified deoxyribose, modified ribose, and modified backbone bonds (e.g., phosphate thioester bonds). Nucleic acids can be of any length and may include one or more of the following: modified ribonucleotide bases, modified deoxyribonucleotide bases, modified deoxyribose, modified ribose, and modified backbone bonds (e.g., phosphate thioester bonds).

[0258] In some embodiments, the payload is an oligonucleotide. In some embodiments, the oligonucleotide may include an oligonucleotide complementary to a gene-meaning sequence, a pre-mRNA-meaning sequence, and / or a portion of a mRNA-meaning sequence, or thereof. In some embodiments, the oligonucleotide may include an oligonucleotide of a gene-meaning sequence, a pre-mRNA-meaning sequence, and / or a portion of a mRNA-meaning sequence, or thereof. In some embodiments, the oligonucleotide described herein may also be a nucleotide-based chemical analog compound capable of binding to a gene-meaning sequence, a pre-mRNA-meaning sequence, and / or a portion of a mRNA-meaning sequence, or thereof. In some embodiments, the oligonucleotide is a meaning oligonucleotide. In some embodiments, the oligonucleotide is an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide may be a single-stranded nucleic acid molecule.

[0259] In some embodiments, the oligonucleotide may be an oligonucleotide sequence of 5 to 100 nucleotides in length, for example, 10 to 40 nucleotides in length, for example, 14 to 40 nucleotides in length, for example, 10 to 30 nucleotides in length, for example, 14 to 30 nucleotides in length, for example, 14 to 25 nucleotides in length, for example, 15 to 22 nucleotides in length, for example, 18 to 40 nucleotides in length, for example, 18 to 24 nucleotides in length, for example, 20 to 40 nucleotides in length, or for example, 20 to 24 nucleotides in length. The oligonucleotide may contain an oligonucleotide sequence complementary to one or more portions of the mRNA sequence.

[0260] In some embodiments, the oligonucleotide comprises one or more ribonucleotides, one or more deoxyribonucleotides, or a mixture of ribonucleotides and deoxyribonucleotides.

[0261] In some embodiments, the oligonucleotide comprises one or more modified nucleosides, such as 5-methylcytidine, 5-methyl-2'-deoxycytidine, deoxycytidine, 5-methyl-2'-deoxycytidine 5'-monophosphate, or 5-methyl-2'-deoxycytidine-5'-monothiophosphate. In some embodiments, the oligonucleotide comprises one or more modified nucleosides, such as 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methylthymidine, 2'-O-methyluridine, or 2'-O-methyladenosine. In some embodiments, the oligonucleotide comprises one or more modified nucleotides, such as 5-methylcytosine or 5-methylguanine. In some embodiments, the oligonucleotide comprises one or more modified nucleotides, such as 2'-O-(2-methoxyethyl)nucleoside, 2'-deoxy-2'-fluoronucleoside, or 2'-fluoro-β-D-arabinonucleotide.

[0262] In some implementations, the oligonucleotide comprises bridging nucleic acid, locked nucleic acid (LNA), restricted ethyl (cET) nucleic acid, tricyclic DNA (tcDNA), 2'-O,4'-C-ethylene linked nucleic acid (ENA), or peptide nucleic acid (PNA).

[0263] In some embodiments, the oligonucleotide may have modified linkages, such as thiophosphate linkages, dithiophosphate linkages, phosphate triester linkages, alkylphosphonate linkages, aminoalkylphosphate triester linkages, alkylenephosphonate linkages, hypophosphonate linkages, aminophosphate linkages, morpholinophosphate linkages, piperazine phosphate linkages, and aminoalkylaminophosphate linkages, thioaminophosphate linkages, thioketone alkylphosphonate linkages, thioketone alkylphosphate triester linkages, thiophosphate linkages, selenophosphate linkages, and / or borane phosphate linkages.

[0264] In some embodiments, the payload is a peptide. In some embodiments, the peptide is a peptide ligand or a binding peptide. In some embodiments, the binding peptide may have a different origin, such as synthetic, human, mouse, or rat. In some embodiments, the binding peptide may be or has been engineered to include one or more (e.g., two, three, four, or five) cysteine ​​or lysine residues exposed to a solvent, which can provide conjugation sites. In some embodiments, the binding peptide may include only naturally occurring amino acid residues, or may include one or more non-naturally occurring amino acid residues. In some embodiments, the binding peptide may be linear or cyclic. In some embodiments, the peptide ligand or binding peptide is a bicyclic peptide. In some embodiments, the binding peptide may be a single-specific peptide or a multi-specific peptide (e.g., a bispecific peptide or a trispecific peptide). A bispecific peptide ligand or a dual-specific peptide ligand may bind to one target at a time or bind to two targets simultaneously. In some embodiments, the binding peptide is a cysteine ​​motif binding peptide. Tyrosinase polypeptide Suitable tyrosinase polypeptides for generating reactive moieties (e.g., ortho-quinones) include tyrosinase polypeptides having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with any of the tyrosinase amino acid sequences shown in any of SEQ ID NO: 1-6 and 52-53. In some embodiments, the tyrosinase polypeptide is a mushroom agaricus tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is a Bacillus megaterium (Bacillus megaterium) tyrosinase polypeptide. Bacillus megaterium ) tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is *Streptomyces marmoratae* (… Streptomyces castaneoglobisporus ) Tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is Citrobacter freundii ( Citrobacter freundii ) tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is Homo sapiens (… Homo sapiens ) Tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is apple ( Malus domestica ) tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is Aspergillus oryzae (… Aspergillus oryzae ) Tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is tomato ( Solanum lycopersicum ) Tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is Burkholderia tsukia (… Burkholderia thailandensis ) Tyrosinase polypeptide. In some embodiments, the tyrosinase polypeptide is walnut ( Juglans regiaTyrosinase polypeptide. See, for example, Pretzler et al., Sci. Rep. 2017, 7 (1), 1810; Ren et al., BMC Biotechnol. 2013, 13, 18; Faccio et al., Process Biochem. 2012, 47 (12), 1749-1760; Fairhead et al., FEBS J. 2010, 277 (9), 2083-2095; Do et al., Sci. Rep. 2017, 7 (1), 17267; Elsayed and Danial, J. Appl. Pharm. Sci. 2018, 8 (09), 93-101; Lopez-Tejedor and Palomo, Protein Expr. Purif. 2018, 145, 64-70; and Fairhead et al., Nature Biotechnol. 2012, 29 (2), 183-191.

[0265] In some embodiments, the tyrosinase polypeptide selectively acts (e.g., to generate a reactive moiety such as an orthoquinone) on a substrate (biomolecule or polypeptide) containing a phenolic moiety (e.g., tyrosine) or a catechol moiety, wherein the substrate is neutral or positively charged within 50 Å (e.g., within 50 Å, 40 Å, 30 Å, or 20 Å) of the phenolic or catechol moiety. For example, a tyrosinase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with any of the tyrosinase amino acid sequences shown in any of SEQ ID NO: 2 and 4-6 may selectively modify the phenolic or catechol moiety on the substrate, wherein the substrate is neutral or positively charged within 50 Å (e.g., within 50 Å, 40 Å, 30 Å, or 20 Å) of the phenolic or catechol moiety. In some embodiments, at least one of the at least two polypeptides of the conjugate contains at least two neutral or positively charged amino acids within 10 amino acids of the phenolic moiety (e.g., tyrosine) or catechol moiety. In some embodiments, at least one of the at least two polypeptides of the conjugate contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 neutral or positively charged amino acids within 10 amino acids of the phenolic moiety (e.g., tyrosine) or catechol moiety. In some embodiments, at least one of the at least two polypeptides of the conjugate comprises the amino acid sequence GGGGCY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

[0266] In some embodiments, the tyrosinase polypeptide selectively acts (e.g., to generate a reactive moiety such as an orthoquinone) on a substrate (biomolecule) comprising a phenolic moiety (e.g., tyrosine) or a catechol moiety, wherein the substrate carries a negative charge within 50 Å (e.g., within 50 Å, 40 Å, 30 Å, or 20 Å) of the phenolic or catechol moiety. In some embodiments, a tyrosinase having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with any of the tyrosinase amino acid sequences shown in any of SEQ ID NO: 2-3 may selectively modify the phenolic or catechol moiety on the substrate, wherein the substrate carries a negative charge within 50 Å (e.g., within 50 Å, 40 Å, 30 Å, or 20 Å) of the phenolic or catechol moiety. In some embodiments, at least one of the at least two polypeptides of the conjugate contains at least two negatively charged amino acids within the 10 amino acids of the phenolic moiety (e.g., tyrosine) or catechol moiety. In some embodiments, at least one of the at least two polypeptides of the conjugate contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 negatively charged amino acids within the 10 amino acids of the phenolic moiety (e.g., tyrosine) or catechol moiety. In some embodiments, at least one of the at least two polypeptides of the conjugate comprises the amino acid sequence GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, SEY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

[0267] method In some embodiments, this document describes a method for covalently linking at least two peptides to at least two payloads. In some embodiments, the method includes contacting a first peptide of the at least two peptides with a first payload of the at least two payloads using a first tyrosinase, wherein the first peptide comprises a first terminal tag comprising (X1). m1X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is a tyrosine, and where m1 is an integer greater than or equal to 0; and using a second tyrosinase to contact a second polypeptide of the at least two polypeptides with a second payload of the at least two payloads, wherein the second polypeptide contains a second terminal tag, the second terminal tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the mixture is a tyrosine, and m2 is an integer greater than or equal to 0. In some embodiments, the method includes contacting a first polypeptide of the at least two polypeptides with a first payload of the at least two payloads using a first tyrosinase, wherein the first polypeptide contains a first terminal tag comprising (X1). m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid in X1-X5 is a tyrosine, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is a tyrosine; and using a second tyrosinase to contact a second polypeptide of the at least two polypeptides with a second payload of the at least two payloads, wherein the second polypeptide contains a second terminal tag, the second terminal tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the mixture is tyrosine, and m2 is an integer greater than or equal to 0. The contact is carried out under conditions sufficient to conjugate the at least two payloads with the at least two polypeptides, thereby producing a conjugate.

[0268] In some embodiments, the subject method of chemically selectively modifying a payload includes contacting: i) a payload comprising a thiol moiety; ii) a polypeptide comprising a phenolic moiety or a catechol moiety; and iii) an enzyme capable of oxidizing the phenolic moiety or the catechol moiety; wherein the enzyme oxidizes the phenolic moiety or the catechol moiety of the polypeptide to generate a reactive moiety, thereby generating a polypeptide comprising the reactive moiety, and wherein the reactive moiety reacts with the thiol moiety, thereby conjugating the payload and the polypeptide to each other to produce a conjugate. In some embodiments, the payload comprises a single thiol moiety.

[0269] In some embodiments, the subject method of chemically selectively modifying a peptide includes contacting: i) a payload comprising a phenolic or catechol moiety; ii) a peptide comprising a thiol moiety; and iii) an enzyme capable of oxidizing the phenolic or catechol moiety; wherein the enzyme oxidizes the phenolic or catechol moiety of the payload to generate a reactive moiety, thereby generating a payload comprising the reactive moiety, and wherein the reactive moiety reacts with the thiol moiety, thereby conjugating the payload to the peptide to form a conjugate. In some embodiments, the peptide comprises a single thiol moiety. In some embodiments, the peptide comprises two or more thiol moieties. In some embodiments, the peptide is modified to include a terminal tag comprising (X1). m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of the amino acids X1-X5 is tyrosine, and where m1 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one of the amino acids X1-X3 is tyrosine.

[0270] In some embodiments, the subject method of chemically selectively modifying a peptide includes contacting: i) a payload comprising a maleimide moiety; ii) a peptide comprising a thiol moiety; and iii) an enzyme capable of oxidizing the maleimide moiety; wherein the enzyme oxidizes the maleimide moiety of the payload to generate a reactive moiety, thereby generating a payload comprising the reactive moiety, and wherein the reactive moiety reacts with the thiol moiety, thereby conjugating the payload to the peptide to form a conjugate. In some embodiments, the peptide comprises a single thiol moiety. In some embodiments, the peptide comprises two or more thiol moieties.

[0271] In some embodiments, this disclosure provides linking a payload containing a thiol moiety to a polypeptide containing a phenol or catechol moiety. In some embodiments, this disclosure provides linking a payload containing a phenol or catechol moiety to a polypeptide containing a thiol moiety. In some embodiments, this disclosure provides linking a payload containing a maleimide moiety to a polypeptide containing a thiol moiety. In some embodiments, the method generally involves reacting a thiol-containing payload with a polypeptide containing a reactive moiety (e.g., an ortho-quinone moiety). In some embodiments, the method generally involves reacting a thiol-containing polypeptide with at least one payload containing a reactive moiety (e.g., an ortho-quinone moiety). In some embodiments, the method generally involves reacting a thiol-containing polypeptide with at least one payload containing a maleimide moiety.

[0272] The method disclosed herein provides a simple conjugation procedure that can site-specifically attach a payload of interest to any location on the surface of a polypeptide, thereby producing a conjugate of interest. The payload can be any of a variety of molecules (e.g., polypeptides; nucleic acids; small molecules; etc.). In some embodiments, the payload is a small molecule (e.g., a cancer chemotherapeutic agent).

[0273] The polypeptide of interest includes antibodies. In some cases, the polypeptide of interest is an antibody fragment or a conjugated derivative thereof. In some embodiments, the antibody fragment or its conjugated derivative is selected from the group consisting of: Fab fragments, F(ab')2 fragments, single-chain Fv (scFv), bivalent antibodies, nanobodies, and trivalent antibodies. In some embodiments, the antibody fragment or its conjugated derivative is selected from the group consisting of: the heavy chain variable region (VH), the light chain variable region (VL), the heavy chain, the light chain, or the constant chain of the antibody.

[0274] In some embodiments, the polypeptide further comprises one or more portions selected from: fluorophores, active small molecules, affinity tags, and metal chelators. In some embodiments, the polypeptide further comprises a fluorescent protein. In some embodiments, the fluorescent protein is green fluorescent protein (GFP). In some embodiments, the polypeptide is an enzyme. In some embodiments, the polypeptide is a receptor.

[0275] The payloads of interest include (but are not limited to) small molecules, peptides, polynucleotides, nucleic acids, carbohydrates, lipids, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs thereof, and combinations thereof. In some embodiments, the payload is a small molecule (e.g., a cancer chemotherapeutic agent); and the peptide is an antibody (e.g., scFv; nanobodies; etc.).

[0276] In some embodiments, the enzyme capable of oxidizing the phenolic or catechol moiety is a phenol oxidase or a catechol oxidase. In some cases, the enzyme is a tyrosinase.

[0277] The term "tyrosinase" in this paper refers to monophenol monooxygenase (EC 1.14.18.1; CAS No.: 9002-10-2) or its derivatives, which are enzymes that catalyze the oxidation of phenols (e.g., tyrosine). They are copper-containing enzymes originally found in plant and animal tissues that catalyze the oxidation of tyrosine to produce melanin and other pigments. Tyrosinases share a binuclear type 3 copper center within their active site. Each of the two copper atoms is coordinated to three histidine residues. Matoba et al., "Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis", J Biol Chem. 2006, March 31; 281(13):8981-90. A three-dimensional model of the tyrosinase catalytic center was published electronically on January 25, 2006.

[0278] In some embodiments, the phenolic moiety of the conjugate is present in a tyrosine residue. In some embodiments, the tyrosine residue is part of the polypeptide of interest. In some embodiments, the tyrosine residue is synthetically introduced into the polypeptide of the conjugate. In some embodiments, the tyrosine residue is linked to the polypeptide via a linker (e.g., as illustrated herein). The tyrosine residue can be introduced using standard recombination techniques, such as by modifying the nucleotide sequence encoding the polypeptide to introduce the tyrosine residue into the polypeptide.

[0279] In some embodiments, the phenolic or catechol moiety is a portion of a non-natural (non-genetically encoded) amino acid introduced into the polypeptide of interest. For example, amber codon (TAG) inhibition can be used to incorporate non-genetically encoded amino acid residues containing either a phenolic or catechol moiety. See, for example, Chin et al. (2002) J. Am. Chem. Soc. 124:9026; Chin and Schultz (2002) Chem. Biol. Chem. 3:1135; Chin et al. (2002) Proc. Natl. Acad. Sci. USA 99:11020; US 2015 / 0240249; and US 2018 / 0171321. As another example, orthogonal RNA synthases and / or orthogonal tRNAs can be used to introduce non-genetically encoded amino acids into a polypeptide, wherein the non-genetically encoded amino acid contains either a phenolic or catechol moiety.

[0280] In some embodiments of the subject method, the thiol moiety present in the payload is part of a cysteine ​​residue. In some cases, the cysteine ​​residue is a naturally occurring cysteine ​​residue. In other cases, the cysteine ​​residue is a residue synthetically introduced into the target molecule.

[0281] In some embodiments, the polypeptide contains a thiol moiety, and the payload contains a phenol or catechol moiety.

[0282] In some embodiments, the reactive moiety is an ortho-quinone or semi-quinone radical, or a combination thereof. In some embodiments, the subject method provides a reaction between an ortho-quinone reactive intermediate and a thiol moiety, as illustrated in Scheme 1 below: Where Y 1 For a polypeptide; L is an optional linker (e.g., as illustrated herein); X 1 Selected from hydrogen and hydroxyl groups; Y 2 The payload is n; and n is an integer from 1 to 3.

[0283] As illustrated in Scheme 1, in some embodiments, a polypeptide comprising a phenolic or catechol moiety (e.g., formula (I)) undergoes activation by an enzyme capable of oxidizing the phenolic or catechol moiety. In some embodiments, activation is achieved with a tyrosinase in the presence of oxygen to generate an intermediate comprising a reactive moiety (e.g., an orthoquinone of formula (II) and / or a semiquinone radical of formula (IIA)), and said reactive moiety reacts with a payload comprising a thiol-based nucleophile (e.g., formula (III)) to conjugate the target molecule to a biomolecule, thereby generating a modified target molecule (e.g., formula (genIV)). In some embodiments, the payload of formula (III) may comprise any payload as illustrated herein. In some embodiments, Y2 in formula (III) is a polypeptide. In some embodiments, the conjugate is described by formula (IV). In some embodiments, the conjugate is described by formula (IV A).

[0284] In some embodiments, the subject method provides a reaction between the ortho-quinone reactive intermediate and the thiol moiety, as illustrated in Scheme 2 below: As illustrated in Scheme 2, in some embodiments, a polypeptide comprising a phenolic moiety (e.g., formula (IB)) undergoes activation by a tyrosinase in the presence of oxygen to generate an intermediate comprising a reactive moiety (e.g., an ortho-quinone of formula (II)), and said reactive moiety reacts with a payload comprising a thiol-based nucleophile (e.g., formula (III)) to conjugate a target molecule to a biomolecule, thereby generating a modified target molecule (e.g., formula (IVM)). In some embodiments, the payload of formula (III) may comprise, for example, any payload as described herein. In some cases, Y in formula (III) 2 It is a polypeptide. In some cases of the (IVM) conjugate, the thiol group is located at the 3 position of the catechol ring. In some cases of the (IVM) conjugate, the thiol group is located at the 5 position of the catechol ring. In some cases of the (IVM) conjugate, the thiol group is located at the 6 position of the catechol ring.

[0285] In some embodiments, the subject method provides a reaction between an orthoquinone reactive intermediate and an amine moiety (e.g., a lysine residue present in a polypeptide), as illustrated in Scheme 3 below: Where Y 1 For a polypeptide; L is an optional linker (e.g., as illustrated herein); X 1 Selected from hydrogen and hydroxyl groups; Y 2 The effective payload is defined as follows: R is selected from hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, and substituted heteroarylalkyl; and n is an integer from 1 to 3. In some cases, R is selected from the amino acids: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0286] As illustrated in Scheme 3, in some embodiments, a polypeptide comprising a phenolic or catechol moiety (e.g., formula (I)) undergoes activation by an enzyme capable of oxidizing the phenolic or catechol moiety. In some embodiments, activation is achieved with a tyrosinase in the presence of oxygen to generate an intermediate comprising a reactive moiety (e.g., an orthoquinone of formula (II) and / or a semiquinone radical of formula (IIA)), and said reactive moiety reacts with a payload comprising an amine-based nucleophile (e.g., formula (III)) to conjugate the target molecule to a biomolecule, thereby generating a modified target molecule (e.g., formula (genIV)). In some embodiments, the payload of formula (III) may comprise any payload as illustrated herein. In some embodiments, Y2 in formula (III) is a polypeptide. In some embodiments, the conjugate is described by formula (IV). In some embodiments, the conjugate is described by formula (IV A).

[0287] In some embodiments, the subject method provides a reaction between an orthoquinone reactive intermediate and an imidazole moiety (e.g., a histidine residue present in a polypeptide), as illustrated in Scheme 4 below: Where Y 1 For a polypeptide; L is an optional linker (e.g., as illustrated herein); X 1 Selected from hydrogen and hydroxyl groups; Y 2 The payload is n; and n is an integer from 1 to 3.

[0288] As illustrated in Scheme 4, in some embodiments, a polypeptide comprising a phenolic or catechol moiety (e.g., formula (I)) undergoes activation by an enzyme capable of oxidizing the phenolic or catechol moiety. In some embodiments, activation is achieved with a tyrosinase in the presence of oxygen to generate an intermediate comprising a reactive moiety (e.g., an orthoquinone of formula (II) and / or a semiquinone radical of formula (IIA)), and said reactive moiety reacts with a payload comprising an imidazole-based nucleophile (e.g., formula (III)) to conjugate the target molecule to a biomolecule, thereby generating a modified target molecule (e.g., formula (genIV)). In some embodiments, the payload of formula (III) may comprise any payload as illustrated herein. In some embodiments, Y2 in formula (III) is a polypeptide. In some embodiments, the conjugate is described by formula (IV). In some embodiments, the conjugate is described by formula (IV A).

[0289] In some embodiments, the subject method provides a reaction between the ortho-quinone reactive intermediate and the amine moiety, as illustrated in Scheme 4 below: In some embodiments, the subject method provides a reaction between the ortho-quinone reactive intermediate and the imidazole moiety, as illustrated in Scheme 5 below: In some embodiments, the method is performed at a pH of 4 to 9, such as 4.2, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.5, 7.8, 8.0, 8.2, 8.5, 8.8, or 9. In some embodiments, the method is performed at a pH of 5 to 8, such as 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.5, 7.8, or 8.0. In some cases, the method is performed at a pH of 6 to 7.5, such as 6.0, 6.3, 6.4, 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, or 7.5. In some embodiments, the method is performed at a neutral pH. As used herein, the term "neutral pH" means a pH between approximately 7.0 and approximately 7.4. The term "neutral pH" includes pH values ​​of approximately 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0290] In some embodiments, the method is performed under physiological conditions. In some embodiments, the method is performed on live cells in vitro. In other embodiments, the method is performed on live cells in vitro.

[0291] In some embodiments, the method may be carried out in an aqueous medium in the presence of one or more buffers. Buffers of interest include (but are not limited to) phosphate buffers, 2-amino-2-(hydroxymethyl)propane-1,3-diol (TRIS), 4-[4-(2-hydroxyethyl)piperazin-1-ylethanesulfonic acid (HEPES), etc. In some embodiments, the method may be carried out in an organic solvent. In some embodiments, the organic solvent is a water-miscible solvent. In some embodiments, the organic solvent is a dipolar aprotic solvent. In some embodiments, the organic solvent is selected from acetonitrile, dimethylformamide, methanol, and acetone. In some cases, the organic solvent is present in an amount of 1% to 20%, for example, 2%, 5%, 10%, 15%, or 20%, relative to water. In some embodiments, the main method is carried out in 1% to 20%, for example, 5%, 10%, 15%, or 20%, of acetonitrile. In some embodiments, the main method is carried out in 1% to 20%, for example, 5%, 10%, 15%, or 20%, of dimethylformamide. In some embodiments, the main method is carried out in 1% to 20%, such as 5%, 10%, 15%, or 20% methanol. In some embodiments, the main method is carried out in 1% to 20%, such as 5%, 10%, 15%, or 20% acetone.

[0292] In some embodiments, the buffer may be 50 mM phosphate pH 6.5, 150 mM NaCl and 2 mM EDTA, or 50 mM acetate pH 5.5, 10% sucrose and 1 mM EDTA. The phosphate in the first listed buffer may be replaced with a HEPES, Tris, acetate and MES buffer (pH 5 to pH 9). In some embodiments, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA may be added to the buffer.

[0293] In some embodiments, the conjugate is a product of dual or triple conjugation (e.g., Reference Formula (IV), collectively referred to herein as a "multiple conjugate product" when n is 2 or 3). In some embodiments, the multiple conjugate product is present in less than 1 part by weight of one or more multiple conjugate products relative to a single conjugate product (e.g., Reference Formula (IV), when n is 1), such as less than 1 part of 20 parts, less than 1 part of 25 parts, less than 1 part of 50 parts, less than 1 part of 75 parts, less than 100 parts, or even less. In some embodiments, the multiple conjugate product is not observed.

[0294] In some embodiments, the conjugate is stable at a range of pH and temperature values ​​and in the presence of multiple additional molecules. In some embodiments, the conjugate is stable at 0°C to 50°C, for example, 4°C to 40°C, for example, 4°C to 37°C. In some cases, the conjugate is stable in a pH range of 4 to 9, for example, pH 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, or 9. In some embodiments, the conjugate is stable in the presence of biologically relevant molecules. In some embodiments, the conjugate is stable in the presence of molecules such as guanidinium groups of arginine residues, primary amines of lysine residues, and aniline moieties. In some embodiments, the conjugate is stable under physiological conditions; for example, in some embodiments, the conjugate is stable in human serum. In some embodiments, the conjugate is stable in human serum at 37°C for a period of at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, or at least 14 days. In some embodiments, the conjugate is stable in human serum at 37°C for a period of about 2 to about 7 days, about 7 to about 10 days, or about 10 to about 14 days.

[0295] This disclosure provides a method for sequentially linking at least two peptides to at least two payloads. As described above, the method utilizes the substrate preference of tyrosinase peptides. The method can be carried out on an insoluble matrix, i.e., an immobilized surface, such as beads.

[0296] Therefore, this disclosure provides a method for linking at least two peptides to at least two payloads, the method comprising: a) contacting a first peptide of the at least two peptides with a first payload of the at least two payloads using a first tyrosinase, wherein the first peptide comprises a first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is a tyrosine, and where m1 is an integer greater than or equal to 0; and b) using a second tyrosinase to contact the second polypeptide of the at least two polypeptides with the second payload of the at least two payloads, wherein the second polypeptide contains a second terminal tag, the second terminal tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10At least one amino acid in the formula is tyrosine, and m2 is an integer greater than or equal to 0. In some embodiments, this disclosure provides a method for linking at least two polypeptides to at least two payloads, the method comprising: a) contacting a first polypeptide of the at least two polypeptides with a first payload of the at least two payloads using a first tyrosinase, wherein the first polypeptide comprises a first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is a tyrosine, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is a tyrosine; and b) using a second tyrosinase to contact the second polypeptide of the at least two polypeptides with the second payload of the at least two payloads, wherein the second polypeptide contains a second terminal tag, the second terminal tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the first polypeptide is tyrosine, and m2 is an integer greater than or equal to 0. In some embodiments, the first polypeptide contains two or more negatively charged amino acids within ten amino acids of the phenolic or catechol moiety, and the second polypeptide contains two or more neutral or positively charged amino acids within ten amino acids of the phenolic or catechol moiety. In some embodiments, the first enzyme is a tyrosinase polypeptide containing an amino acid sequence having at least 75% amino acid sequence identity with any of the amino acid sequences of SEQ ID NO: 1-6, 52, or 53. In some embodiments, the second enzyme is a tyrosinase polypeptide containing an amino acid sequence having at least 80% amino acid sequence identity with any of the amino acids of SEQ ID NO: 1-6, 52, or 53.

[0297] In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide containing an amino acid sequence having at least 85% amino acid sequence identity with any of the amino acid sequences in SEQ ID NO: 1-6, 52, or 53. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide containing an amino acid sequence having at least 90% amino acid sequence identity with any of the amino acids in SEQ ID NO: 1-6, 52, or 53. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide containing an amino acid sequence having at least 95% amino acid sequence identity with any of the amino acid sequences in SEQ ID NO: 1-6, 52, or 53. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide containing an amino acid sequence having at least 99% amino acid sequence identity with any of the amino acid sequences in SEQ ID NO: 1-6, 52, or 53. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence according to any one of the amino acid sequences of SEQ ID NO: 1-6, 52 or 53.

[0298] In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity with any of the amino acid sequences in SEQ ID NO: 2-6. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with any of the amino acid sequences in SEQ ID NO: 2-6. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with any of the amino acid sequences in SEQ ID NO: 2-6. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 99% amino acid sequence identity with any of the amino acid sequences in SEQ ID NO: 2-6. In some embodiments, the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence according to any of the amino acid sequences in SEQ ID NO: 2-6.

[0299] In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence according to the amino acid sequence of SEQ ID NO: 2.

[0300] In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 3. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence according to the amino acid sequence of SEQ ID NO: 3.

[0301] In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 4. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence according to the amino acid sequence of SEQ ID NO: 4.

[0302] In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 5. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence according to the amino acid sequence of SEQ ID NO: 5.

[0303] In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first enzyme and / or the second enzyme is a tyrosinase polypeptide comprising an amino acid sequence according to the amino acid sequence of SEQ ID NO: 6.

[0304] In some embodiments, the phenolic moiety present in the first polypeptide is located in a tyrosine (Tyr, Y) residue. In some embodiments, the phenolic moiety present in the second polypeptide is located in a Tyr residue.

[0305] A first payload can be added to a first polypeptide, and a second payload can be added to a second polypeptide, by alternating use of: a) a tyrosinase that preferentially modifies Tyr residues present in a negatively charged environment (e.g., wherein the polypeptide comprises two or more negatively charged amino acids within ten amino acids of the Tyr residues); and b) a tyrosinase that preferentially modifies Tyr residues present in a neutral or positively charged environment (e.g., wherein the polypeptide comprises two or more neutral or positively charged amino acids within ten amino acids of the Tyr residues). In some embodiments, the first payload and the second payload are the same. In some embodiments, the first payload and the second payload are different.

[0306] In some embodiments, the tyrosinase is inactivated or removed between any two steps of the method and before the addition of another tyrosinase. For example, the second enzyme is inactivated or removed between step (b) and step (c) of the method described above.

[0307] In some embodiments, this document describes a method for selectively conjugating at least two peptides to at least two payloads, the method being carried out by adjusting the temperature during one or more tyrosinase reactions. In some embodiments, a first peptide (e.g., a heavy chain) is selectively modified via temperature control during a first tyrosinase reaction. In some embodiments, the temperature during the first tyrosinase reaction occurs at a temperature of about 0°C to about 18°C, for example, about 0°C to 15°C, about 0°C to 10°C, about 4°C to 15°C, or about 4°C to 10°C. In some embodiments, the temperature during the first tyrosinase reaction occurs at a temperature of about 4°C. In some embodiments, the temperature during the first tyrosinase reaction occurs at room temperature. In some embodiments, a second peptide (e.g., a light chain) is selectively modified via temperature control during a second tyrosinase reaction. In some embodiments, the temperature during the second tyrosinase reaction occurs at a temperature of about 23°C to about 50°C, for example, about 25°C to 50°C, about 25°C to 40°C, or about 25°C to 37°C. In some embodiments, the temperature during the second tyrosinase reaction occurs at a temperature of about 30°C. In some embodiments, the temperature during the second tyrosinase reaction occurs at approximately 37°C. In some embodiments, the temperature during the second tyrosinase reaction occurs at room temperature. In some embodiments, the same tyrosinase is used for reactions at different temperatures.

[0308] In some embodiments, this document describes a method for selectively conjugating at least two peptides to at least two payloads, the method being carried out by deglycosyling one or more of the at least two peptides. In some embodiments, one or more sugars of the one or more peptides are removed (i.e., deglycosylation). In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more than 90% of the sugars of the one or more peptides are removed. In some embodiments, the removal of one or more sugars occurs at a temperature of about 0°C to about 50°C, for example, about 4°C to 40°C or about 4°C to 37°C. In some embodiments, the removal of one or more sugars occurs at a temperature of about 4°C. In some embodiments, the removal of one or more sugars occurs at a temperature of about 37°C. In some embodiments, a deglycosylated antibody is conjugated to one or more payloads at a temperature of about 4°C. In some embodiments, a deglycosylated antibody is conjugated to one or more payloads at a temperature of about 37°C. In some implementations, the deglycosylated antibody is conjugated with one or more payloads at a temperature of approximately room temperature.

[0309] In some embodiments, this document describes a method for selectively conjugating at least two peptides to at least two payloads, said method being carried out by deglycosylating one or more of said at least two peptides. In some embodiments, deglycosylation is achieved by incorporating a mutation into the peptide to remove glycosylation sites. In some embodiments, the mutation includes replacing asparagine with alanine. In some embodiments, the mutation includes replacing asparagine with glutamine. In some embodiments, the mutation includes replacing asparagine with glycine. In some embodiments, the mutation includes replacing serine with glycine. In some embodiments, the mutation includes replacing serine with alanine. In some embodiments, the mutation includes replacing threonine with alanine. In some embodiments, one or more sugars of said one or more peptides are removed by mutation (i.e., deglycosylation). Those skilled in the art will recognize how to use conventional methods to incorporate appropriate mutations into the coding sequence of a peptide to achieve the desired mutation. In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more of the sugars in the one or more polypeptides are removed. In some embodiments, the glycosylated antibody is conjugated to one or more payloads at a temperature of about 4°C. In some embodiments, the glycosylated antibody is conjugated to one or more payloads at a temperature of about 37°C. In some embodiments, the glycosylated antibody is conjugated to one or more payloads at a temperature of about 30°C.

[0310] In some embodiments, this document describes a method for selectively conjugating at least two peptides to at least two payloads, wherein the conjugation occurs in less than about 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In some embodiments, the conjugation occurs in less than about 1 hour, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes, 1 minute, or 30 seconds. In some embodiments, the conjugation occurs in less than about 10 minutes. In some embodiments, the first peptide is contacted with a tyrosinase for 5-70 minutes. In some embodiments, the first peptide is contacted with a tyrosinase for 45 minutes. In some embodiments, the first peptide is contacted with a tyrosinase for 60 minutes. In some embodiments, the second peptide is contacted with a tyrosinase for more than 15 minutes. In some embodiments, the second peptide is contacted with a tyrosinase for 60-160 minutes. In some embodiments, the second peptide is contacted with a tyrosinase for 90 minutes. In some embodiments, the second peptide is contacted with a tyrosinase for 120 minutes.

[0311] Therapeutic uses This disclosure provides a method for treating cancers expressing a target antigen, the method comprising administering to a subject of need a conjugate disclosed herein or a pharmaceutical composition containing said conjugate. In some embodiments, the cancer expresses a target antigen of a polypeptide disclosed herein or a combination of at least two polypeptides.

[0312] This disclosure provides a method for treating HER2-expressing cancers, the method comprising administering to a subject of need the conjugate disclosed herein or a pharmaceutical composition containing said conjugate. In some embodiments, the cancer is a HER2-low expressing cancer. In some embodiments, the HER2-low expressing cancer is breast cancer. In some embodiments, the cancer is a HER2-high expressing (i.e., HER2-positive) cancer. In some embodiments, the HER2-high expressing cancer is breast cancer.

[0313] In some embodiments, the method or pharmaceutical composition comprises 1 mg / kg of the conjugate disclosed herein. In some embodiments, the method or pharmaceutical composition comprises 3 mg / kg of the conjugate disclosed herein. In some embodiments, the method or pharmaceutical composition comprises 8 mg / kg of the conjugate disclosed herein.

[0314] sequence list Example Example 1: Anti-HER2 antibody conjugated with payload This embodiment illustrates the conjugation of two or more payloads to different regions of an antibody.

[0315] An exemplary antibody (e.g., trastuzumab) with a linker is modified as follows: A heavy chain containing a terminal tag ranging from -XY to -XXXXY (where X is any amino acid) is combined at a 1:100 tyrosinase to antibody ratio in the presence of a 5X excess of cysteine ​​nucleophile relative to the antibody. After 1 hour at 22°C, the heavy chain of the trastuzumab antibody shows modification by both catenase and abTYR tyrosinase from Agaricus bisporus. Under similar conditions, a light chain containing a terminal tag ranging from -XXXY to -XXXXY (where X is any amino acid, but at least two Xs are negatively charged amino acids) shows selective reaction with catenase but not with abTYR.

[0316] Nucleophile-loaded molecules were synthesized by combining NHS-Val-Cit-loaded molecules with 2(4-aminoethyl)-aniline or cysteine ​​for 24 hours, and then quenched by dilution with water in a 50 / 50 ratio.

[0317] The antibody light chain is modified to contain a negatively charged C-terminal tag (e.g., -EEEY). The antibody heavy chain is modified to contain a short neutral tag (-SGGY or -SGY). The antibody is expressed and purified according to standard procedures. The antibody buffer is then exchanged for Catenase reaction buffer. Catenase reaction buffer can be 10-50 mM phosphate pH 6.5, 150 mM NaCl and 2 mM EDTA, or 10-100 mM acetate pH 5.5, 0-10% sucrose and 1 mM EDTA. The phosphate in the first listed buffer can be replaced with HEPES, Tris, acetate and MES buffer (pH 5 to pH 9). Additionally, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA can be added to the buffer. As described above, two payloads containing thiols were synthesized and reduced by passing the payloads through an immobilized TCEP resin before use in the tyrosinase reaction.

[0318] The heavy-chain-SGGY tag is ligated by combining the antibody with mushroom tyrosinase at a 3% molar ratio and a 5X excess of payload A relative to the antibody for 1 hour at room temperature with continuous shaking. After completion, the conjugated antibody is purified by hydrophobic interaction chromatography (HIC) or size exclusion chromatography (SEC) according to a predetermined protocol. Protein A columns can also be used for this step.

[0319] To attach the second payload, as prepared above, the antibody modified with payload A was combined with 5x molar of thiol-containing payload B and 1-2% molar of catenase (a modified tyrosinase from Bacillus megaterium). The reaction was run with shaking at room temperature for 1 hour and then purified via a protein A column according to a predetermined procedure.

[0320] This generates antibodies with two different payloads, which are conjugated to different portions of the antibody.

[0321] The modification of the heavy chain was quantified by mass spectrometry. At least 90% of the exemplary heavy chain was modified to allow at least one payload to be conjugated to it via a catenase reaction. Figure 4B (Figure B).

[0322] Example 2: Exemplary antibodies conjugated with multiple payloads This embodiment illustrates the conjugation of at least two payloads with different regions of the antibody.

[0323] An exemplary antibody with a linker is modified as follows: A heavy chain containing a terminal tag ranging from -XY to -XXXXY (where X is any amino acid) is combined at a 1:100 tyrosinase to antibody ratio in the presence of a 5X excess of cysteine ​​nucleophile relative to the antibody. After 1 hour at 22°C, the heavy chain of the trastuzumab antibody shows modification by both catenase and abTYR tyrosinase from Agaricus bisporus. Under similar conditions, a light chain containing a terminal tag ranging from -XXXY to -XXXXXY (where X is any amino acid, but at least two Xs are negatively charged amino acids) shows selective reaction with catenase but not with abTYR.

[0324] Nucleophile-loaded molecules were synthesized by combining NHS-Val-Cit-loaded molecules with 2(4-aminoethyl)-aniline or cysteine ​​for 24 hours, and then quenched by dilution with water in a 50 / 50 ratio.

[0325] The antibody light chain is modified to contain a negatively charged C-terminal tag (e.g., -EEEY). The antibody heavy chain is modified to contain a short neutral tag (-SGGY or -SGY). The antibody is expressed and purified according to standard procedures. The antibody buffer is then exchanged for Catenase reaction buffer. Catenase reaction buffer can be 10-50 mM phosphate pH 6.5, 150 mM NaCl and 2 mM EDTA, or 10-100 mM acetate pH 5.5, 0-10% sucrose and 1 mM EDTA. The phosphate in the first listed buffer can be replaced with HEPES, Tris, acetate and MES buffer (pH 5 to pH 9). Additionally, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA can be added to the buffer. As described above, two payloads containing thiols were synthesized and reduced by passing the payloads through an immobilized TCEP resin before use in the tyrosinase reaction.

[0326] The heavy-chain -SGGY tag is ligated by combining the antibody with mushroom tyrosinase at a 3% molar ratio and a 5X excess of payload A relative to the antibody for 1 hour at room temperature with continuous shaking. Payload A may also be incorporated at native tyrosine residues. After completion, the conjugated antibody is purified according to a predetermined protocol by hydrophobic interaction chromatography (HIC) or size exclusion chromatography (SEC). Protein A columns can also be used for this step.

[0327] To attach the second payload, as prepared above, the antibody modified with payload A was combined with 5x molar of thiol-containing payload B and 1-2% molar of catenase (a modified tyrosinase from Bacillus megaterium). The reaction was run with shaking at room temperature for 1 hour and then purified via a protein A column according to a predetermined procedure.

[0328] This generates antibodies with at least two different payloads, said at least two different payloads being conjugated to different portions of the antibody.

[0329] The modification of the heavy chain was quantified by mass spectrometry. At least 90% of the exemplary heavy chain was modified to conjugate at least one payload to it via a catenase reaction, while approximately 30% of the heavy chain peptide was modified to conjugate at least two payloads A to it. Figure 5 (Figure C). At least 70% of the exemplary light chain is modified to conjugate at least one payload to it via a catenase reaction, while approximately 2% of the light chain peptide is modified to conjugate at least two payloads B to it. Figure 5 (D diagram).

[0330] Example 3: Exemplary antibody conjugated with multiple payloads via temperature control This embodiment describes the use of a similar scheme to that described in Example 1, but with a change in the reaction temperature, and the effect of using Catenase to perform the antibody conjugation step.

[0331] The heavy and light chains of the exemplary antibody were prepared in a manner similar to that described in Example 1. The payload A was ligated to a heavy-chain-SGGY tag by combining the antibody with mushroom tyrosinase at a 3% molar ratio and a 5X excess of payload A relative to the antibody for 1 hour at 4°C with continuous shaking. After completion, the conjugated antibody was purified by hydrophobic interaction chromatography (HIC) or size exclusion chromatography (SEC) according to a predetermined protocol. Protein A columns can also be used for this step.

[0332] To attach the second payload, as prepared above, the antibody modified with payload A was combined with 5x molar of thiol-containing payload B and 1%–2% molar of Catenase (a modified tyrosinase from Bacillus megaterium). The reaction was run with shaking at 37°C for 1 hour and then purified via a protein A column according to a predetermined procedure.

[0333] This generates antibodies with two different payloads, which are conjugated to different portions of the antibody. Figure 6 and Figure 7Example mass spectra of the heavy and light chains are shown before, after, after cleaning, and after the reaction of payload A. The spectra show that the heavy chain is unmodified by payload B after the reaction that conjugates payload B with the light chain.

[0334] Example 4: Exemplary antibody conjugated with multiple payloads via deglycosylation This embodiment illustrates the effect of deglycosylation of an antibody and then conjugation of an exemplary payload with a Catenase enzyme.

[0335] An exemplary antibody was deglycosylated using conventional procedures known in the art. This exemplary antibody contains endogenous tyrosine. The deglycosylated exemplary antibody was then incubated at 4°C with exemplary payload A and catenase enzyme. The resulting mass spectra obtained from analyzing this antibody are shown below. Figure 8 The second graph shows that payload A was successfully conjugated to the antibody. An antibody with natural sugars (glycosylated) was also incubated with payload A and the enzyme, but no conjugation product was observed. Figure 8 (First mass spectrometry). The deglycosylated exemplary antibody was then incubated at room temperature with exemplary payload A and Catenase enzyme. The resulting mass spectrum from the analysis of this antibody is shown in [image / image / data]. Figure 8 The third graph shows that payload A was successfully conjugated to the antibody. The deglycosylated exemplary antibody was then incubated with exemplary payload A and the catenase enzyme at 37°C. The resulting mass spectrum of this antibody is shown in [Figure number missing]. Figure 8 The fifth chart shows that payload A was successfully conjugated to the antibody.

[0336] Example 5: Exemplary HER2 antibody conjugated with multiple payloads This embodiment illustrates the conjugation of at least two payloads to different regions of the HER2 antibody.

[0337] An exemplary HER2 antibody with a linker was modified as follows: A heavy chain containing a terminal tag ranging from -XY to -XXXXY (where X is any amino acid) was combined at a 1:100 tyrosinase to antibody ratio in the presence of a 10X-15X excess of cysteine ​​nucleophile relative to the antibody. After 1 hour, the heavy chain of the HER2 antibody showed modification with both catenase and a tyrosinase (abTYR) from Agaricus bisporus. Under similar conditions, a light chain containing a terminal tag ranging from -XXY to -XXXXXY (where X is any amino acid, but at least two Xs are negatively charged amino acids) showed selective reaction with catenase but not with abTYR.

[0338] Nucleophile-loaded molecules were synthesized by combining NHS-Val-Cit-loaded molecules with 2(4-aminoethyl)-aniline or cysteine ​​for 24 hours, and then quenched by dilution with water in a 50 / 50 ratio.

[0339] The antibody light chain is modified to contain a negatively charged C-terminal tag (e.g., -EEEY). The heavy chain of the exemplary HER2 antibody is modified to contain a short neutral tag (-SGGY or -SGY). The exemplary HER2 antibody is expressed and purified according to standard procedures. The exemplary HER2 antibody buffer is then exchanged for Catenase reaction buffer. The Catenase reaction buffer may be 10-50 mM phosphate pH 6.5, 150 mM NaCl and 2 mM EDTA, or 10-100 mM acetate pH 5.5, 0-10% sucrose and 1 mM EDTA. The phosphate in the first listed buffer may be replaced with HEPES, Tris, acetate and MES buffer (pH 5 to pH 9). Additionally, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA may be added to the buffer. As described above, two payloads containing thiols were synthesized and reduced by passing the payloads through an immobilized TCEP resin before use in the tyrosinase reaction.

[0340] The antibody is coupled to a heavy-chain -SGGY tag by combining it with mushroom tyrosinase at a 3% molar ratio and a 5X-15X (e.g., 10X or 15X) excess of payload A (a small molecule topoisomerase inhibitor) relative to the antibody for 1 hour at room temperature with continuous shaking. Payload A may also be incorporated at native tyrosine residues. After completion, the conjugated antibody is purified according to a predetermined protocol by hydrophobic interaction chromatography (HIC) or size exclusion chromatography (SEC). Protein A columns can also be used for this step.

[0341] To attach the second payload, as prepared above, the antibody modified with payload A was combined with a 5X-15X molar ratio of thiol-containing payload B (a small tubulin inhibitor) and a 1%-2% molar ratio of catenase (a modified tyrosinase from Bacillus megaterium). The reaction was run at room temperature for 1 hour without oscillation and then purified via SEC, HIC, or buffer exchange.

[0342] This generates an exemplary HER2 antibody with at least two different payloads, which are conjugated to different portions of the antibody with different tags.

[0343] The modification of the heavy chain was quantified by mass spectrometry. At least 90% of the exemplary heavy chain was modified to conjugate at least one payload to it via a catenase reaction, while approximately 9% of the heavy chain peptide was modified to conjugate at least two payloads A to it (Figure 9C). At least 93% of the exemplary light chain was modified to conjugate at least one payload to it via a catenase reaction (Figure 9B).

[0344] Exemplary HER2 antibody conjugates were tested in the cell lines of interest. Cell inhibition of each compound across 20 concentration points (n=4) was measured via the AlamarBlue Assay. Figures 10A to 10G Compared to Ds8201a(T-Dxd), the exemplary HER2 antibody conjugates were tested, and the exemplary HER2 antibody conjugates showed a significant increase in total inhibition. The exemplary HER2 antibody conjugates showed significant improvement in cell inhibition in the high-HER2-expressing cell lines BT474 and SKBR3, and the low-HER2-expressing cell line JIMT-1, and had equivalent activity in N87 cells. Both compounds showed high selectivity due to the lack of inhibition in HER2-negative MDA-MB-468 cells.

[0345] The higher stability and low, deterministic response of exemplary HER2 antibody conjugates can be incorporated into more efficient payloads with differentiated mechanisms of action to produce more effective targeted therapies.

[0346] Example 6: Tumor growth in mice treated with an exemplary HER2 antibody conjugated to multiple payloads This embodiment illustrates a mouse study to compare the activity of an exemplary HER2 antibody conjugated with a topoisomerase inhibitor and a microtubule inhibitor with that of Ds8201a (T-Dxd).

[0347] Each SCID beige mouse was injected with 100 μL of Matrigel in 100 μL of PBS containing 5 x 10 g of PBS. 6 JIMT1 cells. JIMT1 is a human breast cancer xenograft model cell line. Tumors were induced to grow in mice until they reached 100 mm. 3 The mice were then randomly divided into 5 groups of 8–10 mice each and treated with control (PBS), Ds8201a (T-Dxd) with a DAR of 8, or MPC (an exemplary HER2 antibody conjugated with a topoisomerase inhibitor and a tubulin inhibitor) with a DAR of 2+2. The treatments for the 5 groups are detailed in Table A below.

[0348] Table A.

[0349] “MPC” represents an exemplary HER2 antibody conjugated with a topoisomerase inhibitor and a tubulin inhibitor.

[0350] Measure tumor volume (length x width x height) twice a week using a caliper (e.g., electronic caliper), and collect a blood (plasma) sample once a week. Measure body weight twice a week.

[0351] Mean tumor growth (mm) 3 The variation over time (number of days after administration) is plotted on Figure 11 In comparison to the control, the exemplary HER2 antibody conjugated with a topoisomerase inhibitor and a tubulin inhibitor showed the same or better activity in slowing tumor volume growth as Ds8201a (T-Dxd). Notably, the exemplary HER2 antibody conjugated with the topoisomerase inhibitor and the tubulin inhibitor had a DAR of 2+2, which is half the DAR of Ds8201a (T-Dxd), which has a DAR of 8.

[0352] Example 7: Exemplary modified antibodies conjugated to a payload under temperature control This embodiment illustrates the temperature-selective conjugation of a payload with an exemplary antibody heavy chain.

[0353] In an exemplary antibody (e.g., trastuzumab), the heavy chain is modified to include an SGGGY terminal tag. The modified antibody is expressed and purified according to standard procedures. The modified antibody buffer is then exchanged for a Catenase reaction buffer. The Catenase reaction buffer may be 10-50 mM phosphate pH 6.5, 150 mM NaCl, and 2 mM EDTA, or 10-100 mM acetate pH 5.5, 0-10% sucrose, and 1 mM EDTA. The phosphate in the first listed buffer may be replaced with a HEPES, Tris, acetate, and MES buffer (pH 5 to pH 9). Additionally, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA may be added to the buffer.

[0354] Modified trastuzumab was combined with an exemplary cysteine-containing drug payload, ethatecan, in a 1:100 catenase (SEQ ID NO: 2) to antibody ratio in the presence of 10X excess of the antibody. The reaction was run with shaking at 4°C for 1 hour. The modified antibody was then purified by ultrafiltration and / or permeation.

[0355] After reacting at 4°C for 1 hour, as Figure 12BThe mass spectrometry data show that essanotecan is conjugated to the heavy chain of the modified trastuzumab via catenase. Figure 12B The mass spectrometry data show that ethatec was not conjugated to the light chain of the modified trastuzumab. Due to the change in buffer pH, a mass difference of -5 Da was observed in the heavy chain from the control (top panel) to the reactant (bottom panel).

[0356] In another exemplary antibody (e.g., trastuzumab), the light chain is modified to include a terminal tag of SSEEEY. After reacting with Catenase at 4°C for 1 hour, essanotecan did not conjugate to the light chain of the modified trastuzumab. Figure 12A ).

[0357] Therefore, when the reaction conditions are 4°C for 1 hour, Catenase selectively conjugates ethathecan to the heavy chain of the modified antibody with the terminal tag ESSY.

[0358] Example 8: Exemplary modified antibodies conjugated to a payload under temperature control This embodiment illustrates the temperature-selective conjugation of a payload with the light and heavy chains of an exemplary antibody.

[0359] An exemplary antibody (e.g., trastuzumab) is used, wherein the heavy chain is modified to have a terminal tag SGGGY, and the light chain is modified to have a terminal tag EEEEY. The modified antibody is expressed and purified according to standard procedures. The modified antibody buffer is then exchanged for a Catenase reaction buffer. The Catenase reaction buffer may be 10-50 mM phosphate pH 6.5, 150 mM NaCl, and 2 mM EDTA, or 10-100 mM acetate pH 5.5, 0-10% sucrose, and 1 mM EDTA. The phosphate in the first listed buffer may be replaced with a HEPES, Tris, acetate, and MES buffer (pH 5 to pH 9). Additionally, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA may be added to the buffer.

[0360] Modified trastuzumab was combined with an exemplary cysteine-containing drug payload, ethathecan, in excess of antibody 12X at a 1:100 catenase (SEQ ID NO: 2) to antibody ratio. The reaction was run with shaking at 30°C for 90 min. The modified antibody was then purified via a protein A column according to a predetermined procedure.

[0361] After reacting at 30℃ for 90 min, as Figure 13BThe mass spectrometry data show that ethathecan is conjugated to the heavy and light chains of the modified trastuzumab via catenase (SEQ ID NO: 2).

[0362] Another modified trastuzumab was prepared, wherein the light chain was modified to have a SSEEY terminal tag. After reacting with Catenase at 30°C for 90 min, an exemplary payload, ethathecan, was conjugated to the light chain of the modified antibody. Figure 13A ).

[0363] Therefore, when the reaction conditions used are 30°C for 90 min, Catenase conjugates ethathecan to the light and heavy chains of the modified antibody containing the terminal tag.

[0364] Example 9: Exemplary modified antibodies conjugated to a payload under temperature control This embodiment illustrates the temperature-selective conjugation of a payload to the light and heavy chains of an exemplary antibody, including non-terminal conjugation sites in the heavy chain.

[0365] The following describes the modification of an exemplary antibody (e.g., trastuzumab). The heavy chain is modified to include the terminal tag ESSY and includes the mutants S298G and T299A to deglycosylate the antibody and expose the non-terminal tyrosine residues on the loop for modification, and the light chain is modified to include the terminal tag SSEEY. The modified antibody is expressed and purified according to standard procedures. The modified antibody buffer is then exchanged for Catenase reaction buffer. Catenase reaction buffer may be 10-50 mM phosphate pH 6.5, 150 mM NaCl and 2 mM EDTA, or 10-100 mM acetate pH 5.5, 0-10% sucrose and 1 mM EDTA. The phosphate in the first listed buffer may be replaced with HEPES, Tris, acetate and MES buffer (pH 5 to pH 9). Additionally, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA may be added to the buffer.

[0366] Modified trastuzumab was combined with an exemplary cysteine-containing drug payload, ethathecan, at a 1:100 catenase (SEQ ID NO: 2) to antibody ratio in the presence of 10X excess of the antibody. The reaction was run with shaking at 4°C for 60 min. The modified antibody was then purified via a protein A column according to a predetermined procedure.

[0367] After reacting at 4°C for 60 min, as Figure 14A and Figure 14BThe mass spectrometry data show that essanotecan binds only to the heavy chain of the modified trastuzumab via catenase (SEQ ID NO: 2). It is noteworthy that, based on... Figure 14A and Figure 14B The mass difference between the mass spectrometry data indicates that only one ethatecan is conjugated to the heavy chain, meaning that only one tyrosine residue was reacted by the catenase, possibly a more readily accessible tyrosine residue on the heavy chain terminal tag. Esaotecan was not conjugated to the light chain of the modified trastuzumab.

[0368] Then, in the presence of 12X excess essanotecan, the modified trastuzumab of this first reaction was combined at a ratio of 1:100 of catenase (SEQ ID NO: 2) to modified trastuzumab. The reaction was run at 30°C for 90 min. After reacting at 30°C for 90 min, as... Figure 14C Mass spectrometry data show that ethatecan is now also conjugated to the light chain of the modified trastuzumab via catenase. Furthermore, mass spectrometry data show that two ethatecan molecules are now conjugated to the heavy chain.

[0369] Therefore, at a lower temperature, Catenase (SEQ ID NO: 2) selectively conjugates ethatecan to a more accessible tyrosine residue on the heavy chain terminal tag, leaving the other tyrosine residue alone. Then, in the second reaction, at a higher temperature of 30°C, Catenase conjugates ethatecan to both the light chain and the less accessible non-terminal tyrosine residue in the heavy chain ring. Notably, the same enzyme is used in both reactions.

[0370] Example 10: Exemplary modified antibodies conjugated to more than one payload under temperature control This embodiment illustrates the temperature-selective conjugation of more than one payload with the light and heavy chains of an exemplary antibody, including non-terminal conjugation sites in the heavy chain.

[0371] The following describes the modification of an exemplary antibody (e.g., trastuzumab). The heavy chain is modified to include the terminal tag ESSY and includes the mutant S298G T299A to deglycosylate the antibody and expose the non-terminal tyrosine residues on the loop for modification, and the light chain is modified to include the terminal tag SSEEY. The modified antibody is expressed and purified according to standard procedures. The modified antibody buffer is then exchanged for Catenase reaction buffer. Catenase reaction buffer may be 10-50 mM phosphate pH 6.5, 150 mM NaCl and 2 mM EDTA, or 10-100 mM acetate pH 5.5, 0-10% sucrose and 1 mM EDTA. The phosphate in the first listed buffer may be replaced with HEPES, Tris, acetate and MES buffer (pH 5 to pH 9). Additionally, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA may be added to the buffer.

[0372] The following provides the protocol followed for conjugating at least two different payloads to a modified antibody.

[0373] DAR2+4 MPC generation scheme 1. Prepare everything except the payload.

[0374] 1. Label the reaction tubes containing buffer and antibody on ice.

[0375] 2. Dilute Catenase (SEQ ID NO: 2) 1:10.

[0376] 3. Prepare antibodies.

[0377] 4. A 1:1 inhibitor mixture (tropolone and L-proline).

[0378] 5. Tris(2-carboxyethyl)phosphine (TCEP) 2. Move the reactants and tubes to a fume hood.

[0379] 3. Add 10 x 1 payload (MMAE) to each reaction tube to induce a reaction at approximately 4°C.

[0380] 4. Ensure the tube has been cooled to approximately 4°C in the incubator.

[0381] 5. Add Catenase (SEQ ID NO: 2) (1x equivalent) to the reactants and run at 200 rpm for 45 minutes with shaking.

[0382] 6. During the reaction run, prepare a 100 kDa, 4 mL Millipore rotary filter (regenerated cellulose membrane) by rinsing it three times with water and then passing some reaction buffer through the membrane.

[0383] 7. After 45 minutes, quench with the inhibitor mixture.

[0384] 8. Take 40 μL of sample.

[0385] 9. Use a 4 mL 100 kDa rotary filter (one filter for every approximately 6 mL of reaction mixture) to perform buffer exchange on the reaction mixture to remove excess Catenase, payload 1, and inhibitor mixture.

[0386] 10. Rotate at 4,000 rcf.

[0387] 11. After each rotation, combine the flow liquid from each filter and measure the absorbance at A280 to track the amount of tyrosine removed from the reaction mixture.

[0388] 12. Before proceeding with reaction 2, ensure that the absorbance reading of the flowing liquid is 0.

[0389] 13. During the final rotation, reduce the volume of each reaction tube to below that of reaction 2 without the addition of the inhibitor mixture, enzyme, and MMAE.

[0390] 14. Measure the accurate volume and adjust accordingly with buffer solution. 15. Take 40 μL of the reactant for online measurements (e.g., yield, gel electrophoresis, mass spectrometry).

[0391] 16. Add appropriate amounts of buffer, payload 2 (15x ethatecan), and catenase (SEQ ID NO: 2) (1x equivalent) to obtain the appropriate final reaction volume and drug equivalent, and run the reaction at 30°C with shaking at 200 rpm for 120 min.

[0392] 17. Take 40 μL of online sample at 90 min.

[0393] 18. Wipe the payload vial and transfer it back to -80°C.

[0394] 19. After 120 min, quench the reaction with an appropriate amount of inhibitor.

[0395] 20. Take 50 μL of the reaction aliquot for online measurement (e.g., gel electrophoresis, mass spectrometry, endotoxin assay).

[0396] 21. Freeze the sample for future use, or store it in a refrigerator for buffer exchange the following day.

[0397] DAR2+2 MPC generation scheme 1. Prepare all items except the payload outside the fume hood: 1. Label the reaction tubes containing buffer and antibody on ice.

[0398] 2. Dilute Catenase (SEQ ID NO: 2) 1:10.

[0399] 3. Antibodies.

[0400] 4. A 1:1 inhibitor mixture (tolphenidone and L-proline).

[0401] 5. TCEP.

[0402] 2. Move the reactants and tubes to a fume hood.

[0403] 3. Add 7 x 1 payload (exatecan) to each reaction tube to carry out the reaction at approximately 4°C.

[0404] 1. Cool the tube to 4°C in the incubator.

[0405] 4. Add Catenase (SEQ ID NO: 2) (1x equivalent) to the reactants and run for 15 minutes without shaking.

[0406] 1. During the reaction run, prepare a 100 kDa, 4 mL rotary filter (regenerated cellulose membrane) by rinsing it three times with water and then passing some reaction buffer through the membrane.

[0407] 5. After 45 minutes, quench with the inhibitor mixture.

[0408] 6. Take 40 μL of online sample.

[0409] 7. Use a 4 mL 100 kDa rotary filter (one filter for every approximately 6 mL of reaction mixture) to perform buffer exchange on the reaction mixture to remove excess Catenase, payload 1, and inhibitors.

[0410] 1. Rotate at 4000 rcf.

[0411] 2. After each rotation, the flow liquid from each filter was combined, and the absorbance at A280 was measured to track the amount of tyrosine removed from the reaction mixture.

[0412] 3. Before proceeding with reaction 2, ensure that the absorbance reading of the flowing liquid is 0.

[0413] 8. During the final rotation, reduce the volume of each reaction tube to below that of reaction 2 without the addition of the inhibitor mixture, enzyme, and payload 2 (MMAE).

[0414] 1. Measure the accurate volume and adjust accordingly with buffer solution.

[0415] 9. Take 40 μL of the reactant for online measurements (e.g., yield, gel, MS).

[0416] 10. Add appropriate amounts of buffer, payload 2 (10x MMAE), and Catenase (SEQ ID NO: 2) (1x equivalent) to obtain the appropriate final reaction volume and drug equivalent, and run the reaction at 30°C with shaking at 200 rpm for 120 min.

[0417] 1. Take 40 μL of online sample at 90 min.

[0418] 2. Prepare mass spectrometry sample spreadsheets and vials during this reaction.

[0419] 11. Wipe the payload vial and transfer it back to -80°C.

[0420] 12. After 120 min, quench the reaction with an appropriate amount of inhibitor.

[0421] 13. Take 50 μL of the reaction aliquot for online measurement (e.g., gel electrophoresis, mass spectrometry, endotoxin assay).

[0422] 14. Freeze the sample for future use, or store it in a refrigerator for buffer exchange the next day.

[0423] After reacting at 4°C for 45 min, as Figure 15A and Figure 15B The mass spectrometry data show that MMAE conjugates only to the tyrosine residue on the heavy chain terminal tag via Catenase (SEQ ID NO: 2). It is noteworthy that, based on... Figure 15A and Figure 15B The mass difference between the mass spectrometry data was due to only one MMAE conjugated to the heavy chain. The MMAE was not conjugated to the light chain of the modified trastuzumab.

[0424] The modified trastuzumab from the first reaction was used in the second reaction with ethatecan. The reaction was run at 30°C for 120 min. After the reaction at 30°C for 120 min, as... Figure 15CMass spectrometry data showed that ethanotecan was conjugated to the light chain of the modified antibody via catenase (SEQ ID NO: 2). Furthermore, mass spectrometry data also showed that ethanotecan was conjugated to cyclic tyrosine residues in the heavy chain. Figure 15D The mass spectrum of the modified antibody after purification is shown.

[0425] At a lower temperature, the catenase selectively conjugates the first payload, MMAE, to a more accessible tyrosine residue on the heavy chain terminal tag, and then uses the same catenase to conjugate the second payload, ethatecan, to the light chain and non-terminal tyrosine residues of the modified trastuzumab. Thus, multi-load antibody-drug conjugates can be generated using the same enzyme by simply adjusting the reaction temperature and reaction time.

[0426] Example 11: Exemplary modified antibodies conjugated with multiple end tags and more than one payload This embodiment illustrates the temperature-selective conjugation of more than one payload with an exemplary modified antibody having a series of terminal tags containing tyrosine residues.

[0427] The reactions described in this embodiment generally use the methods described in Example 10. Modified antibodies with different terminal tags on the heavy and light chains are generated to demonstrate the range of exemplary terminal tags that allow selective conjugation of the heavy and light chains under temperature-controlled reactions.

[0428] Exemplary antibodies with terminal tags on the heavy chain, such as SGGGY, ESY, EESY, ESSY, ESSSY, SSSY, SNNY, SSNY, EGGY, SESY, and SEGY, were generated. Reaction at 4°C with any of these terminal tags and Catenase (SEQ ID NO: 2) for 60 min showed that at least approximately 80% of the heavy chain conjugated to the exemplary payload 1. Figures 16A to 16D Reacting with any of these end labels at 4°C for 60 min showed that at least approximately 70% of the majority of the heavy chains were connate to the exemplary No. 2 payload. Some heavy chains were modified by adding a second No. 2 payload. Figure 16B The second payload may occasionally be added to the same phenol ring as the first payload. This is caused by the re-oxidation of the thiol-modified ring, followed by the addition of an additional thiol to the original modified ring adjacent to or relative to the ortho-quinone ring.

[0429] Exemplary antibodies with terminal tags such as EEEEY, SSEEY, SSSEY, SSSSY, and SSNNY on the light chain were generated. Reaction at 30°C with any of these terminal tags and Catenase (SEQ ID NO: 2) showed that at least approximately 85% of the light chain conjugated to the exemplary payload 1. Figure 16C Reacting with any of these end tags at 30°C showed that at least approximately 70% of the light chains were conjugated to the exemplary payload No. 2. Figure 16D ).

[0430] Example 12: Exemplary modified antibodies conjugated with multiple payloads This embodiment illustrates the conjugation of various payloads with the light and heavy chains of exemplary antibodies.

[0431] An exemplary antibody (e.g., trastuzumab) is modified to include a terminal tag. The heavy chain is modified to include the terminal tag SGGGY, and the light chain is modified to include the terminal tag EEEEY. The modified antibody is expressed and purified according to standard procedures. The modified antibody buffer is then exchanged for a Catenase reaction buffer. The Catenase reaction buffer may be 10-50 mM phosphate pH 6.5, 150 mM NaCl, and 2 mM EDTA, or 10-100 mM acetate pH 5.5, 0-10% sucrose, and 1 mM EDTA. The phosphate in the first listed buffer may be replaced with a HEPES, Tris, acetate, and MES buffer (pH 5 to pH 9). Additionally, up to 15% v / v sucrose and glucose, up to 300 mM trehalose, and up to 5 mM EDTA may be added to the buffer.

[0432] Modified trastuzumab was combined with Catenase (SEQ ID NO: 2) in the presence of an exemplary cysteine-containing drug payload in excess of antibody 5X. Various payloads, including MMAE, doxorubicin, camptothecin, and ethatecan, were tested. The reaction was run with shaking at 30°C for 1 hour. The modified antibody was then purified via a protein A column according to a pre-established procedure.

[0433] After the reaction at 30℃, as Figure 17 and Figure 18As shown in the graphs and mass spectrometry data, all tested payloads were conjugated to the heavy and light chains of the modified trastuzumab via catenase (SEQ ID NO: 2). While most heavy chains were conjugated to one payload molecule, some were conjugated to two or three payload molecules. Some light chains were also conjugated to two payload molecules. As illustrated herein, second and / or third payloads may occasionally be added to the same phenolic ring as the first payload. This is caused by the re-oxidation of the thiol-modified ring, followed by the addition of an additional thiol to the original modified ring adjacent to or relative to the ortho-quinone ring. For the reactions with all exemplary payloads, complete modification (over 95% modification) of both the heavy and light chains was observed.

[0434] Therefore, Catenase conjugates a variety of exemplary payloads to exemplary modified antibodies.

[0435] Example 13: Exemplary modified antibodies conjugated with multiple payloads under temperature control This embodiment illustrates the conjugation of various payloads with the heavy chains of exemplary antibodies under temperature-controlled reactions.

[0436] The following modifications are made to an exemplary antibody (e.g., trastuzumab) to include a terminal tag. The heavy chain is modified to include the terminal tag SGGGY, and the light chain is modified to include the terminal tag EEEEY. The modified antibody is expressed and purified according to standard procedures.

[0437] Modified trastuzumab was combined with Catenase (SEQ ID NO: 2) in the presence of an exemplary cysteine-containing drug payload at 5x excess relative to the antibody. Various payloads, including MMAE, doxorubicin, camptothecin, and ethatecan, were tested. The reaction was run with shaking at 4°C for 1 hour. The modified antibody was then purified via a protein A column according to a pre-established procedure.

[0438] After the reaction at 4°C, as Figure 19 and Figure 20 The data show that the tested payloads were conjugated to the heavy chain of the modified trastuzumab via catenase (SEQ ID NO: 2). While most heavy chains were conjugated to one payload molecule, some were conjugated to two or three payload molecules. Complete modification of the heavy chain (over 95%) was observed in reactions with all exemplary payloads. Small amounts of the modified antibody's light chain were conjugated to the exemplary payloads, for example, less than 30% of the light chain in the reaction with MMAE and less than 10% in the reactions with other exemplary payloads.

[0439] Therefore, under low-temperature (e.g., 4°C) reaction conditions, Catenase selectively conjugates a variety of exemplary payloads to the heavy chain of an exemplary modified antibody.

[0440] The procedures disclosed in Examples 1-123 herein can be performed with any polypeptide (e.g., any antibody) known in the art. Those skilled in the art will understand that the functional groups present on the various parts of the molecule should be compatible with the proposed reagents and reactions.

[0441] Example 14: Synthesis of ethatecan with a thiol-containing linker This embodiment describes the synthesis of ethathecan payload into a linker containing thiols. Exathecan is abbreviated as DXD in the following description.

[0442] Method 1: In step 1, compound A was reacted with DX-8951f in dimethylformamide (DMF) at room temperature for two hours in the presence of N,N-diisopropylethylamine (DIEA) to provide compound B. In the next step, compound B was subjected to DMF containing 2% diethanolamine (DEA) to deprotect the Fmoc group and form compound C. Then, compound C was reacted with bis(2,5-dioxopyrrolidone-1-yl) glutarate in the presence of dimethylacetamide (DMAC) at 0°C for 30 minutes to produce compound D. Next, compound D was reacted with 12 equivalents of 2-aminoethane-1-thiol in the presence of DMAC. The final SH-VC-PAB-DXD compound was purified to 38 mg with 90% purity. [M+H] + 1,014.35.

[0443] Figure 21 The final NMR spectrum of the compound is provided in the image.

[0444] Example 15: Synthesis of MMEA with a thiol-containing linker This embodiment illustrates the synthesis of the payload MMEA into a junction containing thiols.

[0445] Method 2: First, VC-PAB-MMAE was reacted with bis(2,5-dioxopyrrolidone-1-yl) glutarate in the presence of DMAC at 0 °C for 40 min. Next, compound E was reacted with 12 equivalents of bis(2,5-dioxopyrrolidone-1-yl) glutarate in the presence of DMAC at room temperature for 1 h. Finally, the SH-VC-PAB-MMAE compound was purified to give 59 mg. [M+H]: 649.20.

[0446] Figure 22 The final NMR spectrum of the compound is provided in the image.

[0447] Example 16: Synthesis of camptothecin with a thiol-containing linker This embodiment describes the synthesis of camptothecin, a payload, into a joint containing thiols.

[0448] Method 3: First, camptothecin was reacted with 0.5 equivalents of an agent used as a carbonyl source, 1 equivalent of 4-dimethylaminopyridine (DMAP), and 4 equivalents of N,N-diisopropylethylamine (DIPEA) in DCM for 2 hours at 0°C. Next, the resulting compound was reacted with 0.9 equivalents of Boc-Val-Cit dipeptide in DCM and DMSO for 2 hours at room temperature. In step 2, compound F was deprotected to form compound G. In step 3, compound G was reacted with bis(2,5-dioxopyrrolidine-1-yl) glutarate in DMF to provide compound H. In step 4, compound H was reacted with 2-aminoethane-1-thiol in a 1:5 ratio of trifluoroacetic acid (TFA) and DCM for 2 hours at 0°C. Finally, the compound was purified to give SH-VC-PAB-camptothecin.

[0449] Example 17: Synthesis of doxorubicin with a thiol-containing linker This embodiment describes the synthesis of a payload of doxorubicin into a junction containing thiols.

[0450] Method 4 In step 1, the Fmoc-protected VC-PAB compound is reacted with doxorubicin in DIA and DMF to form compound I. Next, compound I is deprotected using DEA and DMF to form compound J. Next, compound J is reacted with bis(2,5-dioxopyrrolidone-1-yl) glutarate, TEA, DIEA, and DMA to provide compound K. In the final step, compound K is reacted with 2-aminoethane-1-thiol to provide the final compound SH-VC-PAB-doxorubicin.

[0451] Based on the teachings contained herein and synthetic procedures known in the art, the procedures disclosed herein can be performed in various ways. In the description of the synthetic methods above, it should be understood that, unless otherwise indicated, all proposed reaction conditions, including the selection of solvent, reaction atmosphere, reaction temperature, experimental duration, and post-treatment procedures, can be selected as standard conditions for the reactions. Those skilled in the art of organic synthesis will understand that the functional groups present on various parts of the molecule should be compatible with the proposed reagents and reactions. Substituents incompatible with the reaction conditions will be apparent to those skilled in the art, and therefore, alternative methods will be indicated. The starting materials used in the embodiments described are commercially available or readily prepared from known materials using standard methods.

[0452] It should be understood that the illustrated embodiments are merely examples of the disclosed products and methods and should not be construed as limiting the scope of the embodiments. Rather, the scope of the embodiments is defined by the claims. Therefore, we claim protection for all contents falling within the scope and spirit of these claims.

Claims

1. A conjugate of formula A', formula B', formula C', or formula D': Formula A' Formula B' Formula C', or Formula D' in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one amino acid from X1-X5 contains tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid from X1-X3 is tyrosine or a portion thereof, where Y a Linked to "S" via the tyrosine or a portion thereof; Y b The second polypeptide contains a second terminal tag different from the first terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in the formula comprises tyrosine or a portion thereof, and wherein m2 is an integer greater than or equal to 0, or X1X2X3, wherein X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof, wherein Y b Linked to "S" via the tyrosine or a portion thereof; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

2. The conjugate of claim 1, wherein the tyrosine or a portion thereof is selected from the group consisting of: , , and .

3. The conjugate according to claims 1-2, wherein the first terminal tag comprises at least two amino acids selected from the group consisting of: aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K).

4. The conjugate according to any one of claims 1-3, wherein the second terminal tag comprises at least two amino acids selected from the group consisting of: aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K).

5. The conjugate according to claims 1-2, wherein the first end tag comprises at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

6. The conjugate according to any one of claims 1-5, wherein the second end tag comprises at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

7. The conjugate according to any one of claims 1-6, wherein the first end tag comprises SGGY, SGY, SGGGY, ESY, EESY, ESSY, ESSSY, SESY, SGGGY, EESY, SSSY, SNNY, SSNY, EGGY, SESY, or SEGY.

8. The conjugate according to any one of claims 1-6, wherein the second end tag comprises EEEY, EEEY, SSSEY, SSEEY, SSSSY, or SSNNY.

9. The conjugate according to any one of claims 1-7, wherein Y a Y b Or Y a and Y b Both are glycosylated.

10. The conjugate according to any one of claims 1-9, wherein Y a Y b Or Y a and Y b Both contain non-terminal tyrosine residues.

11. The conjugate according to any one of claims 1-10, wherein at least one of L3, L4 or L5 is a pyrolytic linker.

12. The conjugate of claim 11, wherein the cleavable connector is an electrophilic cleavable connector, a nucleophilic cleavable connector, a photocleavable connector, a metallic cleavable connector, an electrolytic cleavable connector, an acid cleavable connector, or a protein hydrolytic cleavable connector.

13. The conjugate of claim 12, wherein the cleavable linker further comprises a polyethylene glycol group, a glycosyl group, or a modification to increase hydrophilicity.

14. The conjugate of claim 11, wherein the pyrolytic linker is pyrolytic under reducing and / or oxidizing conditions.

15. The conjugate of claim 11, wherein the pyrolytic linker is pyrolytic under acidic conditions.

16. The conjugate of claim 11, wherein the cleavable linker comprises a disulfide bond.

17. The conjugate of claim 11, wherein the cleavable linker is a protein hydrolyzable linker and contains a protease recognition sequence.

18. The conjugate of claim 17, wherein the protease recognition sequence is recognized by a protease selected from the group consisting of metalloproteinases, cathepsin B, and tobacco etching virus (TEV).

19. The conjugate of claim 11, wherein the cleavable linker comprises a dipeptide, a tripeptide, or a tetrapeptide.

20. The conjugate of claim 19, wherein the dipeptide is a valine-citrulline (Val-Cit) dipeptide, a valine-lysine dipeptide, or a valine-alanine dipeptide.

21. The conjugate of claim 19, wherein the tetrapeptide is a glycine-glycine-phenylalanine-glycine (GGFG) tetrapeptide.

22. The conjugate of claim 11, wherein the cleavable linker is selected from the group consisting of PABC (p-aminobenzyl alcohol), glucuronic acid, and MABC (m-aminobenzyl alcohol).

23. The conjugate of claim 11 or 22, wherein the pyrolytic connector is Val-Cit-PABC.

24. The conjugate of claim 11, wherein "Y1-L4-S-" and / or "-S-L5-Y2" are selected from the group consisting of: S-VC-PAB-DXD, S-VC-PAB-MMAE, S-VC-PAB-camptothecin, and S-VC-PAB-Dorothy Star.

25. The conjugate according to any one of claims 1-23, wherein Y a The variable region of the heavy chain (VH), variable region of the light chain (VL), heavy chain, light chain, constant chain of an antibody or antibody fragment; peptide; or cyclic peptide.

26. The conjugate of claim 25, wherein the peptide is a binding peptide.

27. The conjugate according to any one of claims 1-26, wherein Y b For antibodies, antibody fragments, heavy chain variable region (VH), light chain variable region (VL), heavy chain, light chain, constant chain; peptide; or cyclic peptide.

28. The conjugate of claim 27, wherein the peptide is a binding peptide.

29. The conjugate according to any one of claims 1-28, wherein Y a With Y b Connected via L3.

30. The conjugate according to any one of claims 1-28, wherein L3 comprises a peptide sequence, a dimerization and docking domain, a leucine zipper, or a mortise and tenon structure.

31. The conjugate according to any one of claims 1-30, wherein L3 comprises a peptide bond, a disulfide bond, a maleimide bond, a thioether bond, an azide-alkyne cycloaddition, a cysteyl-DOPA bond, or a hydrogen bond.

32. The conjugate according to any one of claims 1-28, wherein L3 is a connector.

33. The conjugate of claim 32, wherein the connector comprises a sequence selected from the group consisting of: (GS) n3 (G2S) n3 (G3S) n3 (G4S) n3 (G) n3 (GGSGGD) n3 (GGSGGE) n3 (GGGSGSGGGGS) n3 and (GGGGGPGGGGP) n3 , where n3 is an integer from 2 to 20.

34. The conjugate according to any one of claims 1-33, wherein L3 comprises a terminal tyrosine or a portion thereof.

35. The conjugate according to any one of claims 1-34, wherein n is 1, 2, 3, 4 or 5.

36. The conjugate according to any one of claims 1-34, wherein n is 1.

37. The conjugate according to any one of claims 1-34, wherein Y1 and Y2 are identical.

38. The conjugate according to any one of claims 1-34, wherein Y1 and Y2 are different.

39. The conjugate according to any one of claims 1-38, wherein Y1, Y2, or both Y1 and Y2 are small molecules.

40. The conjugate of claim 39, wherein the small molecule is selected from the group consisting of: delutec, ixatec, FL118, irinotecan, topotecan, SN-38, rubotecan, belototecan, letopotecan, gemmatotecan, diflutecan, calontecan, citratecan, nanotecan, ileotecan, DRF-1042, delutec, NSC606985, cimenotecan, ZBH-1205, auristatin (MMAE, MMAF, MMAG, MMAH), chachiomycin, doxorubicin, paclitaxel and paclitaxel derivatives, maytansine (DM1-4), pyrrolodiazepine (PBD), tubulolysin, eribulin, atrazomycin, pyroxine, anthracyclines, and camptothecin (CPT), including the lactone and carboxylic acid ester forms of CPT.

41. The conjugate of claim 39, wherein the small molecule is selected from the group consisting of topoisomerase inhibitors and tubulin inhibitors.

42. The conjugate according to any one of claims 1-38, wherein Y1 and Y2 or both Y1 and Y2 comprise nucleic acids, immune agonists, peptides, cytokines or binding domains.

43. The conjugate according to any one of claims 1-38, wherein Y1 and Y2 or both Y1 and Y2 comprise nucleic acids.

44. The conjugate of claim 43, wherein Y1 and Y2 or both Y1 and Y2 comprise oligonucleotides.

45. The conjugate according to any one of claims 1-38, wherein Y1 and Y2 or both Y1 and Y2 comprise peptides.

46. ​​The conjugate of claim 45, wherein Y1 and Y2 or both Y1 and Y2 comprise a binding peptide.

47. A conjugate of the formula AI, BI, CI, or DI: AI-style Formula BI, CI, or Formula DI; where: Y a' It is the first polypeptide. Each of X1, X2, X3, X4, and X5 is independently any amino acid, where m1, m2, and m3 are each integers greater than or equal to 0; Y b' It is the second polypeptide. X6, X7, X8, X9 and X 10 Each of them is independently any amino acid, where m4, m5, and m6 are each an integer greater than or equal to 0, and where -(X1) is an integer. m1 (X2) m2 X3X4 - Partially different from -(X6) m1 (X7) m2 X8X9 - Partial; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

48. The conjugate of claim 47, wherein -(X1) m1 (X2) m2 The X3X4-part contains at least two amino acids selected from the following group: aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K).

49. The conjugate according to any one of claims 47-48, wherein -(X6) m1 (X7) m2 The X8X9-part contains at least two amino acids selected from the following group: aspartic acid (D), glutamic acid (E), arginine (R), and lysine (K).

50. The conjugate of claim 47, wherein -(X1) m1 (X2) m2 The X3 EDDD, EEDD, RRKK, KKGG, DDGGEES, ESSS, SSS, SNN, SSN, SEG, SSSE, EGG, SSEE, SES, ESS, ES, SR, SK, SN, ER, EK, EG, SE, SHRK, SARK, SPPE, SSEE, SEEE and SSSS.

51. The conjugate according to any one of claims 47-50, wherein -(X6) m1 (X7) m2 The DDD, EEDD, RRKK, KKGG, DDGG, EES, ESSS, SSS, SNN, SSN, SEG, SSSE, EGG, SSEE, SES, ESS, ES, SR, SK, SN, ER, EK, EG, SE, SHRK, SARK, SPPE, SSEE, SEEE and SSSS.

52. The conjugate according to any one of claims 47-51, wherein -(X1) m1 (X2) m2 X3X4 - Parts are SGG, SG, SGGG, ES, EES, ESS, ESSS, SES, SGGG, EES, SSS, SNN, SSN, EGG, SES, or SEG.

53. The conjugate according to any one of claims 47-52, wherein -(X6) m1 (X7) m2 X8X9 - Parts are EEE, EEEE, SSSE, SSEE, SSSS, or SSNN.

54. The conjugate according to any one of claims 47-53, wherein Y a' Y b' Or Y a' and Y b' Both are glycosylated.

55. The conjugate according to any one of claims 47-54, wherein at least one of L3, L4 or L5 is a pyrolytic linker.

56. The conjugate of claim 55, wherein the cleavable connector is an electrophilic cleavable connector, a nucleophilic cleavable connector, a photocleavable connector, a metallic cleavable connector, an electrolytic cleavable connector, an acid cleavable connector, or a protein hydrolytic cleavable connector.

57. The conjugate of claim 56, wherein the cleavable linker further comprises a polyethylene glycol group, a glycosyl group, or a modification to increase hydrophilicity.

58. The conjugate of claim 55, wherein the pyrolytic joint is pyrolytic under reducing and / or oxidizing conditions.

59. The conjugate of claim 55, wherein the pyrolytic joint is pyrolytic under acidic conditions.

60. The conjugate of claim 55, wherein the pyrolytic linker comprises a disulfide bond.

61. The conjugate of claim 55, wherein the cleavable adapter is a protein hydrolyzable adapter and contains a protease recognition sequence.

62. The conjugate of claim 6117, wherein the protease recognition sequence is recognized by a protease selected from the group consisting of metalloproteinases, cathepsin B, and tobacco etching virus (TEV).

63. The conjugate of claim 55, wherein the cleavable linker comprises a dipeptide, a tripeptide, or a tetrapeptide.

64. The conjugate of claim 63, wherein the dipeptide is a valine-citrulline (Val-Cit) dipeptide, a valine-lysine dipeptide, or a valine-alanine dipeptide.

65. The conjugate of claim 63, wherein the tetrapeptide is a glycine-glycine-phenylalanine-glycine (GGFG) tetrapeptide.

66. The conjugate of claim 55, wherein the cleavable linker is selected from the group consisting of PABC (p-aminobenzyl alcohol), glucuronic acid, and MABC (m-aminobenzyl alcohol).

67. The conjugate according to any one of claims 47-66, wherein Y a' The variable region of the heavy chain (VH), variable region of the light chain (VL), heavy chain, light chain, constant chain of an antibody or antibody fragment; peptide; or cyclic peptide.

68. The conjugate of claim 67, wherein the peptide is a binding peptide.

69. The conjugate according to any one of claims 47-67, wherein Y b' For antibodies, antibody fragments, heavy chain variable region (VH), light chain variable region (VL), heavy chain, light chain, constant chain; peptide; or cyclic peptide.

70. The conjugate of claim 69, wherein the peptide is a binding peptide.

71. The conjugate according to any one of claims 47-70, wherein Y a' With Y b' Connected via L3.

72. The conjugate according to any one of claims 47-71, wherein L3 comprises a peptide sequence, a dimerization and docking domain, a leucine zipper, or a pestle-and-mortar structure.

73. The conjugate according to any one of claims 47-72, wherein L3 comprises a peptide bond, a disulfide bond, a maleimide bond, a thioether bond, an azide-alkyne cycloaddition, a cystoyl-DOPA bond, or a hydrogen bond.

74. The conjugate according to any one of claims 47-71, wherein L3 is a connector.

75. The conjugate of claim 74, wherein the connector comprises a sequence selected from the group consisting of: (GS) n3 (G2S) n3 (G3S) n3 (G4S) n3 (G) n3 (GGSGGD) n3 (GGSGGE) n3 (GGGSGSGGGGS) n3 and (GGGGGPGGGGP) n3 , where n3 is an integer from 2 to 20.

76. The conjugate according to any one of claims 47-75, wherein n is 1, 2, 3, 4 or 5.

77. The conjugate according to any one of claims 47-76, wherein n is 1.

78. The conjugate according to any one of claims 47-77, wherein Y1 and Y2 are identical.

79. The conjugate according to any one of claims 47-77, wherein Y1 and Y2 are different.

80. The conjugate according to any one of claims 47-79, wherein Y1, Y2, or both Y1 and Y2 are small molecules.

81. The conjugate of claim 80, wherein the small molecule is selected from the group consisting of: delutec, ixatec, FL118, irinotecan, topotecan, SN-38, rubotecan, belototecan, letopotecan, gemmatotecan, diflutecan, calontecan, citratecan, nanotecan, ileotecan, DRF-1042, delutec, NSC606985, cimenotecan, ZBH-1205, auristatin (MMAE, MMAF, MMAG, MMAH), chachiomycin, doxorubicin, paclitaxel and paclitaxel derivatives, maytansine (DM1-4), pyrrolodiazepine (PBD), tubulolysin, eribulin, atrazomycin, pyroxine, anthracycline, and camptothecin (CPT), including the lactone and carboxylic acid ester forms of CPT.

82. The conjugate of claim 80, wherein the small molecule is selected from the group consisting of topoisomerase inhibitors and tubulin inhibitors.

83. The conjugate according to any one of claims 47-79, wherein Y1 and Y2 or both Y1 and Y2 comprise nucleic acids, immune agonists, peptides, cytokines or binding domains.

84. The conjugate according to any one of claims 47-79, wherein Y1 and Y2 or both Y1 and Y2 comprise nucleic acids.

85. The conjugate of claim 84, wherein Y1 and Y2 or both Y1 and Y2 comprise oligonucleotides.

86. The conjugate according to any one of claims 47-79, wherein Y1 and Y2 or both Y1 and Y2 comprise peptides.

87. The conjugate of claim 86, wherein Y1 and Y2 or both Y1 and Y2 comprise a binding peptide.

88. A polypeptide comprising a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine or a portion thereof; and a second end tag different from the first end tag, the second end tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof.

89. The polypeptide of claim 88, wherein the first end is located at the N-terminus of the polypeptide, and the second end tag is located at the C-terminus of the polypeptide.

90. The polypeptide of claim 88 or 89, wherein the polypeptide comprises a non-terminal tyrosine residue.

91. The polypeptide of claim 90, wherein the polypeptide is modified to expose the non-terminal tyrosine residues, making it accessible to the enzyme.

92. A composition of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of X1-X5 is tyrosine or a portion thereof; or X1X2X3, where X1-X3 are any amino acids, provided that at least one of X1-X3 is tyrosine or a portion thereof; and where m1 is an integer greater than or equal to 0, and the second polypeptide contains a second end tag different from the first end tag, the second end tag containing (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in it is tyrosine or a part thereof, and m2 is an integer greater than or equal to 0.

93. The composition of claim 92, wherein the first polypeptide and / or the second polypeptide comprises a non-terminal tyrosine residue.

94. The composition of claim 93, wherein the first polypeptide and / or the second polypeptide is modified to expose the non-terminal tyrosine residues, making them accessible to the enzyme.

95. The polypeptide or composition of claims 88-94, wherein the first terminal tag comprises at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

96. The polypeptide or composition of claims 88-95, wherein the second terminal tag comprises at least one of the following: GGGGY, RGGGY, RGRGY, RRRGY, RRRRY, EGGGY, EGEGY, EEEGY, EEEEY, GGGWY, GGWGY, RRRWY, RRWRY, EEEWY, EEWEY, DDDDY, SGGY, SGY, KKKKY, RRKKY, RRRKY, EDEDY, EDDDY, EEDDY, RRKKY, KKGGY, DDGGY, EESY, ESSSY, SSSY, SNNY, SSNY, SEGY, SSSEY, EGGY, SSEEY, SESY, ESSY, ESY, SRY, SKY, SNY, ERY, EKY, EGY, SEYP, SHRKY, SARKY, SPPEY, SSEEY, SEEEY, and SSSSY.

97. The polypeptide or composition of claims 88-96, wherein the first terminal tag comprises SGGY, SGY, SGGGY, ESY, EESY, ESSY, ESSSY, SESY, SGGGY, EESY, SSSY, SNNY, SSNY, EGGY, SESY, or SEGY.

98. The polypeptide or composition of claims 88-97, wherein the second terminal tag comprises EEEY, EEEY, SSEEY, SSSEY, SSSSY, or SSNNY.

99. A method for covalently linking at least two polypeptides to at least two payloads, the method comprising: a) Using a first tyrosinase, contact a first polypeptide of the at least two polypeptides with a first payload of the at least two payloads, wherein the first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1). m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of the amino acids X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one of the amino acids X1-X3 is tyrosine or a portion thereof. as well as b) Using a second tyrosinase, contact the second polypeptide of the at least two polypeptides with the second payload of the at least two payloads, wherein the second polypeptide comprises a second terminal tag, the second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof.

100. The method of claim 99, wherein the first tyrosinase, the second tyrosinase, or both the first tyrosinase and the second tyrosinase are Agaricus bisporus tyrosinase (abTYR).

101. The method of any one of claims 99-100, wherein the first tyrosinase, the second tyrosinase, or both comprise a sequence that is at least 80% identical to any one of SEQ ID NO: 1-6, 52, or 53.

102. The method according to any one of claims 99-101, wherein the first tyrosinase, the second tyrosinase, or both the first tyrosinase and the second tyrosinase are catenases.

103. The method of claim 102, wherein the first tyrosinase and the second tyrosinase are catenases.

104. The method of any one of claims 99-102, wherein the first tyrosinase, the second tyrosinase, or both comprise a sequence that is at least 90% identical to any one of SEQ ID NO: 2-6.

105. The method of any one of claims 99-102, wherein the first tyrosinase, the second tyrosinase, or both comprise a sequence that is at least 90% identical to SEQ ID NO:

2.

106. The method according to any one of claims 99-102, wherein the first tyrosinase is Agaricus bisporus tyrosinase (abTYR) and the second tyrosinase is Catenase.

107. The method of any one of claims 99-106, wherein the first tyrosinase and the second tyrosinase are provided simultaneously.

108. The method of any one of claims 99-106, wherein the first tyrosinase is provided first, followed by the second tyrosinase.

109. A method for covalently linking at least two polypeptides to at least two payloads, the method comprising: a) Contacting a first polypeptide of the at least two polypeptides with a first payload of the at least two payloads in the presence of a tyrosinase, wherein the first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5, where X1-X5 are any amino acids, provided that at least one of the amino acids X1-X5 is tyrosine or a portion thereof, and where m1 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one of the amino acids X1-X3 is tyrosine or a portion thereof. as well as b) Contacting a second polypeptide of the at least two polypeptides with a second payload of the at least two payloads in the presence of the tyrosinase, wherein the second polypeptide comprises a second terminal tag comprising (X6). m2 X7 X8 X9 X 10 Among them, X6-X 10 For any amino acid, the condition is X6-X 10 At least one amino acid in is tyrosine or a portion thereof, and m2 is an integer greater than or equal to 0, or X1X2X3, where X1-X3 are any amino acids, provided that at least one amino acid in X1-X3 is tyrosine or a portion thereof.

110. The method of claim 109, wherein the first polypeptide is contacted with the tyrosinase at a temperature between 0 and 15°C.

111. The method of claim 109 or 110, wherein the first polypeptide is contacted with the tyrosinase at a temperature of 4°C.

112. The method of any one of claims 109-111, wherein the second polypeptide is contacted with the tyrosinase at a temperature between 25-50°C.

113. The method of any one of claims 109-112, wherein the second polypeptide is contacted with the tyrosinase at a temperature of 30°C.

114. The method of any one of claims 109-113, wherein the first polypeptide is contacted with the tyrosinase for 5-70 min.

115. The method of any one of claims 109-114, wherein the first polypeptide is contacted with the tyrosinase for 45 min.

116. The method of any one of claims 109-115, wherein the first polypeptide is contacted with the tyrosinase for 60 min.

117. The method of any one of claims 109-116, wherein the second polypeptide is contacted with the tyrosinase for more than 15 minutes.

118. The method of any one of claims 109-117, wherein the second polypeptide is contacted with the tyrosinase for 60-160 min.

119. The method of any one of claims 109-118, wherein the second polypeptide is contacted with the tyrosinase for 90 min.

120. The method of any one of claims 109-119, wherein the second polypeptide is contacted with the tyrosinase for 120 min.

121. The method of any one of claims 109-120, wherein the tyrosinase comprises at least 80% identity with any one of SEQ ID NO: 1-6, 52 or 53.

122. A conjugate of formula E', F', G', H', J', or K': E', Formula F', Formula G', Formula H', Formula J', or Formula K' in: Y a The first polypeptide comprises a first terminal tag, the first terminal tag comprising (X1) m1 X2X3X4X5Y, where X1-X5 are any amino acids, provided that no more than two of X2, X3, X4 and X5 are aspartic acid or glutamic acid, and where m1 is an integer greater than or equal to 0; Y b It is the second polypeptide; n is an integer greater than 0; L3 is an optional third connector; L4 is an optional fourth connector; L5 is an optional fifth connector; Y1 is the first effective payload; and Y2 is the second payload.

123. A compound of formula L1: Equation LI, where R is the effective load.

124. The compound of claim 123, wherein the compound is selected from the group consisting of: SH-VC-PAB-DXD SH-VC-PAB-MMAE, SH-VC-PAB-camptothecin, and SH-VC-PAB-Dorothy Star.

125. A polypeptide conjugate represented by formula S1, S2, S3 or S4, Formula S1, Formula S2, Formula S3, or Equation S4, in: Pp is a polypeptide and contains oxidized tyrosine; n is 1, 2, or 3; Furthermore, the "-S-" of the compound is conjugated to the oxidized tyrosine on the polypeptide.

126. The polypeptide conjugate of claim 129, wherein the polypeptide is an enzyme, an antibody or a portion thereof, a structural polypeptide, a receptor ligand or a receptor.

127. The polypeptide conjugate of claim 129 or 130, wherein the polypeptide is an antibody or a portion thereof.

128. The polypeptide conjugate according to any one of claims 129-131, wherein the polypeptide is an antibody or an antibody fragment with a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain, a light chain, a constant chain; a peptide; or a cyclic peptide.

Images